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Full text of "Report of program activities : National Cancer Institute"

A!S'iA!«YSlS, OF PROGRAM ACTIVmES 
MAJIOKAL mSHTUTES OF HEALTH 



IMATlOHAi. CANCER IMSTITUT'E 



NATIONAL tWSimJTES OF HEAIJTH 
PUBLIC HEMIH SEK.^ICE 
S DKl^ARTWENT OF mMJK EDUCATION, AND WELFARE 






PAGE I 



1. National CaHce'r Institute 2 . Laboratory of Bi ol»gy 

INSTITUTE LABORATORY OR BRANCH 



3. Office of the Chief U. g. ItQQ 

■SECTION OR SERVICE LOCATION (OTHER THAN ©ER^ NO. 

■* BETWESDA). 

6. Biological Studies on the Factors Involved in the Development and 
PROJECT TITLE ' Growth of Tumors in Experimental Animals. '^ 



7. H, B. Anderv« it 

PRINCIPAL INVESTIGATOR (S) 



8. 



OTHER INVESTIGATORS ; 
fi PROJECT DESCRIPTION 

Testicular Tum»rs 



The precsding' report suniinarized an investigation with strain C 
mice designed to utilize testicular tumors to study a dependent tumor. 
The study is now virtually completed and will be published as soon as 
time is available for writing. 

Findings reported last year remain unchanged with the exception 
of those dealing with transplantation. This phase of the study is 
still in progress but the results to date are as follows. 

Sixty-nine tumors were transplanted to ascertain whether they • 
were able to grow in estrogenized hosts only or whether they were a* 
longer dependent upon the hormone. Of PO tumors transplanted fr»m 
mice that Were carrving stilbestrol pellets, 8 failed to grow in either 
estrogenized or nonestrogenized hosts, 2 were dependent and 10 became 
independent. 



h7 

]S^h3.^ PROJECT DESCRIPTI'^N (Cont.) 

r ^ Of U9 tumors that continued to grow after removal of pellets, 

' «^ 5 failed to grow, 2 were dependent and U3 were independent. 

It is seen that as transplantation proceeds more tumors be- 
come independent. Consideralily m^ve work is necessary to analyse 
the. data but the results reported here are sufficient to establish 
cloarly' that earlier reports in the literature concerning the high 
degree of dependency of this type of tumor were probably the result »f 
transplanting tumors before they had achieved independence in their 
original hosts. This finding with testicular tumors raises two questions. 
First, have other types of dependent .t\amors been transplanted too early 
or do they remain dependent in 'their original hosts? Second, what 
factors are involved in the relatively slow development of autonomy 
in testicular tumors? Many skin tumors of mice are known, to evolve 
from papillomas, but the difference between papilloma and carcinoma 
can be detected histologically. Thus far no such difference is 
detectable between dependent and independent testicular tumors. The 
problem is complicated further by the observation that during a 
16 weeks sojourn in mice a stilbestrol pellet produces histologic 
changes in testicles which persist for many months after removal of 
the pellet. Tumors may even develop in a few of these altered testicles. 

xipparently in some tumors the development of malignancy is 
a slow process and such tumors may be used to test the effi. . . • c - 
of preventive procedures. 

The preceding report mentioned three experiments started 
to study testicular tumors. These are still ^in progress, but 
have. progressed sufficiently to supply results. The first was' per- 
formed to ascertain the susceptibility of various F-, .hybrids derived' 
from strain C and other inbred strains. None has proved to be as 
susceptible as strain C but (C X DBA/2)F and (C X '^)F-, hybrids are 
developing testicular tumors in sufficient numbers to indicate their 
value as test animals. I^brids between strain C and strains C57BL, 
I, or RIII, are developing few tumors. 

The second experiment censisted of giving stilbestrol pellets 
to other Inbred strains to compare their susceptibilities to induced 
testicular tumors.. .None has aporoached strain C in this respect. Of 
the strains tested strain C exhibited a unique susceptibility. All 
strain C animals developed testicular tumors before a tumor arose in 
any other strnia. A few tumors have «ccurred in DBA/2 males and one 
in a strain C3F male. Strains RIII, y, I and 0^7 BL are t©o susceptible 



PROJECT DESCRIPTION (Cont. ) ^ .. ^ 

to the toxic effects of stilbestrol. 
- # • 

The third study was designed to ascertain the influence of 
age upon susceptibility. Results between young (2 months old) and 
older mice are not available but mice less than one month of age were 
very svBceptible to toxic effects of stilbestrol and those that 
survi-^ed have not developed tumors. The chief objsctive of this 
experiment, however, is to determine the susceptibilities of 2, 6 
and 12 month old animals. 

Breast Tumors. 

But on§ experiment gave sufficient findings during the year 
to report at%bis time. This experimeRt consisted of administration 
of stilbestrol and .progesterone to agent-free strain C3K females 
to ascertain their influence upon the occurrence of breast tiimors. 
Our untreated breeding females show a breast tumor incidence of. less 
than ^fc. 



Group No. of 
mice 


TJo. with 
subcutane- 
ous tumors 


Average 
age in 
months 


No. 
living 




10'^ stilbestrol 
pellet 


Ul 


10 


2li 


18 




10'? stilbestrol 
pellet plus 
progesterone 


39 


2U 


20 


9 




20^ stilbestrol 
pellet 


37 


10 


19 


XI 





/ 



PROJECT DESCRIPTION ("C^rit.) 

Living mice are between 21 and 26 months of age. Ttmifrs 
are called subcutaneous tumors because they have not been diagnosed 
histologically. 

It is seen that thus far both 10^ and 205? stilbestrol 
pellets have increased the incidence of tumors. Also, progesterone 
plus IC?^ pellets produces more tumors than do 10^ pellets alone. ■ 

This experiment was performed to determine the influence 
of progesterone when administered to agent- free mice. The aim 
of the work was to apply the findings to our colony of wild house 
mice which are known to carry the mammary tumor agent but" develop 
few breast tumors when bred or given pellets of stilbestr»l. It 
is considered essential to obtain breast tumors in these animals 
and to test the tumors for the presence of the agent. Exploratory 
findings with agent-free strain G3F mice suggest the use of b«th 
stilbestrol and pr%.gesteroae in the wild mice. 



PROJECT RE.ORT FOR^ (C«nt.) 

SERIAL no; 



11. 

BUDGET ACTIVITY: 



RESEARCH X ADMINISTRATj ON 

REVIEW •^. APPROVAL TECHIMICAL ASSISTANCE) 

12. 



COOPERATING UNITS, OF T^'E PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIFS, OR PE-^SONNEL FOR THIS PROJECT IN EITHER 
1956 or 1957» 



13. 

IF T~aS PROJECT RESEMBLES, COIr^LEMENTS, OR PAJliLLELS RSSE/vRCH Da-JE 



ELSEvrfHERE IN THE PUBlIC HKILTH SERVICE (¥..T!"OUT INTERCHANGE OP 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESS'RCH: 



i^OJECT REPORT FORM (C»nt. ) 



SER,L\L NO. 



15. , _ 

PUBLICATIONS OTHER THAN ABSTRACTS FROK THIS PROJECT DTJRIM} 



CALENDAR YE/lR 1955 



Andervont, H. B. j Biological background for experimental work •n 

tumors. In; Canadian Cancer Conference, Vol. I; 2-2U. Academic 
Press, Inc., New York, N. Y. 1955» 

\ndervont, F, B. and Dunn, T. B.: Transplantation of hepatomas in 
mice. J. Nat. Cancer Inst., l5t l5l3-l52U, 1955. 



16. 

HONORS AND AWARDS TO PERSONAL RELAtING TO Tf^IS PR-'JECT DURING 
CALENDAR YE\R 1955. 



H. B. Andervont, President, American Association for Cancer Research. 



PAGE 1 

... „ PROJECT REPORT FORM 

1 . National Cancer Institute 2 . Laboratory of Biology \ 

INSTITUTE ~ LABORATORY OR BRANCH 

3 / ^ ' ' ■■"'"':■ . . '' h . , ' ' ' $. hoi 

,$ECTioiJ ok ^mm' ; — ' LocA'rloM(tf 'o^m ium feETH.) ' &tk. Mo. 

6 . Sedimentation behaviour and homngefteity of enzymes and other compounds 
, PROJECT TITLE in crude 'solution (continUeti project). 



7. Drg. G. F. Hogeboom and E.L* Kuff 

PRINCIPAL INVESTIGATOR (3) ., ' , ' 

8. Drs. W. C. Schneider, B. R. Hill, J. L» ^ lrvin; Miss V. F. Embrey 
OTHER IWESTIGATORS 

9. PROJECT .DESCRIPTION 

This. project is a collaborative effort and includes several phases, some 
'of •which are not yet comgleted. 

Methods employed ; As described previously, the method employed involved 
centrifugation under convection free conditions and subsequent sampling 
and analysis of the fluid column at successive levels from top tn 
bottom. The techniques were devised in this laboratory (G. ^. ^ogeboom 
and E. L. Kuff, J. Biol. Chem . 210, 733-7^1 (19^1i)). 

Major Findings and Proposed Course of ^^roject t 

(a) In collaboration with Dr. Borroughs R. Hill of the City of 
Hope Medical Center, an investigation was made of the sedimentation 
behaviour of the lactic dehydrogenase present in the serum of normal 
and leukemic patients. (It had been previously shown that the lactic 
acid dehydrogenase content of leukemic serum was greatly elevated). 
The centrifugation studies conducted in thxs laboratory demonstrated 
clearly that the enzyme present in leukemic serum behaved in exactly 
the same fashion (within the limits of experimental error) as dii 
lactic dehydrogenase obtained from normal serum. It is our under- 
standing that Dr. t'Ih will continue the work, using electrophoresis 
and other methods of fractionation, and that the results will be 
published when this latter phase is completed. 



PROJECT DESCRIPTION (Cont.) 

(b) Several years ago, it was shown in this laboratory that over 
50 per cent of the total nitrogen of isolated liver mitochondria was 
■ in- the form of soluble proteins." It was also shown that the major ta.-"' 
component of the mixture (apparently a monodisperse globulin) was ''■ 
absent from the mitochondria of a hepatoma. A number of attempts to 
determine the significance of the latter finding, using standard 
techniques of protein fractionation, have not met with sufficiant success 
to warrant publication. It is also true that liver mitochondria contain 
a number of enzymes that can be obtained in the soluble phase. With 
the new technique of analytical centrifugation mentioned above, initial 
studies have been made of the DPN-cytochrome c reductase and glutamic 
dehydrogenase released from isolated mitochondria by treatnBnt with 
sonic oscillations. The results thus far indicate that DPN-cytochrome c 
reductase is polydisperse and thus is probably firmly bound to the 
structural fragments pf mitochondria, .At the centrifugal forces thus 
far employed, glutamic dehydrogenase" has not been sedimented'far 
enough to warrant any firm conclusions as to its characteristics. 

During the next year, it is planned to make a further study of the 
soluble fraction obtainable from liver mitochondria, with respect both 
to the enzymes present (e.g., ribonuclease ani desoxyribonuclease) and, 
if possible, to the protein that is absent fr«m hepatoma mitochondria 
but present in l-^^-rge amounts in liver mitoc'- ondria, 

(o) It is also planned to investigate the sedimentatL on behaviour 
of a number, of enz.ynes localized in' the soluble fraction of the 
cytoplasm, including several of those which are involved in purine 
metabolism and have not been obtained in a pure state ("cf. Schneider, 
W. C, and Hogeboom, G. ";i. , Intracellular distribution of enzymes IX. 
Certain purine metabolizing enzymes, J. Biol. Chem . 19$, I6I-I66 (1952)). 

(d) In collaboration with Dr. J..L. Irvin, a study will be made 
of the heterogeneity of desoxyribose nucleoprotein isolated from the 
nuclei of liver cells. Some work on the rate of incorporation of 
labelled amino acids into the nucleoprotein (in collaboration with 
Dr. Stetten) is alsc contemplated. 

Significance to Cancer Research ; It is hoped that the work 
outlined above will provide a rational background to future similar 
studies of neoplastic tissues. 



PROJECT REPORT FORM (Cont'd.) 

10. hOl 

SERIAL NO. 

11. 



BUDGET ACTIVITY;,. 

RESEARCH X AH-lINISTRATION 

REVIEW lit. k^?m'VkL TECHNICAL ASSISTANCE 



12. 



COOPERATING UNITS OF THE PUBLIC HEAIjh SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FCR THIS PROJECT IN EITHER 
1956 or 1957 



13. 



IF THIS PROJECT RESEMBLES, COMPLE^■IENTS , OR P:\R\LLELS RESEARCH DONE 
ELSEWHERE IN THE PHBLIC HEALTH SERVICE (wriHOUT INTSRCHANCE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



PROJECT RE^T'RT FORM (Cont'd) 

m. J4OI 

SERI'^L NO. 

15. : . 

PIIBLIC-TIONS OTHER THAN ABSTRflCTS FROM T'^S PRUSCT DURHJG CALEJID/iR 
YF'VR 1955 

E, L. Kuff, G. 1^'. Hogeboom, and M. J. Striebich, the Sedimentation 
biihaviour of alcohol dehydrogenase and urease in crude solntions, 
J. Biol. Cham, 212, ii39-UU8 (1955). 



16. 

HONORS AND AWARDS TO PERSONNEL RELATING TO TFIS PROJECT DURING CALEND/vR 
YEAR 1955. 

F»r honors and awards, see Annual Report of Kuff, hogeboom, and Dalton. 



PAGE 1 
PROJECT REPORT FORM 



1, National Cancer Institute 2 . Laboratory of Biol»gy 

IICTITUTE LABORATORY OR BRANCH 

3. Cellular Biology Section k ^ $• U02 

SECTION OR SERVICE LOCATION(IF OTHm TH^IN BETHESDAj SER. NO. 

6. The relation between cell chendstiy and cell structure 

PROJECT TITLE ^ ' ' " " 



7« E« L. Kuff, G» ^» Hoge^oom^ A. J. Dalttn 
PRINC IPAL I WEST IGATOR (S ) ^~" 

8. M. F. Embrey 



OTHER IWESTIGATORS 

9* PROJECT DESCRIPTION 

Objective ; (a) To determine the distribution of enzymes and other com- 
pounds of biochemical importance among the particulate structures re- 
leased by mechanical disruption of normal and malignant cellj (b) to 
characterize morphologically the intracellular structures thus released; 
and (c), thus to arrive at an interpretation of the fine structure of the 
cells in terms of biochemical function. Results obtained by the use of 
more precise and sensitive methods for investigating the sedimentation 
properties of cytoplasmic particulate material have strengthened the con- 
clusion that these particles fall into a relatively limited number of 
distinct classes, each of which exhibits a peculiar array of biochemical 
properties. Simultaneously, electron microscopy has greatly broadened 
our knowledge of the fundamental structural units of the intact cell. 
Correlation of these two fields of information is an obvious necessity. 

Methods employed ; The following methods, developed in this laboratory, 
have been employed for the observation and separation of groups of 
cytoplasmic particles from homogenates of tissue: (1) size distribution 
analyses of the particles based upon the rates of sedimentation of their 
specific biochemical characteristics (see the last annual report for 
description of method); (2) differential centrifugation according to 
schedules based upon the size distribution analyses; and (3) density 
gradient fractionation. In the last method, which is currently under 
investigation, a small volume of homogenate is layered over a pre- 
viously prepared continuous sucrose density gradient, and the 
particles centrifuged (in the SW-39 rotor of the Spinco Model E ultra- 
centrifuge) to their iso-density levels. Fractions obtained by any 



PROJECT DESCRIPTIOW (Cont.) 



of the above methods, and characterized biochemically, have been 
prepared for election microscopy, by fixation with osmic acid and em- 
bedding in methacrylate. Structures observed in thin sections of the 
fixed fractions were compared with'lihose found in the whole horaogenates 
and in the intact cells. 

Results ; As descrifesd in the last annual report, size distribution 
analyses of the cytoplasmic particles in rat liver homogenates had 
demonstrated three major classes of particles. Of these, one . undoubtedly 
corresponded to the mitochondria. Electron microscopy has now revealed 
that the mitochondria are well presei^^-ed during the process of homogeni- 
zation in 30=^ sucrose, a fact of some importance in interpreting the ' 
many results hithert^ obtained' *)y differential 'centrifugation. The • 
nature of the other two groups of particles is not yet understood. One 
of these, rich in acid phosphatase and uricase, has been tentatively 
identified with elements of the ergastoplasm of the intact cell. There 
is some evidence that the other group, containing the non-mitochondrial 
DPN-cytochrorae c reductase activity of the cell, may be made up at least 
in part by structures derived from the Golgi complex. 

Considerable attention has been devoted to the question of the 
intracellular distribution ' of ribonucleic 'afcid (RNA) in view of the 
mounting evidence for the participation tf this material in protein 
synthesis. RNA has bten found to be associated with all three of the 
above-mentioned particle groups in liver horaogenates: about 10/^ with 
the mitochondria, 30% with the "aoid phosphatase particles", an4 '^0% 
with the group of smaller particles rich in DPN-cytochrbme c reductase. 
The remainder is riot sedimentable in 30°^ sucrosd at the speeds attain- 
able with the SW-39 rotor. Ifoon severe starvation, the RNA associated 
with the acid phosphatase particles disappears almost completely, while 
that of the smaller particles remains Tit is known that starvation re- 
sults in loss of the ergastoplasmic elements of the liver cell as 
examined in situ). In the very ra-^id growth situation provided by 
Strain L fibroblasts grown in tissue culture ^by the method of Dr. Harry 
Eagle)', preliminary studies have shown that iTiOSt of the crboDlasmic 
RNA is in. the non-sedimentable fraction. 

The sedimentation characteristics of liver gl^roogen as it occurs -> 
in sucrose homogenates has also been studied. If a density of l*$i gm,/cm. 
is assumed, the relatively polydisperse glycogen particles, show an 
average "molecular weight" of about 20,000,000, a value from $ to 10 times 
those reported for liver glycogen isolated bv standard chemical methods. 



PROJECT DESCRIPTION (Cent. ) 



Significance to Cancer Research ; It is felt that a combination of 
the techniques of cellular fractionation and electron microscopy will 
result in a considerable extension of our knowledge of the spatial and 
functional arrangement of intracellular materials. This information, 
p?n*ticularly with regard to components such as ribonucleic acid, may 
ife fel^'ftol ip, u^derstand?^^ tiie. proeesses df .celluLat groi^hj- &M in 
■plr^tidniar, IM &if I'erdTitxa^ing tfeweori ndlr'ffi'ai an'S- afcnofmal ftgefearastfev 
of protein synthesis. In addition, the ability to correlate appear- 
ance under the electron microscope with known biochemical properties 
would greatly facilitate the investigation of many situations not now 
accessible to study - for example, the very early stages of malignancy 
where changes might be restricted to too few cells to analyse by 
presently available methods. 

Proposed course of the project ; During the coming year it is proposed 
to investigate in more detail the sequence of events which occur at the 
time of disruption of cells during the preparation of homogenates. Pre- 
liminary observations of unbroken and partially broken ceils in homo- 
genates indi6«tes that this approach may be the best means of determin- 
ing the intracellular situs of origin of many now unidentified particu- 
lates. This aspect of the project will be carried out with the help of 
a new microtome for thin sectioning and in collaboration with Dr. F. S. 
Sjostrand who will be visiting here from the Karolinska Institute of 
Sweden. 

With the more precise methods of cell fractionation now available, 
it it proposed to continue the study of the major ribonucleic acid con- 
taining fractions of normal cells and to compare them with the correspond- 
ing fractions derived from rapidly growing ceils, both normal and malig- 
nant. An interesting situation is provided by the liver cells of 
animals which have been subjected to severe fasting. As mentioned above, 
the ergastoplasmic elements are l«st from such cells, and there is a 
great reduction in the volume of cytoplasm. TTpon refeeding, theTe is 
a rapid resynthesis of cytoplasm without cell division , together with 
characteristic evolution of the reforming ergastoplasm. It is prooosed 
to compare the cytochemical changes occurring during this period with the 
findings in the more usual situations where growth is accompanied by 
cell division. 



PROJECT RE:^ORT FORM ' (Cont'd) 



10. 1;02 

SERIAL NO. 



11. 



Ri:SEa.RC;' "' ^ ,0'I>;iSTRATi-N 



12 

COOPER^'^TTNG Ili^lTS Oh' THE, p-TELxC iIMLTH SAVICS, OH OTHER ORG.UIiZi-^lUld, 
PROVILING FITOS, FACILITIES, OR PFRSOMSIL FCE T"IS FROJEGT iiv EITHER 
1956 or ]'?57 

D^". F. 3.. o.^o.=^*-,rand, Karolinsk? Institutet^ Sufd-n, 



■"'i? T^^3 mojECT ■Hi!;aii;t'lBfeV'GO>''PI'SS'ENTS, or P\R:\LLELS RESEfJlCF DONE 
ELSF^'-'ERE IN TIE F^'BLiC TTE'-lTt S '^JIVICE, (WITFOIlT INTERCHANGE OF 
PERSONNEL, FACILITIES OR FJltoS), IDENTIFY SUCH. RESE/JICH 5 ■ " 



PROJECT RE^'^RT FORII (Cont'd) 



lii. U02 



SERIAL NO. 
1^. 



RT'ELICATIONo OT^^R T'^-.N /IBSTR'lUTS FR'M T^-IS FRCJZCT DURING C/iLE^TD/Jl 
YE'i-R 10^5 

T-'ogeboom, G, 'W,^ ^.nd Ki:ff, E. L.- , Relat-ion between nell structure and 
cell chemistry. Federation Proceedings lIi ; 633-638 ri955). 



16. 



HONORS AND WARDS TO PERSOM'JEL REUTING TO T'-'IS ^^ROJECT DITRING CALENDAR 
YE!\R 3-955. 

(a) G. '-. Hogeboom: By irvitation, addressed the Open Scientific 
Session of the Federation of American Societies for Experimental 
Biology, San Francisco, April, 1955. (See ref . l5) 

(b) E. L. Kuff , by invitation, presented paper (with G. '-^^ Hogeboom 
as co-author) at Fourth International Sjnnposium on Enzymes: ^"nits of 
Biological Structure "- Function, on "Sedimentation and biochemical 
characteristics of cytoplasmic particulates." (To be published in 
Proceedings of the Symposium) 

(c) G. H. Wogeboom acted as chairman of the Session on Enzymes and 
Cell Structure at the above-mentioned Fourth International S3miposium 
held at the Henry Ford Hospital. 



PAGE 1 



PROJECT REPORT FORM 



1» National Cancer Institute 2 . Lalioratory of Biology 

INSTITUTE LABORATORY OR BRAMCH 

3. Cellular Biology Section h .__ ^ « UoU 

SECTION OR SERVICE LOCATION(IF OTHER THAN BET"ESDA) SER. NO. 

6. 0xidatiY8 metabolism in various cellular structures of normal and tumor 
PROJECT TITLE tissues. 



7. Ruth K. Kielley 

PRINCIPAL INVESTIGATOR(S) 



OT"ER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Title ! Oxidative metabolism in various cellular structures of normal 
and tumor tissues. Xanthine Oxidase. 

Ksthods employed ! Xanthine oxidase and DPNH dehydrogenase enzjrmes were 
isolated from the supernatant fracti'^n of liver cells bjr protein 
fractionation methods described in a previous annual report. The be- 
haviour of these enzymes in response to changes in environmental con- 
ditions was studied by quantitative measurement of their activities* 
Xanthine oxidase activity was measured spectrophotometricaily by 
following the rate of uric acid formation from the oxidation of xanthine 
at 290 mu» DPNH dehydrogenase activity was also measured spectro- 
photometricaily ■fey following the decolorization rate of dichloropheno- 
lindophenol at 600 mu. The extent to which these enz,ymes couple with 
various nitrogen compounds was determined by estimation of either the 
reduction products formed or the reactants disapoearing. The methods 
were chemical, spectrophotometric or enzymatic depending upon the 
nature of the product analyzed. Molybdenum in trace amoimts was de- 
termined by a special modification of the colorimetric thiocyanate 
method. 



PROJECT DESCRIPTION (Cont.) 



Objectives ; (1) To obtain information on the mechanism of reduction 
of various nitrogen compounds by xanthine oxidase and Ijy other as yet 
unidentified enzymes of liver and tumor and the relation of molybdenum 
to the reduction process, (2) to identify and characterize other 
enzymes in liver and tumor tissues which catalyze similar reduction 
reactions and (3) to study ways and means by which knowledge gained 
in these investigations can be applied to cancer problems. 

Major Findings ; It has been found that the xanthine oxidase of liver 
can be coupled to the reduction of various types of nitrogen compounds 
representing a wide range of oxidation-reduction levels. The mechanism 
of electron transfer to these nitrogen compounds appears to require 
molybdenum as a specific metal activator, ^^ophosphate inhibited 
these reductions indicating that the reduction process is metal 
catalyzed. It was shown that the molybdenum present in the enzyme 
is very tightly bound. All attempts to remove it reversibly have so 
far been unsuccessful. Further evidence that the reduction of nitro, 
nitroso and probably other related groups including the azo group 
proceeds through an intermediate step between the dehydrogenase and 
the final transfer of electrons to the N acceptor was obtained b^ the 
demonstration that the rate of reduction was not directly proportional 
to the total dehydrogenase activity (xanthine and DPNH) . In the re- 
action of the enzyme with ox^rgen or dyes such as methylene blue or 
dichlorophenolindophenol on the other hand, the total dehydrogenase 
activity was the rate-limiting reaction. Examples of the types of 
nitrogen compounds reduced by liver xanthine oxidase and their re- 
lative reduction rates are as follows: Inorganic Series ; nitrate 18, 
nitrite 2h, hydroxy lamine 5h. Organic Series ; p-nitrophenol 3$, 
p-nitrosophenol 2^3, p-dimethylaiuinoazobenzene (DAB) 20, 2,$-dinitro- 
phenol 108, 2,It-dinitrophenol 6^, 2,6-dinatrophenol $0, In general, 
it may be said that for the oxidation-reduction levels they represent, 
the organic compounds wore considerably, more reactive than the in- 
organic. The azo group of DAB was significantly reduced although 
at a relatively slow rate. With the dinitrophenols, the position of 
the substituents in the ring greatly influenced the reactivity of the 
compound. 2,5-dinitrophenol containing nitro groups para with respect 
to each other was more active than its isomers. 

Further studies on nitre and nitroso reductases of liver 
confirmed previous suggestions that a second enzyme was present in the 
supernatant fraction of liver cells. The enzyme was found to be a 
flavoprotein specifically catalyzing the oxidation of DPNH in the 
presence of suitable electron acceptors, but unlike the great bulk 
of diaphorase activity found in the liver, this flavoprotein has the 



PROJECT DESCRIPTI'-^N (Cont. ) 



unique property of being able to couple with nitro and nitroso 
compounds (other N-containing groups have not yet been tested). Al- 
though xanthine was not oxidized by this enzyme, there is a suggestion 
that xanthine oxidase is closely associated with this second flavo- 
protein enzyme since the latter is released from crude xanthine 
oxidase preparations by heat treatment. 

The effect of TSPA (N, N' , N' ' -triethylene thiophosphoramide) , 
an anti -cancer agent, on the aerobic oxidase activitj'- and on 
p-nitrosophenol reduction catalyzed by liver xanthine oxidase was 
tested tQ determine whether either or both of these electron trans- 
ferring pathways of the enzyme might be affected. The results were 
entirely negative showing neither inhibition nor activation. 

Preliminary work on the determination of xanthine oxidase 
levels in blood showed, that the activity was limited to the plasma 
component and that it could be conveniently measured spectrophoto- 
metrically provided the sensitivity of the instrument were stepped 
up sufficiently with a photomultiplier. The feasibility of determin- 
ing xanthine oxidase levels in blood was looked into because there is 
some interest in examining the blood levels in cancer patients. 

Significance to Cancer ; Xanthine oxidase as one of the principal 
enzymes of purine catabolism is of special interest in the cancer field 
because of its relation to nucleic acid synthesis and breakdown. Its 
presence in excess 'or absence, its inhibition or stimulation might 
possibly reflect on the chemistry of normal or abnormal growth. Haddow 
et al. found for example, that xanthopterin, a potent inhibitor of 
xanthine oxidase in vitro, often induced hypertrophy of the kidney 
in rodents. He also observed that xanthine oxidase concentrates from 
cow's milk when injected into mice with spontaneous mammary tumors 
produce apparently, anti- tumor effects. 

Recent developments in the chemotherapy of cancer and leukemia 
with various purine analogs point to the importance of xanthine 
oxidase as one of the cnntrolling factors in the effectiveness of the 
treatment. Dietrich and Shapiro found that flavotin, a riboflavin 
analog, potentiates the carcinostatic action of 8-azaguanine through 
inhibition of tumor xanthine oxidase (7^5 mammary carcinoma). This 
inhibition results in the accumulation of xanthine which in turn 
inhibits guanase, an enzyme destroying 8-azaguanine by deamination. 
The over-all effect of flavotin then, is apparently one of preventing 
the destruction of the carcinostatic purine. In this connection one 



PROJECT DESCRIPTION (Cont. ) 



might consider the possibility of limiting the synthesis or activity 
of xanthine oxidase by antagonism of the molybdenum component rather 
than the flavin component of the enzyme. The toxicity experienced 
■with flavotin may very well be due to inhibition of a large number of 
flavoprotein enzymes whose activities are more directly and vitally 
concerned with the energy metabolism of cells than is xanthine oxidase. 
As far as it is known, the only flavoproteins containing molybdenum 
in mammalian tissues are xanthine oxidase and possibly aldehyde oxidase. 

Another aspect of the relationship of xanthine oxidase to cancer 
is the ability of the enz"\Tne to reduce the azo .group of carcinogens. 
After the results on the reduction of DAB were reported, it was learned 
that similar observations were being made at the Chester Beatty Cancer 
Institute where an extensive progr.'^.m of. research on xanthine oxidase 
is being conducted. Furthermore, their observations indicate that 
there is an inverse relationship between rate of reduction and carcino- 
genicity (private communications). Mueller and Miller have shown that 
DAB is also reduced by an enzyme in liver microsomes, presumably 
TPM-cytochrome c reductase, a flavoprotein. It is highly probable 
that other metall«-flavoproteins of the xanthine oxidase type such as 
liver aldehyde oxidase and the DPNH dehydrogenase described in this 
report can also participate in reductive detoxications of azo carcino- 
gens. The relationship between carcinogenesis caused by DAB and the 
riboflavin level of the diet becomes clearer when it is learned that the 
enzymes involved in the reduction of DAB, as far as we know, are 
flavoproteins. 

Proposed research ; On the hypothesis that tumor growth may be influenced' 
by changes in the balance and activities of critical enzymes in t-hese ■ 
tissues, it is proposed that investigations be made on the effect of 
mimosine, an iinnatural amino acid, on animals bearing soontaneous tumors. 
Studies en the physiological effects of t;'is compound are limited to 
observations on rats in which the symptoms were those of a deficiency 
disease including marked alopecia, diminished growth rate and in many 
cases of long duration, cataract. The mode of action in terms of 
enzyme systems affected is unknown, but on the basis of its known 
structure (3-hydroxy, U-Oxy, (Uk), 1-pyridine alanine), several possi- 
bilities can be considered. The most likely sites of antagonism wouJ^d 
appear to lie in enzymatic reactions in which pyridoxine is implicated. 
There is already work by Dietrich and Shapjro on the carcinostatic 
properties of (-'.esoxypyridoxine, a pyridoxine analog and antagonist. 
Other possibilities are that mimosine may interfere with protein 
synthesis or amino acid metabolism involving phenylalanine or tyrosine. 
At the present time, arrangements are being made with Dr. Evan Horning 
to obtain the compound in quantity from imported plant material. 



PROGRESS DESCRIPTION fCont.) 



Some investigations on the xanthine oxidase levels in blood 
plasma of mrmal, non-cancerous and cancerous subjects are being 
considered in collaboration with Dr. F. M. Kalckar, The purpose is 
to determine whether xanthine oxidase levels can be correlated with 
the degree of differentiaticn typical of the tumor. 

From the standpoint of cellular structure and function, it is 
of some interest to know whether enzymes occurring in one part of the 
cell can be coupled to enzymes in another structure of the cell. In 
the intact cell where enzymes are acting in heterogeneous systems, 
such a possibility does not seem to be out of reason. The xanthine 
oxidase system would appear to be an appropriate model for experiment- 
ally testing out such a possibility. There is evidence that in 
mammalian tissues, "xanthine ocidase" consists of two components, a 
dehydrogenase and a terminal oxidase system. The purified xanthine 
oxidase of rat liver is an enzyme no longer associated with its 
natural terminal oxidase system, but in the process of separation and 
purification, has become an oxidase reacting directly with atmospheric 
oxygen. The nature of the natural terminal oxidase system of xanthine 
oxidase has not been determined. It would be of great interest and 
significance if it were localized in the mitochondria for the linkage 
would then provide a possible means for the utilization of a large 
amount of energy resulting from the oxidation of xanthine and hypo- 
xanthine for useful cellular work through the mechanism of oxidative 
phosphorylation. With these ideas in mind, it is proTD: sed that ex- 
periments be tried to determine whether enzymatic coupling can be 
achieved between the xanthine dehydrogenase of the supernatant fraction 
and the electron transport mechanism in mitochondria. 



PROJECT REr-ORT FORM (Cont'd) 



10. 



kok 



SERIAL NO. 



11. 



BUDGET ACTIVITY: 



RESEARCH X AMINTSTRATION 

REVIEW /k A'-^RQVAL TEC^-^ICAL ASSISTANCE 



12. 



CO-OPERATING UNITS OF THE PUBLIC, HEALTH SERVICE, OR OTHER ORGAN IZ -,T IONS, 
PROVIDING FUNDS, FACILITIES, OR "^ERSOMNEL FOR T^'IS PROJECT IN EITHER 
1956 or 1957 



13. 



IF T-'IS PROJECT RESEMBLES, COMPLEMENTS, OR "-ARALLELS RESEARCH BOM 
ELSEnPERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHiVNGE OF 
PERSONNEL, FACILITIES OR FU1©S), IDEivTIFY SUCH RESEilRCH: 



PROJECT REPORT FORM (Cont'd) 



lii. liOii 



SERIAL NO. 



15. ^ 

PUBI.ICAT IO^B OTHER TPAN ABSTRACTS FROM r'lS PROJECT DURING CALENDAR 
YEAR 1955 



Klelley, R. K., Purification of liver xanthine oxidase. Federation 
Proc, lU: 235 (1955). 

Kielley, R. K., Piirification of liver xanthine oxidase. J. Biol. 
Chem. , 216: Uo5 (1955). 

Kielley, R. K., Reduction of 2,U-dinitrophenol by liver xanthine 
oxidase. Third International Congress of Biochemistry, page ^^ 

(1955). 



16. 



HONORS 'IND A¥\RDS TO ^ ^^ONNEL RELATING TO T'-^IS PROJECT DIRING CALENDAR 
YKAR 1955. 



PAGE 1 
. . PROJECT REPORT FORM 
1, National Can<j«r Instituta 2« Laboratory of Biology 

it^tJ i TWE : t^wmfM oft mm 

3. Cellular Biology Section It* $ U04 

3EcT!dK &k SffiVKg ' — t.«)AfTt)H(l^ (^rMi TriAU'BfiM.) Sfcft.No. 

6. Electron mieresawjy and phas^e contrast microacopy of normal and malignan t 
PROJECT T.fTLE cells 

7. A» J. Dalton and Marie D. Felix 

PRINCIPAL INVESTIGATOR (S) ' 

8» J» Bronte Gatenl^, Harry Eagle^ Clifford Grobstein and W» Ray Bryan 
OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION 



A comparative study of the Golgi complex with the 
electron microscope 

The objective of this project is to determine the essential 
variations in the fine structure of the Golgi complex in representative 
cell types in several phyla of the animal kingdom. 

The methods employed are fairly well standardized now - fixation 
in chrome-osmium, double embedding in fom^l-agar and n-butyl methacrylate 
followed ¥y thin sectioning for study with the electron microscope. 

The major finaiings have l|eai the demonstration that (1) the double 
membranes and small vesicle components &f the Golgi comples were 
regularly present Ih all vertebrate cell types studied but the large 
vacuolar component occinred regularly only in highly specialized cell 
types. (2) The fine structiire of the Golgi complex of vertebrate and 
invertebrate cells is basically similar, the double memTirane systems 
and the small vesicle component being present in both. (3) The 
walls of contractile vacuoles of protozoa and soonges possess double 
membrane systems and small vesicles similar morphologically to the 
Golgi complex of vertebrate and invertebrate cells. 



PROJECT DESCRIPTION (Cont.) 

The significance of these findings is related to the over-all ■ 
concept which activates much of the work of the unit - the concept 
that in order to understand the mechanisms involved in the development 
of neoplastic cells, much greater knowledge of the structure and function 
of normal cells is needed. The results obtained from this study suggest 
first that the Golgi complex is universally present in animal' cells 
and second that the Golgi complex is possibly involved in control of 
water balance in cells generally since its structure is so similar to 
that of the walls of the contractile vacuole ^of many protozoa. 

It is proposed to study a series of somatic cells ef inverte- 
brates and the contractile vacuoles of several more protozoa to 
make the above generalizations somewhat more secure. 

A comparison of normal and malignant cells with 
the electron microscope 



The object of this project was to determine whether any 
significant differences in the fine structure of normal and neoplastic 
cells could be detected. 

The major findings have been that while variations in the amount 
of various cell components were noted, neoplastic calls, as might be 
expected possess all the essential components of normal cells. The 
fine structure of chromatin, nucleoplasm and nucleolus was found tc 
be the same in both. Both normal and' neoplastic cells possess 
ergastoplasm in variable amounts but there was a tendency in what 
might be termed anaplastic tumor cells for the small granule component 
of cytoplasm (thought to be responsible for cytoplasmic basophilia) 
to lie free ifi the cytoplasm rather than to be associated with the 
membranes of the eragstoplasm (the usual situation in normal Bells), 
The Golgi complex in tumor cell types which retain some degree of 
function possesses all three c®mponents, (double membrane system, small 
vesicles and large vacuoles) but the Golgi complex of more anaplastic 
tumor cell types possessed only the double membrane system and small 
vesicles. 

• ■ „ 
: While it was not expected that a significant qualitative differ- 
ence between ■'normal ancJ neoplastic cells would be found, such a study 
was indicated since if such a difference were found it would be of 
considerable importance in diagnosis. If a real and consistent corre- 
lation is found between variations in the fine structure of the 
ergastoplasm and Golgi complex and th» degree of anaplasia of malig- 
nant, such a corj-elation could eventually be of some value to the 
CTtopathologist iii'44i^g'^osis. 

In the future i^is proposed to study a series of tumor cell 
types ef varying degrd,p' of anaplasia to determine whether the 
variations described above are consistently associateddin a general 
way with the degree of anaplasia ^ 



PR':^JECT DESCRIPTION (Cont.) 

Phase contrast and electron microscopy of tumor cells developing 
nutritional deficiencies in tissue culture 



The objective of this project is to determine whether there are 
any specific alterations in the fine structure of tumor cells during 
the development of a deficiency state resulting from the absence of a 
specific amino acid or vitamin. 

In addition to thfe usual methods for electron microscopy of 
tissue sections, a method of double embedding, first in formol-agar 
and then in methecrj/late, vias developed.- This double embedding 
method makes possible the accurate orientation of cells, tissues and 
organs and also greatly improves the preservation of cells on free 
surfaces and in tissue culture. During the development of the 
deficiency, living cells have be-n studied with phase contrast and 
comparable cells preserved for study with the electron microscope. 

The major findings to date are; (1) piodification of the 
fine structure of cells appear to be specifically related to a 
deficiency in a particular amino acid. For example, changes in 
mitochondria and the ergastoplasm occur in tyrosine deficiency which 
have not lieen noted in dying cells in control cultures nor in cells 
developing glutamine or folic acid deficiency, Cej.ls developing 
tyrosine deficiency have been studied in detail but the studies on 
glutamine, folic acid and glocuse deficiency are still in progress. 

The significance of these results, which are meager at present, 
is in the suggestion that specific changes may occur under these con- 
ditions and that these changes may be pinpointed in relation to bio- 
chemical and radio-isotope analyses. While these changes may in many 
cases be an "end result" and thus not necessarily indicators of the 
site of utilization or metabolism of a particular amino acid within 
a tumor cell, the very real possibility remains that the combination 
of electron microscopic, biochemical and radio-isotope studies may 
eventually give us this information. 

It is proposed to continue this study with a more detailed 
analysis of the changes induced by glutamine, folic acid and glujose 
deficiencies. This material is already fixed and embedded but not 
sectioned or examined. 



PROJECT ■DESCRIPTION (Cont.) 

A study of the cells native to the peritoneal fluid 

The objective of this project is to develop a greater under- 
standing of the structure, function and origin of cells native to 
the peritoneal fluid. 

In this study living cells ■were examined with the phase contrast 
microscope both as an aid to identification and as a prerequisite for 
study with the electron microscope. 

The major findings have been that the primary cell components 
of the peritoneal fluid are lymphocytes and macrophages. The intro- 
duction of foreign particulate matter to the peritoneal cavity is 
followed by an early rise in neutrophilic granulocytes which is 
followed in t\irn by a more prolonged rise in the number of macro- 
phages. Under these conditions numerous transitions between lympho- 
cytes and macrophages were founri. There was no evidence that meso- 
thelial cells transformed into macrophages. Neither was these any 
evidence that mesothelial cells contribute significantly to the cell 
population of the peritoneal fluid.. 

There has recently been a great increase in the number of 
papers published which described work using ascites tumors as tools 
in cancer research. It is surprising that in none of these has any 
significant attention been paid to the cells native to the peritoneal 
fluid. On the contrary, the idea that the majority of these cells are 
der.ivatives of mesothelium appears to have been accepted by many 
investigators in soite of the lack of evidence for such a derivation. 
In the study of the development of ascites tumors and in the associated 
problem of identification of tumor cells, the information obtainable 
by the application of the newer methods is important. Our work has 
made a beginning in this direction. 

It is proposed to develop this project further by determining 
the reaction of normal cells of the peritoneal fluid to the intro- 
duction of tumor cells and to study the early stages in the develop- 
ment of .ascites tumors, with both the phase contrast and electron 
microscopes. 

Other projects involving electron microscopy but which will be 
covered in detail by the investigators responsible for preparing the 
materials ai«e : 

(a) Electron microscopy of tissue fractions. Drs . Hogeboom 
and Kuff . 

(b) Electron microscopy of the process of embryonic induction. 
Dr. Grobstein 

(c) Electron microscopy of the Rous tumor. Dr. Ray Bryan 



PROJECT REPORT FORM (Cont'd) 

10. Uo6 

SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH X ADMINISTRATION 

REVIEW k k??ROVkL TECHNICAL ASSISTANCE 



12. 



CO-^^ERATiNG UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING RTlvIDS, FACILITIES, OR PERSOMEL FOR THIS PROJECT IN EITHER 
1956 or 19^7 

In cooperation with Prof. J.Bronte Gatenby, University Zoological 
Department, Trinity College, Dublin, Ireland. 



13. _ __ _ 

IF 'T WIS PROJECT RESE^''BLES, COMPLSI^IENTS, OR PARALLELS RESEARCH DONE 



ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT I'JTSRCHANGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



PROJECT REPORT FORM (Cont'd) 

ili. Uo6 

SERIAL NO. 

15. . 

P'^LIC^TIONS OTHER TH^N ABSTRACTS FROM THIS PROJECT DllRING CALEMDAR 
YEAR 1955 

Gatenby, J. Bronte, Dalton, A. J. and Felix, Marie D. ; The 
contractile vacuole of parazoa and protozoa and the Golgi 
apparatus. Nature, 176j 301 (1955). 

Dalton, A, J.; A chrome-osmi-um fixative for electron microscopy. 
.\nat. Rec. 121: 281 (1955). (Note. While this is of abstract 
length it is the definitive article on this subject). 

Felix, Marie D. and Dalton, A. J.: A phase-contrast microscope 
study of free cells native to the peritoneal fluid of DBA/2 mice. 
J. Mat. Cancer Inst. 16: Iil5-hii5 (1955). 



16. 

HONORS \m AWARDS TO PERSONNEL REUTING TO TWIS PROJECT DITRING ' 
CALENDAR YEAR 1955. 

Invited to take part in a symposium on "Mitochondria and other cell 
inclusions" sponsored by the British Society of Experimental Biology 
at Oxford, England, Sept. 19-23. *.t this symposium I was asked to 
serve as chairman of a section on the Golgi apparatus. 

Invited to take part in Symposium on "Cancer Cytology and Cjrbo- 
chemistry" soonsored jointly by the N. Y. Academy of Sciences and 
the Damon Runyon Memorial Fund for Cancer Research. 

Invited to serve as chairman of section on cytology (electron 
microscopy) American issociation of Anatomists Annual Meeting, 
Philadelphia, 1955. 

Invited to take part in symposium on cancer research. Harvard Univ. 
Medical School, Harwich, Mass., June, 1955. 



PROJECT REPORT 



l» National Cancer Instituta 2» Laboratory of Biology 

3 . Cellular Biology Section h . 5 . U07 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETH.) SER. NO. 

6. Precursors of Deox yribonucleic Acid (D NA) in normal, Regenerating an d 
PROJECT TITLE Tumor Tissues. 



?• Walter C. Schneider 

PRINCIPAL INVESTIGATOR (S) 



8. Mrs. Leona W, Brownell 



9. PROJECT DESCRIPTION 

Objective; To determine whether precursors of DNA exist in normal and 
tumor tissues and if so, to isolato and identify the compounds 
present and determine the metabolic pathways in which they aro inter- 
mediates. 

Methods! Tissues, obtained under anesthesia and frozen immediately in 
liquid nitrogen, are extracted with ice cold perchloric acid, Deoxy- 
ribosidic compounds are isolated from such extracts using both ion 
exchange and paper chromatography* The compounds are detected, 
identified and quantitated with the organism lactobacillus acidophilus 
R-26 used in conjunction with spectrophotometric and chemical tests. 

Fajor findings ; Using more refined methods of isolation it has bee;:) 
possible to show that deoxycytidine is present in rat liver and 
accounts for essentially the entire deoxvribosidic activity of 
extracts of this tissue. 

In contrast, extracts of the Novikoff hepatoma showed three 
striking differences in the type of deoxyribose compounds they con- 
tained. The deoxyriboside fraction accounted for only about $0 per 
cent of the microbiological activity of extracts of this tissue and 
contained, in addition to deoxycvtidine deox^oaridine and thymidine. 
The remainder of the microbiological activity has not yet been identi- 
fied but, judging from its behavior on ion exchange resins, probably 
contains either nucleotides, small polynucleotides, or phosphorylated 
([derivatives of nucleotides. 



PROJECT DESCRIPTION (Cont.) 



Studies of rat liver undergoing regeneration have shown' that the 
total amount of deoxvi'ibosidic compounds present is approximately 
doubled as early as 2k hours after partial hepatectomy, -Further- 
more, in contrast to normal liver. 2^-50 per cent of the total 
deoxyribosidic eompoiinds appear to be the unidentified nucleotide 
type . ■ - ; ' 

Significance to cancer research ; The fundamental problem of cancer 
research is to determine why cancer cells are able to multiply 
without restriction whereas the multiplication of normal cells 
occurs at a controlled rate, Deoxyribonucleic acid (DNA), more- than 
any other cellular constituent now known, must be intimately in- 
volved in the solution of these problems for several reasons. In 
the first place, DNA is not only a major comoonent of the chromosomes, 
but also occurs in the cell in a constant amount directly proportional 
to the number of chromosomes present. Furthermore, before cell ., 
division can occur, the entire DNA of the cell must be reduplicated 
to insure that each daughter cell contains the full complement of 
DNA. In addition, DNA alone or in combination with protein has 
been found capable of transforming cells of one genotype to another 
.and even of Inducing cancer. 

The fact that DNA occurs in such constant amounts and is 
capable of inducing heritable changes makes it of the greatest 
importance to determine how this compound is formed in the cell, 
in terms of the prtcursor compounds and enzymes involved. Until 
such inf orma-^j. on is obtained, attempts at blocking DNA sjmthesis, 
and henoe at controlling cell division, must be entirely haphazard. 
The results obtained so far in this project represent a considerable 
advance toward the achievement of this goal since they demonstrate 
that specific nucleosides, hitherto unrecognized as precursors of 
DNA, are widely distributed as tissue constituents. Since these 
compounds are qualitatively different in the normal and tumor 
tissues, the s\Tithesis of DNA in the latter may proceed along a 
different pathway. If this proves to be the case, the possibilities 
of controlling cell multiplication in tumors would he greatly 
increased. The continued study of DNA precursors by means of their 
isolation and identification as well as by the elucidation, through 
enzymatic and isotopic methods, of the metabolic pathways involved 
is consequently of utmost im ortance for cancer research. 

Proposed course: The identification of the nucleotide like 
compounds will receive the greatest emphasis during the ensuing 
year. 4 stirvey of the occurrence of these compounds in various 
tissues is also pla'-^ned and it is hoped that it will also Ise (; 
oossible to begin work on the metabolism of the deoxyribose compounds. 



PROJECT FORM (Cont.) 

10. U07 

SERIAL NO. 

11. 

BITDGET ACTIVITY J ~" ' ' 

RESEARCH X ADMINISTRATION 

REVIEW ^. APPROVAL TEC^'NICAL ASSISTANCE 

12 i ■ - " 

COOPERATING UNITS OF THE PUBLIC HEilLTH SERVICE, OR' OTHER ORGAN " ZATIONS , 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FCB THIS PR'JECT IN' EITHER 
1956 or 1957 ' • 



13. 



IF T"TS PRVECT RESEI'BLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEW'ERE IN THE PnBLIC HEILTH SERvICE (WITHOUT INTERC'-IANGS OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH; 



PROJECT REPORT FORM (Cont'd) 



lii. Uoy 



SERI'iL NO. 



1$. ■■ ■_ •__•'„ 

PITBLICA.TTONS OTHEft T"^N IBSTR/ICTS FROM T^-^IS PRVECT DURING CALENDAR 

YE.AR 1955 

1. Schneider, W. CtS Deoxyril^osldes in animal tissues. J, Biol. 
Chem. 2J6, 287 (1955). 

2. Hogeboom, G. w. and Schneider, W-.C: The Cytoplasm. In The 
Nucleic /Vcids, Vol. 2. Edited by Chargoff and Davidson, 

New York, 1955. 



16. 



HONORS ftND WARDS TO PEftSONl.EL RELATING TO r'lS PROJECT DURE>JG 
CALENDAR YEAR 1955. 



PAGE 1 
PROJECT REPORT FORM 

1 . Na tional Cancer Institute 2 . Laboratory of Biolog y , 

BIc-TITTITE- ' LABOR 'VTORI OR BRIKCH 

3.CellulTi:^ B-L0l og7 Sect ion U- ^ - hOS 

SFCT;~TO:rSE'AniCL LOCAT--TJ?Tf OC^lR TR.N .TETtTIsDA) SER.NO. 

6. studies on t^-e morpVolotj. s- a.id phy3iolo g-.^ c." t'fio lyr:p hrcytic tumors 

PRajLcT tTtTs 



7. Emma ohelto n .■• 

?RlF«IP'iL IIvVESTIGi^.X.!Fs7~ 

8 . 

OTPEi".' Ir'VEiJTIGATORS 

9. PROJECT IESCRIPTjOK 

Objective: To correlate differences in the laorphology and physiology 
oFTwoTymphocytic tumors with the diifer-ences in their behavior in 
the host. 

^'"ethods ; In order to t^.st the ability of the cells of lymphacytic 
ascites tumors to pass t'lrough membranes of known pore size, measured 
numbers of cells are sandwiched betwetn two pieces of Schleicher and 
Schuell membrane filter. Th=:- cells are enclosed by sealing the membrane 
filters to a circle of Ivicite, The sandwicn is placed in the periton- 
eal cavity of a mouse. By making biopsy punctures of the abdomen 
and counting differentially the cells in the ascites fluid it is poss- 
ible to tell with considerable accuracy the time at which the cells ■ 
escape from the chambers ^ 

Major Findings ; 

1. Cells of Lymphoma i2 (diploid tunor, highly invasive) survived 
and grew in chambers (10 mice) for a period of 50 days. The cells did 
not escape from the chambers. When the contents of these chambers were 
injected subcutaneously into mice, tumors were produced in every case. 
Similar results were obtained w?i®n ch^ambers were placed in 17 additional 
mice for periods ranging from 2 to 28 da:(,'S, 



PROJECT DESCRIPTION (Con't.) 



2. Cells of Lymphoma #1 (tetraploid, relatively non-invasive) 
escaped from the chambers and produced ascites tumors in four out of 
a group of 10 mice. In five of the remaining mice killed after 35 
days, a few viable cells were present in the chambers. Inoculation 
of the chamber contents has produced a subcutaneous tumor in the 
host mice in only two cases. In the last mouse, killed aftef $9 days, 
no viable tumor cells were observed in the chamber and no tijimor has 
appeared at the site of the inoculation of the chamber contents. 

The final outcome of the project cannot be foreseen as yet, 
since the results are not clear-cut. The question of leaks in the 
llJambers due to incomplete sealing of the filter is raised. The 
indications are that the cells of L*^'2 have a greater capacity t« 
survive and miiltiply in the chambers than do the cells of L0., 

Significance to cancer research ; The nature of the invasive properties 
of tumor cells is little understood* These experiments are being 
carried out in the hope that they will shed some light on this 
phenomenon. 

Proposed course ; /Idditional experiments with chambers constructed of 
membranes of graded porosity are under way. It is planned to apoly 
this technique to a greater variety of ascites tumors than the two 
mentioned above. 

Cinematographic studies on the locomotion of these cells in vitro 
are planned. 



PROJECT REPORT FORM (Cont'd) 



10, ii08 



SE'^FiL MO. 
11. 



BUDGET ACTIVITY: 

RESEARCH X AD"'"INISTRATION 

REVIEW & APPROVAL TEC^-'i\"ICAL ASSISTANCE 

12. I 

COOFERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORG^NIZ.ITIONS, 
TROVIDING FUNDS, FACILITIES, OR PERSON^ffiL FOR T'-^IS PROJECT IN EITHER 
1956 or 1957 



13. 



IF THIS PROJECT RESETiBLES , COKPLE'ENTS, OR P.'^.RALLELS RESE'IRCH DONE 
ELSElfli'HE'-E IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF 
PERSONl'^'EL, F^'CILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR ITEJ-iS lii, l5 "< l6. 



PAGE 1 
PROJECT REPORT FORM 



1 . TTational C ancer Institute 2 . Laboratonr of Biolog y 

lilSTITTntE ~~ LABOR 'iTORT OR E-:\KOH 

3»Celli-'la:'' Bioiog/ Section k. __5*Ji2^ 

' 3FCf:rrcrSE-'V^Zcl ^^ LOC'\T:.V>Tjn"^' 0':^E'H TH.N :iETTTE3DAj SER.NO. 

6. StucaeP__on_bhejno_rptolu^,r a-id phy3iolcg-,_'-__o/^ t-y ryan.ohc corbie tumors 
HICJLCf TITIE ~ " " 



7 . Emma 3he Ito n . 

PRI-NOIP-'iL IIv\fESTIG'i':'CiT*Sl 

8. — 

Gl'FEr? I: VEdTIa/iTORS 

9. PROJECT "itSC'.IPT.iON 

Obj-ctive: To correlate differences in the morphologrr and physiology 
^T~l^oT.ymphocyt; c tumors with the differences in their behavior in 
the host. 

T'ethods: In order to t-st the ability of the ceils of lymphscytic 
ascites tumors to pass firough membranes of known pore size, measured 
numbers of cells are sandiiiched betWb>on two pieces of Schleichc-r and 
Schuell membrane filter. The cells are enclosed by sealing the membrane 
filters to a circle of lucitj. The sandwicn is placed in the periton- 
eal cavity of a mouse. By making biopsy punctures of the abdomen 
and counting differentially the cells in the ascites fluid it is poss- 
ible to tell -with conside-'-able accuracy the time at which the cells 
escape from the chambers. 

Major Findin ^^st 

1. Cells of Lymphoma f2 (diploid tunor, highly invasive) survived 
and grew in chambers (10 nice) for a period of 50 days. The cells did 
not escape from the chambers. When the contents of these chambers were 
injected subcutaneously into mice, tumors Wo-re produced in every case. 
Similar results were obtained when chambers were placed in 1? additional 
mice for periods ranging from 2 to 28 da:s,'S. 



PROJECT DESCRIPTION (Con't.) 

2, Cells of Lymphoma #1 (tetraploid, relatively non-invasive) 
escaped from the chambers and produced ascites tumors in four out of 
a_ group of 10 mice. In five of the remaining mice killed after 35 
days, a few viable cells were present in the chambers. Inoculation 
of the chamber contents has produced a subcutaneous tumor in the 
host mice in only two cases. In the last mouse, killed aftef 59 days, 
no viable tumor cells were observed in the chamber and no tumor has 
appeared at the site of the inoculation of the chamber contents. 

The final outcome of the project cannot be foreseen as yet, 
since the rosults are not clear-cut. The question of leaks in the 
chambers due to incomplete sealing of the filter is raised. The 
indications are that the cells of Jj}2 have a greater capacity t« 
survive and raaltiply in the chambers than do the cells of L#l. 

Significance to cancer research ; The natiire of the invasive properties 
of tumor cells is little understood^ These experiments are being 
carried out in the hope that they will shed some light on this 
phenomenon. 

Proposed co'^rse ; /Idditional experiments with chambers constructed of 
membrT.nes of graded porosity are under way. It is planned to apoly 
.this technique to a greater variety of ascites tumors than the two 
mentioned above. 

Cinematographic studies on the locomotion of these cells in vitro 
are planned. 



PriOJECT REPORT FORM (Cont'd) 



10. U08 



SE^I/iL NO. 
11. 



BUDGET ACTIVITY: 

RESEARCH X /iD'-'TNISTRAT ION 

REVIEW & A'-^PROVAL TEC'-'NICAL ASSISTANCE 

12. 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OT HER ORG AN IZ iTI OI© , 
PROVIDING FUNDS, FACILITIES, OR PERSON^iEL FOR T'-'IS PROJECT IN EITHER 
1956 or 1957 



13. 



IF THIS PROJECT RESEMBLES, COKPLEi'.ENTS, OR P.'^/ftALLELS RSSEflRCK DON'E 
ELSE'aIHEwE IN THE PUBLIC HE\LTH SERVICE (WITHOUT INTERCHANGE OF 
PE^iSONNEL, F\CILITIES OR FUNDS), IDENTIFY STICH RESEARCH: 



NO ENTRIES FOR ITEi^IS lU, iS "- l6. 



PAGE 1 

PROJECT REPORT FORM 

1» National Cancer Institute 2, Labor attry of Biol»gy 

INSTITUTE ■ ■ LABORATORY OR BRANCH 

3. Cellular Biology Section i b . ; ^ . ItO? 

SECTION OR SERVICE LOCATION (IF OTHER THA.N BETHESDAj SER. NO. 

6. studies on liv«r carcinogenesis in the mouse 

PROJECT TITLE 

7. Emma Shelton 
PRINCIPAL INTESTIGATOR(S) 



OTWER liJVESTIGATORS 



9. PROJECT DESCRIPTION 

Objective; To study the changes occurring in the parenchymal cells 
of the liver of the mouse prior to the formation of a tumor, and to 
develop methods for studying these changes. 

Methods: Nutritional, biochemical, histochemical 

Kajor findings ; A study was made of the tetrazolium method for 
measuring succinic dehydrogenase in order to answer the questions: 

1. Do tetrazolium salts accept electrons directly from 
succinic dehydrogenase? 

2. ^^ow sensitive is the tetrazolium method in comparison with 
oxr</"gen uptake as a measure of enzyme activity? 

3. Can the histochemical tetrazolium method be used as a 
reliable quantitative test for succinic dehydrogenase? 

It was found that: 

1. Since tetrazolium reeuction is inhibited by antimycin, 

the tetrazolium salts do not accept electrons directly from 
succinic dehydrogenase but require the mediation of 
cytochrome b. 

2, The tetrazolium method is a relatively insensitive method 
for measuring succinic dehydrogenase, being in the neighbor- 
hood of twenty-five times less than oxygen uptake. 



PROJECT DESCRIPTION (Cont.) 



3. In spite of the lack of sensitivity of the method and the 
place in the enzymatic chain of elgctron transfer where the 
enz3matic activity is measured, ratios of activity of liver 
to kidney to brain are identical using both tetrazolium 
^ and oxygen uptake. Thus the tetrazolium method can be used 
as a valid and accurate- quantitative measure of succinic 
dehydrogenase. 

The very small tumors appearing in the livers of mice fed 
o-amincazotoluene have a higher succinic dehydrogenase activity than 
do the cells of the surrounding parenchsma as measured hj both the 
tetrazolium method and by oxygen uptake. 

Significance to cancer research ; In order to study the earliest 
changes taking place in livers undergoing the malignant alteration, 
histochemical methods of known accuracy must be developed. In future 
exoeriments, t'ne tetrazolium method can be relied upon to give 
accurate estimates of the succinic dehydrogenase activity of cells 
at the microscopic level. 

Proposed course of project ; Since the strnin A mouse is a poor 
breeder, and is thus not too readily available for use, experiments 
are under way to ascertain whether a high percentage of tximors may 
by produced in the more readily available strain C mouse. 

A quantitative cvtological and histochemical study of the early 
stages of tum»r formation in the livers of these mice is planned. 



PROJECT REPORT FORl^I (Cont'd) 

10. U09 

SERIAL WO. 

11. 

BTTDGET ACTIVITY: 

RESEARCH X ADMINISTR/ITION 

REVIEW "< APPROVAL TECHNICAL ASilSTAUCE 

12. 



Co-operating units of t^e public health service, or othf.r or can izm ions, 
providing ttjnds, facilities, or ^eisonijel for tuts _^roject in either 
1956 or 1957 



13. 



IF TWIS PROJECT RZSE^''BLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSE'.ir"SRE IN THE PTOLIC HEALT^T SERVICE (WITHOUT INTEPC^ANGE OF 
PERSONIMEL, FACILITIES OR FUITOS), IDENTIFY ST^CH RESEARCH: 



PROJECT RE^'iRT FORf: (Cont'd) 

li;. it09 

SFR.I'rL NO. 

15. 

PUBLIC-'.TlONS OTWER TW?.N .\BSTRACTS FROM T"IS PR'^JECT DURING CALENDAR 
YEAR 19^5 

Shelton, E. : i^epatomas in mice. I. Factors affecting the rapid 
induction of a high incidence of hepatomas by c-amincazotoluene. 
J. Fat. Cancer Inst. l6i 107-12?, 1955. 



16. , 

HONORS AND AWARDS TO PKR30NML RELATING TO T^'IS PROJECT DURING 
CALENDAR YE/'iR 1955. 



PAGE 1 
PROJECT REPORT FORM 



1 . National Cancer Institute 2 . Laboratory of Biology 

INSTITUTE LABORATORY OR BRANCH ' ~^ 

3. Cellular Biology Section k- $. UlO 

SECTION OR SERVICE LOCATION (IF OTHER T'-iAN BETHESDA)SER.NO. 

6. Transparent Chamber and Dif fusion Chamber Research 

PROJECT TITLE ' ^ '"" ^ 



7. Glenn H. Algire , 

PRINC IPAL IWEST IGATOR (3 ) ", ~~ 

8 . Doctors C» C. Congdon, Y. J. Evans, L» W. Lav, R« M.Merwin and J.N. Weav er 

9. PROJECT DESCRIPTION 

No.'l 

Research program in Transparent Chamber ■Project 

J^oblem: Developments in Transparent Chamber Methods 

Objectives ; To modify transparent chamber designs to attain increased 
simplicity of construction, and longer useful duration of the chambers. 

Methods employedj Transparent chambers are constructed about an 
internal splint of plastic which provides better support' for the 
skin fold than earlier designs of chambers* 

Major findings : These new forms of internal splint chambers are 
being utilized in current work, in order to determine their 
applicability to problems in which observations must be made over 
periods of several months. 

Significance ; It is hoped to extend the useful duration of 
transparent chambers, so that observations of cellular and vascular 
reactions to carcinogens may be carried on for longer periods of 
time than has been possible heretofore. 

Proposed course ; To utilize these chambers in long term studies 
of cellular and vascular reactions to chemical carcinogens. 



PROJECT DESCRIPT'ON (Cont.) 

Wo. 2 

... problem ; Factors affecting the .survival of heterografts in diffusion 
chambers. 

Objectives ; From preliminary experiments reported last year, it was 
found that himan (HeLa) cells survived for at 'least 50 days in dif.fusioo 
chambers in mice, but after two weeks extensive areas of cellular de- 
generation were found. Current studies are directed toward a study of 
immune factors in the survival and growth of hum.an cells in diffusion 
chambers. Dr. Virginia Evans has furnished the cultures of HeLa cells 
used in these studies. 

• 
Methods ; Human cells (HeLa strain) from tissue culture were placed in 
diffusion chambers using filters of estimated porosity O.ij. microns. 
Observations were made either in vivo using transparent chamber methods, 
or as fixed and stained specimens after intraperitoneal implantation 
and later removal. 

Major findings : HeLa strain of human cells proliferate rapidly in 
diffusion ch-ambers in normal mice for about 2 weeks, after whxch time 
areas of cellular degeneration are noted, but laany surviving cells are 
still present for at least 50 days. Extensive destruction of HeLa 
cells occurs wit'- in 7 davs if placed within diffusion chambers in mice 
which have been immunized by subcutaneous inoculation of this cell 
strain, two we ks prior to implantation of the diffusion chambers, 
indicating that circulating antibodies pass through the filters. 

Significance to Cancer Research ; This is a study of the reaction of an 
animal to grafts of human cells, and the pissibility of eliminating 
destructive effects in order to obtain growth of human tumors in ex- 
perimental animals. ' -ossible application to diagnostic problems of 
cancer will be considered, as well as studies on the; mechanism of 
action of drugs capable of affecting tumor cells. 

Proposed course ; Experiments will be undertaken on the effect of the 
porosity of membranes on survival of heterografts in diffusion chambers. 
This will be done to determine whether it would be possible to find 
a pore size that would allow cell growth, but would not allow antigenic 
material to escape from the chamber and immunize the host. Will it be 
possible to obtain prolonged survival and growth of human cells in 
diffusion chambers by blocking the escape of antigenic material? 

The studies will be extended to other cells of human origin, 
both normal and cancer. 



'^R'^JECT DESCRIPTION (Cont.) 

No. 3 

Problem : Mechanism of action of Chemotherapeutic Drugs on Tumor Cells. 

Objectives ; To determine the mechanism of action of various 
tumor-damaging drugs on tumors in terms of their circulatory effects, 
or direct cvtotoxic properties. 

Methods emrjloved ! Studies of the reaction of tumor cells to injected 
drugs were made under three conditions. 

(1) Using diffusion chambers in which implanted cells are 
isolated from contact with host tissues by porous membranes; 

(2) Solid vascularized tumors where circulatory effects may 
be involved J 

(3) Solid vascularized tumors, as influenced hy injected 
drugs, plus procedures to alter the circulatory response. 

I^ajor findings ; ^;ouse sarcoma cells growing in diffusion chambers 
placed intraperitoneally are not ai'fected by bacterial polysaccharide 
injected intraperitoneally. Subcutaneous injection of podophyllo toxin 
results in mitotic arrest without extensive necrosis of cells growing 
in diffusion chambers in the absence of blood supply. This result is 
in contrast to the hemorrhage and necrosis wh .ch occurs in solid 
vascularized tumors following injection of either of these drugs. 

Preliminary evidence has been obtained that increased blood : 
flow caused by local warming of tissue may orevent the tumor-damaging 
action of podophyllotoxin. 

Significance to Cancer Rtjsearch t Evaluation of therapeutic agents in 
cancer depends upon understanding their 'mode of action. Transparent 
chamber methods for microscopid observations in living mice, 
supoleniented by diffusion chamber procedures, help to determine 
whether an agent acts directly upon the living cell or indirectly 
through its effects on the circulation of the host animal. 

Proposed Course of ^reject: 

1. To apply the diffusion chamber methods to studies 

of the effects of various chemotherapeutic drugs on cells isolated 
from host tissues with particular reference to leukemia and to 
human tumors, 

2. To continue studies on the role of circulatory responses 
in the action of tumor-damaging agents. 



PROJECT DESCRIPTION (Cont.) 

No. h 

Problem ! Mechanism of radiation' protection by bone marrow. " 

Objectives ; Lorenz and coworkers have shown that injections of 
isologous or homologous bone marrow will protect mice against amounts 
of X-radiation which are lethal to uninjected animals. The mechanism 
of protection is not clear. One hypothesis is that the injected bone 
marrow cells survive and proliferate to take over the function of the 
marrow of the irradiated mice; another-, that protection results from 
some as yet unidentified humoral factor. The object of these experi- 
ments was to determine whether radiation protection can be brought 
about by bone marrow cells enclosed within diffusion chambers. A 
positive result would support the humoral theory; a negative result 
would be in accord with the cellular rfepopulation theory. This work 
was done in cooperation with Doctor C. C..Congdon. 

Methods ; Determination of the minimal effective number of marrow cells 
necessary for protection when given intraperitoneally; inclusion of 
this number of cells in diffusion chambers (and multiples of this 
dosage) for intraperitoneal implantation into irradiated mice and 
determination of the effect on survival as compared with controls. 

T^jor findings ; It has-been found that marrow cells grow'within 
diffusion chambers which are placed intraperitoneally. The dosage 
required for protection when marrow cells are injected directly into 
the peritoneal cavity has been found by Congdon to be approximately 
5,000,009 cells. This number of cells and twice this population en- 
closed within diffusion chambers, were placed intraperitoneally in 
previously irradiated mice. The results were negative, that is, no 
protection was a'fcrded by bone marrow cells enclosed within diffusion 
chambers and placed intraperitoneally. In following up this negative 
result, the volume of fluid in which the cells were suspended proved 
to fee an important factor, since Congdon found that protection was not 
obtained after i.p. injection if the cells were suspended in the same 
volume of fluid (.02 cc) as used (necessarily) in diffusion chambers. 

Significance to Cancer Research; Studies on radiation protection are 
of importance in enhancing the value of X-radiation as a therapeutic 
tool in cancer. 

Proposed Course; This aporoach has been discontinued. 



PRnjECT DESCRIPTION (Cont.) 

No. 5 

Problem ; Invasiveness of leukemia. 

Objeetives ; F'ouse leiicemia cells differ greatly in their capacity to ■ 
invade the tissues of the host. Studies ■were undertaken to determine 
whether this property is correlated mth the ability of these cells 
to pass through filters of graded ooroSityr Parallel studies of . 
the migrator^,^ behavior of leukemic cells are being carried out using 
time-lapse motion pictures. ' 

Methods ; Fragments of leukemia 1210 were placed in diffusion chambers 
formed from filters ranL,'ing from h' millimicrons to O.U microns 
aver-ige pore diameter. Bi'^logieal and histologic studies were made 
to determine the fate of the cells. Siinilar studies are in progress ■ 
using leukemia 5l78, a less invasive neoplasm. These tumors are pro- 
vided by Doctor Lloyd Law. 

Major Findings ; In the systems so far tested, if has not yet been 
possible to consistently prevent the development of leukemia in the 
hosts. Current studies are devoted to finding out whether leukemic 
cells pass through the pores of the filters or through the seal 
uniting the parts of the chamber. Alternative exolanations for these 
results are poftsible and are being tested. 

Significance for Cancer Research ; These studies are concerned with 
factors which promote the spread of cancer cells in their invasion 
of normal tissues. Results of such experiments, using a- series of 
filters of graded porosity and leukemlas of diverse invasive properties, 
will contribute to an understanding of the mode of transmission of 
leukemia through membranes, and to a characterization of membranes 
in terms of their properties in a biological system. 

Proposed Course; Studies will be continued as described above. 



PROJECT DESCRIPTION (Cont.) 

No. 6 

Problem ; Studies in carcinogenesis using diffusion chamber methods. 

Objective: The diffusion chamber method for "tissue culture in vivo" 
provides a new approach to certain problems of chemical carcino- 
genesis. Earle and associates have found that normal adult fibroblasts 
grown in tissue culture in a completely heterologous media in the 
absence of deliberately added MCA give rise to sarcomas when inoculated 
into mice. One possible explanation for these results has been "growth 
in the heterologous media," This explanation would be ruled out if 
normal or embryonic mouse tissue underwent a malignant change when 
enclosed within diffusion chambers. 

Goldblatt, following, experiments and theories of Warburg ''1927), 
has presented evidence that normal fibroblasts in tissue culture undergo 
a malignant change if subjected to conditions of intermittent anoxia, 
'■'■hrough selecting filters of increasing thickness, and of consequent 
greater diffusion distance, one could study the effect of this variable 
on neoplastic change. 

The immediate objective is to ascertain whether aiult or embryonic 
subcutaneous tissue enclosed within diffusion chambers undergo neoplastic 
transformation. 

I^ethods employetl ; 

1. To determine whether the materials (plexiglas II, and 
Millipore filters) are carcinogenic for mice - (by intraperitoneal 
and subcutaneous implantation). 

2. Fragments of muscle from young mice (strain C3w) were 
enclosed v:ithin diffusion chambers and placed intraperitoneally 
into adult hosos of the same strain. After 6 months these were 
reimplan^-ed (open) into new hosts which had received UOO-I4.25 r total 
body irradiation. Another series will be done after 12 months. 

In other experiments, subcultures were made each month for 6 months 
before testing for neoplastic transformation. 

Major fin dings; It has been found that fragments of muscle from 
young mice produce a vigorous outgrowth of connective tissue cells, 
with numerous dividing cells. Adult connective tissue cells also 
migrate out in diffusion chambers. 



PROJECT REPORT FOM (Cont'il) 



10. - Uie • 



SERIAL NO. 
11. 



BUDGET ACTIVITY: ■ , ■ 

RESEARCH X ADMINISTRATION 

REVIEW & APPROVAL - ■ TECHNICAL ASSISTANCE 



12. 

COOPERATING UNITS OF THE PUBLIC- HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR T-'IS PROJECT IN EITHER 
1956 or 15^7 



13, 

IF TWIS PROJECT RESE?''BLES, C0MPLE1«ENTS , OR PAR^fiLLELS RESEARCH DONE 
ELSEVcHERE IN THE P^IE^LIC HK4LTF SERVICE (WITHOUT I1^!TERC"-'ANGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



PROJECT DESCRIPTION (Cont. ) 



Normal connective tissue cells have been carried in diffusion 
chambers in mice for 6 months. Reimplantation (open) into irradiatei- 
hosts has not led to the production of tumors. 

Normal connective tissue cells have been carried in diffusion 
chambers with subculture at intervals of 1 month, and biological tests 
were made for the development of malignancy at the end of 6 months. The 
results have been negative. 

No mice have developed tumors as a result of subcutaneous 
implantation of materials used in making diffusion chambers. These 
experiments are not yet completed. 

Significance to Cancer Research ^ -Increased understanding of factors 
involved in the transformation of normal to neoplastic. cells is of 
fundamental importance in cancer research." 

Proposed course of project; This experiment is still in progress. 
Various chemical carcinogens and related but inactive compounds will 
be used in studies of the effects on cells and organs using diffusion 
chambers. 



SERIAL NO. 



15. 



PUBLICATI'^NS OTHER THAN ABSTRACTS FROM THIS pR'^JECT DURING CALENDAR 
YEAR 19?^ 



Weaver, J. M. , Algire, G. H», and Prehn, R. T. ; The growth 
of cells in vivo in diffusion chambers, II. The role of cells 
in the destruction of homografts in mice, J. Nat. Cancer Inst. 
15: 1737-1767 (1955). 

Algire, G. 't., and Merwin, R. M. : Vascular patterns in tissues 
and grafts within transparent chambers in mice. Angiology 6; 
311-318 (1955). 



16. 

HONORS AND AWARDS TO PERSONNEL RELATING TO T'-^IS PROJECT DURING 
CALEIWAR YEAR 1955 • 



Page 1 
project report 

National Cancer Institute 2 . Lab oratory of_ Biology 

INSTITUTE L.-.BOF.-.TORY OE'BRt.WCH " " 

Cellular Bi olog.y Section 4. 5. 414 

SECTION OR SERVICE LOCATION (IF OTHER THAN EETHESDa) . SEFJAL NO 

Tran sp arent Chamber Resear ch ' 

PROJECT TITLE """ ' ' ■" " ' " 

Ruth M. Merwin 



PTJNCIPAL HvIvESTIGaTCRCS) 



Dr. C« C. Congdon, Dr. K. K. San ford and Dr. G. H. .-.Igire 
OTHER INVESTIGATORS 



PROJECT DESCPJPTION 



Problem #1. Fate cf homologous bone marrov; cells injected intravenously into 
irradiated mice. Ruth Merwin and Charles C. Congdon 

Objective; To study the mechanism by which marrow injected intravenously pro- 
tects mice that have been given vdiole body irradiation. It has not yet been 
proven whether the introduced cells repopulate "the tissues of the irradiated 
animals or whether the injected, suspension provides soaie substance that enables 
the -irradiated cells to. recuperate. If cells from, an animal not genetically 
like the irradiiited one were injected and if one/2Jtefttify these introduced 
cells and estimate their numbers it snould be possible to find aut whether the 
injected cells repopulate the marro'w or whether the introduced cells only tide 
the animal over until its ov.ti ceils can divide. 

j,eth»d : Mice of the LAF strain were irradiated with 900 r and each was pro- 
tected from this otherwise lethal dose by an intravenous injection of homo- 
logous juarrow from a mouse of the C3li strain. At various intervals thereafter 
the marrows of the protected LAF mice were tested to see if homologous cells 
ViTere present. Tue iriarrows were injected into LaF iiiice each of which carried 
a nonvascularized homograft of Harderian gland tissue from a C3H mouse. Non- 
vascularized homograft do not initiate imir.unity in the LaF host but if the 
injected marrow being tested contained homologous cells immunity vrould be 
initiated in the h»st and -vvould cause the graft to disintegrate. The percent- 
age of the grafts disintegrating and the interval before disintegration pro- 
vided a means of determining v;hethsr the injected cells incre^.sed in number. 

jiajor f indings : aU of the marrows taken from the protected LaF mice whether 
they were taken within 6 hours or not until 150 days after irradiation and 
protection showed the presence of homologous cells except about a third of 
those taken within 4 days. 

An increase in the number of these homologous cells vias indicated by 
12 days after injectien, and no decrease in number seemied to take place before 
1$0 days, the longest intej;;val after which the marrows were tested. 



Page 2 



A few tests of the blood, spleen, lymph nodes, and thymus of the pro- 
tected mice indicated that homologous cells were present in them also. 

Significance ; It is important to understand the mechanism by which injections 
of bone marrow protect anijnals that have been giveh an otherwise lethal dose 
of irradiation. 

Proposed course ; The data obtained will be prepared for. publication. 

Problem #2, The effect of local roentgen irradiation on the formation of nevr 
capillaries after injury, Ruth Merwin • ' 

Objective : In a previous study it vj-as found that the capacity of vessels to 
form new sprouts v«fas affected ty doses. of irradiation over 500 r. The. interval 
before new vessels invaded the coating of exudate around wires placed in the 
skin became longer as the dose increased. At present a study is being made 
using a higher dose to find out v;hat. dose, will completely prevent the formation 
of new capillaries. Also the growth of new capillaries around wires placed 
in the skin a year after irradiation is being studied. to find out whether tne 
endothlium will have regained its capacity to grow without delay into an in- 
jured area. , ■ . ; .... 

Methods ; The growth of capillaries into the coating of exudate that forms 
around wire stitches placed in the skin of mice is being studied microscopic- 
ally using transparent chambers. 

Major findings : Preliminary findings indicated that a dose of 2,000 r com- 
pletely prevents the growth of new vessels around some, but not all, wires. 
One year after local irradiation the delay before sprouts form in an injured 
area is approximately the ssjne as when an injury is made immediately a:fter 
irradiation. 

Significance ; An understanding of the changes in normal tissue irradiated* , , 
locally is of importance because it is difficult to irradiate a tumor without 
including the surrounding normal tissues. The deleterious effect of irradia- 
tion on nornial tissues sometimes limits the dose that can be given to a tumor^ 

Proposed' course : To Continue as stated above. 

Problem #3. Tumor growth and vascularization,- Initial vascularization of 
transplants of tumor and normal tissue, Ruth Mei-win and G-lehn Algire 

Objective : The object of this study is to evaluate the significance of the 
activity of the graft and host vessels in the initial vascularization of grafts. 

Methods employed ; Microscopic observations were made on grafts placed in 
transparent chambers in living mice, With the usual methods of studying 
grafts one must focus through the graft in order to see the area in vAich the 
graft and host vessels establish connections. Therefore, grafts were placed 
so the area in which these changes take place would be beside rather than 
under the graft. 

Major findings ; The surviving vessels in grafts ff normal tissues produce 
sprouts which may be active in establishing corinections between the vessels 
of the graft and those of the host. On the otiier hand, the vessels in grafts 



Page 3 

of tujnor tissue survive only in some grafts and do not produce new vessels. 
Grafts of nonnal tissue of the size used did not cause host vessels to form 
new sprouts, whereas grafts of a siriiilar size of a mammary adenocarcinoma 
caused a rich network of new sprouts to form. 

Significance ; The methods used here provide a means by which comparisons 
could be made between' the effect of various kinds of stimuli and various 
kinds of tamers on blood vessels. 

P roposed course : It is planned to compare various typss of tumors in their 
capacity to stimulate host capillary proliferation. 

Probl em /f4 t Effect of irradiation on the capacity of mice to develop immunity 
to homografts. Ruth Merv/in and Ks-thsrine Sanford 

O bjective ; The objective of this study was to find out how long after irrad- 
iation mice are incapable of d;;veloping Immunity to homografts. 

M ethods ; A nonvascularized homograft will net initiate immunity but it will 
be destroyed if the host becomes immune. The interval after irradiation and 
after the injection of an ijni'aunizing dose of homologous cells before a homo- 
graft disintegrated was taken as the interval during vjhich mice could not 
develop immunity. 

Major findings ; Preliminary results indicate that there is much variation 
between mice but that mice can acquire imjnunity as soon as '11+ days after 
900 r and protection by isologous marrow or as soon as 11 days after irradia- 
tion with 450 r. 

Sign i ficance ; To help identify the natur-e of the changes taking place in 
cells that become neoplastic (see the report of Dr. K. K, Sandford) 

Proposed course ; To continue as stated above, 

414 

SERIAL NO. 



BUDGET ACTIVITY: 

EES^AECH X ADiZNISTR^.TION 

RSVIM & APPROVAL TECHMIC^ ASSISTANCE 



12, 



COOPEIxiTIKG UNITS OF THE PUBLIC PCi-.LTH SERVICE, OR OTHER OIiGaNIZATIONS, 
PROVIDING FUNDS, F..CILITIES, OF PERSONNEL FOR TliiS PROJECT IN EITHER 1956 
or 1957 
Dr. C. C. Congdon is at Oak Ridge National Laboratory, Oak Ridge, Tenn, 



IF THIS PROJECT RESEiiBLES, C0.v.PLEl,ESIT3, OR P.iRALLELS RiiSEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE C.ITHOUT INTEFJCH.\NGE OF PiFSONNEL, FACILITIES 
OR FUNDS), IDSiMTIFY SUCH RE3E.-.RCH; 

414 

SERIAL NO. 



15. 

PU^^LICaTiUNS OTH.R TILU\f ABSTFuiCTS FROM THIS Pi>,0J3CT DUPING CaLE'.NDaR Yli^.R 1955 

Viiscular patterns in tissues and grafts within transparent chambers 

in mice, Glenn H. jilgire and Ruth M. MenA^in, 

;jigiology 6: 311-318, 1955 ' ' ' 

16 , . 



H0M01.S .nlO HVlAicDS TO PERSONITfiL RELATING TO THIS PROJECT DURING CALENDAR YEaR 
1955. 



PAGE 1 

PROJECT REPORT FORM 

1 . National Cancer Institute . 2 « Laboratory of Biology 

INSTITUTE . , laboratory" OR BRAICH 

3. Cellular Biology Section 1;. , ^ ^ ' ^^^ 

SECTION OR SERVICE ' ~ LOCATION(IF OT"^R THAN BETHE3DA.) SER. NO, 

6» The mechanism of destruction of homngrafts 

PROJECT TITLE 

7. J» ''''» Weaver . 

, PRINCIPAL INVESTIGATOR(S) 



1. G. ".Algire 

OTMER ItJVESTIGATORS 



9. PROJECT DESCRIPT-'^'iN 

Objectives ; To elucidate, the mechanism of homograft destruction. Work 
published in 1951; (J. N.C.I. IS: ii93-^07 and 509-5l7) suggested that 
such grafts are nrt destroyed by circulating antibodies and further 
data published in 19$5 (J. N.C.I. 15: 1737-1767) suggested that they are 
destroved by contact with host l^/mphocjrtes. There was some evidence in 
the latter work that these lymphocytes release a diffusible specific 
cytotoxin f antibody?) at the site of the graft. The purpose of further 
work in 1955 was to see if thi-S was so. 

T^iethods employed ; Single and double diffusion chambers, tissue cultures, 
serologic methods, transparent chambers and conventional cytologic 
methods fsee following section for further details). 

Fajor findings ; "'"odified intraperitoneal diffusion chambers were 
employed with two adjacent comoartnents separated by cell-impenetrable 
membranes with a pore size of about one micron. Lymphocytes from 
C57BL mice previously immunized with C3H tissue were combined in one 
compartment with C3'~' "target" cells (spleen or tumor). 
'*s expected from previous work:, this resulted in the death. of botto 
the l^TOphocytes and the target cells. Similar "immunized" lymphocytes 
alone or target ceils alone had been placed initially in the second 
compartments. Both of these two types of cells died in these latter 
compartments whereas cc-itrol nonirrmuniaed lymphocytes or control 
G57BL target cells survived here. 



PROJECT DESCRIPTION (Cont.) 

Furthermore, target cells combined with dead "immunized" 
lymphocytes (frozen and thawed) in intraperitoneal diffusion 
chambers with one compartment did not survive whereas such target 
cells did survive when combined with control dead nonimmunized 
lymphocytes. Whether, as might be expected, dead target cells will 
destroy "immunized" lymphocytes in diffusion chambers has not yet 
been studied. 

These results can be interpreted most simply as signifying 
that homografts are destro\'Bd by antibodies which are bound to host 
lymphocytes until these i-\Tnphocytes contact the grafted cells and, 
in addition, that this destruction of the graft is accompanied by 
the death of the host lymphocytes which is caused by antigens released 
from the dying grafted cells. 

Further attempts to destroy target cells in vittio by combining 
them with living or dead "imm.xmized" lymphocytes and efforts to 
visualize in vivo by means of modified transparent chambers the 
interaction of living graft and host l-/mphoc^^es have been unsuccessful 
to date — quite p<3ssibly for various technical reasons. 

Proposed Course ; The above findings will be extended and methods 
will be sovght to visualize in vivo the interaction betwesn living 
grafted cells and host lymphocytes and also to cause this to occur 
in vitro where the mechanism involved can be more easily analyzed. 



PROJECT REPORT FORt^ (Cont'd.) 



10.l£l6 

SERL\L NO. 



11. 

B'HXJET i'.CTIVITY: 



RESE/IRCH" -X ADfaniSTRATlON 

REVIEW k A PROVAL TECH1IIC\L \S3I3TANCE 



12. 

CO-JPERATIMG imiTS OF THE PUBtIC I-^ALTH SERVICE, OR OT'-'ER ORGAri.'ZATION, 
"PROVIDING FUrroS, FACILITIES, OR ?£?^.SON:IEL FOR T'-I5 ^^'"JECT I'! EITHER 
19^6 or 19^7 



13._ ^_^ 

IP Tf:5 ^RoJfic^T tt^3Et^6Lr.5, Col^^^LKi.ErTS, or P.-.R-JLLEi3 ftESi'-^dp? COilE' 

ELSEV^'JERE II'" THE PUBLIC HEMTH SEP.7ICL (w THOIJT IIJTERCH'k'GE OF 
^EPoGlf-IEL, F.-^CILITIES OR FUi-JDS), IDENTIFY SUCH RES-',RCH: 



PROJECT REPORT FORM (Cont'd) 

Hx. hl6 

SER.IAL NO. 



15 . 

PUELiCATIOtJS OT^ER TT-AN ABSTR/^.CT5 FROM T"IS PROjSCT tillRING CALENDAR 



YEAR 1955- 

Weaver, Algire, Prehn; J. M.C.I. l5: .1737-1767, 1955. 



16. 

HONORS AND AWARDS TO ^IRSOKJEL, RELATING TO T^^-'IS PROJECT DURING CALENDAR 
YEAR 1955. ■■■ ■ • ■ • 



SUPPLEMENTAL PROJECT DESCRIPTION OF SERIAL NO. Ul6 LABORATORY OF 

BIOLOGY - DR. JAMES M. WEAVER 
PROJECT DESCRIPTION (Addition) 



Significance of work ; "Cancer iinmunity" has long been a subject 
of considerable interest and of intensive investigation. Latency 
of cancer and occasional Spontaneous regressions of cancer in man 
may well be manifestations of an immime reaction of the host to 
the tumor. If such reactions could be inteisified at will, the 
benefit to the host is obvious. However, the mechanism of re- 
jection of homologous tumor grafts in animals and of homografts 
in general has long been disputed. This mechanism has certain 
features in comnion with better understood phenomena of immunity, 
such as reactions to bacteria or foreign proteins, but the search 
for antibodies to these tissues has usually been to no avail. 
TTntil the nature of this mechanism is clarified, it seems unlikely 
that attempts to utilize it to combat cancer in man will be success- 
ful. It is hoped that the research program described above will 
contribute toward an imderstanding of this mechanism. 



Sni. 19T:!'B'J'-' 



PROJECT REPORT FORM 
1. National Cancer Institute 2, Laboratory of Biology 



INSTITITTE LABORATORY OR BRANCH 
3. General Biology Section i;. 5. ^^'^ 



SECTION OR SERVICE LOCATION (IF OTHER THA1«I BETHESDA SER,# 

6, Investigation of the role of genes and their relationship to -n«n-genetic 

PROJECT TITLE 
factors in the development of cancer* 

7. W. '£♦ Heston 

PRINCIPAL II'JT?ESTIGATOR(S) 

8, Margaret a, Deringer, Th e lna E. Dunn, Delta Uphoff, George Vlahakis 

OTHER im'ESTIGATORS 

9. PROJECT DESCRIPTION, 

Objectives; To determine the effects of genes on the occurrence of tumors, 
particularly of the lung and of the mammary gland, of the mouse. To obtain 
some indication of the number of genes involved and where possible to ident- 
ify specific genes. To ascertain the manner in which the genes are related 
to non-genetic factors in the induction of tumors. 

Methods employed: This report covers only certain faicets of a long term 
program in which each year certain sub-projects are completed and certain 
new ones are initiated. Methods employed in various facets of the program 
may be much the same with new procedures interjected when needed. 

In the study of pulmonary tumors a number of Ihbred'- 'trains of mice 
are available with specific incidences cof pulmonary tumors ranging from 90 
percent in strain A to less than 1 percent in strain C57L, Seven specific 
genes located on five specific chromosomes have been shown to be associated 
with the fccurrence of pulmonary tumors - some through the effect of the 
gene itself and some through linkage* The occurrence of these tumors can 
be increased by extrinsic factors including the carcinogenic hydrocarbons, 
urethan, nitrogen and sulfur mustard and radiation, and the nuinber of tumors 
occurring offers a valuable quantitative measure of response. Through proper 
matings specific desired genotypes can be produced and these can be studied 
in relationship t» any of the extrinsic factors. Through transplantation 
of lung tissue various genotypic combinations between host and lung tissue 
can be created. The carcinogens can be introduced to shorten the duration 
of the project and also to study and compare their specific effects in com- 
bination with specific genotypes. Gold thioglucose has been injected t« 
increase body weight comparable with effect of the lethal yellow gene. 

In the study of mammary gland tumors a number of inbred strains are 
available with certain incidences of mammary tumors and differing in ability 
to transmit the mammary tumor agent. The line of strain C57BL here at the 
NCI is outstanding and valuable in our studies in that as shown by Andervont 
it will not transmit the agent for more than one generation. Through ap- 
propriate matings and foster-nursing of young the relationship of the geno- 
type t» the agent can be studied. 



Page 2 

Major findings: - Lung Tumogs. 

In an attempt to determine whether or not the lethal yellow (A/) gene 
of the mouse increases the •ccurrence of pulmonary through some general effect 
related to its increasing body weighty non-yellow a a sibs have been injected 
with gold thioglucose. Following this treatment the aa mice gained weight so that 
'their average weight was approximately equal to that of the A^a mice without ^'old. 
Furthermore the average number of lung tumors observed in aa mice injected with 
gold and later with dibenzanthracene was about the same as that of the A^a jnice 
without gold but with the dibenzanthracene. ■ There are some unexplained sex differ- 
ences in average number of tumors which may be clarified with further experimenta- 
tion. Before publishing we want als» to get data on effect of gold on eccurrence 
of lung tumcjTS in Ay"a mice and also the effect on occurrence of lung tumors •! 
holding dowri the weight of Aya mice. through dietary restriction. This study is 
underway. 

In the' study in which we' were attempting to determine whether or not 
the effect of the A^ gene in increasing occurrence of pulmonary tumors was local- 
ized in the lung, the results were somewhat irregular. This was done by trans- 
planting AJa lung into aa hosts as well as Aya hosts and transplanting aa lung 
into A^a hosts as well .as aa hosts,' The greatest number of t'omors occurred in 
the transplanted lung when the genotype of the transplant and that of -the host 
were the same. The' highest incidence of tumors occurred in aa transplants in aa 
hosts and the next highest in Aya transplants in Aya hosts. The least incidence 
•ccurred in Aya transplants in aa hostg and next to the host occurred in aa trans-' 
plants in Aya hosts. This experiment will be repeated before publication, 

■Reticulosis involving- particularly the axillary and inguinal nodes 
has been found in a high percentage of these AYI''y mice. Although scattered cases 
of this condition have been observed previously in a number of our stocks this is 
■the first time we have noted it in a high incidence in any group. It has not 
been recorded in such an incidence previously in AYF;j_ mice. The cause of the con- 
dition* is not known. 

In further dose-response studies on the induction of pulmonary tumors 
in strain A mice with dibenzanthracene th© nature of the response curve has been 
determined in the region of very minu+e doSes, A straight line response has been 
noted between dose ,5 in^. and dose .01 rag. with the point for zero dose deviating 
upward from the extension of this"" straight line. The points for dose .00^ mg, 
and dose ,0025 mg, also deviated upward. Response was measured in terms of aver- 
age number of nodules, and the lower points that deviated upward represented 
group's in which not 100 percent of the animals had one or more nodules,' It may be 
that some theoretical res^xmse points can be established statistically assuming 
variation in the animals having no tumors and these points may not deviate from 
the straight line response. 

Such dose-response studies have also been made on the induction of 
pulmonary tumors in strain C3Hf mice with urethan in doses ranging from 1.2^ mg. 
t»*hO mg. No increase in tumors was noted below the dose of 10 mg, and at doses 
10, 20, and UO mg, the response was so slight owing t» the resistance of the 
strain that it -was impossible to determine the dose-response relafp.onship.'' 



Page 3 

The outstanding observation in this c^roup of C3^'h' mice was definite evi- 
dence of increase in incidence and average number of hepatomas with ini;rease in 
do>sage of urethan. This was noted in the males only. This is the first obser- 
vation, to ovir knowledge, of the induction of hepatomas in mice with urethan. 
An experiment has been set up to verify the observation before publication. 

Manjm ary ula n d Tumors ; 

otudy of mammary g'land tumors in strains without the mammary tumor agent, 

Ln oui- C3Hf strain, mammary tumors are continuing to arise with an inci- 
doncp of approximately 25 percent and an average tumor ape of approximately 20 
months. -Although our colony is now in Ahe 20th and 30th generation of inbreeding 
since the original foster-nursed litter from which this strain was started, we 
still havi^ seen no evidence of high tumor lines segregating out as wfuld be ex- 
pected wore the agent present. 

In July 19'i3, a litter of Andervont's subline C3HfAn, that in his labora- 
tory had shown a considerably lower manunary tumor incidence than had our C3Hf, 
was obtained, and from that litter a colony has been bred. Of 23 C3Hf/An breeding 
females tl'.at have been autopsied, 6 or 26,i have had mammary tumors at an average 
at;;e of 13i3 months, the remainder died without mammary tumors at an average age 
of I'i months. It, tlierefore, appears that in r>ur laboratory the incidence of 
tumors in his line is going to be comparable with that in our line and significan- 
tly higher than that in his line in his laboratory.- Explanation for this differ- 
once piay be one or more of the following. 1) In our laboratory' th^ animals are 
fed Peerwood diet whereas in his the diet is P;?.rina chow, 2) in our laboratory 
the females have been bred throughout the year whereas "they are not bred during 
the winter months in his laboratory. 3) in our laboratory the females are housed 
in the same large room with animals ^dth the milk agent, although on opposite 
sides of the room and on different racks, whereas in his laboratory agent fre« 
animals ai'e not kept in the same room with animals with the agent* Since breed- 
ing has been shown t© be an important factor in producing mammary tumors in our 
C3Hf line (the virgins having an incidence of less than 5 percent whereas the 
breeders have 25 percent or more) the second possible explanation is probably the 
most important. The third explanation would imply infection with the agent and 
as indicated above we have seen no avidence of this in our own C3Hf line. 

The large mass of data on the relationship of the genotype to the milk 
agent yias now been prepared for publication and sh%ul'*' be submitted in the near 
future. The genetic control over the propagation and transmission of the agent 
is very pronounced, the agent being eliminated by the third backcross generaticn. 
Once eliminated the agent cannot be revived by again introducing a suitable geno- 
type. The agent cannot be caused to arise de novo- . The agent can be transmitted 
by the males and once introduced into the line in this way is immediately built 
up to create a high tumor line, 

Tfc test further for the presence of the agent tJhcse data were tabulated 
to show parent-offspring correlation* A positive correlation appeai-ed only where 
it was certain from other observations that the agent was present. ^ In other 
lines although quite a number of tumors were present there was n« parent-off- 
spring correlation which was in line vjlth the previous conclusion that these 
turners arose in the absence of the agent. 



1 Page U 

The data also were tabulated to determine whether or not more tumors 
occurred in females of later litters than in those of early litters. With the 
exception of the few females that wer« infected by the male and were infected 
after the first or second litter was born so tumors occurred more in later 
litters, there was no difference between early and late litters either in groups 
where the agent was known to be present or in groups where it was absent. 

Cleft palate and harelip. 

In I'^aser's interpretation of his results on induction of cleft palate and 
harelip in strain A mice with cortisone he has contended that the cortisone did 
more than increase the occurrence of an anomaly that occurred spontaneously in 
the strain. His arguinent was that with cortisone he got some cleft palate with- 
out harelip, whereas cleft palate without harelip never occurred spontaneously. 
Since I had contested this j.nterpretation end s'ince we had the opportunity to 
observe a large number of newborn strain A micfe we have gathered data on these 
anomalies. Of 1313 males born 8.3 percent ha/a both harelip and cleft palate, .6 
percent had harelip without cleft palate and .23 percent had cleft palate with- 
out harelip. Of 1351 females born 6.29 percent had harelip and cleft palate, .37. 
percent had harelip without cleft palate and .22 percent had cleft palate without 
harelip. Thus, without treatment of the mother the animal can have a cleft palate 
without evidence of the harelip. 

Significance to program of the Institute: One of the approaches to the control 
of cancer in man is through the study of the factors in the development of cancer 
in mice, the manner in which these factors become effective and the way in which 
they are related to each other. Basic among these factors .are the genes, but the 
observable effect of the genes is greatly influenced by nongenetic factors. The 
results reported help to round out our knowledge in this area and it is hoped that 
such knowledge will be of assistance in understanding the factors causing cancer 
in man and in directing investigations on cancer in man. 

Proposed course of project. 

The studies will be continued much along the same lines as in the present 
report. Linkage studies that were not at a stage suitable for reporting now will 
Ite continued and possibly concluded in the coming year. Data such as those on 
the transplanted foetal lungs will be written up for publication as soon as Dr. 
Steffee has finished going over the slides. 

The experiment for verification of induction of hepatomas in mice with 
urethan will be carried on and possibly concluded. This will be extended to in- 
clude repeated doses as well as single graded doses. 

The dose-response studies on the induction of pulmonary tumors are being 
extended to urethan in strain A mice, the strain C3H having been too resistant 
to give analyzable results for the dosages used. It is necessary to get results 
from some carcinogen in solution since it is realized that the straight line 
relationship observed with the dibenzanthracene dispersion may have represented 
only distribution of DBA particles. 

Studies on the effect of the A^ gene in increasing pulmonary tumors in 
mice are being extended to include data on AJa mice treated with gold and also 
on i^Pi. mice whose weights are held comparable with that 'of their aa sibs through 



Page 5 

The experiment attempting to localize the action of the A^ gene in in- 
creasing the occurrence of pulmonary tumors will be repeated before the results 
are published. 

A new but related project on the effect of concentration of oxygen on the 
occurrence of pulmonary tumors in strain A mice is getting under«^ in collabor- 
ation with Dr. Arnold W, Pratt. This study is based upon the observation ef in- 
creased rate of radiation induced mutations in Drosophila and other microorganisms 
with increased concentration of oxygen, increased melanotic tumors induced with 
radiation in Drosophila larvae with increased oxygen, and increased spontaneous 
melanotic tumors in the larvae with increased oxygen but without i-adiation. The 
studies will include 2 month old strain. A mice injected with dibenzanthracene 
and expose* to various concentrations of ©xygen, and als» newborn strain A mice 
exposed to various concentrations of •xygen but not injected with dibenzanthrac- 
ene. 



10. 



11. 



12. 



13. 



15. 



16. 



U17 



SERIAL NO. 



^,UDGET ACTIVITY: 

RESEARCH ^ Administration 



P.EVIEVif & APPROVAL Technical Assistance 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDIi-lG RTNDS, FACILITIES, OR PERSOMeL FOR THIS PROJECT IN EITHER. 1956 
or 1957 



IF THIS PROJECT RESEl-BLES, COMPLEMENTS, OR PAFiALLELS RESEARCH DONE ELSE>fHERE 
IN THE PUFiLIC HEALTH SERVICE ('.-JITHOJT INTERCHANGE OF PSRSONNE;., FACILITIES 
OR FUNDS), IDENTIFY SUCH RE^-LAHCH: 



lU. U17 

SERIAL NO. 



PL^r^-LICA.IONS OTIiER THAN ABSTRACTS YRCH THIS PROJECT DURING CALENDAR TuAR 
1955. 

Deringer, Margaret A,, and W, E. Heston 
Development of pulmonary tumors in mice segregated with respect to the 
three genes: dominant spotting, caracul, and fused. J. Nat. Cancer Inst, 
16: 763-768, 1955. 



liONORS"AND~AWARDrTO PEPSCN^^IEL REUTING TO THIS PROJECT DURING CALENDAR 
yf:ar 1955. 



PAGE 1 



PROJECT REPORT FORM 



1 . N ational Cancer Institute 2 . Laboratory of Biology 

INSTITUTE LABORATORY OR BR.ANCH 

3» General Biology Section h - 5' Ul8 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SER. NO. 

6. Host-tumor Relationships ^ ■ 

PROJECT TITLE . "^ 



7« M» '(. Barrettj ^I. B. Kelroy, M. K» Deringer 



PRINCIPAL INVESTIGATOR (S) 



OTHER IWE3TIGAT0RS 



9. PROJECT DESCRIPTION ' • 

In multicellular organisms the morphology and physiology 
of each cell and each tissue is strongly influenced by other cells or 
tissues in its environment. There seems to be a cellular ecology which 
maintains organization and balance from earliest differentiation to 
death. Ample experimental evidence for such intercellular influences 
exists. 

Cancer may be vievjed as a disturbance of su^h an ecological 
balance and from such a view one may derive a definition of cancer. A 
definition in these terms is the only definition of w.hich I am aware 
that does not require frequent important qualifications. For example, 
some normal tissues have enormous proliferative oower and may even 
be invasive 'whereas some malignant cells multiply slowly and may remain 
localized indefinitely but in the end the normal usually responds to 
controlling influences and the malignant does not.' 

Such a definition is primitive but its constancy is a recom- ,. 
mendation. The primitive quality is due to a lock of knowledge of the 
nature of the forces or influences that modify the intercellular 
balances. At present we c.?ll these influences "imrr.uno gene tic", for 
want of a better term. Q-or research is directed at learning more 
about the nature of these influences. The possibility of final 



PROJECT DESCRIPTION (Cent.) 

practical application of the knowledge is suggested liy the occurrence 
of spontaneous remissions and even regressions in cancer of both man 
and animals - regressions that appear to be brought about by a 
resistance on the par-t of the host. 

Methods 

The basic procedure is transplantation of standard tumors 
into hosts of known genetic constitution and studying the effect of 
exoerimental variations on the host, the tumor., or both* - The dis- 
ciplinary techniques involved are those of genetics, immunology, and 
■experimental surgery r 

Major Findings 

A review of this field, including our major findings and their 
place in the general perspective was reported in press last year. This 
paper entitled "The nature of tumor immunity" has now appeared as a 
chapter in a book; see publications. 

We previously reported that a host can be made immune to the 
implantation of a tumor by prior inoculation of washed red cells or by 
stromata but not when the cells or stromata"have been broken up by 
•relatively gentle procedures. We also reported that the antigen was 
easily destroyed by heat or by formalin. Others have strongly sug- 
gested that the antigen concerned is a hemagglutinogen. Available 
data indicate that, genotypically, these antigens are related but 
we believe that ph ^otypical differences exist and may be important. 
Accordingly we have accumulated unpublished data that indicate such 
differences* Hild sonic vibration destroys the "resistance" antigen 
but not the hemagglutinogen for mice (dextran technique). This work 
has -been hampered by "histocompatibility" limitations which prevented 
observation of the 2 types of phenomena in the same mouse. We have 
now succeeded in adapting a tumor so that future observations can be 
made in the same mouse,- The "resistance" antigen (for mice) is 
destroyed. by heating for 30 minutes at U2-I4.U C whereas the hemagglutino- 
gen (for rabbits) rem.ains potent after 30 minutes at 56 C. In one 
strain of mouse a high degree of resistance can be induced by certain 
red cells but such mice do not have hemagglutinens demonstrable by 
the new techniques. (Mr, Melroy has done most of the serologic work). 
Another aspect of the differential characterization of this antigen 
is found in a study of incubation peri-ods. We reported a freakish 
incubation curve that seemed to characterize the development of 



PROJECT DESG^IPTI"N (Cont.) ^ , 

resistance following inaculation of red cells. This -work is very 
laborious but we have made some additional progress. The first 
notch (decrease in relative immunity during the 3rd week) in the 
curve has been see'n again and additional evidence for the second 
notch (5th week) has been obtained. Tha^'"^' gain-loss-re gain" phenomenon 
has not been reported in immunology. We do not know what it means 
but it seems to have something to do with the particular antigen be- 
cause it does not occur when tumor is used as the antigen. This 
might represent an obscure biologic difference between normal and 
malignant tissue but, because the cell type was different, this is 
uncertain, 'vll these points permit us to continue with the hypothesis 
that there is something peculiar about this type of immunity snd the 
antigens that generate it, 

A paper reporting the effect of multiple inoculations of a 
tumor and the implications of the result^ in interpreting Lewis'' 
"breaking down pf resistance", Greene's "Constitutional factors 
peculiar to a tumor bearing host" and Casey's "secondary XYZ" effects 
appeared in the December 19'?h issue of J. N.C.I, which came out in 
January, 1955» Tfee crux of the matter is that the cachectic host' " 
cannot muster such .immunologic defenses .as it. previously possessed and 
this loss is nonspecific. As an outgrowth of that work we reir^vesti- 
gated the effect of incision and partial excision on the progress of the 
disease. The work with tumors transplanted within strain and out. of 
strain has been concluded* Neither simple cruciate incision nor partial 
excision increased the number of metastases and the latter prolonged 
the survival of the hosts. (Details given in previous report). We are 
lifiw observing the effect in spontaneous mammary tumors. Accumulation of 
a properly matched series is very slow. So far the results are in 
agreement with those in transplanted tumors. These results are contrary 
to several reports in the literature, including one in the J. N.C.I, for 
19h9 and one in Krebsartz fer 1952. 

Following our discovery of a maternal influence on growth of 
a tumor and the specificity of "adaptation" we attempted to find a 
clear extrachromosomal (aside fr^m milk agent (a)) or X-Y chromosomal 
inj;^luence on turner growth. A special tj'pe of F^' hybrid produced ty. , 
Dr.' Deringer furnished a very elegant substrate for this investiga£ioh 
but we were unsuccessful in demonstrating an effect. The experiments 
have been abandoned. , . 



PROJECT DESCRIPTION (Cont.) 

Several years ag» Cl»udman reported that mice ,ff strain 
C3Fe, obtained by transfert-ing fertilized C3H ova into 'C57BLK mothers, 
were tolerant to a tumor of C57BIK origin. If this were true it would 
furnish an interesting and instructive variation to the other known 
examples of "pseudohybridization" or "acquired tolerance." However, 
Cloudman reported tolerance in generations F^ to F . Tolerance in 
the parental generation woul* be easy to believe btrt not tolerance 
in filial generations. The istablishment of strain C3He by Dr. 
Deringer gave us an opportunity to reinvestigate this. A total of 
I7U C3He mice of generations Fg to R failed to yield any takes of a 
tumor of C57BIK origin an^ there also were none in 1; mice of the 
parental generation. ThuiS no confirmation of Cloudman's report was 
obtained. A manuscript reporting these negative findings is in 
preparation. 

I participated in the Gordon Cancer Conference as an invited 
discusser but no manuscript for publication was involved. 

A considerable amount of time has been spent in reviewing 
manuscripts, ap plications for" fellowships, and grant-in-aid applications. 

Significance " ' , 

The field with which this woi*k is concerned is so difficult 
and obscure as to make cle^.r definitions and outlines almost impossible. 
Nev..rtheless there is a slowly growing body of fevidence which indicates' 
that there are interactions betw.cn tissues and betwefen tissue and its 
host which involve critic^J. factors in the phenomena of cancer, embryology, 
and transplantation of normal tissues. A "defensible definition of cancer 
cannot be given today except in descriptive and biologic terms. The 
terms usually include things related to the experiments that we have been 
describing. The experiments may be significant in several ways both 
practical and theoretical. 

The most important implication is that some of these results 
suggest progress toward understanding ©f what controls the grewth of a 
cancer and how this differs from normal tissue. We have already seen 
that a tumor's power to overcome host resistan<?e can be altered; its rate 
of growth, tendencj'- t^ regress, and tendency to metastasize are all 
puljject to experimental change. The host's reaction can be altered, 
ex^ei-imentallv, not only between similar hosts but also at different 
times and sites in the same host. The changes in the tumor have eluded 
morp'-ologic, chemical, or serologic definition. One of the antigens 
which changes the "host would l>e misgad. by current chemical methods. 
'Jowever one might attain a biolog4<C' description of the factors that 
sometimes cause snontaiieous tumorS of man and animals to regress and th^t!, 
centrol metastasis. It has already been obaaj^^?^ in this laboratory 
Tand confirmed elsewhere) that such factors materially effect the outcome 
of many chemotherapeutic experiments. 



PROJECT REPORT FORM (Cont'd) 

10. Ul8 / 

SERI1L NO. 

11. 

BUDGET ACTIVITY: ' 

RESEARCH X ADMINISTRATION 

REVIEW & APPROVAL TECHNICAL ASSISTANCE 



12. 

COOPERATING UNITS OF THE PUELic HE/iLTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, F\CILITIES, OR P2RS0WJEL FOR TKLS PROJECT IN EITHER 
1956 or 1957 



13. 

iF TT^tS pftojEC* kfiSffifeLK, toMt^LEiEN'rS, oK mALLELS'RTSEAftcH bofffi 

ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOIT: IKTiIflCHANGE OF 
PE[iSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH} 



PROJECT DESCRIPTION (Cont.) 

The experiments on incomplete excision may lessen the fear 
of some surgeons'in attempting palliation in certaxn selected cases , 
and thus lead to amelioration of a few. 

Relation to' others 

There is a complex theoretical irterlocking ^^^ween this 
«ork a«d the work of Drs. Algire, Grobstein, Prehn, and Sanford. 



PROJECT RE^^ORT FORM (Cont'd) 



lU. iil8 

SERIAL NO. 



1?. ^ 

PUBLICATIONS OTHER THAN ABSTRilCTS FROH T"IS PROJECT DURING CALEIO/m 
YEAR 1955 



Barrett, M. K. and Hansen, W. H.: Resistance of mice to multiple 
inoculations of a transplantable tumor. J, Nat. Cancer Inst. 15: 
Ull-h20, 195U (A:;Teared in 1955). 

Barrett, M. K.: The nature of tumor immunity. In Origins of 

resistance to toxic agents, M. G. Swag, R. D. Reid, and 

0. E. Reynolds, eds. 'icademic Press, New York, p. 308-333, 1955* 



16. 

HONORS AND AWARDS TO ^E^^SONNEL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1955. 



PAGE 1 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Biology 

ITSTITUTE ' LABORATORY OR BRAWCH 

3. General Biology U . 5» Ul9 

SECTION OR SERVICE L0CAT10N(IF OTHER T^AN SEE. NO. 

BETHESDA) 

6. Gastric Cancer Investigations _^ 

PROJECT TITLE 

7 . M. K. Barrett ^ 

PRINC IPAL INVESTIGATOR (3 ) ' ' 



OTHER INVESTIGATORS " 

9. PROJECT DESCRIPTION 

QlBjectives 

Efforts have been tiireeted at finding methods f tr producing 
or studying some of the supoasedly etiologic factors in gastric 
cancer. Many etiologic or predisposing factors have been suggested 
but laboratory investigation of them has been held back by a lack of 
adequate methods for producing them in animals. Our work has com- 
prised a series of attempts to produce starting points from which 
gastric cancer studies could go forward, Tn addition, we have 
attempted to stimulate work in this field generally through th<? 
activities of the Gastrointestinal Cancer Committee. 

T^ethods 

New techniques of experimental surgery (some developed in" 
this laboratory) and physiology have been used to study gastric 
polyps iu dogs and peptic ulceration in rats. 

Findings 

It was reoorted last year that when artificial polyps are 
constructed in a dog's stcanach the mucosa of the polyp undergoes de- 
generative changes ending in atrophic gastritis. A paper reporting 



PROJECT DESCRIPTI'^N (Cont.) 

this appeared in Gastroenterology for March 1955. 

In keeping with our general ideas regarding tissue 
specificity, we adopted an hypothesis that the stomach does not 
digest itself because some "recognition factor" prevents it. A 
recognition factor or specificity might exist between the enzymes 
and the tissue, or between some element of the mucus and the tissue, 
or among all three. If this were so the mucosa, which is relatively 
immune to the corrosive action of its own secretions, might be 
readily attached by foreign secretion. Under properly controlled 
conditions gastric juice was transferred between strains of rats. 
It was found that, although each strain was resistant to its own,., 
juice for a period of U hours (or more), the glandular mucosa of "■ 
each was vulnerable to the juice of the other during a period of 3 
hours (or less). A manuscript reporting these data has been sub- 
mitted to Gastroenterology. , . . _ : "' ~ ": ',' 

Significance 

The experiments on gastric polyps should influence the 
clinical management of polyps, in the direction of radical treatment, 
gsner^.lly. They also provide the long sought method for producing 
atrophic gastritis regularly, although better methods are still 
desirable. ,• • • 

The studies on peptic digestion may have far reaching im- 
plications, which ai present are speculative; One might ask some 
questions. Is the pepsin specific in an'immuno^enetic sense? If so,' 
do other enzymes have a biological specificity in the natural state 
and do they lose this specificity in vitro ? (As pepsin may do). 
Is a different enzyme from pepsin involved in the initial attack 
upon the mucosa? (If so, the specificity remains"). Does the mucus 'v,- 
(jontain a mucopolysaccharide which is antibodyrlike in function? : If so, 
is this where -lysozyrae enters the field in peotic ulccr r.ad ulcera- 
tive colitis? Does gastric mucosa sometimes contain groups of 
cells whAch are immunogenetically aberrant? (Most clones of cells 
do contain atypical cells). Is the chronic ulcer case an example 
•f this with an area of permanently lowered tolerance and the acute 
ulcer case merely a case of temporary imbalance of tolerance for 
transieftt causes? If so the first, should always be excised for 
cure, the second may get well without surgery. Is this the reason 
that nearly all gastric cancers (at the surface) ulcerate? If so, 
one might be justified in deliberately augmenting ulceration in 



PROJECT RE^CRT FORM (Cont'd) 



10. Ul9 

. SERIA.L NO. " 

11. 



BUDGET ACTIVITY: 



aESEARCH X ^DMINISTR;'VTION 
REVIEW k APPROVAL " TECHNICAL ASSISTANCE 



12. . 

. COOPERATING UNITS OF TITE PUBLIC HEALTH SERVICE, OR OTHER '" 
ORGANIZVT '^NS, PROVIDING FUI^IDS , F.\CILj.TIES, OR p■SRSONI^IEL FOR 
THIS PROJECT IN EITHER 1956 or 19$ 7 



13 . 

IF TWIS PROJECT RESEMBLES, COMPLE-'ENTS, OR ? JIALLELS RESKIRCH DONE 
ELSEWHERE IN THE PLTBuIC HEi'iLTH SERVICE (WITHOUT INTERC^'iNGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESMRCHt 



PROJECT DESC'.IIPTION (Cont.) 

some small lesions - a long shot. At any rate, this appears to be 
a new and provocative idea in gastric digestion - and there has not 
been too many such during a century of investigation. 

Ga strointestinal Cancer Committee work . 

\ large amount of time has gone into work with this 
Committee. A small Si'-mposium was held in Stone House near the end 
of last year. At the request 6f the editor of Gastroenterology I 
prepared a short summary of the proceedings and this appeared in 
Gastroenterology for June 1955 • There has been a demand from 
abroad for this reprint. I don't know why. 

I carried the major burden in organizing the 6th National 
Gastrointestinal Cancer Conference which was held in New York in 
April. Editorial work and proof reading of the proceedings have been 
time consuming but are completed and the complete Proceedings appeared 
in Gastroenterology for October, 1955. This is much more rapid 
publication than was obtained previously. 

Proposed Course 

We are planning tec'riniques whireby we may be able to deter- 
mine whether the specificity of gastric juice for gastric mucosa 
also extends to unrelated tissues. If the specificity resides in the 
enzymes, such an extension could be very important. Other attempts 
will be made to see if stronger implication of the enzyme can be 
obtained. 



PROJECT REPORT FORM (Cont'd) 



lU. Ui? 

SERilL NO. 



15. 

PUBLICATIONS OTHER THAN ABSTR.'ICTS FROM THIS PROJECT DURING 



CALENDAR YEAR 1955 



Parrett, M. K., Lcfco, T., and Hansen, W. H. : Degenerative 
changes in the mucosa of artificial gastric poljrps in dogs. 
Gastroenterology 28: 393-Uol, 1955. 

Barrett, M. K.: Summary of gastrointestinal cancer symposium, 
November 19, 195U. Gastroenterology 28: 969-971, 1955. 



16. 

HONORS AND AWARDS TO PERSONNEL RELATII\IG TO THIS PROJECT DURING 
CALENDAR YEAR 1955. 



PAGE 1 
PROJECT REPORT FORM 



1. National Cancer Inst. 2. Laboratory of Biology 
IM3TITUTE ' LABORATORY OR BRANCH 



3. General Biology Section i;« ^ . 1|28 (a) 

SECTION OR SERVICE LOCATION( IF OTHER . THAN . BETHESDA ) SER. NO. 
Studies •n the Rous sarcoma virus. I. Development of th« 
6» virus-chicken teat-system for general use in basic studies tn 
PROJECT TITLE carcinogenesis and other' tfio logical reactions. 



7. W. Ray Bryan 



PRINCIPAL I ^iVEST IGATOR ( S ) 
8. John B. Moloney, Dorothy Calnan 



OTHER INVESTIGATORS^ 
9. PROJECT DESCRIPTION, 53 y^ X'-'td'Cioo 

Objectives ; To bring about adequate control of the biological and 
experimental factors which cause excessive variations and interference 
with orecise systematic investigations involving the Rous sarcoma 
virus-chicken test- system (a valuable system for crucial studies on 
carcinogenesis, particularly, from the yiewppint of; viral etio.iagy). . 

Methods employed t Sinc,e satisfactory methods have now been worked , 
out and the principal objectives noted above have been accomplished!, , 
this project, is currently of minor importance, consisting only in . ,_ 
the further refinement of existing procedtres. The, details of 
methods are therefore not reiterated here '(See IRSli report under 
this heading). 

Major findings ; The major findings developing from this study, 
which had been in progress for about U years, have been detailed 
in a comprehensive review of the subject published during the past 
year )see references 1 and 2 below). The contributions of this 
laboratory to the problem are summarized as follows; 

1. Control of the variations in initial potency of 

extracts was accomplished by showing a correlation be- 
tween tumor initiating dose of virus, and the '/leld 
of virus in the tumor tissue extracts. By using 
only tumors initiated by strong doses of virus, the 
tumor source material can be kept highly potent, and 
predictable . 



PROJECT 3DESCR I PTION 

2, Control of the variable loss in biological activity 
of different virus oreparations was accomplished by 
the use of citrate reagents, in which p-itency remains 
stable for at least 7 days at ordinary refrigerator 

temperatures.,. 

Preservation for long periods of time (at least 
■' * . 2 years) was accomplished, by freezing in citrate 

solutions and storage in a COj ice chest. This find- 
ing permits the use of standard virus preparaticfns 
■'' . * for quantitative biological studies and inyostigations . 
extending over long periods of 'time i''-*^ .^di'i 

3. Control of variations in susceptibility among different 
test lots, of chickens employed at. different times was • 
accomplished by the use of stable standard preparations 
of virus (see 2) and bioassay methods based upon refer- 
ence to standard preparations. 

li. The variations in susceptibility among individual - 

chickens of a common lot was brought under statistical 

control by studies on the distribution of chicken 

sensitivities and the selection of appropriate 

statistical methods applicable thereto. 
mor-^"- ■ ■ ■ ■ . " , 

Future course of project; It is planned to initiate sometime next 
spring or summer, a study on' different types of host chickens in 
their responses to the Rous sarcoma virus. The study will involve 
various inbred lines of c'nickens derived from the Regional Poultry 
Laboratory, U.S.D.A., at East Lansin'g, Michigan, and will be in 
collaboration with Dr.' B.H.- Burmester of' that laboratory. The ob- 
jective will _ be to find lines' of high, intermediate and low genetic 
susceptibility to the virus'^ for further use in contemplated studies 
on virus-host interaction. 



■ ^ir" y'.i 



PROJECT REPORT FORM (Cont'd) 



10. 1^20 (a) 
SERIAL NO. 



11. 



BUDGET ACTIVITY 



RESEARCH X ADMINISTRATION 
REVIEW & APPROVAL TECHNICAL ASSISTANCE 

12. — — 

COOPERATING UNITS OF T HE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS , 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 or 1957. 



13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUfeLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



PROJECT RS^nRT FORM (Cont'd) 

lU. U20 (a) 

SERIAL NO. 

15. 

PUBLICATIONS OTHER T'^AN ABSTR.\GTS FROM T^'IS PROJECT D'lRING CALEfffi-'iR 
YEAR 1955 

W. R. Biyan; Biological Studies on the Rous Sarcoma Virus. 

I, General Introduction. J. 'Jat. Cancer Inst. i6: 285-286, 1955. 

W. R. Bryan: Ilsidem. II. Rgvisw of Sources of Experimental 
Variation and of Methods for their Control. J. Nat. Cancer Inst. 
16: 287-315, 1955. 



16. 

FON'-^RS AND Wi'^RDS TO ^/ aSONwEL RE_-iTING TO T' " IJ PROJECT DURING 
CALENDAR YE\R 1955. 



PAGE 1 
PR'-iJECT REPORT FORM 



1. National Cancer Institute 2 » Laboratory of Biology 

Institute ' ^ laboratory or branch 

3, General Biology Section h- g. U20 (b) 

section or service location (IF oti-ter t^'An BETH.; ser.no. 

6. studies o n the R ous Sarcoma virus. II. Pvirifi cation of the virus. 

PR''^JECT TITLE 



7. John B. ^ioloney and W. -Ray Bryan 



PRINCIPAL INVESTIGATOR (S) 
8. Dorothy Calnan 



OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives ; 

To obtain the Rous sarcoma virus in sufficient quantity, and in 
a sufficiently high state of purity, to permit its physical and 
chemical characterization, as well as its general use in investigations 
on mechanisms of carcinogenesis. 

Methods employed ; 

(a) Enzymatic degredati-n of impurities by the use of 
hyaluronidase and trypsin, (b) precipitation of the virus by 
protamine sulphate, and (c) differential ultracentrifugation, were 
combined to produce a purification techaique which yields a product 
of h to 12 fold higher purity Con a nitrogen basis) than methods 
previously developed. A report of the procedures has been published 
(see reference, below). 

It is apparent that these same procedures, carried through 
2 or more additional cycles, might accomplish final purification 
of the virus if stronger sonrce materials were available for their 
application. (See further discussion under "Proposed course of 
project", below). 



PROJECT DESCRIPTION (Cont.) 

Significance to cancer research ; The rapidity of action of this tumor 
virus (microscopic tiutiors in 2 or 3 days, gross tumors in 5 or 6 days) 
indicates that it may enter directly into the intracellular reactions 
involved in malignant transformation. This virus may therefore be 
capable of guiding the investigator directly to the elements or re- 
actions involved in the transformation from the normal to the neoplastic 
ptate^ Studies on the chemical interactions are dependent upon the 
availability of practical quantities of essentially pure virus. 

Proposed course of project ; (a) Further refinement of present success- 
ful separative procedures with emphasis on increasing the final yield 
so that additional purification cycles can be undertaken, (b) the 
overwhelming amounts of non-virus material ("impurities") to virus 
in the present starting extracts of tumor tissue cause relatively 
great losses by any method of separation. Efforts are therefore being 
made to increase the virus content of the tumor tissue to be used as 
virus source material (See under project III). 



PROJECT REPORT FORM (Cont'd) 
U20 (b) 

10. SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH X ADMINISTRATION 

REVIEW ^ APPROVAL TECMICAL ASSISTANCE 

12. ______ 

co6*ffiA'ftW6 ilWiM oP fM 'mtit HEAttH SffiVtdE,' 6ft ofH^TR okGAiiiZAf ^'onS, 

PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 or 1957 



13. 

IF THIS PROJECT RESE-fitES, d'6l^^KEiN'iS , Oft PARALLELS ftfi^fiAR^J^ tloM 
EISEW"ERE IN THE FiBLIC HEALTH SERVICE (WITHOUT ItJTERC"ANGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR ITEMS lli, 15 and 16 



PAGE 1 
PROJECT RE'^ORT FORM 



1. National Cancer Institute2. Laboratory of Biology 

INSTITUTE LABOR.^TOR^' OR BRANCH ' 

3. General Biology Section h • S . Ii20 (c) 

SECTION OR SERVICE LOCATION (IF OTHER T".\N BETHESDA) SER. NO. 
Studies on the Rous sarcoma virus. III. Ihe quantitative relation- 

6. ship between inducing doses of virus and the amount of virus ^ 

PROJECT TITLE extractabl^ from experimental tumors. 



7. W. Ray Bryan 

PRINCIPAL INVEST IGATOR(S) 



8. Dorct hy Cal np.n and J ohn B. Moloney 

0T^"ER~IWESTii';Af0R3 ' ■ , 

9. PROJECT DSSCRTPTiON 

Ob jectives ; The original objectives of this researc>i w.:.re accomplished 
dm-jng the past year ind a report of the firdings was given in the 
publication listed below. 

M.^jor finding? ; The anioant of virus extractable from experimental 
iu'iors vas found to >>': related to the dose of virus usal for 
initiating the tumor. Above certain initiating dose l2\/-els (2^0 or 
more ED^O units), the virus rields ap:)roached an upper limit asymptoti- 
cally, but at lower virus initiating doses the yields were highly 
correlated with initiating dose. Doses of 1 ED^O unit, or less, 
procLuced tumors from which very little or no virus could bo recovered 
on ext.actj.on. 

Significance to cancer research ; In addition to the practical useful- 
ness of the information, which permitted methods for pi educing source 
tissue of constant high potency (see project I), the results of this 
study have imoortant implications with respect to concepts in cancer 
research. For example, it has been generally assumed by workers in 
this field that tumors having a viral etiology should yield demon- 
strable virus on extraction, and that the failure to demonstrate 
virus in filtrates of turners is justification for regarding them 
as being of nonviral etiologyt The above findings show decisively 
that this is not a valid assumption, and that under certain re- 
producilile experimental conditions even the most potent and most 



PROJECT DESCRIPTION (Cont.) 

rapidly acting tumor virus known is capalile of initiating tumors 
from which little or no virus can >»e recovered on extraction. 

Since the frequencies of experimental tumors are also 
related to initiating dose, the probability of detecting virus 
in tumor tissue extracts decreases as the frequency of the tumors 
decreases. A.t the extremely low frequencies with which "spontaneous" 
tumors occur in nature, therefore, one would not, on a basis of these 
findings, expect to demonstrate the presence of a viral agent even 
if the tumor should be of viral origin, 

Proposed course of project ? The developments in this project have 
opened up new areas of investigation. The most significant problem 
to be investigated is that of the reason for the observed correlation 
between initiating dose and virus ^/ield. The final answer to this 
question will, of course, involve both the mechanisms of virus re- 
production (in this particular case) and of neoplastic transformation. 
>Tany ye=>rs would probably be required, therefore, for substantial 
progress toward this end. 

The present efforts on this project are confined to: (a) 
The development of biological materials which will be necessary for 
the more 'hasic studies, such as a transplantable tumor L'.ne of low 
dose origin which will provide ample amounts of tumor tissue yielding 
little or no virus on extraction; and similar stable tumor lines 
yielding extremely high, as well as Intermediate potencies, (b) 
Exp'i.oratory experiments on possible mechanisms which rould explain 
the correlation; such as the presence of inhibitor substances 
(suggested by certain results of earlier investigators), or differ- 
ences in amounts of virus actually reproduced (electron -micrographic 
studies with Dr. Balton - preliminary). 



PROJECT FORM (Cont'd.) 

10 .j£20_j£)____ 
SERIAL NO. 

11. 

BirOGET ACTIVITY; 

RESEARCH X AmiNISTR\TIO.N , 

REVIEVJ ^. APPROVAL TECKMICAL ASSISTANCE 



12. 



COOPERATING UNITS OF THE ^UBLIC HEALTH SERVICE, OR OTHER ORG.INIZ'TIONS, 
PROVIDING FUNDS, FACILITISS, OR ^ERSONl^EL FOR T'-'IS PROJECT IN EITHER 
1956 or 19^7 



13. 



IF T'-'IS PR-^JECT RESEIIBLES, COK^'LEI'iENTS, OR ^'-R'-LLELS REoEAxxCH DONE 
ELSEWHERE IN THE PT^BLIC HEALTH SERVICE (WITHOUT II'TERCH'INGE OF 
PERSOi^-^L, FACILITIES OR FiTNDS), IDENTIFY STTCH RESE'RCH: 



PROJECT REPORT FORI>^ (Cont'd) 

lU. HgQ (c) 

SERI'vL NO. 

Ig. 

PUBLIC/\T10NS OTHER THAN ABSTRACTS FROM T^IS PROJECT DIEING CALENDAR 
YEAR 19^5 

Bryan, W. R,, Calnan, D. and Moloney, J. B. : Biological studies 
on the Rous sarcoma virus. III. The recovery of virus from 
experimental tumors in relation to initiating dose. J. Nat. 
Cancer Inst. 16: 317-335, 1955. 



16. ■ . 

HONORS AND AWARDS TO "fRSONlviEL RELATING TO T^'TS PROJECT DURING 
CALENDAR YEAR 1955. 



PAGE I 

PROJECT pj:port 

• * National Cancer Institute '_ 2 , Laboratory o f Biology 

INSTITUTE . ■ LABOIUTORY OR BRANCH 

I* General Bio logy Section I4. 5. I 42I 

SECT ION "CR 'SERVICE ' LOC.iTION'^ (IF OTHER TIliiN SERIAL NO 

EETHESDA 

) , Genetic Studies in Mice ' A. Genet i c Studies of Tumors B. Ctenetic Study of a 

PROJECT TITLE ' ' ' "' • 

Mutant. .Urogen ital Abnormalities in Stra in A x C Line 9 93$ Rats. 

' . Margaret K. Deringer 

PRINCIPAL i:TE3Tir-AT0P(37 " ' 

i. Dr, V. E. Hest on - Dr. M. K. Barrett ^ ] 

OTHER INVESTiaiTORS • ■■ 

'. PROJECT DESCRIPTION: 

Objectives; 

A. Genetic studies of tumors 

. li To study the effect of the injection of the mammary tumor agent into 
agent-free strain C3Hf mice duririg the .late stages of pregnancy at which 
time the agent has been reported to disappear from the blood of mice 
which carry the agentj and to see if the agent can be transferred 
tlirough the uterine wall to the fetus (in cooperation with Dr. Heston). 

2. To study the effect of the subcutaneous injection of 2~f^™ii^oszotoluene 
on the prQcluction of hemangioendotheliomas in strain Hn mice. It has been 
observed that hemangioendotheliopias developed spontaneously in l8.'i of a 
group of untreated strain HR mics» No other strain of mice has been des- 
cribed in which hemangioendotheliomas occur frequently but they have been 
produced by subcutaneous injection of o-aminoazotoluene in strains BALB/c, 
A, C3H, and C5'7BL. 

3. To study the incidence of mammary gland tumors in virgin, in breeding, 
and in forced-bred females, and in stilbestrol injected males of strain 
C3HeC57BL (a substrain developed in this laboratory by the 'transfer of 
fertilize* ova from a strain C3H mouse to a strain C57BL mouse). 

U. To determine whether a mammary tumor agent is pi'esent in strain BL 

(Bagg L) and in strain S'^ (Swiss) (in cooperation with Dr, Heston). 

$, To study the. relationship of certain known genes (W - dominant spotting, 

Ca - caracul, and Fu - fused) in mice to tjie production of pulmonary 

tumors induced by the injection of a hydrocarbon (in cooperation with Dr* 

Heston). 

6, To produce special types of animals for collaborative experiments 

with Dr. I^'i, K^ Barrett (see Dr, Barrett '.s report for details), 

B. Genetic study of a mutant 

1. To test a mutant, phenotypically-likc.waltzer which arose in strain 
A mice maintained in this laboratory (in cooperation with Dr. Heston). 

Urogenital abnormalities in -strain a x C line 993$ rats. 



i'ACE 2 

lit autopsy contreiiital absence of one kidney was observed in a rat of 
the strain A x C lino 993$ colony* Subsequently, a number of rats of 
this strain wore observed to have one kidney missing, and a number were 
observed to have othor urogenital jibnorinalitiL-.G. The extent and inci- 
ili'iu'i' of those nbnovma.l conditions hars bc>'n obnorvcd. 

Method H omployc d : 

Al 1. llroups of strain C3Hf mlro have boon injected intraporitoneally with 
0»5 ^'0« O-'C f S% cell free extract of a mJUiimary t^iland tumor, from a strain 
C3H mouse. The [.^roupn of animals were injected 1-3, U-6, and 7-9 days 
boforo the birth of tho youncj immodiately followin^^ the birth of the 
young; and when thco first litters wore weaned. Two groups, maintained 
as virgins, have been injected at h$ and 65 days of ape respectively. 
Unlnjooted female mice have been allowed to produce one litter each and 
then have been retired at the time at which these litters wore weaned. 
Approximately 200 female sff sprint;; of mothers injected before the birth 
oj" the yonnf^ have been kept as virgins. The animals in this experiment 
will bo maintained as long as possible and a study of the occurrence of 
mammary i:l''^nd tumors and of other typos of tumors will be made. 
2» One hundred retrain HR mice (equally distributed as to sex and as to 
profjonoo or absence of hair) wore injected with 100 mt^:, of o-aminoazo- 
toluone dissolved in olive oil. The injectioiis were startecf when tho 
animals were 2 to 2 -."V months of age and were given in monthly doses of 
10 mj% each. One hundred uninjcctod animals served as controls, illl •f 
tho animals were observed for tho development of tumors and of any un- 
usual lesions. 

3. One hundred C3Hi females have been set aside as virgins in order to 
study the' development of maiumary gland tumors in them. One hundred C3He 
females are being forced-bred for the same purpose. These animals are 
isolated when prcgnaat .'iiid nre rc;turned to the breoding cage within 2U 
hours ot less following the birth of the litter. One hundred males have 
been injected subcutaneously in the right axilla' with 6-8 mg. pellets of 
10^ stilbestrol in chclestrol for the purpose of studyiiig the development 
of nvuim*\ry gland tumors. This will aliso be determined in the case •f tho 
brooding females which are used to maintain the colotiy of C3He mice. 
Ij, Newborn strain C3Hf or C3He femnlu mice are foster-nursed on strain 
BL and on strain S^.'JY( females. The foster-nursed animals are maintained 
as virgins v-md are observed for the development of mammary gland tvunors.^ 
^', The mice \iBod in this exvxrimont were olfspring produced by mating 
F-, hybrids of strain IJCaT'u and A with strain 119li» Tho animals were 
injected intraperitoneally with O^S mg, of 1,2,5,6-dibcnzanthracene at 
two months of age. They wore autopsled at 10 months of age and were 
observed for the development of pulmonary tumors and of aixy unusual 
lesions, 

P, 1, Stivnin A mados, , -howing the waltaiiir; chi^.racter were outcrossed to 
strain 119l4> F]_, F2* ^'^'^ backcross generations wore produced and data 
were collooted regarding the presence of the charaott.r in the different " 
groups of animals, 

A rtuttnu .saarch for atawrrmfilities of the urogenital system was made 
at autopsy in youii^.-; animals (1 to i8 days of age) and in adult animals 
(2 to 17 months of a^e) of the -strain A x C lino 9935 rats. 



P.UJE 3. 

Majo r findings; ■ 

jv, l.'Tn the group injected 1-3 days bcforo the birth of thtj young, 11 of 
a group' of 57 females have dovclopod mammary !,':1-.uki l.mnora at an avorago 
ago of 20,9 months; the non-tumorous ajiynals diofl at an average age of 
21,1 months. In thu group injected [|-6 days before th^,. birth of the 
young, 13 of- a group, of $3 females have developed maimnary gland tumors 
at .an average age of 20.8 months; the non-tumorous animals died at an 
average a^ of 20,2 months. In the group injected 7-9 days before the 
birth of the young h of a group of 37 have developed majranary gland tumors 
at an average age of l6.0 months; the non-tumorous animals dying at an 
average age of 19.8 months. In the group of animals injected at the wean- 
ing of the first litter 5 of a group of 52 animals developed mammary gland 
tumors at an average age of 2lj..l,|. months; the non-tumorous animals died at 
an average age of 20.1 months. One fumale of a group of l6 injected 
following the birth of their young developed a m<-jnuna.ry gland tumor at 25 
months;. the non-tumorous animals died at an average aije of 19»9 monthn. 
Nine of 195 of the offspring of injected mothers developed riKimmary yland 
tumors at an average age of 20.1 months; the non-tumorous animals died 
at an average age of 20,6 months. Three of 57 of the females, retired 
after weaning of their first litter, developed mammary gland tumors at 
an average age of 18,3 months, the non-tumorous animals died at an aver- 
, age age of 21.1 months. In the groups injected at kS '^nd 65 days of age, 

5 of Ul females in the former and 2 of k3 females of the latter group 
developed mammary gland tumors at average ages of 15..0 and 19.0 months 
respectively; the non-tumorous anim.'ils died at average agc_,s of 20.0 ,and 
20,3 months, 

2, The incidence of hemanyiioendoiholi^mas yind of hepatomas has bi,un in- 
creased in. the animals which have been injected with o-riminoazoioluene 
as compared with the untreated animals. Hemangioendotheliomas were ob- 
served in 13 of 23 injfoctcd haired a.nd ,12 of 22 injected hairless males 
as compared with 8 of 2k untreated haired and 2 of 16 untre .ted hairless 
males. They were observed in 13 of 22 injqcted haired and in 17 of 25 
injected hairless females as compared with 6 of 23 untreated haired and 

6 of 25 untreated hairless females. Hepatomas were observed in 8 of 23 
injected haired and in 6 of 22 injected hairless males as compared with 

3 of 2[|. untreated haired and 1 of l6 untreated hairless .males. They were 
observed in lli of 22 injected haired and 23 of 25 injected hairless fe- 
males as compared with 1 of 23 untreated liaired Lnd none of 25 untreated 
hairless females. 

Multiple tumors of both therje types •ccurred more frequently in the 
injected animals, 

3, No results to date, 

Ii. Six of a total of l6 strain C3Hf or C3He females, fo;iti..r-nursed by 
strain 3L, have developed mammary gland tumors. The tumors have appeared 
at an average age of 13.0 months, the nontumorous animals have died at an 
average age of 15.0 months. All of tl^ C3Hf or C3He females, foster- 
nursed on strain SWl,^, are still alive, 

5, In th>; outcross resulting from crossing (A x WCaFu)Fi and ('iCaFu x u)F-| 
animals with strain 119)4, the segregants that Wk,re fused were less sus- 
ceptible to induced pulmon^iry. tumors than were the segregants that were 
nonfused. No significant difference in susceptibility was observed be- 
tween the segregants with dominant spotting and those without spotting 
or betwe(;n the caracul and noncax'acul .'segregants, (Geventy-five of IlO 
animals exhibiting domina,nt spotting developed pulmonary tumors following 
intraperitoneal injection of dibcn'/,o.nthracone. Sixty-nine of 107 animals 
without Roottlng developed pulmonary tumors,, The difference between the 



PAGE h. 

two is not significant)! X - .33, P is botw-cn O.^j iind 0.7. Thirty-one 
of 58 animals with caracul coat and 2? of h2 animals with normal coat 
developed pulmonary tumors. The difference between thciTi is not sig-nifi~ 
cantj X^ - .05, P is between 0,8 and 0,9, Twenty-seven of ^0 animals 
with fused tail and 39 of 52 aniiuals with normal tail developed pulmon- 
ary tumors,' The difference between the two is significant! X = h»9't P 
is between 0,02 and 0,03, ) Growth curves showed that the average weight 
of mice that were fused was less than that of mice that were nonfuse^l. 
and the mice that were caracul weighed less than those that were' non- 
caracul.' There was no difference betx^een the average weights of the 
males with and without dominant . spotting, but the females with spotting 
weighed more than those without, 
B, 1, In the (II9I4 x i.)F2 animals, 75 of a total of 63h animals (11,8-0 
exhibited the waltzing character and in the (119U x j4)-iij3C animals, 97 
of a total of 251 animals (38. 6;^) exhibited it, .These results indicate 
that possibly this character is due to a single recessive gene,. No 
further results at present available, 

.ib normalities of the urogonitaJ. system in strain A x C line 993$ rats 
were observed in 13ii of a total of 7U8 females and in 137 of a total of 
813 males autopsied at 1 to 18 days of age,. In animals autopsicd at 2 
to 17 months of age, Ii5 of I89 females and 12 of 62 males were abnormal, 
•The principal manifestations were the absence of one' kidney, or the' pres- 
ence of one cystic kidney. These were accompanied' generally by other 
abnormal conditions,. In females, they included a missing ureter, uterine 
horn, oviduct, ovarian capsule, or ov;iry; an incomplete ureter or uterine 
hornj a cystic ureter, uterine horn, or ovarian capsulej or hypertrophy 
of the remaining kidney, with vaji'ia^tion in the degree of abnormality, ' 
In males, they ijicluded a missing ureter, epididymis, vas deferens, or " 
vesicular glandj atrophy of the te,stis,- vesicular gland, or. epididjanisj 
cystic ureter; undescended testis; and hj'pertrophy of the remaining 
kidney, all also varying in degree. 

Significance to can c er r esco-rch; . " 

A, It is already well ^established that the genetic constitution influences 
the development of tumors, .ill facets of this project will add more infor- 
mation to that. already existing concerning the mode of inhcritiince of var- 
ious types of tumors, 

B. The study of the mutant may be of significance in providing another 
known character which will be of use in the linlcage studies of factors in- 
fluencing susceptibility to various' types of tumors. 

Proposed course of the project ; 

It.. 1. This study will continue through 1956, 

2, This study will be completed early in 1956 (four experimental females 
remain to be autopsied) and the results x\rill be prepared for publication, 

3. This project will continue tlirough 1956, 

U, It is anticipated that this experiment will continue through 1956, 
5» The results of this experiment will appear in the December 1955 issue 
of the Journal of the N-ational Cancer Institute, . 

6. Special types of animals will coatinue to be produced for experiments 
with Dr, Barrett, 
B, 1, Project will continue through 1956, 

The results of this experiment have been i^rritten up and the manuscript, 
has been submitted to the Editorial Eoar«l for appro\'a,l for publicfrtxon in 
the Proceedingpof the Society for Experimental Biology and Medicine, 



PAGE 5. 



LO. J42I 

-?— pj '-J :jr, ^ ■ 



LI. 



BUDGET ..CTI\r[TY: 

rese;jrch X /JDraraSTIt.TION 

KEYim & .iPPROV;A TECHNICAL .,.SSIST;.NCE 



L2. 



-3. 



COGPEIi^TING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER 0RG;.NIZATI0NS, 
PROVIDIi''G FUNDS, F.XILITIES, OR PER30:.;NEL FOR THIS PROJECT IN EITHER 19^6 
OR 1957, 



IF THIS PROJECT RESEIMBLSS, C'HPLEISNTS, OR P.JIALLELS RESE.JiCH DONE ELSHaPTERE 
IN THE PUBLIC ESilLTH SERVICE (iflTHOUT INTERCFLJ^GE OF PERSOiiNEL, FACILITCES 
OR FUNDS), IDENTIFY SUCH RESEARCH: 

.I4. U21 



SERL:.L NO. 

5. 



PUBLICx.TIONS OTHER THx.N .JBSTR.'>.CTS FROM THIS PROJECT DURING C.^LEND..R YE..R 1955 

1. Deringer, M. K,, and Lorenz, E.: Results of exposure of newborn HR 
mice to X radiation. J. Nat. Cancer Inst. l5: 923-929, 1955. 

2. Deringer, M. K., Lorenz, E., and Uphoff, D. E, : Breeding behavior and 
tumor development in (C57L x ^x)Fi^ hybrid mice receiving X radiation to 
ovaries shielded. J. Nat. Cancer Inst, 15: 931-9l4l, 1955» 

3. Deringer, M, K«, and Heston, vj. E.J Development of pulmonary tumors 
in mice segregated with respect to the three genes: dominant spotting, 
caracul, and fused. J. Nat, Cancer Inst. I6: 763-768, 1955. 

h» Deringer, M, K,, and Heston, W. E.i Abnormalities of the urogenital 

system in strain A x C line 993$ rats, (Submitted to the Editorial Board 
of the Journal of the National Cancer Institute). 



P^GE 1 



PROJECT RE-'ORT FORM 



1. national Cancer Institute 
Tf^fTfUTE ^ ~~" 



2 . Laborator;/ of Biology 



LABORATORY OR BRANCH 



3« General Biolog;'- Section 
SECTION OR SERVICE '' 



U._ -5. Ij23 

LOCATION) IF OTHER THAN BETH.) SER. NO. 



6. Developmental Physiology- 



PR'-^JECT TITLE 
7'. Clifford Grobstein 



PRINCIPAL INVESTIGATOR(S) 



OTHER INVESTIGA.TORS 



■9. ■ PROJECT DESCRIPTION 

■4. Objectives:' To increase 'understanding of the interaction of tissues 
and the corrfponents of rudiments in normal development as these relate to 
the differentiation of new cell types, and to' apply iiiformation obtained 
to pathological problems, in particular to problems of tumorigenesis and 
malignancy. 

B» Methodst Developing rudiments of mouse organs, e.g. kidney, salivary 
gland, and axial skeleton, are separated into their components and 
cultured in isolation and in various recombinations to determine the 
degree to vjhich their development is interdependent and the nature of 
the interactive mechanisms involved. Development is observed in vitro 
and/or on reimplantation into the artterior chamber of the eye. 

Major findings; Study continues of trans-filter induction between 
embryonic spLnalcord and me tanephro genie mesenchyme. Methods have 
been developed, in collaboration with Dr, Dalton, for electron micro- 
scopy of the filter during induction. Cvilture methods have been further 
refined to permit elimination of the clot, thus making possible study 
of the induction in defined nutrient media as these become available. 
Preliminary studies on intact rudiments with the amino -acid- vitamin 
mixture of Eagle indicate that it can be used if supplemented with 
l|orse serum or mouse ascitic fluid. 

The nature of the trans-filter inductive influence cannot yet be 
specified. Filters in contact with inductively active tissues may show 
three types of penetration depending on tissue type, filter type, and 
duration of contact: 1) Cytoplasmic processes identifiable with the 
electron microscope; 2) Granular or filamentous material demonstrable 
by staining or phase contrast; 3) Diffuse material present after 
alcohol-formalin fixation and digestible by tr-'/psin. Efforts are 



PROJECT DESCRIPTION (Cont.) 

continuing to determine possible correlations between the several 
kinds of penetration and biological activity, and to "trap" biological 
activity in the filter in the absence of living tissue, 

Metanephrogenic mesenchyme, which never forms epithelial tubules 
by. itself in vitro, does form tubules when implanted into the eye or 
brain of adults - despite the fact that tissues from these sites are 
negative as inductors in vitro. 

Parotid epithelial rudiments undergo characteristic parotid-type 
branching in sub-mandibular mesenchyme - suggesting that the mesenchyme 
of the two salivary rudimentis shares a common property not found 
previously in mesench-^mtie from non-salivary sources, and that the 
difference in type of branching is a function' of epithelial rather 
than mesenchymal properties. 

Preliminary experiments by Dr. Robert Auerbach, N.I.H. fellow, 
indicate that lens induction by the optic vesicle in the mouse can be 
obtained in vitro and that the process may be able to be analyzed by 
methods similar to those used for kidney, salivary gland and cartilage. 

D. Significance to cancer research: A.s noted in last year's report 
these studies bear on the nature of factors which sta'kilize or disrupt 
tissue architecture. 

E. Proposed course of project: Continued study, is planned of the 
nature of the mechanisms of induction in the several in vitro systems 
available. Approaches in progress include; l)Electron microscopy 

of the filter betwe^;n interacting tissues; 2) Variation of filter 
oorosityand other .experimental conditions in an effort to separate the 
three t:/pes of filter penetration for correlation with biological 
activity; 3) Determination of the nature of the factors responsible for 
tubule formation in implanted kidney mesenchyme; ii) Culture of the 
interacting. system in defined media so as to simplify identification 
of active materials. 



PROJECT REPORT FORM (Cont»<l> 

10. U23 

SERIAL NO. 



11. _ 

BUDGET ACTlVmi 



RESE?JICH I AD-'^'INISTRATION . ,, 

REVIEv^ ^ APPROV/i TECHNICAL ASSISTANCE 

^ '• fo6p^^ilmrmin:$, OF the V nBLici hil/.lt h ~5e^vice, or .6ther oRGaNr^vi-iuiNS , 

raovSiNG FUNDS, FACILITIES, OR pERSONIJEL TOR T"IS PR'aJECT IN EITHER 
1956 or. 1957: 



' if T-lS PRijteCT R-^ryBLga, doM "'LEMEN TS, Ok -:^!\RJl'LLEI5 R'^SL-lkuH 1^)1^^ 
ELSK^-ERE IN THE PUBLIC HEALTH SERVICE (WiTHOUT INTERCHANGE OF 
PERSONNEL, FACILITIES OR FUNDS) , IDENTIFY SUCH RESEARCHj 



PROJECT REPORT FORM (Cont'd) 



111. It23 

SERIU NO. 



'pTIBLICATIONfe OTHEft TF/IN fiBSTRICTS FROM T^iiJ t'KOJECT DURING GALJiNUAk 
TEAR 19^5 

1, In vitro studies of cartilage induction in mouse somite mesoderm 
(with ". uoltzer) J. Exp. Zool. 128: 333-358 19$$ 

2, Tissue disaggregation in relation, to detemir.ati->n and stability 
of cell type. tan. i^. Y. Acad. Sci. 60: ,1095-1106 19$$ 

3, Tissue interaction in the morphogenesis of mouse embryonic 
rudiments in vitro. In "Aspects of synthesis and Order in 
Growth", PrincetoR nniv. Press 19$$ 



16 . : , : 

HONORS AND AVJ'iRDS TO -PERSONNEL HiiLATiNG TO T'lB PRr^JECT DURBIG 
CAiENDAR YEAR 19$$. 



PAGE 1 
PROJECT REPORT FORM 



1, National Cancer Institute 2, Laboratory of Biology 



INSTITUTE LABORATORY OR BRANCH 

3. General Biology Section - li« ___^ ^' U2I). 

SECTION OR SERVICE - . LOCATION (IF OTHER THAN BETH. SERIAL NO, 

6, Tissue Compatibility Studies 

PROJECT TITLE 

7. Richmond T. Prehn 



PRI 'CIPAL IN\^ESTIGATOR(S) 
Joan PI. Main 



OTHER IIWESTI GATORS 

9. Project Description 

Object; The long range objective is^ of coarse, the understanding of the 
physiology of cancer, which in turn will some day lead to control of the disease. 

It is axiomatic that the well-being of the cancer. cell is dependent upon 
its social acceptability to the normal cells of its host - i.e. upon its recog- 
nized failure to arouse a significant ■ immune response or foreign body reaction. 
The cancer cell, in order to survive, produces little or no detectable response 
of an antigenic nature, but is apparently .accepted by the host organism as a 
legitimate, if somewhat undisciplined part of itself. 

This fact stimulates the question: why are some tissues or cells antigenic 
and others not? The facile. answer that, "only foreign cells are antigenic," not 
only begs the question - it is net .correct. The discovery of a variety of auto- 
antibodies demonstrates the cogency of the question. It is not irrational to 
believe that the understanding of growth m general and of cancer in particular 
may await a better understanding of why cells are antigenic in some cases and 
not in others. Such knowledge might make it possible to produce autoantibodies, 
i,.e» to induce imjnunological growth inhibitors artificially against cellular 
types such as cancer which probablj'' have little or no natural antigenicity. 

The immediate object of t.'ie project is therefore to investigate the causes 
or sources of cellular antigenicity or its lack - to investigate the origin of 
the ability of the organism to "recognige" some cells as endogenous and others 
as foreign and x\Thy it sometimes makes mistakes. 

Methods and Results: The immediate work of th.e. project can be conveniently 
divided into three related categories: 

I. The phenomenon of specific aq^iired tolerance: — The original observa- 
tion was that when strain DBA mrlce were exposed to an X-ray dosage of 
800 V and then protected from the lethal effects of the radiation by 
the intravenous injection of (E>lLB/c x DBa),Fi bone marrow, the DBA 
mice, throughout their subsequent life were tolerant to skin grafts 
from BALB/c donors. Subsequently, it was found that bone marrow from 



:^age 2 

BALB/c rather than F-] hybrid mice would acconplish the same result. 
The effect was profound, almost 100,1 of the homografts surviving as 
though they were autografts. The tolerance produced was apparently, 
specific for the antigens contained in the bone, marrow, since resist- . 
ance to ot'fier antigens such as those of a C57-L skin graft, returned 
as soon as the mice recovered from the acute effects of the radiation. 
Further studies suggested that. second set, htmografts are "accepted as 
yell- as first ..set by "tolerant" mice* To-date, attempts to reverse 
the tolerance by injections of im/iiune splenic tissue have failed. - . 
Preliminary results w?ugge.st that the tolerance producing' factor ,may 
be subcellular and 'pcrhaps capable of Sijrial transmission in irrad- 
- ■ irited hosts. The phenomenon is not limited to strain BALB/c grafts . 
to strain DBA recipients, but has been demonstrated wi'th (CxDBA) F]_ 
grafts to (Lxi\).F]_ mice and' with B'ALB/c grafts to C3H, It is apparent 
however, that certain strain peculiarities do exist although these ^ 
have not yet been fully explored, A qualitatively similar though 
quantitatively much weaker effect 'has been obtained by substituting 
L.D. 50 dost.s of nitrogen mustard for the radiation^ 

II. Antigenicity of methylcholenthrene induced sarcomas* /b r.umbcr 

of investigators have reported the apparent production of isologous 
immunity to methylcholanthrone induced sarcomas. Since it is the 
prevailing opinion that tumors are non-ant igenic in the animal or 
strain of origin, the results of these investigators have been attri- 
buted to an "unrealized genetic heterogeneity of materials. However, 
it has now .bee"n found in this laboratory that such immunity can be 
regu,larly produced by MCA induced s.arcomas but not by spontaneous 
sarcomas or mammary carcinomas. It is, therefore, a,pparent that if 
the immunity produced is duo to the small residual genetic heterogen- 
eity, then MCA indufced sarcomas are for some reason peculiarly sus- 
ceptible. The only other possible? explanation would seem -to be that, 
due to some peculiarity 01 the HCA, those tumors may differ from the 
others by being antigenic in th© animal of origin - i.e. they may be 
capable of producing' true autoifiraunization^ Fortunately, experimental 
means are available to distinqaish between these alternative hypotheses, 

III, riiscellancous: A number of miscellaneous but related experiments have • 
been done during the past year: a» The growth of strain C skin grafts 
in (CxDBA) D'A backcross mice has been studied. Results indicated that 
5 or more histoconipatibilitv genes were segregating. , b. The claim 
of Hardin and "Jerder that multiple successive skin homo-grafts could 
produce a state of acquired tolerance was inv^^stigated. The results 
did not substantiate this claim,, c. Red cell a;._glutiaation within 
di.lfusion chambers was studied. Fed cell agglutinins could not be 
demonstrated in the chambers in vivo even in immune mice, d. The 
immunising power of red Qells in diffusion chambers was investigated. 
It was foimd that the diffusion chamber prevented immunization even 
though the red cells themselves remained potentially antigenic, e. It 
was found in a preliraiu-iry expcrimtn,t .that, contrary to the results 
obtained at other sites, intra-diffusion chamber homologous cells were 
killed when diffusion chambers were implanted intra- spleMcally in 
immunised mice, f. An attempt with the aid of X-radia-oion and bone 
marrow to produce an effect in mice by the Rous chiaken tumor agent 



Page 3 

was an apparent failure, g. An attempt to "adopt" tumor cells to 
a foreign strain by prolonged growth of the tumor cells within diifusior, 
chambers in the foreign strain gave possibly slight but generally in- 
conclusive results. This despite the exposure of the cells to the 
foreign medium for a period of 9 months, h. A preliminary study of 
the efficacy of various routes of administration in producing immunity 
to homologous red cells has suggested that the subcutaneous is signifi- 
cantly poorer than the intravenous, intraperitoneal or Intracutaneous 
routes, i. A statistical study of the influence of the occurrence of 
a mammary tumor upon the probability of the occurrence of a second in 
the same mouse was undertaken in high tumor strain C3H mice. The re- 
sults are not yet apparent, j, A study was made of the effects of 
trypan blue on MCA carcinogenesis. This was inconclusive, k. Pre- 
liminary results suggest that the "immunization" of adult BALB/o mice 
to the mammary tumor agtnt interferes with the growth of transplantable 
BALB/c mammary tumors. 

Major Findings and Significance: Two findings are possibly of major importance: 

a. The production of specific acquired tolerance by X-radiation and bone 
marrow administration. This is a significant advance in our under- 
standing of the mechanism by which an animal is capable of distinguish- 
ing foreign from endogenous cells. It is too early to say whether 
this work will in addition, have any direct practical application 
though it is apparent that a useful investigative tool has been dis- 
covered. 

b. The antigenicity of isologous MCA induced sarcomas. This work demon- 
strates an app.irently unique characteristic of such tumors. It is, 
however, too early to forsee where this observation will lead. 

Proposed Course of project: 

A, With regard to the specific acquired tolerance produced by radiation 
and bone marrow administration, it is planned to investigate the 
follovring. Some of these studios arc already under way, 

a. Farther studies of specificity and possible strain limitations. 

b. Effects of varied dosages of radiation and of marrow on the 
phenomena, 

c. Effect of varying the period between radiation and antigen admin- 
istration, 

d. Can tolerance be produced in immunized mice? 

e. Effect of different routes of foreign antigen administration. 

f. Influence of tissues other than marrow as a source of foreign 
tolerance producing factor. 

g. Study of possible changes in transplantation specificity of the 
skins of tolerant mice or of grafts in tolerant animals, 

]|. Fnjrther study on the coll free nature of the tolerance factor and 
its serial transmissibility. 

^* Can tol6rahce"'fe«;''irt'Vferscd by itilirrbw andX radiation treatmeht. 

■ ■ ■ • ■" ' ■•':,;:'^; ■■■'■■' d.^ ■ ,^ ■','■.' ; 

It is doubtful if time will permit all of these studies to be initiated, let 
alone completed during the follotxing year. 

B, With regard to the antigenicity of HCA induced sarcomas, a initial ex- 
periment is now in progress to determine v;hether autologous MCA tumors 
will show the same antigenicity as isologous. The future of this 



Page U 
investigation must await the results of that experiment. 
10. U2li 



11. 



12, 



13. 



SERIAL NO. 



BUDGET ACTIVITY: 

RESE: RCH X /iDMINISTRiiTION 



REVIEI'J & APPROVAL TECHIJICAL ASSISTANCE 



COOPERATING UNITS OF THE PUBLIC HEAL-TK SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONIffiL FOR THIS PROJECT IN EITHER 
1956 OR 1957, 



IF THIS PROJECT RESEMBLES, C0HPLEI-1ENTS, OR P.^JIALLELS RESEilRCH DONE ELSE- 
^dHERE IN THE PUBLIC HEiVLTH SERVICE (liTITHOUT INTERCHi^NGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTIFY SUCH RESE^iRCH: 



lii. U2U 

15. 



SERI/iL NO. 



16. 



PUBLICATIONS OTHER THAN ABSTRi.CTS FROM THIS PROJECT DURING CALENDiiR 
YEilR 1955. 

1. Main, J. M., and Prehn, R. T,: Successfull skin homogrr.fts after 
administration of high dosage x-ray and homologous bone. marrow. 
J.N. C.I. 15: 1023 - 1028 (1955). 

2. '/feaver, J, M., Algire, G. H., and Prehn, R. T.: Growth of cells in 
diffusion chambers II. J.N.C.I, l5: 1737-176? (1955). 

3. Prehn, R. T., Algire G, H., and VJeaver, J. M.: Diffusion chamber in 
homograft research Trans. Bull. 2; Hi? (1955). 

k» Prehn, R. T., and Main J. M. : Lack of immunizing capacity of homologous' 
cells within diffusion chambers J.N.C.I. - In press. 



HONORS ;JJD AteiRDS TO PERSOMIEL RELiJTNG TO THIS PROJECT DURING CALENDiiR 
YEAR 1955. 



Pa,^e 1 
PROJECT REPORT FORM 



■^» Nation al Can(^er\^In£tvtute 2. , Laboratoj^ y of r^.xj^lo^y 
insfitute ~ '" ' ' LAEOPATORY OR BRANCH 



3. C-eneraT Biology'- Section h, . ^'______ 

SECTION OR SERVICE LOCATION (IF OTHER THi-'N EETHDSDA) SERIAL NO, 

6. '^ • "Eff'fects' of the a'^ and"»b Genes on'Mouse Metabolis m and Physiology" 

■ "' PROJECT TITLE — — 

7. George L. v jolff ' ^| ^ '_ 

PRIl'CIPAL I!"/ESTTGAT0RCs1 " "' " "*~ . 



OTHER IN'^ST I GATORS 

9, PROJECT DESCRIPTION; 

Objective: The_ objective of this project is to determine the^ primary aad plei»- 
tropic effects of the A^ (lethal yellow) and ob (obese) genes on mouse metabolism 
and physiology, especially as related to the increased lung cancer incidence in :■. 
iVfa mice and to the hormone balance in mice carrying the A^ and/or the ob genes. 

Methods Employed: An inbred strain of mice carrying the -AJ and ob genes is being 
developed in order that the effects of these genes^ both singly and in combina- 
tion, may be s\.udied against the identical genetic background. 

The anaerobic glycolysis of Harding-Passey melanom.as (in the presence or 
absence of exogenous insulin and/or testosterone) after transplantation to normal 
and gonadectomized, male and. female, Ob- and obtb , A-^a and aaj mice is determined 
by means of a" Warburg re'spirometer apparatus. 

The anaerobic glycolysis of kidney minces (in the presence or absence of 
added insulin and/or testosterone) of male and female Ob- and obob mice at various 
ages, after growth of the Karding-Passey melanoma, or after exposure to high 
temperature (35°c). has been studied by the same method. 

Acid and alkaline phosphatase activities of A^a and aa mouse lung and Harding- 
Passey melanoma homogenates are being determined. 

The possibly differential resistance of Ob - (non-obese) and obob (obese) 
mice to high temperature stress is being studied by exposiag the animals to 35°C 
for 2ii hours. 

Major Findings: The yellow obese mouse phenotype (presumably A^^aobob) has been 
produced and found to be viable. 

The rate of e'stablishment and growth of the flarding-Passey melanoma, six 
weeks after inoculation, has been found to be higher in obob mice than in Ob- 
mice. 

The in vitro rate of anaerobic ^glycolysis of the Harding-Passey melanoma 
grow-n in obob females was found to be higher than that of melanomas grown in any 
other sex-genotyne group, including ovariectomized obob "females. In the presence 



Page 2 

'.' . . 
of exogenous testosterone this difference has a probability of less than ,91 of 

being duo to chance. 

Stimulatory effects of exogenous insulin on melanoma glycolysis were'-tb- 
served in the presence of added testosterone. 

In vitro Fridney mince r;lycolysis of. obob mice has,,^ in general, been f»und t» 
be higher than that of Ob- mice at 30 and 63 days of » age and after 2U hours,' ex- 
oosn.re to 3$°C. """eraale kidney glycolysis in all of these categories tended t» be 
higher than the corresptndi .g male kidney glycolysis. 

Growth of the Harding-Passey melanoma depresses the in vitro kidney mince ■ 
glycolysis of obob mice, but seems to have no effect on that of Ob- mice. 

Testosterone inhibits in vitro kidney mince t.lycolysis of Ob- and obob mice, 
while insulin has been found to stimulate kidney mince glycolysis, especially 
after the mice had been exposed to 35°C. 

The partial contrel »f the potential glycolytic capacity of mouse kidney tnd 
Harding-Passey melanoma b - an insulin: anti-insulin sj'-stem and a pleiotrspic 
effect of the obob genotype on this system is suggested by the data (sbtained. 

Significance to Cancer Research: In order to obtain an understanding of cancer, 
it is necessary to learn how genes control the balance of hormones regulating 
normal and tumor cells so that any difference between the latter may be explftitei 
in the treatment of neoplasms. 

To explain the relatively high incidence of lung tumors in mice carrying the 
Ay gene as opposed to a relatively lower incidence in aa littermates, it is nec- 
essary to know which specific metabolic reaction is under the control sf this 
gene. The solution of this probl>..ra might ultimately rovide a method of prevent- 
ing lung tumors and of treating Ivng tumors cheinotherapcutically. 

The use of the Harding-Passey melanoma as a bio-indicator of the hormonal 
balance and the study of the combined and separate effects of the Ay and ob genes 
on this hormonal balance are designed to provide clues to possible relationships 
between altered metabolism and lung cancer. 

Proposed Course of Project: The determinations of alkaline and acid phosphatase 
activities in A -'a and aa mouse lung homogenates and in Harding-Passey melanomas 
grovm in Aya and aa mice will be continued and extended to determine specific 
phosphatase activities. 

As socn as the new strain carryir.g bcth the Ay ana ob genes is sufficiently 
inbred, determinations of the in vitr o r tes of glycolysis of Harding-Passey 
melanomas grown in normal and gonadectomis,ed, male and female mice of the four 
iiJ f c rent phenotypes ( AyaOb- , A/a obob , aaob-, aaobob ) will be begun. Similar 
determinations will be made on non-maliv^nant tissues, e.g. kidneys, of these 
mice . " 

The role of adrenal steroids in the insulin;. :inti-insulin system in relation 
t» the Ay and- ob genes xv^ill be investigated. &'-• 



12. 



13. 



page 3 



10. h26 

SERIAL NOT 

11. 



BUDGET ACTIVITY: 

RESEARCH X ADFUNISTRATION 



hSVISVJ & APPROViiL TECKNICiiL ASSISTANCE 



COOPILRATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORCrANIZATIONS, 
PROVIDING FUIMDS, FACILIT.TES, OR PERSOMEL FOR THIS PROJECT IN EITHER 19^6 



OR 1957. 



IF THIS PROJECT RESEIIBLES, CG:'IPLSr'IENTS, OR P^.RALLELS RZ'^'Eii-RCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (VflTHOUT Ii\ITERCHAtU.E OF PERSONNEL, FACILITIES 
OR FUNDS), IDE^'TIIFY 8UCH RLSE/aRGK: 



NO ENTRIES FOP ITEMS lU, l5 and 16. 



PRCJECT UEFORT F03f.: 

1, National Cancer Institute ' .2, Laboratory of Biology 

IIISTITUTE L/i30R/.TCaY GS 3Ri;r!CH 

3. Leukemia Studies Section 4, _____^______________ 5. 427 

SECTICrJ CR SERVICE LOC^iTION (IF CTHER HWIl SERIAL f!C. 

SETI-ESDA) 

6. Studies on the etiology and chemotheraoy of experimental lymphomas, 

7, Lloyd '/J. Law 

PRINCIPAL If IVESTIGATCR ( 3 ) 



8, ['.lichael Potter in certain collaborative proiects. 

OTFoER inVESTIGATCRS 

9. PROJECT DESCRIPTICn 

Our investigations in leukesriia and other lymphomas in mice come 
under two major headings: 1) Studies of the etiology and pathogenesis of 
the disease , end 2) Studies of mcch::':isns involved in inhibition of 
leulcemic cell ■"rovjth throu;;h the -.'.se of certain selected compounds, 
particularly such antirxtabolites as ontifolics, antipurines, and 
antipyrirnidines, g.c; ,. methotrexate, 6-mercaptopurir;c, 8-a3sguanine, 
azaserine, 6-azaur3cil, and the pyrazolo pyrimidines. 

Heretofore major etiiphasis has been placed upon lymphocytic neo- 
plasms. As may be seen fror/i this report, our interest is now extended 
to include etiology and therapy cf otlier raorphologic forms, such as 
Type A and Type 3 neoplasms (Dunn), plasma cell neoplasms, etc. 

1) Studies of the etiolotry and rjathoienesis of experimental leukemias . 

A. The r5le of the thymus in spontaneous cases . 

Additional data are novj at hand concerning the role of thymic 
tissue in the production cf lymphocytic neoplasms in (C-SM x AKR)F]^ 
mice, AKR thymic fragments significantly increase the incidence 
and time at death from leukemia and, in the majority of cases (57 
of 77), the transplanted thymic tissue is neoplastic, C3Hf 
thymuses have n^: such influence and none of the recovered thymic 
frngnents were found to be neoplastic, AKR spleen likev;ise is 
found to be ineffective. This study has been extended (with Or, 
f.l. Potter) to include the role cf AKPi thymic tissue in thymectomized 
(AKR X C3H) mice. It is knov;n (from a small series) that the 
incidence of lyir.phomas in thynect"mi?.ed (AKR x C3H) mice is 56% 
(conpared to 53% in intrct controls), but the average age at death 
follo7;ing thymectcny is increased from 14.8 to 19,0 months. 
Furthermore, lymphocytic neoplasms have net been found in the 
thynectomized mice. 



(Project description continued) 



SERIAL m, 427 . p^j^gg 2 

3. ?he influence of ^irafted thymic tissue following exjosiire 
of recipient mice to ^c-^radiation . 

Certain data are rovj ^vriiable concernin'j the influence of • 
C573L thymic tisst'.e rr.fted into thynectcmiied, irrndisted 
_ . ^ ;: ^25 r (C573L x A)Fi nice: 

1, Thyme c tome ctomy ccnr;letely inhibits the indnction of 
lymphocytic neoylasrns (but not ty-;e A reticulum cell neoilasrcis) . 

^.. 5r^fted thymic -tissue, from 1 tC'IC" 'da'y""''-C'idC573L mice, 
becosiiec Iculreuiic in 3".% o:: the esses if transfer is made either on 
day 1 or day 7 after ::-rcdiatioi: . 

3. TrsTiSplcntEtion studies of these induced neoplasms indicate 
that those arising early, rt 5 snd 6 months (4 cases), transplant 
to C573L and the F}, r/lorcas those arising loter, C to 10 months 
(9 cases), g^ovj progressively only in the "Ft^ mouse (no-t in C57BL,- 
origin of thymic tissue). 

These results provide the information suggested by I'^aplari of 
an "indirect mechanism of x-ray induction" dependent on the post- 
irradiation state, but also provide further data on the thymic 
"sphere of influence", indicating that this tissue is capable of 
inducing lymphocytic neoplasms fcllc-r/ing repopulatio-n vjith F^- 
recipient round cells. 

The follovjing additional e>: ;eriments -r.si-ng this model are 
nov/ underway: • ' 

a) Influence of strain A thymic grafts of various ages 1^ 10, 

30 days on the inductih: of lymphocytic neoplasms. 

b) Study of the pcrsisteiice of the post-irradiation state. 
Thymuses from 1-7 c\-3y Old/SL 'onors have- been grafted at 1, 7, 14, 
and F;o days. 

c) Influence of G673L P.nd A bone marrow on the" induction of 
leu'cemia in grafted G573L thymuses in thymoctcmized, irradiated 
(C573L X A) mice.' ■■ ... 

C. Influence of thymectomy i n CSM/Fg subline . 

nearly 30/o lymphomor. have been observed in a milh-agent free 
subline of the C3H strain obtained from Ur . Figge. Appro'dmately 
one-third of these r,?opli, sms are lymphocytic but. very fevj involve 
thymic tissue, Cn tiie othar hand, fractionated ;c-radiation (4 ;: 
■90 r, weekly), produces a preponderance of lymphocytic neoplasms. 
Thymectomy has been accomplished in 2 groups: those observed for 
the - spontaneous disease and a group recei.vir.g x-radiaticn. 

LJ . The role of cell-free materials in the ^ induction of leu'cemia and 
parotid gland neo'^lasms . 

In a study conducted jointly with Dr. Thelma Dunn, Pathology, 
results have hceii published concerning the incidence of leukemia, 
parotid gland neoplasms and other neoplasms in C3Iif/Lw, 
(C3K y. AKa)Fi and' (G3K x C3H/Fg)Fi mice following introduction of 
leukemic materials (extracts, centrifugates, and filtrates) from 
the high-leuhemic lines AKR, C56, and C3Hf/Fg» The incidence of 

(Project descriptiors continued) 



SERIAL nc. 427 PAGE 3 

' leukemia and the age at death were found not to be influenced 
by the introduction into young '(;<:. 24 hour old) mice. 

Certain other observations, however, v/ere of interest: 

1» Pcrotid gland neoplasms were found in all 3 groups 
of test mice, especially in the (CSH x C3H/Fg)Fi cross. 

2, In association vdth the parotid neoplasms in this latter 
group (25%) nearly all the test mice shovjed hinhly invasive 
subcutaneo^us tumors. 

3. Many other uncommon forms of neoplasms vjere found, etq ». 
adrenal medullary tumors, early mammary tumors, but these 
occurred among the control as v;ell as the experimentel mice. 

Since Gross has suggested that subline differences may play 
a part in the response to a leu':emogenic agent, this v/ork has now 
been extended to include 105 C3H/Bittner subline mice inoculated 
< 24 hours with centrif ugates (9500 R.Ti) and filtrates (3erkfeld 
and Selas) from 12 different spontaneous AKR leukemias. These 
same preparations have been given simultaneously to 85 C3I-]f/Lw 
subline mice. 

.7e had observed, at the last annual report, t'lct a transplan- 
table adrenal medullary neoplasm gave nearly 40% parotid gland 
neoplasms in C3M and (C3H x A'-R) mice v;hen tested at the G2, G3, 
and G8 transfers. Consequently, a series of studies v/ere com- 
menced relating to: 

1. Influence in different inbred mice; 

2. Effects of different types of filtration; 

3. Tests for activity after various periods at -60''C. ; 

4. Titration of stored material. 

More than 250 mice were set-up and, to date, after 11 months, 
no parotid tumors have been observed and the ability of L5665 
(the adrenal medullary neoplasm) seems to have disappeared with 
the later transfer generations. 

Crosses between the 2 sublines discussed above, C3Kf/Lv; and 
C3H/Fg, have now been made reciprocally, and inoculations of cell- 
free materials from AKR and G3H/Fg leukemics are being accomplished 
into infant mice to study the unusual occurrence of a high incidence 
of parotid gland neoplasms associated vath subcutaneous neoplasms. 
The subcutaneous growths appear to differ morphologically and bio- 
logically from those seen spontaneously; for example, in old 
C3H strain mice. 

E. Study of certain -/rotectivc factors in induced and spontaneous 
tyrnghomas . 

1. 3one marrow repressive factor . 

a) Data are now nearly completed on the influence of high- 
leukemic (AI'R) bone marrow and low-ieukcmic (CSIlf) bone marrow on 

(Project description continued) 



SERIAL UC. 427 PAGE 4.. , ,..v 

spontaneous and x-royed induced ieukemia in (CSHf x AKR)Fi mice. 
It is clear that C3Hf bone mferrov; represses lymphomas in these 
hybrids i36'0 whereas AKR bone marrov; influences the incidence 
and mean age of leukemic death in o positive direction (75%), 
It is clear also that 4 monthly IV inoculations is most effective 
in' repressing lymphomas in the F^ hybrid (7% at 12'. months com- 
pared with 27% in controls). 

b) This work is being extended (by liiss Deita Uphoff) to 
study the influence of CSlIi bone marrov/ in t!ic AKR strain. 
Periodic inoculations of bone marrow into AXS mice of various 
ages from 2 vreeks to 3 months have been commenced. 

c) It has been fcund (Uphoff) th:t iatact bone morrow 
colls from certain H-2 lines will protect against other 11-2 
irradiated mice (mortality), e.g. , CSS bone marrovj gives maximum 
protection to (C3Mf x AI-R)Fi mice. This v;ould appecr to be a 
good experimental model to sti'.dy the relationship of protection 
against 1) mortality; 2) thymic cegcneration;and 3) induction of 
lymphomas by radiation. Such studios are nov; in progress. 

d) The role of bone marrov; in protectioh against x-ray 
induction of Type A rnd B reticulum laeoplasms is being investigated 
in D3A/2 thymectomized, irradiated mice, and in BAFj thymectomi:3ed, 
irradiated mice. In both these groups there appears to be an 
induction by x-rays of Type A end B neopljsn'is. 

2» Liaternal resistance influence ( i.iRF ) ■ 

An attempt to further characterize an influence of re- 
sistance to leukemia in AKR and C5G mice by foster-nursing uoon 
old (> 32 wee!:) STOLI mothers. 

Although AKR mice in most instances show less leukemia, 
yjhich appears late in life (beyond 1 year),, there are found 
litters which develop tlie disease as expected even though 
fostered by old STCLI mothers, 

F2 and F3 mice obtained from AKR mothers^ fostered on old 
STCLI mice, are now under observation to determine if LBF can be 
passed on in successive generations, 

Follo'ving reciprocrJ. cresses are nov; being accomplished 
in the hope of studying the influence of iLRF oti radiation-induced 
lymphomas : 

(Old) STCLI X C57X/::a 

(Young) 3TCLI x C57a./Ko 

Fractionated irradiation at 90 r x '', of 7 days, and at 

22,5 r X 4,. of 7 days, ' ,, ..; 



(Project description continued) 



SERIAL NO. 427 PAGE 5 

2) Studies of mechanisms involved in inhibition of leukemic cell growth , 

A. Studies are now underway to develop resistant and dependent variants 
of other lymphocytic neoplasms and, in addition, of type A and B 
reticular neoplasms. Cur interest here is to determine if cross- 
resistance and collateral sensitivities are the result of specific 
drugs used, or are dependentin part on the neoplastic cell population. 
Dr. Li. Potter has established, in ascitic form., a battery of 15 such 
neoplasms from vjhich selection will be mode. From this group an 
Azaserine-resistant plasma cell neoplasm 7C429, which exhibits a new 
pattern of resistance, has been established. This resistant popula- 
tion of cells will be of interest since specific sites in biochemical 
pcthvrays at Vijhich Azaserine acts are Itnoiw (in pigeon lines and 
E, coli systems). The response to various antileukemic agents is 
being studied of neoplasms which do not show exponential growth in 
the ascitic form, 

3, In collaboration with Dr, Arnold ".'Jelch, Yale University, studies ore 
in progress of several urr.cil and orotic acid analogs which have shovm 
great promise as inhibitors of bacterial grcvjth. Three of these com- 
pounds: 6-az3uracil and the methyl and benzyl derivatives of 6-uracil 
sulfone have been shown to give striking inhibition of the neoplasms 
L1210, L4946, and L5178 only if given at least 3 times daily. It 
has been found also that if given in the drinking H;:0 (5 mg/ml) 
optimal inhibition is attained, thus simplifying the projected work 
of 1) developing resistance to these compounds and 2) studying reversal 
mechanisms, 

C. In collaboratiw work with Dr. Charles Elichol of Yale University studies 
are being made on 1) content of PGA; 2) capacity to alter PGA to 
compounds measurable as OF; an:' 3) differences in sensitivity to PGA 
antagonists in in vitro systems employing ascitic forms of our L1210 
resistant and dependent variants. 

Certain findings to date are of interest: 

1, An extremely high activity of ascitic cells in contrast 
to lymphomatous cells he s been found, making possible the de- 
velopment of cell-free systems to study such changes as cell 

permeability and differences in enzyme systems as they relate 
to resistance. 

2, Antipurine variants have a far greater capacity to convert 
FGA to CF-like compounds in comparison with the sensitive ascitic 
cells, and also shov; a striking sensitivity to inhibition of 
this conversion by A-methopterin. These results fit in v/ell 
with studies on formate incorporation, and suggest a shift in 

the metabolic pattern from utilization of exogenous metabolites 
to one involving de novo synthesis from precursors in the cell. 



SERIAL []G. 427 FA3E 6 

10. 427 
SERIAL f]C. 

11. . 

BUDGET ACTIVITY: 

RESE/iRCH . /T7 ADini-!I5TR/iTI0N /~7 

REVIEW & APPROVAL Z7 'lECHrJICAL ASSISTAHCE Z7 

12. Laboratory of Fatholoav" 

CCOFERATIIJG UNITS OF TFE PUBLIC HE/iLTH SERVICE, OR CTEER OSGArilZ/iTICIlS, 
FROVIDIHG FUriDS, FACILITIES, OR PERSOIIMEL FOR THIS PROJECT lU EITIiER 

1955 or 1957 

* Provided personnel for collaborative worl^ in certain projects. 

13. [■lone, 

IF THIS PROJECT RESELiXES, C0L;PLEL;EI ITS , CR PARALLELS RESEARCH DONE ELSE^JIiERE 
IN TI-IE PUBLIC I-IEALTH SERVICE CVITMCUT INTERCII/iNGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDEr^TIFY SUCH RE3ERACH: 



P/.9E 7 

14, 427 
SERIAL fJC. 

15. 

PU3LICATIGII3 CTHER THAN ABSTRACTS FROU THIS PROJECT DURIilG CALEIIDAR YEAS i9J 



Mandel, U. George and Lloyd 7J. Lav/; "The Effect of 4-AGino-5-ir.-iicl£:scie- 
carboxamlde oii tl;e Carcinostatic Action of S-Azagwanine." Cancer Resesroh 
14 ; COP-11, December 1954, 

LavJ, L. v7, :"Stiidies or; Trsnsformations to ResistsTiCt and De:jende::ce 

iii Leid:emiG Cells," Origins of Resistai.ce to Toxis Ano-"ts , pp. r68-?;S6, 

1955, published by Acedemic Press, Inc, New York. 

Law, L. :'J, , Thelma 3, Dunn, "nd Feter J, Boyle: "IJeopfesiris in the C3H 
Strain sad in Fj Hybrid ilce ef T\70 Cr'/sses Fcllo^/iiing Intrcduction of 
Ex"^rEcts and Filtrates of Leidie-inic Tiscaes," J, list. Cancer Inst, 16(2) : 
495-539, October 195:3. 



16. Hone. 



HCHORS ArJD AWARDS TO FERSCUIIEL RELATIi'C TG T^IC FRCJECT DjRIIIG CALErDAR 
YEAR 1935, 



PAGE 1 

PROJECT REPORT FORK 

1, National Cancer Institute 2. Laboratory of 3ioloqy 

INSTITUTE UBORATORY OR BRANCH 

3. Leukemia Studies Section 4. _______________________ 5. 427(a) 

SECTION OR SERVICE LOCATION (IF OTHER THAN SERIAL NO. 

BETHESDA) 

6, Studies on the etiology and chemotherapy of experimental iTinphomas. 

PROJECT TITLE 

7, Luchael Potter 
PRINCIPAL INVESTIGATCR(S) 

8, _j: 

OTHER INVESTIGATORS 

9, PROJECT DESCRIPTION 

1) Leukemoqenesis : chemical 

F/ 
DBA 12 mice have been painted with 0»2% methycholanthrene in 

ether 3 x /wk in various experiments, Liany of the experiments in 

progress here have revealed that this mouse develops many different 

morphologic forms of reticular neoplasms, mainly Type A and 3 reticulum 

cell sarcomata. Some of the information obtained, it is hoped, will 

reveal a method for inducing such types of reticular disease with 

greater regularity, 

A. Path of action • 

/Jithin cages, mice have been selected and painted, while others 
in the same cages h^ve not been painted. This study has been de- 
signed to determine the role of absorption through the skin as the 
path of introduction of the carcinogenic material. If the non-painted 
cage mates develop leukemia to the same degree as the painted mice, 
greater emphasis will be placed on studying the oral route of this 
material in leukemogenesis. Ten cages have completed the painting 
schedule; in these cages there are 32 painted and 28 non-paintedmice, 

3, Crude tobacco smoke condensate 

Crude tobacco smoke condensate has been obtained and is being 
painted on the skin of D3A^/2 mice. The carcinogenic activity of 
this crude material, if based on the presence of hydrocarbons, may 
increase the incidence of lymphocytic neoplasms in these animals 
prior to one year of age. The mode of action of known carcinogenic 
hydrocarbons in this mouse, i.e-. . a cumulative action on a distant 
system, administered during a susceptible physiologic age, suggests 
this type of biologic test may indicate the presence of materials 
leukemogenic to the mouse. Through the cooperation and kindness of a 
commercial firm a large supply of crude tobacco smoke condensate 
is being made available, and with the supervision and cooperation of 
Dr. J, Hartwell of this Institute, concentration of hydrocarbons 
by vacoum distillation will be carried out and this material will 
also be tested. In experiments thus far, the presence of nicotine 
has limited the use of this material, and probably obscures the 
action of substances which may be present. The complexity of any 
(Project description continued) 



SERIAL NO. 427(a) PAGE 2 

biologic test of this material is taken into consideration, 

C. The influence of bone marrow on . methylcholanthrene-induced 
lymphocytic neoplasms . 

DM ^/2 mice painted in the routine manner for 10 or 20 
treatments have received 2 intravenous bone marrow injections 
follovang the last treatment. In each case the bone marrow is 
of DBA V2 origin. The dose of bone marrow consisted of that 
amount of bone marrow obtained, from 2 femora., 2 humeri, and ... . . 
" tibia from one mouse. The injections vrere given on the 2nd 
and 16th day following the last painting. Ninety-six mice are 
involved in the experiment: 48, 10 painting; 48,,. 20. painting; 
each group further subdivided in half and half treated and not treated 
v;ith bone marrow, 

D* The comparativ e action of x-radiation and me thylcholanthrene in the 
induction of reticular neoplasms in the DBA ^V2 mouse . 

The D.3A ^^ mouse develops lymphocytic neoplasms in the thymus 
and in other organs in the reticular system. This mouse also has 
a moderate incidence of spontaneous reticulum cell sarcomata, of 
the A and 3 type, Gonadectomy in this mouse has been reported by 
Kirschbaum to have tvjo effects on raethylcholanthrene-induced lympho- 
cytic neoplasms in the D3A/2 mouse: (1) increase the incidence of 
lymphocytic neoplasms; (2) to overcome the physiologic barrier of age. 

In order to test in the same strain mouse whether the mechanisms 
of leukemogenesis are the same, B3A ^/2 mice have been irradiated 
tvith 125 r every 7 days, for 4 exposures; subgroups of thymectomized 
mice, and gonadectomized nice, have been included. Other pertinent 
groups of mice are included, luore animals are being added to the 
series. 

Group At 10 



- 12 


months 




63 


13 


20.3% 


21 


5 


24 % 


42 





% 


19 





% 


30 


12 


40 % 



Intact X-ray 

Gonadectomized X-ray 

Gonadectomized None 

Thymectomized X-ray 

Intact tiCA 

It has been found by examination of the autopsy data, and / leukemia 
tissue sections, that the character of methylcholanthrene/is quite 
different from that of x-ray/ A-fadistion appears to produce a 
monotonous type of lymphocytic neoplasm in the thymus, whereas in 
the methylcholanthrene group lymphocytic neoplasms, in combination 
with type 3 reticulum cell sorccmsta, have been found v;ith great 
frequency. Gonadectom.y thus far has not greatly augmented x-radiation- 
induced leukemia in the DBA f"/2 mouse. Further studies are in 
progress, 

(Project description continued) 



SERIAL NO. ^-27 (a) 



PAGE 3 



2) Genetic Studies 

In 1946 Furth described multiple osteomata in thymectoinized 
AKa mice. Those tumors appeared in animals of 12 months of age or 
over. Normally this type of growth would not be seen in these mice 
because tlie mice v/ouid be dead of leukemia before the osteomata could 
appear. Eaployincj papain digestion of mouse carcasses, skeletons of 
(C3H X AXR)F-, mice 12 months and older were found to contain those 
neoplasr^is. They ore multiple; commonly appear on the skull, pelvis 
and loirer extremity; almost never in the upper e::tremity; more common 
in females (over 80?^ of females hove one or more of these lesions 
after 14 months). An interesting finding has been that these lesions 
have not been seen in only 2 of 37 (AKR x STOLDFj and (3TCLI x AKR)Fi 
mice of similar age. This problem is of real interest since the inci* 
dence of leukemia in the 2 AKR hybrids is also different, 3TGLI is 
known to contribute a nursing influence which delays and inhibits 
leukemia. Further studies employing other A!<;R hybrids, and genetic 
studies of backcross animals, are undervjay. 







Osteoma 


ta 






Strain 


Sex 


No, 




No, of 
animals 
bearing 
tumors 


Total 

number 
tumors 


C3H X AI<R 


2? 


27 




21 


95 




db" 


28 




15 


32 


STOLI x AKR 


99 


17 




2 


3 



a& 



3) Hormonal Studies 



A, Gardner has reported that estrogen treatment of C3H mice, of 

the Strong subline, developed thymic neoplasms follov;ing estrogen 
treatment, .'Jeekly doses of !^0 mgm of estradiol benzoate are 
being administered to 2 sublines of C3H, The G3H F/Lw and the 
Z^ line from Bittner, Gross has reported that subline differences 
exist, ivhen he attempts to induce leukemia with extracts of AKR 
leukemic tissue. It is of interest to determine if a similar 
difference exists with estrogenic induction of thymic tumors, 

3, A study of leukemoqenesis in (DBA x CE)F i and (CE x D3A)F j^ mice . 
Virgin females of this hybrid type develop hyperestrogenism, 
characterized by a proliferation of the endometrium. Reciprocal 
hybrids of these strains have been produced, though it has taken 
some time to get enough CE mice, and the first group has been 
painted v;ith methylcholanthrene. Lymphocytic neoplasms in the 
virgin females will be transplanted to determine if these neo- 
plasms are dependent on this abnormal hormonal state. The role 
(Project descri{)tion continued) 



SERIAL no. _ 427 (^ .) PAGE 4 

of rjenetic facttfrs, goneid^t^oKij ^ etc* Virill be evaluated. To 
date, no lymphocytic neoplasms Kav« been found in the D3ft x CE- 
hybrid mice, 

4) The development _o|, transo^la ^ ntabl^ retl,ejAlaj ^ n g ^ cji l nsms of the mouse^ 
in ascites form , for the study of chernqtherapcatie ffl,echg.n,ismG « 

A spectrum of transplantable tuniors,principally in D3A/2 mice, 
has been developed in the last year in order to: 

a. increase the range of morphologic forms vvhich can 
be studied vath chemotherspeutic agents; 

b. to test differences within morphologic classes of response 
to the antimetsbolites, azaserine, methotrexate, 

, 6-mercaptGpi5rine. 

For this purpose the follovjihg tumors have been established; and 
sorae of their ciiarecteristics ore listed, 

A. Lymphocytic Ileo'^lcsrr.s 

A series of 10 ascitic lymphocytic neoplasms, originating in 
the D3A/2 mouse, has been developed. l\!ith these ascitic tumors 
studies on drug sensitivity Vi/ere carried out; further, using cell 
doses varying from 10 to 10"' cells, titrations of these tumors 
were undertaken. In order to establish true sensitivity or resistance 
to a given antimetabolite, vjith survival time ys the end point, the 
cell population must be uniform. Studies o f ti tration reveal that/ Semi- 
unifcrmity is acquired in most Ccses gradually,/ ^trrig;,t line re- log- 
lationships between cell dose in log units r;nd days of survival arith- 
sre the criteria for unifornity. Si-uh relationships have been mic 
found to hold for Ll2i0, and for G of the turiicrs in this series, 
P268, F312, P335, These s.t,rBight^, lines have been found to ex- 
tend from cell doses of TO" to IC, Some tur.iors have never shown 
this character or^have developed it very- slowly, • L517S---3rrd-F330, ■ 
c.nd the plasma cell tumor, 70429, are examples. 



In many experiments employing antiraetaooiites it has 'oeen diffi- 
cult to obtain good results with early transplant generations, 
principally because the tumors take too long to kill, thus allowing 
the animals to live after. the maximal optim?! dose of the anti- 
metabolite can be given. 

From .this work facts regarding the adr.dnistration of antiinetabolites 
to animals bearing tumors of a logarithnic -rr.d a non-iogarithmic 
character are being learned. 



(Project description continued) 



SERIAL no. 427(e) 



PAGE 5 



I, Lymphocytic 



Mo. 



IndEJ.ction 

or 

oriciin 



Strain 



288 


fviCA'" 


312 


I,!CA 


335 


L.CA 


ope 


LiCA ■ 




LICA 


433 


Z-ray 


5178 


ncA 


553 


Spent. 


421 


X-ray 


413 


LICA 



3A/7: 



RETICUL/lR I!E0PLASI.:3 



Time of death from 
doses of 1-3 :: 10^ 
ascites cells 
At Days 

generations survival 
7-11 13.6 - n. 



II. Reticulum Cell Sarcomata (D3A/2 ) 



228 
329 



KCA 
hiCA 



II, HodqIcins-li::e Lesion 

195 MCA " 
L7235 Filtrate ? C3H 

IV. Plasma Cell Tumor 

70429 Spent. G3H 

V. Granulocytic Leu!:emia 

I-I5530 SpoRt. CSe^ 



7-11 



— 7 '■ 

4 



1/ 
17 

42 



- 13.1 

- 11.7 

- 16.7 



About 30 



i!ot ascitic 



20-40 days 



Trans:?lgnt gener? 
tior. at which 
converted to 
ascitic ti'.mor 



days 



*Fie t iiy 1 c h o 1 a n t h r e n e 



(Project description cc;itin«ed) 



SERIAL NC. 4r,7(a) PAGE 6 • 

3» Tiie Ret id; I am Cell Sarcomata (Type A snd 3) 

Dunn in her review described z classiiicrtioa of retieular 
neoplGsms of the moi-se. An im'jortsnt category of neoplsrns in the 
mouse has not been evaluated es to the relationship to humsH disease,. 
cell type involved, and response to chemotherapy. The relationship 
of these neoplasms to the more chronic lynijihornats of mail has been 
repeatedly cited. Type A and 3 neoplasms hEve been transplanted in 
DBA/?, mice. Ten neoplrisms sre Egsiti available and have been trans- 
plsnted. These necplEsras sre being converted to the sscitic form 
where the cells arc studied i/ith ohsse micrescopy. Two Type A neo- 
plasms have been converted to sisGites turners, Ti\'o Type 3 neoplssms 
have been carried for 3 gerierstions. These neoplssms ^rc sppearing 
so&ner and it is hoped this most interesting cell type '.jill be avail- 
able in the ascitic form. Studies thus far reveal these Type A tutaors 
to bo sensitive to triethylene rx-iamine. The problems of adrainis- 
tering-this drug effectively s-;;;iin hsve isportant bearing on human ^ 
chensothejjapeutic problems. 

TmQ Type B neoplasms, L.7':.35 in C3H, and P195 in the D.B/i/" mouse, 
are imiqwe in their transplant ch&r?'cter. These tunors require many 
months to develop, .'teen they do appear, it is first in the spleen 
and then generalises in the lyivph nodes. Studies of the morphologic 
changes" during transplaiitatiosi are being carried out with Dr. Clyde 
Dawe and Dr. Thelina Durn of the Patholo^/ Section, These tumors have 
not yet been tested v/ith cheraGtherapeutic agents, 

C. Plasma Cell Tumor 

The plasma cell turnGr #704-19 arose ir- :_',3 ileocecal region in 
a C3K/He mouse, and originally was a plasmj c^ii neoplasm. Following 
20 transplant generations as r solid tumor, it v;as converted in one 
transplant generation to an ^iseitic tumor. The ascitic tumor cell 
no longer resembles a differontiotcd plasms cell. At ascitic genera- 
tions 3^ 9, snd 17 this tumor ?;as titered by inoculating doses of 
cells which varied from 10? to 10", Cell doses of lO'*', 10*^ cells gIo 
not hill anymore rapidly than doses of 10*. In 5 experiments utili- " 
2ing 165 animals, employing doses of .SS " 2,?.5 x 10^ cells., 87 per- 
cent of control animals were dead by the 40th day, whereas only 15 
percenit animals treated with ?.0 or more daily doses of 5 mgm/kg b.w, 
azaserine were dead at the 40th day. It has been found by these and 
other experiments that: 

1, Aza serine is a povjerful inhibitor of this tumor, 
?:, Aza serine is relatively non-toxic and osn be given for 
extended periods, thus providing a chtmothvirapeutic model 
for the study of a) the developmtnt of resistance, and 
b) the reasons for eventual curative failure; c) the 
mechanism of action of asaserine; d) some- crude idea of 
the rate of resistant lines developing in a group of 
animals can be explored. 

3. Tv7o resistant and probably dependent lines of this tumor 
have been isolrtcd. Others are being tested. 

4. Azaleucinc, anotrier analog, hrs been obtained and has 
been found to else be inhibitory to this tumor. Studies 
on dosage, cross resistance, and other properties, are 
undervrey, 

(Project description continued) 



SERIAL no. 427 (a) FA3E 7 



D. Granulocytic Leukemia 

This turner was at first z- ohlororna and hss only recently 
developed. It has developed virulence rapidly. It is in ascitic 
form and consists mostly of myeloblasts i/ith some myelocytes and 
more differentiated forms. Tic studies have as yet been undertslcen. 

5) Radiation Induced Leu.':ernia 

A. The pathophysiologic mechaiiism hj which thj^mic lymphocytic neoplasms 
are iriduced' in C57X/Ka mice are not clearly defined. The most 
strildng influence thus far depends upon fractionation of tlie x-ray 
exposures. This suggests that an inj ury-regeneration phenomenon may 
be involved. The fractionation procedure has been carried out by 
Kaplan at 4, 8, and 16 C^y intervals with little difference in the 
incidence of leukemia. An experiment is underway in which groups 
of C57X/Ka mice are receiving '^:2Z r four times at intervals of 
16, 30, and 45 days. This proiongstioa of the interval is designed 
to break up the' injury-regeneration time-relationship, if such a 
relationship is the determinin'j factor, 

3. The development of the Rf str:;in (with Zr . L, il . Law) 

The Rf strain of mice has been reported to have a moderate 
incidence of granulocytic lei:;:e;.iie , following the e::posi;re tc single 
doses of 128 r. Rf mice hr.ve been bred in this laboratory for the 
last year, and good breeding colony hcs been established. Liice from 
. this colony have been used for prilirainary , confirmatory experiments 
regarrling the incidence of granulocytic leLncemia, The mice have hee-a 



!)laced in the following groiv^s 



Leuhemo.^enic Treatment 



1. Intact L-6 r single dose 

?. Intact 128 r, 4 doses, 7 day intervals 

3, Thymectomized i'-.G r single dose 

4, Thymectomized 1"S r, 4 doses, 7 day intervals 

5, Intact Fainting with methycholanthrene 

6, Intact :Veekiy injections of 25 y estradiol 

7, Intact breeding colony. 

Leukemias are beginning to appear^ Several chioromas and non- 
thymic leukemias have appeared in various groups. This mouse may be 
the instrument ijhereby further information on the etiology of granulo- 
cytic leukemia will be forthcoming. 



PAGE 8 



10. 427(a) 

SERIAL m. 



11. , ^___> 

BUDGET ACTIVITY: 

RESEARCH / x / AD1;!I1!ISTRATICI! / / 

REVIE.J & APFRCVAL Z7 TECMTIICAL ASSISTAFICE / / 

* 
j9^ Doctors Clyde Davje and Thelma Dunn, Lab. of Prithology, . • • 



COOPERATiriG UniTS OF TItE PUBLIC "EALTH SERVICE, OR OThER ORGArllZATICnS.PRO- 
VIUiriG FU[1DS, FACILITIES, OR PERSONIIEL FOR THIS PROJECT II! EITHER 1956 or 

1957 

* AH aspects regard! nrj the ;-r;t!iology of reticular disesse. 

13. Hone. 

IF TlilS PROJECT RESEK3LE3, CCFiPLEiJEnTS, OR FAR/iLLELS RESEARCH DONE ELSE.VMEl 
IN TI-E PUBLIC HEALTH SERVICE (.riTI^CUT INTERCHAMGE OF PERSGNriEL, FACILITIES 
OR FUNDS), IDENTIFY SUCH RESEARCH/: 

14, 15 & 16: NO ENTRIES FOR ITEMS 14, 15 & 16. 



PAGE 1 



PROJECT REPORT FCRf.i 



1 « National Cancer Institute ^ . Laboratory of 3iolor]7 

iriSTirjTE LA30PJ\TCRY Cri 3RAMCII 

3, Leukemia Studies Section ^-.__ 5, -127(b) 

SECTICn OR SERVICE LOCATICri (IF OTHER THAN ISLUll*) SERIAL NG. 

6« Studies on the etiology and chemotherapy of exnerimental lyrnrhomas, 

PROJECT 7IILE 

7, Bernard Shacter 

PRI11CIPAL iriVE3TIGAT0R(3) 

e. - ^ 

CTI-ER iriVESTI GATORS 
9. PROJECT DESCRIPTION 

Project ; Biochemical raechanisms of resistance to metabolic antagonists in 

chernotherr! py of experimental lymphomas. 

Objectives : 

The purpose of this project is to discover the mechanisms in- 
volved in the development of resistance to the nrowth inhibitory action 
of agents initially active against experimental lymphomas, 

methods enroloyed ; 

The effects of kno'/m c'r.emotherapGutic agents on various metabolic 
activities of lymphoma cells crc determined, in order to establish possible 
differences in the action of the agents on sensitive as opposed to resis- 
tant cells, f/.etabolic activities studied include uptake of radioactive 
precursors i;ito proteins and nucleic acids, effects on significant enzyme 
systems, and effects on concentration of important cellular metabolites, 

Liajor findings ; 

In the course of determir.ing the effect of amethopterin on 
glutathione and ascorbic acid levels of sensitive and resistant leukemic 
cells, it was found that there was a decrease in liver glutathione levels 
cf animals bearing the rapidly groiving lymphocytic leukemia L4946 in 
ascitic form. Administration of 3 mg/!:g amethopterin to animals bearing 
rapidly gro'ving tumors vjas iollov;ed by a cessation of tumor growth, as 
measured by change in total volume of ascitic oelis, and by a return of 
liver glutathione levels to considerably above normal. The techniques 
of measuring changes in total volume of ascites cells and changes in 
liver glutathione levels are being applied as a simple yet precise means 
for determining action of other possible chemotherapeutic agents on the 
growth of experimental lymphomas. 



(Project description con'td.) 



SERIAL NO. 427(b) PAGE 2 

SiqnificaHce to cancer research ; 

One of the major problems in the use of chemotherapeatic agents 
for the clinical control of leu'conun is the eventual development of re- 
sistance to the action of the drug, such that the leukemic process can no 
longer be controlled. If it v/ere possible to establish the mechanisms 
involved in development cf resistance, it might be possible to institute 
measures to either avoid this undesirable effect, or else to circumvent 
it, perhaps by combined therapy. 

Proposed course of project ; 

During the next calendar year it is proposed to investigate some 
of the mechanisms luhich have been suggested as leading tov/ard resistance. 
These include a study of deaminction of S-asoguanino by lymphomas sensitive 
to, resistant to, and dependent on the agent, since increased deamination 
of asaguanine has been proposed as a mechanism for resistance to the agent. 
The primary effort will be directed tor/ard a search for alternative pathi/ays 
of synthesis of esseutir.l products by resistant cells, as the possible 
mechanism for development of resistance. 



3ESIAL no. 427(b) PAGE 3 

10. 427(b) 

3ERI/J. no. 

11. 

BUDGET ACTIVITY : 

RESEARCH Rl ADUiniSIMTICrJ 

REVIEV; & APPROVAL TECI-SIICAL ASSISTAIJCE 

12. None 

COCPERATiriG UIJITS CF TrE FU3LIC HEALTFI GERVICE, OR CTKER CRGArJIZATICNS, 
FRCVIDIllG FUI-©3, FACILITIES, OR PERSONNEL FCR THIS PROJECT If] EITHER 
1956 or 1957 

13. None 

IF THIS PROJECT RESELIBLES, CCi.iPLEl.IENTS, OR PARALLELS RESEARCH DONE ELSE- 
WHERE IN THE PUBLIC [EALTH SERVICE CJITKOUT IMIERCI-IANGE OF PERSCFINEL, 
FACILITIES OR FUNDS), IJENTIP/ SUCH RESEARCH: 

14. No entries for Items 14 & 16, 

15. ^ \ \ 

PUBLICATIONS OTIER TI-IAN ABSTRACTS FROU THIS PROJECT DURING CALEfSAR YEAR 
1955. 

Shacter, 3,, Interrelations in Respiratory, Phosphorylative and Nitotic 
Activities of Ehrlich Ascites Tiimor Cells: Influence of Dinitrophenol, 
Arch. Biochem. and Biopliys. 57, 3C7-4G0 (1955). 



PROJECT REPORT FCRl/i 



PAGE 1 



1, Mational Ccncer Institute 

INSTITUTE 



?. Laboratory of Biclocry 



UBCRATORY CR 3RANCH 



Leukemia Studies Section 
SECTICri CR SERVICE 



5. 427(c) 



lccatigm (if other ti-ian serial lic. 
beth;) 



6. Studies on the etiolociv and chenot.'ieraoy of experimental lymphomas. 
PROJECT TITLE 

7. S.E. Reauae 

PRIIJCIPAL II]VESTIGATGR(S) 

8. None. , 



OTHER INVESTIGATORS 
9. PROJECT DESCRIPTION 



Project : Studies on the etiology and chemotherapy of experimental lymphomasi 

Objectives: Investigate genetic and biochemical mechanisms involved in 
the development of drug-resistance and -dependence in riiouse leukemias, 

fviethods employed ; 1) Model bacterial systems have been employed for 
genetic studies of the frequency and mode of development of resistance to 
purine analogs v;hich are effective antileukemic drugs, 2) In vitro studies 
have been made of specific biochemical reactions considered likely to be 
involved in the development of resistance to craethopterin, 3) Patterns 
of activity of a number of antipi'.rine drugs h::ve been studied with respect 
to their effect on a number of drug-sensitive, -resistant and -dependent 
leukemias. 

Ka.lor findings : 1) No organisms tested heve proven to be satisfactory for 
the genetic investigations, 

2) The reaction of most interest in this phase of the 
work has been the cleavage of aminopterin at C-9 to yield pterdine and 
p-3minobenzoylglutamate (measured as diazotisp.ble amine). Although such 
cleavage has been reported ic a number of microorganisms, no such activity 
has been detectable in aminopterin (or amethopterin-)-resistant leukemic 
lines, Leukemias sensitive to this drug, as well as those dependent upon 
it, appear also to lack cbility to cleave it. Liver, kidney, spleen, and 
skeletal muscle, :;rom both normal and leukemic mice ^ shovired no such activity i 

3) The adenine analog, 4-aminopyr3zolo-(3,4-D)-pyrimidine 
(APP), and guanine analog, 4-hydroxy-6-anilncpyra2olo-(3,4__)-pyrimidine 
(riAPF), have been investigated for their ability to inhibit various leu- 
kemias or to satisfy the drug requirement of anti-purine-dependent lines. 
APP inhibits all sensitive, resistant and dependent leukemic lines tested 

to the extent of 40 to 60 percent, including one (L-4946) which is naturally 
resistant to other antipurines, AFP does hot substitute for 8-azaguEnine 
or thioguanine in lines dependent upon these drugs. Purified KAFF does 
not inhibit any of the tumors inhibited by APP; an unpurified sample had 
shown some such activity. An interesting point is that HAP? appears to 
satisfy the antipurine requirement of an 8-azagu3nine-dependent leukemia, 
AFP inhibits the growth of an amethopterin-^dependent line. Other, 
(Project description continued) 



SERIAL lie. 427(c) PAGE 2 

variously substituted, members of this series of compov.nds, as vifell as the 
(4, 3-d) congener of APP, liave shown little or no antileukemic activity. 
APF is quite toxic to mice (C/Daft)Fi, producing; detectable weight loss at 
o'oses of 7,5 mg/hc X 3. At levels of 15-20 mg/!cg X 5, severe extraorbital 
hemorrhages may occur. 

All data obtained so far apply to activity in terms of y/eight of 
tumors grown as subcutaneous lymphomas. Survival times of nice carrying 
the same leukemias as ascitic tumors are not prolonged by APP. 

Significance to cancer research ; 1) If one or more bacterial strains 
can be found which imitate the responses of our leukemir. s to the pyraaoles 
and other antipurincs, it is felt that much c?^n be learned about the 
genetic aspects of resistance and dependence which, thus for, has not been 
amenable to a direct analysis in the leukemic material, Cf particular 
interest in this respect are dcta on the frequency of mutation to dependence 
and resistance; allelic relationships among genes affected; genie interactions 
such as suppression v;hich may occur. At present, available technics are 
inadequate to permit a direct attack on such problems involving mutation in ■ 
leukemic cells. 

2) Detailed knowledge of biochemical events involved in the expression of 
dependence or resistance offer expanded hope of effective chemotherapeutic 
attack against fulminating or refractory leukemia encountered in the clinic. 
Negative information, vjhile rarely conclusive, can help narrov/ the area 

in which fruitful exploration is li!:cly to be made. It is tentatively 
concluded that, in our leukemic m.aterial, cleavage of aminopteria does 
not play a role in the expression of resistance to this drug, 

3) The activity df 4-aminopyra3olo-(3,4-d)-pyrimidine against antipurine- 
resistont mouse leukemia suggests that it might be of interest clinically. 
Little appears to be known concerning its pharmacology or toxicology. 
Given such information, it night prove satisfactory for trial against 
antipurine-refractory leukemias, or used in combination with antifolics 

or other an ti purines. 

The fact that APP inhibits leukemias resistant to other antipurines, 
whereas other antipurines show cross-resistance omong one another against 
these same leukemias, suggests that AP? is active at a biochemical locus 
different from that of other purine rnalogs. The failure of APF to substi- 
tute for 8-asaguarjine for the 8-a3aguanine-dependent tumor is consistent 
with this view. These and other data suggest several different points 
of action of purine analogs in the sensitive, resistant and dependent 
leukemias. Further analysis of the interactions and cross-resistance 
patterns should yield insights into the biochemical mechanisms underlying 
theip, as i>;ell, perhaps, as specific points vjhere rational chemotherapeutic 
attacks might be made. 

Proposed course of sroject ; 1) Further attempts will be iTiade to Obtain 
suitable bacterial strains to serve as genetic models as discussed above. 
Emphasis will be placed on obtaining antipurine-sensitivo, -resistant 
and -dependent lines of organisms which provi'r'e opportunity for relatively 
extensive genetic analysis (e.g., Escherichia coli . Salmonella spp,)« 
^^ Iji vitro studies will be concentrated on the problem of pinpointing 
the loci of action of APP and IiAPF in the leukemic lines in which they are 
active i^ietaboT ically. An attempt will be made to follcvj, qualitatively, 
purine synthesis by tumor homogenates and ascitic cell suspensions and ~ 
the effects produced by the pyrs^soies in t!iese systems. If these surveys 
are successful, it may bo possible to extend and quantitate them by means- 

(Project description continued) 



SERIAL I!C. 427(c) PAGE 3 



of radioisotope studies in collaboration vjith someone competerit In scsh 

technics. If such brei sr.d cell suspensions prove satisfactory, tiiey 

could be used in plsce of the intact animals now being used in studies 

of reversal ox activity of APF and FIAPP. 

3) Many difficulties have been encountered in attempts to study the effects oi 

the i:yrozoles combined vjith normal purine bases or other purine analogst 

Those problems arise as a result of the lov^j solubilities of the compounds, 

toxicity of many combinations, etc., so that activity measurement in 

terms of tumor vjcirht often become unreliable at best and impossible at 

ivorst. Shacter has shown that, under certain conditions, ascitic tumor 

packed cell volume can be correlated vjith antileul:emic activity. Host 

liver glutathione levels con also be used as a measure of such activity. 

It is planned to investigate the extent to which these criteria can be 

used to replace tumor weiqht as a measure of intileuhemic action. 



SERIAL Ho. 427(c) PA3E 4 



10. 427(c) 

SERIAL MC, 



11. 



3UD3ET ACTIVITY: 

RESEARCH fT] ADLlIIlISTR/iTia! /~7 

REVIEW & APPROVAL /~7 ■ TECIirilCAL ASSISTAFICE / / 

12. Mono. ^ 

COOPERATIMG UIIITS CF T!-!E PaSLIC MEALTH SERVICE, CR OTIIES CRGAniZ/iTICnS, 
FROVIDIMG FUfSS, FACILITIES, t'R FERSCIIIEL FOR TI-IIS FRCJECT III EITIE,^ 

1956 or 1957 

13, none. 

IF TI-IIS PROJECT RESEI.:BLES, COf.^LEl.lEf .TS , CR PAR/iLLELo RESEARCH DOilE 
EL3EV/IIERE III TI-IE PU3LIC I-EALT:-! SERVICE (?JITI-!GUT inTERCrlAIIGE OF FERSOIinEL, 
FACILITIES OR F'JIIDS) , I,";EI!TIFY S'JCM RESE/.RC:^: 



NO ENTRIES FOR IlEI.iS 14, 15 & 16. 



Genoral Project 1|28 

Serial Nc. 



Study cf chemically defined medium and cell nutrition. 



Page 1, 



PROJECT REPORT FORM 



1, N ational C a ncer 2, Biology 



Institute Laboratory or Branch 

3. Tissue Culture h^ Bethesda, Md« 5. [i 28(a) 

Section or Service Lo.caticn Serial No, 

6. Development of a defined medium for the cultiva ti on of s tr ain L ce lls in 
Project Titles vitro. 

7. V« J . Eva-ns _^ ^ 

Principal Investigator (sp 

8. J. C. Bryant, W» R. Earle, K, K. Sanford, B. B. Westfall, M.C, Fioramonti, 

and W. T. McQuilkJJi, . 

Other Investigators 



9. Project description: 

Objectiv e; To develop a satisfactory defined medium adequate for pro- 
ducing proliferation of strain L mouse cells in order to study their 
nutritional and metabolic behavior* 

Methods employed ; Replicate cultures are prepared and enumeration of 
nuclei are made after exposure tc defined media for varying intervals. 
Such media were devised from the collected information on the nutrition 
of bacteria, mammalian and .avian cells in vitro end the data from physi- 
cal and chemical fractionations of naturally occurring medium on which 
the strain L cells are maintained. 

Major findings t Several media have been devised and tested. Media have 
been tested containing amine acids, amides glutathione, vitamins, coen- 
zymes, unsaturated fatty acids, desoxyribosides of nueleic acids, car- 
bohydrate sources and inorganic salts, No antibiotics, fungicides or 
protein sources are included. A medium has been devised which allows 
a continued (11 months to date) reasonably rapid rate of proliferation 
for clone L cells. More recently the unsaturated fatty acids have been 
omitted from these media with no deleterious effect upon proliferation. 
Vitamin B 12 has been added wi.th a resulting small increment of additional 
growth. In a continuation of this ^^rork the essential amino acid require- 
ments of strain L have been determined as well as the essentiality of the 
coenzjrmes group and the nucleic acid derivatives group. 



Page 2, 

Significanc es A chemically defined medium is essential for determing 
difference s~in the physiological between normal and malignajit cells 
and for "research in the nature and origin of malignant transformation, 
The present medium^ essential in all respects, serves as a prototype 
medium for a prototype cell in tissue culture. Such a medium also 
makes tissue cultured cells a most valuable tool for chemotherapeutic 
and virological studies. Actually the best chemically defined medium 
described for strain L cells so far, from these studies has already been 
found to be an exceptionally excellent medium for virus studies with 
human cell strains, when it is supplemented with heterologous serum in 
, small volumes* 

Projected s tu dies ; Continued research will be done to determine the ' 
essentiality of each of the groups of components as well as the in- 
dividual members of the group and to fortify the medium with additional 
known growth promoting material. At the same time metabolic studies 
are in progress using this medium, (See report B, B, Westfall) 

10, U28(a) 

Serial No, 

11. 

Budget Activity: 

Research x Administration 

Review & Approval Technical Assistance 

12. ; 

Cooperating units of the Public Health Service, or other organizations, pro- 
viding funds, facilities, or personnel for this project in either 1956 or 
1957, 

Medium has been made available to cooperating units of the Public 
Health Service such as the Microbiological Institute, 

13. ' \ 

If this project resembles, complements, or parallels research done else- 
■ where in the Public Health Service (without interchange of personnel, 
facilities or funds), identify such researchj 

The organization rf Dr. Harry Eagle in the Micrc^iolcgical Institute is 
also working on cell nutrition in tissue culture, 

lU. U28 (a) 

Serial No, 



15. 



16.. 



Page 3 . 



Publications other than abstracts from this project during calendar year 
1955. 



Studies of Nutrient Media for Tissue Cells in Vitro . I, A protein -free 
chemically defined medium for cultivation of strain L cells. Cancer 
Research i6sOO-00 Jan 1956, V.J. Evens, J. C, Bryant, M.C, Fioramcnti, 
W. T, McQuilkin, K.K, Sanfcrd, and ¥. R, Earle, 

Studies of Nutrient Media for Tissue Cells in Vitro . II, An improved 
protein-froc chemically defined medium for long term cultivation of 
strain L 929 cells, V. J, Evans, J, C, Bryant, VJ. T. McQuilkin, M.C, 
Fioramonti, K. K, Sajiford, B. B. Westfall, and W, R. Earle, Cancer Res, 
16 1 00-00, Jan. 1956.t 

Honors and awards to Personnel relating to this project during calendar 
year 1955* 



Page 1, 



PROJECT REPORT FORJI 



1, Natirnal Cancer 2, BiolcQT 



INSTITUTE L/vBGRATORY OR BRANCH 



Tissue Culture h* Bothesda, Md. $, U28 (b) 

Siictirn or Service Lrcr?-ticn Serxal Nc, 



6 . A study rf the stability cf prctcin-frce chemically defined medium. 
Frcject Title 



7, V. J. Evans, 

Principal Investigator (s) 



!. W, T« McQuilkin 

Other investigator (s) 



9. Project description: 

Objective ; To determine whether the chemically defined medium is stable 
biologically after being maintained in solution at 5 C for a prolcpgej^ 
period cf time, ' ',' 

Methods employed; Cultures of strain L-929j mouse fibroblast cells were 
maintained for a period cf six weeks in protein-free chemically defined 
medium NCTC 109 the component solutions of which had been stored at $'^C - 
as long as 3 to ii months. The cultures were fluid changed and serially ■ 
transferred in the manner cf routine cultures. 

Major findings ; The morphology and cell proliferation of the cultures 
appeared excellent and equal to stock cultures maintained in chemically 
defined medium that wrs made up fresh every twd weeks. 

Signiflcsnce ; Reports in the literature of the development of chemically- 
defined culture media and accepted data regarding the stability of certain 
cf the component solutions indicate rather extreme instability cf certain 
cf the components of the medium. According to this information such 
constituent elements as glutathione, coenzjTnes, glutamine and vitamiji B- 
vitamin B-12 solutions must be kept fresh or in a frozen state until im- 
mediately before use. To know that the chemically defined medium is not 
so biologically unstable mptes possible substantial saving of labor in 
the repetitious preparation of the medium. It also makes possible a wider 
range of use of the medium in experimentation. 

Proposed course of project; To study other aspects of the stability of 
the medium with the view toward further simplification of its preparation. 



10, U28 (It) 

Serial No, 



Page 2, 



II, 



Budget activity: 

Research x 



Administraticn 



Review & approval 



Technical assistance 



12, None 



Cooperating units rf the l\iblic Health Servicej or other organizations, pro- 
viding funds, facilities, cr personnel for this project in eithep 19SS cr 
1957. 



13 • None 

If this project resembles, complements, or parallels research dene elsewhere 
in the Public Health Service (without interchange of personnel, facilities 
cr funds), identify such researchi 



1^- U26 (^) 

Serial No* 



1^, '.None 



.Publications other than abstracts from this project during calendar year 



16 , Nono 

Honors and awards 'to personnel relf.ting to this project during calendar 
year 1955. 



i'agc 1 



1, National Cancer ^ 2. Biolog[__ 



Instituto Laboratory or Branch 

3, Tissue Culture h. Bethesda, M d. 5. i|2 8(c) 

Section cr Service Lccaticn Serial Wc. 

6 . The effect of horse serum residue and ce rta in chemically defined supplement s 
Project Titlet on pro lie ration of strain L clone 929 cells from the >■ 

mouse, 

7 . V, J> Evans ■ 

Principal Investigator (s ) 

8 . M. C. Fi oramonti, ^ K, K. Sanford, W. R. Earle, J. C ♦ Bryant, W. T.McQuilk in. 
Other investigators 

9« Project description; ..,..• 

Objective To develop a partially defined medium incorporating washed 
serum residue and free amino acids as well as other growth factors that 
is capable of supporting proliferati.cn of strain L cells as the whole 
serum. 

Methods employed; The replicate culture techniques accompanied by 
nuclei enumeration methods were used to obtain data on the action of 
horse serum residue obtained by ultrafiltration supplemented with certain 
chemically defined media. The horse serum residue was obtained by ultra- 
filtration procedure as described by Sanford et al. The supplements vjere 
those of Morgan, Morton and Parker's mixture 199» 

Major findings ; Quantitative experiments were used to test the effect 
of supplements to a basic medium of the residue fraction remaining after 
ultreJiltraticn of horse serum. This stu.dy was carried out on washed cell 
suspensions of clone 929 strain' L cells, origjjially obtained from a 
strain C3H mouse t. These cultures were planted in T-l5 flasks on glass 
substrate and changes in population levels were determined by enumeration 
of the nuclei at seven, ten, thirteen, fourteen and twenty-one day inter- 
vals. The unsupplomented fraction of the horse serum was incapable of 
maintaining the inoculum level beyond seven days. This residue medium, 
siipplemented with the amino acids, amides and amine of horse scrum was less 
effective in maintaining population levels. The further addition of niacin, 
p-aminobenzcic acid, niacinamide, pyridoxine HCl, thiamin ,HC1, d-Ca pento- 
thencitej i-inositol, 'choline chlrridej riboflavin, ascorbic acid, gluta- 
thione, cysteine ;HC1, biotin, folic acid, vitsmin A, vitamin D(calciferol), 
tween 80, menadione, vitarain E and ATP as contained in mixture 199 of 
Morgan, Morton, and Parker gave population levels superior to those. obtained 
by use of unfractionated horse serum. Under the experimental conditions 
used, addition of the other components of mixture 199 to this medium gave 
no added increase in proliferation and, in fact, indicated a possibly 
inhibitory action. 



15. 



16. 



Page 2 

Si gnificance s Development cf a chemically defined medium is cf major 
Importance for ultimate comparison of normal and malignant cells 
and this was one step in the development of such a medium. 

Proposed course 

.of project: Continued study to develop a chemically defined 

medium 'oF'general use. 



10, U 28(c) 
11. 



Serial Mo, 



Budget activity; 

Research x Administration 

Review & Approval Technical Assistance 



12 i None 



Cooperating units of the Public Health Service, or other organizations, 
providing funds, facilities, or personnel for this project in either 
1956 or 1957. 



13 » None 



If this project resembles, conplements, or parallels research done 
elsewhere in the Public Health Service (without interchange of per- 
sonnel, facilities or fimds), identifj^ such research: 

The organization cf Dr. Harry Eagle in the Microbiological Institute 
is also working on cell nutrition in tissue culture. 



lU. I428 (c) 



Serial Mc, 



Publications other th^'H abstracts from this project during calendar 
year 1955. 

The effect of horse serum residue and chemically defined supplements 
on proliferation of strain L clone 929 cells from the mouse. Cancer 
Research v, l5: 00-00, 1955. M.C. Fior?iiicnti, J.C. Biyant, IaT. T, Mc 
Quilkin, V„ J, Evans, K. K, Saufcrd, and ¥,R, Earle, 



Honors and "wards to personnel relating to this project during calen- 
dar year 1955* 



Page 1 
PROJECT REPORT FORM 
1. National Cancer 2. Biclcsr 



Institute Lnboratcry cr Branch 



3, Tissue Culture Section ii. Bethesda, Md, g« 1^28 (d) 

Section or Service Location Serial Nc» 



6, Si mplification of a pr otein-free chemically defined medium NCTC 109 
Project Title 



7. V. J. Evans 

Principal Investigator (s ) 



8 , J. C. B ryant, N.M. Hawkins end M,C, Fiorrjfionti, K, K. Sanfcrd sxid lAj^RtSarle 
Other Investigators, 

9» Project Descriptioni 

Ob ject ivet To determine the essentiality of individual members of ccrapcn- 
ent groups of coenzymes mixtures nlucurcnic acid mixtures and fatty acid 
new in the complex chemically defined prctein-frce medium NCTC 109 used tc 
grew strain L-929 cells. 

Methods employe d: Replicate cultures of strain L cells are preparecl and 
enumeration of nuclei are made after exposure tc the test medium for vary- 
ing intervals. From chojiges in the number of nuclei en test medium in 
relation tc t he complex but complete medium containing at least all the 
essentials and perhaps some non-essentials for strain L it is possible 
tc determine the essentiality of each individual component. 

Major findings . Exploratory data indicate that it may be possible to omit, 
Glucuronclactcne, methyl linclenate, methyl arachidcnate, diphcsphopyridine 
nucleotide, cocarbcxylase, flavin adenine dinuclectide, uridine triphopyri- 
dine nucleotide. 

Significance i The data indicate that it is possible tr prepare for practical 
purposes a simpler medium. Further, it is learned that the substances 
which can be omitted are not essential nutritional requirements for this 
strain of cells because their absence does not result in lessening of growth 
or cause death of the cultures. 

Pr ojected studies ; Continued siinplificaticn of the medium will be pursued 
and the data will serve tc act as a basis for determining the nutritional 
essentials for other normal and malignant coll strains. 



Page 2 



10« ^28 (d) 

Serial Nc, 



11. 



Budget activity! 



Research x Administraticn 

Review & Approval . ■, , Technical Assistance 



12. None 



Cooperating units of the Public Health Service, or other crgrnizationS, 
providing funds, facilities, cr personnel for this project in either 
1956 or 1957 



1 3 . 

If this project resembles, complements, or parallels research done else- 
where in the Public Health Service (without interchrnge of personnel, 
facilities or funds), identify such research} 

The organization of Dr. Harry Ergle in the Microbiological Institute is 
also working on cell nutrition in tissue culture, 

li^» 1^28 (d) 

Serial No, ', 



15. None 



Publications other than abstracts from this project during calendar year 

1955. 



l6 « None ^ 

Honors and awards to personnel relating to this project during calendar 



Page 1 



PROJECT REPORT FORM 



1, National Crncer 



Institute 



2, Biology 



Laboratory or Brajich 



3» Tissue Culture 



lit Bethesda, Md, 



Section cr Service 



Location 



Serial Mo* 



6« S upplemental studies of the ef fe ct of ultrafiltrat e of whole chicken-egg 
Project title J extract on the cultivation of cells in vitro. 



7 « V« J. Eva-ns 

Principal investigator (s) 



8 , W.TtMcQuilkin, J. C . Bryant, W* R, Earle, K. K, Sanford, M,C. Fioramonti. 
Other investigators 



9« Project descriptions 

Objective ; To supplement the study of ultrafiltrate of whole chicken -egg 
extract that was carried cut and reported in 19^h> by studying certain 
aspects of the stability of the ultrafiltrate. 

Me thods employed i Replicate cultures of strain L cells were cultured 
on egg ultrafiltrates that had been 1) heated to 6^0 for 1 hour, 2) 
dried and reconstituted, 3) frozen for over two months and ij.) a compari- 
son was made in long term cultures between the ultrafiltrate made from 
fertile eggs and that made from non-fertile eggs. 

Major findings ; The proliferative ability of the ultrafiltrate was not 
diminished by heating cr by drying and reconstitution. The two-^onth 
old frozen ultrafiltrate had a proliferative ability comparable with 
that of fresh embryo extract medium. The fertility or non-fertility of 
the eggs used as a source of the ultrafiltrate appeared to make no dif- 
ference in its proliferative ability. 



Signif icr.nc e: These findings emphasize the biological stability of egg 
extract ultrafiltrate and its potential usefulness as a substitute for 
embiyo extract in tissue culture media. 



Page 2 



Prop osed crurse of projectt Stock and experimental cultures of various 
cell strains have been and wi.ll increasingly be carried on this medium, 

10. 128 (e) 

Serial No» 



11. , : 

Budget Activity 5 

Research x Administration 

Review & Approval Technical Assistajice 



12 , None 

Cooperating units of the Public Health Service, or other organizations, 
providing funds, facilities, or personnel for this project in either 
1956 or 1957. 



16.. 



13. None , , , _ . • "' • 

if this project resembles, complements, or parallels research done else- 
where in the Public Health Service (without interchange of personnel, 
facilities or funds), identify such research s 



Hi. ii28 (o) 



Serial Mo, 
15 . In Preparation 



Publications other than abstracts from, this project during calendar year 

1955. 



Honors and awards to personnel relating to this project during calendar 
year 1955. 

¥, T. McQuilkin used this material as a basis for a thesis submitted to 
George Washington University in partial satisfaction of the requirements 
for the degree of Master of Arts in Zoology, which was awarded in October, 

1955. 



Pago 1 
PROJECT REPORT FORM 



1, Naticnal Cancer 2, Biclcgy 



Institute Labcratcry cr Branch 

3» Tissue Culture li. Bethesda, Md. $, li28 (f) 

Sccticn or Service Lccatirn Serial No, 

5, D evelrpment cf a defined medium frr the cultivaticn cf human skin cells. 
Project Title 

7, V. J« Evans and W. R. Earle 

Principal Investigator (s) 



5, See Itcm'l 2« 

Other Investigators 



?, Project; description: 

Ob jective ; To develop a satisfactory defined medium adequate for producing 
proliferation of the human skin strain, §Yl6h.m This is essential for use 
cf this cell strain in problems of cajicer research, homcgrajCting and viral 
research, 

"- Major Findings { Exploratorj'' data recently obtained indicates that the ' 
human epidermal cell strain #1769. will grow in the chemically defined 
medium NCTC 109 as already worked out in this laboratory provided there is 
human or horse serum supplementation at low concentration, 

Significg.ncet On these media, NCTC 109 previous exploratory virological 
studies have' been successful. For cajncer research and humaji transplanta- 
tion work such a defined medium is imperative. Information resulting from 
studies of such a human strain may also serve as exploratory data for pre- 
paring a tissue preservation medium for the living cells. The implication 
cf usefulness in nutritional and meta.bclic studies in relation to skin 
cancer are ma.nifold and obvious. 

P roposed ocurse of project ; a) With this strain as with all the others, 
The essentiality of all the individual components and component groups cf 
medium NCTC 109 are being investigated tr improve the chemically defined 
portion of the medium} b) Similarly the action cf the horse serum is in- 
vestigated to detcCTiine its biochemical and biophysical function} c) The 
chemically defined media will be used tr study long term preservation cf 
normal tissue at refrigeration temperatures] d) Tr use NCTC 109 as medium 
for studying (the biochemical products and - the rejectirn- 
accoptance phenomenon, (See separate project) 

^0. U28 (f) 

Serial No, 



•5561 -ics^ 

3UJN •91 



•5561 
s'uoN •5X 



l£li_LEH£S 



jqoj-cosoj: qons j^Tq.uop-t: *(spunj ao soTqixxoisj 

*XSuuos:t©d JO o2uuqoaoq.uT q.nji{q.xtt) ooxAjog qq.X'eoH ofl^li^ ^^1 ^ saoiiK 

•0SX9 oujp qa jposoj sxoxiTSJBd jo 'sq-ugiuoxdmoo, ' saxq^^osoj q.09Cjjd sxq^ JI 

ouoM 'CX 



• saoq.^§xq.soAux jjxuss **I*0*W^^ 

uoxsxAjodns aopun xo"^-3s«^Sc! sjucg ongsfi £q ouop jCxi^^U'i.oTi sbw >iajjM. sxitq.,jo 

qoTiH •aeq.uoo X'^'^TP^W T^-^^K I^ujxcj.-aM *looqog x^^TP^H X'-^-^^^M 'Jl-Ucg onssfi 

*i.56l -i^ 

95X iioLtq-xo ux q.ooCoj:d sxi^q. joj x^^^^'^saod aj *soxq.xxX3i2J 'spimj SuxpxAjad 

^sujxq.pzxuBgjj jcqq.j gj ^ooXA-iag qq-X'-^sH oxxqtij o qq. jj sq.xun guxq.-o'jgdjjo 



> uoxq.-aaci.sxuxiirpv x qojcuosoH 
^ ;.A!q. xAxq.O'e ci.ggpna 



Page 1 



PROJECT REPORT FORM 
Naticnrl Cancer ^ 2, Biology 



Institute Laboratory cr Branch 

3, Tissue Cultur e k- Bcthesda, M d. g . I;28 (g) 

"Section or Service Location Serial No, 

6, The effect of scrum fractions on the proliferation o f strain L cells in vitro . 
Project Title 

7. K.K. Sanfcrd and B. B. Westfcll. 

Principal Investigator (s ) 

S • W. R, Earle, J, C. Bryant, V,J, Evans, E. V. Peppers, M« C. Ficramontij and 
Other Investigators W.T, McQuilkin, 

9» Project Descriptions 

Obje ctive t To examine those coraponents of the large molecular portion 
of serumThat appear to be essential for the rapid proliferation of 
strain L mouse cells in vitro , with the ultimate objective of isolating 
components that could be added to a chemically defined medium for the 
purpose of increasing the rate of cell proliferation. 

Methods emplcyed t Horse serum was fractionated by a modification of Cohn's 
low -temperature procedure. Method No, 10, The effect of four serum fractions 
on the rate of proliferation of a clone of mouse fibroblasts was tested 
in quantitative experiments. 

Major findings: All protein fractions isolated from horse serum were found 
to promote growth of strain L cells. When tested in a protein-free basal 
medium, the gross -globulin fraction could be substituted for the large 
molecular portion of horse serum to yield comparable rates of increase in 
cell numbers. After removal of the gamma globulins from the gross 'globulin 
fraction, the residual globulins could also be substituted for the proteins 
of whole serum with only slight decrease in numbers of cells, A lower 
rat'e 'of increase in "cell numbers was obtained with the albumin and gamma 
globulin fracti'ons tested at a concentration of 1,3 percent; when tested 
at a higher concentration, how'ever, (2,6^)- the' rate of increase in numbers 
of cells grown in gamma globulins was the same and in the albumins was less 
than that of cells grown in the same concentration of the unfrattionated 
serum proteins. 

Significance to cancer research t Although a protein-free chanically de- 
fined medium has been developed for strain L cells, at least four other 
cell strains in this laboratory grow in the defined mediura only when a 
small amount (0,0^ to 1 percent) of serum protein is added. To pursue 
carcinogenic, metabolic and nutritional studies on these strains, defined 
serum fractions appear to be essential at the present time. 



Pag© s 



Prrposed course cf this project ; , Experiments will be continued testing 
the relative value of certain commercial serum fractions prepared. as ■ 
nearly as possible according to the methods developed in the present stu(^, 

10, U28 (g) 
Serial Mo, 



11 . . 

■ Budget activity: 

Research X Administration 

Review & Approval Technical Assistance 

12, None " . \ ■ ^' ' , 

Cooperating Units of the luijric Health Service, or other organizations , pro- 
viding funds, facilities, or personnel for this project in either 1956 or 
1957. 

If. this; proj6ot~relemblei3, ccmplcments, or parallels research done elsc- 
where in the Public Health Service (withotit interchange of personnel, 
facilities or funds), identify such research: 

The orgajiization of Dr. Harry Eafvlc in the Microbiological- Institute 
is also working on cell niitrition in tissue culture. 



13. 



lU, U28 (g) 

Serial No, 



15. 



Publications other than abstracts from this project during calendar jcdt 
.1955, 

The effect of ser-um fracticns en the proliferation cf strfein L mouse 
cells in vitro . K, K. Sriifcrd, B. B. Westfall, M.G. F^cramonti, W,T« 
McQuilkinj J, C, Bryant, E.V. Peppers, V, J. Evans and WJl, Earle, 
J, National Cancer Inst, 16j789-802, 1955. 

l6. None ^ _.____>__^ 

Honors *and awards to personnel relating" tr this project during calendar 
year 1955. 



Page 1 
PROJECT REPORT F0RI4 
1, Nationr-1 Cancer 2'i, Biclcgy 



Institute Laboratory or Branch 

3, Tissue Culture It, Bethesda, Md, ^5, k2Q /y^-^ 

Section or Service Location oerial No, 



6 , S tud y of c h emically defined mediwii and cell nutrition for clone 92.9 cells-, 
Project Title 

7. K . K. Sanford 

Principal Investigatcr(s } 



8 , M «C. Fiorajionti, V/.T. McQuilkin, J,C. Bryant, V.J. E vens an d ¥,R.E arle, 
Sther Investigators 



9» Project descriptiont Attempts to simplify and improve a protein-free 

chemically defined culture medium, NCTC-108 for the proliferation of strain 
L mouse cells. 

Objective ) To determine which components of the chemically defined mix 
are essential for cell proliferation and to establish the optimal con- 
centrations of these components. 

Methods employed } The effects of varying concentrations and depletions of 
components on the proliferation of strain L cells have been determined by 
quantitative replicate culture procedures , 

Major findings ! Of the 26 amino acids, amines, and amides of the protein- 
free medium NCTC 108, those essential for proliferation of strain L, cells 
^ have been determined. From dose-response curves on those essentials, new 
mixtures have been devised and are now being tested, 

•An analysis is being made of the effects of the desojqj^ribosides on cell pro- 
liferation. 

Proposed course of project; This project will be ccntinued until the 
effect of the dcsoxj'ribosides and other nucleic acid derivatives on, -cell 
proliferation have been established* The study of the amino acid mixtures 
will be completed scon, 

•U>, Significance to caneer research ; A simplified chemically defined medium 
"Is iof primary significance in order to determine differences that may 
exist in the nutritional requirements of malignant cells dji vitro as com- 
pared with the normal cells from wi:ich they arise. 



10, U28 



Seri 



ial No, 



Page 2, 



11. 



Budget activity { 



Research x 



Adrainistraticn 



Review & Approval 



Technical Assistance 



12, Nrne 



13, 



Cccpcrating units of the Public Health Service, cr ether organizations, pro- 
viding funds, facilities, or personnel for this project in either 19$6 or 
19^7. 



If this project resembles, complements, or parallels research done elsewhere 
in the Public Health Service (without JJiterchange of personnel, facilities 
or funds), identify such researchs 



The organization of Br. Harry .Eagle in the Microbiological Institute 
is alsr working on cell nutrition in tissue culture. 



lU. U28(h) 



Serial No, 



1^, None 



Publications other than abstracts from this project diiring calendar year 

1955. 



16, None 



Honors and awards to personnel relating to this project during calendar jqpt 

1955. 



Page 1 
PROJECT REiX3RT FORM 
1, Naticnal Cancer 2. Biclcgy 



Institute Labcratrry cr Branch 

3. Tissue Culture U. Bothesda, Hd, 5. ii28 (j) 

Secticn cr Service Location Serial Nc, 

6 , A study of the preservation and storage r f strain L cells. ' 

Project Title 

7, V. J. Evan s 

Principal Investigator (si 

8, W.T. KcQui lldji 

Other Investigators 

9, Project description; 

Objective t To determine how long strain L cells could be preserved at 
5^C with no_. cr infrequent, fluid changes. 

Methods employe d} Flasks of stock cultures were placed in the cold room 
for periods ra-nging from three tr eight weeks during which times the 
medium was either not changed at all or was changed at infrequent inter- 
vals. When cultures were withdrawn from the cold storage, they were fluid 
chajiged imraediately and then maintaiiied in the usual manner to determine 
whether the cells wruld revive. 

Major findings } All cultures in cold storage fcr 3 weeks survived and 
recovered satisfactorily -generallj'- within one week aJTter removal from 
cold room and return to routine procedures. Cultures could survive and 
recover from an additional three-week cold storage period if returned 
to normal treatment fcr about a week beforehand. No cultures survived 
when stored at ^'^C fcr 5 weeks cr longer even with fluid changes at the 
end of the third cr fourth week. 

S ignificance ; The usefulness of such preservation methods was under 
consideration for the mcintenance of strains at minimal expense. This 
work partially corroborates the work done during the same period by 
Swim and Parker and reported in Prcc, Sec. Exp, Biol, and Med,, vol, 89j 
Aug, -Sept, 1955. 

10. 1428 (i) 

Serial No. 



Pago 2. 



11; 



Budget Activity t 



Research x 
Review & Approval 



Administ ration 
Technical Assistance 



12 , None 



Cooperating units of the Public Health Service, or other organizations, pro- 
viding funds, facilities, or personnel for this project in either 1956 or 
1957. 



13. None 



If this project resembles, complements, or parallels research done else- 
where in the Public Health Service (without interchange of personnel, 
facilities or ftinds), identify such researcht 



1^. 128 (i) 

Serial No, 



15. 



Iniblications other than abstracts frrm this project during calendar year 
1955. 



l6. None 



Honors and awards to personnel relating to this prcject during calendar 
year 1955. 



Page 1 



PROJECT REPORT FORiM 



1, National Ca.ncer 2, Bi elegy 



Institute Laberatery or Branch 

3, Tissue Culture li, Bo thcsda, Md. g. U28 (j) 

Section or Service Location Serial No, 

6, DeveloFment of a defined raediuin for the cultivation of mou se liver 

Project title: epithelial cells. 

7 , V. J. Evans ' ' 

Principal Investigator (s) 

8 , W. R. Earle and N. M. Haw kins ■_ 

Other Invjstigators 

9, Project description: 

O bjective ; To design and test a defined medium which will support prolifer- 
ation cjid allow determination of effect of individual components of the 
medium on substrains of mouse liver cells of low and high tumor incidence, 
(designated //II4.69 aJid #1795 respectively). 

M ethods employed ; Replicate cultures are prepared and enumeration of nuclei 
are made after exposure of various intervals to the defined media. Such 
media are devised from collected data on the nutrition of bacteria and 
mammalian and avian cells in "vitr o and also from the data on physical and 
chemical fractionation of naturally occurring media. 

Major findings ; The prototype media NCTC 107, IO8 , 109 for strain L 
cells h'ave been found unable to support continued proliferation in the 
normal clone liver strain lii69« However, the incorporation of serum 
fraction such as gross globulin as well as, in as little as O.Og gm, 
percent of amino acid free horse serxm residue produces minor prolifera- 
tion of the cells. The essential amine acids and amides for this strain 
have been determined. Exploratory data indicate to be identical with those 
required for strain L cells. VJith this medium, glucose and amino acid 
utilization, keto acid formation, and formation of lactic acid on both 
the high and low incidence tumor cell have been determined in replicate 
cultures, (See report of B, B, Westfall) 

Exploratory data from cultures of the high tumor producing strain .of 
liver cells when subjected to medium NCTC IO8 in which the amount of 
glutajTiine has been decreased show almost twice the proliferation that 
'the low tumor producing strain shows. 



Page 2 

S ignificance i A partially defined medi-um for studying the behavicr cf 
liver epithelial cells has been devised, A slightly mcdificd medium 
will support prclif eraticn alsc cf a strain cf liver cells cf high 
turner incidence. This suggests a difference between a ncrmal and malig- 
nant cells in tissue culture. It must be learned if this explcratcry 
data can be confirmed and whether additional differences can be demcnstratcc 
in vitro from a nutritional point of view. 

P roposed course of project; Efforts iidll be mrde to substitute for and 
augment the effect of the 0,0^ gm % of amino acid-free protein in this 
■ medium. It is proposed tc do this by introducing further purified 
fractions cf serum proteins and tc -supplement the medium w-ith single 
materials which can be closely attached tc the protein residue. It is 
further proposed to continue tc demonstrate nutritional differences 
between the two cell lines, .and to constantly attempt' tc devise .media 
free cf any protein for these strains also, 

10. Ii28 ( j) 

■ Serial No, . ■ ' .. .■ 

11. ,^ \ , . 

Budget activity? 

Research x Administration 



Review & Approval ■ Teclmical Assistance 



12. ^- _ • - • '" " • ' •• • ,, 

Cooperation units of the' Public Health Sgrvice, or other organizations, 

providing funds, facilities, or personnel for this project in either 
19^6 or 1957. 



Laboratory cf Clinical Investigation, National Micrrbiclogical 
Institute , 



,13, ' None* 



If this project roserablcs, complements j or parallels research done else- 
where in the Public Health Service (without interchange of personnel, 
facilities or funds), identify such research:. 



1^* ^28 (j) 



Serial Mo, 
l5« None 



Publications other than abstracts from this project during calendar year 
1955. ■ ■ , 



16, None 



Honors and awards tc personnel relating to this project during calendar 
year 1955. 



Generc-l Project I|.29 



Serial Nc, 



Studios cf the influence cf cell pcpulp-tirn en prcliferaticn, 



Page 1 



Project Report Form 



-^* -Ngitiicna,!, riancp.r. ,.i. 



^ 



stitute 



2. 



BJcJ.r. 



hij CI r.gy r~ 

Laboratciy cr Branch 



3« Tissue Culture 



Secticn cr Service 



U» Bp-h.hp.ciHa _, MH-, 



Location 



5* li29fa) 

Sdrial No, 



6, Studies of the influence of cell pcpulr?.tion on prclif eraticn« 
Project Title 



7. K. K. Scnford 

Principal Investigator (s) 



9. 



G, L. Hobbs and W,R, Earle 



Other investigators 

Project descripti on; Single cell studies and the development of cell 
clones. 



Objective { To develop clones of human skin, mouse riypmary carcinoma, 
and four strains of mouse fibroblasts. Each of the^iS clones is desired 
for specific research projects which will be considered •under other 
headings. The development of a clone of mouse manimary carcinoma is for 
use in a collaborative study with Dr, H, B, Andervont on the relation 
of the milk factor to the m'-.llgnant cell in tissue culture.. 

Methods employed; A sieved cell suspension is prepared and drawn into 
capillary pipettes . Capillary segments are cut, each segment contain- 
ing one isolated cell. The segments are embedded in plasma clot in a 
Carrel flask, and the culture fluid is renewed thrice weekly. 



Major findings; Attempts have been made to define more accurately the 
conditions allowing the proliferation of single isolated cells. Some 
progress has been made. 

Significance to Cancer Research tThe development of clones of cells is 
of fundamental importance for future studies in carcinogenic cell 
transformations among populations of cells in vitro , virus studies, and 
in studies of the differences in the physiology and nutrition of malig- 
nant cells and the normal cells from which they arise in culture. 



11. 



Page 2 



Proposed course ■ Renewed efforts will be made to develop clones cf 
of pr oject { the several cell strains listed -above, and to define 
more accurately the conditions that allow the proliferation of single 
isolated cells. 



10. ' h29 (a) 

Serial No, 



Budget activity; 

Research x Administration 
Review & Approval Technical Assistance 



12, None 



"Cooperating Units of tho Public Health Service, or other organizations, 
providing funds, facilities, or personnel for this project in either 
19^6 or 1957. • ■ 



13. None 



If this Project Resembles, Complements, or Parallels research dene else- 
where in the Public Health Service (without interchange of personnel, 
facilities or funds) identify such research. 



lU. U29 (a) 



Serial No, 
1$, None 



Publications other than abstracts from this project during calendar year 
1955. 



l6 , None 



Honors and awards to personnel relating to this project during calendar 
year 1955. 



.PROJECT REPORT FORM 
1, National Cgjicer 2. Biolcgy 



Institute ■ Laboratory or Branch 

3. Ti.ssne Ciil ti^rfi .Section . ^^ ^' - ■ ■ Rp,t,hP.,sdn. . Mfi . _- — ^' li?.9 (b) 

Section or ScrvicG Location (if other than B 

6. Growth .of massive fluid suspension cultur'es of animal tissue cells. 
Project Title 

7. W. R. Earle ■ . ' ' 

Principle Investigator (s) ~ i 

8. Jay C. Bryant, E. L. Schillin g, B, B.' Westf all, Ellison Peppers and V, J. Evans. 
Other Investigators 

9» Project Description: Growth of massive fluid suspension cultures of ; 

both normal and malignant animal, tissue cells. Application of these 
methods in defining factors resDcnsible- for proliferation of represent- 
ative tissue cell types in such .cultures ,' 

Objectives } The general objective of this project is to develop methods 
and equipment for growing massive fluid suspensions cultures of animal 
tissue cells. Factors receiving attention' in ' the attainment of this 
objective include particularly various types of fluid media, control' of , 
air inflow, control of pH,' and regulation of volume and compositions of 
culture fluid in relation to nvLmbors of cells arid rate of proliferation. 

Methods Employed ; The standard Brxmswick type platform shaker enclosed in . 
an incubator box, which had been developed previously, was used for con- 
tinuing studies with massive cultures. The shaker was operated at about 
12,000 revcilutions per hour, and the size of culture flask used was l|- liter. 
Continuous aseptic flow of 'a gas mixture cf S% GO2, 20^ cxygen and 7$t nitro- 
gen, saturated with water vapor, was maintained through each culture flask. 
The rate of gas flow through each flask was held constant at either llj.0 or 
280 ml, per hour. Phenol red at a concentration of ,002 percent was used in 
all of the culture fluid,* as a pH indicator. The fluid was either renewed 
periodically without appreciable loss of cells or was increased by increments 
of fresh fluid. The rate of proliferation of cells in suspension was deter- 
mined periodically by nuclei counts of carefully sampled aliquots. 

Major Findings: Considerable progress has been made during the year in 
attaining better control of the major factors governing the growth of cell 
suspension cultiires in shaker flasks. With all cells used the concentra- 
tion of glucose in the medium has been increased up to several times the 
coneentration in Earle 's normal saline in order to maintain cell growth 
during intervals between fluid changes unlimited ty exhaustion of glucose. 



General Project h30 



Serial Mc. 
Studies en the cultivation of epithelial cells* 



Page 1 
PROJECT FORM REPORT 



1. National Cancer ^^ 2, Biolcgy 



Institute Laboratory cr Branch 

3. Tissue Culture U. Bet hcsda, Md, 5. U30(a) 

Section or Service "Tccaticn Serial No, 

6, Stiidy of a str ain of H-uman Liver Cells « 

"^ "'Prc3e5t; Title: 

7, V. J. Evans . 

Principal Investigator (s) 

8 , N, M, Hawkins, W. R, Earle, and B, B. Westfa ll ^ 

Other Investigators 

9» Project Description: 

Objective: To cultivate a strain of hvu-aan liver cells for long term 
stiidies for ultimate ccmpariscn of normal human cells and malignant 
cells derived from it. 

Methods Minced human liver prepared by trypsin treatment together 
with stirring and subsequent tissue culture cultivation in human serum 
and chick embryo extract, has yielded a strain of cells. 

Ma jor findings: For the first time a hximaji liver strain gives some 
promise of continuing tr grow in vitro » This is contrary to data from 
■ ^ cr 6 other endeavors tc cultivate human liver for any prolonged inter- 
val. To date this strain of cells subcultures readily in fluid suspension 
and exploratory data indicate that it may grew in shaker cultures thus 
facilitating metabolic studies. 

Significance to Cancer Researc h; In addition to significance tr cancer 
research the strain of cells should be an invaluable tccl to virc-logists . 
Techniques developed in establishing this strain are of usefulness in 
establishing human liver strains for comparative studies in the pl:ysiology 
of the normal and malignant cell. 

Prop osed course of proje ct: Since adequate riiicunts of tissue and fluids 
are obtained from the large culture those -urill continue tc be used for 
glucose and glycogen analysis and for comparison with similar studies on 
the ether stx*ains of mcuso and human epithelial cells in the laboratory. 
Attempts tc produce a malignant transformation of this cell in vitro will 
be made. Since the HeLa strain of cells from a human cervical carcinoma 
has given rise tc small identifiable grotrths in the anterior chamber of 
the mouse eye similar techaniques may be worthy of consideration for other 
strains of cells developed and in particular for this human liver epithel- 
ium, :^ '-rain of cells will be cloned as scon as feasible. 



Page 2, 



10« f}30 (a ) 

Serial Nc, 



11« ^ ■ 

Budget Activity? 



Research x Administraticn 

Review . & Approval Technical Assistance 



12. None 



Cccperating Units cf the Public Health Service, or other organizations, 
providing funds, facilities, or personnel for this project in either 1956 
or 1957. 

13. None 

If this project resembles, ccmplements, or parallels research done else- 
where in the Riblic Health Service {without interchange of personnel, facil- 
ities or funds), identify such research: 

la. ^30 (a) 

Serial No. . . 



15. Hone . ^ " ^ 

Publications other than abstracts frciii this project during calendar^ year 1955 



16, Mone ^ 

honors and awards to personnel relating tc this project during calendar year 



1955. 



PROJECT REPORT FORM ^^S® ^ 
1. National Cancer 2, Biology, 



6. 



INSTITUTE UBOR/iTORY OR BRANCH 



3» Tissue C-ulture ll, Bethesda, Md. 5. 1|30 (b) 

Section or Service. Location . . Serial No, 



Preservation cf culture cells. 



Project Title 



7. V^ J. Evans and W. R. Earle, 
Principal Investigators. 



See Item 12 

Other investigators. 



9» Project description: Now that cells can be cultures in substantial 
amounts is it desirable to be able to preserve these cells in a latent 
state, for extended periods cf time. The most premising method reported 
to date, has been reported by Polge, C. (Nature l6i|j 666; l9i;9) who 
used glycerine impregnation for pjreservaticn of bull spermatazoa,. The 
Tissue Bank, Naval Medical School has utilized this method for the pre- 
servation cf skin and cornea. It is desirable to learn the length of 
preservation; whether this inexpensive material (dry ice and glycerine) 
is satisfactory; and, whether this material is less toxic to cells than 
other materials. Tissue culture viability of preserved tissue will serve 
as the index of the physiological conditions of the cells. 

Methods employed; Rabbit comea cultures were planted in a thin 
plasma clot under perforated cellophane in Carrel 3.5 flasks, A 
nutrient medium of horse serum and which embryo extract lA Earle's 
saline was used. One half of a rabbit cornea was used in each culture 
flask. Initially fresh medium and wore planted within 30 minutes from 
the time of removal from the eye. Soaked corneas were placed in test 
tubes containing 1^% USP glycerine by volume ±a Earlets saline> or, 
in Ringer's saline for 1 hour. Division, explantation and incubation 
of unfrozen corneas took place within an hour. Frozen comea were 
frozen either with (l) no soaking, (2) after soaking in glycerine in 
Earle's balanced saline for 1 hour and (3) after soaking in 1$% glycer- 
ine in Ringer's saline for. 1 hour. The excess fluid was decanted and 
■ the tubes of corneas were first immersed in a container of carbon 
dioxide alcohol slush at -760C for 3 minutes. At the end of 1 hour 
■ at -76°C the tubes containing corneas were removed, thawed by immers- 
ing in a water bath at 38'^C and corneas were divided, explanted and 
incubated , 



rage x 



Page 2 



In the exploratory skin study to date, the prccediires were approxi- 
mately similar to that used to the cornea study, except that human 
senm was substituted for horse serum, and the skin used, was of ... 
human origin. 

M ajor findings t In the study on rabbit comea. the fresh cornea 
showed excellent migration of epithelial and fibroblastic cells in 
all instances within U8 hours. Corneas which were soaked in dilute 
glycerine or soaked and then frozen showed a slight lag in migration 
but were soon indistinguishable in migration and cell appearance from 
fresli corneas. Corneas frozen without glycerine protection showed no.' 
migration in 28^ of the cult\ires. The remainder showed retarded mi- 
gration and severe cell injuzy. Corneas preserved by the 1$% gly- 
cerine, in S^% Earle's saline by volume, and frozen and preserved 
at -76OC. have been fctmd tc be viable in tissue culture up to 6 s 
months. Exploratory data, obtained tc date on skin preserved ty these 
methods does not appear tc be viable in tissue culture. 

Significance ; Before beginning the study the feasibility of using 
human corneas was considered. Since insufficient numbers could be 
obtained, the rabbit cornea was selected since most experimental 
work to date has used this source. The tissue viability test was 
used to test the physiological condition of corneas preserved by the 
above methods because it was considered of greater critical useful- 
ness than either the clarity of the graft after transplantation or 
tests of the respiratory enzyme systems. The results. :0f, the study 
demonstrated for the first time that rapidly frozen .glycerine im- 
pregnated corneas were consistently viable, -Fovr -factors rather than 
any independent one appears to be responsible for thisj (l) glycer- 
ine soaking, (2) fast freezing, (3) fast thawing and {k) the use of 
a highly buffered balanced physiological saline, such as Earless 
saline .rather than Ringer's Salfjie, 

.Corneal endothelial cells (which are reqixired in a viable state for 
successful keratoplasty) may assist in establishing criteria for the 
preservation of cells cultured in , large masses and stored in glycer- 
,ine. If it can also be demonstrated that corneal endothelial cells, 
which are preserved by glycerine impregnation and stored at dry ice 
temperature, exhibit viability in vitro , possibly, cells of less 
specific function may be preserved 'in a like manner. The human skin 
which has been preserved by a modified glycerine method serves well 
as a clinical homograft dressing. In spite of the failure to date to 
establish viability in vi tro , it still appears important to learn 
whether or not improvements in methods of preservation of skin, so 
that viability in vitro is demonstrable, will yield a superior .dress- 
ing. 

Serial No, 



Page 3 



11. 



12. 



Budget Activity 

Research x Administration 
Review & Approval Technical Assistance 

Cccperating Units of the Public Health Service, or other organizations, 
providing funds, facilities, or personnel for this project in either 
19^6 or 1957. 

a) Tissue Bank, b) Dept. of Ophthalmology, c) Naval Medical Research 
Institute, National Naval Medical Center, 

13. None 

If this Project Resembles, Complements, or Parallels Research Done 
Elsewhere in the Public Health Service (without interchange of person- 
nel, facilities or funds), identify such research. 

Jh, U30 (b) 



Serial Mo, . 

15. 



Publications other than abstracts from this project during calendar year 
1955. 

The viability of fresh and frozen corneas as determined in tissue 
culture. Mcpherson, S. D,, Draheim, J. W., Evans, V, J, and W. R, 
Earle, American J, of Ophthalraclcgy - In press. 



16, None 



Honors and Awards to Personnel relating tc this Project During Calendar 
Year 1955. 



Page 1 
PROJECT FORI! REPORT 



1, National Cancor 2, Biclcgy 



Institute Lc.bcratcry cr Branch 

3, Tissue Culture il. Bethesda. Md, 



S. h30 (o) 

Serial 'Nc 



Sccticn cr Service Lccaticn Serial Nc 

6, Studies en the cultivaticn of human cndccrine secr eting ti ssue i n vivo, 

SojecT~Title 

7, V . J. Evans 

"Principal Investigator (s ) 

8, N. M. HaiJkins, W. R. Earl e 

ether Investigators 

9» Project Description: 

Objec tive : To cultivate cells of endocrine tissue as made available 
by the EnH'ocrinclogy Branch of the National Cancer Institute in order 
to learn the extent and duration ci hormone secretion of isolated cells 
in vitro. 

Methods em p loyod i Additional islet cell tumor tissue from the pancreas 
of a clinically diagnosed oasc of carcinoma of the pancreas has been 
obtained from the Endocrinology Branch, of the National Cancer Institute, 
Dissociation of the tissue tc small but multiple colonics for explanta- 
tion of cultures has been done. This method has been successful for 
cultivation of other humaji epithelial cell strains. 

Maj or findings } Presently this new strain of cells is still in an early 
stage of cultivation in vitro. 

Significance to cancer research ; The development of techniques for 
growing human endocrine tissues in vitro so that they continue to _ ■ 
function should have considerable practical value for therapy. From the 
point of view of cancer research any endocrine assays or data from histo- 
chemical assays should serve as valuable indices of the changes in cells 
in vivo and in "^{itro both in clinical and experimental conditions. 

Proposed course of project : Experiments will be continued tc culture 
various tissues of endocrine origin as they are made available from 
services in the Clinical Center and other sources. Methods must be 
developed for culturing such highly specialized cells. The duration and 
amount of hormone secretion and study of the morphology of the tissue 
growth V7ith photographic recordings will be made. Histochemistry, bio- 
chemical analyses and morphology and mouse chajiiber techniques will be 



Page 2 



employed and correlated with function and therapy. Attempts will be made 
to devise new methods of assay of fiinction by grafting as suggested by 
mouse chajnber techniques and as justified by the growth of cells in vitro, 



10. U30 (c) 



Serial No, 



11 • 



Budget Activity} 

Research 



Administration 



Review and Approval 



Technical Assistance 



12. 



Cooperating Units of the Public Health Survicc, or other organizations, 
providing funds, facilities, or personnel for this project in either 1956 
or 19?7. 

Endocrinology Branch, National Cancer Institute, and Transparent Chamber 
Unit, Laboratory of Biology, National Cancer Institute, 



13. None 



If this project resembles, compleiiionts, or parallels research done else- 
where in the Public Health Service (without interchange of personnel, 
facilities or funds), identify such research: 



Hi'. U30 ( c) 

Serial No, 



15. None 



PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 

1955. 



16. N6ne ' , 

Honors and Awards to Personnel Relating to this Project during Calendar 
Year 1955. 



Page 1 
PROJECT REPORT FORI>^ 
1, Naticnal Cancer 2, Biolc^ 



Institute Laboratory or Branch 

3t Tissue C ulture It, Bethesda, Md. 5» I|.3 0(d) 

"Section or Service Location " SeriaT'No, 

6. 



7. 



A study of long term human skin strains. 






Project Title 






V. J. Evans and W. R. Earle 






Principal Investigators 


..tn ■■>,:■;; 




See item 12, 







Other investigators 

9. Project Descripticnj Objec tive : a) To produce and maintain long 
term human epthelial cell tissue culture. This will tend to an 
understanding of tissue culture cell metabolic needs as a.', preliminary 
to transplantation studies, b) To determine by continuous in vivo 
transplantation of cells, the possibility of spontaneous in v itro 

— malignant, transformation of human skin cells. 

Methods emplc^^ ed j a) Human skin from a 65 year old man obtained by • 
the Tissue Bank, Naval Medical School, was separated into dermal and 
epidermal elements, A cell suspension of epidermal elements was planted 
in human sei'um and has^bisen carried in vitro now for over 2 years, A 
subline ^^ras established in horse serum for experimental work where human 
serum is undersirable, b) Shaker studies were established according 

—.to the procedures of Earle, Biyaiit and Schilling, (c) The strain 1769, 

'^''^ "*'•■• human skin has been implanted in the anterior chamber of the G3H mouse 
- eye, and the pouches of golden hampster. Mice were irradiated and 
cortisine treated, as described by Toolan. 

M ajor findings: Human skin has been successfully cultivated over two 
years m human soru.m. The subline on horse serum has been carried now 
for over 6 months. The details for preparation of the strain, descrip- 
tion of the cells and related use of the cells have been reported in 
a manuscript. In exploratory studies the strain on human serum when in 
■the presence of vi.able homogenous tissue will elicite what appears to 
be a rejection-acceptance phenomenon (see report on rejection-acceptance 
" phenoraen in vitro), b) The complete detail of methods employed vnd 
size of cultures attained in the report of Earle, Bryant, and Schilling, 
"Growth' cf '.massive fluid suspension cultijres of animal tissue cells". 
Fluid and cells from these were made available to Dr. lAfestfall (see 
report of Westfall and Peppers) for studies on utilization of glucose, 
storage of glycogen and production of keto acids, etc, c) No overt 



Page 2 



turners were found in the anterior chamber cf the mouse eye or in the 
hampster mcuth pcuch. This '^as contrary to the findings cf tumor 
tissue when HeLa (Human cervical. carcincma) was implanted. 

Significance ; For the first time a strain rf human skin cells has been 
cultured in vitro for over twr years and continues to grow luxuriantly, 
resembling morphologically epithelial cells, Nimiercus types of studies 
have been made with it. It is known to support some l5 viruses. The 
strain can be grown in amovints adequate for metabolic studies. It has 
been possible to initiate problems related tc hcmotrans plantation. 
The cells have possible application in clinical use, particularly in 
reparative surgery. They may have additional significance in investi- 
gations on cancer therapy, both because it supports viruses, and as 
a stable strain the^,- may be of value in screening chemotherapeutic agents, 
Comparative studies on their metabolism with that cf epithelial- cancers. 
might be significant. 

Pr oposed course of study t Tc continue studies on the nutrition and metab- 
olism of the cells for ultimate use in clinical work. To confirm studies 
on homcgraft response and to continue studies on large shaker flasks tc 
facilitate the above. 



10, J|30 (d) 



Serial No, 



11. 



Budget activity: 

Research x 
Review & Approval 



Administrative x 
Technical Assistance 



12. 



Cooperating units of the Public Health Service, or other organizations, 
providing funds, facilities,, or personnel for this project in cither 
19^6 or 1957 



Much of the work on this problem has been dcno ly -personnel of the 
Tissue Bank, Naval Medical School, National Naval Medical Center, 

13 » None ' 

If this project resembles, complements, or parallels research done 
elsewhere in the I-\iblic Health Service (without interchange cf per- 
sonnel, facilities or funds) identify such research; 



Page 3. 



1^» |f30 (d) 

Surial Mc, 



15. 

FYiblicriticns rthjr than abstracts frcra this project during calendar year 

Perry^ V. P., Evans, V. J,, Earle, W. R., Hyatt, G. ¥. and Bedell, W,C,- 
Lcn,;;; Term Tissue Cultxire of Human Skin, Am, J, cf Hyi^iene, Jan, 19^6. 

Bassett, C.A.L,, Evans, V,J,, CaTipbcll, D, and Earle, W. R. -Character- 
istics and Potentials rf Lcnn-term Culture of Human Skin. N,M,R, I, 
Project Report 00? 081.10. 11. 

l6. Secretary of Navy Cominendation Medal tc Hospitalni?Ji Vernon P. Perry IC for 
for his participation in this work as carried en under our supervision in 
the Tissue Culture Section^ Laboratory of Biology, 



PROJECT REPORT FORM 



1, Naticnal Cancer 2. Biology 



Institute Laboratory or Branch 

I, Tissue Culture it. Be thesda, Md. , 5« U30 (e) 

Section or Service Location Serial No, 

6 , To ad apt strains o f cells to media of heterologou s compo s ition , 

Project Title 

7, V« J. Evans , 

Principal Investigator (s ) 

8, W. R. Earle and see item 12 

Other investigators 

9« Proje c t Descriptio n < 

Metho ds a) Present experimental studies include! subjecting human malig- 
nant cells and huraan cells from non-malignant skin to media of various 
combined mixtures of human and horse serum until horse serum alone could 
be used. The cells were maintained in Carrel D-3.5 flasks and Earle T-30 
and T-60 flasks. After intervals of 120 hours or more, the less foreign 
and it is presumed less toxic serum concentrations permit subculturing of 
substrains of cells for study. 

Major Findings: It has been found possible hy the method described above, 
that cultTores of human malignant tissue (HeLa l88l) initially propagated 
in human serum, can human scrum, can be adapted to heterologous horse 
serum medium to produce HeLa strain 1985. By a similar procedure a strain 
of normal human skin epithelium grown in human serum and designated strain 
1769 has been adapted to horse serum. This adapted strain was designated 
strain 2198. Both horse serum adapted strains have been found of usefulness 
in homografting problems in mouse chamber work (see report Algire et al.), 
and for cultivation of hujiian viruses (see report Habel and JlcBride, National 
Microbiological Institute). 

Slgniflcance i Both these strains have already been found of significance 
in studies of cultivation of human viruses, (Strain 1985 has already been 
used in studies on mechanisms of homografting. The mechanism of adapta- 
tion should be an interesting situation for study by the immunologist in- 
terested in the problem of homografting as well as by the protein chemist, 
Habel, Gregg ajid McBride of the Microbiological Inst, and Dental Listitute 
reported results on large numbers of horse serum adapted cjiltures submitted 
to them, from this laboratory, In one experiment cells --dapted to horse 
serum were less susceptible than those grown in human serum and less sus- 
ceptible thaji fresh kidney to poliomyelitis virus. However, a second ex- 
periment indicated rn equal susceptibility of all three types of tissue' 
culture to the three types of policmyse litis, HeLa cells grown in horse 



serum were just as responsive to pclio virus isolated from 23 separate 
stock emulsions of polio virus as those grown in human serum. 

HeLa cells grown in horse serum in suspended cell shaker type of culture 
and then carried in a maintenance medium for 2[i hours pnd tested for pclio- 
iryeletis as efficiently as those grown in human serum. This indicates that 
viruses are able to propagate in cells growing in the sho.kor type culture 
and an advantage is gained where hcrse serum is used both, because of its 'i* 
availability and because the specific antibiodies of hum&i serum may be 
eliminated. 

Pr ojected pla nt Further study of problems of homografting in mouse diffu- 
sion chambers with both strains of cells and their respective substrains 
to determine likenesses and differences in behavior will be made, 

10. It30 (e) 
Sbriar"No, 

11, ^wm^'^^ ' 



16. 



Budget Activity: 

Research x Administration 

Review & Approval ' Technical Assistance 



12. 



Cooperating Units of the Public Health Service, or other organizations, 
providing funds, facilities j or personnel for this project in either 1956 
cr 1957 

Laboratory of Infectious Diseases, National Microbiological Institute; 
Tissue Bank, Naval Medical School, National Naval Medical Center; Trans- 
. parent Chamber Unit, Laborafrry of Biology, NCI 



13., None 



If this .Project Resembles, Ccmplomonts, cr Parallels Research done elsGwhon 
in the Public Health Service (without iiitcrchans^^c of personnel, facilities 
or funds), identify such research: 




Publications other than abstracts from this Tofoject during calendar year 
1955. ,. ., 

Perry, V.P., Evans, V. J, and Earlc, VJ, -'R. Cultivation cf large 
cultures cf HeLa Cells in hcrse serum. Science 121$ 8o5, 1955. 



Honors rnd awards to personnel relatin,-;,' tc this project during calendar 
years 1955. 



Page 1. 



PROJECT REPORT FORM 
1, National Cancer 2,' Biology 



Institute Laboratory or Branch 

3. Tissue Cultur e ii, Beth csda, Md,. 5. J^30(f ) 

Section or Service Location Serial No, 

6, Studi es on the utilizaticn of a long term strain of skin epithelium for pro - 

Project Title pagation of virus, 

7, V . J. Evans, '^ '2 

Principal Investigator (s) 



¥, R. Earle. - Also see item 12 



Other Investigators 



Project descri pt ion ; To test whether hu.man skin epithelium will support 
cultivation of certain human viruses. 

Met hod - More than 2000 roller tube cultiure of a long term strain of humrji 
skin "cells from a 6$ year old man were studied for cytopathogenic effects 
and production of complement fixing rntigens when exposed to numerous sig- 
nificant viruses . 

■ Majo r Findings } To date those viruses apparently multiplying in this cell 
strain are; adenoid?! pharyngeal conjunctival viruses types 1, 2 and 3j 
Coxsacki virus tjpo B-3j herpes slmplexj B virus j vaccinia j mvimpsj en- 
cephalcnsrocarditios virus j' lymphocytic choriomeningetis virus; St, Louis 
encephalitis virus; and yellow fever virus. 

Significanc e; This strain of ^^ells for virus studies has significance 
because of its being an epithelial coll of human origin. Many viruses 
have not been propagated in vitro since this cell strain grows luxuriantly 
in large amcunts, and maintains many viruses in rapid proliferation, it 
offers substantial potentialities both as a basic research tcol and as a 
practical basis in large scale p«^:ducticn of viruses. Already, this cell 
strain,_ coji be maintained in a defined medium supplemented with only limited 
amcunts of serum for these specific studies on viral propagation. In other 
cases, where desired, an entirely heterologous serum may be used. 

This tissue may be assumed to be non-malignant until exhaustive studies prove 
otherwise. 



12. 



16. 



Page 2, 



Prcposed course rf p roject : Exploratory data on the cultivaticn of this 
strain cf cells in mass amounts in the shaker type culture will be con- 
firmed for other studies but the obvious value of this strain of cells 
from a normr.l tissue source useful for vaccine production is suggested 
for consideration by virologists, end will not be studied by our group, 
Continuaticn of efforts to demonstrate any potential malignancy must be 
pursued. Methods presently available for doing so will be tested and new 
ones will be sought. Methods of controlling possible development of malig 
nacy. in tissue culture must be tried and their validity tested. 



10. h30 (f) 



.. Serial No, 
11 • None 



Budget Activity: 

Reserxch x Administratirn 

Review & Approval TechnJ.cal Assistance 



Gcoperating Units of the Public Health Service^ or ether orgrnizaticns^ 
TDrcvidin? funds, facilities, or perscnnel for this project in either 1956 
ov 10^7 / 

L.-^.bci-c tc?.y cf Infectious Diseases, National Hcrphr logical Institute^ 
Clinical Investigation, National Institute cf Denta] Research; Tissue 
E,.\nk Naval Medical School, National Naval Medical Center. Over 2000 
cultures have been furnished the first twr of those for virological re- 
search: 



13-. N-nc 



It tiiis pr.-.^GcL resembles, ccmpl'j.ients- or piv-diGis research done elsc- 
wnere-in the "^nblic Health Service (without ii;.terchange of personnel, 
facilities cr funds), identify such research. 



lit. U30 (f) 

Serial Nc , 



15. 



fublicaticns other than abstracts from this project during calendar year 
1955. 

Long Term Tissue Culture of Human Skin, Perry, V. P,, Evans, V, J,, 
Errie /J. R,. Hyatt, G, W,, Bedell, \i, C. Am. J. of %giene, (Jan) 
1956.' 



Honors fnd awards tc personnel relating to this project during calendar 
year 1955. 



Page 1 
PROJECT REPORT FORM 



1, National Cancer 2, Biology 



Institute Laboratory or Branch 

3 • Tis3»e Cul ture ___;_ _ _ _ -V- - h . Bethesda, Md. ^ . h30 (g) 

Section or Service " Location Serial Mo, 

6, Rejection-A cceptance, phenomenon in vitro, 

Project Title 

7, V. J. Evans and W. R. Earle 

Principal Investigator (s) 

8, S ee item 12, 

Other Investigators 

9, Project description t Tc determjne if viable and non -viable tissue frag- 
ments in vitro will elicite an in vivo homog raft -like reaction. 

Method: Fresh and freeze dried tissue have been, procured from the 
Tissue Bank, Naval Medical School and planted in Carrel D-3#5 flasks, 
_.-.: with plasma and a fluid medi'tn phaie ' ccntairting st^'ain 1769 (long term 
human epithelial) and allowed tc clot. Observations were made at 14.0,96 
and 120 hours tc determine if rejection of strain 1769 has taken place. 

Major Findings: In an exploratory series, observations suggest that there 
is a recognizable horaograft-like reaction in vitro. The 214-72 hour re- 
jection phase of strain 1769 is evidenced by the formation of a definite 
zone of partial or complete growth inhibition at the periphery of the 
fresh tissue explant. This inhibition zone has not been observed with 
freeze dried tissue explants. 

Significance to Cancer Research: If these proliminaiy observations can 
be confirmed they might well be most sighificant in further elucidation 
of the hcmograft response that occurs in vivo . 

Projected Plans: To repeat and expand these findings with different types 
of tissue jboth viable and non-viable, of homogenous and heterogenous 
origins, 

10, l|30;(g) 
Serial No, 

11, 

Budget Activity: 

Research x Administration x 

Review and Approval Technical Assistance 



12. 



Page 2 



Cooperating Units of the Public Health Service, or other organizaticns 
providing funds, facilities, or personnel for this project in eithor 
1956 or 1957. 

> f . ., 

Tissue Bank, Naval Medica:l School, National Naval Medical Center. Much 
of this work was actually done by Tissue Bank Personnel under supervisici 
of N.C.I., senior investigators. 



13, None 



If this project resembles, complements, or parallels research dene 
elsewhere in the Public Health Service (without interchange of person- 
nel, facilities or funds), identify such research; 



lli. ' il30 (g) 

Serial No, 



l5 , None 



Publications other than abstracts from this project during calendar 
year 1955. ■ 



16. Wone „ 

Honors and awards to personnel relating to this project during cal- 
endar year 1955. 



Goneral Project _Ii31 

Serial No. 



Studies cf cell trans fcrmaticn in vitro . 



, - . . Page 1 

• ' ■ PROJECT REPORT FORM 

1, Nat irnal Cancer , 2. Bic logy 

institute laboratory or Branch 

3« Tissue Culture lu Eothesda, Md. g. U31 (a ) 

Section or Service " Location Serial No. 



6, Studies of coll transformation in v itro . 



Project Title 
7. K. K, Sanfcrd 



Principal Investigator (s) 
a, G, L. Hcbbs, M. Fiorajncnti, and W. R. Earle 



Other Investigators 

9. Project description: Characteristics of two ].inos of cells originating 
from a single adult mouse cell, , 

O bjective ; l) To define quantitatively the sarcoma-producing capacity 
of cells of these two lines as dependent on the numbers of cells injected 
into mice end as influenced by the period of cell groirt.h in vitro , 2) To 
determine whether the difference in sarcoma-producing capacity of these 
two coll lines is related to a difference in immimolrgic properties affect- 
ing their transplantability t'" the mouse strain of origin and to define 
the autonomy and immunologic specificity of the cell lines through hcmolo- 
■■ -gcus transplajitaticn studies. 3) To compare the proliferation rates in 
vitro of the two cell lines, k) To compare the growth in v ivo of these 
two cell linos, g) To ostablish and analyze the chango in^sarccma-producing 
capacity induced in these cell lines by mouse passage, 6) To determine 
differences in oxidative ' metabolism of those two lines of cells, 7) To 
deterrainc differences in the nutritional requirements of these two lines 
of cells. 

Methods employed; Quantitative procedures in vjhich leicwn numbers of cells 
were injected into both X-irradiated and ncn-irradiated, immunized and ncn- 
iramunizcd mice of the inbred strain of origin have been used. Injections 
of cells were also made into foreign inbred strains of mice. Proliferation 
rates in a stock horse serum-chick embryo extract culture medium have been 
determined md nutritional requirements are being studied by the use of 
quantitative replicate culture procedures . In collaboration with Dr, Mark 
Woods the oxidative metabolism of the two cell linos is being investigated. 
In collaboration with Dr. Ruth Morwin an attempt is being made to determine 
the la,tent period for sarcoma production from cells of these two lines in- 
jected into mice treated so as to be unable temporarily to develop a homo- ■ 
graft reaction. Cell growth in vitro has also been studied ty implanting 
known numbers of colls into diffusion chambers, each plaood in the perito- 
neal cavity of a mouse. 



11. 



Page 2 

Major findings ? 1) Marked differonces in the sarcom?-producing 
capacity of these two cell lines were demonstrated, 2) The sarcoma- 
producing capacity of cells of these two lines was found tc be depen- 
dent on the numbers of cells injected into mice and was influenced by 
the period of cell growth in vitro. 3) Both lines of cells were found 
to be antigenic tc the strain C3H mouse. Immunologic cross reactions , 
occurred between these two linos rnd also between these lines and a 
different clone of cells, clone 929 of strain L. h) The cells of these 
two lines grew specifically in the strain. C3H mouse and not to any ' . 
extent in other strains of ■mice' tested, 5) In mice treated so as to 
be unable temporarily to develop a hcmogrsft reaction (see report by 
Dr. Ruth MertTin ) marked differences were observed in the latent periods 
for tumor development from injections of equivalent numbers of cells 
of the two lines. These data indicated that the main difference in 
sarcoma-producing capacity of these two cell lines was not a difference 
in their trans plan tability tc the mouse but was a difference in their 
ability tc survive and grow in a non-resistant host. 6) The malignancy 
of cells of the low sarcoma-producing line could be markedly increased 
by animal passage. After one generation in a mouse, the cells were - 
changed. This cell transformation lasted for approximately 6 months of 
growth in vitro for the one tissue cultiu'e line studied, 7) The prolifer- 
ation rate of cells of these two lines when cultured in a horse serum- 
chick embryo extract mediiim was not significantly different, 8) Cells 
of these two cell linos could not proliferate on chemically defined 
mixture 'ICTC 107-108 tinless 0,5 to 1 percent serum proteins were added. 
The amine acid requirements appeared to be the same as those of strain L 
cells. 9) Initial studies by Dr. Ifcods indicated a marked difference 
between these two cell lines in their rates of aerobic and anaerobic 
glycolysis and in their rates of respiration. 



Signific ance to cancer rcse arch;_ This analysis of the behavior of 
cell lines that djjifer markedly in their ability to produce sarcomas 
in the strain C3H mouse and that have transformed from nonnal to tumor- 
producing cells should provide some information as tc. the type of 
metabolic change that these cells have und.:rgone in their transformation, 

Proposed cours e of project; Tc complete the studies outlined above and 
■ to pursue particularly the biochemical studies in an effort tc detect 
the mecho.nisras underlying the changes in malignancy of these cells. 



10. 1|31 (a) 

Serial No. 



Budget Activity: 

; Research x Administration 

Review and approval Technical Assistance 



12'. 



Cooperating Units of the Public Health Service, or other organizations, 
providing funds, facilities, or personnel fcr this project in either 
19^6 or 19^7. 

Cooperation with Dr. Dean Eurk, Dr. Mark Woods, Labcr"tcry of Biochemistry 
and with Dr. Ruth Merwin, Laboratory of Biology, N.C.I. 



13. None 



If thn.s r>rcject resembles, compleracnts, or parallels research done else- 
where in the Public Health Service (without interchange of personnel, facil- 
tics or funds), identify such research; 



lli. hn(a)_ 

Serial Ho. 
l5. None 



Publications other than abstracts from this project during calendar year 
1955. 



l6. None 



Honors and awards to personnel relating to this project during calendar 
year 1955. 



PROJECT REPORT FORM 
1. Naticnal Cancer 2, Biology 



Institute Laboratory or Branch 

3, Tissue Culture U. Bothesda, Md $. I|.31 (b ) 

Socticn cr Service Location ' Serial 



No 



6» studios of cell transformations in vitro 



Project Title 
7. K.K. Sanford, 



Principal Investigator (s ) 
1, G. L, Hobbs and I'J.R. Earlc , 



Other investigators. 

9. BrojGct Description; ' A study of the tumor-producing capacity of clone 
y29 of strain L. 

Objective :, To test, the sarocmarproducing capacity of a clone of cells, clone 
929, derived from a single cell' 6f strain L. Strain L was originated in 19iiO 
from an explrnt of subcutaneous connective tissue taken from a strain C3H mouse, 
After two yerrs rf growth iii vitro, cultures of strrin L gave rise to sarcomas 
when injected into strain 0311 mice. The incidence of mice developing sarcomas 
in 19ii3 from the injected cells was 68 percent. Throe years later in 19li6, 
this incidence hrd dropped to 1 percent. The object of the present study was 
to explain this change in sarcoma-producing capacity of the cells. 



Methods emplcyed ; The sarcoma -producing capacity',. of the cc^s Was been tested 
by injecting the cells intraniuscularly intf^ X-irrJidiatpd' and non-irradiated 
strain C3H mice as well as into foreign strains df mice and strain' C3H mice 
immunized with clone 929 cells. 

Ma j or findings ; VJhen clone 929 cells were injected into X-irradinted" strain ' 
C3H mice, the percentage of mice developing sarcomas was comparable to that 
obtained in 19li3 when strain L cells wure first tested for sarcoma-producing 
capacity. An injection of clone 929 cells was found to induce an immune re- 
action in strain C3H mice that completely prevented the ■ growth- of clone 929 
cells subsequently injected. It was thus demonstrated th-^t the apparent decrease 
in sarcoma-producing capacity of these cells resulted from the development of 
an incompatibility between the tissue culture cells and the strain C3H mouse 
rather than from an,y dembnstrablc ch.r'nge in the mclignancy of the cells. Two 
sarcomas derived from clone 929 cells 'were foiind tr grow iti all strain C3H 
mice injected after serial transfers in vivo. A specificity of the sarcoma 
tissue for the strain C3H mouse was also demonstrated, 

Signific-'^nce tc cancer research ; At the present the only method for deter- 
mining the malignancy of tissue culture cells is to inject the cells into 
animals of the inbred strain from which the tissue originated. The signi- 
ficance of the present study is to point out that negative results of such 
a test do not necessarily indicate a lack of malignancy of the cells tested. 
It has been demonstrated that an incompatibility may develop between the 
cultured cells and the mouse strain of origin such that the cells become 
antigenic to the host. The change observed earlier in the sarccma-producing 



Page 2 



capacity of this tissue culture strain has thus been explained as a 
change in the transplantability of the cells and not as a change in 
their malignancy,. It has also been demonstrated that cells grown for 
10 years in a heterologous culture medium have not lost their specif icty 
for the animal strain of origin. 

Proposed course of sub-project: This sub-project will not be continued. 



10. ii31 (b) 



Serial No,. 



11. 



Budget Activity J 

X Research 

Review ?Jid approval 



Administration 
Technical Assistance 



12 , None 



Cooperating Units of the Public Health Service, or other organizations, 
providing funds, facilities, or personnel for this project in either 1956 
or 1957 



13. No'" . 

If this project resembles, complements, or parallels research done else- 



where in the Public Health Service (without interchange of personnel, 
facilities or funds), identify such research j 



^' ^31 0>) 

Serial No. 



15. 



Publications other than abstracts from this project during calendar year 1955. 



The tumor-producing capacity of strain L mouse cells after 10 years i n vitro . 
K,. E, Sanford, G«L. Hobbs, and W.R. Earle, Cancer Research, In press. 



l£ • , N one. [ ^ 

Honors end awards to personnel relating to this project during calendar year 
1955. 



Page 1 

PROJECT REPORT FORM 



1, National Cancer 2, Biology 



3, Tissue Culture h» Bethesda Md. ^ 5» Lgl (c) 

Section or Service Location Serial No. 

6, Study o f two st rains of mouse liver epithelium frcm the ssme single cell. 

Project title* c^ 

7 • V» J « Evans, 

Principal Iftveatigatoris) 

8. N ,M» Hawkins and W.R.Earle 

Other Investigators 

9« project Descriptions 

Objective ; To study two substrains, arising from a common clone of mouse liver 
epithelium. One of these strains (1795) is of high tumor producing ability the 
other is low in tumor production. The primary objective is to have available 
two such strains for comparing their metabolic and physiologic characteristics. 

Methods employed { Cell cultures have been studied in vitro and have been 
continuously injected in both x-irradiated and non-irradiated C3H mice. The 
behavior of these cells in replicate culture has been studied for their re- 
sponse metabolically. 

Major findings ; Mouse liver clone (li|.69) was originated from a single cell 
of strain 721 in April 19li.8, Metabolic studies will be dealt with in detail 
by Dr« B,B» VJestfall, This clone cell strain has shown a low capacity for 
developing tumors on injection, only 1 tumor has been found in non-irradiated 
mice vjhile 2 have been found in x-irradiated mice in over 200 animals. Practi- 
cally 100^ tumor incidence was obtained frcm one of these lots on trocar in- 
jection into both non -irradiated and x-irradiated mice. Tissue culture from 
one of these tumors were grown to produce strain 17 95 • This strain developed 
a much enhanced capacity in a much shorter interval for its cells to give rise 
to tumors on reinjection into C3H mice. The incidence of takes vTith this strain 
is 81.0^ on injection in x-irradiated C3H mice and 69,8^ by injection in non- 
irradiated C3H mice. Tumor tissue on subinoculation into irradiated mice was 
100^ while those in non-irradiated mice was only 10^, 

A manuscript is in preparation deseribing these two cell strains. 

Significance to Cancer Research ; A comparative study of the metabolic and 
physiologic behavior of these two strains in tissue culture appears to be 
of significance in understanding the characteristics of transition from a 
cell strain able to give rise to practically no tumors to one able to give 
rise to a high percentage of tumors. 



Page 2 

Projected work: Clone cells of this high incidence tumor strain will 
he used for experimental work tc compare and to deteimine if tiffliors can 
be obtained from the second pure cell strain as well as the mixed cell, 
strain (179^), Studies are still in progress to determine if the mixed 
cell strain (17 95i) continues to give the same high incidence of timors or 
whether this incidence is lessened ty continued proliferation in vitro.« 



10. " U31 (c>> 
11. 



Serial number 



Budget activitys 



12 , None 
Cc 
■ ' \1 



X Research Administration 

Review and approval Technical Assistance 



None I . • 

Cccporating Units of the Public Her^lth Sorvlce , or other organizations, pro- 
\ldtog funds,' facilities, or pnrscnnel rcr this project jji epither 19^6 or 



13. "N^re- „._'.■ ■ . ^ ■ 

■'If trd;! prefect re^orobles, complements, - or :para'j.lel3 research done elsawhere 
-■in the l-u.jlic Keallth Service (without interchange of personnel, facilitie's 
or lunda ) „ identify such resear':;h , , , , . 

ll;. 1|31 'jt' ) ._. '■ 
Serial' iiC, ■ ■ 



15.- ' Ncne-" - ^ .■__ ■ ■ ' ■ , ^ ' 

•■Piib-lications other thaii atj tracts' from 'i.his project .during calendar year ,1955 ■ 



1.6. None ' ■■■ ' ■ ' _ . ^ ■ . . - 

HOTiors and awards to persomiei relating to this project during calendar year 
1955. 



:i<iS. C^Md: 



General Project l|.32 



Serial Nc, 
Ccmpariscn micro cinematography • 



• Page 1 
PROJECT REPORT FORM 
1, National Cancer 2, Biology 



Institute Laboratory or Branch 

3 » Tissue Gultnre k , Bethesda ' 5:« :'i|-32 (a) 

Location. '" ~ ' /Serial No, 

6, Studies with the Comparison Micrccinematograph. 



7. 


Projec 
. W. R. 


t Title. 
Earle 








Princ 


ipal Invest 


,igator 


■ (s) 


8. 


, W. T. 


McQuilkin 


(part 


time) 



Other Investigators 

9» Project Descriptionj 

A three optical system time lapse cinematograph has been developed and con- 
structed in the NIH Instrument Shops. This is to be used in comparative 
study of normal and malignant cultures, and comparison of cultures under 
different experimental conditions. Particular attention will be paid to 
factors which influence population changes in cultiires, 

, One person was given preliminary training in use of this instrument. Final 
adjustments were begun on the instrument after the long delay reported last 
year. Several experimental film strips were run on behavior of various cell 
types. Person being trained recently resigned to take outside position at 
a much higher salaiy. No replacement has been authorized, but Ifr, McQuilkin 
(Biologist GS-7) is much interested in the equipment and is carrying out 
further part time work on it. This equipment needs full time work on it to 
make it of practical usefulness however, 

S ignificajice to Cancer Research } This camera is a powerful instrument in 
¥tucfy and comparison of various normal and cancer cell types ■under controlled 
experimental conditions. 

Proposed course of project . As technical assistance is available the studies 
projected arc as follows: l) Comparison of cell strains with various nutri- 
ents missing from medium, 2) Transition of epithelial cells to more spindle 
shaped forms, allowing possible clearer understanding of transition of car- 
cinomas to "sarcoma" and transition of epithelial cells to "fibroblasts" • 
like forms, 3) Influence of cell population density on manner of migration 
and the morphology of epithelial cells, ii) Action of specific reagents on 
cells , 



U 32 (a ) 



10. Ii32__ 

N:: 



'^- ^ Page 2 

11. 

Budget Activity: 

. Research x Administration 

Review and approval Technical Assistfjice 

,12, _ None 

---"-• C cooperating Units of the Public He a It'h Service, cr other organizations, pro- 
•*' ■ viding funds, facilities, or personnel for this project in either 1956 or 
1957 



13. N one ' " ' • 

If this project resembles, complements, or parallels research done elsewhere 
in the Public Health Service (without interchange of personnel, facilities 
or funds), identify such researchs , . 

11;. Ii32 (a) 

Serial No. 

l5. No ne, '- ^ ' I ' 

Publications' other than abstracts frcji this project during calendar year 1955 



l6. None 



Honors, and 'Awards to Personnel relating tc this project during calendar year 

1955, ■•-'•'?^' •;, 



FkyE 1 
PROJECT REFCRT FCRk 



1. national Cancel" Institute ?:, Laboratory of 3:.olocr/ 

inSTITUTE L'lBCR/.TCRY OR 3R.'".rc:-! 

3. Leulrernia Studies Section 4, . 5. 433 



SERIAL 



6. Studies on t!;s eticlocp/ of nio;.'.se leu!:emias end neoolasns of the ogrotir. 

FRCJECT TITLE and adrenal gisnds. 

7. Sargh E. Ste:^fsrt 

FRII !CI?AL If] VESTIGATGR (3 ) 

6, [lone 

GTrlER If!VE3TI3ATCRS 

9. PROJECT DESCRIFTICn 

Froject: Studies on the etiology/ of iriouse leulienias and neoplasms of the 
parotid and adrenal glr.nds. 

Obiectives ; To find good sources for the parotid gland tumor agent and 
the leukernis agent in order to recover them and determine if they are' 
identical. 

I^iethods emjlcyed ; 

I. rievjborn mice were inoculated v/ith cell-free extracts prepared 
from iei'henuc mouSe tissues, from parotid gland tumors, and 
from other tumors arising in mice having received e;:tracts 
of leukemic mouse tisst^es. 

For controls, similar nice r/ere inocLilsted viith the xollov;ing: 

a) Extracts prepared from tisst'.es of normcl strain C3M mice. 

b) Extracts from C3H mammary tismors carrying the mil!': agent, 

c) Extracts from human lymphomas, 

d) An active mumps virus, 

e) Hethylchoianthrene applied to the s!:in of newborn mice. 

II. 3y using a method prcvionsiy described (Stewart, Proceedings of 
American Assoc, Cancer Research, April 1955) , attempt to recover 
a virus from transplanted ien!:emias, parotid gland tumors and 

adrenal gland t-amors and test these for carcinogenesis, 

III, Study the histopathclcgy observed in the organs of mice (irradi- 
ated" and ncn-irradiated) carrying transplanted mouse tumors, 
and of mice inocs^icted with- the virus recovered in II to note 
if there are similarities. 



,'!5C r total body radiation. 

(Project description continued) 



SERIAL r!0« 433 PAGE 2 

Ks.jor lindings : 

I. Two good sources for the parotid glsnd tumor ogent have been found; 
one is AKK leukemia »60, extracts of which, on repe?.ted tests over 
s ?-yeaif period, have yielded an incidence of 50 to 80 percent 
parotid glsnd tumors, some of the mice have had both leukemia snd 
parotid tumors "nd others only ieu'^emia. The other, CSH leukemia 
*19, has yielded Bbout a 30 percent incidence of parotid gland 
tumors and adrenal tumors over a period of 3 years. 

Other sources for the parotid gland tumor agent have been an AKR 
leulcemia #RIL6, CSI-I leul:emia #11?'4, a paraganc'liomn , snd. a mamriiary 
tumor. The last two arose in (C3Hf :: AKR) hybrid mice as a result 
of inoculating with AFCR Icukemir: extracts. 

rione of the mice inoculated with extracts prepared from carotid 
gland or adrenal gland neoplasms from normal tissues, from human 
lymphomas, or from mill: agent msniraary tumors, have developed 
parotid gland tumors or lcu!:emias, 

iviice receiving mumps virus, ' nd those receiving methylcholanthrene, 
have remained free of leuicemis and parotid gland tumors, 

II. A virus has been recovered from the following leul-emias; AKR lou- 
kemias »60 and ffRIL6, and frca G3H leu'cemias »11!^;4 and #19. These 
4 leukemia s have repeatedly been gocd sources fcr the p:"rotid gland 
tumor agent-. It has not been possible to recover a virus from the 

.parotid gland tumors, or from the adrenal tumors. Extracts prepared 
from these tumors have not yielded parotid gland tumors. ' ' ' 

This virus is highly lethal for nev/b. i v.iicc-, especially for strain 
C3H. A few (C3I-!f x AKR)Fi I'.ybrids have survived the inoculations, 
and 4 out of 15 which received virus after serial passage developed 
parotid gland tumors in from 5 to 12 months. 

III. Five C3H parotid gland tumors transplanted into irradi"ted mice 
grew in from 50 to EC percent of the first passage transplants 
and retained their original histology even after repeated trans- 
planting. Cne tumor has been carried for 3 years r'P.i 2 for 2 
, years, i'.iice carrying these tumors lived for from 3 to 6 months and 
•developed marked hepatomegaly, cardiomegaly, hemorrhagic adrenal 
glands, -enlarged spleens, . and edematous peripheral nodes. The 
microscopic findings were as fcllov/s: The sinusoids cf the adrenal 
cortex and cf the liver were so greatly dilated with blood that in 
many_ there was almost complete atrophy of the parenchymal cells. I;iarke' 
myeloid metaplasia and erythropoiesis were observed in the spleen 
c.n;d liver. Congestion of the kidney gioneruli and other organs was 
,. alsa observed. From the histologic findings a diagnosis of hypervolemi; 
has, been made (Dr. Tlxima 2vSin) » Atrophy cf lymphoid tissue w"s 
generally found, i.xtastasis of the parotid gland tumor to the lungs 
has been a common findin-';. 



(Project description continued) 



SERIAL MC. 433 PAGE 3 , . 

These same tumors, vihen i.:ioculated into no::-irrsdiated mice, 
failed to grow in a high i^ercent of transplants, aud vj'ien they 
did grow they were rapidly transforraed to ssrcornos, liice 
carrying the sarcomas did not develop hypcrvoleirJ.a, This vras 
also true in those instances v;here the tumor crowth was slow and 
the mice remained alive 4 to 5 months (tumors prinoipslly sarco- 
matous). The myeloid metaplasia end lymphoid atrophy observed 
in the irradiated mice was also found here. 

All mi".e v;ith the transplanted tumors, whether sarcomas or 
parotid gland tumors, developed a ::Tanulo-:ytosis; the white 
blood counts were frequently over lC0,0C0/ci5bic mm. Also, 
all developed a severe anemia, the hemoglobin frequently droooing 
to 3 or 4 grams/lCO cubic ml. 

The histologic changes observed in mice that developed an acute 
infection, after inoci'lrtion vjith t!!e filterable agent (Pro- 
ceedings Am. Assoc, Cs::cer [{esearch, April 1955), were similar 
to those observed in mice with the parotid gland tumor in that 
myeloid metaplasia, lyr:phcid atrophy, and hyperemi?; of the 
different organs ivere also noted. Severe anemia with a granu- 
locytosis u'os also present, 

Significance to cancer rcsaarch: 

It is felt that this study has contributed to the study on the etiology 
of the parotid gland tumor end leuheniias. 

Proposed course of oroject ; 

To be continued with more emphasis on demonstrating lec'cemo genie 
activity of the agent. 



SERIAL no. 433 FAGE 2 

fa.ior lindincis ; 

I. Two good sources for the parotid glcnd tumor agent luwe been found; 
one is AKR Inukemifi w&G, extrccts of which, or. repe'ated tests over 
a 2-year period, l:>cive yielded an incidence of 50 to 80 percent 
parotid glnnd tumors, some of the mice hcve hod both leuliemiei snd 
pnrotid tumors nnd others only leukemia. The other, CSH leukemia 
#19, has yielded about a 30 percent incidence of parotid gland 
tumors ond odrcncl tumors over :i period of 3 years. 

Other sources for the parotid gland tuiViOr ogcnt have been an AKR 
leukemic W:RIL6, C3H leu!:c!Tii? #11?.4, a paragnnqliomn , and a mammary 
tumor. The last two arose in (C3Hf y. AKR) hybrid mice os a result 
"of inoculating with AKR Iculcemia extracts. 

None of tiie mice inoculated with extracts prepared frcra parotid 
nland or adrenal r;lar.d neoplrsms frotn normal tissues, from human 
lymphomas, or from mil!: agent laammai-y tumors, have developed 
parotid gland tumors or lcu!:cmias, 

iviice receiving mumps virus, '.nd those receivinc mothylcholanthreno, 
have remained free of leukemia and parotid gland tumoi's, 

II, A virus has been recovered from the following leulcemias; Alui Icu- 
kemias #60 and »RIL6, and frcn C3H Icukemias »11S4, and ^l*?. These 
4 leukemia s have repeatedly been good sources for the parotid gland 
tumor agent'. It has not been possible to recover a virus irom the 
.parotid gland tumors, or from the adrenal tur.iors. Extracts prepared 

from these tumors liavc not yielded parotid gland tumors. 

This virus is highly iothrl for nv,'.;b: i i.iicc, especially for strain 
C3H. A few (C3Hf x AKR)?"; liy.rrids have survived the inoculations, 
and 4 out of 15 which received virus after serial passage developed 
parotid gland tumors in from 5 to 12 months. 

Ill, Five C3H parotid gland tiiniors transplanted into irradiated mice 
grew in from 50 to CC percent of the first passage transplants 
and retained their original histology even after repeated trans- 
planting. Cne tumor has been carried for 3 years z\it 2 for 2 
. years. IVdce qarrying these tumars lived for, from 3 to 6 months and 
'developed marked hepatomegaly, cardiomegaly, hemorrhagic adrenal 
glands, enlarged spleens,, and edomatous peripheral nodes. The 
microscopic findings were as fcllov;s: The sinusoids of the a'drenal 
cortex and of the liver were so greatly dilated with blood that in 
many_ there was almost complete atrophy of the parenchymal cells, L.arke- 
myeloid metaplasia and erythropoiesis were observed iu the spleen 
Ein.d liver. Congestion of the kidney gloneruli bvs\ other organs was 
a 1 SO; observed. From the histologic findings a diagnosis of hypervolemia 
has. boon made (Dr. T!:clma Dunn). Atrophy of lyr.phoid tissue w"s 
generally found, littastasis of the parotid gland tur.or to the lungs 
has been a common finding, 

(Project description continued) 



SERIAL NC. 433 PAGE 3 



These same tumors, when inoculated into no::-lrrc:!iated mice, 
failed to cjrovv in a high ;^';Grcent of transplants, and ivhen they 
did grow they were rapidly tremsiormed to saroowos, liice 
carrying the s"rcomas die not develop hypervolemia. This vjas 
also true in those instances where the tumor growth vjas slow and 
the mice remained alive 4 to 5 months (tumors principally sarco- 
matous). The myeloid mctaplosia and lymphoid atrophy observed 
in the irradiated mice was also found here. 

All r;:i",e v;ith tl;e transplanted tumors, whether srrcomac or 
parotid gland tumors, developed a ^'ranulo^ytosis; the white 
blcod counts were frequently over lGO,?CO/cuhic mm. Also, 
all developed a severe anemia, the hemoglobin frequently dropping 
to 3 or 4 grams/lCO cubic ml. 

The histologic chan{;e3 observed in mice that developed an acute 
infection, after inoculation vjith tlic filterable agent (Pro- 
ceedings Am. Assoc, Ca:-.cer ['{esearch, April 1955), were similar 
to those observed in mice v/ith the parotid gland tumor in that 
m.yeloid metaplasia, lyr.ph.oid atrophy, and hyperemia of the 
different or;;;ans wore also :";Oted, Severe anemia with a ;]ranu- 
locytosis u'os also present, 

Significance to caiiocr rcsoarch; 

It is felt that this study has contributed to the study on the etiology 
of the parotid gland tumor and leuhomias. 

Proposed course of 'roiect ; 

To be continued witli more emphasis en demonstrstinr; lci'''.emo3enic 
activity of the agent. 



PAGE 4 

10. 433 

SERIAL, NO. 



11. 

3UBGET ACTIVITY : 



RESEAPvCn /x~7 ADL'IIIIISTR/vTIOn fl 

REVIEsV & APFRCVAL [J_ TECI-INICAL ASSISTAflCE /~7 



12, None, 



COOPER/iTiriG UTIITS OF THE PU3LIC HEALTH SERVICE, OR OTHER CRGANIZ/^TIOIiS, 
PROVIDING FUrSS, FACILITIES, OR FERSCMNEL FOR THIS PROJECT IN EITMER 

1956 or 1957. 



13, None, 



IF THIS PROJECT RESEMa.E3, COI^'iPLEtiEMTSi CR PARi'XLELS P^SEARCH DOI'E ELSE- 
WHERE IN THE PUH.IC HE/i,TH SERVICE (/JITMOU? INTERCH/'iNGE OF PE3SCNFIEL, 
FACILITIES OR FUNDS), IDENTIFY SUCI^I RESEZ-.RCK: 



PAGE 5 
14. 433 

SERI/iL r!C. 



15. 

PUa^ICATIOIIS OTHER 7F.LV. k3STR{XT3 FRGL, THIS PROJECT DURIHG CALEriDAS YEAR 

1955 



Ste?;art, Ssrah E. : "[leoplssr.i.s in mice inoculated ^ith cell-iree 
extracts or filtrates of leu!:emic mouse tissues, I. r.'eopiesrns of the 
carotid and adrenol glands," J. Net. Cancer Iiist, _15: 1391-1415, 
April 1955. 

3tew"rt, Sarah E. ; "Heoplssms in mice inoculated with cell-free extracts 
or filtrrtos of leu'cernic mouse tissues. II. Leircernia in hybrid mice 
produced b'j cell-free filtrates," J. Hnt, Cancer Inst, j^: '1.1-53, 
August 1955. 



16. 



HCriCRS AMD A:a/ARDS to FERSCIIIiEL iJELATIHG TO THIS PROJECT DURIFIG CALEHDAR 
YE/iR 1955. 



General Project 1^3l4 

Serial No. 



Metabolic studies on tissue cells in vitro, 



Pr.ge 1, 
PROJECT REPORT FORM 
1. Naticnal Cancer 2, Biology 



Institute Laboratory or Branch 

3, Tissue Culture h* Bathes da, Md. 5« 1|3L (a) 

Section or Service "Location Serial No* 

6, Metabolic Studies on Tissue Cells in vitro 
Project Title 

7, B, B. Westfall ; ^ 

Principal Investigator (s) 

8, E . V. Pep pe rs, V,J« Evajis, K.K. Sanford, N. M. Hawkins, J.C, Bryant, 
E, L. Schilling, M,C, Fioramonti, G, L. Ho bb s, and W.R. Earle, 
Other Investigators . 



9. Pro ject descr iption ; Alpha-keto acids in tissue cultures, 

Obj ectj.v ,-;; No information was ?vr.ilable on the behai/ior of alpha-keto 
acids ill "the medium during growth of the cells in tissue culture, and 
since these are important substances in the metabolic cycle it was 
deemed desirable to study certain key ones both to ascertajin any changes 
that might be taking place d\3ring groirth of the cells and to see if they 
might substitute for the comparable a-minr acids in culture as certain 
ones have been found to dc in mammalian growth. 

Method s; Existing methods for the estimation of the alpha-keto acids 
were modified end adapted so as to give good separation of the alpha- 
ketoglutaric acid, oxal-acetic acid and the two geometric isomers of 
pyruvic acid din itropehnylhydraz ones # The methods wore then applied 
to the ultrafiltrates of the horse serum and chick embryo extract and 
tc the protein -free supernatant s from the media after growth of the 
cells in the media. 

Major findings ^ The medium supplied to the cells was f oimd to be low ' 
in keto acids, but in those media showing good growth of the cells 
the cells were quickly able to restore the level to a more ne?-rly plasma* 
like concentration. 

Si gnificance s This study was partly an orientation work aimed at getting 
methods well in hand and determining concentrations of the keto acids 
supplied to the cells. Further work is planned with different strains 
and various media to see what effect these combinations have. This is 
part of the program concerned with the general metabolism of the cell, 
It is therefore proposed to extend the study as indicated. 



10. Lt3U (a) 
Serial No« 



Page 2, 



11. 



Budget Activity; 

Research x 
Review & Approval 



Administration 
Technical Assistance 



12 » __ None 

Cooperating units of the Public Health Service, cr other organizations, 
providing funds^ facilities, or personnel for this project in either ' 
19^6 or 19^7. • ' ' 



13 • No resemblance or complementation, ^ ■ ^ 

If this project resembles, complements , or parallels research done 
elsewhere dn the Public Health Service (without interchange of person- 
nel, facilities or funds), identify such research: 



^» U3i; (a) 

Serial No, 



15. . 

Publications other than abstracts from this project during calendar year 
1955, 

"a-Keto acids in tissue culture,, I, The concentration of pyruvic and 
a-ketoglutaric acids in the ultrafilt rates from horse serum and chick 
embryo extract. B.B. Wostfall, E.V. .Poppers, and JAf.R. Earle. J ^ National 
Cancer Inst, l6;l: August 1955* 

l6. None 

Honors and awards to personnel relc?ting to this project .dui'ing calendar 



year 1955. 



Page 1 

PROJECT REPORT FORM 

1, Natirnal Cancer 2, Biclogy 

Institute Labcratrry cr Branch 

3, Tissue Culture k» Bethcsda, Md. 5. k3h (b) 

Section cr Service Location Serial Nc» 

6, Metab olic st udy en tissue cells in vitro, '_ 

7. B. B. Westfall _^ 

"Principal Investigator (s) 

8 , E,Vv Poppers . K.K. Srnfcrd, V. J. Evans, N, M. Hawki ns^ and W.R.Earle. 
Othor investigators 

9. Project description: Fraction of horse serum proteins for use in 
cultivation of cells in vito. 

Obje ctiv e: It had been shown horse serum freed of its readily diffusi- 
ble substpjiccs by ultrafiltration when properly supplemented gave ex- 
cellent growth of the tissue cells in culture. As a first attempt to 
see if the growth was perhaps associated with some one of the more or 
less well recognized discrete fractions, separation by means of a 
modified Cohn low-temperature technique was carried out and the fractions 
tested for growth by Dr* K. K, Sanford, If one of these contained all 
the growth stimulating factors it would have been a distinct forward 
toward getting a well-characterized medium for growth studies. 



Methc^d s i' As noted above, • 

" Major findings ; Growth was reasonably good with all fractions, but 
better, with some than ethers; the gross globulin fraction was that, 
one giving as good proliferation as the original serum residue. The 
study indicated that growth was not necessarily associated with any 
particular moiety, or if such should be the case that it was effective. 
If time and suitable facilities are available further work on frac- 
tionation is contemplated. It is hoped that outside sources may be 
found that might undertake such fractionation. 



10' U3U (b) 

Serial No, 



Page 2. 



11 . ■ 

Bndgot activity: 



Research x Administraticn 
Review & Approval Technical AssistsnCG 



12. None 

Cccperating units cf the Public Health Service, cr ether crganizatirns, 
providing funds, facilities, cr personnel for this project in either- 
19^6 or 1957. 



13. 



If thl:^ project resembles, complements, or parallels research dene, 
elsci-huxo in the Public Health Service (without interchange of personnel, 
facili-r"e3 or funds), identify such research i 

It has been ruinored that Dr. H. Eagle's group and Dr. H. Stoinman 
have been working along similar lines, but we have no direct infor- 
mation , 

Serj.aj i<'c^ 



15. 



Publications other than abstracts from this project during calendar 
year 1955. 

Effect of serum fractions on the growth of strain L cells, K.K.Sanford, 
B.B.Westfall, M.C, Fiorr^nonti, W.T, HcQuillcin, J.C.Bryant, E.V. Peppers, 
V,J» Evajis and W.R.Earlc. J. National Cancer Inst. l6:3s Dec. 1955. 

l6 . None . • - 

Honors and awards to personnel relating to this projecst' diiring calen- 
dar year 1955 



Page 1 
PROJECT REPORT FORM 



!♦ National Cancer 2, Biology 



Institute Laboratory of Branch 

3» Tissue Culture ! ;. Bethesda, Md. $ , ii3li (c) 

Section or Service Location Serial No, 

6, Metabolic studies on tissue cells in vitro. 



Px-oject Title. ■ 

7, B.B. Westfall 

Principal Investigator(s) 

8, E.V, Peppers, V.J. Evans, K, K. Sanford, M, M, Hax-zkins, J. C. Bryant, 

■ E« L. Schilling, M. C, Fioramonti, G.L« Hobbs, and W,R. Earle, 

Other investigators, 

9, Project description: Chemical studies on the changes in the nutrient 

■ mediwii when cells are grown in large quantity and volume of the shaker 
flask. 

Objective } No information was available as to change in nutrient in 
the medium during grcvrth of cells on the large shaker apparatus. Also 
since growth was very good end it was known that the quantity of certain 
of the amino acids were supplied in low a-moimt it x-jas deemed desirable 
to see if any of these were limiting in concentration since Fischer had " 
demonstrated in 19^3 that the free amino acids lysine, histidine, arginine, 
valine, leucine, is oleucine, threonine, phenylalanine, tryptophane, methio- 
nine, cystine and the amine glutamino were needed for good growth of cells. 

Major Findings ; In one study with the HeLa. strain of cells it was found 
that ty using the medium made up of human placental fluid and whole egg ■' 
extract ultrafiltrate with their attendant low concentration of rjnino 
acids, certain of the amino acids appeared to be exhausted from the medium 
in a 3-day interval with a heavy inoculum and good growth. 

Significance { This study was primarily part of a program tct the study 
of cellular behavior under different conditions, ca-ncer being thought 
of as mainly a phenomenon in the growth of cells. The strain of cells 
under study were human malignant cells of epithelial origin. 



Page 2. 



Methods i The cells were grcwn en the shaker flasks with ccntinucus 
gas exchange and ocntinucus shaking. The mediijin was examined for free 
amine acids by paper chrcmatography; for alpha-keto acids by extrac- 
tieri, and paper chrcmatography, f cr. lactic acid and glttccse by adapta- 
tion of existing spfectrcphotometric .methods. 

It is proposed to continue and extend similar studies to other cells and 
to various conditions of maintenance. 

10, _i:t3U (a) 

Serial No", 



11. 



1^. 



Budget activity: 



Research x 'Administration 

Review & Approval Technical Assistance 



12, None 



Cooperating Units of the 1-ublic Health Service, cr other crganizati'^ns, 
providing funds, facilities, or personnel for this project in either 
1956 or 1957. 



13, . No rescmblarice or complementation known. ■ 

If this project resembles, complements, or parallels research done else- 
where in the Public Health Service (without interchange of personnel, 
facilities cr funds), identify such researchj 

i^« lilh fc) ■- 

Serial No, 



Publicr.ticils other than abstracts from this project during calendar 
year 1955. 



"The change in concentration of certain constituents of the medium during 
growth of the strain HeLa Cells." B. B. Westfall_. S.V.Peppers and W.R, 
Earle.r fhe American J. of Pfygione, l6{3{ April; 1955. 

l6. None 

Honors and awards to personnel relating to this project during calendar 
year 1955* 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. laboratory of Pathology , 

INSTITUTE LABORATORY OR BRANCH 

S'oo 

3. Cancer Pathology Section 4. 5. NCI §^» 

SECTION OR SERVICE LOCATION(IF OTHER THAN BETHESDA) SERIAL NO. 

6. Histogenesis and pathology of induced and spontaneous tumors of laboratory 

anim als . 

PROJECT TITLE 



7. Harold L. Stewart 



PRIHCIPAL IN v-ES'.ClGATOR(S ) 
8. Katharine C. Snell 



OTHKR INVESTIGATORS 



PROJECT DESC^IIPTION .: , - 

Object: To gain knowledge of . the etiologic and pathogenic factors 

involved in the production of neoplasms in experimental animals, 
and to make information obtained available to other investigators. 

Methods: 1. A fascicle on transplantable and transmissible tumors of 

animals has been prepared for publication in the Atlas of Tumor 
Pathology under the auspices of the Subcommittee on Oncology of the Committee 
on Pathology of the National Research Council. This fascicle Includes the 
history, transplantation behavior, detailed pathological description, and 
microphotographs of 50 of the most widely used animal tumors. (Dr. Lucia 
Dunham has collaborated on this work.) 

2. A study is being made of the pathology of spontaneous and Induced tumors 
of the liver of animals. This study will form the basis of papers to 

be presented at the Symposium, on Liver Cancer at Kampala, Uganda, and 
Leopoldville, Belgian. Congo^ in August 1956 under the auspices of the Unio 
Internationalis Contra Cancrum. It will also be published as a chapter in 
the revised edition of The. Physiopathology of Cancer (F. Homburger and 
W. H. Fishman, editors). 

3. A series of untreated rats of seven strains, males and females, breeders 
and non-breeders, is being necropsied at various age intervals and 

subjected to detailed pathological examination in order to enumerate and 
classify spontaneous tumors, to study tissue changes with increasing age, 
and to furnish controls for present and future experiments. 



Coo 

SERIAL NO. NCI 509* PAGE 11 

PROJECT REPORT PORM (Cont'd) 

9. PROJECT DESCRIPTION (Cont'd) 

4. An attempt Is being made to produce adenomatosis of the lung . by feeding 
l,2;5^6-dib'3nzanth3:acene to DBA mice. 

5. Monkeys that received injections of carcinogenic svbstances into the 

, wall of the stomach several years previously are being necropsied and 
studied to ascertain whether these substances may have produced gastric 
neop'.a^ms, ■■•■ '- -■ ■ • ■ , 

6. A study is being made of the tuiftOrs occurring ir. the !1H0 strain of 
irice. Th«^;se mice are of interest because of the fact ihat their 

ancestors received a single doee of methylcholanthrene in an effort to 
proiucc: an, hereditary gastric carcinoma. 

Major ♦:Jn-iirigr,: 1. Fascicle on Transplantable and Transmissible Tumors 

of Auiinals. The manuscripts on 50 tumors, together with 300 micro- 
pbotogrpph*i, havrt been completed and these have baen approved for publicatiort 
by the M.I.H. Editorial Board. 

2. The study of liver tumors is in progress and microphotographs and 
lantern slides are being prepared, i 

3. A total of 404 untreated rats have been necropsied, the series of ■ 
necropsies is complete on the Marshall 520, the AXC, and the Osborne- 

Mendel strains. Microscopic evaluation of the material is in progress. 
Unusual tumors noted have been carcinomas of the adrenal cortex^ a 
carcinoearcotaa of the jejunum, and tumors of the lung and testis. 

4. Necropsies and evaluation of sections of the DBA mice fed 1,2,5,6- 
dibenzanthracene are being made, but it is too early in the experiment 

to report any significant findings. ^ ..„.». 

. .,;■ . V ■■' , •; " ■ ' '■■■"■ " '■ •■ ' ■ •■•orfuci'.Oiir: 

5. No monkeys necropsied to date have shown gastric carcinoma nor any ^' -'.v' 
lesions that can be attributed to the injection of carcinogenic 

substances into the wall of the stomach. '<< •<• > "- ./ ! . 

. 'Gin.brr. :iw Tnvii ^irfrj J 

6. Various tumors of the ^HO strain of mice have been obseifWiff, amJ' 
these are being studied histologically and evaluated. There have been 

no carcinomas of the glandular stomach, but tumors of the nervous system^ ' 
lung, uterus, and reticulo-endothelial system have been found. ■ 'iv .. ■,. . 

Significance to Cancer Research: The knowledge gained from a study of 

neoplasms in laboratory animals should provide a basis for 
further work on the mechanism of carcinogenesis in the human being. 



erial No. NCI -&ef PROJECT REPORT FORM (Cont'd) PAGE III 

. PROJECT DESCRIPTION (Cont'd) 

Proposed Course of Project: It Is expected that the fascicle on Transplant- 
able and Transmissible Tumors of Animals will be published in 1956. 

The study of the pathology of liver tumors will be completed and ready for 
publication by the summer of 1956, 

Experiments dealing with carcinogenesis (enumerated as parts 3-6) will be 
continued. 



PAGE IV 



PROJECT REPORT FORM (Cont'd) 



10, NCI ^e^- 



SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH ADMINISTRATION /T 
REVIEW & APPROVAL fj TECHNICAL ASSISTANCE £1 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



13 » None to my knowledge 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE V 



PROJECT REPORT FORM (Cont'd) 



L4. NCI '%m- 
SERIAL NO. 



L 5. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 

Stewart^ H, L. and Hare, W. V. Chronic gastritis of the glandular stomach, 
adenomatous polyps of the duodenum and calcareous pericarditis in strain 
D.6.A. mice. J. Nat. Cancer Inst. In press. 



L6. None 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955. 



PAGE I 



PROJECT REPORT FORM 

1. National Cancer Institute 2. Laboratory of Pathology '• - 
INSTITUTE LABORATORY OR BRANCH 

3. Cancer Pathology Section 4. New York, Israel, Washington^D.C. 5. NCI -54^ 

SECTION OR SERVICE LOCATION (IP OTHER THAN BETHESDA) SERIAL NO. 

6, Geographic patholdgy field studie's on uterine cancer in New York City, 

Israel, and Washington, D, C. ' ^ -' ' ' ' • " ' "' ■-■■■■ 

PROJECT TITLE 

ti??; Lacia J. Dunham, Harold F. Porn, Harold L. Stewart ' 

PRINCIPAL INVESTIGATOR(S) - 

8. John H. Edgcomb, Louis B. Thomas 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Field studies of uterine cancer (cervix and corpus) in New York City, Israel, 
and Washington, D. C. ' -rfs^'Hiv- 

Objectives; The objective of this project is to complete an extensive 

questionnaire study of women with uterine cancers in different 
geographic areas and racial groups, and to analyze the data obtained with 
a view to identifying factors suspected of predisposing to or causing 
female genital cancers. Also, basic data on the incidence and pathologic 
(microscopic) diagnoses of the canci?.rs are obtained. 

Methods: For the area under study and the period of study all patients 
possible with the diagnosis of uterine cancer are intervievjed, 
and an equal number of control patients as well. The confidential data 
recorded include social, medical, and surgical history, and menstrual, 
marital, and pregnancy data. Records are kept of the small number of 
patients not interviewed, their diagnoses, and the reasons for failure to 
interview. Pathologic material is studied in all cases. 

A coding statistician and two medical social worker interviewers are 
currently eoployed in the project. 

Major findings: 1. Data from about 2,000 interviews with Jewish women in 
Israel have been reviewed and coded. 

2. Data from about 2,000 interviews with Jewish and 
non- Jewish white women in New York City have been reviewed and coded. 

3. Data from about 400 interviews of non-white women in 
New York City have been reviewed and coded. 

4. Data from about 20 interviews of non-white women in 
Washington, D, C, have been reviewed and coded. This study was started 
late in 1955. 



SERIAL NO. NCI h^ PAGE II 

PROJECT REPORT FORM (Cont'd) 

9. PROJECT DESCRIPTION (Cont'd) •)>«.!' t 

5. Pathologic material has so far been examined in about 
■j,i' ^P;. percfnt_ of . the above cases. ..,-.■ 

' 7''r:A ■> . H.;/n ' 6* The data from apqiitJZ, 500 cases have been transferred 
to punch cards for IBM analysis. 

7, The medical literature on geographic ■ and enytronaje|»t;al, 
factors in uterine cancer has been reviewed, ,j 

Significance to cancer research: The significance of the study lies in the 

hope of supplying a sound scientific basis for the understanding 
of factors of custom, habit, or environment as they may be related to ■ 
relatively high frequencies of uterine cancers in some groups of women as 
contrasted to intermediate or relatively low; frequencies in other groups.. B 

Proposed course: The proposed course is to tabulate, analyze, and appraise 

the material referred to above, and to prepare.reports of these 

studies for publication. At the conclusion of the project it is planned 

. to undertake clinical studj,es. in geographic pathology on patients with,/ 

cancers of the esophagus, stomach, and large bowel. . .■;.-,v? hr,.:; 






■.'5 J X-i ■■ 
- -:f!4 h: 



i .v.-l:.a 



PAGE III 



PROJECT REPORT FORM (Cont'd) 



10. NCI -^J^- 
SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH /^ . ADMINISTRATION ^ £7 
REVIEW & APPROVAL £7 TECHNICAL^ ASSISTANCE £37 



12. Office of Biometry. Office of the Director, National Institutes of Health 
COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



■lAi i-J~> 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE IV 



PROJECT REPORT FORM (Cont'd) 



14.__NCI_5i5u_ 
SERIAL MO. 



15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 

Dunham^ L. J. and Dorn, Harold F. 

Techniques in the Geographic Pathology of Cancer : '5 V'i3aH 

Schw. Zelt. fUr Allg. Pathologie und Bakterlologie, Vol. 18, No. 4, 1955 

Printed in Swtt«erland. • v«iiy.'S,: 



16. None 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955 






PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology 
INSTITUTE LABORATORY OR BRANCH 



nnnr 



3. Cancer Pathology Section 4. 5. NCI -^a^ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Geographic Pathology. Experimental studies relating to the geographic 

pathology of mouth cancer. 

PROJECT TITLE 

7. Lucia J. Dunham 

PRINCIPAL INVESTIGATOR(S) 

8. Marvin S. Burs tone ' ' • ' ■ • '- 

. - OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Experimental studies on mouth cancer. 

Objectives: The objective of this project is to examine the mucous membrane 

of the hamster cheek pouch for proliferative changes that may 
develop when test substances are chronically maintained in contact with these 
surfaces. Substances used are environmental materials suspected of being 
mildly carcinogenic for man. 

Methods: The pellets which have been prepared for insertion in the cheek 

pouch of the hamster include the tobacco^ lime, areca nut, and 
pepper leaf of the far Eastern "betel quid," the plastic material used in 
preparing "false" teeth, and tobacco tars and control materials. A technical 
assistant is retained part-time for help in the study. 

Major findings: Since the project is a long-term one, there are no major 

findings at present. It is hoped to maintain the experi- 
mental animals for at least two years before gross and microscopic study 
of the pouches. The operations used for testing substances has so far been 
performed on 107 hamsters. 

Significance to cancer research: The significance is to reach an improved 

understanding of chronic irritant factors as they may relate to 
cancer of the mouth in man. 

Proposed course: The study will be extended to more animals and to a wider 

variety of suspected substances. Current experimental 
animals will be studied with care, when illness or death intervene. In 
addition, improved techniques of study will be attempted. 

."ilJ/Tra 



PAGE II 



PROJECT REPORT FORM (Cont'd) 
SERIAL NO. ' 



ill.. 



iiaai 



BUDGET ACTIVITY: 

.,.. qi .,->. ,,, j^ggg^jjcH /x7 ADMINISTRATION ff']^^^^:- 

REVIEW & APPROVAL /~7 TECHNICAL ASSISTANCE 7~7 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PROVIDIN 
FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 OR 1957 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS > OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, F/^ILITIES OR 
FUNDS) ^ IDENTIFY SUCH RESEARCH 



ft^ o« 



NO ENTRIES FOR 14, 15, or 16 



PAGE I 



PROJECT REPORT FORM 



1 . National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

3. Office of the Chief 4. 5. NCI 40^ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Histogenesis and histopathology of cutaneous neoplasms. 

PROJECT TITLE 



7. John H. Edgcomb 



PRINCIPAL INVESTIGATOR(S) 
8. None 



OTHER INVESTIGATORS 
9. PROJECT DESCRIPTION 

During the past year the project has included the following activities: 

A. Studies of diseases of man (collaborative research). 

i. Mycosis fungoides 

The reaction of three patients with mycosis fungoides to electron 
beam therapy has been studied pathologically. This type of therapy causes 
remissions of cutaneous tumors in this condition, 

ii. Melanomas 

Naevi from the Skins of patients with malignant melanomas have 
been examined. It appears that more naevi from such patients show junctional 
activity than is the case of naevi from control patients. 

B. Studies of diseases in animals 

i. Further studies are being made on C strain mice bearing Dr. R. 
Bryan's transplantable squamous carcinoma. Many of these mice develop 
severe leukocytosis. Attempts to transform this disease into granulocytic 
leukemia have been unsuccessful, 

ii. An attempt to produce melanomas in guinea pigs is being 
continued. As yet (2 years) no melanomas have developed. 



PAGE II 



PROJECT REPORT FORM (Cont ' d ) 



10. NCI •&£?- 
SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH ■ /x7 • AD^ : [j 

REVIEW & APPROVAL £7 TECHNICAL ASSISTANCE £7 



12, None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH 



NO ENTRIES FOR 14, 15, or 16 



PAGE i 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Patholoav . . 
INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. &. Pathogenesis 4. 5. NCI -^^ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAt NO. 

6. Accumulation and dissemination of information relating to the normal and 
pathologic anatomy of laboratory animals, particularly mice, with special 

emphasis on neoplasms. fj j.- :■ •^;i:.r ■....:..,...,. . , 

PROJECT TITLE M/l-wbgat . 



7. Thelma B, Dunn 



iiinjs^ 



PRINCIPLE INVESTIGATOR(S) 



8. None 



OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION 



.*i:„i(i- 



Objectives: To accumulate information relating to the normal and pathologic 

anatomy of small laboratory animals, and to publish it in such 
a form as to be readily available to laboratory workers throughout the world. 

Methods: Individual study of particular phases of pathologic anatomy of ; ., 

laboratory animals; collaboration and consultation with other 
scientists at the National Cancer Institute in projects requiring the review 
of histologic material obtained from experimental animals; review of iqaterial 
sent in for an opinion by scientists from outside laboratories; carrying in 
transplant of unusual neoplasms in mice, so they can be kept for continued 
study; and accumulation of literature and references relating to the subject. 

Major findings: The present project has been carried on through a number of 

years. During the past year a paper has been published 
with Dr. Law describing the morphology of an unusual neoplasm of the parotid 
salivary gland in mice, with a review of previous reports of salivary gland 
tumors in mice. Several transplantable tumors are being carried, which have 
not been described before in the mouse. These are a uterine adenocarcinoma, 
a parathyroid neoplasm, an actively secreting adrenal cortical neoplasm, 
and a mucoid adenocarcinoma of the ovary. 

Significance to cancer research: Since the mouse is used so regularly in 

cancer research, it is necessary that the normal and pathologic 
anatomy of this species be well understood, if reliable conclusions are to 
be drawn from cancer studies. 



SERIAL NO. NCI 568- 



PAGE II 



PROJECT REPORT FORM (Cont'd) 



9. PROJECT DESCRIPTION (Cont'd) !,ii«Gi_i.??J!l' 

Proposed course of project: Work on this project will continue as^it has 
in the past. - Phases of the subject now under special study are. 

a. Systematic review of the :endocrliie: system; of the mouse rv:#, Ross ^ 
MacCardle is collaborating in this by carrying ""^^f "^^««/^l^^^^ ■''°. „ 

the pituitary. A. study of the endocrine organs of different inbred strains 
at different ages has been bfegun. ■ ■ ^mpji' 

b. Continuing collaborative study with (1) Dr. Lloyd I^" «^.'"J^^>^"=^^;;Jj|^ 
various filtrates, and of mice developing leukemxa^ (2) Review^of stmilfx 

material from Dr. Sarah Stewart; (3) Dr. W. E. Heston of ^/'>^.J^^^ ^^^""^^^l . 
strain C3H and C57BL in which a variety of tumors appeared; (4) Dr. H. u. 
Andervont on mammary tumors in mice, and the pathologic changes in wild :^ 
mice and (5) reviev, of small numbers of slides from Dr. Margaret perirjgar,,, 
Dr. Elizabeth Jones, Dr. Michael Potter, Dr. Vincent Price, and Dr. Robert . 
Greenfield, and others. 

c. Continuing review of unusual lesions sent from outside^sputG|s^ , Amot^^ '' 
those recently received were several from Dr. W. U. Gardner of Y^le,; 

Dr. Michael Klein of Florida, Dr. P. Loustalot of Switzerland, and Dr. 
H I. Pilgrim of California. Opportunity to review these uousual lesions 
Is of great value since it gives a chance for comparison with other 
-labotatdrieis;^.^'-. U'-^axo;-! a;lt tyj- i^niin.isrs no'.- .:'mit:>of} o'"' : ■■• • 

.ai Continuing observation of unusual transplantable tumprs. Important ^^ , 
information has often been gained by observing the behavior of transplanted 
tumors, especially where they have a hormonal effect. , • : ;...,.T •.; use;:^:!^-^ 

e; Systematic review of literature relating to enjiocripe,organp inmlp,^,.^,, 

- •haS'^been- started, ....■■■ . .;.. . .:.;.iii j:..^ii.x.!;j,4-ici 

'..k y-i.':- >r ■■ ■ . ' :;■■;. .io sal fs;/> im^ yo-f- iii \-in -^9 

..iaaj;d*f'« .-aill ..'.-fu'lun 'br. : ; ■■ ^.l^«ii'uml'>■o■^■^■ -hrsii -yuti i-.! 

J-; ■..rxjfr^o.o ui i-fgooTifJ j.'-j hyii.-i'x:x- : r.^ ^.lasauii?- :6>rfT .: aa«ibHi>.- totfiS' 

■ '■:.. ;•; ...iioivotq 3-. . .. . : sv- , .... .,;; ffi: -tossls Y'T^vlTr.i? 

,;- 0-- •■•:'■■■' .■ ■'••■■ ■"■;■■■>' '.3 sob ti'jod Jl<-ia 

hivx'idHintv; i'. 

:,:■,■ . • ■ - ". , •■' b'--«iJj'iTI -P- f5i"!0- 

;■ ■ ■ ' :■ ... ■ .,.' ,, ,' ;,.. , ;. .;.jif£.-J,l-liH-'giS. 

' : , ■ . . .ij'Mi-'O • ' ' ■■ ■■■ 

..,■ .;,..J.ji:'!-! .!:■- '^O yW(03Si/Ti; 

■jfcbu3e t: ' ' ■■ nvCitb .xi 



PAGE III 



PROJECT REPORT FORM (Cpnt ' d) 



So 3 

10. NCI -^oe- 

SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH . iW: ADMINISTRATION £7 

REVIEW & APPROVAL /"7 TECHNICAL ASSISTANCE /~7 



12. None 



COOPERATING U'JITS OF THE PDBLIG HEALTH SERVICE^ OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OK PERSOUNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



13. Not known. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE IV 



14. NCI 



PROJECT REPORT FORM (Cont'd) 
03 



SERIAL NO. 



15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 

Dunn, T. B., Morphology and a Cure for Cancer, Journal of the American 
Medical Women's Aseaclation. Vol. 10: 101-109. April 1955. 

Law, L. W,, Dunn, T; B., arid Boy Ie> P. J. Neoplasms in ttid C3H strain and 
in F, hybrid mice of two crosses following introduction of extracts and 
filtrates of leukemic tissues. J.N.C.I., 16: 495-539. 1955. 

Dunn, T, B.,. Mammary Tumors in Laboratory Rodents. A lecture to a class 
in vetefinary pathology in a course oti Diseases of Laboratory Animals. 
Delivered December 5, 1955. This lectuife to be published in a textbpok 
related to the course, 

Andervont, H. B., and Dunn, T. B. Transplantation of hepatomas in mice. 
Published in J.N.C.I., 15: 1513-1524, from Proceedings of the Conference 
on Experimental Hepatomas. 



16. HONibRS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955: ■■• ■ . ' ■■■■-■.■ '^V 

Elected Secretary-Treasurer of the Washington Society of Pathologists, 1955-56, 



PAGE I 



PROJECT REPORT FORM 



I. National Cancer Institute 2. Laboratory of Pathology __j 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue', fy. Pathogenesis 4 . 5. NCI -»ib^ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. An electron microscopic investigation of normal and neoplastic tissues. 

PROJECT TITLE 

7. William G. Banfield ^; i ^ 

PRINCIPAL INVESTIGATOR(S) 

8. None 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives: To describe the ultra structure of selected neoplasms and to 
compare it with suitable normal and control tissues. 

Methods: The usual electron microscopic methods will be used. 

Major findings: The microscope is not yet in operation. 



PAGE II 



PROJECT REPORT FORM (Cont'd) 



10. NCI -5^-?- 
SERIAL NO. 



11. 



BUDGET ACTIVITY: 



RESEARCH /x/ ADMINISTRATION: , X-A 

REVIEW & APPROVAL /"7 TECHNICAL ASSISTAnC^ /T' 



12. None ' ' ': .:'.:,.,.. .... ..-^ . 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS tRO- ' 
VIDIN!?, FUNDS, FACILITIES, OR PERSONNEL, FOR TH,IS PROJECT IN EITHER 1956 or 57 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR 14, 15, or 16 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2, Laboratory of Pathology 
INSTITUTE LABORATORY OR BRANCH 



3. Of flee ;of the Chief 4. . 5. NCI 50 6 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHBSDA) SERIAL NO. 

6. Pulmonary Tumor ;__ . . - ; 

PROJECT TITLE 



7. C. Harold Steffee ,. -. ; ' 

PRINCIPAL IKVSSTIGATOR(S) 

8. None ^ 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION .. ; , . 

objectives: (1) To gain, basic information on biologic aspects of cancer, 
using pulmonary tumors as a tool in this endeavor. (2) To 
gain information regarding the etiology and pathogenesis of human lung 
cancer, and to determine, the carcinogenicity of various substances 
suspected as etiologic agents in human pulmonary cancer. This requires 
finding a method for producing similar tumors in animals. 

Methods: (1) Biologic studies have been done using tumor transplantation 

and genetic techniques. (2) Attempts to induce experimental 
bronchogenic neoplasms in animals haye used embryo transplantation techniques 
and also the intratracheal administration of known carcinogens and suspected 
carc^ihogens in adult mice. : 

Major findings: The study of, the effect of size of the original lung tumor 

and the s.ize of the injected fragment upon transplant- 
ability has yielded -the following information. The data suggest that th^re 
is a critical size of pulmonary tumors; smaller neoplasms transplant poorly. 
When pieces of varying size of large tumors are transplanted the percentage 
of "takes" is essentially the same for all sizes except the very smallest. 
During the course of this experiment; appvoxiraately 20% of the animals 
died over ^weekends and were too autolyzed for definitive autopsy. Because 
of this and also because the results obtained are of border-line statistical 
significance, it was decided to repeat this experiment, checking the 
animals on weekends. Insufficient time has elapsed with the second running 
of this experiment to permit accumulation of definitive data. 



SERIAL NO. NCI. 506 PAGE II 

PROJECT REPORT FORM (Cont'd) 

9. PROJECT DESCRIPTION (Cont'd) 

The hypothesis that malignant ' cells dbisdrved in the sarcomatous trans- - 
formation of the alveologenic adenoma of the mouse are derived from host 
stroma has been tested. It ha^ been found that this hypothesis is incorrect, 
(2) Previous studies with embryo mouse lung transplants, using methylcholan- 
threne as a carcinogen, produced 7 sarc0fflas, 1 squamous-celi carcinoma, . . 
and 2 adenocarcinomas in a total of 25 such transplants. Repetition of 
these studies with refinements in technique by which it is hoped more 
epithelial tumors may be obtained have not been completed as yet, A 
similar study using cigarette tar as th6 "carcinogen" has failed to 
produce any gross tumor in 13 such transplants. 

An experiuent, in which several types of cigarette tars have been adminis- 
tered intratracheally to mice together with suitable control aobstances 
has not yet bean completed. Mice which received intratracheal cigarette 
tars and died during the course of the experiment to date, have shown no 
increase .in pulmonary tumors. 

Collaborative studies on the carcinogenicity of various chromate fractions, 
carried on with Dr. Anne Baetjer of The Johns Hopkins University have not 
yielded gross tumors to date. Microscopic studies have been completed 
on approximately half of the animals so exposed, and these sections show 
.chiefly a non-specific inflammatory reaction and rarely the formation of 
tiny granuloraata. - ., 

...■.■' ■ ■■ ■■<'•■■- . . ;:;/i!SjJOd:t/!<. If;;- Xo'3-*^-"' . .■ ■ ■■ ^■■■- ■'•'■ii 

Significance to "cancer' teseatch;- Study bf the relationship of tumor slJse 

to transplantablllty is a problem in the fundamental nature 
of malignancy. Definition of the exact point at which benignity becomes 
malignancy will sharpen the focus for further biological and biochemical 
r studies. The fact that the sarcoma cells which often arise on serial 
transplantation of mouse lung tumors are not derived from the host serves 
, to eliminate this one possibility in the search for the source of these, :.: 
cells. The importance of this search to the general problem is one of .-.. ■ 
understanding the fundamental cell behavior as well as ap understanding 
of the occasional carcinosarcomas which are observed in the human. The 
Other experiments performed in this project represent a more direct attack 
on the human cancer problem. Bronchiogenic carcinoma is now in many areas 
the commonest malignancy of men, and seems to be increasing in frequency. 
Many laboratories throughout the country are engaged in the search for ; 
etiologic agents but all of this work is seriously hampered by the lacl^ 
of a suitable ..test ohject. Thus, we have placed much emphasis on trying 
to produce an analagous tumor experimentally. Simultaneously, we have ;.;■ 
studied, the effects on experimental animals, of two substances which haveth 
been implicated in human neoplasia^ namely cigarette tars and chromateis. 
Though these are but two pf the many suspected etiologic agents, they iat 
least represent a patt of the attack on the vast problem of human. luhjg;,ci 
cancer. u io 



JERIAL NO. NCI. 506 PAGE III 

PROJECT REPORT FORM (Cont'd) 

). PROJECT DESCRIPTION (Cont'd) 

Proposed course of the project. Since the principal investigator is 

leaving the National Cancer Institute in December of 1955, many 
of these studies of an exploratory nature will be terminated. The animals 
bearing transplanted pieces, of lung tumors in the study of the, effect of 
transplant size and tumor size upon transplantability will be allowed to 
live out thcair life span in order that this data and this experiment can 
be completed. In the study on sarcomatous degeneration of transplanted 
lung tumors, no further transplants will be done even though all of the 
tumors have not yet undergone the degenerative changes. I believe 
sufficient data is already at hand to support the conclusions given above. 

Experiments on embryo transplant techniques and with intratracheal _^_ ,\ 

adinini is t ration of cigarette tars wLH be terminated and the results- ' • • "i 
prepared for publication if this seemis Warranted . The remaining slides 
in the collaborative study with Dr. Baet jer Will be studied but no hew 
experiments are planned in this area since the grant to Dr. Baetjer will 
terminate in 1956. Another collaborative study has been planned with the 
Medical Director/ Randall" B. Haas, Vf, S. Public Health Service Hospital, 
Manhattan Beach/ to inveistigdte othier possible medhariistns of pulmonary 
carcinogenesis. These experiments will include' intratracheal administration 
of carcinogenic hydrocarbons adsorbed on carbon particles, to tuberculous 
and non-tuberculous animals', 'to iietdrmlne the lipoid solvent effect of 
caseous necrosis. These experiments will also include studies on certain 
enzymes which have been reported to cause squamous metaplasia of bronchial 
epithelium in humans. The animal work in these experiments will be done 
at Manhattan Beach; the pathologic material will be examined by the principal 
Investigator in his tiieW'locatiari. .-•.It' is- expected that this experiment will 
get underway shortly after' the fir6t Of the hSxt daleridar, yegir.. We haye 
prepared the carbon particles' for these exp'eirlments, hdve tested their 
toxicity on guinea pigs, and will shortly ship them to Dr. Haas. 



:J yiri^'i 



PAGE IV 



PROJECT REPORT FORM (Cont'd) 



10. NCI 506 
SERIAL NO. 



•f' 



11. 






' , ^:;i:> ■'So ;■;*«:.-■ 



BUDGET ACTIVITY: 'io ■•v-ki.3'S tsffS wi :s';j-.: aJ -aiitj; : .:t'.j -../OiUq • 
«;*.:.': -RESEARCH /^ADMINISTRATION 



A./ 



'; / , REVIEW & APPROVAL £;/ TECHNICAL ASSISTANCE? 7^/' = ^ 

COOPERATING UNITS OF -THE PUBLIC HEALTH SERVICE, OR; OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES/ OR PERSONNEL FOR THIS- PROJECT IN EITHER 1956 or 1957 

1) PHS Gratit C'-603, providing funds to Dr. Anne. M.; Baetjex, Department • 
of Environmental Medlfeine, The Johns Hopkins University, 615 North Wolfe 
' Street, Baltimore, Maryland, f or experlTOents on inhalation- and intrat . 
tracVieal' injection O'f;.chromates,i ', rsrsio o'^BfjiiEjftswuiOdi ' .rfijaaS isi.aS: '. ■■'■■'' 
• ■ '■- ■.■•:;..,., V ;. . ■■;. I w';t>?j;v;'. :;■.■.«.■■■; ■ s*^'-dT ■ :,^{M.n..'noni^:\:■■■ 

2) U. S. Public Health Service Hospitdl, Manhattan Beach, Brooklyn 35>N.Y. 



'>j3*':*fswl»«.r'oaa!--. iiii4 sfj- 



"iip^rifi • »s.iaoitoan' &|j\)::^^i:i 



13« No similar research kndwn^ to be in progress elsewhere. - . 

IF THIS PK)JECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH 



NO ENTRIES FOR 14, 15, or 16 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of PatholoRy 

INSTITUTE LABORATORY OR BRANCH 

3. eancer loduc. & Pathogenesis 4. 5. NCI -^.Qg- 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Mitochondrial changes in the liver of X- Irradiated mice. ■■ . 

. PROJECT TITLE 



7. Ross C. MacCardle and Charles C. Congdoo 



PRINCIPAL INVESTIGATOR(S) 



8. 



OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION: 

Objective: To determine whether cytoplasmic elements are the primary focus 
of initial damage in X radiation injury, and to study the 
anatomical basis of recovery from X radiation injury that 
follows injection of bone marrow into irradiated mice, we 
studied mitochondria and other ;CytQlogical structures by the 

Methods: usual methods at early periods postirradiation. 

Major findings: It was found that mitochondria of the liver certainly are, 
altered long before the animal succumbs, and the mitochondria - 
recover their normal state after bone-marrow treatment. This 
may be a factor in X radiation injury. This has led us to 
study the state of the hepatic and serom phospholipids and 
choline, the mitochondria after x-raying the liver only, and 
after shielding the liver. 

Significance to cancer: Having found a definitive change in mitochondria, 
we now proceed to study the mitochondria of the reticulo- 
endothelial system in spleen, bone-marrow thymus, and lymph 
nodes. We are studying the thymus of C57B1 and LAFj^ mice in 
which X radiation produces lymphocytoraa. This involves a 
cytological study also of thyraectomized mice in which X radiation 
fails to produce tumors. 

Proposed course: We wish to finish the study of the liver and bone-marrow 
after X radiation, and study the other cytological structures 
which we have already preserved in this project. We wish also 
to make a comparative cytological study of the thymus of 
various strains of mouse in respect to their susceptibility to 
formation of tumors at different ages. 



^7 

10. NCI 403- 
SERIAL NO. 



PAGE II 



PROJECT REPORX FORM. (Cont'd) 



IX. 



BITOGET, ACUVJtY.: 



RESEARCH [^1 ADMINISTRATION /_/ 
REVIEW & APPROVAL [J TECHNICAL ASSISTANCE 7j7 



12. None '.-.:V •.■'• :'^ ...■:;. ,:;/■■• . r. , ' ■^:;\; ■..,;:: ■ 

COOPERATING UNITS OlF THE PUBLIC HEALTH SERVICE, OR ,<)THER ORGANIZATIONS, .PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



13. None 



IF THIS' PROJECT :RBSEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH i DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTEI^CHANGE OF. PERSONNEL, FACILITIES . 
OR FUNDS),; IDENTIFY SUCH RESEARCH: r : , . : ' 



PAGE III 



PROJECT REPORT FORM (Cont'd) 



14. NCI »ee- 

SERIAL NO. 



15. 



PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 

The present part of this project in respect to tumors will be done probably 
in conjunction with Dr. Lloyd Law of NCI. The remainder of it is being 
done with Dr. Congdon v;ho is now at Oak Ridge National Laboratory in 
Tennessee. 



16. None 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955 



H'jjr. 



PAGE I 



PROJECT REPORT FORM 



I Mo^^nna ^ rancer Institute 2. Laboratory of Pathology __ . 

• INSTITUTE LABORATORY OR BRANCH 

I ■ ■ 5 NCI "^'0^ 

^- SEmON OR^kvtcE"'" -^"^^ ' location (IF OTHER T HATlEfttESDA) ' SeHa^ 

6. Comparative ^tudy of the macroscopic fortn and cytological structure of 
normal human epidermis and human hair follicles to evaluate changes in 
pathologic states such as in basal-cell tumors, squamous-cell tumors, 

intraepidermal tumors, acne, mycosis fu neoides. and other diseases _^_„ 

PROJECT TITLE - H , ;■ -^^ - > . • ,-• 



7. Ross C. Macr.Ardle with Dr. E. J. Van Scott of NIK 
PRINCIPAL iNVESTIGATOR(S) 



8. 



OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION 



Wooden models are being made of whole hair follicles in health and 
disease that reveal striking differences in size and Pattern. Minerals 
and athercytoiogical constituents :wiil:he studied at different levels of 
the follicle. This study of models has ;|ipt been attempted previously. 



PAGE II 
PROJECT REP.QRT FQRM (Cont'd) 



11. 



;\;BWKJEX ACTIVITY: ., 

RESEARCH /x7 ADMINISTRATION £7 
REVIEW & A^PRdVAL' £7 . TECHNICAL ASSISTANCE £7 



^^ • COOPERATING DNITS OF THE PUBLIC HEALTH SERVI CE, OR OTHER ORGANIZATIONS, FRO- 
SSlwf FUNDS, FACILITIES, or PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



ITAOIXisiiVfeiii iSiiilj" 



"'Tr?r:^;3fi tDUC4>: 



^^'tp^THTS project RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DQNE ELSEWHERE 
iS TOE Pmic HeS^^^^ (WITHOUT iNTERCHANGE OF PERSONNEL, FACILITIES OR 



FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR 14, 15, or 16 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology - 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. & Pathogenesis 4. ' 5, NCI SC t 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Relationship of Np.di oxidase (cytochrome oxidase) granules to mitochondria 

in tiora al and malisnant tissue - 

project' title 



Ross C. MacCardle 



PRINCIPAL INVSSTIGATOR(S) 



None 



OTHER INVESTIGATORS 



PROJECT DESCRIPTION 

During the past two years, it wae discovered by the use of ultracentri- 
fugation that the Nadi oxidase granules and mitochondria resident in the 
living cytoplasm are anatomically independenu of each other in situ in the 
normal intact cell of enithalrvn of the eoidtdymis, kidney and liver of 
mouse. The form of the Nadi o;.iJase granule is- distinctly ..different from 
that of the mitochondrion. It ha& now been found that in hepatoma of the 
mouse they are also anatomically separate. However, in the case of' the 
malignant dependent thyrotropliin-secreting transplant tumor of the pituitary 
gland, the Nadi oxidase granules can be dislodged by ultracentrifugation to 
one pole of the cytoplasm along with the mitochondria, whereas the granules 
of the normal pituitary gland cannot be moved by the same centrifugal force 
required to move the mitochondria -- the mitochondria of a normal gland 
thus being moved to one pole of the cell leaving the Nadi granules unmoved. 
This may not necessarily mean that the mitochondria and Nadi oxidase 
granules of a tumor are anatomically associated, however. It may indicate 
that the viscosity of the malignant protoplasm is less than in normal cells, 
thus permitting the granules to be moved with greater ease, or it may mean 
that the weight of the granules of this tumor differs from that of the 
granules of the normal gland. 



PAGE II 
PROJECT REPORT FORM (Cont'd) 



10. NCI BQir ^ 
SERIAL NO. 



BUDGET ACTIVITY: '■■ ■ ^ - ■ ■.^. •■-,■; r.r"."-^"":Tr^n""' 

RESEARCH /x7 ADMINISTRATION £7 
REVIEW, & APPROVAL £7 TECHNICAL^ ASSISTANCE £7 



12. None 



COOPERATING U>[ITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEl'IENTS , OR PARALLELS RESEARCH DONE: ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCH^^NGE OF PERSONNEL, FACILITIES OR 
FUNDS); IDENTIFY SUCH RESEARCH: 



s^rortf- 



NO ENTRIES FOR 14, 15, or 16 



PAGE 1 



PROJECT REPORT FORM 
1. National Cancer Institute .. 2. Laboratory' of Pathology 

institute; . , . laboratory or branch 

3. Cancer Sd^uc . &. Path oi^eaesis 4. . S. NCI-^W- 

SECTION OR SERVICE " LOCATION (IF OTHER THAN BETHESDA) SERIAL NO 

6. Intracellular cytology of thyrotrophln- secreting tumors of the anterior 
lobe' of the. pituitary gland of radiothyroidectotnlzed mice, and of human 
pituitary tumors » . 

PROJECT TITLE, 

7. Ross C. MacCardle . . ■ , . ,. 

PRINCIPAL INVESTIGATOR(S.) 

8. 

OTHER INVESTIGATORS 



9. PR9JECT DESCRIPTION: 

Object ; The object of this study is to ascertain and define the earliest 

morphological alterations that occur in cells of tissues in their 
change from the normal to the malignant state of protoplasm in respect to 
a possible analysis of the underlying cause of the malignant transformation. 
This tumor of the mouse represents one of the most valuable experimental 
tumors because it resembles pituitary tumors of man. It clearly consists 
of a single^ hlstologically-identifiable type of tissue. One or another 
of the constituent cells of the anterior lobe proliferate in different 
states of hyperplasia and malignancy. The gland is stimulated to become 
hyperactive in producing the thyroid-stimulating hormone for a thyroid 
gland that has been essentially destroyed by treatment with radlolodine. 
Finally, the hyperplastic tissue becomes malignant to produce 10 times 
as much hormone as the normal gland. The amount and kind of hormone 
produced by a tumor can be determined, and the cytologlcal alterations of 
the same tumor can be identified and related to the function. 

Methods ; Cytologlcal methods can detect alterations in cellular minerals 

(microincineration), mitochondria, Golgl apparatus, nucleoprotelns, 
cytochrome oxidase granules. 

Findings ; The major findings so far indicate that the Golgl apparatus 

first becomes hypertrophied and more readily metallophilic as 
oore cytoplasmic iron becomes deposited in the vicinity of it. The 
staining properties of the mitochondria of the tumor are different so that 



SERIAL NO: 



NCI ,&ee- ^ 



PAGE II 



PROJECT REPORT FORM (cont'd) 

9. PROJECT DESCRIPTION (Cont'd) 

they cannot be destainediais easily as/thdse- of the< ttorttial gland. ;Most-of . ; 
the cells of the malignant ,thyrotrc>phin'-secretlng tumor appear to'cirlgihate 
from alpha cells^ thus suggesting that TSH may be formed in these cells. At 
least in the malignant state. The cellular constituents of the hyperplastic 
tissue have not been identified with production pf-TSHi and there, is no. .; 
! evidence that alpha ceiis of the normal gland produce thyrotrophinii : :■ ' 

Proposed Course ; The proposed course of the project is to vtudy the fetal, 

:i t 1' adult -and hyperplastic. glaiiid to compare the relation of .. 
cytologicali changes to prdducti6n> of hormones with those of : primary and . 
transplant malignant tumprs!. It is clear that a study of the. effect of . ; 
TSH on the cells of an already developed TSH-tumor should be made to . s 
determine the cellular organoids first affected when the activity of the 
tumor begins to diminish in the presence of TSH. Comparative cytological 
studies of human and animal pituitary tumors should be accoinplished.; ; It: . i 
is proposed to study the effect of radioiodine on young -and .adult dwarf -> 
homozygous mice that lack pituitary alpha cells. 



HCMa:* 



\Wi^T^'mm^. s^^i'^y.^- , 






?.:t!«;3fcj; 



i.;;^jl[5, 3lfc!' 






PAGE III 



PROJECT REPORT FORM (Cont'd) 



10. Nci-»ee- 

SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH /^ ADMINISTRATION fj 

REVIEW & APPROVAL £7 TECHNICAL ASSISTANCE £7 

12. None 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 OR 195/ 



13. No 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE tV 
PROJECT REPORT FORM (Cont'd) 



14. NCI -^ear 

SERIAL NO. 



15. 



PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 195 

MacCardle, R. C, and C. C, Congdon, 1955: 

"Mitochondrial changes. in hepatic cells of X-irradiated mice." 

Am. J. Path. 31: 725-745 



MacCardle, R. C. 1955: 

"Characteristics of mitosis in tumor cells." 
In press : Annals of N. Y. Academy of Sciences. 



16 . None 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1955. 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. &, PatboReneais 4. 5. NCI 4et- 

LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Writing a brief Monograph entitled: "The Tumor Cell: Characteristics of 
Malignant Prot op ?.p.sm " as part of a series of volumes of An International 
Treatise on Physiology of Protoplasm "Handbuch der Protoplasmaf orschung" 

being edited by Heilbrunn and Weber. 

PROJECT TULE 



7. Ross C. M^cCardle 

PRINCIPAL INVESTIGATOR(S ) 



OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION: 

Object: The object of this monograph is to summarize and correlate our 

knowledge of the properties and possible identification of a 
malignant cell of a tumor. 

Method: By reviewing the literature and relating some new data found by the 
author^ it is clear that although a single white blood cell of the 

Major findings: 

circulating blood of a leukemic mouse can upon injection into a 
normal host produce leukemia (notwithstanding the serum injected 
with the cell), such a cell possesses no anatomical features by 
which it can be distinguished from its normal cell of origin. The 
most malignant tumor constitutes several types of cells, some of 
which may have nothing to do with malignancy and none of which can 
be detected as a malignant cell. The transplantability of the 
malignant tumor cells is discussed, and the cytology of several 
tumors is described for the first time. 

Significance to cancer: This paper will bring together old and new data 
in an effort to evaluate the status of our knowledge of the 
mechanism of the transformation of normal protoplasm to malignant 
protoplasm. 

Proposed course: I propose that it now be printed in book form, and 

it is nearly ready to go to the printers. I was invited to write 
It, and I understand that it was accepted in advance. 



PAGE II 



PROJECT REPORT FORM (pont'd) 



10. NCI-404r / 

SERIAL NO. 

11.- ■■ ;,..■' ■, . ■ 



BUDGET ACTIVITY: 



RESEARCH /x/ ADM7.MISTRATI0N /_/ 
REVIEW. & AFFROVAL £7 . TEGU.NXCAL ASSISTANCE /j' 



12. None 



COOPERATiKG CNITS OF THE PUI^LIC RRALTH SERVICE, OR O'/HER ORGANIZATIONS /PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



13. None 



IF THIS PROJECT RESEME.LES, COMPLEMENTS, OR PARALLELS, RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE Of' PERSONNEL, PACILITiES 
OR FUNDS), IDEHTIFV SUCH RESEARCH: 



NO ENTRIES FOR 14, 15, or 16 



PAGE I 



PROJECT REPORT FORM ■ 

1. National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. & Pathogeoesis 4 . 5. NCI -&e4- ^ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6 . Intracellular cytology of human and animal tumors. 

PROJECT TITLE • . . 



7. Ross C. MacCardle 

PRINCIPAL INVESTIGATOR(S) 

8. ' 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION: 

Object: Little is known of the basic intracellular cytology of the 

cytoplasm of human or animal tumors. Most of the modern studies 
deal with electron microscopy (a method limited to tissue fixed 
in one or two ways) and certifugal isolation of mitochondria 
of a polyglot tumor (hepatoma) of the- mouse (the "mltophondria" 
being of questionable integrity and identify after crushing the 
cell). 

Method: Using light microscopy and ordinary cytological methods, I have 
been studying the minerals, Golgi apparatus, mitochondria and 
mitotic figures of various tumors in an attempt to evaluate some 
of the remarkable features found by electron microscopy in tumors 
that have not been studied by ordinary methods. I have been 
making a comparative cytological study of white blood cells of 
circulating blood in human leukemia in relation to the structure 
of cells of the bone marrow. I have found that the Golgi apparatus 
is in the form of widely dispersed dictyosomes in the argyrophobic 
cytoplasm of the young transplant Sarcoma 37 in mice, whereas it 
is a small compact network in richly argyrophilic cytoplasm of the 
older vascularized transplant tumor. This seems to indicate a 
detectable difference in slowly-growing and rapidly-growing 
malignant tissue. I am also studying basal-cell tumors, squamous- 
cell carcinomas, superficial intraepidermal tumors, Bowen's 
disease, and Xeroderma pigmentosum in the skin of man. 
Proposed study: I expect to continue this study along the same lines. 



Page II 
PROJECT REPORT FORM (Cimt'd) 



10. NCI -see^ / 

SERIAL NO. 



11. 



BUDGET ACTIVITY: ■ ' ' '!■^■ 

RESEARCH /x7 ADMINISTRATION fj 
REVIEW & APPROVAL £7 TECHNICAL ASSISTANCE /[J 



12. 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 195^ or 1955 



13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR 14, 15, or 16. 



FAOE I 



PROJECT REPORT FORM 

1. National Cancer Institute 2. Laboratory of Pathology '- ■ :' " ■ 

INSTITUTE LABORATORY OR BRANCH 

3 . Cancer Pathology Section 4 .- • ■ ;.5 . NCI - S ^ aa - 

SECTION OR SERVICE LOCATION (IP OTHER THAN BETHESDA) SERIAL NO. 

6. Mechanisms of tumor invasivenesa> and the influence of chemical agents upon 

them. __«________«-.^ ' ■ 

PROJECT TITLE ■, : .-■.;:? i ■ >v .i^li*i '?«•;■ »» ss ;i: ii i ^ *; 3 ;,«.,.,.■ 

7. Joseph Leighton . ' . . . .,/; V;, •; j ■■^:- ■■■ ■. \ _______ 

PRINCIPAL INVESTIGATOR(S) . ^«- t-. 

8. Murray Belkin (Laboratory of Chemical Pharmacology), Ira Kline (Laboratory 

of Chemic a l Pharmacology) 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives: (1) The further development of quantitative methods for eva:laiating 

a) the destructive invasion of normal human tissues by human 
cancers in tissue culture, and b) the interaction in vitro of host cells and 
tumor cells. 

(2) The study of the mechanisms of local invasion and of 
metastases in tissue culture with special emphasis on the metabolic require- 
ments of these malignant processes using as experimental tools biochemical 
agents such as cortisone, hyaluronidase, stilbesterol and analogues of 
metabolites. 

(3) The appraisal of the influence of chemical agents on tumor 
invasiveness in which the pharmacologic effect is considered in terms of 

a) altered rate of invasiveness, b) changes in the organization and differen- 
tiation of the tumor, and c) the responses of the host tissues which may 
tend to limit the spread of the tumor. 

(4) The discovery of the conditions which must be supplied for 
the tumor in tissue culture so that progressive growth may be obtained from 
a large percentage and a wide variety of types of clinical cancers, making 
chemotherapeutic studies possible. 

(5) Cultivate tumors from the operating room to observe the 
extent of differentiation in vitro as a supplementary aid to the evaluation 
of sections made of biopsy and autopsy material. 

Methods; Sponge matrix methods for tissue culture are being used in this 
work. 

Major findings: (1) Tissue culture studies on the destructive Invasion of 

normal tissues by cells of human cancer have . been further 
extended. Invasion by several cancer cells has been observed including 
a) strain HeLa (Gey), which arose from an epidermoid carcinoma of the , 



dV<5> 
SERIAL NO. IICI-$«- |>A^ IX 

tioiia uron 9»iii (€«i»t*4> 

9. PROJECT DESCRiniOK <ewH*0 

cervix> b) «tr4to KB |Batl«> «%l«| M^if .fv«a « •ieclnoiiia of t^^ 

c) strain J-96 (08go«4> vkUl «Im« twom k iiibna«jrtlc Uwhcmia. A variety 

of normal huiRantl9s««8 4t« t*«iitfif4 ijnf r«fUce# by chtie tumor cells. 

(2) further ttvilif oi ir«t4>tlom of sponge matrix methods 
have been developed vliicli fCMilk tie ifB«ilit«#BB* •i Ike speed of the 
invasive process. 

p) The spontas«»«fl t««Mlora»«ioit of normal human connective 
tissue cells into a histolof Uallf MUgMftt lwiior>a8 been observed. A 
paper describing this event tf l» fr«M to 2£i£S££> Studies are in progress 
on the biologic malignancy •# tM«ji«li i^e in eultures where it is 
combined with normal human tts«««s awl 6sk Heterologous animal transplantation. 

(4) A study OR one foleiitial chemotherapeutie agent ^ NCI #3022, 
a water soluble podophylletextn Aevivatlve has been completed and a 
manuscript describing our experlnsMs is being prepared. 

(5) Several **iienMl** tell lines originating in other labora« 
tories have been ejcamined in s|««g« «8t«ls« In some instances these "normal*' 
cells: are morphologically iftdlttlaguifhaliie from highly malignant cancer. 

'The; biologic properties of thes* tells in terms of their invasive capacity 
is being studied. 

Significance: The productivity of this ftoject has In large measure; 

developed from the aalMre of its organization. It has been 
one of close coilaboratioA ani iiilegfation of efforts of personnel of two 
laboratory departments. Pathology and Chemltal Pharmacology. 

Methods have been developed vhieh fermit the experimental 9t;udy of the 
invasive powers of human eaneer so that the metabolic requirements of 
invasion as well as potential inhibitors of invasion can be studied. 

Observations have been made in combinations of tumor with normal tissues 
that may contribute directly to our understanding of the invasive process. 
Cells growing out of several normal tissues invade tumor cell masses to an 
extent comparable to the invasion by the tumor of other normal tissues. , 
This raises the possibility that in some instances normal cells in the body 
may enter and divide a tumor noJule giving rise to several new centers of 
tumor growth. A second observation which may be significant is a negative 
one. In Invasion experiments, one almost never sees degenerating forms 
among the normal cells in the areas being invaded by tumor. It is as if 
the normal cells disappear completely, either by migration or very rapid 
dissolution. 

Proposed course: The principal, investigator, has planned to accept a position 
at the University of Pittsburgh in May> 1936, where he will have 
an opportunity to teach as well as to continue his rftsfearch work. The project 
will be pursued there, and the facets of it' that are of direct interest to 
the Laboratory of Chemical Pharmacology will continue to be studied at the 
N.I.H. It is planned and expected that many aspects of the fruitful collabora- 
tion existing up to now between Dr. Belkin| Mr. Kline, and the principal 
Investigator will contisuf , regardless of the distance between cities. 



PAGE III 



PROJECT REPORT FORM (Conf^d) 



10. NCI %i9r- 

SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH ZH/ ADMINISTRATION fj 
REVIEW & APPROVAL /~7 TECHNICAL ASSISTANCE /T 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



1 3 . No similar rea e arch is known to be in progress elsewhere. 

IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH 



PAGE IV 



PROJECT REPORT FORM (Cont'd) 



14. NCI-&g'g— 
SERIAL NO. 



15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING , CALENDAR YEAR 1955 

Major contributor to An Introduction to Cell and Tissue Culture , by the 
staff of The Tissue Culturie Course, Cobjperstown, New Vork> 1949-53, 
Burgess Publia^jlng Company, ,1955,. 

r'N'saw.,.. , • ■■■■■■■■■■■■■'■ \,j: •'-^-.-sr-'- ,/■*;;,■•-;■ :i ;> ^ 



16. HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1955 

Guest Lecturer and Consultant on Tissue Culture to the Department of 
Pathology, New England Deaconess Hospital, July 11 & 12, 1955. 

Lectures at Camp De trick, Maryland; Naval Medical Research Center, Bethesda; 
and Tissue Culture Course at Cooperstown, New York. 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology '• 

INSTITUTE LABORATORY OR BRANCH 

\ .■',■■ vT// 

3. Cancer Indue & Pathos enesis 4. . ; 5 . NGI-4a3- 

SBCTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Effect of neoplastic diseases and of various stresses on the pathophysiology 

and morphology of certain endocrine organs in man and in animals. 

PROJECT TITLE 

7. E. M. Nadel . ' ; 



PRINCIPAL INVESTIGATOR (B) 
8. None (See Item #12) 



OTHER INVESTI.GATORS 

9. PROJECT DESCRIPTION w 

Quantitative -measurement of polar reducing corticosteroids in man and in 
animals by the use of recently developed paper chromatographic, fractionation 
techniques. 

Objectives: To quantitatively measure the .polar corticosteroids present 
in the bloody urine,, and tissues of man and animals, (normal, 
stressed^ and tumor-bearing) and to qorrelate where possible the clinical, 
course with the adrenal and other morphologic alterations and with specific 
corticosteroid excretion patterns* . ; 

Methods: The term stress, is used in its widest concept for the purposes of 

this report and. includes dietary deficiency and excesses as stresses, 
hormonal excesses, and deficiencies as stresses, x-ray irradiation effects, 
stress by physical and microbial agents, carcinogens, and the effect of 
carcinoclastic and potentially carcinostatic drugs. 

Animals: Stressed animals, normal: controls and tumor-bearing animals are 
prepared biologically and the clinical course followed. Tissues, blood, 
and urine are collected during the course of the observation period and at 
post-mortem examination, for chemical, histsOchemical, and histopathological 
study. Extraction) purification and analysis are done by specific applica- 
tions of chromatographic and spectrpphotonietric methods. 

Man: Clinical patients from the Leukemia Service, NCI, Surgical Service, NCI, 
and Psychosomatic Service, NIMH, are being studied in addition to non-patient 
controls. Analyses for polar corticosteroids are performed on the urine, of 
patients before, during, and after the administration of ACTH. 



SERIAL NQ. mi ^2^ PAGE II 

PROJECT REPORT FORM (Cont'd) 

9. PROJECT DESCRIPTION (Cont'd) 

Major findings: The pattern of exexetlon of the new tiolar corticosteroids 

has now been studied extensively in guinea pigs with 
leukemia, with liposarcoma, following stllbesterol treatment, following 
- ACTH administration, following x-radiation,- etc< . (1) There are qu^intitative 
differences in the excretion of 6-^ hydroxycortlsol, Steroid. lla, and: 
Cortisol in neoplastic disease. (2) There is a decrease in 6-p hydroxylation 
in scurvy in guinea pigs. (3) Stilbesterbl treated scorbutic guinea pigs 
respond to ACTH: less than 'do conCrbi guinea pigs.' (4) There are strain 
differences in the response of guinea pigs to ACTH. (5) There are 3 polar 
blue-tetrazolium reducing corticosteroid zones in the urine of man in 
contrast to 3 such zones in the urloe. of guinea pigs as determined by. paper, 
chromatography after ethyl acetate extraction, (6)' Th6 response of clinical 
patients to ACTH is by an increased extraction of both free and of conju- 
gated corticosteroida. The principieknoftn free eprttcoBteroids are 6-P'^' 
hydroxycortlsol and tetrahydro-F and Cortisol (F). '- ' 

Significance to cancer research: The presence of compouridS^iiriore polar> " 

than Cortisol in the urine of man and of guinea pigs, indicates: 
(1) Conclusions based oh th6 excretion of cfarticosteroids without specific 
reference to their identity must be re-evaluated, e.g. the principle^ ; . 
corticosteroid in the albino Hartley guinea pig is Cortisol, whilethe. . . 
principle corticosteroid in Strain 2 and Strain 13 guinea pigs is not 
Cortisol. (2) When hydro lyzed urines are extracted atid theit glycogen r 
potency compared with that of extracted unhydcoLyzed tiritie there is little 
difference in the biologic activity of the 2 extracts; Inasmuch sts the : 
free v,ncohjugated polar urinary corticosteroids are the biologically active 
materials responsible for nearly all of the glyoogenlt activity of hydrolyzed 
or unhydrolyzed urine it now becomes important to study these free corticos- 
teroids in some detail. (3) Up to the present insufficient attention had^: 
been given to these new polar corticosterdid« for 2 reasons: a)i they were not 
khowh' to be present prior to their recent discovery by us in the urine of 
goinea pigs and of man; and b) previous methods of extraction .actually 
destroyed them. 

Proposed course: It was thought prior bo 1950 that Vitamin C def icient - 
; : , (scoxbutlc) guinea plg« had poor adrenal function.. We, 
showed that adrenal activity was actually excessive in scurvy by (a) measuring 
the corticbid in ' the urine, (b)me?asuring It in the blood, (c) demonstrating 
the biological activity of purified' extracts, (<J) isolating and crystallizing 
the major product, (e) identifying it; as hydrocortisone (compoaod ,F) and 
(f ) by histological study. These studies exploded previous speculations 
"i/and served to seifc up hew problems,' •' ' ' '^ ; .:;;;} , ' 

; Despite a. high circulating blood tltre of glucocorticosterolds, the glycogen 
in the liver of Vitamin C deficient guinea pigs is paradoxically low. It 
was found that the guinea pig cannot deposit glycogen in its liver in the 
absence of Vitamin C, even though Cortisol were administered together with 
glucose or fructose. This is being further explored. 



PAGE III 

v^. ISA^ PROJECT REPORT FORM (Cont'd) fj:-- _fSi« ^^f :;. ^/--fU 

9. PROJECT DESCRIPTION (Con,t.,'d)...»s3:) l»®&1 ?S?^a tE^l./.^^ 

Further studies on the guinea pig indicated six, oth^i;: upsuf pej:ted;;Urinary 
corticosteroids were present. All have since been isolated and all but 
one identifiiddv One of, these is, a, previously unknown, compound, 6 BOH Cortisol, 
This new corticosteroid has now been found in human; urine together. v;ifh a 
new series of C2iOg compounds. Isolation of these new steroids is in 
progress to provide sufficient amounts for biologic testing.-- :_ " .>;;;?: 

The;; quantitative pattern- of the corticosteroid excretion .is, currently being 
investigated in guinea pigs under various stress combinations;,, sych as 
x-radiation^ dietary deficiencies, drug effects, neoplastic diseases, 
environmental alterations,, .and an attempt; is being made tp, corarelate adrenal 
morphology with the excre.t,ion of specific cor tico^sterpid's., This, typ^ of 
investigation will later b^ extended, to other" endocrine organs. , ^ 

A clinical study developing from the observations in guinea pigs and 
utilizing Clinical Center material from the Leukemia Service and Surgical 
Service, NCI, and from the Psychosomatic Service of the NIMH has been 
started. The purpose is to study the alteration in the excretion of specific 
corticosteroids during the course of leukemia, e.g. in remission, in relapse, 
under therapy, while intervening infection, during active disease, and to 
note whether the pattern changes which the adrenal is challenged with ACTH. 
These patterns are compared with those of non-neoplastic patients and patients 
with neoplastic diseases other than leukemia. 

Autopsy material from the patients who have died with leukemia is being 
collected for a histopathological study of the effect of leukemia on 
various endocrine organs. 

Biosynthesis of steroids in normal and tumor-bearing animals is being 
studied in vivo with a Visiting Scientist. New methods of detection are 
being developed with the aid of radioisotopes. In vitro studies utilizing 
a simple perfusion apparatus are contemplated. 

A "library" of frozen urine aliquots and bloods from clinical patients 
and animals is being collected for future study. 

In addition, a long-term study is planned on the corticosteroid patterns 
occurring following the use of methylcholanthrene, total body radic^tion, 
and other potentially carcinogenic agents, in order to detect any specific 
quantitative and qualitative changes in corticosteroid excretion pattern 
in carcinogenesis. A related study on humans exposed to radiation has been 
proposed. 



4V/ 

SERIAL NO. NCI %idr PAGE IV 

PROJECT REPORT FORM (Cont'^) 

9. PROJECT BESGRIPTION (Cont'd) '-^^i ^' ^ n :- 

A continued istvidybn the effect of vatious stressed (see Methods Jabbve)rvo 
Is contemplated, oh the guinea pigs* "iiS"^ wr? anrf bi^^ 03 vfs« eWT 

The effect of various potentially therapeutic agents on the course of 'i'; 
cancer in the guinea pigs is being studied with the hope of picking up 
alterations in the specific corticosteroid excretion patterns associated; 
with c^rcinostasis. ''^ 

■. 1 i '•■'.■■ ■ ' . SK.'.: I:- 'bi- ; ■ .v 

A continued study on carbohydrate n»etabolistn in the guinea pigs -is being 
pursued witli the hope of applying this fundamental knowledge to the in vitro 
perfusion studies that are planned when facilities become available; 

: . . :iai«r'iD J,sa.i:ffi.jD jii 

.., ' ■ \ ' '■ i-'.r-l i-Mi^ . ^'V1 

oiilaijqu' 1;. . . 
,jS!qBiu;:, ni . , . : 

oJ hmr, 1^3? , ■ ' ■•!•.■■.• 

.nyjr- ^}'<>:_'h . :. . ■ ■ '■■.., 

■ \if.i:'- . ■ . ■ ., ■ . .. - . ■ 



10. NCI -5*3- 



SERIAL NO. 



PAGE V 



PROJECT REPORT FORM (Cont'd) 



11. 



BUDGET ACTIVITY: 

RESEARCH /x7 
REVIEW & APPROVAL /"7 



ADMINISTRATION h,l 
TECHNICAL ASSISTANCE 7~7 



12 . E. Frei. ClinlGal Center. NCI. L. Cramer, Clinical Center, NCI. 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. None to my knowledge. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH 



PAGE VI 
PROJECT REPORT FORM (Cont'd) ^' 

14. NCI-5£» 
SERIAL NO. 

15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 

Effect of neoplasms and of stresses on the excretion of specific corticos- 
teroids in the guinea pjig, 
Nadel, Burstein/ and Doirftnan. 
Clin. Res. Proc.^ Feb. 1955 (Prpc. Am, Fed. Clin. Res/f^ 

Urinary excretion of Cortisol, 6-p hydroxycortisol and an unidentified 
steroid (Steroid Ha) by guinea pig with leukemia or with liposarcoma. 
Nadel, Burstein, and Dorfman. 
Proc. Am, Assoc. Cancer Research 2: 37, 1955, 

.Corticosteroids in the urine of normal, and. scorbtitic gulin^i pigst 'isolation 

■"'^■'arid ffuantitative determination;, ■■"■' ' 'pV-^ ;■''*'-• i ' ' ' '^'-.^'•j'H, ?'•'"•-■'■■ ' ''■' 

BUratein, Dorfman/ and Nadel. ' ' , ' i HP .^ .mtlJIim- .mn^^^mmy 

J, Biol. Cheira. April 1955. 

Isolation of polar reducing corticosteroids from human urine. 

Nadel, Burstein, and Dorfman. 

Arch. Biochera. & Biophysics., Accepted October 7, 1955. 

Dissimilarity in alkaline phosphotase staining of Keratohyaline granules. 
Nadel and Wodinsky. 
..„ Jj Histochem. & Cytpchem. S.ept. 1955. 

' Tfon«pdrallel ' changes in ' changes in cholesterol and ascorbic^acid^-in the 
' adrenals of malaria parasitized' chicks, ■;•' ;;'■,, «.Vp,'>'f' ' /■?:'• '■:y~ l■xy^s.,^^.'. 

Taylor, Greenberg, Josephson, and Nadei. 

J. Clin. End. & Met. June 1955. (Proc. Endocrine Society) 

On the defect in glycogen deposition in the livers of scorbutic guinea pigs. 
Nadel, Mulay and Saslaw. 
Endocrinology May 1955. 

16. None __«______->_ 

HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955. 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology . ■i'y.^X.'O^" . 

INSTITUTE LABORATORY OR BRANCH • 

■ ' ■ -y; ' S*^ 

3. Cancer Indue. & Pathogenesis 4. , 5, NCI -S-^^^ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BBTHESDA) SERIAL NO. 

6. Effect of microbia'l and other agents on the development and course of 

neoplastic disease In animals. . ^' ^ • ' ■ ' "'"••■' 

PROJECT TITLE : '^ 

7. E. M. Nadel 



PRINCIPAL INVESTIGATOR(S) 
8. None (See Item #12) 



OTHER INVESTIGATORS 

PROJECT DESCRIPTION , , - 

The present project title includes 3 sub-.projects gathered' under the project 
title of! (a) Mutual aspepts of the- pathophysiology of leukemia and malaria 
in animals. When it became lobvio^s that the project on perfusion studies 
could not begin because of the administrative uncertainty of ispace;, facilities 
and moving date to the Clinical Center laboratories, 2 other projects related 
to a) were undertaken. ..... .- i • ,. . .■ . -^ ■ 

These projects include: (b) The effect of certain viruses on the course of 
cancer in animals and . (c) the, mode of: action of certain living and chemical 
agents on the developinent; and cou?:se of cancer in animals. 

Objective: (1) To explore the activity of certain potentially carcinostatie 

and carcinogenic agents. (2) To. explore certain immunogenetic 
and endocrine aspects of resistance to malaria infection and to viruses 
helpful in a study of the carcinostatie potentiality of these microbial 
agents. (3) The mode of action of certain virucidal and raalarlacidal drugs 
as carcinostatie agents. 

Methods: The overall problem is studied by the combined use of techniques 

in pathology, microbiology, hematology, genetics, chemical 
pharmacology, and immunology. Inbred, hybrid and specific backcrosa animals 
are studied for resistance to micro-organisms such as the malaria parasite, 
neurotropic viruses, etc., and the effect of these on the course of trans- 
plantable tumors is studied. Combinations of drugs and of viruses are 
studied. 

Major findings: (1) Guinea pigs with leukemia lived significantly longer 

after they were experimentally infected with the virus of 
lymphocytic choriomeningitis. (LCMV). (2) Guinea pigs made immune to the 



SERIAL NO. NCI -^^Sr" PAGE II 

PROJECT REPORT FORM (Cont'd) 

9. PROJECT DESCRIPTION (Cont'd) ■ , -:;-.;. 

,LGM virus wer.e afforded no protection when the virus was injected after 
transplantation of the tumor, (3) Four drugs (ethionine, anjethopterin, and 
" a :purine and pyrimidine ati^Iogue) were studied to find that dose at which 
each individual drug was ineffectivfe. Combinations of these drugs at their 
individually ineffective dosages successfully proloriged the life of tumor- 
bearing mice, more than 100% in comparison to controls. (4) Repeated 
therapy with the combination of 4 drugs to tumor-bearing mice resulted in 
the development of a resistant tumor. This resistant tumor is now being 
used as a means of studying the mode of actioii of other potentially 
carcinostatic drugs. .■[■■^T\bIT-'.S\>Vii 

Significance to cancer research: (1) A neurotropic virus (LCMV) niade 

relatively benign by repeated passage to mice (300 generations) 
was able to prolong the life of leukemia guinea pigs from 19 to over 30 days. 
The virus did not of itself appear lethal to guinea pigs though other 
-strains of the virus apparently are. This increase in survival is greater 
^ . than that previously reported frqia this project, on the inhibitory effect 

- of malarial infection on the cc)urs6 of Leukemia in mice. This type of 
study has therapeutic implications for use in mati. (2) , By judicious use 
of a tumor, resistant to four different types of- chemotherapeutic agents, 
the mode of action of potentially carcinostatic drtjgs, with. different 
chemical structure can be studied. ,; 

Proposed course: Attempts to enhance the duration of the inhibitory effect 
of the microbial agents under study are being made. 
! These include the judicious use of hormones, xrray irradiation, drugs and 

- multiple sequential use of microbial agents, various pyrogenic materials 
(e.g. polysaccharides, etc.). The possibility of the use of viral therapy 
in the form of the LCM virus for leukemia in nian must be considered. 
Continued studies on the possible similarities in the genetics of .resistance 
in mice to malaria and to leukemia, and the role of endocrines in , such 
resistance are underway. 



■a: i-ui .-,■:. :■. .^ftiPiivv..- j.i<p:ri: 



PAGE III 
PROJECT REPOKT FORM (Cont'd)' 



10. NCI »£A— 
SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH /x7 ADMINISTRATION fj 
REVIEW & APPROVAL /"7 TECHNICAL ASSISTANCE /T 



12. 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 

Cooperating personnel assigned to the staffs of Dr. Greenberg, Chemotherapy 
Section, Laboratory of Tropical Diseases, NMI, Dr. Haas, Office of the 
Director, NMI, Dr. Jay, Laboratory Aids Branch, Office of the Director, NIH. 



13. None to my knowledge ■ ■ ■ 

IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH 



PAGE IV 
PROJECT REPORT FORM (Cont'd) 

14. NCI-S-^^- 
SERIAL NO. 

15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 

Effect of malaria on leukemia in mice. 
Nadel, Greenberg, Coatney. 
J. Infectious Diseases. 

Differences in survival of several inbred strains of mice and their hybrids 
infected with Plasmodium berghei. 
Greenberg, Nadel^ Coatney, 
J. Infectious Diseases. 

Synergistic inhibitory action of amethopterin and a diaminopyrimidine 
Oh leukemia L 1210 in mice. 
Nadel, Greenberg. 
Cancer Research, 

Increased resistance to malaria of certain inbred strains of mice^ their 
,. hybrids and backcrosses, 

Nadel, Greenberg, Coatney, and Jay. 
Am, J. Path. 

Resistance to quadruple combination therapy in leukemia L 1210 in mice. 

Nadel and Hilgar. 

km,. J. Path,, June 1955. (Proc. Am. Assoc. Path. & Bact.) 

Inhibitory effect of lymphocytic ehromomeningitis virus in thg course of 

leukemia in guinea pigs, 

Nadel and Haas. 

Fed. Proc. 14: March 1955. (Proc. Am. See. Exp. Path.) 

Backcross studies on the genetic of resistance to malaria in mice. 
Nadel, Greenberg, Jay, and Coatney. 
Genetics, Sept, 1955. 



16. None 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

6'/3 

3. Cancer Indue. 6e Pathogenesis A. 5. NCI ' 525- 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Use of isolated organ perfusion techniques in a study on the pathologic 
physiology of tumor-bearing animals. 

PROJECT TITLE 

7. E. M. Nadel 

PRINCIPAL INVESTIGATOR(S) 

8 . None \ ' 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objective: Study changes in physiology accompanying tumor growth to learn 

how tumors divert energy sources away from the host^ as a means 
towards the control of tumor growth. 

Methods: The perfusion methods used have been used successfully in the hands 

of Dr. Leon Miller, v/hile the micro-methods to be used have been 
described by Drs. Scholander, Kirk, and Natelson. 

Major findings: There have been no major findings inasmuch as work on this 
project has been delayed by unforeseen circumstances. 

Proposed course: It is hopefully anticipated that work can be started on 

this project shortly after the contemplated move from 
Building 8 to Building 10. 



PAGE II 
PROJECT REPORT FORM (Cont'd) 



10. NCI •&£»• 
SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH /z/ ADMINISTRATION £7 

REVIEW & APPROVAL /"7 TECHNICAL ASSISTANCE ' l~l 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH 



NO ENTRIES FOR 14, 15, or 16 



PAGE I 



PROJECT REPORT FORM 



National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABOR/iTORY OR BRANCH 

3. Cancer Ifldue. & PathoRenesis 4. . ^ ^ 5 . NCI -§4:9- 

SECTION OR SERVICE LOCATION ( IF OTHER THAN BETHESDA) SERIAL NO. 

6. Induction of adrenocortical tumor. in the rat and study of the adreno-cortical 
steroids of the tumors, thus induced. Also, the effect of these steroids on the 

steroid metabolism of the host. 

PROJECT TITLE 



7. A. S': Mulay 

PRINCIPAL INVESTIGATOR(S) 



8. W. H. Eyestone 

OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION 

Objective; To find out if the steroids produced and the enzyme systems 

involved, in the adrenal cortical tumor differ either qualitatively 
or quantitatively from those in the adrenal gland. 

Methods: Transplanting and harvesting of the adrenal tumors. Determining 

activities of enzymes like choline esterase, adenosine triphosphate, 
phosphomonoesterase, and alkaline phosphotase. Quantitative and qualitative 
metabolism of steroids by tumor slices under different given conditions, on 
fresh tumor tissue. Extracting steroids from harvested tumors stored in 
deep freeze (when enough material is collected), purifying the extract, 
separation of various steroid fractions by chromatographic methods, and then 
their characterization by various physical and chemical methods. 

Major findings: Adreno-cortical adenocarcinoma has been induced in 9.57o of 

the female Osborne-Mendel rats, under experimental treatment. 
This adrenocortical tumor has pronounced effect on the adrenal glands of the 
host. Considerable quantity of formaldehydogenic steroids have been found 
in the extracts of these tumors. 

Significance: Successful conclusion of this program may give us some insight 

of chemical and enzymatic changes which transform normal 
adrenal gland into adrenocortical adenocarcinoma. 



SERIAL NO. NCI *i6 PAGE II 



PROJECT REPORT FORM (Cont'd) 



9. PROJECT DESCRIPTION (Cont'd) 

Proposed Course: A considerable portion of the next calendar year will be 
devoted to separation and identification of the steroids 
in this tumor. This involves growing and collecting the tumor material, 
extracting the steroids, separating different steroid fractions chromato- 
graphically, and collecting enough of each fraction for further separation 
and then physical and chemical characterization. At the same time a 
technician is being trained in enzymologic and metabolic procedures for 
working on fresh tumor tissue. 



PAGE III 



PROJECT REPORT FORM (Cont'd) 



0. NCI -5^:6- 
SERIAL NO. 



1. 



BUDGET ACTIVITY: 

RESEARCH /x7 ADMINISTRATION [j 
REVIEW & APPROVAL l1 TlECHNICAL ASSISTANCE l~l 



2. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATION, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



3, As far as I know, it cjoQS not. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE IV 
PROJECT REPORT FORM (Cont'd) 

14. NCI Hfr^ 
SERIAL NO. 

15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 

Mulaj, A. S. and Schlyen, S. M. 

Les:'.cn5 induced in C573R mice with,galliux citrata and -nethylcholanthrene. 

Am. J. Pathol. In prass. 



16. HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1955 

Elected to imerabership in Society for Experimental Biology and Medicine 
(Rational) " ' 



PAGE 1 



PROJECT REPORT FORM 



National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

Cancer Indue. & Pathogenesis 4 . 5 . NCI 512- 

SECTION OR SERVICE • LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 



6. Sex difference in the incidence of hepatomas in rats, fed a carcinogenic 
azo dye, and study of hormonal and other factors responsible ;for this. • 

difference. ___«__^ ________ 

PROJECT TITLE 



7. A. S. Mulav . : . '/ ^ ■ ■ ■"'.-■ ■ ■■ : '. ■"■'■■ : • :■' ' .- - ■■'- '^ ^ 
PRINCIPAL INVESTIGATOR(S> -• 

8. None 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION ;•::-.:■• 

Objective: To determine the, role played by sex hqrmones on the -Incidence of 

hepatomas induced in rats fed carcinogenic azo dyes^ and see what 
part, if any, is played by other so-called "residual sex differences." 

Methods: Male and female rats, intact, gonadectonized, and with or without 
estrogen or androgen treatment, are fed a synthetic diet of known 
composition containing £-dioethylaminoazobenzene. All groups of rats thus 
treated are kept under identical conditions, and a few animals from each 
group are sacrificed at definite time intervals. Livers of these animals 
are examined for macroscopic and microscopic hepatomas. From these observa- 
tions, effect of the treatment on the relative induction time and the 
incidence is calculated. 

Major findings: Intact male and female Osborne -Mendel rats fed a carcinogenic 

azo dye in multiple deficiency synthetic diet of known 

composition, for 10 months, showed a marked sex difference in the incidence 
of hepatoma. 

Significance: Evidence thus gathered will forge a link in the chain of 

environmental factors necessary to induce neoplasm in animals. 

Proposed course: Experiments on various treatments to different groups of 

animals are just starting and will run through the next 
calendar year. 



PAGE II 



PROJECT REPORT FORM (Cont'd) 



10. NCI-^t^- 
SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARGH • /x7 ■ AMIINISTRATION : ■ ■■ [j 
REVIEW & APPROVAL H TECHNICAL ASSISTANCE /~. 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. As far as I know, it does not. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS , OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 






.>y t&hnoi:. 



PAGE III 



PROJECT REPORT FORM (Cont'd) 



14. NCI-§4^ 
SERIAL NO. 



15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DDRISG CALENDAR YEAR 1955 



Symeonldls^ A. and Mulay^ A. S. 

Histopathology of the adrenal glands of rats fed a low protein, low riboflavin 

diet alone, or with £-dimethylaminoazoben2ene. 

J. Nat. Cancer Inst. In Press. 



16 . None 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955 



■) mj^k 



,0M J. 






.ji 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

3. Office of the Chief 4. 5. NCI •S^'fr- 

SECTION OR SERVICE - LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Induction, Pathogienesis and Morphological Alteration of Tumors Arising in Bone 
PROJECT TITLE 

7. Albert W. Hilberg 

PRINCIPAL INVESTIGATOR(S) 

8. None '■ ' ■ " - ' - 

OTHER INVESTIGATORS - 

9. PROJECT DESCRIPTION • . 

Objectives: To develop a consistent method of producing bone tumors in 

animals which are similar, morphologically, to those in man 
and to study the events leading to tumor formation or alteration of tumors 
as affected by transplantation. 

Methods: Animals have received Injection of beryllium oxide by various 
routes such as intravenous, intramedullary and intratracheal. 
At intervals skeletal surveys by x-ray examination and blood serum phosphatase 
levels are determined. All control animals and injected animals are autopsled 
and bony tissues examined grossly and microscopically. Special studies of 
tissues are made using histochemical and x-ray techniques. 

Transplantation of spontaneous bone tumors into various sites such as liver, 
kidney, subcutaneous tissue and intraperitoneal spaces is earried out to 
study effects on morphology of tumors. 

Major findings: The rabbits injected with beryllium have developed a 

lymphoma, 2 bone sarcomas, and 1 bone sarcoma developed 
in a mouse and is being successfully carried in serial transplantation. 

Morphological variations in spontaneous bone tumors by selective site 
transplantation into kidney have revealed a differentiation of undifferen- 
tiated cells into bone after bone had failed to develop in subcutaneous 
transplants. This represents a possible redifferentiation of anaplastic 
cells. 

To date some 40 spontaneous tumors primary in bone in mice have been found 
and classified by morphological type. A detailed description is being 
completed. 



SERIAL NO. NCI -Si*- PAGE II 

PROJECT REPORT FORM (Cont'd) 

9. PROJECT DESCRIPTION (Cont'd) 

Significance to cancer research: Bone tumors are said to be among the most 
common neoplasms in children. These tumors develop at any age 
and represent a major problem since cures are rare and treatment usually 
mutilating. Attempts to learn oore a^out the development of bone tumors, 
Including contributing factors in development and the biological and 
morphological behavior of these tumors can give valuable information toward 
the goal of prevention, treatment) and cure of bone tumors. Information 
concerning specific substances, such as beryllium, which produce bone. tumors, 
may be of value because of the industrial uses of beryllium and its compounds. 

Proposed course: Continuation of the study of animals injected with 

beryllium oxide. Continuation of selective Bite transplan- 
tation studies of spontaneous tumors arising in bone, with particular 
emphasis on transplantation to the liver. The classification and description 
of all spontaneous tumors arising in bone in mice is a continuing process as 
these tumors are received from many sources within the various laboratories 
of the National Institutes of Health. 

Collaboration in beryllium studies in intratracheal injections and in 
studies of Rous sarcoma will continue until completion. 



PAGE III 



PROJECT REPORT FORM (Cortt'd) 



10. NCI •aiO' 
SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH /x7 ADMINISTRATION £7 
REVIEW 6. APPROVAL £7 TECHNICAL ASSISTANCE £7 



12. None to my knowledge 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



13. None to my knowledge 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE IV 
PROJECT REPORT FORM (Cont'd) 



14. NCI-§4^ 
SERIAL NO. 



15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 195! 

Hilberg; Albert W. 

Morphological Variations Itt an Osteogenic Sarcoma of the Mouse when 

Transplanted to the Kidney 

J. Nat. Cancer Inst, Inpress. 



16. None 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology ' 

INSTITUTE LABORATORY OR BRANCH 

3. Pathological Technolosy Sect. 4. 5. NCI 



SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Pathological Technology Section 

PROJECT TITLE 

7. Mr. J. M. Albrecht ' ■ - ■ ■ 

PRINCIPAL INVESTIGATOR(S) 

8. None ■ • 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Stained tissue sections are the fundamental basis of all clinical and 
experimental studies of cancer. The Section prepares histological sections 
for all the investigators of the National Cancer Institute. It makes available 
all the established routine and special stains and in addition develops and 
provides the current experimental methods of tissue preparation such as 
enzyme stains and specific histological stains. 

January 1. 1955 - December 31. 1955 

Number of Investigators 67 

Number of Pieces of tissue 105,361 

Number of Bottles of tissue 15,921 

Number of blocks cut 52,356 

Blocks cut serially .................. 464 

Frozen blocks cut 722 

Number of autopsies 14,635 

Number of recuts 920 

Number of slides stained H&E 67,562 

Number of slides stained special ..... 13,701 

Number of unstained slides ......... 7,805 



SERIAL NO. NCI -frtS" 



PAGE II 



PROJECT REPORT FORM (Cont'd) 



9. PROJECT DESCRIPTION (Cont'd) 



aJwlMafii 



Photographic Service rendered to the Laboratory of Pathology, .January 1. 1955 
to December 31, 1955 ' 



GROSS 



• Black & White _.•,, . ....:/ 'S'^^'-il^;::"^...^ ■ .■■•84 

Color . , 94 



MICROS 

Black & White , 380 

Color 333 

- LANTERN SLIDES ' "" " ^ , -'^r . ^v. 

Black & White .......,,..., 103 

Color .......,.,,,....,.. ,, ,. ; ; 195 

PRINTS 



^ X 5 , .; .1548 

8 ^ IP . . 32 

14,x 17..,,. ... .^, ..,..,>..,,,. ,;.-... vr. ■,... ./v.'^. ...■ :;'5 ■■ 
.Mounted. ..,.:.,.,:..,. ..,..-..,, >, .^.,; .::; .. :.--*-v; ..<> . .^^ •■28a\ ^' 



10. 



r/7 

NCI rH:3- 
SERIAL NO. 



PAGE III 



PROJECT REPORT FORM (ebnt-»d) 



11. 



BUDGET ACTIVITY: 



RBSBARCH /x/ 
REVIEW 6> APPROVAL [J 



ADMINISTRATION /_/ 
TECHNICAL ASSISTANCE ll 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEM"ENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERK)NNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE IV 



PROJECT REPORT FORM ; (Cont ' d) 



14. NCl-§4» 
SERIAL NO. 



15. None 



PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CAUNDAR YE^ 1955. 






16. 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955. 



Willie D. Morgan received cash award June 1955 for a vacuum device and 
siphorvacuum cap which aids ah; the Cfl^ utility bottles frprt 

"large cans or bottles ot; reagents. ; ■ ; . . .» .. . ■. ,■'•.. . ,^\ 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute. 2. Laboratory of Pathology ' ■ , 
INSTITUTE LABORATORY OR BRANCH , , 

3. Office of the Chief 4. ■ . . .:" - ' 5. NCI-»6^ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO, 



6. Heterologous Transplantation 



PROJECT TITLE 



7. C. Harold Steffee and Katharine C. Snell 



PRINCIPAL INVESTIGATOR(S) 
8. None 



OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives: To produce neoplasia of human tissues. 

Methods: Human fetal tissues have been transplanted into the cheek pouch 
of the hamster, the host animal being treated with cortisone. 

Major findings: The transplanted fetal tissues generally persist for a 

reasonable time in the hamster cheek pouch but no evidence of 
neoplasia of these tissues has been obtained, A major obstacle in this 
program has been the development of dissecting aneurysm in many of the 
cortisone-treated hamsters with premature death of the animal resulting from 
hemothorax or hemoperitoneum. Similar lesions have been found in the control 
animals treated with cortisone but not given any fetal transplants. That 
this finding is not limited to hamsters is evidenced by the discovery of 
degenerative changes in the aorta of each of two mice bearing functional 
adrenal cortical carcinomas (Dr. Thelma B. Dunn). 

Significance to the program of the Institute: The original objective of 

these studies was to produce human carcinoma in organs which 
•re difficult to study in experimental animals because of the lack of 
analagous neoplasms in the animal. These sites include the lung and the 
gastrointestinal tract. Had we succeeded in this endeavor we would have 
had an extremely valuable tool for testing a number of suspected carcinogens 
for these tissues, and a tool which would have been much more comparable to 
the human situation than is otherv/ise possible. Perhaps additional studies 
will permit us to attain this objective. The significance of the aortic 
lesions to cancer research is at best rather nebulous. It may, however, 



SERIAL NO. NCI •5f>t- 



PAGE II 



PROJECT REPORT FORM (Cottt'd) 



9. PROJECT DESCRIPTION (Cont'd) 

provide us with further insight into the fundamental effects of adrenal 
cortical hormones on the connective tissues of the body. This, then, is 
an area in which we are seeking fundamental knowledge with no immediate 
applicability to the cancer prohlem. 

Proposed course of the project: A number of hormones related to cortisone 
will be tried in an attempt to attain. our original objective. . 
Additional studies will also be carried out to try to define the precise 
relationship of cortisone to the development of the aneurysm. 






PAGE III 



PROJECT REPORT FORM (Cont'd) 



10. NCI -SOJ- 
SERIAL NO. 



a. 



BUDGET ACTIVITY: 

RESEARCH /x7 ADMINISTRATION [j 
REVIEW & APPROVAL [1 TECHNICAL ASSISTANCE fj 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



13 . None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR 14, 15, OR 16 



^b' ffjoO) ;cv;-.-'{ r>;r';rH>i 






)rTjv.TTDA -ndirM 



'i^^'iHf. / viMTVa« 






■■■: V jKf t^I 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology 

^ INSTITUTE LABORATORY OR BRANCH 

^f 

3. Cancer Indue. &. Pathogenesis 4. 5 . NCI 4-H- 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Chemical and histopathological changes induced in the adrenal glands of rats 
fed a deficiency diet. (This diet is usually used in conjunction with azo 

dye feeding for the induction of hepatoma in rat.) . 

PROJECT TITLE 



7. A. S. Mulay and E. M. Nadel 
PRINCIPAL INVESTIGATOR(S) 



A. Symeonidis 



OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION 

Objective: To find the factor or factors absent in the deficient diet, 

which are responsible for the induction of the histopathological 
changes observed in the adrenal glands of rats kept on this deficient diet, 
and others deficient only in one or two known factors. To study the 
chemical and metabolic changes in the adrenal glands of these animals, 
concomitant with the histopathological changes. 

Methods: Starting with a complete synthetic diet of known composition, 

different diets are prepared from which a known factor or factors 
are withheld. Such diets are fed to different groups of rats and the 
chemistry and histopathology of their adrenal glands is compared with that 
of the adrenal glands of rats on complete diet of known composition. 

Major findings: Histopathology of the adrenal glands of rats fed a 

multiple deficiency diet of known composition (one used 
in connection with hepatoma induction in rats with azo dye feeding) is 
described in our preliminary paper "Histopathology of the adrenal glands of 
rats fed a low protein, low riboflavin diet alone or with p-dimethylamino- 
azobenzene" in J.N. C.I. In press. 

Significance: The role played by the adrenal gland in the induction of 
hepatoma in rats fed multiple deficiency diet containing 
carcinogenic azo dye, has been demonstrated in our laboratory (J.N. C.I. 
14: 805-817, 1954, and Endocrinol. 57: 550-558, 1955) and in others 



SERIAL NO. NCI 54* PAGE II 



PROJECT REPORT FORM (Cont'd) 



9. PROJECT DESCRIPTION (Cont'd) 

(Richardson: Cancer; 6: 1025-1029, 1953, and Griffin: Cancer Research 13: 
77-79, 1953). Knowledge of the factor or factors responsible for these 
changes will take us a step nearer to the understanding of the neoplastic 
process. 

Proposed course: Critical experiments to confirm our first obserVati&tts -. 

on the altered histopathology of the adrenal glands of 
rats on multiple deficiency diet of known composition are in progress. 
Better part of the year will be taken up in confirming the histopathology 
picture and analyzing for concomitant chemical changes in these altered, v, 
adrenal glands. Experiments with complete diets of known chemical 
composition, deficient in only one or two known factors have just been 
started. 



PAGE III 



PROJECT REPORT FORM (Cont'd) 

LO. NCI Jh^ 
SERIAL NO. 



LI. 



BUDGET ACTIVITY: 

RESEARCH /x7 ADMINISTRATION fj 
REVIEW & AP?WVhL /~7 TECHNICAL ASSISTANCE /"7 



L2. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. As far as I know, it does not. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: , 



PAGE IV 
PROJECT REPORT FORM (Cont'd) 



14. NCI 



^ 



SERIAL NO. 



15. PUBLICATIONS OTHER THAN ABSTRACTiS FROM THIS PROJECT DURING, CALENDAR YEAR 1955 

Nadel, E. M., Mulay,. Av S.> and;;S,aslaw,. L. D. 

On the failure of glycogen deposition in the livers of scorbutic Guinea Pigs. 

Endocrinol, 56: 584-589, 1955.- i'; J. : , ■. 3 • 

Symeonidis, A., Mulay, A. S., and Trams, E. G. ■ 

Effect of prolonged pretreatment with desoxycorticosterone on the liver of 

hepatectotnized rats. 

Endocrinol. 57; ,550t55B, ,195^;: v;^ 

Mulay, A. S. and Eye stone, W. H. 

Transplantable adenocortical adenocarcinoma in Osborne -Meade 1 rats fed a 

carcinogenic diet. 

J. Nat. Cancer Inst. 16: 723-739, 1955. 



16. •^■■^" ■■ 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955. 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. l^aboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. & Pathogenesis 4. 5. NCI -§49- 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Tissue Culture of Mouse Leukemia s. '■ ^ ^. - • 

PROJECT TITLE 



7. C. J. Dawe 

PRINCIPAL INVESTIGATOR(S) 



8. Michael Potter and Joseph Leighton 
OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION 

Objectives: Development of methods for isolation and continuous cultivation 
in vitro of mouse leukemia cells. 

Methods: Methylcholanthrene-induced leukemias in ascites form in DBA mice 

are used as a source of leukemia cells which are propagated 
alternately in gelfoam matrix in roller tubes and in mice. By inoculating 
the tissue cultures intraperitoneally into mice at successively increasing 
time intervals after explantation, it is possible to determine the survival 
time of leukemia cells in tissue culture and to perpetuate the cell time^ 
which otherwise dies out in tissue culture alone. 

Major findings: In the four months during which this project has been 

underway, it has been possible to carry one line of 
lymphatic leukemia through 6 tissue culture passages and an equal number 
of mouse passages. During this time the maximal survival time in tissue 
culture has increased from 7 to 13 days. The possibility that a morphologic 
change may have occurred in the leukemia cells is under study. 

Significance to cancer research: For studies of growth characteristics, 
nutritional requirements, and susceptibility to therapeutic 
agents, it is desirable to be able to cultivate pure populations of leukemia 
cells in relatively controlled environment such as is obtainable in tissue 
culture. The purpose of this project is to determine whether leukemia cell 
lines can adapt to continuous tissue culture propagation as a result of 



SERIAL NO. NCI «3- ■'■■^^ 'X.iiW\!r- ■: PAGE II 



PRO JECt REPORT FORM (Cont'd) >™H£ii-r£:'_ll:; 

9. PROJECT DESCRIPTION (Cont'd) 

repeated exposure to' tlssbeeiil'ttlire' environs If this prdves to be so 
the method can then be applied more generally to the isolation of various 
types of leukemia cells in tissue culture. At the present time^ no 
satisfactory method of achieving this end is available. . 

Proposed course: The method described will be continued for at least a 

year^ at which time the results and possibilities of the approach 
will be evaluated. Additional cell lines will be added to the. experiment.'.. 



'=Ji)Si,-i3,- 






■ •. i ■ ■ ^■^:^^Z>^Li::' ■■■ ■ ■■:'■' 

■.■'■' . iu\^-:jj ..b <: • ■ ■ ■ • ■ 






■3/3 utjQuni: 



PAGE III 



PROJECT REPORT FORM (Cont'd) 



10. Nci-^ty 

SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH /x7 ADMINISTRATION [j 
REVIEW & APPROVAL [j TECHNICAL ASSISTANCE [j 

12. None 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR 14, 15, or 16 



■i» ■?«■ 









■'^^T> »'SI'T>iT-ff.H?^ 



iO '8'Sjtti,!)S;jivv ., -.',.■■■> .;/?Ai:iJ}M^i:iS.i ^Y^iMi.: 






PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology ■" ■ ■ ''• 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. & Pathogenesis 4. ■^_^ 5. NCI -§4-»' 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Tissue culture in a perfusion chamber designed to use whole blood as a source 

of nutrient. .■■■.-. - ■■- ' ^" ' " ' ' ' ' ^__ 

PROJECT TITLE 

7. C. J. Dawe 

PRINCIPAL INVESTIGATOR(S) 

8. None . _. 

OTHER INVESTIGATORS : • : . • T ' ' 7'. " 

9. PROJECT DESCRIPTION; .:;.;;'. w r^ ■ 

Objective: To determine the applicability of a new type perfusion chamber 
to special problems in tissue culture. 

Methods: A lucite perfusion chamber has been designed and constructed to 

permit culture of tissue explants in a flowing plasma medium 
derived by membrane filtration of whole blood, circulated through the 
chamber under physiological pressures in a gravity system. The chamber 
permits continuous microscopic observation of the cultured cells. 

Major findings: This iapparatuB has hot yet been tested in relattoh to ;it;s 

effects on all viability and growtli'. The hydraulics have , 
been shown to function satisfactorily as outlined above.' 

Significance to cancer research: Using fresh heparinized whole blood as the 
circulating medium it becomes possible to bathe tissue explants 
in an environment presumably very similar to that provided by the plasma 
component of the circulating blood in vivo. Oxygen tensions of the medium 
can be controlled by varying the oxygenation of the circulating red cells. 
The investigator's objective in developing this chamber is to study the 
effects of the medium so derived on the survival and proliferation of 
leukemia cells in vitro. The extent to which leukemia cells can proliferate 
while actually within the plasma of the peripheral vascular system is at 
present unknown. Other applications of the apparatus are apparent. For 
example, it would be possible to observe the effects of metabolic products of 
one cell type on another cell type by connecting the chamber in series. 

Proposed course: The growth promoting or inhibiting effects provided by 

this system will be studied primarily on mouse and human leukemia 
cells, using human blood as the circulatory element Initially, 



PAGE II 



10. mi iVr- 
SERIAL NO, 



PROJECT REPORT FORM (Cont'd) 



si ISSK 



11. 



:?{:*:* !■ 



BUDGET ACTIVITY!' 



RESEARCH /x/ ADMINISTRATION i_l 
REVIEW. &. APPROVAL, /"7 . TECHNICAL ASSISTANCE /"7 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES^ OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS-, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



■\^«'.-liK^ 



I ■^ A * .. 



. . ■■ <^:\ hitkl^i/ i': ' 




:.;:;■$/- !>'*<> 3 fjg,,r 




^ •■••I'Aijt*/- Sif:? ■ : 




^. . "f'J ■fiMs 




■ ■ aiuow 1 




:. -n: -fto' ^q- 




■^^-iU a^ ::3asu<': 




■■ ! ■ •■■■ •■I^-^« :Sjyjli i 




• ■■■irimiiii'ghi.r.i.i 


vH' 



PAGE III 



PROJECT REPORT FORM (Cont'd) 

14. NCI -^rtir 
SERIAL NO. 

15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 

Cytologic studies of sputum, secretion, and serous fluids of patients with 

malignant lymphoma. 

C. J. Dawe, T. B. Woolner, E. M. Parkhill, and J. R. McDonald. 

Amer. J. Clin. Path. 25: 480-488, May 1955. 



16. HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955: 

Certified in Pathologic Anatomy by American Board of Pathologists (April 1955) 
Completed all requirements, including thesis and examinations for degree of 
Ph.D. in Pathologic Anatomy from University of Minnesota. Degree to be 
conferred March 1956. Thesis title: "Hodgkin's Disease and Its Inter- 
relationships with Other Disorders," 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. & Pathogenesis 4. . : . . . 5. NCI Sifn 

SECTION OR SERVICE LOCATION (IF OTHER THAN, BETHESDA) SERIAL NO. 

6. An investigation of the collagenous connective tissues. 

PROJECT TITLE 

7. William G. Banfield 

PRINCIPAL INVESTIGATOR(S) 

8. None ; , ■ ■ __^__ ■ . . ■ 

OTHER INVESTIGATORS ' . . 

9. PROJECT DESCRIPTION 

Objectives: The immediate objectives, are: 

1. To elucidate the age changes in collagen. 

2. To uncover mechanisms affecting the usual state of the 
collagen in the body. 

Methods: 1. Collagen from human skin^ scars and Achilles tendons are 

subjected to dilute acetic acid in order to establish more 
definitely the normal limits for the age-swelling pattern of Achilles tendon 
and to measure the amount of soluble collagen present in theee tissues. 
Variations in the reaction of skin and Achilles tendon from the normal are 
investigated through the patient's history and possible causes of such 
variation are subject to animal test. The maturation of scars is studied 
with respect to changes in acid soluble collagen and histology. 

2, Hamsters are tested for changes in skin collagen after 
hormone treatments or gland removal. 

3. A successful search has been made for a stain which will 
react differently to collagen from a young Achilles tendon than to collagen 
from an old Achilles tendon. 

Major findings: 1. A possibility has been found that chorionic gonadotropin 

and an adrenal cortical hormone each may increase the 
solubility of human skin collagen. 

2. A method for differentially staining collagen in 
young and old tendons and in young and old scars has been developed. 

3. Recent scars contain a large amount of acid soluble 
collagen whereas old scars do not. 

Significance to cancer research: A knowledge of collagen metabolism 

including collagen maturation, may help in the understanding 
of connective tissue tumors. Such knowledge should also contribute to an 
evaluation and possible control of the stroma which accompanies and even 



SERIAL NO. NCI -Hrfr- PAGE II 

PROJECT kEPORT FORM (Cont'd) 

9. PROJECT DESCRIPTION (Cont'd) 

seems ne^cessary for the continued growth of many tumors. Staining techniques, 
especially if placed on a hi stoch^nflcal basis, would be invaluable for 
studying the collagenous components of tumors. 

Proposed course: The measurement of theacid soluble collagei^'ln scars will 

be continued until all stages in the maturation of a 
scar can be represented. The scar specimens will be studied using the 
newly-developed differential staining technique for cqllageh and cohv6tltlonal 
histologic stains. The screening of skin and tendons for their content of 
acid soluble collagen will be continued and leads will be followed by 
animal experimentation. Animal experiments to determine, possible effects 
of gland removal on collagen development will be continued. The mechanism 
by which old and young collagen is differentially stained will be investiga- 
ted and an attempt will be made to improve the method. The electron 
microscope will be used to further the project. 



PAGE III 



PROJECT REPORT FORM (Cont'd) 



10. 



SERIAL NO. 



LI. 



BUDGET ACTIVITY: 



RESEARCH U/ 
REVIEW & APPROVAL /~7 



ADMINISTRATION L_l 
TECHNICAL ASSISTANCE /T 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHi\NGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE IV 



PROJECT REPORT FORM (Cont ' d) 



14. NCI -6^6- 
SERIAL NO. 



15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1W5 



Banfield, W. G. 

Width and length of collagen, fibrils during the development of human skin, 

in granulation tissue and in the skin of adult animals. 

Journal of Gerontology 10: IS-'l?, 1955,: 



16. HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1955. 

None. 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. & Pathogenesis 4. '" "' " ' 5. NCI 5 2€" - 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Effects of Ionizing Irradiation with emphasis on the morphologic alterations 

and neoplasms '■ ' 

PROJECT TITLE 

7. Richard L. Swarm . ■ ■ " . ■ . ■ '• ' 

PRINCIPAL INVESTIGATOR(S) 

8. None ] ^ - '■ ■ ' ; - - . 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives: The collection of additipnal information regarding the develop- 
ment of neoplasms in man and laboratory animals following 
exposure to radiation injury is one general objective of this study. Some 
interest in the type of irradiation change other than that which is manifested 
in the formation of neoplasms is maintained. The different effects produced 
by different types of x-radiation. Alpha irradiation and Beta irradiation 
are of particular interest. A knowledge of the changes effected by different 
types of radiation in existing tumors will be sought. 

Methods: Three types of approach have been made. (1) Participation in the 

Surgical Pathology and Post-Mortem pathology division of the 
Pathologic Anatomy Department of the Clinical Center. Here a review of 
accessioned specimens is made possible by actual participation in the study 
and diagnosis of specimens as they are received. An effort is made to 
select and categorize anatomic material showing radiation change. (2) Detec- 
tion of neoplasms and other changes in animals who have been given whole 
body irradiation of varying amounts at significant intervals prior to 
anatomical study. (3) A study of the distribution and effects of some 
radioactive isotopes given by different routes to animals and therapeutically 
or for diagnostic purposes to man will be made. Here the emphasis has been 
on the effect of I-V administered colloidal thorium dioxide. 

Major findings: A new project. Of interest, however, is one human case 

which was studied during life by the General Medicine Branch 
of NCI and which was later studied anatomically. Study revealed the presence 
of neoplasms in the liver, spleen, and bone of a patient who had stored 
thorium dioxide in the reticulo-endothellal system for many years. Studies 
of this are as yet incomplete, however, from the observations made In this 
case and other reported cases, an etiologic relationship between the storage 
of thorium and the development of neoplasms in man seems to exist. 



SERIAL NO. NCI -We-- PAGE II 



PROJECT REPORT FORM (Cont'd) 

9. PROJECT DESCRIPTION (Cont'd) 

Proposed course: (1) An interest in the morphologic effects of radiation 
on human tissue and in particular on previously existent 
neoplasms will be maintained and further developed. Particular emphasis 
will be placed on the study of the effects of different types, and energies 
of irradiation. Study of human case material (speciii^ens) from the Clinical 
Center will be the important phase of this activity, (2) The anatomic 
study of animals exposed to whole body irradiation will be continued. 
(3) The study of the distribution and morphologic changes effected by the 
administration of colloidal thorium dioxide given intravenously to man for 
diagnostic purposes and experimentally to animals will be, continued. 
Knowledge of the pattern of storage and effects in man will be furthered 
by a proposed istudy, of human material .at, .the Clinical, Center, The Armed 
Forces Institute of Pathplogy, and, of material submitted by Dr. William 

LOOney,-. .;■;, >:,.'? ,, ■ ,,.,..,:•. i = :.-;, :: .■!„ ; .i^w. .• ^ ■ ■,,;•.:.. 






^X'nz -U: 



PAGE III 



PROJECT REPORT FORM (Cont'd) 



10. HCl.^2S- 
SERIAL NO. 

11. 



BUDGET ACTIVITY: 

RESEARCH /x7 ADMINISTRATION £j 
REVIEW & APPROVAL /~7 TECHNICAL ASSISTANCE /"7 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 195fr or 1957 



13. IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 

(1) The work up of anatomic specimens from patients who have received 
irradiation in the Clinical Center will complement the work of the clinical 
services particularly that of the Radiation Therapy Branch. (2) The 
anatomic study of laboratory animals given whole body irradiation has been 
limited to hamsters supplied by Dr. W, Smith, Radiation Branch, NCI. Other 
pathologists have assisted Dr. Smith in this study. (3) Research on the 
morphology of damage in animals produced by I-V administered thorium dioxide 
has been made by many investigators in this country and abroad. Only two 
European investigators have succeeded in producing neoplasms in animals 
following I-V administration of colloidal thorium dioxide. Experiments 
designed to confirm or refute these findings are contemplated. To my 
knowledge, no study of the morphology of thorium aggregates in human tissues 
like that proposed has been undertaken elsewhere. 



PAGE IV 



PROJECT REPORT FORM (Cont ' d ) 



i'a.3 

14. NCI.5^-S— 
SERIAL NO. 



15. None 



PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 



16. HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1955 



Elected to Fellowship in the American Society of Clinical Pathologists and 
to membership in the Washington Society of Pathologists and in the D. C, 
Section of the Society for Experimental Biology and Medicine. 



:>;i^3HHSi L. . .,- ; TDSLOKf aiHT ^J[ 






PAGE I 
PROJECT REPORT FORM 

1. Nationa l Canc er I nstitut e 2 .JLaboratory of.2.?tholog^ 

"institute ' ' LABORATORY "or branch" 

3._0ffice of tjbe. Chief 4. 5 .NCI_f*9-;;i_ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BEfflESD^ SERIAL NO. 

6. Morphologic and histochemical study of a human carcinoma of > the floor of ■ 
the mouth in material from the patient, from long term tissue culture of 
the tumoF and from heterologous transplantation of tissue culture 
material to the cortisone treated rat. 



PROJECT TITLE 
Alan S . Rabson 



PRINCIPAL INVESTIGATOR (S) 

8 . Gerald "Suskind . , . . ■...; ^^ _„ , , j_ [■■ ] ■ '"• '-■■..■■ _ ._____„_____ 

OTHER INVESTIGATORS ' 

9. PROJECT DESCRIPTION 

Objectives: The objective of this project is to determine whether or not 

there are significant morphologic and histochemical differences 
in tumor cells after prolonged grov/th in tissue culture and in heterologous 
hosts . 

Methods Employed: In 1954, a tumor of the floor of the mouth was excised 

from a patient at the Clinical Center and cells from this 
tumor have been grovm in large quantity in tissue culture as recently 
reported by, Eagle; (Strain K-B) . The original histologic sections of this 
tumor as well as subsequent recurrences are available, in the files' of the 
Pathologic Anatomy Branch), for morphologic and, histochetftlcal Study i The 
cells in tissue culture will be studied on cover-slip preparations and in 
sponge matrix tissue culture. Solid tumors in cortisone treated rats have 
been produced by injection suspensions of the cells in tissue culture, and 
are being studied with a variety of fixatives and stains. 

Major Findings; The project has only been in progress for a short time and 
no significant findings are available. 

Significance to Cancer Research: The potential value of cell lines of human 

tumor cells in biological and chemothera- 
peutic studies has been questioned on the grounds that the cells are variants 
of the original tumor and have only a limited relationship to it. It would 
seem to be of considerable interest if it could be demonstrated that cells 
grown for many generations in tissue culture and subsequently in heterologous 
hosts are morphologically and histochemically similar to the original tumor 
from which they were derived. 

proposed Course of Project: As described above under Methods Employed, the 

morphology and histochemistry of the original 
carcinoma of the floor of the mouth will be compared morphologically and 
histochemically with the tumor cells in tissue culture and in cortisone 
treated rats . 



10 ■ NCI $^^9- 
SERIAL NO. 



PAGE II 



PROJECT REPORT FORM (Cont'd) 



11, 



BUDGET ACTIVITY: 

RESEARCH , , /xj 
REVIEW & APPROVAL /^ 



AiDMINISTRATION /_/ 
TECHNICAL ASSISTANCE /~7 



12, 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE/ OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 or 1957 



13 



,IF THIS PROJECT- RESEMBLES , COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HE-UTH SERVICE (WITHOUT INTlERCH/iNGE OF 
Personnel, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE III 
PROJECT REPORT FORM (Cont'd) 



14. NCI j ^^ 
SERIAL NO, 



15. 



PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 
1955 

Pretest of Forms and Field Techniques For Use in the Detroit -Windsor 
Air Pollution Study by A.F.W. Peart, C.P. Anderson, A.S. Rabson and 
W.L. McEwen, AM/i Arch, of Indust. Health 11: 47, 1955. 

The Effect of Gamma Globulin on Subclinical Infection in Familial 
Associates of Poliomyelitis Cases II. Serological Studies and Virus 
Isolations from Pharyngeal Secretions, G.C. Brown, A.S. Rabson, and 
J.H. Schieble, J, of Immunology 74; 71, 1955. 

C-Reactive Protein in Serum of Patients with Leprosy. A.S, Rabson, 
International J. of Leprosy 23: 155, 1955. 



16. 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1955. 



PAGE I 



PROJECT • REPORT FORM 

1. National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

3. Office of the Chief 4. 5. NCI 5-3^6— 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Attempt to induce carcinoma of the bladder in hamsters by infection with 
Schistosoma haematobium 

PROJECT TITLE ' 

7. Dr. Eloise Cram and Dr. Louis B. Thomas 

PRINCIPAL INVESTIGATOR(S) 

8. None - 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

This research project is in collaboration with Dr. Eloise Cram, NMI, and 
consists of studying chronic infection with S. haematobium (Gold Coast strain) 
in hamsters; particularly with reference to the induction of bladder 
carcinoma, 

(a) 217 hamsters were exposed to approximately 300 cercariae each early 
in 1955. 

(b) Approximately 177 infected hamsters are still living and will be 
allowed to live as long as possible, 

(c) Forty infected hamsters have died after 162-305 days after exposure 
to cercariae. These animals have all had heavy Schistosomal 
infection of the intestines and liver, but only slight infection 

of the bladder. Pathological study of these animals is incomplete 
at this time. No lesions suggestive of neoplastic change in the 
bladder have been seeo. 

Significance to cancer research: S. haematobium infection has been found 

associated with bladder carcinoma in several parts of the world and 
is thought possibly to be a cause of bladder carcinoma. This 
chronic infection study is possible because of Dr. Cram's work 
in getting S. haematobium established in hamsters. 

Proposed course of the project; 

(1) A continuation of the study of Gold Goast strain S. haematobium 
in hamsters, 

(2) Similar study of chronic infection with Egyptian strain S. haematobium 
when infected snails become available in Dr. Cram's laboratory. 



10. N CI »3e— ■ ■ 
SERIAL NO. 



11. 



PAGE II 



PROJECT REPORT FORM (Cont'd) 



!(>!ii3iO 



BUDGET ACTIVITY: 



RESEARCH /x/ ADMINISTRATION. . [J 
REVIEW & APPROVAL [j TECHNICAL ASSISTANCE [j 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



:.;fi=, ;!: 



-t sl^T 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS),' IDENTIFY SUqn RESEARCH : .; 



s t-ftoni'.- 



NO ENTRIES FOR ,14, 15, or 16 



PAGE I 

PROJECT REPORT FORM 

1. National Cancer Institute 2. Laboratory of Pathology 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. & Pathogenesis 4. 5. NCI 526 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Metabolism of Cortisol into higher hydroxylated derivatives in guinea pigs 

with and without neoplastic disease. [ 

PROJECT TITLE 

7. E. M. Nadel and S. Burstein 

PRINCIPAL IN\nESTIGATOR(S) 

8. None 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives: This title includes three sub-projects t (1) Isolation and 

elucidation of structures of new Cortisol metabolites in vivo 
(Dr. S. Burstein). (2) Study of enzymatic transformations involved in specific 
tissues. (3) The incorporation of C''-^ acetate into Cortisol and metabolites. 

Methods: The methods previously utilized include, extraction, paper and 
column partition chromatography, infra-red spectrometry, ultra 
violet spectrophotometry, incubation techniques using slices and homogenates 
of liver, adrenals, and other organs, use of radioisotopic techniques. 

Major findings; (1) 6-p hydroxycortisol and Steroid Ila (as yet unidentified) 

have been isolated from the urine of guinea pigs. (2) 6-p 
hydroxycortisol, Steroid 2 (as yet unidentified) tetrahydrocortisol have 
been isolated from the urine of man. (3) Radioacetate is incorporated in 
increased amounts into Cortisol and corticosterone in the adrenals of 
scorbutic guinea pigs, and both steroids have been identified for the 
first time in the tissue of the guinea pig. 

Significance to cancer research: This work provides the background material 

for the continuation and extrapolation of similar studies on the 
tissues of tumor-bearing guinea pigs. 

Proposed course: In this collaborative project we will endeavor to complete 

such studies during the coming year. Such a study is now 
feasible because of the closer association with Dr. S. Burstein on the 
reservation as a Visiting Scientist from the Worcester Foundation for 
Experimental Biology. 



PAGE II 



PROJECT REPORT FORM (Cont'd) 



10. NCI 526 



SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH M ADMINISTRATION [j 

REVIEW & APPROVAL /"7 TECHNICAL ASSISTANCE /~7 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER ORGANIZATIONS PRO- 
VIDING FUNDS^ FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. None 



IP THIS PROJECT RESEMBLES; COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH 



NO ENTRIES FOR 14, 15, or 16 



PAGE I 



PROJECT REPORT FORM 



1. National Cancer Institute 2. Laboratory of Pathology ■ '". ■ ■■. 

INSTITUTE LABORATORY OR BRANCH 

3. Cancer Indue. & Pathoaenesis 4. 5. NCI ^34:- 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Morphology, pathogenesis aihd tra'nsplantability of spdntanedus neoplasms 

within the canine species. . ., 

PROJECT TITLE 

7. Louise S. Lombard _^ . ___ 

PRINCIPAL INyESTIGATOR(S) 

8. None , ., ' ■ .".••;■•' •- ' - . ; ■' ■ ' ', ' ^ ^ [ ■ ' . ■ ■ ■' ^ .• •:- 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives: The objective of this project is to study the morphology and 
pathogenesis of spontaneously occurring neoplasms within the 
canine species and to obtain transplantable canine neoplasms which could 
be utilized in morphologic, chemotherapeutic, metabolic, endocrinologic 
and irradiation studies. 

Hethods: Through- the use of' cortisone and/or ^t^ irradiation, a 

spontaneous ianaplastic thyVo'id carcinoma was transplanted by 
various routes in heterologous puppies and studied motphologically. 

Major findings: The transplantable canine thyroid carcinoma was grown in a 

wide variety of tissues with growth occurring in almost 
1007o of the irradiated heterologous puppies. The morphology of the tumor 
remained essentially unchanged throughout the 35 serial transplant genera- 
tions. Lymph node and lung metastases were found in irradiated animals 
receiving either fresh or frozen tumor tissue (the thyroid carcinoma was 
preserved by storage at a -60 to -70°C. for several months). 

Significance to cancer research: Transplantable malignant neoplasms in the 

dog would offer tumors in a larger host for morphologic, 
metabolic, biochemical, hormonal, and irradiation studies, as well as 
including another species for the testing of chemotherapeutic substances. 
The transplantable canine thyroid carcinoma, intracerebrally inoculated, 
is now being used as a test tumor for the efficacy of radioactive boron in 
the treatment of brain tumors (Univ. of Penna.). 

Proposed course: The transplantable thyroid carcinoma will be transplanted 

to untreated, closely inbred puppies and in irradiated 
puppies receiving cysteine and bone marrow. Morphologic studies and 
transplantation experiments will be performed utilizing other spontaneous 
canine tumors, especially leukemia. 



PAGE II 



PROJECT REPORT FORM (Cotit'd) 



10. NCI ^a^- 
SERIAL NO. 

II. 



BUDGET ACTIVITY: 



RESEARCH . /x/ ADMINISTRATION r; ' 7_/ 
REVIEW & APPROVAL [j TECHNICAL ASSISTANCE £7 



12. School of Veterinary Medicine^ University of Pennsylvania Facilities, 

-Personnel (Dr» Mark Al lam,. Dean) 1956. . . . :— , 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE OR OTHER 'ORGANIZATIONS' PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 57 



13. None ' 



IF THIS PROJECT RESEMBLES, COMPLEMENTS,- OR PARALLELS RESEARCH DONE ELSEWHERE ' 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FAClLlTlEfS OR- 
FUNDS), IDENTIFY SUCH RESEARCH 



PAGE III 
PROJECT REPORT FORM (Cont'd) 



JTAy 

14. NCI- 521" " 
SERIAL NO. 



15. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 



Urbain Leblanc 

Early Veterinary Pioneer in Cancer Research 

J. Amer. Vet. Med. Assoc, 126: 363-365, 1955. 



16. HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955 

Society of Phi Zeta, May, 1955. 

American College of Veterinary Pathologists, Nov., 1955. 

Washington Society of Pathologists, Nov., 1955. 

Conference of Research Workers in Animal Diseases in North America, Dec, 1955, 



Note : Collaborative research is being done with Drs. H. L. Stewart and 
H. B, Andervont on adenomatous gastric lesions in strain I mice; 
with Dr. W. R. Bryan on rapid and slow growing Rous Sarcoma; with 
Dr. H. P. Morris on experimentally produced pituitary adenomas and 
hepatomas in rats. 



M'^ 



ii;-'-;joru 



.1 Ti>SL-o>j; 



;r ■ r.^jj'^: ^^'iii-v^ ^iiroAirff'ii/^ <. 



PAGE 1 
PROJECT REPORT FORM 



1,' National Cancer Institute 2, Radiation Branch 

INSTITUTE L.'.BOPJiTORY OR BR/UMCH 



Radiation Biology Section h» 5» 62U 

SECTION OR SERVICE LOCATION (IF OTHER THi'.N BETHESD/J SERI/iL 

NO. 



6, Description and Treatment of the High-Radiation-Doso Syndrome 
PROJECT TITLE 

7« H. L. Andrews and K. C. Brace 
PRINCIP/iL INVESTIGATQR(s) 



8. H. Gm p 



OTHER INVESTIGATORS 

9, PROJECT DESCRIPTION 

Objectives ; 1, To characterize the syndrome produced by x-ray doses up 
to about 100 times that lethal in 30, days. Emphasis in this project is 
on function rather than on pathology, 2, To study agents capable of 
modifying the high-dose syndrome, 3. To study the effect of dose rate 
and fractionation on the high-dose syndrome, , 

Methods employed ; Doses from 1000 r upward are administered with the 3 Mev 
x-ray generator. The guinea pig is the animal of most interest because of 
the sharp break in signs of radiation injury at 6^000 r. Typical studies 
made before and after radiation are blood coimts, blood electrolytes, pain 
threshold^ pinna reflex, electrical impedance of body tissues. Survival 
time s are carefully recorded. 

Major findings ; Using 200 KVP x-rays at 55 r per minute we have found in 
the guinea pig: 

1, At 6,000 r there is a sharp change from a 5 day death in depres- 
sion to a death in less than 2lt hoiars with marked signs of increased 
central nervous system excitability, 

2, lAJhen barbiturates are given prior to irradiation, irradiation of 
even 15,000 r produces only the depression normally seen with less than 
5,000 r and the survival time is about k days instead of the 1 day or less 
obtained ^^^ithout medication, A series of depressant and anti-convulsant 
drugs are without effect on survival time although some prevent the appear- 
ance of the high-dose syndrome. 

Significance to cancer research ; Any increase in knowledge of the biological 
effects of radiation are of potential value in radiation therapy. It is of 
interest that the large doses used in this project are not large in terms of 
local doses delivered for therapeutic purposes. 

Proposed course of project ; Much of the work already done will be repeated 
using the 3 Mev generator to obtain a greater relative radiation dose to 
underlying structures in the central nervous system. The high dose-rates 
obtainable with this generator i\Tiii be studied for biological effectiveness 
and will permit an extension of studies of agents modifying the high-dose 
syndrome. 



lo.Nei- 



62h 



SERL'JL MO. 



PAGE 2 



11, BITOGET ACTIVITY: 

Research - /C7 
Review & Approval /~7 



Administration /^ 
Technical Assistance /~7 



12. COOPEIL'.TING UNITS OF THE PUBLIC HE/iTH SERVICE, OR OTHER ORGi.NI CAT IONS, 

• PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PliOJECT IN EITHER 
1956 or 1957: 

None 



13. IF THIS PROJECT RESE!'IBLES, COi^IPLEIlENTS, OR PiiR.'XLELS RESEj^CH DOI^ 

ELSEVJIIERE IN THE PUBLIC HEALTH SERVICE (WITHOUT IKTERCaiNGE OF PERSOl'MilL, 
FACILITIES OR FTOIDS), IDENTIFY SUCH RESE/JICH: 

None 



NO ENTRIES FOR ITEMS U4, l5 & I6, 



PAGE 1 
PROJECT REPORT FCRIl 

1, National Cancer Institute 2. Radiation Branch 



INSTITUTE LABORATORY OR BRANCH 

3« Radiation Biology Section h^ , 5. NGI-627 

SECTION OR SERVICE LOCATION(IF OTHER THAN BETHESDA; SERIAL MO, 

6, Change in Tissue Constituents by Radiation 
PROJECT TITLE 



7. H, L, Andrews and E, J. Liljegren 
PRINCIPAL INVESTIGATOR(S; 



None 
OTHER II\rVESTIGATQRS 



9, PROJECT DESCRIPTION 

Objectives ; To determine changes in amino acid concentrations produced by 
x-irradiation in various body tissues of the guinea pig. 

Methods employed ; Assays of various tissues were made by separation 
columns and paper chromatography. Normal ahimals were compared with 
those receiving various doses of x-rays« 

ria.ior findings ; Experimental work has been completed and the results 
are being analyzed. There are changes in amino acid concentrations but 
it is premature to discuss them in detail now. 

Significance to cancer research : Any increase in our knowledge of the 
biological effects of radiation is of potential importance in radiation 
therapy, 

Proposed course of project ; Unless the data are more striking than 
presently appears this project will be terminated with publication of the 
findings « 



10, NCI.627 

SERIAL NO. 



PAGE 2 



11. BUDGET ACTIVITY: 

Research /^ 
Review & Approval f^ 



Administration /V 
Technical Assistance £J 



12. COOPERATING UI^ITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONIffiL FOR THIS PROJECT IN EITHER 

1956 OR 1957. 

None 



13.. IF THIS PROJECT RESEMBLES, CQlPLEIffiNTS, OR P/iRALLELS RESEARCH DONE 

ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 

None 



NO EOTRIES FOR ITEMS li;, l5-& l6i 



PAGE 1 



PROJECT REPORT FORPI 



1, National Cancer Institute 2» Radiation Branch 

., INSTITUTE * LABOR/iTORY OR BRilNCH ■ 

3. Radiation Biology Section h, ___, 5« NCI>628 

SECTION OR SERVICE LOCATION (IF OTHER TH/JM BETHESDAJ StlRI/iL NO, 

6, Dosimetry of High Energ;y Radiations 
PROJECT TITLE 

7* H. L, Andrews 

PRINCIPAL INVESTIGATOR ( S ) 

8. R« E, Murphy 



OTHER INVESTIGATORS 



9k PROJECT DESCRIPTION 

Objectives : To develop methods and instruments for determining the radia- 
tion doses delivered to tissues at energies up to 3»S Mev and at high dose 
rates. 

Methods employed : The usual types of ionization chambers used for radiation 
dosimetry are not designed to read correctly at the x-ray energies avail- 
able from the 3*5 Mev generator and are probably incapable of measuring 
the high intensities anticipated when the generator is run at full output. 
Chemical dosimeters appear to have a response independent of photon energy, 
and to be capable of accurately recording high dose rates. Work has con- 
centrated on two chemical systems, the production of HCl from chloral 
hydrate and the oxidation of ferrous iron to ferric, 

i^fa..1or findings : As an extension of the main problem the chloral hydrate 
system has been adapted to depth-dose determinations by the addition of a 
gelling agent. With this dosimeter a beam of radiant energy can be visual- 
ized and measurements of local radiation doses made with a probing pH 
electrode. The gel absorbs radiation almost exactly as does water, and 
hence its response will be a good indicator of tissue dose in complex 
structures not amenable to calculation or to measurement with other methods. 

Significance to cancer research ; A most basic requirement for good radia- 
tion therapy is that the tumor dose be made as high as possible relative t« 
the dose delivered to healthy tissue. Any method which can improve the 
measurements of dose delivered to deep body struct-ures should improve the 
ability of the therapist to keep the tumor/tissue dose ratio high. 

Proposed course of project ; Measurements at high dose rates have not been 
made because of target failures when attempts were made to operate the 
generator at high power. As this difficulty is overcome chemical dosimetry 
will be applied to high dose rates and to short pulses of both x-rays and 



PilGE 2 



MCI-628 

SERI/iL NO, 



PROJECT DESCRIPTION (CONT.) 

elGctrons, These measurements- will then provide a basis for the extension 
of research in radiation biology into a dose-rate range never before reached 
in ,the. laboratory. 



10. MCI-628 
SERL'iL NO, 



PAGE 3 



11, BUDGET ACTIVITY: 

Research /T/ 
Review & /^jproval /V 



Administration ^£7 
Technical Assistance /^ 



le, gooperj;tihg mits of the public he/xth service, or other orgaotzations, 

PROVIDING FUIJDS^ FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 or 1957: 

None 



13. IF THIS PROJECT RESEMBLES, COMPLEffiNTS, OR PjJUXLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEj'XTH SERVICE (WITHOUT INTERCHi^NGE OF 
PERSONNEL, FACILITIES OR FUIffiS), IDENTIFY SUCH RESE/JICH: 

None 



NO ElMTRIES FOR ITEIIS lij, 15 & l6. 



PAGE 1 



PROJECT REPORT FORM 



1, National Cancor Institute 2, Radiation Branch 

INSTITUTE L'xBOPu.TORY OR BRilNCH 

3. Radiation Biology Section h, g. MCI-629 

SECTION OR SEliVICE LOCATION (IF OTHER TIL'iN BETHESD/J ' SERIAL WO, 

6, The effect of ionizing radiation on amino acids 

PROJECT TITIE 

7. Charles R. Maxwell 

PRINCIPAL INVESTIGATOR(S; 



^« Dorothy C, Peterson 



OTHER INVESTIGATORS 



9, PROJECT DESCRIPTION 

Objectives; The objective of this project is to determine the mechanism 
of the chemical reactions induced by ionizing radiation in aqueous solu- 
tions of amino acids. This is part of a long range progrrj-n to accmnulate 
information on siraple systems of biological interest so that general 
principles may be ascertained and applied to complex systems which are 
not amenable to thorough, direct investigation. 

Methods employed; Solutions of rmino acids are irradiated with ionizing 
radiation and then analyzed for the products formed. Since the products 
are sensitive to radiation it is necessary to determine the yield of 
each product as a function of dose to as low a dose as possible and to 
extrapolate these values to obtain the initial yield of the product at very 
low doses before secondary reactions distort the picture. 

Most of the study is done with $0 iW X-rays as the ionizing radiation. 
The effect of dose rate and ion density is investigated by irradiating 
with electrons and alpha particles. Protons and neutrons will probably 
be used in the , future. 

Ma j or findings ; Earlier work has shown that x-rays induce four reactions 
in aqueous solutions of both glycine and alanine. One of these reactions 
was shoT>rn to result directly from the absorption of energy by the dissolved 
moleculej the other three were shown to be indirect, e,g,, the res'ult of 
energy absorbed by the water which is generally considered to produce H and 
OH free radicals and H2O2. 

The work this year has been concentrated on studying the role of the 
above active intermediates, 

1) Irradiations in the presence of dissolved oxygen have shown that the 



Page 2 

NCI-629 
SERI/X NO, 



PROJECT iiEPORT FOM (Cont.) 

reductive doamination of glycine to acetic acid is the action of the H free 
radical, . 

2) Experiments using OH free radicals produced chemically from Fe"*"*" and 
H2O2 have shown that the oxidative deamination of glycine to glyoxalic 
acid is the action of the OH free radical* 

3) Failure to observe the formation of formaldehyde as the result of 
OH radicals distributed uniformly in the solution by the reaction of 
Fe"*"*" and H2O2 has lead to the conclusion that the reaction producing 
formaldehyde is peculiar to the high local concentrations of radicals 
along the discrete tracks of ionizing particles, 

k) Investigation of the effect of alpha particles from Po^-^ upon 
aqueous solutions of glycine has shown that this densely ionizing particle 
induces the same reactions as docs x-rays and electrons but in a different, 
ratio. The relatively high yield of formaldehyde is in agreement with 
finding 3). 

5) The kinetic measurements associated with obtaining result 3) have 
provided considerable insite as to the mechanism of the "catalytic" 
action of Fo"*"*" ion on the action of H2O2 on aiuino acids and will result 
in a separate publication. 

Significance to cancer researc h; Although not directly connected with 
Cancer research, it is of great interest because it seeks an understanding 
of the mechanism for the effects of radiation which is used empirically 
as a tool in the clinical treatment and laboratory study of cancer. 

Proposed course of project; Continuation of the work will be directed to 
completing thu glycine and alanine investigation and extending the work 
to the, effect on more complicated amino acids. 

Specifically; 

1; The mechanism for the large effect of very small concentrations of 

dissolved oxygen upon the glycine reactions will be studied, 

2) Measvirements upon the influence of glycine concentration and of 
temperature upon the relative frequency of the various reactions will be 
refined, 

3) Studies upon the effect of pH upon the reactions will be made, 

k) Studies upon the. effect of x-rays on phenylalanine and tyrosine will 
be initiated. 



PAGE 3 



PROJECT ICEPOKT FOlttl (Cont.) 



10. MCi-629 

SmiiiL NO, 



11. BUDGET ACTIVITY: 

Research /X/ 

iieview & Approval /^ 



Administration // 

Technical Assistance /~7 



12, COOPERi'JING UiMITS OF THE PUBLIC Haj;j.K SEIIVICE, OR OTHER ORGANIZilTIONS, 
PROVIDING FUNDS, FACILITIES, OR PEi"iSOm\JEL FOii THIS PicOJECT IN EITHETt 
1956 OR 1957: 

None 



13, IF THIS PROJECT IcESiilMBLES, CQ'IPLEMENTS, Oi P;ju.LLELS RESEiffiCH DOME 
SLSEVJHERE IN THE PUBLIC HEi'.LTH SERVICE (WITHOUT INTERCHANGE OF 
PETiSONHEL, FACILITIES OR FU1\^DS), IDENTIFY SUCH I^SEi'.RCH: 

None 



PAGE k 
PROJECT ilEPOItT FQiHi (Cont.) 



lii._NCI-629_ 
SERIAL NO, 



15, PUBLia.TIONS OTHER THAN ABSTR/lCTS FROM THIS PiiOJECT DURING CALE^D;il 
YEAR 1955: 

The Effect of Ionizing Radj.ation on /imino Acids 

II,' The Effect of X-iiays on Aqueous Solutions of Alanine, 

N. E. Sharpless, A. E. Blair and C, R, Maxwell, Radiation 
Research 2, 135-lJ-ti4 (1955) 

III, The Effect of Electron Irradiations on Aqueous Solutions of 
Glycine, C, R. Majaiell, D, C, Peterson^ and W. C, VJhite, 
Radiation Itesoarch 2, h31-k3Q> (1955) 

16, HONORS AND AWJiDS TO P:'iR30Hl\IEL RELATING TO THIS PROJECT DUIiING 
C/iLEND/Ji YEiui 1955: 

None . 



PAGE 1 



PROJECT REPORT FOiM 



1, National Cancer Institute 2, Radiation Branch 

INSTITUTE LilBORATORY OR BR^INCH 

3. Radiation Biology Section k, . 5» NCI-63I1 

SECTION OR SERVICE LOCATION(IF OTHER THAN BETHESDAj SERIAL NO* 

6t Late effects of irradiation in anijnals protected from early death 

by bone marrow or antibiotic treatment 

PROJECT TITLE 

7» Willie W, Smith, pathological studies by George Brecher (Clinical 

Center), Katherine Snell, Richard Swarm 

PRINCIPAL INVESTIGATOR(Sj 

8, Ilo M. Alderman, Ruth Gillespie 
OTJffiR IWESTIGATOR ( S ) 



9, PROJECT DESCRIPTION 

Objectives! To determine the extent to which life expectancy is reduced 
in animals which have been exposed to severely damaging radiation and then 
treated in such a way as to prevent early death from infection, hemorrhage 
or anemia and to determine where possible, the cause of death in these animals. 

Methods employed; Animals exposed to radiation which causes death in 
the absence of treatment are treated with bone marrow or streptomycin 
and are then observed for the remainder of their lives. At death 
autopsies are performed and histopathological studies made. Various 
treatments are applied with the object of prolonging life. 

Ma j or findings ; (l) In hamsters surviving the first month following 
exposure to x-radiation the median survival time appears to be linearly 
related to the radiation dose rather than to the proportion surviving the 
first month. Treatment with streptomycin or marrow appears to prevent 
early death but not to promote total recovery, (2) Three treatments 
applied to hamsters surviving a month after irradiation have failed to 
alter significantly the survival time, (3) Histopathological studies 
have shown a high incidence of gastric ulcer, pneumonia, vascular and 
renal changes and neoplasm, depending upon the initial radiation dose 
and the time of death. 

Significance to Cancer Research; (l) An evaluation of the total 
efficacy of treatments which may be used to reduce the deleterious effects 
of therapeutic treatment with x-ray; (2) an estimate of the incidence 
and type of neoplasm which may be expected after whole-body exposure 
to varying doses of radiation; (3) an estimate of the physiological condi- 
tion of the animal after exposure to varying doses of radiation. 



PAGE 2 

SEilKL NO, 
PROJECT iiEPaiT FOrxl'I (COW.) 



Proposed cou rs o ox project; In addition to continuing this project 
r.long the lines indicated above, functional tests will be applied to the 
animals surviving various exposures and treatments. These will probably 
include resistance to oxperiraental infection, ability to mobilize leuco- 
cytes, ability to respond with polyerythcmia to repeated exposures to. ' 
hypoxia, resistance to toxins and possibly liver and kidney function 

"t/GSuS « 



PAGE 3 



PROJECT REPORT FORM (CONT.) 



10. NCI-63I1 

SERIAL NO. 



11, BUDGET ACTIVITY: 

Research /^ 

Review & Approval f~] 



Administration // 
Technical Assistance /V 



12. COOPERATING UlilTS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOIi THIS PROJECT IN EITHER 
1956 OR 1957: 

Dr. George Brccher, Clinical Center Pathologist, 



13, IF THIS PROJECT RESEMBLES, GOMPLPilENTS, OR P.jRj'.LLELS RESE/JiCH DONE 

ELSEWHEiiE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, 
FACILITIES OR FUITOS), IDENTIFY SUCH RESK'JICH: 

None. 



PAGE li 
PROJECT REPORT FOmi (CONT.) 



lii. NCI-63li 
SERIAL NO, 



15, PUBLICATIONS OTHER THAN ABSTIi..CTS FROfl THIS PROJECT DUIiING C;.LENDjdR 
YE/Jl 1955: 

X- Irradiation in Hamsters r-nd Effects of Stroptoinycin end I'larrow- 
Spleen Horaogenr.te Treatment, W. W. Smith, R, I, I'fcrston, L, Gonshery, 
I. M, Alderman and H, J, Ruth, Im. J. Physiol. l83, 98 (1955). 

16. HONORS AND AWARDS TO PERSONNEL RELVTING TO THIS PROJECT DURING 
CALEND/Jl YEIJi 1955: 

None, 



PAGE 1 

PROJECT REPORT FOIiM 

1, National Cancer Institute 2, Radiation Branch 

INSTITUTE LABOjrt.TORY OR HRi'.NCH 

3, Radiation Biology Section h, " g, NCI-636 

SECTION OR SERVICE .LOCATION (IF OTHEii THAN BETHESDA; SEiilAL NO. 

6, The Lethal Effects of Visible Radi a tion on a Strain of Haploid Yeast 

PROJECT TITLE 

7, Dr. Mort i mer M. Elkind 

PRINC.LTi.L INVESTIGATOR(S j 

8, None 

OTHER IfJVESTIGATORS 



9, PROJECT DESCRIPTION 

Objectives; The objectives of this investigation are to delineate the 
parameters which control the sensitivity of yeast cells to visible 
radiation, and, where possible, to correlate the effects observed with 
processes within the cell. 

Methods employed; Actively growing (log phase) yeast cells (haploid 
Saccharomyces cerevisiac) are harvested from a liquid growth medium 
(yeast extract plus dextrose), washed by centrifugation, and resuspended 
in a potassium phosphate buffer. Immediately after resuspension, the 
cells are essentially insensitive to the emission from a 300 watt, incandes- 
cent lamp slide projector (3^0 m/u to 750 m/u), VJith time, the population 
becomes increasingly light sensitive to this emission as measured by the 
ability of the cells to grow into visible colonies x^rhen plated on agar 
containing growth medium. The cells are irradiated in the same buffered 
solution and are kept at a temperature of from 1,0-2, Co C during irradiation. 

Jfcjor findings; A, For a given composition of the buffer solution and a 
given temperature of storage, with time the colls become progressively more 
light sensitive, B, For the saiue length of time of storage the sensitivity 
is strongly dependent on the temperature of storage. For instance, 2 hrs, 
of storage at 30° C will produce the same sensitivity as about 28 hrs, 
storage at l-g-° C, C, For the same length of time and temperature of 
storage, the sensitivity is strongly dependent upon the pH of the buffer 
solution and to a lesser extent upon the molarity of the buffer solution, 
D, Suppression of oxygen tension of the buffer solution at the time of 
irradiation decreases the sensitivity, E, CompoTed to log phase cells, 
resting cells do not develop appreciable light sensitivity in the course 
of storage. 



PAGE 2 
NCI -6 36 
SERI/iL NO, 

PROJECT REPORT FORf'f (Cont.) 



S ignificr.ncG to cancer research; This research is a phonoinonological 
study' ox a process present in a biological system which, if at all, has 
not received rauch attention in the past. In addition to the technical 
need for a knox^rledge of the scope and extent of this effect as it might 
present itself a,s an artifact in other radiation studies with this organism, 
this work is related to oancer research as basic biological research in 
general is so related. 

Proposed course of project; 0[3timumly the culJiiination of this work would 
probably consist of an identification of the biological processes responsible 
for light se;isitivity. With this in mind it is planned to complete the 
exploration for the apparent pertinent parameters involved, and to examine 
the action and absorption spectra of these cells within the limitations 
of available equipment and techniques. 



10, NCI-636 
SERIAL NO. 



PAGE 3 



11. BUDGET ACTIVITY: 

Research f^ 
' Review & Approval / / 



Administration fl 
Technical ^issistance ri 



12, COOPEitATING UNITS OF THE PUBLIC 1Ij^.AL^j:'H SERVICj^], Ou OTHEA ORGANIZATIONS, 
PROVIDING FUNDS, I'ACILITIES, OR PERSONNriL FOR THIS PROJECT IN EITHER ' 
1956 wR 1957: 

None 



13. IF THIS I'ROJECT RESEMBLES, CQIPLEIIENTS , a.. PiR^ALLELS RESEJJiCH DONE 
ELSafflERE IN THE PUBLIC HE^'XTH SERVICE (WITHOUT INTERCHANGE OF 
PEliSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEiARCH: 

None 



PAGE k 



1^* MCI-636 

SERI/J. NO. 



1^. PUBLICATIONS OTHEii THj-il\I ABSTIiACTS FROil THIS PK0J3GT DURING CiiEND/Ji 
., TEiiR 1955: 

Mortimer M, Elkind and Carl A, Beam, "Variation of the Biological 
Effectiveness of X-Rays and Alpha-Particles on Haploid Saccharomyces 
cerevisiae j" Radiation Research , 3, 88-1 OU (1955). 

16. HONORS AND AW.mDS TO PERSONNEL REUJING TO THIS PROJECT DURING 
C/iLEND/J{ YE/iR 1955: 

None « 



PAGE 1 
PROJECT REPORT FORM 



1, Mational Cancer Institute 2, Radiation Branch 

INSTITUTE L/iBOFu'^TORY OR BR/J^CH 

3, Radiation Biology Section k» 5» NCI-637 

SECTION OR SERVICE LOCi.TION(IF OTHER Tm'.N BETHESDA; SERI/J. NQ, 



6, Development of High-Intensity X-ray Sdiifce 
PROJECT TITLE 



7i H. L, Andrews _«___^ 

PRINCIP/iL I1^VESTIGAT0R(S; 



R. E. Murphy 



OTHER IIWESTIGATORS 



9. PROJECT DESCRIPTION 

Objectives ; To obtain from the 3 Mev generator the x-ray output which it 
should be capable of producing. 

Methods employed ; The 3 ^'fev Van de Graaff generator given to NIH by the 
Liggett and i'lGycrs Tobacco Co. is capable of producing x-rays at intensities 
never before reached in the laboratory. It had never been used as an x-ray 
generator and when attempts were made to utilize its capabilities x-ray 
targets failed by melting. Failures occurred at only about l/k of full 
power and hence the possible generator capability is seriously restricted. 
Since target fadlure allows cooling water to enter the accelerating tube 
each failure represents a minor disaster. Pending remedial steps the 
generator has been operated conservatively to insure continuity of service. 
Improved operation is to be expected from: 1, higher voltage operation, 
2, high frequency target scanning, 3. use of targets of high atomic number, 
hi improved target cooling. 

Major findings ; A study of failures of the gold targets, and the general 
theories of x-ray production have suggested the following: 

1, Since the efficiency of x-ray production increases with voltage 
operation at the highest possible voltage will give increased output for 
equal target heating. By careful attention to details we have raised the 
routine operating voltage from 3»0 J'lev to 3.5 I'fev, For speeial purposes 
operation at 3,8 Mev appears possible but can not be counted on for daily 
use, 

2. All target failures appear to be due to a burst of high current 
lasting for perhaps one microsecond. If this is the case sweeping the 
incident electron beam over the face of the target at very high speed 
should reduce lofal heating. Preliminary experiments with 300 kilocycle 
scanning indicate its feasibility and indicate the direction for future 
equipment design? 



FIJJE 2 



NCI-637 
SERIAL NO. 



PROJECT DESCRIPTION (CONT.) 



3» Since x-ray production is proportional to the atomic number of 
the target material steps have been taken to replace the gold (Z=79) 
targets with thorium (Z=90), Thorium .targets have been obtained from the 
AEC, and will be installed when suitable welding techniques are proven 
satisfactory, 

U. Delivery of cooling water to the back surface of the target has 
been improved and steps are planned to reduce the trauma caused by target 
failure. 

Significance to cancer research ; With the high outputs potentially 
available from this generator radiation doses can be administered at dose 
rates never before available. It is possible that quite ' different biological 
results will be obtained when a given radiation dose is given at say 10,000 
r/rain rather than at the more usual ^0-100 r/min. 

Proposed course of pro.jcct i These were covered under "major findings," 

This project moves slowly to avoid disruption of existing radiation schedules 

but within 6 months operation at full generator power can be expected. 



PAGE 3 



10. "CI-637 

SERIiJ. NO, 



11. BUDGET ACTIVITY: 

ResesTch /TJ 
Review & Approval ri 



Administration f^ 
Technical Assistance f~] 



12, CDOPERATING UNITS OF THE PUBLIC HEALTH SERVIClI, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSOMEL FOR THIS PROJECT IN EITHER 
1956 OR 1957: 

None 



13. IF THIS PROJECT RESEl'lBLES, COIiPLEi'ISNTS, OR P/HALLELS RESE.'JICH DONE 
ELSEVJHERE IN THE PUBLIC HE.'.LTH SERVICE (WITHOUT liWERCHANGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESE/JICH: 

None 



NO EI\ITRIES FOR ITEMS II4, l5 & I6, 



PAGE 1 



PROJECT REPORT FORM 



1, National Cancer Institute 2, Radiation Branch 

INSTITUTE Li^BOR/xTCEY OR BR/.NCH 

3» Radiation Biology Section h, __, ^ 5. NGI-638 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDAj SERIilL NO. 

6, A study of the neuropathology of massive doses of x-rays in the 
guinea pig 



PROJECT TITLE 



7. Kirkland C. Brace 

PRINCIP/iL INVESTIGAT OR ( S ) 



8« Howard L. Andrews 



OTHER IIWESTIGATORS 



9, PROJECT DESCRIPTION 

Objectives ; To determine the effect of massive doses of x-rays on the 
central nervous system of the guinea pig. 

Methods employed; Guinea pigs are exposed to various doses of x-rays 
and the brains removed at various intervals after the exposure. The 
tissues are examined microscopically to determine the time course of 
and type of pathology that could be observed. 

Major findings; The only readily observable changes with doses of less than 
25 J 000 r of x-rays are in the granule cells of the cerebellura. Doses of 
less than 6,000 r do not show any changes in the central nervous system. 
Doses of in excess of 6,000 r produce a peculiar pyknosis of the granule 
cells which appears about an hour after the exposure at a dose rate of 50 r 
per minute. Almost 90 per cent of the cells are involved after 8 hours. 
Death occurs at about 2k hours. The neurological symptoms observed are 
closely associated with the amount of pyknosis present. 

Significance to Cancer Research; It has long been reported that the non- 
dividing cells of the central nervous system are higlily resistant to radia- 
tion. It appears from this data that the cerebellum is particularly radio- 
sensitive in the guinea pig. This may explain the particular sensitivity of 
the medulloblastoma which is derived from the same anlage as the granule 
cells. 

Proposed course of project; With the availability of new equipment we 
expect to repeat this study at much higher dose rates to determine if 
there is any change. We also plan to extend the study to some other species 
of animal. 



PAGE 2 



PROJECT REPORT FCRii (cont.) 



10, NCI-638 

SERIAL NO. 



11, BUDGET ACTIVITY: 



Research /6c7 
Review & Approval f] 



Administration /~] ': 
Technical Assistance ^ /~7 



12. COOPEIL\TING TOJITS Oi^ THE PUBLIC HE/iLTH SERVICE, OR OTHER ORG/J^J'IZATIONS, 
PROVIDING FIMBS, FACILITIES, OR PEi^SOWNEL FOR THIS PROJECT IN EITHER 
19^6 OR 1957: . 

National Institute of Mental Health 
(This project was reported last year by NINDB, lA that time Dr. Aivord 
was principal investigator along with Dr. Brace, Dr. Aivord has left NIH, ) 

13, IF THIS PROJECT RESEMBLES, CaiPLEi'IENTS, OR PAR,'.LLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HE/iLTH SERVICE (WITHOOT INTERCHANGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEi.RCH: 

None I 



NO ENTRIES FOR ITEflS lli, l5 & 16, 



PAGE 1 

PROJECT REPORT FORM 

1, Mationg.l Cr.ncer Institute 2, Radiation Branch 

INSTITUTE UBOII/.TORY OR BR.'iNCH 

3. Radiation Biology Section h. 5. NCI-639 

SECTION OR SEIWICE LOCATION (IF OTHER TffixN BETHESDiJ SERIAL NO. 

6 . A study o.fetiiT:.-ULUiUJijja ^aB3^a§y o f life span of the nucleated red cell 

PROJECT TITLE 

7. Kirkland C, Brace 



PRINCIPAL INVESTIGATOR! S) 



8, Paul D. Altland, NliJ'ID 
OTHER INVESTIGATORS 



9, PROJECT DESCRIPTION 

Pro.ject; A study of the life span of nucleated erytlirocytes of the birds, 
reptiles, and amphibians using dU, 

Objectives; A better understanding of the remarkable resistance of the 
lower forms and particularly the poikilotherms to the effects of ionizing 
radiation. 

Methods employed; The erythrocytes of various species of animal are 
labeled in vivo by the intravenous, or intraperitoneal injection of glycine- 
2-Cli4, The methyl carbon is incorporated into the hemoglobin of the 
erythrocyte and is carried with the coll until it is destroyed. The 
determination of the life span is based upon the interpretation of the 
fall in the specific activity of the hemoglobin following injection of 
the glycine, 

!lajor findings; The life span of the bird erythrocyte has been determined 
by several other methods. We have repeated the determination to check 
the validity of our method. The value of I|.2 days we obtained for the 
duck erythrocyte confirms previous work. The specific activity of the 
hemoglobin of the box ti:irtle remains almost unchanged after 500 days 
after injection and the specific activity of the hemoglobin of the toad 
remains unchanged after 270 days after injection. We can only interpret 
this data as showing that these cells are extremely long lived. 

Significance to Cancer Rasoarch; An understanding of the effects of 
ionizing radiation on a cell depends on knowing the normal physiology of 
the cell. Those extremely long lived cells of the poikilotherms may 
partially explain the resistance of these cells to effects of radiation and 
could explain the general radio resistance of the animal. 



PAGE 2 



serl;l no, 

PliOJECT REPORT FORM (corit.) 



Proposed course of project: The work on the turtle and toad has been set 
up to continue for several more years, We hope to determine if a finite 
life span of these cells does exist. If possible, this study will be 
extended to somo other species especially the fish. 



PAGE 3 
PROJECT REPORT FORM (cont.) 



10. NCI^639 

SERIJ.L NO. 



11, BUDGET ACTIVITY: 

Research /S7 Aijministration / / 

Review & Approval f~ ] Technical Assistance / / 

12, COOPERATING UNITS OF THE PUBLIC HEALTH SElRVICE, OR OTHEit ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 OR 1957: 

National Institute of /arthritis and Metabolic Diseases 



13, IF THIS PROJECT RESEI'IBLES, CQiPLEi'lENTS , Ou P;j"ulLLELS RESEiu'iCH DONE 
ELSEVniERE IN THE PUBLIC HEiXTH SERVICE (WITHOUT INTERCHANGE OF 
PERSONNEL, FACILITIES OR FLWDS), IDENTIFY SUCH RESJiU'iCH: 

None 



1$. PUBLICATIONS OTHER TH.uN ABSTRACTS FRQl THIS P.tOJECT DUilING Ci.LENDiJi 
YE.'-at 1955: 

I^rkland C. Brace and Paul D, Altland, 7ted Cell Survival in the 
Turtle, The i'merican Jom-nal of Physiology 183 , 91 (1955 )» 

16, HONOItS AND AWixRDS TO PERSONNEL REL/.TING TO THIS PiiOJECT DURING C;XJ»JD;jt. 
lE/ii 1955: 

None, 



PAGE 1 



PROJECT REPORT FORM 



1, National Can c er Institute 2, Radiation Branch 

INSTITUTE L/.BORjVrORI OR BR/.NCH 

3. Radiation Biology Section k, ^ 5, ^^^-^^Q 

SECTION OR SERVICE LOC;iTION(IF OTHER TH/J BETHESDAj SERLIL NO. 



6, The effect of x-radiation on the anaphyla ctic response in mice 
PROJECT TITLE 



?♦ Falconer Smith __^_^ 

PRINCIPAL IIWESTIGATOR ( S ; 

8 , t'larie M. Grenan, (Hazel P. G vunp, not presently attached to this p roject) 
OTHER INVESTIGATORS 



9, PROJECT DESCPIPTION 

Objectives ; a) Obtain a quantitative expression in terras of combining 
ratios of antigen and antibody for the increases susceptibility of the 
irradiated mouse to anaphylaxis, b) Laprovo the tissue specificity of 
leucocyte and other tissue antigens and study the effects of their 
homologous antibodies on irradiLated mice. 

Methods employed; The responses of passively iminunized, irradiated mice 
to varying concentrations of antigen are tested using hen's egg albumin 
and its homologous antiserum (rabbit) as test prepare.tions. In addition, 
tests are carried out with mice using intravenous injections of mouse- 
leucocyto antiserum and erythrocyte preparations. 

Major findings; Rabbit, mouse leucocyte antiserums (prepared from peritoneal 
exudates) given intravenously is more harmful to irradiated mice than to 
their nonirradiated controls. Since a similar result was observed with 
hen's egg albumin antiserum, additional v'jtudies of a quantitative native, 
using this preparation will be made. 

Significance to Cancer Research ; Anaphylaxis is a coinmon response to 
protein by appropriately sensitized mammalian tissue and is apparently 
enhanced by radiation. It is considered possible that specific tissue 
sensitivity can be obtained which in turn may produce an additive effect 
when combined with x-radiation. In addition, studies of the effects of x- 
rays on the anaphylactic response jirovide information on the biochemical 
behavior of the reactive tissue. 

Proposed course of project; Improvement in specificity of tissue antigens 
by the isolation of specific cell types, the preparation of antisera to 
these and testing of the antisera will occupy a major portion of the 
calendar year assigned to this project. 



PAGE 2 



PROJECT REPORT FORM (cont.) 



]_0, NCI-6iiO 



SERI/J. NO. 



11. BUDGET ACTIVITY: 



Research fxj 
Review & Approval f~J 



Administration £J 
Technical Assistance f~] 



12, COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUl^DS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER , 
19^6 OR 1957: 

No other units cooperating, 

13. IF THIS PROJECT RESEt'lBLEG, COMPLEMENTS, OR PARi'iLLELS RESK.RCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (VJITHOUT INTERCILa\lGE OF PERSONNEL, 
FACILITIES OR FU1\IDS), IDENTIFY SUCH RESE/JICH: 

No knowledge of parallel studies elsewhere. 



NO ENTRIES FOR ITEMS lit, \$ & l6. 



PAGE 1 
PROJECT REPORT FORJl 



1, National Cancer Institute 2, Radiation Branch 

INSTITUTE LIBORATORY CR BRANCH 

3, Radiation Biology Section h* g, NCI-6Ul 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDAj SERIAL 

.. NO. 

6, The Effect of Irradiation on Susceptibility to Viral Infections 
PROJECT TITLE 



7. Willie W. Smith, Bernice Eddy (L.B.C.) 
PRINCIP/vL INVEST IGATOR(S) 



8, Ilo M, /ilderman, Ruth Gillespie 
OTHER IIWESTIGATORS 



9, PROJECT DESCRIPTION 

Objectives; To deterraine whether or not exposure to x-radiation alters 
susceptibility to various viral infections. 

Methods employed; The response to challenge with poliomyelitis or 
influenze virus in mice given just sublethal irradiation is compared 
with that of controls. 

Major findings; The results thus far indicate that mice exposed to 
just sublethal radiation are no more susceptible to the challenging 
injection of polio virus than are controls. Experiments with influenza 
are in progress. 

Significance to Cancer Research; To promote a more complete understanding 
of the effects of irradiation and to enable one to anticipate possible 
deleterious effects of irradiation used therapeutically. 

Proposed course of projects This project will be continued along the 
lines indicated. In addition, we plan to study the effects of sublethal 
radiation on response to several other noxious agents. 



PAGE 2 
PROJECT REPORT FORM (Cont.) 



^Q NCT-6i;l 

SERIAL NO. 

11, BUDGET ACTIVITY: 

Research /x7 Administration /V 

Review & Approval /~ / Technical Assistance /V 

12, C00PER.;TING UI^ITS of the public HE.^LTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONl^L FOR THIS PROJECT IN EITHER 
1956 OR 1957: 

Dr. Bernice Eddy,. L, B, C. 

13, IF THIS PROJECT RESEMBLES, CaffLSI'lENTS, OR P/Jli'iLLELS RESE/^RCH DOME 
ELSEl'JHERE IN THE PUBLIC HK\LTH SERVICE (V/ITHOUT lOTERCHANGS OF 
PERSONlxIEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESE/JlCH: 

None. 



NO ENTRIES FOR ITEl'lS lU, l5 & l6, 



PAGE 1 

PROJriCT REPORT FORM 

1, National Cance r Institute ^ 2, Radiation Branch 

INSTITUTE LABOR/.TORY OR BRANCH 

3, Radiation Biology Section h, 5. NCI-61;2 

SECTION OR SEjIVIGE LOCATION (IF OTIiER THAN BETHESDAj SERIAL 

NO. 

6, Antibody production in irradiated mice: Effect of fractional body 
shielding on hemolysin production and of radiation dose rate on 

hemolysin production 

PROJECT TITLE 



7* Falconer Smith 

PRINCIPAL IWESflGATOR(Sj 



8, H, Jeanette Ruth 

OTHER INVESTIGATORS 



9, PROJECT DESCRIPTION 

Objectives; The objective of this project is to determine the effect of 
x-radiation administered under the indicated conditions on the immune 
response in mice. 

Methods employed; Peak anti sheep erythrocyte hemolysin titers of 
individual mice are determined at weekly intervals following exposure to 
x-rays, radiation dose rate being varied in some instances while in others 
the volume of dose of x-rays is varied. 

Ma j or findings ; It was recently shown that bone marrow or spleen homogenate 
was ^^^ithout effect on radiation damage to the immune response. Shielding 
varying fractions (60-90^) of the body provides varying degrees of protection 
to the inmune response at a ;:;iv3n total body dose as determined by serum 
hemolysin titers at one to three weeks after x-ray exposure. 

Significance to Cancer Research; Antibody production reflects one aspect 
of protein synthesis by living tissue and is therefore directly related to 
cell metabolism, a problem of much interest in cancer research. 



PAGE 2 



PiiOJECT REPORT FOffl'I (cont.) 



10. NCI~6It2 
SERIAL NO. 



11, BUDGET ACTIVITY: 

Research /x7 

Reviex^r & Approval [^ 



Administration f~] 

Technical Assistance /] 



12, COOPER/xTING UI\IITS OF THE PUBLIC HIL'iLTH SERVICE, OR HER ORGANIZ..TE ONS, 
PRO IDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 OR 1957: 

No other units cooperating. 



13, IF THIS PROJECT RESIilBLES, COfffLaiENTS, OR PiiR^lLLELS RESE^'iRCH DONE 
ELSEWI-iERE IN THE PUBLIC HEj\LTH SERVICE (WITHOUT INTERCH.;NGE OF 
PERSONNEL, FACILITIES OR FU"NDS), IDENTIFY SUCH RESE/JiCH : 

No knowledge of parallel studies elsewhere. 



PAGE 3 
PROJECT REPORT FCRM (CONT.) 



ll;, NCI-6U2 
SERI/iL NO. 



1^, PUBLICixTIONS OTHER TIL'.N ABSTR/.CTS FRQl THIS PROJECT DURING 
Cj.LEl®/Ji YE/Jt 1955: 

Falconer Smith and H, Jeanette Ruth, Hemolysin Production in 
Irradiated Mice Given Spleen or Bone Marrox\r Homogenate, 
Proceedings of the Society of Experimental Biology and Medicine 
90, 187 (1955). 

,16, HONORS AND AW/J1DS TO PERSONNEL REK.TING TO THIS PROJECT DURING 
GALEND/iR YEiJi 1955; 

None, 



PAGE 1 



PROJECT REPORT FORM 



1. National Cancer Institute 2, Radiation Branch 



BISTITUTE LABORATORY OR BR^^NCH 

3. Radiation Biology Section k. ^ ^NCI-6i|3 

SECTION ..OR SERVICE . LOG TIONIIF OTHER THM BETKESD* ) SERIAL MO. 

,6. Long Term Survival and Tumor Incidence Follomng Acute or Chronic 

Irradiation 

PROJECT TITLE 

7. Joanne Hollcroft 

PRINCIPAL INVESTIGATOR! S) 

8, Eliza Miller, Charles C. Congdon 

OTHER INVESTK.'ATORS 



9, PROJECT DESCRIPTION 
Objectives : 

A, To study long term survival and carcinogenesis of: 

1) Chronic irradiation of guinea pigs T-jith or without injections of 
bone marrow and determine the effect of bone marrovi treatment in 
aleukemic leukemia. 

2) Acute radiation doses i«dien the animals are protected from the 
acute irradiation effect, 

B, Metho(i employed: 

1) Family 2 guinea pigs were exposed to 8,8 r per day gamma irradiation, 
•when their hematocrit number dropped to about 25 they wers removed from the 
radiation field. At this time half the pigs were given intravenous injections 
of bone marrow. Survival and tumor incidenee were studied. 

2) C^Hb mice were x-iiradi;^t cd under the folloijing conditions: 

(a) liOO r at birth, 

(b) laOO r with sham spleen shielding, 

(c) ijOO r v.d.th spleen shielding, 

(d) 900 r -with spleen shielding, and 

(e) 900 r iidth chemical protection of cystiene and anoxia 



PAGE 2 



NCl-61i3 

SERIAL NO. 

PROJECTION DESCRIPTION (CONT.) 
C, Major findings: 

1) Intravenous injections of bone marrow suspensions decreased the 
number of early deaths following limited chronic irradiation. These 
injections may have prevented so' e aleukemic leukemia in male guinea pigs 
but seemed to have no effect on aleukemic incidence in female guinea pigs. 
The hematocrits of the animals developing aleukemie leukemia appeared to 
drop lower after removal from the radiation field than animals lAioh 
recovered more completely. Of the animals which recovered from the chronic 
irradiation the bone marrow treated animals .seemed to have a longer 
survival time, 

2) Survival time following acute ejcposures was not influenced by spleen 
protection or the fact that the animals were irradiated at birth. The , 
mice receiving cystienc and anoxia protection, lived longer than the spleen 
shielded animals. In the mice irradiated with 900 r an increase in the 
following lesions appeared: 

(a) Adrenal tumors, 

(b) ovarian tumors, 

(c) glorerular sclerosis, 

(d) rp'^clofibrosis as noted in the sternum, 

(e) lens damage to the eye. An increase in the number of 
reticular endothelial neoplasms T,Tas noted in the animals protected 
chemically and an increase in miscellaneous carcinomas and sarcomas icls 
seen in female spleen shielded mice. In both groups pyelonephritis occurred 
less frequently than in the controls, 

Significanee to cancer research : Irradiation is a well knoT«/n carcinogen. 
The long-term effects of aci'te doses of total body irradiation are of 
consequence in considering the role of radiotherapy in treatment of caxicer. 

Proposed course of project : 

1) Influence of hematocrit number and white blood count as i-ell as 
total dose •n development of aleukemic leukemia, 

2) Completion of histologic studies. 



PAGE 3 



10. NCI-61;3 
SERIAL WO, 



11, BUD"'-5T ACTIVITY: 

Research /x7 
Review 4 Approval/^ 



Administration f~~J 
Technical Assistance f~~J 



12, It is hoped that Dr, Congdon of the Oak Ridge National Laboratory will 

continue ^^d-th the pathologic diagnoses on these studies. 

CGOPSRATIMG UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZ lTIONS, 
PROVIDBTG FUNDS, FACILITIES, OR P5RS0WNEL FC"'. THIS PROJECT IN EI'r^ER 
1956 or 1957. 



13. 'None, 

IF THIS PROJECT RESEI-ffiLES, CCMI'LMEi^ITo, OR PARALLELS RSSE/\.RCH DONE 
ELSK'HERE 13 THE PUBLIC HEALTH SERVICE ('-'I'fflCUT IWTERCHtNGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDi^ITIF^^ S' CH RESE/iRCH. 



NO ENTRIES FOR ITEI"?S lij., l5 & l6. 



PAGE 1 
PROJECT PlEPORT ^OKl 

1. ?Tational Cancer Institute 2. Padiation Branch 

E'^STITUTE IJIBORATORY OR BRAJICH 

NCI-6UIi 



3, Radiation Biology Section h, ^* 



SSCTia^ OR SERVICE LOCATION (IF OT^'KR Tl-Uil'-J BETHESDA) SERIAL NO. 



6. Modification of X-irradiation Treatment of Localized Tumors. 
PROJECT TITLE 



7, Joanne Ilollcroft 

PRINC IPAL INVESTIGATOR! Sj 



8, None ^ 

O'fflER BIVESTI'-ATORS 



9. PROJECT DE'^CRIPTION 

O bjectives ; To deterinine in what manner the tumor reacts to ionizing 
radiation cind in what manner the radios ensitivity may be inci'eased,. 

Methods employed ; Vary the physiology of the host prior to, during or 
follo^cLng irradiation of a transplanted lymphosarcoma L-1, follow tumor 
gro-wth , 

Major findings ; 

1, Attempts todiange blood supply to tumor by administration of 
adrenalin, histamine, priscoline or diathermy did not greatly alter the 
response of the tumor to x-irradiation, 

2, VTien a single dose of cortisone was admjjiistered either prior to 
or following irradiation the increase in response was found to be additive, 

3» Thiotepa may increase the response if given at the proper time 
prior to irradiation. 

hf Fractionation of a dose of UOOO r into k equal doses given 2 minutes 
apart produced the same regression as the same dose given daily but if the 
dose is given at 2 day intox'vals the effect is less* 

Significance to Cancer Research ; To find methods of increasing usefulness 
of radiotherapy. 

Proposed course of project : Continued studies on fractionation and 
comparison of fractionation treatm-ont of radiosensitive tumors id-th that of 
radioresistant tumors. Studies on radiation induced radioresistance. Use of 
antimetabolite and cytotoxic poisons to potentiate radiation effects. 



PAGE 2 



10. yCI-6iii| 
SERKL NO. 

11. BUDGET ACTIVITY: 

Research /x / 

Review & Approval /"^ 



Administration /V 

Technical Assistance /^ 



12. GOOPERATBIG UfllTS OF TKE RJBLIC HKILTH SiiRX^ICE, OR OTHER ORGANIZATIONS, 
PROVIDDIG FUI'^DS, FACILITIES, , OR. PERSONNEL FOR THIS PROJECT IN EITHER 
1956 OR 1957: 

None 

13. IF Tl-nS PROJECT RSSETiBLES, COI-ffLEt'IENTS, OR PARilLLELS RESKIRCK DONE 
ELSKNEl^E IN THE PUBLIC HE.\LTH SERVICE ('TITKOUT INTSTiCPLuNGE OF 
■r-ERSOM'EL, FACILITIES OR FUNDS) IDS^TTIFy SUCH RESEARCH: 

None 



NO ENTRIES FOR ITHfS ik, l5 & l6. 



PAGE 1 



PROJECT REPORT FORM 



1, National Cancer Institute 2, Radiation Branch 

BISTITUTE MBOPATORY CR BRANCH 

3.. Radiation Biology Section li. g,WGI-6i|g 

SECTION OR SERVICE LCGATIO]\t ([F OTHER TE:\N BST^-'ESD.OSERIAL NO, 

6, Effect of X-irradiation on Disseminated Lymphosarcomas, L#2 and L2C» 
PROJECT TITLE 



7. Joanne Hollcroft 



rREICIPAL raVESnGATORTsy 



8, Charles C. Congdon 

OTi-IER INVESTIGATORS 



9, PROJECT DESCRIPTION 

Objectives ; To determine the LD 100 g, L-2 tumor and devise a feasible 
treatment schedule, Treatmen"-. of L2C guinea pig leukemia. 

Methods employed ; A/HeN males bearing L-2 lymphosarcoma were irradiated 
starting on the 10th day after transplant. A bio-assay of the liver spleen 
and tumor from these mice x^as performed by innoculating A/HeN males ' stib- 
cutaneously i^rith these tissues at various times follomng treatment, 

A/HeN males bearing L-2 tumors were x-iraiiated and treated subsequently with 
bone marrow. 

Family 2 guinea igs bearing L2C lymphocytic leukemia were treated T>jith 
x-irradiation plus bone maA-rowj thiotepa, x-irradiation clus bene marrow 
or thiotepa alone, (This work was started by Dr. Congdon,) 

Ifajor findings ; Kith doses greater than 2000 r the greatest number of "takes" 
occurred when 'the tumor fragments were transplanted 1, 2 or 3 days after 
treatment. Few transplants grew from tissues taken immediately or h hours 
after treatment. Two daily doses of 1000 r or li daily doses of 500 r were 
found equally effective in inducing the transplantability of tumor fragments 
1 day after the end of treatment. No tumor fragments grew after transplantaticn 
from animals receiving I1.OOO r, >Jhen tumor fragments were irradiated in vitro 
id.th itOOO r 1^% of the tumor transplant grew. 

In treating mice bearing L-2 tumor with irradiation followed by bone marrow 
longest survival time (11 days) occurred when the animals were irradiated 
with 5000 r to the tumor and 900 r to the body given in 2 equal doses h hours 
apart. 



PAGE 2 

NCI-6It^ 
SKR.nL NO, 

PROJECT DESCRIPTION (COWT.) 

Family 2 guinea pigs bearing L2C lymphocytic leukemia have been treated 
successfully mth 3' x iiOO r x-irradiation plus bone marrow, thiotepa or 
a combination of thiotepa and x-irradiatlon. 

Si gnif ioaiee to Cancer Research ; Radiotherapy of leukemia. 

Proposed course of project ; Use of the bio-assay technic to study radio- 
sensitivity of other disseminated neoplasms. Study of the number of cells 
innoculated vs time to death in hopes to extrapolate this information to 
the effectiveness of radiation treatment. Continuation of radiation 
treatment of guinea pig leukemia. 



10, NC I -6ii5 

SmiAL NO. 



PAGE 3 



11. 



BUDGET ACTIVITY": 

Research /x/ 

Review & Approval /~J 



Administration /~ 7 

Technical Assistanoe /^ 



12. COOPER'.TING UNITS OF THE PUBLIC HKILTH SERVICE, OR OTHER ORGANIZ.\TIOMS, 
PROVIDING FUNDS, FACILITI'S, OR PERSONNEL FOR r^IS PROJECT IN EITHER 
19^6 OR 1957 : 

None 

13. IF THIS PROJECT RESEMBLES, COl^^LEI'MT TS , OR PARALLELS RESKIRCH DONE 
ELS0«IHEB1E IN 'THE HIBLIC HKJAm SERVICE (^^^ITH UT DJTmCHAJ^TGE OF 
PERSONNEL, FACILITIES OR FUNDS) IDEf^TIFY SUCH RESK'lRCH: 



NO E[\ITRIES FOR ITEMS \\x, 1^ ^'. l6. 



PAGE 1 
PROJECT ItEPORT FOM 



1, National Cancer Institute 2. Radiation Branch 

INSTITUTE L/iBOPu.TORY OR BPuliICH 

3. Radiation Therapy Service h, , ^ 5» NCI-6'^0(C ) 

SECTION OR. SERVICE LOCATION (IF OTHETi THAN BETHESDRj SERIAL 

NO. 

6, Sorvlco Puidiation Therapy 

PROJECT TITLE 

7, J, Robert Andrews, IWD,, and Philip Rubin, M«D, 

PRINCIPAL IblVESTIGATOUSj" 

8, Robert W. Swain 



OTHKi INVESTIGATORS 



9, PROJECT DESCRIFTION 

Objectives; To provide radiation therapy for those patients requiring 
such in the course of the disease for which they are being investigated 
by research groups other than the itadiation Branch, 

Methods employed; Consistent with contemporary radiotherapoutic practice 
employing a wide range of photon or other energies, ^ radium and radio- 
isotopes as indicated. 

Patient material; Those patients present with a wide variety of neoplasms 
for which radiation therapy might be of palliative or other value. 

Ila. j or findings ; Variable as would be expected from the diverse nature 
of the material studied and the widespread diseases generally present. 

Proposed course of project; To continue. 



PAGE 2 



PROJECT REPOiiT FOitfl (Cont.) 



10. NCI-6gO(C) 

SEicIAL NO. 



11. BUDGET ACTIVITY; 

Research /~7 
Review & Approval [^ 



Administration /V 
Technical Assistance /~ 7 



12. COOPEtiATING UNITS OF THE PUBLIC HEi'.LTH SERVICE, Oit OTHER OliGANIZATIONS, 
PROVIDING FTOIDS, FACILITIES, Oli PERSONNEL FOR THIS PROJECT IN EITHER 
1956 Ou 1957: 

None 



13,. IF THIS P..OJECT F^SSEMBLES, COMPLEMENTS, Ou P/JL'iLELS RESKJtCH DONE 
ELSEMlEIci^. IN THE PUBLIC HEALTH SEIi.VICE (WITHOUT INTERCH..NGE OF 
PETtSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESE/JiCH: 

None 



NO EOTRIES FOR ITEMS lli, l5 & l6. 



PAGE 1 

PROJECT REPORT FORM 



1» National Cancer Institute 2, Radiation Branch 

INSTITUTE LABORATORY OR BRANCH 

3. Radia ti on Thoraov Serv ice h* S, NCI..6gl(C) 

SECTION OR SERVICE rOCATION (IF OTHER THAN BETHESDA; SERIAL NO. 

6, Electron Beam Radiation Therapy 
PROJECT TITLE 



7. J. Robert Andrews, M.D, 

PRINCIPAL I1WESTIGAT0R(S} 



8. Philip Rubin, M.D», and Robert W. Swain 
OTHER INVESTIGATORS 



9, PROJECT DESCRIPTION 

Objectives: To deliver effective doses of ionizing radiation to the 
epidermis and corium by means of controlling energies and character 
of the ionizing radiation to limit the effects to this zone. 

Methods employed; Ionizing radiation is an effective treatment for a 
variety of multiple, malignant, superficial, cutaneous neoplasms including 
mycosis fungoides. Bowing's disease and, possibly, Kaposi's sarcoma. 
Ionizing radiation may be administered as x-ray therapy but in this case 
there is generally such absorption of radiation in deeper tissues, including 
the radiosensitive bone marrow, as to limit the amount of radiation to 
less than an effective dose. The absorptions of electrons in the energy 
range (<. 2,0 Mev) available is, however, limited to less than 1 em, of 
tissue. This makes possible the administration of effective doses of 
radiation to superficial neoplasms without affecting deeper structures. 

The physical factors associated x^ith the production, dosimetry, 
and clinical use of an electron beam arc being thoroughly studied. Clinical 
studies of skin erythema and skin blistering doses arB being performed and 
compared with x-ray effects as to relative biological effectiveness. The 
effects of various doses on superficial cutaneous neoplasms are being 
studied. In addition to clinical observations, appropriate biopsy material 
is being obtained and all observations arc being carefully documented. 

Patient material; Cases of mycosis fungoides. Bowing's disease, Kaposi's 
sarcoma and other primary or secondary superficial malignant neoplasms, 

Maj or findings : This project was initiated only in the last six months. 
The response of the limited niunber of patients so far available for study 
indicates that this type of ionizing radiation is clinically effective. 

Proposed course of project; As indicated in methods above. 



PAGE 2 
PROJECT REPORT FORM (cont.) 



10. NCI-6'^1(C) 

SERIAL NO. 



11. BUDGET ACTIVITY 

Research f~ J Administration /V 

Review & Approval /~] Technical Assistance £J 

12, COOPERATING MITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FIM3S, FACILITIES, OR PERSOMEL FOR THIS PROJECT TO EITHER' 
1956 OR 1957: 

None 



13, IF THIS PROJECT RESEi'lBLES, CQlPLEilENTS, OR PARALLELS RESEARCH DONE 
ELSEIfffiERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCH/ii'^GE OF 
PERSONNEL, FACILITIES OR FUITOS), IDENTIFY SUCH RESE/iRCH: 

None 



NO ENTRIES FOR ITEMS II4, l5 & I6, 



PAGE 1 
PROJECT liEPORT FORM 



1, National Cancer Instituto 2, Radiation Branch 



INSTITUTE LABORATORY OR BRANCH 

3, Radiation Therapy Service it, ^, NCI>6g2( C) 

SECTION OR SERVICE LOCATION(IF OTHER THAN BETHESDA; SERIAL 

NO. 

6, The Relative Biological Effectiveness (RBE) Factor for the Destruction 

of Human Carcinoma ^ ^ 

"PROJECT TITLE 



7. J, Robert I>xidrews, H«D. , Philip Rubin, M,D. , and Robert VJ. Swajn 
PRINCIP;i INVESTiaATOR(S} 



8, Eugene J, Van Scott, M,D., a.nd Richard P. Reinertson, M«D. 
OTHER liWESTIGATORS 



9, PROJECT DESCRIPTION 

Objectives ; To determine the relative biological effectiveness (RBE) 
factor for the destruction of hur,ian carcinoma. 

Methods employed; Human multiple, superficial cutaneous neoplasms are 
treated with a range of doses by a variety of ionizing radiations (including 
low voltage unfiltercd x-rays, medium voltage filtered x-rays, 2 Mev x-rays, 
the gamma rays of radium, and 0,9 and 1,5 Mev electrons) and the cancerocidal 
dose determined for each. In addition to grossly observable clinical effects, 
microscopic histological studies have also been undertaken. 

Patient material; Cases of multiple, superficial, epithelial cancers of 
the skin. 

Major findings ; Major findings can not be reported at this time because 
this project was initiated only within the last six months. 

Proposed course of project; As indicated in methods above. 



PAGE 2 
PROJECT REPQIT FORM (cont.) 



10. NCI-6^2(C) 
SERL'X NO, 



11. BUDGET ACTIVITY 

Research fl Administration fl 

-Review & Approval /V Technical Assistance /V 

12. C00PER.;TING units of THE PUBLIC HEALTH SERVICE, OR OTi-iER ORGi'.NIZi.TIONS, 
PROVIDING FUITOS, FACILITIES, OR PERSOMEL FOR THIS PROJECT IN EITHER 
1956 OR 1957: 

None 

13. IF THIS PROJECT RESEilBLES, COflPLEl^iENTS, OR P.iRiULELS RESEi'JlCH DONE 
ELSEl'JHERE IN THE PUBLIC HE^'XTH SERVICE (WITHOUT iNTERCHiUMGE OF PEIiSOIWEL, 
FACILITIES OR FUl\fDS), IDENTIFY SUCH RESK'uRCH: 

None 
NO ENTRIES FOR ITEMS lit, \$ & 16. 



PAGE 1 
PROJECT REPORT FORM 



1, National Cancer Institute 2, Radiation Branch 

INSTITUTE LABORATORY OR BRANCH 

3. Radiation Therapy Service U, gt NCI-653(C) 

SECTION OR SERVICE LOCATION(IF OTHJR THAN BETHESDA; SERIAL NO. 

6, The Influence of Oxygen in Modifying Radiation Response 



7, J. Robert Andrews, M. D. 

PRINCIPAL investigator(s; 



8. Philip Rubin, M. D, , Robert W. Swain 
OTHER liWESTIGATORS 



9. PROJECT DESCRIPTION: 

Objectives ; To investigate the possibility of modifying response of 
neoplastic cells to ionizing radiation by altering the oxygen content of 
blood» 

Methods Employed ; Appropriate patient material (see below) is 
irradiated over bilaterally more or less symmetrically enlarged Ijmiph nodes 
with equal doses of ionizing radiation (2^0 KV X-rays) under different 
conditions of oxygen satiu-ation of hemoglobin and dissolved blood oxygen. 
One bilaterally symmetrical area is irradiated under conditions of 
normal atmospheric press\ire and oxygen tension. The other bilaterally 
symmetrical area is irradiated under conditions of normal atmospheric 
pressure with lOOJ? inspired oxygen under which conditions the differential 
oxygen tension is increased by a factor of approximately 5» 

If a differential effect is observed it is proposed to extend the experi- 
ment by irradiation of similar patient material under conditions of re- 
duced oxygen tension (normal atmospheric pressure but reduced differential 
oxygen tension) and under conditions of increased oxygon tension to above 
norraal atmospheric pressure by radiation in a pressiu'e chamber. It is 
further proposed to investigate directly the oxygen tension in the tissues 
exposed to ionizing radiations by the use of appropriate instrviments. 

Patient Material ; Cases of Hodgkin's disease or lymphosarcoma with 
bilaterally and symmetrically enlarged lymph nodes in previously untreated 
areas. 

Ma j or Fj.ndings ; Major findings can not be reported at this time because 
this project was initiated only within the last six months. 

Proposed Course of Project ; As indicated in methods above. 



PAGE 2 



10. NCI-6g3(C) 
SERIAL NO. 



11. BUDGET ACTIVITY: 

Research ff ■ Administration f~J > 
Review & Approval f^ Technical Assistance ri 

12. COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 0THJ3R ORGANIZATIONS, 
PROVIDING FU'NDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 OR 19^7: , _ . . 

None 

13. IF THIS PROJECT RESEflBLES, CQIPLHIENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF 
PERSONNEL, FACILITIES OR FOTJDS), IDENTIFY SUCH RESEARCH: 

None 
NO Ei^RIES FOR ITEMS lU, l5 Sc 16, . : 



PAGE 1 



PROJECT REPOPiT FORM 



1, National Cancer Institute 2, Radiation Branch 

INSTITUTE LABORATORY OR BRANCH 

3, Radiation Therapy Service h* S, NCI-6^1i('C ) 

SECTION OR SERVICE LOCATION(IF OIliER THAN BETHESDA) SERIAL NO. 

6, Tiine as a Modifier of C l inical Fuadiation Response 

7» J. Robert Andrews, M. D. 
PRINCIPAL INVEST IGAT OR (Sj 

®» RTJlip Rubin, M . D, an d Robert '-' . Swain 
OTHER IWESTIGATORS 



9. PROJECT DESCRIPTION 

Objective ; To evaluate the prolongation of treatment time as a modifier 
of clinical response to ionizing radiations. 

Methods Bnployed ; Irradiation of appropriate patient material (see 
below) to tumor doses confirming with the equation, 

D = 2750 tO.23 
over approximately a 100 day treatment period. 

Patient Material ; Cases of squamous cell carcinoma of the head, neck, 
larynx and uterine cervix in Stages II and III, 

Major Findings ; Major findings can not be reported at this time because 
this project was initiated only vrithin the last six months. 

Proposed Course of Project ; As indicated in methods above. 



PAGE 2 



10. NCI-6gi;(C) 
SERIAL NO. 



11. 



BUDGET ACTIVITY: 

Research [J Administration fl 

Review & Approval [J Technical Assistance f~] 

12. COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER, 
19^6 or 19^7 s 

None 

13. IF THIS PROJECT RESKIBLES, COIIPLEMENTS, OR PARALLELS RESEARCH DOM 
ELSEV/HERE IN THE PUBLIC HEALTH SERVICE (VJITKOUT INTERCHANGE OF 
PERSONl^L, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 

None 



PAGE 3 
PROJECT REPORT FORM (cont.) 



11;. NCI..6g[;(C) 

SERIAL MO. 



15. PUBLICATIONS. OTHER THAN ABSTRilCTS K^OIi THIS PROJECT DURING CiXEND^Jl 
121 Ji 1955: 

J. R. /mdrews and Jog M, Moody, The Dose-Time Relationship for the 
Cure of Squamous Geil_CaiicinDma: Presented at the Inter»-/unerican 
Congress of Radiology, V/ashington, D, C, 1955j Accepted for publica- 
tion by the /merican Journal of Roentgtinology and Radium Therapy, 

16. HONORS ;.ND AVL'J^DS TO PERSOM^IEL RELATING TO THIS PROJECT DURING CALEi©/Ji 
lam 1955: 

None 



PAGE 1 



PROJECT REPORT FORM 



1, National Cancer Institute 2, Radiation Branch 

INSTITUTE LABORATORY OR BRANCH 

3. Radiation Therapy Servlco k, g^ ;'-TCI-6 55(C) 

SECTION OR SERVICE LOCATION(IF OTHER THi^N BETKESDA; SERIAL NO, 

6, Detormination of Radiation Tolerance to the Brain by ileans of 
Electroencophalographic ^ Studies 

PROJECT TITLE 

7. Philip Rubin, M.D., and J. Robert Andrews, M,D. 

PRINCIPAL INVESTIGATQR(S; 

8« Dr« Cosimo Ajmone Marsan 
OTHER INVESTIGATORS 



9, PROJECT DESCRIPTION 

Objectives: The use of electroencephalographic studies while treating 
various brain tumors will be employed to determine if any damage has been 
done to normal brain tissue. It is assumed that physiological changes 
reflected in the electroencephalogram may precede the severe structural 
anatomical changes. No attempt has been made to correlate electroencephalo- 
graphic changes in definite areas of the brain with isodose curves which 
will give exact dosage to these points. 

Methods employed; Serial electroencephalographic studies will be done 
during the course of treatment, beginning with a base line study. Aberra- 
tions in brain waves in certain areas will be correlated with the dosage 
level at these points. With the sharp beam alignment and available isodoee 
c\irves this can be calculated with sufficient accuracy to have considerable 
correlative values. 

Patient material; Appropriate case material which includes a wide range 
of brain. tumors which have proved to be inoperable, but show some evidence 
of radioresponsiveness on the basis of the literature. 

Major findings; Major findings can not be reported at this time because 
this project was initiated only within the last six months. 

Proposed course of project ; It is proposed that in the next year this study 
will be considerably amplified in conjunction with an expanded activity in 
brain tumor research in the NINDB, 



PAGE 2 



PROJECT REPCSRT FORI'f (cont.) 



10. 



NCl-655(c) 

SERIAL W07 



11. BUDGET ACTIVITY: 

Rcsuarch [j 
Review & Approvr.l // 



Administration [J 
Technical Assistance [J 



1956 OR 1957 5 ■ ■ '■ ■ - 

None 

t. TE THIS PROJECT RESEMBLES, COiffLElENTS, OR P/JL'XLELS RESEARCH DONE 

''• eL™^e'S1he'public HkLTH service (wnwri™^^^^ OF 

PERSOOTMEL, FACILITIES OR FUNDS), IDENTIFY SUCH R^Sl^iJlCH: 

None 



NO EOTRIES FOR ITEMS lU, l5 & l6. 



PAGE 1 

. PROJECT REPORT FORM 

1, Nation al Ce.ncer Institute 2. Radiation Branch 

INSfifUTE " LABORATORY OR BR/JJCH 

3. Radiation T herapy Service 1;. g. MCI-656(^ 

SECTION Ce SERVICE LOCATION(IF OTHER THAN BETIIESDAj SERIAL NO. 

6, Attempt at Radiotherapy vrith s35_Sulfate 
PROJECT TITLE . 



7. Philip Rubin, M, D- and J« Robe r fei/.hdi-ews, M, D. 
PRINCIPAL INVEST IGAT OR ( S ) 



J. A. Holler oft, Marion Matthews, Raymond Gottsch.^.lk, I1.D« 
OTHER irVESTIGATORS 



9. PROJECT DESCRIPTION 

Objectives ; s35 given as sulfate selectively concentrates in cartilag* 
and tissues containing abundant sialfated mucopolysaccharides. Since the 
beta radiation of S35 is very weak (in the neighborhood of pO lev, ) it 
must be determined first vrhetiier this can destroy cartilage cells. The 
s35 is laid doi'm. in the ground substance of cartilage, a chondroitin sulfuric 
acid polysaccharide. It is postulated that the path of the emitted electrons 
will be adequate to reach the cells* To date, no evidence of cell destruc- 
tion has been shown with the dosage used in adult cartilages. It seems 
essential before one embarks on the use of large amounts of this radio- 
isotope which will mean considerable expense, that evidence of some radio- 
toxic effect should be shown on cartilage. 

Methods employed ; The follovxing animal experiment was therefore decided 
upon and is being carried out as outlined below: 

1, Thirteen litters of white suckling rats were utilized weighing 
between 8 to 25 gms, 

2, Varying amounts of radioactive sulfate were injected ranging from 
the toxie level (2,0 Mc/gm, ) to the postulated therapeutic level (0,1 Mc/gm, ) 

3* The animals that have died during the procedure were bottled in 10^ 
formalin and are to be examined at a future date by Dr. Gottschalk for the 
pathology and tissue assays which are pertinent to the procedure, 

1;, Weights of the animals will bo taken every week to obtain a growth 
curve, 

5, X-rays of all the bones in the body will be made every two weeks to 
determine the effect of the administered S35 on epiphyseal centers and bone 
growth. 

Patient material ; Application to Patients with Chondrosarcoma; If the 
above work suggests that groiving cartilage can be destroyed by radiosulfur, 



PAGE 2 

NCI-6^6 (130 
SERL'A NO, 
PROJECT itEPOuT FORM (cont.) 

ari ottempt vill.be made to utiliZG this agent on a clinical basis. Only, 
niticnts w;th advanced chondrosarcoma who have been treated with surgery 
and external radiation previously will be candidates for this treatment. 

I'laior findings: Definite disturbances in the epiphyseal and metaphyseal 
i^feiT^J^^n'of long bones have been observed. This remains to be 
correlated with tissue assay and radiomicrographs of tissue sections. 

Signif icance tq3'.ncexJl^s_3archi A new method of treating chondrosarcoma 
ij; the ani'iiar~jxporiraents prove successful. 

Proposed course_o^£ro,loc:y. Continuation as outlined above until studies 
are completed which will be in three months. 



10. Mr.T-yA rX^ 
SETtlAL NO. 



PAGE 3i 



11. BUDGET ACTIVITY J 

Research f^ 
Review & Approval /"^ 



Administration f~ ] 

Technical Assistance f^ 



12. CCX)PER/i.TING Ul^IITS OF T ;E PUBLIC HEALTH SERVICE, OR OTHER ORGMIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONI^JEL FCR THIS PROJECT IN EITHER 
1956 OR 19571 

None 



13, IF THIS PROJECT RESEifflLES , COiiPLEilENTS, OR PiLRilLLELS RESE/JiCH DOIME 
ELSEWHERE IN THE PUBLIC HE.'J.TH SilEVICE (V/ITHOUT INTERCHANGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDEOTIFY SUCH RESE^'J^CH: 



NO ENTRIES FOR ITEMS lU, l5 & l6. 



PAGE I 
PROJECT REPORT F-^RM 
PROJECTS WHICH ARE BEING DISCONTINUED 
N.C.I. 2. General Medicine Branch 



INSTITUTE LABORATORY OR BRANCH 

Nutrition and Metabolism h. ____«_______^ 5* NCI~70l(C ) 

SECTION OR SERVICE ' LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

A. Meticorten: Metabolic and clinical effects of massive doses of 

PROJECT TITLE Prednisone in subjects v^ith malignant disease 

Donald M. Watkin 

PRINC IPAL INVESTIGATOR ( S ) 



OTHER INVESTIGATORS 



i^OJECT DESCRIPTION 

Objectives : To quantitate the effect of massive doses of Prednisone,, a 

synthetic steroid reputedly lov; in midesirable side effects, in 
malignant disease not amenable to more conventional therapy. 

Methods Employed: Metabolic balance technique I -^ labelled human serim 
turnover . 

Renal function studies . 
Electrophoretic analysis of serum protein. 

Patient Material : Two women^ one with multiple myeloma and one with 

lymphosarcoma, and three men, one with Hodgkins Disease, 
one with multiple myeloma, and one with prostatic 
carcinoma. 

Major Findings : All subjects demonstrated negative nitrogen balance; all 

demonstrated retention of sodium and chloride. Theoretical 
phosphorus balances could not be reconciled with calcium 
and nitrogen balances. Potassi\Jm balances were negative. Uric acid 
excretion was slightly elevated in three and dramatically elevated in 
two patients. Marked negative phosphorus and nitrogen balances and 
ma:rked elevated urinary uric acid excretions were associated with 
objective diminution in tumor size in lymphoma and Hodgkins Disease. 
I ^ albumin turnover rates were accelerated by Prednisone therapy in one 
patient, genal function studies before and during treatment indicated 
a marked reduction in renal plasma flow apparently due to reduction in 
cardiac^ output, an example of forward failure. Electrophoretic studies 
of plasma proteins revealed for the first time a change in the charac- 
teristic pattern in a patient with multiple myeloma. 



NCI-701-(C) PAGE 2 

SERIAL NO. 

PROJECT REPORT FOEM (conb'd) 
f • ■ ■• 

Numerous side effects including acute psychosis, congestive heart fail\ire, 
paroxysmal tachycardias^ systemic moniliasis, pulmonary embolism and 
severe acne were observed during massive Prednisone therapy. 

' Significance to Cancer Research ; Prednisone, because of its potency greater 

than Cortisone and freedom from side effects 
when given in smaller doses, suggested a means of giving enormous 
corticoid doses to patients with' hopeless malignancy. The -reporttjd •:■• 
studies indicate that in massive doses the hormone has numerous 
undesirable side effects, that it is effective against malignancies in 
the lymphoma group, and that it can induce some changes hitherto un- 
reported in multiple myeloma:. Except for experimental purposes,; however. 
Prednisone in massive doses cannot be recommended as a therapy for 
malignant disease. 

Proposed Course Oj' Project ; The discrepancies, between theoretical and actual. 

phosphorus balances, changes in myeloma proteins, 
changes in resistance to disease, and alterations in intracellular 
electrolytes, especially in the myocardiim, are avenues which may be 
followed as suitable patients present themselves and As new synthetic 
hormones are developed. 

B. Metabolism of nitrogen, calcium, phosphorus, electrolytes in metastatic- 
prostatic carcinoma. 

Objectives ; Accumulation of metabolic data in patients with extensive 

metastatic prostatic maligna.ncy be.fore, during and after various 
therapeutic maneuvers. 

Methods Employed ; Metabolic balance technique. Red cell turnover deter-' 

minations . 

Patient Material ; In 195^ j two patients were studied before and after 

orchiectomy. In 1955; one of these returned one year 
after operation for a follow-up metabolic study. In addition^ two 
other patients with extensive metastatj.c disease, both several years 
post-orchiectomy, were admitted for study of the effect of hormone 
administratjon. 

Major Findings ; The beneficial effects of orchiectomy in reducing calcium 
and protoplasmic loss were ma-intained one year after 
operation. Stilbestrol administration was of equivocal value. Massive 
Prednisone dosage superimposed on stilbestrol administration in one 
patient produced a temporary clinical improvement, but in the long rion 
resulted in marked wasting of bone and protoplasm. 

Incidental Findings; The patient treated with Prednisone demonstrated a 

unique afibrinogenemia which was followed throughout 
his course and which seemed to improve under Prednisone therapy. This 
was not due to prostatic fibrinolysin but to a lack of fibrinogen 
production. 



PAGE 3 
CI-701(C) 
ERIAL NO. 

PROJECT REPORT FORM (cont'd) 

Significance to Cancer Research : Quantitative evaluation of the natural 

course of the disease together with 
quantitative measurements of the effects of therapy. 

Proposed Course of Project ; Continued search for more patients with 

extensive prostatic disease, follow-up on patients 
already under study, further investigation of the relationship of 
prostatic malignancy to afibrinogenemia. 

C. Metabolism of fluoride in leukemic subjects with and without chronic 
fluorosis 

Objectives : Investigation of possible role of fluoride in genesis of 
leukemia . 

Methods Employed ; Metabolic balance technique. 

Patient Material ; During 195^ two leukemic patients with fluorosis, one 

leukemic without fluor-os is and one non-leukemic without 
fluorosis were studied. In 1955 j three patients without fluorosis, 
two normals and one with multiple myeloma, were studied. 

Major Findings : On an intake of distilled water, patients with fluorosis 
excrete fluoride in the urine. Patients with and without 
fluorosis excreted a fixed percentage of fluoride intake. Ljukemia or 
multiple myeloma did not alter the fluoride excretion pattern. Balances 
of nitrogen, potassiiwi, phosphorus, calcium, sodium and chloride were 
not significantly altered by fluoride administration at levels of 
5 mg. per day. 

Significance to Cancer Research: The essentially negative results of these 

studies should help allay the fears of many ". 
laymen that fluoride in drinking water m.ay prove harmful. No evidence 
has been obtained in these studies to indicate any harmful effects 
of fluoride administration. These studies of course do not rule out 
fluoride as a carcinogen but do demonstrate the absence of any measurable 
toxic effect. 

Proposed Course of Project ; Any further pursuit of this subject will depend 

on the ability of NIDH to perform fluoride 
assays. At the present, no extension of the program has been planned 
as far as NCI is concerned. 

D. Role of B complex vitamins in tissue anabolism 

Objectives ; To observe the utilization of B complex vitamins in tissue 
anabolism induced by steroids or hyperalimentation. 



PAGE k 
fGI-70l(C) 

>:rial no. 

PROJECT REPORT FORM (cont'd) 

Methods Employed ; Metabolic balance technique. Bioassay for vitamins in 
food and urine. 

Patient Material ; One patient studied initially in 195^ was carried over 
into 195,^ 

Major Findings : No direct relationship was noted between vitamin retention 
and tissues' anabolisra. The small quantities of vitamins 
required and the inaccuracy of the bioassay technique 
may account for the negative findings. 

Significance to Cancer Research ; The domonstratiun of the essentiality of 

certain vit;arains in the growth of normal 
or tumor tissue could lead to development of effective antimetabolites. 

Proposed Course of Project: The bioassay procedures originally conducted 

by the Endocrinology Branch are no longer 
available, he>nce, no continuation of this project is contemplated. 

E. Metabolism of human cerum albumin administered intravenously in large 
doses to subjects with malignant disease. ■ 

Objectives : Quantitative measurasBiipnt of the metabolism of large amounts of 
human serun albiwiin given intravenously. 

Metboda Employed : M'-tabolic balance technique. 

Eluctrophoretic analysis of serum. 

Turnover of l''-3-'- labelled human serum albumin. 

Patient Material ; One normal, one subject with Hodgkins Disease^ one patient 
with multiple myeloma,, one v;ith lung cancer and one with 
face cancer. 

Major Findings ; Balance data indicate that the albumin was gradually 

utilized over a period of about- h weeks. Isotope studies 
show an increased rate of albumin turiiover luring and after albumin 
administration. 

Significance to C a ncer Research ; Albumin is readily available plasma 

~ constituent frequently low in patients 
with advanced cancer. 

Proposed Course of Project : Further evaluation of the data compiled in 

these studii.:s. Wo new studies planned at 
this time. 






PAGE 5 



PROJECT REPORT FORM (cont d) 



LO. NCI-70L(C) 



SERIAL NO. 



LI. 



BUDGET ACTIVITY: 

RESEARCH [YJ ADMINISTRATION / / 
REVIEW & APPROVAL I 7 TECHNICAL ASSISTANCE 



L2. 



COOPERATING UNITS OF THE PUBLIC HEALTH SlilRVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



L3. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HE/VLTH SERVICE (WITHOUT INTERCmvNGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR ITEMS i'+;, 15 & l6 



PAGE I 

PROJECT REPORT FOM 

1. N. C, I. ' 2. General Medici ne Bra nch 

INSTITUTE LABORATORY OR BRANCH 

3 ■ __ 4. _ „ _: - 5." NCI..702XCJ 

SEGTIWJ OR SERVICE LOCATION (IF oflKR flBN BETHESDA) SERIAL NO. 



6 . Metabo lic St udy of Se rum Proteins . , — . 

TrOJ^GT TITLE 

7. J. L. Fahey. J. L. Stei nfeld. J. M. Watki n. H. A. Sober and E,_A^_Petersgn_ 
PRINCIPAL INVE^.TIG;iTOR(sT 



OTHER INVESTIGATORS 

9A. PROJECT 'DESCRIITION: Electrophoretic Studies of the Serum Proteins in Neo- 
plastic Disease States. (J. L. Fahey) 

Objectives ; The objective of this project is to electrophoretically quanti- 
tate the serum protein changes occurring in neoplastic states 
and to correlate these changes with specific aspects of the 
clinical status of the patients. 

Methods Employed ; Development and refinement of a quantitative method of 
zone (paper) electrophoresis was completed during the 
year. Systematic follow-up of patients admitted to the 
Metabolic, Acute Leukemia and Solid Tumor Services was instituted so that 
serum protein data can be correlated with a clinical evaluation of the type 
and extent of disease, disease activity, nutritional state of the host and 
therapy employed. Sequential observations in the same patients are an im- 
portant aspiect of this study. 

Patient Material ; Patients admitted to the Clinical Center for other pro- 
jects were utilized in this study. 

Major Findings ; Zone (paper) electrophoresis can be utilized for quantita- 
tive measurement of serum proteins. , 

Long term patient follow-up was instituted. A study of 
the serum protein changes during immune response to antigen challenges in 
leukemic and control patients (in collaboration with the Acute Leukemia 
Group) revealed no striking correlation of gamma globulin changes in pre- 
liminary evaluation. However, the study is not yet complete. 

Genei"al assistance to all clinical groups has been utilized to evaluate 
serum proteins of patients with multiple myeloma and of patients in which a 
gamma globulinemia was suspected. None were found, however. 



NCI-702(C) PAGE 2 

PROJECT REPORT FORM (Cont'd) 

Signi ficanc e to Cance r Research ; Delineation of the sequence of plasma pro- 
tein changes in neoplastic disease is 
needed, for this is an aspect of tumor- 
host relationship which lends itself to quantitation. Also, the effects 
upon the serum proteins of anti-tumor therapeutic regimens is generally . 
unknown. 

Pr<; ' p osed Course of Project ; Emphasis will continue to be upon serial deter- 
minations throughout the course of illness in 
the patients studied, and upon correlation with 
clinical data. The immune studies will be completed. Evaluation of the 
effects of cortisone, similar steroids and other chemical agents will 
continue. , 

9B.. PROJECT DESCRIPTION: Measurement of Albumin Turnover and Total Exchange- 
able Albumin in Patients with Cancer, Control Patients 
with Weight Loss and Volunteer Normal Subjects. 
(J. L. Steinfeld) 

Ob jectj-ves ! To determine whether the decreased serum albumin concentration 
found in patients with cancers is due to an increase in plasma 
' ' volume or failure of albumin production to keep up with albumin 
degradation or metabolism - and further to ascertain if the metabolism of 
albumin in cancer patients proceeds at a normal, increased or decreased 
rate. ■■:-■■..-' 

Metho ds e mployed ; Commercially available (Abbott Laboratories) I ■'albumin 
is checked' for free radioactivity and satisfactory prepa- 
rations (one milligram of albumin nitrogen and 100 . Iv^ 
are injected ihtravenoucly into patients oh relatively constant caloric 
,';'■ and nitrogen intake. Frequent serial sampling of serum radioactivity as 
■ ■• ^ well as daily determination of total urinary radioactivity permits calcu- 
, latibn of total exchangeable albumin and albumin turnover. 

"^l " f^atient Material ; Adult female 11 admissions 308 patient days 
'■■ "' ' ■ Adult male 5 admissions 14.0 patient days 

Out patients 3 admissions 

Major Fi ndings; The major findings of this project during the pt si year 

have been significantly decreased total exchangeable albu- 
,,^^....j^^_ , . min found associated with relatively normal plasma volumes 

"'" and the decreased or low normal turnover rates of albumin in oancercus 

patients. This is evidence against a much increased albumin turnover with 
failure of albumin production to keep pace with the increased destruction 
..being' the factor responsible for the low serum albumin concentrations 
i^een clinically. 



'io 



Significanc e |to Cancer Research; In order to understand the physiological 

relations between host and neoplasm it is 
necessary to investigate the possible 
Sources of tumor food supply. Since serum albumin concentration is low in 
cancerous patients, one might hypothesize trapping of .serum albumin by 
tumor and use of that albumin for metabolism or tumor growth. It is of 



NCI-702(G) 



PAGE 3 
PROJ^T REiORT FORI-l (Cont'd) 



importance to learn which nutrients tumors can and cannot use since such 
understanding may contribute to the development of effective anti-tumor 
agents . 

Prop os ed Course of Pro.iect ; In collaboration with Dr^ Robert Milch, the 

tissue distribution of I"'-^ -^albumin in tumors as 
compared with normal tissues will be determined 
through the counting of J-^^-'-alburain found in various tissues obtained at 
operation or at autopsy. These determinations will be corrected for the 
II3I blood content of the various tissues. 

Also the distribution and turnover of serum albumin and other plasma pro- 
teins in cancer patients will be investigated using endogenously labeled 
plasma proteins as with S^^ or C-^ amino acids, 

90. PROJECT DESCRIPTION: Fate of Intravenously Administered Albumin. 

(J. L, Fahey) 

Objectives ; llie objective of this project is to characterize Che dis- 
appearance rate and distribution of the products of albumin 
loads administered intravenously to patient in various neo- 
plastic and nutritional states. 

Methods Employed ; Complete metabolic balance techniques have been utilized 
to follow nitrogen and, in certain instances, calcium and 
phosphorus balances in selected pateints. Intravascular 

distribution of albumin and other serum proteins have been measured by 

electrophoresis and radioactive iodinated albumin. 

Patient Material ; Average Stay 

No. Days 

Admissions: Adult Male 4 35 days 

Adult Female 2 25 days 

Ma.ior Findings ; Intravenously administered albumin is partially available 
as a source of nitrogen for tissue and tumor needs. 

Significance to Cancer Research ; Patients with neoplasm develop low serum 

albumin levels. The cause of this pheno- 
men is obscure. Determination of the fate 
of intravenously administered albumin loads under several conditions 
should help to clarify this problem. Nutritional evaluation of intra- 
venously administered albumin can be undertaken at the same time. 



Proposed Course of the Project; Early completion of analytic data is anti- 
cipated. Correlation and evaluation of 
findings can then be completed. 



NCI-702 (C) PAGE k 

PROJECT REPORT FORM (Cont'd) 

9D. PROJECT DESCRIPTION: Chromatography of' Serum Prbte ins. (J. L. Fahey) 

Objectives ; The objective of this project is to further characterize the 
' ii serum proteias frOiB normal and tumor-bearing patients by ap- 

plication of new methods of protein separation. ' ,'J.Li 

Methods Et-aployed ; Drs. Sober and Peterson of the Laboratory of Biochemis- 
■; tr-y, NCI^ have designed systems of substituted cellu- 

lose columns on which protein components move differen- 
tially under pH and salt gradients in controlled temperature conditions. 
. . , iif.' Individual serum protein components are identified by electrophoretic 
, , j and spectrophotometric -means. ' ' ' • 

Major Findings ; Within the past year equipmerrt has been assembled' and the 
fj^-.tj. chromatographic procedure establishei in the clinical 

area. Further work on the method has resulted in some re- 
duction in the time and complexity of the procedure required to carry 
„ .,_ j out an analysis. 

Significance to Cancer Research ; The alteration of body protein metabo- 
lism in neoplastic states is reflected 
by serum protein 'changes. These have as 
yet been only grossly defined by other analytical means. Fxorther identi' 
f ication and characterization of the serum proteins themselves Are 
,. necessary in order that the causes of protein abnormality may be studied 

Proposed Course of the Project ; Further effort will be devoted to develop 

ment of a modified, more rapid adaptation; 
yj... of the present column chromatographic 

'. technic. When this is achieved a clinical survey will be feasible in 
,.., which sera from patients with a variety of neoplasms and in various 
. states of disease activity will be tested. 

.9E. PROJECT DESCRIPTION: Evaluation of Protein Metabolism by Means of Isotop< 

Labelled Amino Acid. (J, L. Fa^iey) 

ft -...- wol "^^^^ study has been delayed because of the basic de- 

.v. ;,; cision to change the method of protein fractionation 

'• . . from the cold-alcohol m:thocl of Cohn to the substi- 

tuted-ceiluiose column technic of Sober and Peterson, However, this 
method requires further development before it can be efficiently applied 
to the requirements of such a study. Work with the method is already 
underway as noted in report. 



NC 1-702 (C) P^GE 5 

PROJECT RErORT F0R14 (Cont'd) 

10, NCI-702(C) 



SERIAL NO. 



A, B. C. D and E 



BUDGET ACTIVITY: 

RESEARCH ^ AlI'iINISTI^iTION O 

REVIEW a APPROVAL O TECHNICAL ASSISTANCE [J 



GOOPERi^TING UNITS OF THE i^UBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 

1957. 

A, The Acute Leukemia, Solid Tumor and Surgical Services of the NCI have 
made available blood samples from patients admitted to study on those 
services and have collaborated generously in providing the clinical in- 
formation necessary for adequate interpretation of tlrie serum protein 
data, 

B, None, 

C, None. 

D, Drs. Sober and Peterson of the Physical-chemistry Section of the Labora- 
tory of Biochemistry, MCI, have been responsible for the development of 
this chromatographic method and are actively collaborating in the appli- 
cation of this technic to clinical studies, 

. E, None. 

13. ^ 

IF THIS PROJECT RESEI-iBLES, CO^iPLEilENTS OR PARALLELS RESEARCH DONE ELSEVfflERE 
IN THE PUBLIC HMLTH SERVICE (WITHOUT INTERCIi'iNGE OF PEn.^ONNEL, FACILITIES OR 
FUNDS), IDENHF? SUCH RESEARCH: 

A. None. 

B. None, 

C . None , 

D. None. 

E. None. 

No entires for items 14, 15 and 16. 



1-. N.C.I. 
INS TI TOTE 



PROJECT REPORT FORM 
2, General Medicine 



PAGE I 



LABORATORY OR BRANCH 



3. Metabolism 



SECTION OR SERVICE 



h. ^. NCI 703(c) 

LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 



6, Estimation of Red Cell Survival In Patients With Advanced Neoplastic 

Disease Before and After Treatment. ■• 

PROJECT TITLE 



7. J. L. Steinfeld 

PRINCIPAL INVESTIGATMTsT 



8. None 



OIKFil INVESTIGATORS 



9. PROJECT DESCRIPTION 

Objectives ; To determine if measurement of red cell life would be a 

useful parameter for ascertaining the value of a proposed 
therapy for cancer by first correlating such studies of 
red cell life span vjith approved methods for clinical and 
laboratory evaluation of cancer therapies. 



Methods employed: 



100 micro-curies of Sodium Ghromate^l in 8cc of ACD 
solution are incubated with 50cc of patient's blood 
and after washing throe times with saline to remove 
free radioactivity, the labelled red cells are 
reinjected into the patient from whom serial samples 
of blood are taken, radioactivity measured and red 
cell life span is estimated. 



Patient Material; Admissions 



Adult male 
Adult Female 
OPD Visits 



3 

1 

20 



90 pt-rdays 
30 pt-days 



Major Findings : Twenty studies in eighteen patients have shown that red 
cell life span is variably decreased in cancer patients. 
In only two instances was the patients' status stable 
enough to perroit two full studies of one month each 
to be carried out before and after therapy. No 
conclusions may be drawn at this time as to value of 
this technique for judging efficacy of cancer therapy. 

In a collaborative effort with Drs, H, Chaplin and 
P. Schmidt of the Clinical Center Blood Bank, red 
cell survival of blood stored and frozen at -1;5°C for vary- 
ing periods of tirae xi^as found to be in the normal range. 



NCI-703(C) PAGE 2 

PROJECT REPORT FORM (Cont'd) 

Significan ce to Cancer Research ; Objective measures of judging the 

— . ^ destructive effects of cancer therapy 

on cancers and beneficial effects' on 
the host are urgently required at the present time. Such techniques 
as this - although involved and time consuming - would be of value in 
select cases if correlation were high between red cell life span 
and clinical status.. 

Proposed Cour se of Project ; To continue as outlined above and to pursue 

~ further with Drs, V. Price and R, Greenfield 

studies of the tissue distribution of 
labeled red cell components at surgery and at autopsy, comparing 
normal and cancerous tissues after correction for the blood content 
of the tissues • 



NCI-703(C) 



10, NCI-703(C) 

SERIAL NO, 



PAGE 3 



PROJECT REPORT FORM (Cont'd) 



11. 



BUDGET ACnVITY": 

RESEARCH fTT 

REVIEW & APPROVAL / / 



ADMINIGIRAHON / / 

TECmaCAL ASSISTAKC E/ / 



12, None 



COOPERATING UNITS OF IHE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSOmiEL FOR THIS PROJECT IN EITHER 
1956 or 1957. 



13. None 



IF THIS PROJECT RESEI-fflLES, COl-iPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WIIHOUT INIERCHANGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RKSEARCH: 



No entries for items lU, l5 and l6. 



PAGE I 
PROJECT lilCPOKT FOm 

L, Nj^.Ij 2. General M edicine Bran ch : .-__ 

INSTITUTE " LABOMTOkY OK BMNCH 

5. A. , 5. N0I::204Lia 

SECTION OK SERVICE LOCATION (IF OTHER THAN BETHESDa) SiiiRIAL WO. 

The Influence of Delta Amino Levulinic Acid and Some of Iti; Analogues on 

j. Tumo r Growth, ^ 

PROJECT TITLE 



D. P. Ts chudy 



PRINCIPAL INVESTIGAT(m(S) 



OTHER IN^/ESTIGATORS 



PROJECT DESCRIl-TION 



Objectives; Since delta ajnino levulinic acid is an intermediate in porphyrin 
synthesis and one carbon transfers we are attemptinp to demon- 
strate a growth promoting action of this compound in tumors, 
along with direct demonGtratlon in tumora of the enzyme which converts delta 
amino levulinic acid to porphobilinogen. We are attemptinf^ to inhibit tumor 
growth by means of chemical analogues of this compound. 

Metho ds emp lo y ed; Organic synthe.'Jis, tumor transplantation, tumor growth rate 
methods, paper chromatograptiy, extraction of enzymes from 
turaors, piianuacological methods and histologic methods. 

Patient MatqriaJ. : 

Ma.ior Findings; Delta amino levulinic acid has been synthesized by a now 
method, A number of chemical analogues of this compound 
have been prop'. red, some of which had never previously been 
synthesized. Pharmacologic and enzyme aludiojj in animals are not completed. 

Significance to Cancer R ea earch ; If inhibition of "one carbon fragment" utili- 

zabion can be attaine(J by this mo't lod along 
with partial inhibition of porphyrin syn- 
thesis, antitumor activity may result. S/nergistic increase of anti-tumor 
activity of folic acid antagonists may also be produced. 

Propos ed Cou r se of Project; If we are successful in demonstrating the enzyme 

synthesizing pori)hobilinogen, we may attempt to 
demonrrtrate its intracellular localization. 

Growth inhibitory activity of any of the compounds will be studied in other 

systems including human tumors. 



PAGE 2 
PROJECT REPORT FORM (Cont'd) 



10. 


NCI-70A(C) 

SSitlAL WO. 












11. 
















BUDGET ACTIVITY 














■ -RESEARCH 




HI 


ADMIillSTRiTION 


D 






REVIEW & AI^ 


r'ROVAL 


D 


TECHNICAL AS3ISTAWCE 


D 




12. 















13. 



COOPERATING UNITS OF THE PUBLIC HE/iLTH SERVICE, OR OTHER OHSAWIZATIONS, PRO- 
VIDIWG FUl'IDS, FACILITIES, OR PERSOm\!EL FOR THIS PROJECT IN EITHER 1956 or 
1957. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEi^RCH DONE ELSEWHERE 
IN THE PUBLIC HE^lLTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES , 
OR FUNDS), IDENTIFY SUCH RESEARCH: 



No entries for items 14., 15 and 16» 



PAGE I 
PROJECT REPORT FORM 
N.C.I. 2. General Medicine Branch 



INSTITUTE LABORATORY OR BRANCH 

3. ^. 5- NCI-706(c) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. study of the Value of a Skin Test Using Polysaccharide in Cancer and 

PROJECT TITLE Non-malignant Diseases 

7. John H. Tuohy and Murray Shear 

PRINCIPAL INVESTIGATOR (S) 



OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION: 

Objectives : To examine the sensitivity and specificity of the test for 

human malignancy in which a small measured amount of bacterial 
polysaccharide is injected intradermally into the skin of 
humans with and without malignant disease. 

Methods Employed : As indicated above. 

Patient Material : None . 

Major Findings : This study has been in abeyance during the reporting year due 
to the requirements of the Solid Tumor Chemotherapy Program. 

Significance to Cancer Research : Implicit in the objectives listed above. 

Proposed Co\irse of Project : This study may be abandoned in view of the 

increased activity in the Solid Tumor Chemo- 
therapy Program. 



PAGE 2 
PROCfECT REPORT FORM (cont'd) 



10. NCl-7o6(o; 

SERIAL NO. 



BUDGET ACTIVITY: 



RESEARCH JTJ ADMINISTRATION /~7 

REVIEW & APPROVAL / 7 '^ ^L ASSISTANCE / / 



12 . 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 195 6 or 195' 



13- 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEyVRCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR ITEMS 1*4, 15 & 16 



PAGE I 
PROJECT REPORT FORM ^ 

fj^Q_j^ 2. Gctif.Tal Modiclru.' Branch 



INSTITUTE ' LABORATORY OR BRi^NCH 

k. ' ___^ !3. NCI-707(C ) 

SECTION OR SERVICE LOCATION (IP OTHER THAN BETHESDA) SERIAL NO. 



Evaluation of Blood and Plasma Water Content in Patients with Malignant 
PROJECT TITLE ' Neoplasms . 



John H. Tuohy 

PRINCIPAL INVESTIGATOR (S: 



OTHER INVESTIGATOR:; 



PROJECT DESCRIPTION, 

Objectives : To determine the relative difference, if any, between the 
moisture content of the blood of patients with malignant 
disease, patients with other forms of acute and chronlo illness, 
and normal individuals. 

Methods Employed : The chemical determination of moisture content of whole 
blood, serum, plasma, and tissue by the use of the 
Carl Fischer rea^orit. 

Patient Material: None . 

Major Findings : This project has been temporarily abandoned and was not 
active during the reported year. 

Significance to Cancer Research : Carried to completion with a positive rftsult, 

t'hls would furnish a diagnostic test for 
malignant disease . 

Proposed Course of Project: Indicated above. 



10. NCI-707(C) 
SERIAL NO. 



PAGE 2 



PRtJECT REPORT FORM (cont'd) 



11. 



BUDGET ACTIVITY: 

RESEARCH 

REVIEW & APPROVAL 



fTJ ADMINISTRATION / 7 
I 7 TECHNICAL ASSISTANCE / / 



12. 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 1957 



13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES . ITEMS \h, 15 & i6 



PAGE I 
PROJECT REPORT FORM 

1. N.C.I. 2. General Medicine Branch 

INSTITUTE LABORATORY OR BRANCH 

3. Dermatology h. '>. NCI-708(C) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Dermatologic Research 

PROJECT TITLE 



7 . E» J. Van Scott, Richard Reinertson, C. B. McCall; Jack Walte, Ross McCardle 
PRINCIPAL INVESTIGATORS 



OTHER INVESTIGATORS 



A. PROJECT DESCRIPTION - Statistical Incidence of Types of Pigmented Lesions 

on the Palms and Soles - E. J. Van Scott;, 
Richard Reinertson, and C. B McCall 

Objectives : To determine the incidence of pigmented lesions on the palms 

and soles J and to determine histological types of those occur- 
ring on these sites. 

Methods Employed: Examination of palms and soles, of randomly selected group 
of individuals, for presence of pigmented lesions. 
Biopsy removal of random group of such lesions. 

Patient Material : Random selection from patients in Clinical Center^ em- 
ployees, juvenile home. 

Major Findings : Approximately 2% of over 7OO individuals examined have 

been positive for pigmented lesions on the palms or soles. 
Approximately ^5 lesions have been excised from 40 

individuals and have been exEunined histologically. Over 50^ of these 

lesions have been found to be "Junction nevi." 

Significance to Cancer Research : Si'ice Pack reported that the incidence of 

nevi on the palms and soles is less than 
l^j and since k to 12^ of melanomas are sail 
to occur on the foot, an accepted ciorrent therapeutic procedure is to 
prophylactically excise any pigmented lesion from the palms and soles 
on the rationalization that such lesions are unduly prone to develop 
into melanomas. 

The data of this project questions the validity of such reasoning 
and opens for re -evaluation the role of the ,;-inction nevus in the 
development of melanoma. 



NCI-708(C) , , PAGE 2 

SERIAL NO. ' 

PROJECT REPORT FORM (CONT'd) 

Proposed Course of Project : This project is completed, pending the final 

compilation of the data and its review by- 
statistician. 

9. '\B. PROJECT DESCRIPTION - Relationship of Junctional Activity in Pigmented 

Nevi to Development of Melanomata - E. J. Van Scott 
and Jack Waite 

Objectives ; To determine incidence of activity, junctional or otherwise, of 
nevi in patients with and without melanoma. 

Methods Employed : Nevi on the backs of three groups of patients will be 
nxjmbered by a constant method. The three groups of 
patients will be: 

a) Normals (patients without melanoma). 

b) Patients with metastatic melanoma. 

c) Patients with primary operable melanoma. 

Randomly selected nevi will be excised from the three groups of 
patients for histological examination. 

Patient Material : Patients hospitalized in the Clinical Center or followed 
in Admission and Follow-up Clinic. 

Major Findings : Nevi from three individuals have been excised to date. No 
findings to report at this time. 

Significance to Cancer Research : This project attempts to establish whether 

or not there is some generalized bodily 
influence which promotes malignant change in 
"benign nevi" found on the skin of patients with melanoma. 

Proposed Course of Project: Continuation as outlined above. 

9. C. PROJECT DESCRIPTION - Histologic and Histochemical Anatomy of Certain Skin 

Lesions, as Correlated with Gross Anatomy - E. J. 
Van Scott and Ross McCardle 

O'ff-jectives : To establish the gross and microscopic anatomical changes which 
occur in the skin and its appendages under normal and pathologi- 
cal conditions. 

Methods Employed : Punch biopsy specimens are serially sectioned and stained 

with different stains . Sections are examined microscopical, 
ly. Balsa wood models of cutaneous structures are made 
from projections throvigh a microscope of these structures; resultant 
models are 72 times normal size. 



PAGE 3 
fCI-7Q8(c) 

;erial no. 

PROJECT REPORT FORM (cont'd) 

Patient Material : Skin specimens obtained from patients in Clinical Center; 
Employee Health Service. 

Major Findings : Reco istruction models thus far made: 

Condition No. of models 

Normal hair follicle of back . 2 
Follicle' involved with acne (back) - ,. ■^.rh 
Follicle involved with basal cell 

carcinoma (back) 1 

Normal follicle of beard 2 

Beard involved with alopecia areata 2 , 

Follicle of normal scalp 1 
Follicle involved with alopecia 

areata (scalp) 2 
Follicle involved with early male 

baldness ... 2 ■ , 



Total number of models ,l6 

' Corirelati'bn of inicro-scopic changes vith gross changes CQncurrently 
- being- mad^. 

Significance to Cancer Research : ■ This' study relates to: 

1. ' Elaboration of anatomical changes taking piace in. the skin at 

the' time of development of skin, cajacer. . ; 

2. The study of controlled growth and .quiescence of the. germinative 
cells' in the hair bulb,i 

3. The effects of the hormonal milieu on the growth . of hair and 
sebaceous glands .' ■ ' 

k. Suggests the possibility of the study of hair growth in tissue 
cxilture as a means of studying certain condition& which may 
affect cellular mitosis. 

Proposed Course of Project ; Continuation as outlined above. 

). D. PROJECT DESCRIPTION - Amino Acid Content and Enzymatic Activity of Skin 

and Mucous Membranes - E. J. Van Scott and 
Richard Re inert son: 

Objectives: Quantitative determination of certain amino acids and arglnase 
activity in the various layers of normal and pachologic skin. 

Methods B uployed: Tissue to be analyzed includes epidermis and corium^ 

'^' mucous membrane of upper esopha-gus and vag"Lna^ and liver, 
obtained from autopsy material; also includes scales from 
patients with exfoliating skin diseases, malignant tissues from skin 
of' patients with either primary or metastatic disease; hair, nails. 



PAGE h 



NCI-708(C) 
SERIAL NO. 

PROJECT REPORT FORM (cont'd) 

Chemical methods; 

1. Arginase: Method of Krebs and Henseleit, modified. 

2. Cystine: Okuda titration of hydrolyzed tissue. 

3" Arginine; Sakaguchi method, as improved by Sakaguchi. 

k. Other amino acids: Paper chromatography of hydro- 
lyzed tissue, elution of color from spots on paper and 
quantitative estimation by colorimeter. 

Patient Material : 

Major Findings : Determinations of arginase activity, arginine, cystine, and 
free siilfhydryl have been made on several specimens of 
psoriatic scales, plantar calluses, epidermis, and scales 
from exfoliative dermatitides. The amount of arginine in psoriatic 
scales has been found to be significantly less than that found in 
plantar calluses. 

Method for analyses by paper chromatography is being refined. 

■ Significance to Cancer Research : The amino;, aeid pattern of total whole skin 

of a nima ls has been reporte.d. to^^hange when 
the skin undergoes malignant chaxige. The 
amino acid content of the various layers of the skin is not known 
and particularly is it unkno\m for human skin. This project attempts 
to partially define the values for various chemical constituents in 
discrete layers of normal and abnormal skin. Changes in the 
chemical composition of cancerous skin may be accounted for by volume 
changes of certain layers of the skin rather than elementary changes 
brought about by the malignant, growth per se. On the other hand, the 
actual chemical composition of specific cellular elements, or layers, 
may change under pathological conditions. This project attempts 
to clarify such points. 

Proposed Course of Project : Continuation as above. 

9. E. PROJECT DESCRIPTION - Chemotherapy of Skin Cancer and Skin Diseases by 

Iontophoresis - E. J. Van Scott 

Objectives : Evaluation of local effect of drugs in skin tumors and skin 
diseases of substances introduced by electric current. 

Methods Employed; Introduction of substances into skin by iontophoresis 
apparatus; observations of gross changes. 

Patient Material: Incidental procedure on patients hospitalized for other 
major purposes. 

Major Find.ings : No demonstrable effect has been shown to occur in normal 
skin, psoriatic skin^ and in tumors of mycosis fungoides 



PAGE 5 

JCI-706(C) 
5ERIAL NO. 

PROJECT REPORT FORM (cont'd) 

following the iontophoretic introduction of thallium, mangamese, 
cobalt , methotrexate . 

Significance to Cancer Research : Project attempted to illicit any direct 

effect of substances introduced locally into f 
skin tumors. 

Proposed Course of Project; Discontinuation as a discrete project. 



PAGE 6 
PROJECT REPORT FORM (cont'd) 



10 . NCI-708(C) 
SERIAL NO. 



11. 



BUDGET ACTIVITY 

RESEARCH JYJ ADMINISTRATION / 7 

REVIEW & APPROVAL /~7 TECHNICAL ASSISTANCE / J 



12. None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 1956 or 
1957. 



13. 

IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCIiANGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR ITEMS 1^1, 15 & l6 



PAGE I 
PROJECT REPORT FORM 
N.C.I. ■2 Genera! Medicine Branch 



INSTITUTE LABORATORY OR BRANCH 

Acrtc Leukemia ^ • 5 ■ NCI- 709(C) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

The Chemoi-herap y of A cute Leukemi a 

'PRO.JECT TITLE 



E mil. Frei , III 

PRINCIPAL INVESTIGATOR fgy 



E . J. Freirelch, J ames Stengl e, Richard T- SUver, G- Lennard QoJ.d 
OTHER INVESTIGATORS 



PRO.TECT DESCRIPTION- 

Ob.'jectives ■ To study the effects of chemical com-DOunis on the course of 
acute 'eukemia. 

Methods employed- I. All patients i-eferred to the Clinical Center with 

the diagnosis of acute Leukemia are admitted to the 
Gti'.dy. (for participating hospital units see #12). 

2. Objective criteria I'or the e\/-aLua';ion of disease 
activity have teen developed and applied 

3- One of the following methods xor evaluate n-^, drug 
effect wl 11 be app ; ied depcniini upon the degree 
pf kiowa antitumor ac.;ivlty 

a) Pi '.ot studies in a few patients with varying 
dosages for dru-^s of potential antitumor activity. 

b) Sequential aria lysis i e.. administration of the 
drui to the number of patients necessary to 
establish with confideiK-e limits thai, it is_, or 

■is not, influencing the course oJ' the disease and. 

c) comparative .studies of dru-^'s of proven human 
antiiiojnor activity in an effort to detertiii-ie whether 
3i,:,njf leant dif.t'erenceg e/cLst 

h. Combinations and varyi.i/j, dosa/^e schedules will be 
stu'iie'-l when laboratory evidence or th'ioretical 
factors sugjesi. possible incrf.-ased antitumor activity. 



WCI-7Q9(C) PAGE 2 

SERIAL NO. 

PROJECT REPORT FORM (cont'd) 

Major Findings ' 1 A preliminary review of our data indicates that 

. combined Metho .rexate and 6 Mercaptopurine therapy . . 

is not superior co either one above. 
2. The evidence as to whether the administration of 
Methotrexate at Less frequenc intervals alters the 
therapeutic toxic ratio is inconclusive. 
3» Pilot studies using MetHoorexate at intervals frequent 
enough to mainoain constant blood levels have revealed 
enhanced therapeutic effect 

Significance to Cancer Research - Trial in humans of potentially effective 

chemical compounds is of obvious importance. 
Quantitative comparative studies designed to detect 
s i.ight but statistically significant differences are 
essential to direction of drug; development into 
profitable areas. 

Proposed.; Course of Study The most intense activity will obviously focus 

on the drugs that show major activity in the 
animal screen. Leads that develop as a result 
of studies by the pharmaco logy group such as dosage 
schedules routes of administration, etc. will be 
exploited. 

In an effort '.o increase the number of clinical trials 
other hospital units have been inc Luied in the study 
and it is hoped that more will be added. 



PAGE 3 

PROJECT REPORT FORM (cont'd) 

NCI-709(C) 
SERIAL NO. 

BUDGET activity! 

RESEARCH ^TJ ADMINISTRATION ' ' 

REVIEW & APPROVAL ' ^ TECHNICAL ASSISTANCE f ' 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER '-956 or 95^^ 

Department of Pediatrics 
University of Buffalo 
Buffalo^ N Y, 

Roswell Park M morial Institute 
Buffalo 3, New York 



3. None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCRANGE OF PERSONNEL, FACILITIES 
OR FWros), IDELITIFY SUCH RESEARCH- 



NO ENTRIES FOR ITEMS L^l '5 & l6 



FAOJ.0 I 



H;ai,,CT U^lJ'OiiT FOluM 



1 . NC.J 2, Uumr.Ml Medici luHrnnc li 

1 N;'T.LTUTK Ly\iK)o'A'ri) iY OK Blu\llCV 



3. Non 



Jone ]|. None ''.Nt; 1-71.0(1;) 

-,CT.10N o;< Si'^mciv I'A.. .Mi'U (.11-' (':Tnp;i'! Tiiy^i^: rn';'ri)i':;">l")A) ;.>.,;;:Ia1, Wo. 

ltit:4)rjti. uLlai'v ^iota,boliSlll I11 M.md 

"OTfFimT! — 



!■ :iN(;iPA.i., iNV;';;;T;i:CATo;;,(t:i) 



1. Alton lM.'i;;lec, MJI., n(ni.n(.l Tseluulv, M.1I. 



OT!M,;' .ilvv.';oTK;Atw,;;; 
rivui i',(.;t n ; c.i.MrTniM 

Project : .Inheriiu'di .'vry Mel.Mluil, i :iiii In l''i.'in 

Ob. juctives ; At. v.'irieu;! i.j 111 ;: 111, nili'.'vr of Uw ncvncl 1 iii I'.'i 1. In'/incii. ;; col. I .'tborato 
wi 111 IIk' cl:lnical {.roup .i.n 'Ti attempt to m.'ike pronliie biochem- 
\r.:\\ litudion on pationts with known deJ'ecto in Jnturmed;l,'ir" 
motaboliriin, or to uxploro pospible diriturbnnno;i in 1 nt.rnn. 'di /M-y 
mi.;tai.)olism in pftientn w' th nuopl.-uitic diwu.'uie, 

fi tl od;; : Inxr]] pati^Jitti .'ire ndridited io tJi. (.'iMi' t.m 1 iiiiMi.lcin'' nurvlcie ru'id 
th ) details of the prof.rnnis, ond thoir J.ntopration with probluma 
of patiunt caro, ary worked out by consultation between iiKiiribura 
of the f-enoral medicine staff, and th>.i nonclinicfil inventit'.ntor. 
The apocifio 1; 'bora tor \' luetbodfi utiliiBed ni-'e r/ither variable, 
nnd depend on tJw f.yp.> nl' in-nblom nndor atudy. In goMeral, they 
ha.vo followed l.li. p,'i|l,Mni nl' ;;imple i.ixploratlon o,l' the propunce 
or ab.'ionci' in hhiMd 'Mid nrin o\' |in.dii(' I.;: id' 1 nl.oriiiiid 1 ary 
metaboli sin. 

i'ati> nt Mat.irlal : {199S caieiKlar year) j\i timuS it in possible to under- 
take- ntudios in patients nlreadv on tho survico. At oUier tinuig 
special recruitment ia noedod. For oxamplo: The donire of 
Dr. Muisttir to study the antlno pcid pattorns in phan;ylpyruvic 
olipophrenia waa li.d to nucli fjpecial rnrrui tiii' nl,. 

Admissionn: Ne. Averwr.^ ;;tay 
Day.? 

Childr>:n male 1 2Q 

Child rni I'Miiiale 1 20 



Page 2 



PROJECT REPORT FORM (Cont'd) 



Major findings ; These are reported. by the separate laboratories collab- 
orating in the studio,?. 

Patients, knovm to have phenylpyruvic oligophrenia, were 
admitted on February 28, 19$^ for studies to be carried out by 
members of this Section in collaboration with Dr, Samuel Bessman 
(Associate Professor of Pediatrics, University of Maryland Medical 
School, Baltimore) and Dr. Sidney Udenfriend (National Heart Institut. 

AdmJ.ni strati on of flutamine- (7.6niMole per kilo) led to a 
dramatic drop in phenylpyruviC' acid excretion — to one-third to 
onti-fourth of th^j control level, 

a. Oral administration of glutamine and aspara£,ine (7.6 
mM/kilo) does not lead to dangerously high levels of 
blood ammonia nor to very freat e::cretion of ammonia,, 

b. Blood levels of glutamine, 5 times the norms! level, 
were maintained for about 3 hours. 

c. Glutamine administration does not appear to have affected 
the excretion of phenylacctylglutamine, or of creatinine. 

It may tentatively be su' gestc^d that the strikinf- decrease . 
in phenylpyruvic~"acid excretion after glutamine administration is 
due to transajnination of this keto acid to phenylalanine. 

Significance to CANCER Research ; This program is of importance to the 

National Cancer Institute program because it represents an attempt 
to utilize directly the newest developments in the nonclinical 
branches, in the study of the natural history of neoplastic disease. 
In thi? way, the theory and the technique of biochemistry, tissue 
culture, biology of groT^^bh and radiobiology can be made available 
to the clinical studies at an early stare of development. It is 
hoped that from such active collaboration will come new leads as 
to the pathogenesis of neoplasia. 

Proposed cour s e of project ; At the present time no specific projects are 
being carried on, since the ones proposed last joar have either 
been completed or developed into full projects of their own. 



PAGi:; 3 



PROJECT RiiPOilT FOrn^i (Cont'd) 



0. 



NCI-710 (C) 



1. 



BUDGi:;! ACTIVITY: 



RESEARCH £3^ aDMNI :.T?UiTION r~~7 

REVIii;;>[ & APPROVAL /~7 TECHNIOii ASSISTANCiI; /~7 " 



From time to time patients iidll be studied in cooperation mth 
other Institutes at the National Institutes cf Health, but i-jithout 
2, specific allocation cf other research funds, 

COOPERATING UNIT3 OF TKj. PUBLIC h.. ,1/111 SERVICE, OR OTHER 0.-;rANI2ATI0NS, 
PROVIDING FUNDS, FACILIT1.,S, OR PER30KK;:.L FOR THE PROJECT IN EITHER 19$6 or 

1957 



3, Does not apply 



IF TEI':. PXJ.^CT RE3Ei'IBL..,S, CriiPL.EEETS, OR PARALL.!,LS RESEilRCL DONE ELSE- 
WHERE IN TH^ PUBLIC KEl/n; ECl^VICi:, (vlTEOUT INTERCIlAKG. OF PERSONNEL, 
F.lC1LITIES or funds), IDx^NTIFY SUCI: RESEi.RCH: 



NO ENTRIES FOR ITEMS ll;, li;; & 16 



PAGE I 



N.C.I. 



PROJECT REPORT FORM 



2. General Medicine Branch 



INSTITUTE 



LABORATORY OR BRANCH 



3. Solid Tmor Chemotherapy k. 5- NCI-71l(C) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) . ^ERIAL NO. 

S. Assay of Compounds for Antitvimor Effect by Injection into Skin Metastases 
PROJECT TITLE 



7. T.:C. 0. BrindLey 

PRINCIPAL INVESTIGATOR ( S ) 



OTHER INVESTIGATORS 



PROJECT DESCRIPTION 



Objectives : To develop a method of assay of compounds for antitumor effect in 
humans by the injection of these materials into subcutaneous 
tumor masses. 

Methods employed : Needle and syringe injection of lesions; determination of 

antitimior effect by direct caliper measurement, comparative 
photographs and histological study. A patient having a 
remission of her malignant disease was studied by endocrinological survey 
and a search for immune response to the tiamor was made by serological 
tests and by transfusion of this patient's blood or plasma to other 
patients with similar malignancy. 



Patient material- 



Admissions ' 



Adult males 
Adult female 



No. 



7 



Average Stay 
Days 

52 



Outpatient; 



NiJmber of patients 
Number of visits 



10 
20 



Major findings - The study so far has shown that (l) extensive histological 
change may be caused by antitumor agents before changes in 
size can be demonstrated by caliper measurement or changes 
in appearance occur, and (2) the quantity of antitumor agent which must 
be injected locally into tumor masses in order to cause regression in 
size of these masses is relatively large and approaches the systemically 
tolerated dose in some cases. 



NCI-711-(C) PAGE 2 

SERIAL NO. 

PROJECT REPORT FORM (cont'd) 



One patient in the study had a complete regression of all metastatic 
lesions. 

Significance to Cancer Research - For many agents the- effects on biological 

systems and the antitimor effects in 
animals are not closely correlated with anti- 
tumor effects in the human. The number of agents to be tested is large. 
So a method whereby agents can be safely tested within a short period 
of time in humans woyld be useful. The possibility that the injection 
of subcutaneous metastases could be developed into such a method should 
be explored. 

Proposed course of the project : For the past several months suitable patients 

for this study have not been available. The 
project has been temporarily stopped for this 
reason. When such patients become available in quantity, it is planned 
that the project will be resumed. The patient having the complete re- 
mission of her disease will continue to be studied for possible causes 
of this remission. 



10. NCI-711(C) 
SERIAL NO. 



PAGE 3 



PROJECT REPORT FORM (cont'd) 



11. 



12. 



BUDGET ACTIVITY 

RESEARCH JT] ADMINISTRATION /~7 

REVIEV7 & APPROVAL / 7 TECHNICAL ASSISTANCE / 7 



None 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATION, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 195^ or 195 



13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC H^LTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTIFY SUCH RESEARCH; 



Project 700C was a similar study as carried on by 
Dr. R. D. Sullivan. 



NO ENTRIES FOR ITEMS lU, 15 & l6 



PAGE I 



PROJECT REPORT FORM 



N.C.I. 

insttttte" 



General Medicine Branch 



LABORATORY OR BRANCH 



i. Solid Tumor Chemotherapy 
SECTION OR SERVICE 



5- NCI-712(C) 

LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 



A. Controlled Studies in the Chemotherapy of Solid Tumors 

B. Preliminary Studies of Chemo therapeutic Agents on Human Solid Tumors 
PROJECT TITLE 



A. C. 0. Brindley; B. I. Shnider, J. H. Tuohy 

B. C. 0. Brindley, B. I. Shnider^ J. H. Tuohy 
PRINCIPAL INVESTIGATOR(S} 



None 
None 



R INVESTIGATORS 



A. PROJECT DESCRIPTION ~ Controlled Studies in. the Chemotherapy of Solid 

Tumors 

Objectives : These are two-fold: (l) To test the validity of the experimental 
design of a cooperative randomized controlled study of chemo- 
therapeutic agents on a comparative basis, and (2) to determine 
the relative effectiveness of a test chenlotherapeutic agent against a 
standard drug in the treatment of a number of selected solid tumors in man. 

Methods employed : (l) Determination of experimental design including the 

format of the cooperative study. 

(2) Selectioa of patients. 

(3) Selection of test and standard drugs and their admini- 
stration according to an established protocol. 

(k) Antitumor effect is judged only on the basis of 

objective measurements. 
(5) Collection and synthesis of data from all cooperating 

groups. 



Patient material: 1955 (April-November) 



Admissions: 



No. 

Adult male 20 
Adult female 31 

Children, male 1 
Children, female 



Average stay 
days 

90 . 
90 



PAf E 2 
NCI-712 (e) '■ 

SERIAL NO. *:• - ' • . 

PROJECT REPORT FORM (cont'd) 

Outpatients: 

In Admissions & Follow-up Department 
Patients 77 
Visits 122 , „ ... V ..,;, V 

In home or outside hospital 
Patients 20 
Visits 20 

Major findings : The study is now in its formative stage. At present 
Triethylene Thiophosphoramide as the test drug and 
HN2 as the standard are being compared according to the 

design indicated above. Twenty-nine- (29) patients have been, or are now 

in the study. : • : . ■,.■. 

Significance to Cancer Research : By matching patients and the drugs being 

compared in a completely randomized manner , 
and by basing anti-tumor effect solely on 
objective criteria, this study represents an entirely new departure in the 
field of cancer chemotherapy. It is the first major effort to transfer the 
experience in experimental therapeutics in other fields to study of chemo- 
therapeutic agents in malignancy. The cooperative endeavor of which this 
project is a part has achieved acceptance by the Clinical Panel of the 
National Cancer Chemotherapy Center. It is being suggested as a pattern of 
study for other cooperative groups. There is hope that in this way drugs 
Showing promise in more preliminary studies can be critically and rapidly 
evaluated in large numbers of human cases . 

Proposed course of project : In the course of the next calendar year, it is 

anticipated that five other institutions will join 
in this study following the experimental design 
originated by the Solid Tumor Chemotherapy Group of NCI at the Clinical Center. 

Our own activities here will consist in increasing our experience and parti- 
cipation in this study by expanding our out-patient activities and bed 
capacity. 

9. B. PROJECT DESCRIPTION - Preliminary Studies of Chemotherapeutic Agents on 

Human Solid Tumors 

Objectives ; To test rapidly agents which have shown promise in other studies 
(animal screening, pharmacological, etc.) for their toxicity and 
antitumor effect in humans . 

Methods employed : Agents will be studied in selected patients using sequential 
analysis and objective criteria. 



PAGE 3 

rci-7i2(c) 

)ERIAL NO. 

■ PHOJECT REPORT FORM (cont'd) 

Patient Material : (Estimated - calendar year 1956) 

8 patients per agent x 5 agents = Uo patients..,.. _..,,-.'■... , 
Average days/patient « 60 days 

Patients participating in "A" may also be included in this study upon 
completion of their participation in the Controlled projestT-'. ,-.-",-,. 



Major Findings : None ... 

Significance to Cancer Research : . A logical part of a drug development program 

and sequel to animal screening is a rapid 
preliminary method for the assay of an 
agent's toxicity and anti-tumor activity in humans. It is a necessary pre- 
liminary to the final critical evaluation of any chemotherapeutic- material. 

Proposed course of project : As agents are furnished from intramural and outside 

sources, they will be systematically evaluated by 
this technique. 



PA(5E k 
PROJECT REPORT FORM (cont'd) 



10. NCI-712(C) 

SERIAL NO. 



11. A & B 



BUDGET ACTIVITY: 

RESEARCH /xj ADMINISTRATION / / 

REVIEW & APPROVAL f^ TECHNICAL ASSISTANCE / J 

12. 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 or 1957. 

A. Cooperative Groups; 

By virtue of requested grants it is expected that the following 5 
institutions will contribute to the cooperative study of which this 
project is a part. 

Department of Medicine, University of Miami, Miami, Florida 
Roswell Park Memorial Institute, Buffalo, N. Y. 
Lemuel Shattuck Hospital, Boston, Mass. 
Johns Hopkins Hospital; Baltimore, Md. 

Georgetown University Medical Division, D. C. General Hospital, 
Washington, D. C. 

B . None 

13. A & B - None 

IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OP 
FUNDS), IDENTIFY SUOH RESEAROH: 



16. 



PROJECT REPORT FORM (cont'd) 



Ik. NCI- 712(C) 
SERL\L NO. 



15. A & B - 



PUBLIC OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1955 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 
1955 

A. Acceptance by Clinical Panel, National Cancer Chemotherapy Center and 
its establishment as model for other such projects. 

B. None 



PAGE I 



PROJECT REPORT FORM 

1. N.C.I. ^^ 2. General Medicine Branch 

INSTITUTE LABORATORY OR BRANCH 

3. h. 3. NCI-7.13(C) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Energy Metabolism and IV Fat Emulsion 

PROJECT TITLE 

7. C. B. McCall 

PRINCIPAL INVESTIGATOR (S) 

8. Pr. Donald Watkin 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives : To measure the effect of hyperalimentation with intravenous 
fat emulsion on respiratory quotient and energy expenditure 
in patients with inoperable cancer. 

Methods employed : After suitable training period, by an open circuit 

method employing high velocity low resistance values and 
Douglas bags, measure Og and COp in expired air and 
calculate RQ, oxygen consumption, and calories/square meter/hour 
during three periods: ..l) control, 2) while receiving IV fat, and 
3) follow up after IV fat discontinued. 

Patient materia L : Patients on Dr. Watkin 's metabolic service admitted 
for IV fat study. 

Major findings : Five individuals have been studied to date. There has 
been no consistent pattern diiring control period, 
precluding any statement as to the effect of the extent of the 
tumor. 

Two patients diiring the second period (i.e. receiving IV fat) 
showed an abnormal i.y low RQ to a level suggesting ketone body 
formation. There was no consistent BMR change. In one case 
fasting RQ was relatively high with subsequent fall while receiving 
IV fat. 

One individual in follow up showed normal RQ. 



NCI-7 3(C) . PAGE 2 

SERIAL NO. ■" ■ , , 

PROJECT REPORT FORM (cont'd) 



Significance to Cancer Research : This study may help in part to answer 

the question, whether one can protect 
a patient from his cancer by supplying 
calories and how is IV fat emulsion used by body. 

In -general, this type study might prove useful on a number 
of metabolic patients under a variety of conditions, such as, 
type alimentation, extent of tumor, and nutrition. 

Proposed course of project : ■ Finish the patients already started and 
' ' add others until we have information as 
outlined complete on 6 patients. 



PAGE 3 
PROJECT REPORT FORM (cont'd) 



10. NCI-7L3(C) 
SERIAL WO. 



11, 



BUDGET ACTIVITY: 



RESEARCH .pCJ... ADMINISTRATION r—j 
REVIEW & APPROVAL _/" J_ TECHNICAL ASSISTANCE / / 



12. NONE 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 or 1957. 



13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTJFY SUCH RESEARCH: 



NO ENTRIES FOR ITEMS ik, 15 & 16 



"vrr 



PAGE I 



PROJECT RaORT FORM 
1. H.C.I. 2. General Medicine Branch 



INSTITUTE UBORilTORY OR BR-'IWCH 

3. Leukemia Section k. 5.NCI-7 1A ( C) 

SECTION OR SERVICE LOCATION (IF OTHER TliilN BETIiSSm) SERIAL NO. 

6. Iron Metabolism. 



PROJECT TITLE 



7. James Stenglc 



PRINCIPAL IiIVESTIG.iTORCS) 



OTHER IlWSSTia.TORS 



9. PROJECT DESCRIPTION 

Objectives : a) Does the iron metabolism of the patient with malignant neo- 
plastic discasj differ from that of the normal? 
b) Is the leukemic cell or solid tumor competing for iron at 
the expense of normal body needs and may this be a partial 
explanation for the anemias characteristic of these 
diseases? 

Background ; Recent work in the National Cancer Institute 

(Price) indicates that certain mouse tumors have a 
high iron content. It is commonly noted in bone 
marrow observation that stainable iron is lacking during an 
acute exacerbation of leukemia but may reappear during re- 
mission. It has been reported (Simmons and Everett) that 
normal rat loulvocytes incorporate considerable amounts of 
radioactive iron administered orally or parente rally. 

Methods Employed ; In leukemic patients serial serum iron o.nd leukocyte iron 
determinations will be made and correlated with hemoglo- 
bin and leukocyte counts. 

In vitro studies of normal and leukemic leukocyte uptake 
of radioactive iron will be made . 

In solid tumor patients seruin iron and hemaglobin values 
will be followed and surgical and autopsy specimens analysed for iron 
content. 

Some of the above work will be carried out in cooperation 



Page 3 

PROJECT RETOltT FOHM (cont'd) 



10. WCI - 7 U (C ) 

SERIAL NO. 



11. 



BUDGET ACTIVY: 

RESEARCH /X7 ADMINISTRiTIOW [J 
REVIEW & APPROVhL [J TECffiTIC>.L ASSISTANCE fj 
12. 



G00PE.<ATING Ui\IIT3 OF THE PUBLIC HE;.LTH SERVICE, OR OTHER ORGANIZATIONS, Fi^0- 
VIDING FUNDS, FACILITIES, OR i- EitSONNEL FOR THIS PROJECT lij EITHER 1956 or 1957 



13, 

IF THIS PROJECT RESEI'iBLES, C0I%^L5i«iErir3, OR P-uULLELS RESE^iRCH DONE ELSEiJHSRE 
IN THE PUBLIC HFALTH SERVICE ('.v'lTHOUT INTERCH.1NGE OF lEr.SONNEL, FACILITIES OR 
FUND) , IDENTIFY SUCH RE3S.11CH: 

No entries for items, 14, 15, and 16. 



PAGE I 
rROJECT liEt^ORT FOhJyi 
1- N.C.I. 2. General Medicine Branch 



INSTITUTE lABORiTOx{Z OR BRANCH 

3. Leukemia Section U. 5. NCI-715(C) 

SECTIOil OR SERVICE ~ LOCATION (IF OTHER TE'iN BETHESDA) SERIAL NO. 

6, Th e Hemorrhagic Diathesis Associated with -cute Leukemia. 

PROJECT TITLE 

7. E. J. Freireich 



PRINCIPAL liWE 3TIGAT0R( S) 



Emil. ■Freii::JII 



OTHER li^IVESTIGATORS 



9. PROJECT DESCRIPTION 

Objectives: a) To identify factors responsible for precipitating gross 
hemorrhage in already thrombocytopenic patients, 
b) To study the effectiveness of fresh blood, platelet trans- 
fusion, and other blood products in controlling gross 
hemorrhage in leukemic patients. 

Me thods 'Emp loyed ; In vivo tests of bleeding tendency, in vitro tests of 

blood coagulation, clinical observation and measurement 
of gross bleeding. 

Pa t ient Material : 

Ma.ior Findings : Major effort thus far has been directed toward setting up 
techniques and procedures. 

Signi ficanc e to Ca n cer Re sea rch ; Hemorrhage ranks with infection as the two 

major causes of death in acute leukemia. 
At oresent no effective prophylaxis or 
therapy for thrombocytopenic hemorrhage in leukemia is available. Many of 
the now chemotherapeutic agents for leukemia as well as other tumors cause 
throm-boponia and bleeding. Any affective therapy of thrombopenic bleeding 
could greatly prolong life of acute leukemia patients as well as improve 
the effectiveness of chemotherapeutic agents. 

Proposed Course of Projec t : Observations of coagulation mechanism will be 

made periodically in acute leukemia patients in 
an attempt to define any changes associated with 
the onset of clinical hemorrhage. Patients suffering from hemorrhage will 
be given fresh blood transfusions and other blood products and the effect on 
the clinical bleiding and coagulatio'.: mechanism assayed. 



."AGS 2 
PROJECT REI-OHT FOWi (Cont'd) 



10. iIOI::a^(G}., 
SERIAL NO. 



11. 

BUDG"'T ACTIVITY: 



RESSilRCH Jl^ ADl-ulMI3TlmTI0N ' /27 

RHVia; ;^. ilPi ROVhL /S/ TSCHl^IIGAL aSSISTaIICE £} 

^^^ Division of Biologies 'Gontfol g^^^^ ^^^^ 



COOPER.iTING UNITS OF TM PUBLIC Hm iLTH SERVICE, On OTHER ORGANIZATIONa, PRO- 
VIDING FUNDS, F-iCILITIES, OR PERSONNEL l^'OR THIS PnOJEGT IN SITiIER 1956 or 1957 



13. 



None' 



IF THIS Pi.OJECT RESEMBLES, GOi-lTLi^-iJlNTS, OR P.iR-LLELS ESSE iRCH DONE ELSEWHERE 
IN THS PUBLIC HEiLTH SERVICE (l/ITHOUT lilTERCEii'JGE OF rSRSONNSL, FACILITIES OR 
FUiMDS) , IDENTIFY SUCH RESE.\RCH: 



No entries for items 14, 15 and lo. 



PAGE I 



PROJ'^CT REPORT FORK 
1. l^u.I. 2. General I'ledici ne Branch 



INSTITUTE LrlBOE-.TORY OR BR-iNCH 

3. Leukemia Section ^A. 5. NC 1-716 (C' 

SECTIOii OR SEiWIGE LUGiiTION (IF OTHER TliAN BETHE^M) SERIAL NO. 



6. Nutritional Aspects of the Th e rapy of Acute Leukeraia 

PROJEGT TITLE 



7* Emil F rei II I a:a4,,.Emi'l^ F3rei r.elch . 
PRINCIPAL INVESTIG..T0R( 3) 



OTHER INVESTIGATORS 



PROJEGT DESCRIPTION 

Objective; To determine the effects of nutritional factors contained in 
animal tissues on the course of human acute leulcemia. 

Methods Empl oyed; Various animal tissues are emulsified, analyzed for chemi- 
cal composition and presence of infectious agents, and if 
tolerated by experimental animals, are administered to 
to acute leukemia .atients. The course of tiie disease is followed by clini- 
cal observation of the patient and various hematological deterroinations on 
blood and bone marrow. 

Patient M ateri al : None . 

Major Findings; Emulsified mat-^rial has been prepared from hog tissues and 
stored at -50°G.- Studies in animals are in progress and no 
evidence of toxicity has been found to date. 

Significance t o Cancer Research; The major approach to the therapy of neo- 
plastic diseases has been and continues to 
be the use of cytotoxic agents. As a new 
approach, based on the assumption that the neoplastic cells are capable of 
maturing and returning to normal, nutritional non-cytotoxic factors will be 
used in the treatment of acute leukemia. The discovery of a factor or fac- 
tors that would promote the maturation of leukemic cells would have immense 
significance . 

P roposed C ou rse of Projec t; The pig tissues now available, if found satis- 
factory after screening in animals will be ad- 
ministered to acute leukemia patients that have 
been proven refractory to conventional forms of therapy. This will be 



NCI-716(C) 

PAGE 2 

PROJECT REPORT FORM (Conf) 

administered by nasogastric tube and the course of the disease carefully 
observed. If any changes occur, further separation of component tissues 
will be used to identify the responsible factor. If no changes occur, 
other tissues and other routes of administration will be attempted. 



PAGE 3 



PROJECT Ri^ORT VQRA (Cont'd) 



10. NCI -716(C) 

SERIiiL NO. 



11. 



BUiXJST ACTIVITY: 



RESilARCH /x/ 

REVIEW & ..IPi^ROV.xL /J 



ADMINjoTRiTION /Z/ 

TEOHiaCAL ASSISTANCE /^ 



12. 



U. Z. Department of 'griculture - heats Research Division, Beltsville, Md, 

Dr. gili s an d Dr. Hiner supplied an d prepared hog tissues. 

COOPER-TLfc Ul'JITS OF TI€^ PUBLIC HS.iLTH SERVICE, OR OTEER' ORGiUlIZ^iTIOi'lS," PRO- 
VIDING FUIIDS, F.iCILITIES, OR PERSONNEL FOR THIS i ROJEoT IN EITH-R 1956 or 
1957 



13 . None 



IF THIS PROJECT RESE1-':3LES, G0MPLEi£NT3, OR FARhLLSLS RESEaRCH DOjIE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (iJITHOUT INTERCaUIGE OF PERSONNEL, F.xCILITIES OR 
FUi'iDS), IDENTIFY SUC;I RESEiiRCH: 



No entries for items 1^., 15 and 16. 



I PAGE I 
PROJECT REPORT FORM 
K.C.I. 2 . General Medicine Branch 



INSTITUTE LABORATORY OR BRANCH 

Acute Leukemia h ___^ ^ . NCI-7'-7(C) 

SECTKJN OR SERVICE ' LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

Infections, Fe ver, and H ost Resistance in Acute Leukem ia^ 
PROJECT TITLE 



Richard T. Silver 



PRINCIPAL IlWESTIGATOR(Sj 

]. Dr. John Utz, NMI Dr. E.:dl Frei : Dr. John Fahey; Dr. Grace Beal. NMI- 
OTHER INVESTIGATORS Dr. Robert Kolb L.B.C. 



PROJECT DESCRIPTION 

Objectives' 1. To obtain data relating to the natural history of fever and 
infections in acute leukemia. 
2. To define the mechanisms of impaired host resistance to . 
infection in acute leukemia. 

Methods employed- Fevers of known and unknown origin have systematically 
been studied according to a previously defined protocol 
in over 30 patients with acute leukemia. Comprehensive 
bacterial and viral data have been collected and analyzed in conjiinction 
with a group in the Nationa'. MicrotiologicaL Institute. 

In order to define the capability of '.e'okemic granulocytes to phagocytize 
opsonocytophagic determinations have been made in 11 patients. The 
antibody response to 6 challenge antigens has been measured in 11 Leukemic 
patients and in 7 "normal controls." Alterations in serum proteins 
d\iring the immunization procedure have been measured by paper electro- 
phoresis 

Major Findings: The tabulation of the relative incidence of fever of known 
and unknown origin in the leukemic patient has not been 
completed. Of major importance in problems of patient care 
is the finding that many fevers previously ascribable to leukemia per se> 
have been demonstrated to be due to a complicating and remedial bacterial 
infection. The marked susceptibility to bacterial infection does not 
seem to apply to viral diseases . 

The opsonocytophagic tests have been concluded. No difference in the 
phagocytic activity of the mature polymorphonuclear cell in leukemic 



NCI -7 1-7 (C) - ?AGE 2 

SERIAL NO. 

?R©JECT REPORT F9M (cont'd) 

blood during active disease, bacterial infection or in hematological 
remission (as compared to nonrKiL) was noted. Hiagocytosis by the 
more iicffiature celLS of the granulocytic series of both Leukemic aftd 
normal blood is minima. This confirms previous reports in the 
literature, The susC2ptibiIity to infection does not bear a direct 
relatio-iship to the absolute mature polymorphonuclear count; but 
■ rather to a ratio of mature poly^totaL WBC Significant differences . ■ 
in the response to challenge antigens and in the electrophoretic 
pattern of the serum proteins have not been observed., although 
final results must await completion of the immunization procedures in 
the remaining normal controls/ and statistical analysis.. 

Significance to Cancer Rssearch - In spite of the voluminous leukemia 

literature, there have been no comprehea- 
'" sive observations relating to fever and ■ 
infection in these patients. Although there have been many individual 
■ studies pertaining to phagocytosis antibody response and electrophoretic 
patterns in leukemia there has been no study to the best of our know- 
ledge^ which has attempted to correlate all these parameters with serial 
clinical observations. Thus, it is hoped that information obtained from 
this study will augment our understanding of the natural history of 
acute leukemia and aspects of host resistance in general, and aid in the 
further improvement in the clinical management of the leukemic patient. 

Proposed Course of Study The present study should be concluded within the 
" next 6 months . 



PAGE 3 

PROJECT REPORT FORM (cont'd) 

NCI-7I7(C) 

SERIAL NO, 



BUDGET ACTIVITY: 

RESEARCH JTJ ADMINISTRATION I / 

REVIEW & APPROVAL I 7 TECHNICAL ASSISTANCE / 7 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER L956 or L957 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDEl^ITIFY SUCH RESEARCH)- 



NO ENTRIES FOR ITEMS 1^, 15 & I6. 



:) \Am 



PAGE I 
PROJECT REPORT FORM 

1. N.C.I. 2. General Medicine Branch 

INSTITUTE LABORATORY OR BRANCH 



h. ^. NCI-7'.8(C ) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 



6. Pulmonary Physiology 



PROJECT TITLE 



7. C. B. McCall 



PRINCIPAL INVESTIGATOR(S) 



OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION - Ventilatory studies on patients with airway, lung, 

or pleural involvement by neoplasm. 

Objectives : To study and correlate changes in tumor size with pulmonary 
function and blood gas studies and clinical course. 

Methods : Obtain baseline determinations of cotaL vital capacity, timed - 
VC, maximum breathin^-j capacity,, residual air, total lung volume, 
I nitrogen rinse out, and fluoroscopy. These determinations will 
be repeated at suitable intervals and correlated with X-ray size 
of tumor, sput\:iBi, wheezes, chest pain and dyspnea. 

Patient material : All patients on Solid Tumor Chemotherapy Program with 
suitable pulmonary lesions. 

Major Findings : A major portion of my time in past five months has been 
devoted to first, learning techniques and determinations; 
second, learning their proper interpretation and 
evaluation and thirdly, calibrating instruments and developing 
more accurate methods. In addition, during this time I have 
answered k2 consults from NCI - these tests being especially 
valuable in patients before and after pulmonary surgery. 

As to the above project, I have to date only baseline 
determinations from which no conclusions can be drawn. 



WCI-T '-8(C) PAGE 2 

SERIAL NO. 

PROJECT REPORT FORM (cont'd) 



Significance to cancer research : Such studies may shed some Light on 

'the effect of various tumors and their 
growth on pulmonary physiology, parti- 
ticularly ventilation^ and possibly provide some clue as to the 
cause of dyspnea. 

Proposed course of project : Perform studies as indicated on all suitable 

patients admitted to So '.id Timor Chemotherapy 
Program. 



PAGE 3 
PROJECT REPORT FORM (cont'd) 



10. NCI-7l8(.C) 



SERIAL NO, 



11. 



12. 



13. 



BUDGET ACTIVITY: 

RESEARCH [~T~ ] "ADMINISTRATION j -J 

REVIEW & APPROVAL / /_ TECHNICAL ASSISTANCE /~7 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 OR 1957. 



IF THIS PROJECT RESEIffiLES, COMPLEMENTS, OR PARALLELS RESKf\RCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR ITEl/B lU, I5 & I6. 



PAGE I 
PROJECT REPORT FORM 
N.C.I. 2. General I'-L. 1-1 c I. le Branch 



INSTITUTE L^BOR'^TORY OR BRANCH 

3. Acute L^ukeuia 3. 5- NCI-T19(C ) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL WO. 

b. A StuJ-y of the Clinical Ma:iiibstatiOiis of Acutg Leukapia 

PROJECT TITLE 



7. Enil Frsi, III . 

PRINC IPAL lOTESTIG/iTOR ( 3 ) 



8 : Richard T. Silver, Richard Svarm, E. J. Van Scott, Charles Wells 
OTHER IWESTIG/lTORS 



9. PROJECT DESCRIPTION 

Objectives: To deliaeate ebiologlc a-t-id pathogei:etlc iactcrs of the 

various ma'iifestatioas of acute leukenia with particular 
emphasis on the oral, neuroloj;ic, hepacic, rc^al, and 
osseous lesio s and the ab..orTial uric acid metauollsiii. 

Methods employed ; The coraprehensive application and correlation of 

clinical, heroa to logical, radiologic, chemical, micro- 
biologic, and histologic methods to the above manifes- 
tations in patieiits vjlth acute leixlceiriia . 

Major Findings ; Though data concernii g the above has bee:i collected, 

it has not as yet been analyzed and correlated. It is 
appare^it that multiple factors may contribute to these 
manifestations, e.g. the leukemic process per se, antimetabolite 
therapy, steroid therapy, infection, anemia, etc. 

Significance to Caxicer Research: A study of the imtural and modified 

history of acute leukemia and its 
complications is of obvious importance 
to cancer research. 

Proposed Course of Project ; It is anticipated that some of the information 

gained from this study will have practical 
application in the clinical management of 
this disease. Therefore, at least portions of the study will continue 
indefinitely. Moreover, additional diagnostic and e.cperimental 
procedures will be applied when it is apparent that the present methods 
are inadequate to explain the observed manifestations. 



10. NCI-719(C) 
SERIAL NO. 



PAGE 2 



PROJECT REPORT FORM 



11. 



12. 



BUDGjiT ACT:-VI'iT 

RESEARCH Jxj ATMIND.S'rPATICN / / 

RE\^Fv/ S: APPROVAL / / TEOffl^ICAL ASSISTAITCE / 7 



C0OP£;P.4T:r;v:i UirriS O? the public HMLQH service, or OTIiER ORGANIZATIONS, PRO- 
VIDING FlTIl^JS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER I956 or 
1957. 



13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSE- 
WHERE IN THE PUBLIC HEALTH SERVICE (V^THOUT INTERCHANGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTIFY SaCH RESEARCH: 



NO ENTRIES FOR ITEMS \h, 15 & I6 



\.bi\n -i: 



PAGE I 



PROJECT REPORT FORM 



N.C.I. 



INSTITUTE 



2. General Medicine Branch 



LABORATORY OR BRANCH 



k. ^___ 5. NCI-720(C) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 



Svalual Aiaino .Aci.d Metabolism InlHum an 'Subjects by Utilization of 
PROJEC' jE Parenteral Routes of Administration 



John L. Fahey 

PRINC IPAL INVESTIGATOR ( S ) 



OTHER INVE3TIGAT0F.S 



I. PROJECT DESCRIPTION 



Objectives : The general objective is to study the human metabolism of intra- 
"~~~~~~~~~ venously administered amino acids in normal and disease states. 
Specific objectives are (a) to establish the identity and amount 
of individual amino acids essential to achieve positive nitrogen balance, 
and (b) to approach the problem of determining the role of individual amino 
acids by observation of the effects of incomplete amino acid mixtures. 

Methods Employed : Optically-pure 1-amino acids prepared by the methods of 

Greenstein and obtained from him have been placed in aqueous 
solution and administered intravenously at a constant rate of 
infusion. By means of complete collection techniques metabolic balance 
data have been obtained. 

Total nitrogen, ammonia nitrogen, urea nitrogen and alpha amino nitrogen 
have been determined on appropriate samples of urine, stool or plasma. 

Patient material: 



Admissions: 



No. 
2 



Total Stay 
300 



Major Findings : 1. Mixtures of pure 1-amino acids containing all of the 
"essential" amino acids (Rose) were adequate to achieve 
nitrogen balance in the single malnourished adult main- 
tained on a balance study. 



NC I-720(C) PAGE 2 

SERIAL NO. 

PROJECT REPORT FOR!/I (cont'd) 

2. '5inission of .l.-Arginine from the intravenous mixtiore was capable. of 
producing convulsions and coma and, in preliminary dog experiments, 
has been lethal. 

3- Administration of the toxic (arginine-def icient) solutions resi^Lted in 
a marked rise in blood ammonia level of the dogs which coincides with the 
development of convulsions and coma. The rise in blood ammonia can be 
prevented by coincident or prior administration of 1-arginine. 

Significance to Cancer Research: Further information on amino acid disposi- 
tion and nitrogen transfer within the 
intact subject will provide a better base 
from which to investigate, tumor metabolism and the metabolism of. the 
tumor- containing host. Ciinically, development of. means of managing 
the toxic effects of elevated blood ammonia levels would be important 
in the management of hepatic damage states and in conditions of alkalosis 
in which ammonium chloride administration is indicated. Safe, profitable 
intravenous administration of amino acids is important in the general 
medical care of many patients with neoplasm. 

Propos'id Course of the Project: A. Further investigation of the conditions 

determining 1-Arginine requirement (i.e. 
anesthesia, rate of amino acid administra- 
tion or ammonium administration, and possible similar protective action 
of other amino acids) are to be continued in preliminary dog studies. 
Extension of these observations to human subjects in normal and 
pathologic states is planned with special interest in hepatic dys- 
function states resulting from cirrhosis, tumor invasion of liver, and 
from the presence of tumor in the host. 

B. Further efforts to establish the requirements for other amino acids 
will proceed after clarification of the role of 1-arginine. 



12, 



13. 



PAGE 3 
PROJECT REPORT FORM (cont'd) 



10. NCI-720(C) 
SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH ]TJ ADMINISTRATION / 7 

REVIEW & APPROVAL HI TECHNICAL ASSISTANCE / / 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER I956 or 1957 

Drs . Jesse Greenstein, M. Winitz and J. Birnbaura in the Laboratory of 
Biochemistry, NCI, are investigating amino acid requirements and metabolism 
in rats utilizing intra-peritoneal administration. Constant consultation 
and exchange of information as these studies have progressed have been of 
mutual benefit. 

The Laboratory of Biochemistry has also prepared, and made available, the 
purified 1-amino acids utilized in this study. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FUNDS,) IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR ITEMS lU, I5 & 16. 



PAGE I 
PROJECT REPORT FORM 
N.C.I. 2 • General Medicine Branch 



INSTITUTE LABOMTORY OR BR/iNCH 

Nutrition and Metabolism h. 5- NCI"72l(C) 



SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

3. Metabolism of Nitrogen, Minerals and Vitamins in Subjects with Malignant Diseas e 
PROJECT TITLE 

^ Donald M. Watkin, M.D. and Donald P. Tschudy. M. D. 

PRINCIPAL INVESTIGATOR(S) 

3. Jesse L. Steinfeld, M D ; John L. Fahey, M.D.; and Donald P. Tschudy, M.D.; 
OTHER INVESTIGATORS in addition, Residents Montague Lane and Charles B. 
McCall and Clinical Associates Wroth, Roush, Mohler, 
Gold, Silver, Schroeder, Fritz, Landau, Levine, 
Forkner, Schick, Goulian, Flick and Paton 

). PROJECT DESCRIPTION 

Objectives : Investigation of the over-all and intermediary metabolism of 
protein, fat, carbohydrate, minerals, electrolytes, vitamins, 
calories and water in the natural course of malignant disease 
and in the response of the disease to nutritional, chemothera- 
peutic, endocrine, surgical or radiologic therapy. 

Methods employed : The metabolic balance technique is used to identify changes 
in tumor and host by measuring quantitative differences 
between the intake and output of body constituents. 
Quantitative measurement of water balance together with analysis of 
expired air (Dr. McCall) are used to estimate energy expenditure. Radio- 
isotope techniques are utilized to measure albumin turnover and red cell 
survival time (Dr. Steinfeld). Electrophoretic partition of plasma 
proteins is used to identify abnormal plasma protein patterns and observe 
changes during the course of a study (Dr. Fahey) . Heavy isotope (N-'--') 
techniques are used to study the metabolism of urea and uric acid 
(Dr. Tschudy). 

Patient Material : 

In patients January 1, 1955 1 

In patients admi-tted January 1, 1955 - December 31^ 1955 

Initial Clinical Center Admissions l8 

"Second Clinical Center Admissions h 

Third Clinical Center Admission 1 

In patients other than above studied during 1955 - H 

Total Deaths 3 

Total Autopsies 3 



PAGE 2 

NCI-7gl(C) 

SERIAL NO. PROJECT REPORT FORM (cont'd) 

Outpatients; 

Screening 8 

Follow-up 6 

Research 1 

Total patient days attributable to patients studied on this project- 3692 

Major Findings : Because of the large number of different studies which 
were completed under the general heading of NGI-70l(C), 
a separate report on each study is given herein. 

A. Nitrogen, albumin, mineral, electrolyte and energy metabolism during 

hyperalimentation by intravenously administered fat emulsion in subjects 
with malignancy and cachexia; Donald M. Watkin 

Objectives : Quantitation of effects of excessive calories supplied by 

fat administered intravenously on host and tumor/ evaluation' 
of caloric hyperalimentation as a therapeutic measure; 
exploration of the relationship between energy metabolism, pro- 
tein metabolism and tumor growth. 

Methods employed : a) Metabolic balance technique utilizing liquid constant 
diets , 

b) I -^^ labelled human serum albumin turnover. 

c) Open circuit measurement of respiratory quotient 
{B.M-) and basal metabolic rate (B.M.R.). 

Patient Material ; Two men and one woman with inoperable carcinoma, one man 
with sarcoma,, and one man with post-gastrectomy cachexia. 

Major Findings : All subjects maintained weight on liquid diets during control' 
periods . Fat emulsion intravt,nously increased caloric intake 
by 1/3^ resulted in weight gains, retention of protoplasmic 
constituents, and an increase in total albumin and rate of albumin turn- 
over. R.Q.'s in patients with rapidly progressing carcinoma were so 
low as to suggest ketone formation during control periods. All patients 
showed low R.Q.'s during fat emulsion infusions. Side effects from 
infusion of emulsions were back pain with the initial infusion, BSP 
retention, and a moderate anemia during the infusion. A striking 
finding of general interest was the ease by which cancer patients can 
consume extremely high caloric intakes when the combination of liquid 
diets and intravenous fat emulsions are used. In one patient, hyper- • 
alimentation was associated with objective increase in the rate of 
tumor growth. 

Significance to Cancer Research: Practically, these studies show the feasi- 
bility of feeding adequate calories to 
cachectic patients. From a theoretic point 
of view., they suggest the presence of a defect in carbohydrate metabolism 
in patients with advancing cancer. They show that fat supplied calories 
can be utilized to spare protein in cancerous subjects. 



PAGE 3 

NCI-7gl(C) 

SERIAL NO. PROJPJCT REPORT FORM (cont'd) 

Proposed Course of Project : The abnormality in carbohydrate metabolism 

suggested by R.Q. studies v/ill be actively 
pursued. New emulsions of fat will be tried 
with the ultimate goal of providing adequate nutrition completely by 
the parenteral route and hence eliminating cachexia and starvation 
as a cause of death in cancer patients. Abnormalities of energy 
metabolism during hyperalimentation in cancer patients will be 
studied. 

B. Renal excretion of uric acid at various plasma uric acid levels in 
subjects with leukemia and other malignancies: Donald M. Watkln 

Objectives : To investigate the relations among the renal tubular reabsorption 
of uric acid, plasma uric acid level and uric acid production. 

Methods employed : R nal clearance technique. 

Patient material : Two patients with leukemia, two with multiple myeloma, 
and one with reticulum cell sarcoma. In two, serial 
studies were performed. 

Major findings : One subject with chronic myelocytic leukemia studied 

before, during, and after treatment with myeleran demonstrated 
a depression in mrlc acid clearance with no change in plasma 
uric acid level during therapy followed by a marked increase in uric 
acid clearance, a decrease in the absorption of filtered uric acid, and 
a decline in plasma uric acid level after completion of treatment. 
This was accompanied by an increase in glomerular filtration rate, 
renal plasma flow and TmPAH. The reciprocal relationship between the 
tubular transport of uric acid and PAH is an incidental observation 
corroborating similar findings made by NHI workers utilizing rabbit 
kidney slices. 

Significance to Cancer Research : Uric acid is in man the end product of 

purine metabolism. In leukemia and certain 
allied malignancies plasma uric acid levels 
are high. This level is determined in part by renal tubular reabsorption 
of uric acid, in part by uric acid synthesis, and in part by the release 
of uric acid from tissues undergoing destruction. The renal contribution 
to the maintenance of a plasma level must be known before any interpre- 
tation on uric acid production or release from tissue can be made. 

Proposed Course of Project : Renal function studies de'signed to measure 

glomerular filtration rate, renal plasma flow, 
TmPAH, loric acid clearance and renal tubular 
transport will be conducted in subjects with leukemia or other malignan- 
cies associated with abnormalities in uric acid plasma levels. Every 
effort will be )iiade to study these subjects serially before, dui'ing and 
after successful chemotherapeutic, endocrine, radiologic or sxirgical 
therapy . 



PAGE k 
TCI-7gl(C) 
■5ERIAL NO. 

PROJECT REPORT FORM (cont'd) 

In addition, the information acquired will serve as guides to more 
elaborate studies of the uric acid pool and uric acid synthesis 
carried out with the aid of heavy or radio isotopically labelled 
uric acid and its precursors. 

C. Vitamin 3^2- Vitamin Big metabolism in normal subjects of various ages 
and in subjects with malignant disease; Donald M Watkin 

Ob jectives : ^Qualitative and quantitative observations on the over-all 
metabolism of vitamin By^^i its levels in blood plasma, its 
distribution in the bodies of normal subjects and those with 
cancer, and its excretion by the kidney. 

Methods employed : Microbiological assay for^itamin B-jo i"^ blood plasma and 
urine . Measurement of CO labelled radioactive B^ p in 
blood, urine, tissue and feces. 

Patient Material ; To date all studies have, been conducted in roughly 250 
presumably healthy individuals without known malignant 
disease . 

Major Findings: Normal values for blood levels of B-^o ^^ various age cate- 
~ gories have been established. Absorbtion of vitamin 'Bj_2 

from the G.I. tract, after oral ingestion has been related to 
dose, age of subject and the subject's gastric acidity. Urinary excretion 
following various intramuscular doses of B^p has been quantitated for 
presumed normals aged 20 - 100 years. Renal clearance of B-, p in various 
age groups has been quantitated at low and intermediate plasma B-ig 
levels . Recent studies have demonstrated the mc-chanism of B-i o clearance 
at high plasma levels. 

Significance to Cancer Research : Vitamin Bnp pl^-ys 'in indispensable role in 

the metabolism of all foodstuffs and in 
hematopoiesis. It has been used with ■ ■■ 
reported success in treating neuroblastomas. Plasma B, ,-j levels in 
leukemia are extraordinarily high. B-]_2 antagonists are^being developed. 

Proposed Course of Project : The techniques for the microbiological assay 

using L. Leichm nil and for the analysis of 
C0°'-' labelled radioactive vitamin B^o s-re being 
developed. Bloods will be collected from all patients with cancer 
admitted to the Clinical Center, assayed for B-j^gj ^^^ analyzed 
electrophoretically for plasma protein. In selected patients, B^o 
■distribution studies using the renal clearance technique will be 

riO ^ 

performed. Distribution of radioactive CO" labelled B-,p in normal 
and tumor tissue will be determined in surgical and autopsy specimens. 



PAGE 5 



rci-7gi(c) 

JERIAL NO. PROJECT REPORT FORM (cont'd) 



D. Leukemia: Donald M. Watkin 

Objectives: Characterization of over-all metabolism in chronic and acute 
leukemia and the changes induced by anti-leukemic therapy. 

Methods employed,: Metabolic balance technique. Discrete renal function studies. 

Patient Material: In 195^ three patients with chronic and one with acute 

leukemia. In 1955 ^ one patient with chronic myelocytic and 
one with acute lymphocytic leukemia as in patients; one 
patient with chronic myelocytic leukemia as out-patient. 

Major Findings: Myeleran therapy in chronic le-ukemia results in a marked 

early increment of phosphorus and uric acid excretion followed 
by a reduction in uric acid excretion to bslow-control values 
with continued therapy. Methotrexate therapy in acute leukemia induces 
an increase in phosphorus and uric acid excretion. 6-Mercaptopurine 
therapy was associated with a temporary diarrhea, although no definite 
evidence of a sprue-like syndrome could be proven. Hydrocortisone in 
combination with 6--MP induced a remarkable increase in uric acid 
excretion. 6-MP over a prolonged course reduced plasma and uric acid 
levels to extremely low values . 

Significance to Cancer Research : These studies provide quantitative means of 

evaluating the rate of progression of the leuke- 
mic process and the extent to which this 
process may be altered by antimetabolite therapy. Thuy point toward 
possible mechanisms of action of antimetabolites in leukemia. 

Proposed Course of Project : Selected leukemic patients who are sufficiently 

well to cooperate in balance and renal studies 
will be studied with particular emphasis on the 
excretion of uric acid and on calcium and phosphorus metabolism. 

S* Nitrog en Utilizatio n; A Study of Nitrogen Utilization in Human Tumor 

Patients by Means of N-^^ Labelled Amino Acids. 
(D. P, Tschudy) 

Objectives: To study the effects of malignant tumors on the organism with 
respect to over-all rate of protein synthesis, size of nitro- 
gen metabolic pool, rate of incorporation of nitrogen into the 
tumor and other aspects of nitrogen metabolism. 

Methods Empl oyed; Synthesis of isotopically labelled amino acids, isolation 
of compounds from blood and urine, preparation of samples 
for mass spectrometer. 

Patient Material : Noj. Aver, stay 

Admissions: 3 5 

Ma ,ior Findi ngs; Tumors contain higher levels of isotopic N than serum pro- 
teins examined at the same time. Rate of labelling of urea 
and ammonia is about the same in tumor and non tumor 



WCI-721(C) PAGE 6 

PROJECT REPORT FOR^i (Cont'd) 

patients. Some cancer patients may excrete less isotope than normals. 

Significance to Cancer Research ; Following the mathematical model of Ritten- 

berg and San Pietro we will quantitate the 
effects of cancer on the protein synthesis 
rate and size of the nitrogen metabolic pool in patients. 

Proposed Course of Project ; Data is being applied to various mathematical 

models for calculations of both rates and rate 
constants for the over-all organism with re- 
spect to nitrogen metabolism. The physiological validity of these models 
may be tested by direct experimentation and measurement of certain rates. 



Page 7 

PROJECT REFOxlT FOM (Cont'd) 



10. 


NC 1-7 21(C) 
SERIAL NO. 




1]. 








BUDGET ACTIVITY: 






RESEARCH 


HI 




REVIEW & API ROVAL 


D 



12. 



13. 



ADMINI'-^TRx.TION [J 

TECHI\fICAL ASSISTANCE [J 



COOPERATING UIMITS OF THE PUBLIC HKiLTH SERVICE, OR OTHER ORGANIZilTIONS, PRO- 
VIDING FUi^DS, Facilities, or personnel for this project in either 1956 or 
1957. 

A, B, C, D and E - none 



IF THIS PROJECT KESEf'iBLES, COFlPLEMENTS, OR rAR-xLLELS RESEiiRCH DONE ELSEWHERE 
IN THE PUBLIC rISALTH SERVICE (WITHOUT INTERCHiWGE OF PERSONNEL, FACILITIES OR 
FUI^DS), IDENTIFY SUCH ftSSEARCH: 

A, B, C, D and E - none. 
No entries for items 14, 15 and 16, 



PAGE I 



PROJECT REPORT FORM 



1, N, C. I. 



INSTITUTE 



2, General Medicine 



LABORATORY OR BRANCH 



3. Metabolism 



SECTEON OR SERVICE 



U. , 5, NCI-72£(C) 

LOCATION (IF OTHER .THAN BETTIESDA SERIAL NO. 



6, Measure of Blood Volume, Red Cell Mass and Total Circulating Albumin in 

Patients with Cancer. 

PROJECT TITLE 



7; J. L. Steinfold 



PRINCI PAL INfc tTGATORTsT 



8, None 



OTHER INVESTIGATORS 



9. PROJECT DESCRIPnON 

Objectives: To determine whether there is a consistent decrease in 

patients in the various stages of carcinoma growth in the 
red cell mass, since conflicting reports in the literature 
state that there is and that there is not a decrease in 
the red cell mass in patients with carcinomas ♦ 

To determine whether the decreased concentration of serum 

albumin seen so frequently in patients with cancers is a 

result of an absolute decrease in albumin or is the result 
of an increase in plasma volume. 



Methods employed; 



Patient Material: 



Major Findings; 



Blood Volume, Red Cell Mass and Plasma Volvme are 
determined using the isotope dilution method with 
II3I albumin or red cells labeled indth sodium 
chromate^l, 

80 Patients Admitted for other NCI clinical research 
programs. 

Red Cell Mass was consistantly decreased in 80 studies 
in 60 patients with advanced carcinomas. Plasma 
Volume - in the absence of congestive heart failure - 
was within norma.1 limits. 

Recalculation of the conflicting data available in 
the literature using the concept of "body hematocrit" 
and a body hematocrit: peripheral venous hematocrit 
ratio of 0,91 reconciles the reports so that the 
available published data are consistent with the 
present findings at the NCI, 



NCI -722(0) 



PAGE 2 



PROJECT PEPORT FORM (Cont'd) 



Significance to Cancer Research ; In order to adequately characterize 

a metabolite it is essential to know- 
not only its concentration (previously 
available as hemoglobin or albumin concentrations) but also its 
volime of distribution* To further characterize any metabolite the 
rate of production and rate of metabolism also must be determined, 
however, this project is concerned with only the first of the 
above two requirements. 



P roposed Course of Project ; To extend these observations to include more 
~ patients with cancer in various states 

( extent ) of disease and to make repeated 
observations on the same patient, throughout the course of his illness. 



PAGE 3 



PROJECT REPORT FORM (Cont'd) 



10. NCI-7£2(C) 
SERIAL NO. 



11, 



BUDGET ACTIVITY: 

RESE.'^JICH fT~J 
REVIEW & APPROVAL/ 7 



ADMINI S TRAT ION / / 
TECHNICAL ASSISTANCE J 7 



12. None 



CCOPERATIMG UNITS OF THE PUBLIC HEALTFI SERVICE, CR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONN-EL FOR ffllS PROJECT IN EITHER 
1956 or 1957. 



13. None 



IF THIS PROJECT RESEMBLES, COMPLEI^'IENTS, OR PimALLELS RESEARCH DONE 
ELSElfflERE IN THE PUBLIC HEALTH SERVICE (WITIiOUT INTERCH;J^IGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



No entries for items lii, l5 and l6. 



PAGE 1 
PEDJECT REPORT FORl^I 



National Cancer Institute 2, Surgery Branch 

INSTITUTE lABORATORY OR BRANCH 

• . , NCI 

. ^. ■ - 5._JZ5QiCi_ 

SECTION OR SERVICE LXATION SERIAL NO. 

Removal of Cancer and Other Tissues for Histologic, Biochemical and Other 

Studies as Required by Scientists and Other Investigators for Correlated 

PROJECT TITLE Studies. 



7. , , Dr, R.R. Smith. Dr. J. H. Waitet Dr/ W. E. Schatten 
PRINCIPAL INVESTIGATOR(S) " 



Dr. A, Ship Dr. R. Miller Dr. W. Kramer 
8 . Dr. R. Milch Dr. L. Cramer Dr..H. Herbsman - 
" OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION" ■ . - 

Ob.iectives ; 

The object of this project is to supply human tissue for histologic, 
histochemical, biochemical and biophysical studies to scientists working 
in various branches of the Cancer Institute who require tissue for 
studies. As a rule, such studies are done as cooperative projects with 
various investigators but on occasions specific types of tissue are re- 
quired for specific studies. For example, one investigator requested 
that specimens of skin and its associated neoplasm be made available for 
keratinization studies, and various muscle biopsies and /or biopsies of 
liver tissue under specific conditions have been requested. These 
materials are obtained during operations for standard therapeutic reasons. 

Methods Employed ; 

Methods employed are standard surgical procedures. 

Patient Material ; 

Material for this project is mostly obtained from patients 
hospitalized for study in other projects. An example of such a case is 
a patient with an islet cell adenoma of the pancreas. This patient was 
admitted and studied mostly by the Endocrinology Branch but material was 
used in a number of correlated biochemical and histologic studies. 
K patients with extensive melanomas and/or basal cell carcinomas were 
admitted especially to obtain tissue for special studies by investigators 
in other branches. It is difficiilt to differentiate patients admitted 



PROJECT REPORT FORM (Cont'd) 



PAGE 2 



NCI~750(C). 



SERIAL m . 



Patlenlii, M aterial ; (Continued) 

specifically for this project. However, 127 minor procediores were performed 
for other branches of the Cancer Institute, and 62 minor operative pro- 
cedures were performed for other institutes on patients admitted by those ; 
institutes to obtain tissue for biopsy and/or biochemical, biophysical or 
histochemical studies. 

Ma.ior Findings; 

Presented in reports of other projects by other investigators. 

Significance , to Cancer Research ; 

This project is. more or lesjs a service function for other branches 
of N.C.I, and N.I.H. 

Proposed Course of Project ; 

The number of such cases will continue at about the present level 
in proportion to the patient load. 



11, 



BUDGET ACTIVITY 



RESEARCH /X: 

REVIEW & APPROVAL / . "7 



ADMINISTRATION 

TECHNICAL ASSISTA1>ICE /Z7 



(No entries for items 12, 13, 15 & l6) 



PAGE 1 
PROJECT R:PORT FORM 



1. Matlona l C ancer Institute 2. Surgery Branch 

■ p:sTiTuTE ' ■ ij:Bmnmrm~mKEm 

3. h. 5. NGI-75l(C) 

SECTION OR SER.'Kfe LCC/iTlOlvi (lE OTiiER T^L^J? j^^Tilii^M) SERIAL JJO. 

Evaluation of Radical Surgery and the Development of New Surgical 
6. Techniques as a Therapeutic Ifcans of Palliating or Definit ive Therapy 
PROJECT TITLE ' ; of Advanced Cancer. 



7. Dr. Robert R. Snith 

PRIMCIPAL IWESTl(lVrCR(S ) ■ 



Dr. John 11. Waite - Dr. Williaj-n E. Schatten 

OTHER i-westig;.t:rs " 



9. PROJECT DESCRIPTION 

Objectives : The objectives of this project are to develop methods of 
obtaining increased palliation and/or definitive cure of 
advanced crncer, present day therapy depends u.pon the 
complete removal of cancer or its local destruction by 
physical agents J I.e., svrg^r^j, radiation, or chemotherapy. 
Salvage by any of these methods is low (l5-30%), and points 
to the ineffectiveness of the tools that we now have avail- 
able. Examples of this type are found especially in cancers 
of the pelvis and in the head and neck areas. Clinical 
material from these two areas is readilj/ available. 

^iethods employed : Patients with advanced cancer of the pelvis and/or head 
and neck areas are admitted for study. These patients do 
not. have dissemination of their disease beyond the opera- 
tive area. This project is in realitjr composed of a number 
of projects of specific sites but because of the relative 
infrequency of cancer of specific areas, studies must be 
arranged to take advantage of the clinical material as it 
becomes available. In the report of a v-ear ago, study of 
paranasal sinus cancer was Mentioned. Seven patients with 
this tjn^e of cancer were admitted for surgery and in only 
two were conditions right for the more radical type of 
surgery to be performed. This surgery consisted of resection 
of t: e floor of 'the anterior cranial vault including the 
cribriform plate, contents of the nose, the orbit, and the 
involved antrum nnd/or hard palate. T\irenty-eight primary 



PROJECT REPORT FQffi4 (Cont'd) PAGE 2 



HCI-75l(C ) 
SERIAL NO. 

tlethods employed ; (Continued) 

carcinoinas oj? the cervix were admitted,- The rndicsl 
surgery consisted of pelvic lyraphadenectomy associated 
with the radical hysterectomy and/ or pelvic exenteration. 

Patient material ; As, noted in the previous project reports there is . . ' • 
considerable overlap of patient materialj that is, a 
patient is used in more than one project. There were, 
however, 7 patients admitted for the prim?r antrum 
study and 28 patients adinitted for study ox cervix canccr» " • 
Tht. total number of hospital days for thxs project was 
l666 with 102 out-patient followup visits. 

Major findings; Of the., 28 peryix,.gast,.s, adiiiitted, some of which were • 
admitted and treated in the previous 3''ear but never 
reported, 17 pelvic exenterations were performed. An 
additional 8 patients had radical hysterectom;/ and node 
dissections, and 1 patient had.:a. radical resection of a 
cervical stump cancer, plus radical iliac node dissection. 
Complete analysis of this group of patients is not possible 
at this time, mainly because a sufficient length of time 
has not elapsed since surger",- to allow adequate evaluation. 
It is possible at this time to note that in our hands as 
the surgery is perf orrbed at this hospital the operation is 
relatively safe. There has been one postoperative death 
in this group.- This patient died about ten days after 
radical pelvic exenteration. She died as a result of 
intestinal obstruction complicated b^r peritonitis. We 
feel that for the tyoe of clinical material presented at 
this hospital the radical surgical approach to advanced 
pelvic cancer gives worthwhile palliation and increased 
longevity. More complete analysis of this group of patients 
- • should be available x-jithin the next year. 

Of the 7 paranasal sinus cancer patients, only 2. of them 
■ were suitable for the radical resection described above. 
Both of these patients have done x^fell and are still free 
of disease after a relatively short postoperative period. 
Our experience with this small num.ber has shown that the 
operation is safe. It does not produce objectionable deformi- 
ties and is well received by the patients. In 2 of the 
patients admitted for this stud;/- the disease was found to 
extend into the middle cranial fossa and definitive surgical 
procedure could not be performed. In one of the 7 antral 
cases, the patient died with disseminated cancer throughout 
his lungs and mediastinum. This patient had a radical 
resection of the maxilla but not the anterior cranial fossa. 
The other patient had a modified radical antrum resection 
and is free of disease at the present time. The 7th patient 



PROJECT REPORT FORM (Cont'd) PAGE .3 



NCI-75l(C) 

SERi^'.L WO. 



Major findings ; (Continued) 

has had surgery performed only a short time and is not 
available for evaluation. 

Significanco to Cancer Research : We believe that the developraent of 
effective means of palliating and/or curing advanced 
cancer is one of the fundaraental purposes of the Cancer 
Institute's program. Documentation of the course of 
cancer of these areas as the patients continue through 
the course of their disease adds considerably to the 
total knowledge of the natural behavior of cancer. It 
should also be pointed out that this project provides 
considerable material for both excised adjacent normal 
tissues as well c?.s the cancer itself for scientists to 
use in other projects in the Cancer Institute. 

Proposed course of project : During the coming year it is proposed to 

contijiue the studies as outlined above. It is anticipated 
that in the future as in the past, one of the m.ain problems 
with pelvic cancer which requires the excision of the urinary 
bladder is a means to control the urinary stream. Studies 
are underwaj'^ at the present time in which the ureters are 
placed in ?n isolated loop of bowel. This looks promising 
but is still not the ideal method of maintaining adequate 
urinary drainage. It is hoped that additional cases of 
advanced but locally operable paranas.-'l sinus cancer will be 
obtained in order to f-urther study the neuro-physiolor-ical 
changes involved in this type of radical resection. As in 
the past, it should be pointed out that the neurosurgical 
portion of paranasal sinus study was undertaken in conjunction 
with neurosurgeons from the Institute of i'eurological Diseases 
and Blindness. 

11. 

BUDGET ACTI\^ITY: 

RESEARCH /x/ ADMDJISTRiiTION £7 

RE\?IE\'if & APPROWvL [J TECHNICAL ASSISL'.NCE [1 

MO ENTRIES FOR ITEMS 12, 1,3, lU, 15 & l6. 



PAGE 1 
PROJECT REPORT FOM 



1. NationaJ. Cancer Institute ...,, 2, . Surgery Branc h 



INSTITUTE LABORATORY OR BRANCH 

3. 4, _»«__ 5. NCI-752(C) 

. SECTION OR SERVICE LOCATION {IF OTHER THAN BETHESDA) SERIAL NO. 

6. Clinical Investigation in the use of Viruses in the Treatment of Human Cancer . 
PROJECT TITLE 



7. Dr,. R. R. Smith and Dr. R^ J« Huebner 

PRINCIPAL INVESTIGATOR(S) 

8. Dr. W. E. Schatten. Dr. L. B. Thomas and Dr. W. P. Rowc 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives: 

To develop means of treating human cervix cancer using cultin'e fluid 
containing live virus, attempting to reproduce in vivo the in vitro 
observation of the destructive action of viruses on cells in tissue culture. 

Methods Employed ; 

APC (adenold-pharyngeal-conjunctlval) viruses grown in 5% chick serum 
in Hanks-Simms solution containing HeLa cells was filtered through a 
sintered glass filter and tested for sterility and safety. This fluid 
was then injected by direct needle injection into the cervix tumor mass 
via the vagina, and from above directly into the mass, or by intra-arterial 
cannula passed up through the femoral artery into the lower abdominal 
aorta. It was necessary to determine the antibody levels of the serum of 
the patients Injected to make sure that the proper virus was used.. 
Following the injection it was necessary to obtain culture swabs from the 
vaginal fluid and/or biopsy tissues or cervical scrapings. Blood cultures 
were taken regularly. The patients were thoroughly studied for evidence 
of systemic manifestation of a virus disease. At the start of this inves- 
tigation, it was determined that using this agent in susceptible individuals, 
a local necrosis could be produced without the production of any recogniz- 
able systemic disease. In a number of instances, following the use of the 
local chemotherapeutic agent, more definitive surgical therapy was insti- 
tuted. In 8 patients radical pelvic exenteration was possible following 
the virus therapy. In an additional 3, radical hysterectomy and node 
dissection was performed. These were standard operative procedures which 
have been modified as described in Project 751(c), 



PAGE 2 
PROJECT REPORT FORII (Cont'd) 



SERIAL I-D, 



Patie nt Material ; 

During the past twelve months 19 patients were admitted especially for 
this study, for a total of 224-2 patient days. 35 out-patient visits were 
made by this group. As pointed out before, this patient material was avail- 
ble for a number of correlated biochemical and histological studies as well 
as the radical pelvic surgery study as described in Project 751(C). 

M a.ior Findings : 

To date, 28 patients have received virus fluid for therapeutic studies 
of their cervix cancer.- kn. additional 6 patients received control material 
consisting of the tissue culture fluid containing everything but tlie virus. 

The findings during the past year have confirmed those given in the 
report last year; that is, patients whose sera does not contain antibodies 
to APC virus develop an area of necrosis at the site of injectionj when 
antibodies are present such necrosis has not been observed. In 24- patients 
that have been more thoroughly evaluated, injection of these viruses in 
amounts up to ADO cc. of ctilture media did not produce appreciable systemic 
disease. On three occasions, an influenza-like disease was produced, con- 
sisting of general malaise, photophobia, and fever. These symptoms dis- 
appeared without therapy in a few days , There was no pharyngitis or con- 
jionctivitis as observed in upper respiratory infections caused with APC 
viruses occurring spontaneously. Local necrosis progressed to extensive 
cavity formation in the vaginal pelvic portion of the cancer in 20/5 of the 
patients. There was moderate slough of the lesion in 52^ of the patients, 
in 20^ a slight to questionable necrosis was observed, and in S% no response 
was observed. Injected virvis was recovered from tissue biopsies and/or 
vaginal smears as late as 17 days after the treatment, A prompt antibody 
rise was observed in all cases. Extensive cyi;o-histologic studies of • 
repeated biopsies and smears failed to show aiay specific recognizable effect 
that could be attributed to the virus. Increasing amoiints of necrosis were 
observed on biopsies taken during the period of slough but rtb inclusion 
bodies or other specific viral effects were demonstrated. In no instance 
was there complete destruction of the cancer observed, Regrowth of the tumor 
was observed" in the area of slough as early as 10 days , 

It soon became apparent that the drug did produce local changes which 
we are convinced have a specific effect upon the cancer tissue. No obser- 
vable effect has been noted oh the other tissues of the body. It is apparent, 
However, that the prompt rise of antibodies prevents the continued growth of 
the virus in the tissue, thus limiting its effectiveness as a destructive 
agent. Attempts have been made to use cortisone in increasing amounts. This 
did not prevent the rise of antibodies. Its effect on the local lesion in 
conjunction with the virus used remains to be settled. It did seem, however, 
that the best results of the virus were obtained in patients that were closer 



PAGE 3 
PROJECT REPORT FORM (Cont'd) 

NCI-75 2(C) 

SERIAL NO. 

M a.ior Findings: (Cont'd) 

to the terminal portion, of their disease j that is , that they were debilitated 
and more serio-usly ill. This suggests that the nonspecific defense mechanism 
of the body is .probably a factor in the limitation of the use of this agent 
as it exists today. - ' ' '. 

Significance to Cancer Research ; 

This program helps to carry out one of the fundamental aims of the 
Cancer Institute, that is, of developing new and more effective methods of 
therapy of common malignant neoplasms. It also helps to identify and 
categorize the newly discovered APC viruses and helps to delineate the re- 
action on human tissue of the virus n^od in the experiment. If a method 
can be developed to allow the propagation of this virus in the tumor tissue, 
it is reasonable to expect that the continued propagation, not only locally 
but in the disseminated disease, might be developed into an effective means 
of controlling cancer. 

Pro posed Cou rs e of P ro.iect: 

During the coming year it is hoped that this project can be pursued 
more vigorously. Ehjring the past year and a half it was recognized that 
this was a new approach and that caution was necessary to prevent misi:aider- 
standing in using human material in this type of study. We are sure now 
that this material is not harmfiol, tliat it does not produce aggravation of 
the growth of the neoplasm, and that we are doing these patients a service 
by studying them and providing the care that is available. It is believed 
that the course the project should take is as follows: 

(l) As noted in the i^ vitro study of the effect of this virus on 
HeLa cells, the degree of destruction is directly proportionate to the 
concentration of the virus present in the fluid. It is hoped that this 
virus can be concentrated 100 to 1000 times so that tremendous amounts 
of this material can be given to the patients in a smaller volume of 
fluid. This is a problem in production of virus which is being studied. 

(2) The serial use of APC viruses whose antigenicity is not exact 
and will allow the use of multiple viruses through multiple injections 
to produce the same type of effect. 

(3) Continued studies to alter the host resistance to infections in 
an effort to produce a viremia which would allow contamination of all 
of the cancer cells and a more complete destruction. 

(4) Repeat the histologic study in an effort to definitely detennine 
the mode of action of this material. 



PAGE i 
PROJECT REPORT FORM (Cont'd) 



-JJCJr25£(£l 

SERIAL NO. 



11. 



BUDGET ACTIVITY 

PkESEARCH' ■ ■ . /Tj ADMINISTRATION - /~7 

REVIEW & APPROVAL /~7 TECHNICAL ASSISTANCE /"~7 



(No entries for items 12, 13, H and 16) 



PACE 1 
PROJECT REPORT FORI! 



1. National Cancer Institute' 2. Surgery Branch 

INSTITUTE lABOPJlTORY OR BRANCH 

3. ■ 4.._ . 5. NCI=2i _ 

SECTDN OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL 10 . 

Cytologic Determination of Number of Tumor Cells and/or Recurrences 
6. -"Recover ed or Fou nd_.ln an Operati ve Vtound Following Removal of a.. Primary. 
""project TITLE Cancer in Continuity with its Regional Lymph Drainage 

■ Area Containing Ibtastascs . 



7. Dr. R. R. Smith 



PRINCIPAL DIVESTIGATOR(S) 



3. Dr« A. ]/l. Kilbcr&-. Dr. J. . K. V/aite and. D r. W. E. Schatten 
OTHER INVESTIGATORS 



9, PROJECT DESCRIPTDN 
Ob.icctives : 

This project is the clinical part of PrdjectNo. 754 dealing with 
the seeding of operative wounds ty cancer cells. Its primary objective 
is to demonstrate the frequency and the nature of tumor cells found in 
the washings obtained from operative vrounds from which operable cancers 
have been removed. 

Methods Employed; 

..•Patients with locally advanced priinary cancers of the head and neck, 
• pelvis, or other appropriate sites, with regional lymph node metastases j 
have .been admitted for primary surgical therapy. For , the most; .pair t, , ,.• 
these cases have been those with extensive metastases, in -other words, 
• the borderline operable ones. The surgery performed is mostly the standard 

operative procedures accepted as treatment for the specific cancer sites. 
. In part, these same patients provided material for Project 751(C) in 
which the limits of opcrability have been f lurther defined by a more exten- 
sive resection with and \./ithout primary reconstruction. 

Following the removal of the tumor and its metastases, the- ctperative 
-area is thoroughly washed \;ith- a fine saline spray. The aspirate is 
fixed in an alcohol-ether mixture and in the Pathology Lab is centrifuged 
down, tiie sediment being treated by laking the red cells which are present 
in the fluid. The remaining sediment is then smeared and studied in the 
usual Papanicolaou technique and/or clTompod together in a block and the 
usual histologic sections prepared. 



PAGE 2 
PROJECT REPORT FORI! (Cont'd) 



NCj:~753(C) 

SERIAL NO. 



Patient !'atcrial; 

25 patients were admitted specifically for this project for a total 
of 1737 hospital days, 100 outpatient visits were made by these patients. 
It should again be pointed out that nearly all these patients were used 
in other studies, and in some patients admitted for other projects, wound 
washings were taken. This was demonstrated by the fact that during the 
calendar year of 1955, 72 different patients had wound washings studied 
in the Cytodiagnostic Laboratory. . . ._ 

M ajor Findings ; 

Complete analysis of all 72 cases is not available at this time, "The 
results of a study of 36 of these cases have been tabulated and was pub- 
lished in the December 1955 issue of the J.N.CI, 

In summary of the 36 cases, woimd washings were positive in 9 instances 
and in an additional 5 cases the washings contained suspicious cancer cells. 
When the wo\and wasliings were positive, local recurrences had already 
developed in 2 of these patients arid in an additional case in which the 
washings were suspicious. In 2 instances where the washings were negative, 
local recurrences had already occurred. 

The finding of clumps of tumor cells in 25^ of this small series 
would certainly make one suspect that this figure \Jould represent a minimimi 
number of cases that could be expected to demonstrate local recurrence of 
tumor. However, it should be pointed out that the finding of tumor cells 
in a wound is not necessarily assiorance that a recurrence will develop. 
It is possible that washing of the woond would destroy the implants, and 
the host factor which allows the recurrence to develop could possibly con- 
trol the remainder. On the other hand, the finding of negative wound wash- 
ings would be no guarantee that recurrences would not develop because of 
the difficulty in assin-ing ourselves that the washings are truly negative. 

The important point of this work to date has been that malignant cells 
in wound washings can be relatively easily identified. Most of the washings 
that were examined contained no isolated tumor cells but small fragments 
which could be readily identified. In one instance this contained a piece 
of cancer one-tenth of a millimeter in diameter. Cell structure could 
very easily be identified in this case. In all instances cells or clumps 
of cells seen in the wound washings were comparable when compared with 
the tissue biopsies of the surgical spcciiiicn. In all cases in which the 
washings were considered positive, the tumor cells identified corresponded 
in morphological characteristics- with the tumor cells seen in the tissue 
section. 



rJIJ'JECT REPORT ETCRM (Cont'd) 

PAGE 3 



NCI-753(C) 



SERIAL m . 



Significance to Cance r Research; 

Demonstration of cancer cells in operative wounds again points up the 
deficiency in our present methods of cancer therapy. On the positive side 
it demonstrates a possible cause of failure of this type of therapy, and 
certainly suggests a means of approaching the problem from a chemotherapeutic 
standpoint. 

£rgpP8e^ Cpuygg of.Prp.Jep.t; 

It is proposed to continue this Study as outlined above, further delin- 
eating the frequency and the characteristics of a seeded wound. It is hoped 
that this series will be allowed to increase to the point where it will be a 
statistically significant one. It will be necessary to continue to follow the 
patients that have been so treated over the next 2-3 years to further determine 
the ratio of local recurrence and failvire of therapy to the finding of tumor 
cells in an operative wound. 

It is hoped that within this next year the results of the laboratory part 
of this experiment (Project 754) will have progressed to the point where chemo- 
therapy of operative wounds can be brought to the operating room. 

Ll, BUDCET ACTIVITY; 



RESEARCH Al ADIIINISTRATION 

REVIEW & APPROVAL /Zj TECmilCAL ASSISTANCE 



.5. 



PUBLKATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR ZEAR 1955 



Paper entitled "Cancer Cell Seeding of Operative Wounds" 
By: Robert R. Smith and Albert W, Hilberg 
Published in J,N.C,I., Vol, 16, No. 3,- December 1955 



(No entries for Items 12, 13 and l6) 



PJ.CiE 1 



PROJECT REPCRT FORM 



1. National Cancer Institute 2. Surgery Branch 



3. h. ■ 5. NCI-75U 

SECTIOfI OR SEli;Kii ' LOd/.'i'lOM (Ih' Ol.m mU\T BETHSSDa) fJi^Ria Mu . 

Evaluation of the Nature, Cause, and Means of Preventing Seeding 
6, of Operative Wo unds Using Tra n splan table Animal Tumors. 

PROJECT TMLE 



7. Dr. Robert R. Smith 

PR Wc ipA L t^iV£6Tli\T0R( S ) 



8. Mr. Richard V. Eck - Dr. John H. Waite - Dr. Ross Miller - ^ 

OTHER IlWESTId'.TCRS Dr. Arthur Ship - Dr. William Kramer 

9. PROJECT DESCRIPTION 

Objectives ; 

The objectives of this project have been broadened during the past 
year from methods of producing experimentally in animals multiple 
recurrent tumors in operative wounds, to the establishment of the 
standard experimentally seeded operative wounds which provides methods 
of testing the efficacy of various therapies aimed at preventing the 
wound seeding. 

Methods employed ; 

As pointed out in Project No. 753(C) a frequent finding in radical 
surgical therapy of cancer is the presence of tumor implants in the 
operative wound. In trying to evaluate the effectiveness of any treat- 
ment applied to an operative wound, and in order to demonstrate the 
safety as well as the effectiveness of any given chemotherapeutic regime, 
it is believed necessary to develop in the animal a situation which is 
comparable to that in the human. This would allow a statistical analysis 
of a given form of therapy over a short period of time. , In order to 
develop this experimental tool the V^ carcinoma in rabbits has been 
utilized to produce in our hands a seeded operative wound which in practi- 
cally 100>? of the cases produces multiple tumor implants in the operative 
wound within a 2 to 3 week period. K ascites tumor injected intraperi- 
toneally produces numerous implants in the peritoneal cavity. S-91 mouse 
melanoma produces multiple implants in the lungs when injected intravenous!-, 
or subcutaneously in thst area, Mr. Richard Eck has developed a method of 
raising an air bubble on the back of a mouse by injecting air in a subcu- 
taneous pocket. Turaor suspension injected into this area, following which 



RiGE 2 
PROJECT RiiPCRT FOM (Cont'd) 



NGI-75U ■ 

Methods employed ; (Continued) 

2 

the air is removed, produces a wound with multiple implants. With V 
carcinoma, dissection of the axilla and removal of the pectoral muscles 
produces a setting very similar to that seen in the operating room in 
which a radical mastectomy was done. 

During the past year the procedure has been perfected to the point 
where the three types of tianor described above have been standardized 
so that in our hands they produce consistent results. The three trans- 
plantable tumors provide the opportunity to test the cancer study 
qualities of a compound or a procedure in vitro as well as in vivo . ^ 
mixing in the test tube the compound to'Te tested with the inoculum, allo-v 
ing it to remain in contact for a stated period of time and then applied 
in the air pocket or intravenously or intraperitoneally, would allow a 
fast screening method to determine the carcinolytic properties of a given 
compoimd.'*^ . - . • 

Ano-jiher factor which requires study is the safety of the use of a 
given compound oij tissue. It is believed that any compound that would 
be of any value clinically, inust not damage to any degree large blood 
vessels or adjacent nemres. Any extensive necrosis of normal tissue 
would contra indie ate its use in such a study. To test the efficacy of 
this a system has been developed whereby the brachial plexus of the mouse 
and rabbit is exposed and the chemical under study is applied directly to 
the brachial plexus and observations made in this manner. An additional 
test for the safety of the drug under study was obtained by washing the 
operative wound on one side of the animal and leaving the opposite side 
contaminated with tumor tissue to grow untreated. In most instances 
these animals acted as their own control. 

Major findings ; 

1. Using the methods outlined above it has been shown that the washing 
of the wound with a saline spray caused a decrease in the number of 
implants that grew, but in no instance was it possible to prevent the 
development of tumor implants by the spray washing or blotting of the woui: 
with saline, ' ■ 

2. A group of animal tumor implants were tested with podophyllin resin 
drugs, pod'ophyilin, and alpha peltatin. Toxicity studies showed that 
over 10 rag. per kilo of alpha peltatin produced severe generalized tcaic 
manifestations and local toxicity consisting of d^^mage to nerves was 
apparent with as little as 1 milliliter of f mg.^ alpha peltatin producing 
nerve paralysis. Mere concentrated solutions of th-e drug produced a co- 
agulating type of necrosis involving tte entire axilJLa. In l6 animals ir. 



PAOE 3 
PROJECT REPORT FORK (Cont'd) 



mi-ish 
SERIAL MO. 

Major findings ; (Continued) 

which the wound was washed with alpha peltatin solution, allowing the 
drug to remain in the wound, the tumor continued to grow luxuriantly 
in all dilutions well into the range where toxic symptoms occurred. In 
fact, it was the observer's impression that in certain instances the 
alpha peltatin seemed to cause the tumor, if anything, to grow better 
on' the treated side as compared with the controlled side. 
3. Additional chemicals - NCI - 1136, 3022, lQ9U, podophyllotoxin, 
alpha peltatin and crude podophyllin resin, were all ineffective in 
reducing the number of lung tumors in S-91 melanoma injected intravenously. 
In some experiments these compounds caused an increase in the number of 
lung tumors up to 100%. The possibility of this being due to a stress _ 
phenomena seemed to be borne out when the same results Xirere obtained with 
heat stress, cortisone or systemic formaldehyde stimulation. The possi- 
bility of using systemic podophyllin-t^npe drugs in conjunction with local 
washing was tried but inconclusive results were obtained. Because of the 
extensive local tissue damage it was suspected that this drug in its 
present form has very little to offer at the clinical level at this time. 
h. Formaldehyde seems to be the most effective m.eans yet available to 
prevent the seeding of operative woionds. Toxicity studies in mice and 
in rabbits as outlined above show that in mice 1% formaldehyde left in 
contact x^rith the brachial plexus for five minutes produced no symptoms. 
In rabbits, using a |% formaldehyde, no damage could be demonstrated to 
the brachial plexus in the 11 animals tested. 1% solution left in contact 
with nerves for 20 minutes produced nerve damage. |^ formaldehyde solu- 
tion prevented the growth of tumor implants in 3 of 5 animals tested and 
allowed a single implant to develop into others. In saline control ani- 
mals, consistent results were obtained in growing large numbers of implants 
in the area. 

5. 27 different chemicals have been tested, both in vivo and in vitro 
studies. The ones that show promise of results and possible application 
at the clinical level are (1) citric acid, a 1% solution which lowers the 
pH to 2 which increases to 2.5 in contact with tissues. A 1;^ solution 
was between 90% and 95% effective in preventing the growth of K ascites 
tunor. At the other end of the spectrum, sodium carbonate with a pH of 
11.2 dropoing to 10.9 when in contact with the tissues produced no appre- 
ciable nerve injury when left in contact with the plexus for 5 minutes, 
and was lOO/o effective in the prevention of growth of the K ascites tumor. 
(2) Ethanol, a 23/^ solution was also 100^ effective in the in vitro test. 
It has not been tested as yet in the actual operative wound seeding, 

6. f^ertonic solution, such as 10^ sodium chloride, is also effective 
in diminishing the number of tumor implants. 



,^ 



PAGE h 
PROJECT REPORT FORM (Cont'd) 

NCI-75U 

SERL-.L NO. 

PROJECT DESCRIPTION (Continued) 

Significance to Cancer Research ; 

With the development of methods to artificially create a seeded 
■operative wound it has been shown that a standard formaldehyde solution 
produces results which will, if applicable to humans, greatly increase 
the effectiveness of cancer therapy as it exists today. The immediate 
applicability of this type of procedure to the clinical cancer problem as 
it' exists offers opportunities for immediate benefits even though the 
ijltimate answer for cancer will probably not reside in this study. 

Proposed course of project ; 

It is proposed to continue the studies of delineating the effects 
of these chemicals on seeded wounds, seeking to find the ideal chemical 
which will prevent 100^ the seeding of an operative woiind and at the 
same time leave the wound in a condition which will allow primary heal- 
ing. 

It is recognized that eveh though therapy of an operative wound would 
become 100^ effective in preventing thirMevelopraent of tumor implants, 
therapy of cancer would still not be 100^ effective because of the disser " - 
nation of the tumor implants which occurs at the time of surgery or shortl;/ 
thereafter. It seems reasonable to expect that the ideal therapy would 
entail a drug which could be used systemically and would be effective in 
preventing the implantation of lung or liver metastases, and locally to 
prevent the implantation of tumor cells in. the tissues of the operative 
wound. Dr. William Kramer is investigating the use of colchicine -like 
drugs and Dr. Roes Miller the use of several arsenic compounds to try 
to fulfill these criteria. 

11- 

BUDGET AdTlVlTY: [ 

RESE/iRCH /X7 ADMINISTR^iTION £7 

■ REVIEW k APPR0V;.L /~7 TECHNia.L .ISSISTANCE £7 

NO ENTRIES FOR ITEMS 12, 1.3, lU, l5 & l6. 



PAGE 1 



P.iOJjiCT RLPOPiT FORM 



1. National Cancer Institute 2. Surgery Branch 

-TFHTTfTJTE " LA.JQRATQHY OH bHAKCH 



3. '• U. 5. NCI-735(C ) 

SiiCtiffl OS S,:ft\/lCr. LOGATIOi: (IF OTHLaii THAij BETHESDAi SERIAL NO. 

The Development of New Methods for Treatment of Lung Cancer Using 

6. Regional Chemo ther apy : Clinical Research 

PROJECT Ti'i'L^ 

7. Dr. John H, Waite 



PRKCIpAL liJ7ESTllrATUk(b) 
Dr. William Kramer 



9. PROJECT DESCRIPTION 

Objectives ; The chief p-urpose of this project is to improve treatment 
of those ccncers of the lung which are found to be beyond 
hope of surgical cu.re b^' resection. 

Methods employed ; The plan of attack is one of combined surgery and 
chemotherap3^. l/hen open thoracotomy reveals a tuaor not 
amenable tO' surgical resection^ catheters are implanted in 
the vascular channels supplying the diseased lung, and 
exteriorized thr'ou?;h the chest wall. After the patient has 
recovered from his thoracotomy; a potentially chemothera- 
peutic agent such as nitrogen mustard is injected thorough 
the catheter. Serial rnessurements of the si7;e of the x-ray 
shadoi'j cast by the tunor are made. Changes^ if observed, 
are compared with the changes in other lesions in untreated 
portions of the lung fields if other lesions exist. Although 
the project was conceived primarily to get a high dosage into 
primary lung carcinoma, a certain percentage of which have 
been shown to be slightlj"- susceptible to general chemotherapjr, 
it was also planned to stud^^ metastatic tumors when discovered 
at open thoracotomy and unsuitable for surgical excision. 

Thus far, we have followed the policj^ that it would be 
VLnfsir to subject inatients with definitely known inoperable 
t'omors to the risk of thoracotomy when the possibility of 
benefit from regional chemotherapy is as vet unproven. 



■^ROJZCT R.POilT FORK (Cont'd) PA(S 2 



^imm HO. 



Methods Employed : (Continued) 

Therefore, ^^re have careful .y worked up every case before 
surgery, rejecting as unsuitable those patients with 
obviously non-resectable tumors. Most of thes^ non-explor-. 
able patients are placed on a suitable study progran in ihe 
General Medicine Branch while receiving- palliative x-ray 
. , therapy. Lung tunors found to be resectable at open opera- 

tion would of coarse be -dven-' the ■ benefit., of , the best 
excisionsl sia^ger^'- availaole, tlius viaking this sub-group 
also unavailable for regional chemotherspj'' studies. The 
excisional surgery patients vjould contribute to lung cancer 
studies ^oj stiiiiulating -our thoughts toward better surgical, 
procedures, by being available for wound seeding studies 
under Project no. 753(C), ard as subjects for early diagnosis 
studies. 

One of the ajor points to be worked out is the exact 
placement of the catheters with regard to the blood supply 
to the neoplasm, Tliree sets of vessels, viz, the pulmonary 
vein, the puLmonary artery and the bronchial arteries, are 
available. Zxpeririients under Project no. 7$6, Dog Research 
in Catheter Implantation, are being used to develop a tech- 
nique for these different implantations. 

Patient i'aterial ! 

m patients have been studied during a total, of 20 admissions. 
l5 of these admissions were durjng the current calendar year. 

No. Average Stay Days 

'Admissions: Adult males 9 SO 

Adult females '5 60 

Outpatient: Number of patients 2.0 

NuTiiber of visits 1)5 

!1ajor Findings ; 

Seven patients have been found to be suitable for the stucfy 
in that woncup conf jj^raed the presence of lung tumor and 
possible curability hy excisional surgery. Of these seven 
patients, two proved to have a bronchogenic carcinoma suit- 
able for pneumonectomy, and hence not available for catheter 
implantation. No urinary bronchogenic patients have yet 
been found in whom thoracotom;^/ was indicated, but who were 
found inoperable. 

In five patients metastntic tumors were found. Two of 
these metastases were ap-iarently solitarj'- and it appeared - 
best to. treat these" patients by excisional surgery which was 
accordingly performed. This left tturee patients with metastatic 



PAGE 3 
PROJ..:CT REPORT FORM (Cont'd) 



I\TCI-755(C) 



?''ajor Findings ; (Continued) 

tumors available for catheter implantation, the catheter 
being; implanted in the pulmonaiy arterjr in two cases and 
in the pulmonar?/- vein in one case. Nitrogen mustard in- 
jected daily has been the chemotherapeutic agent studied 
so far to establish base line values. One patient, with 
metastatic adenocarcinoma from a colon primary showed no 
apoarent change in the size of her tumor until the time of 
death from a cerebral metastasis , six weeks later. The 
second patient, also with metastatic adenocarcinoma from a 
colon primary/, has shown a slight regression of the treated 
pulmonarj'- metastasis during the same tjjne that a new meta- 
stasis has appeared in the contra-lateral lung and grew 
constantly. Perfect access to the pulmonary artery bed was 
maintained in this patient for the nine months that lie lived 
after catheter implantation. Both of these patients have 
shown minimaL- bone raarroX'J changes compared with the changes 
usually seen after intravenous administration of nitrogen 
mustard. In one patient, with metastatic cancer from the 
cervix uteri, the catheter was implanted in a radical of 
the pulmonary vein and the pulmonarj^ vein tied off between 
catheter implantation site and the heart with a thought to 
gain access thereby to the bronchial vascular bed. On 
injection of nitrogen mustard into this patient, she developed 
an abscess of the treated portion of the lung which appeared 
to start first at the tuj^ior site but later spread to the 
entire lobe requiring subsequent lobectomy. Seven of the 
lit patients worked up have been not suitable, because of 
proven metastases, or because of the disease found was other 
than a suitable carcinoma. Three of the four far-advanced 
patients were fo^ind suitable for m.etabolic studies on Dr. 
Watkin's service. 

Significance to Cancer Research : Continuing increase in the incidence and 
death rate due to lung cancer demands maximal efforts to 
control the disease. The primary or basic science approach 
involves efforts to determine the biolocic nature and environ- 
mental relationships of lung cancer. While of great hope, 
this research has yet to produce applicable results, A 
second phase is seen in programs aimed at the discovery and 
treatment of lung cancer in its earliest stages. This attack 
has succeeded in curing up to ^% of all patients seen by 
extirpative surgery, the only curative treatment now known. 
The third phase of lun:; cancer research involves improvement 
of treatment of the disease when it is past its earliest 
stages, and the large nuimber of well-advanced lung cancer 
patients no\<t seen provides an urgent motivation for the clini- 
cal investigator to search for an approach such as described 
in this project. 



PROJECT REPCET FORM (Cont'd) RiGE k 



NGI-75g(C) 
SERIAL KO. 



9. PROJLCT DESCRIPTION (Continued) 

Proposed course of project ; Thus far it has been shown that catheter 
implantation ' into the pulmonary vessels is a feasible 
■ procedure and that it provides accurate access to the vas- 
cular bed for a prolonged period of time. It is planned 
to continue the studj'- as outlined, using patients with 
operable metastatic and broncho-enic carcinomas. It is 
hoped to get a number of bronchogenic carcinomas to test 
this method inasmuch as this is the type of turaor which 
has been shovm to be slightly , sensitive even to intra- 
venous a dill in is trat ions of mustard. \Je are also considering 
whether it might not be justifiable to perform thoracotomy 
with catheter implantation even on patients with clinically 
inoperable tumors, inasmuch as so little of honestly thera- 
peutic value is available to this group today. 

k new method of gaining djjrect access to the bronchial 
artery bed is under investigation in the animal laboratory. 
Project 756, and may be available soon for clinical evalu- 
ation. 



11. 

SufiGET ACTWlT/: ' ' — — ^ 

RiiSLARGH /x7 ADil IN IS TUITION AJ 

REVIEW 5: , I.??[i mh L /y TECHN ICA L ASS ISTANCE A7 

NO EKTRIES FOR ITEiIS 12, 1.3, Ik, l5 & 16. 



PAGE 1 
PHOJECT REPORT FCBIi 



1. National Cancer Institute 2, Surgery Branch 

Institute L^son^TokY on i^mm 

3, h. - 5.- NCI-756 

gi^CTION OH ^^UVKJE L'Mr.TICN (IF OTiGR m.f dilTiiEiJDA) sekl.l ^iu. 

Development of New llethods in Treatment of Lung Cancer using Regional 

6. Chemotherapy: Labora tory Research 

PROJECT TiTi^' 

7. Dr« John H. Waite 

8. Dr. William Kramer - Dr. I- filliam B anfield 

OTHER IMVSS'iia'.iOR!:; ' 

9.. PROJECT DESCRIFnON 
Objectives ; 

This has been a key project for the development of techniques which 
were later used in the human lung cancer project 755(C). Using the dog 
as an experimental animal, various methods for the catheterization of 
pulmonary vessels at open operation were studied, in an effort to potentiate 
the known effect of parenteral nitrogen mustard on bronchogenic tumors and 
to provide a satisfactory- route icr evaluation of new possible chemothera- 
peutic agents as they are developed. 

Methods employed ; 

Fine polyethylene catheters were introduced at thoracotomy into a 
bronchial artery or into a branch of ? pulmonary artery or pulmonary 
vein. The catlieters were brought out throu.^ii the chest wall and allowed 
to remain in situ for a long period of time. The continuing patency of 
the catheters was tested and possible deleterious effects on the surround- 
ing tissue were watched for. By injectJjig diodrast, . serial angiograms 
were obtained under various experim.;ntal conditions and studies on approxi- 
mately 50 dogs. It was also attempted tc deten.iine x^hether the differential 
damage to normal dog lung structures could be accomplished bjr the injection 
of extremely high doses of nitrogen mustard. 

Major findings : 

Implantation of catheters at open thoracotomy was perfected. Catheters 
can be left in for periods of time as long as six months providing continued 
access to the pulmonary' circulation. The feasibility r.nd harmle.ssness 



PROJECT REPORT FOmi (Cont'd) R'^GE 2 



NC 1-756 

^rl;l no. 



Major findings : (Continued) 

of the catheterization, technique was demonstrated satisfactorily, 
enabling us to bring it to the human project. Previous experiments had 
shoxm that differential damage to the bronchial -lucos? could be obtained 
hj injection of large quantitities of mustard directly into the bronchial 
arteries, which vessels are knuwn to be the direct blood supply to broncho- 
genic carcinoma in humans. Direct application of this technique to humans 
did not seem possible because of the variability of bronchial vascular 
supply in humans, and because of . the likelihood of finding difficulty in 
the dissection of finer hilar structures in advanced carcinoma of the 
bronchus. We have continued the studies with attempts to gain access to 
the bronchial vascular beds. 

Injection into the pulmonary artery branches failod to produce specific 
burning of the m.ucosa, although of course this does not ni^eessarily mean 
that mustard so injected in huraans would not cause a greater effect on 
the bronchogenic carcinoma which is much more sensitivo than the normal 
tissues of dogs. In another attem.pt to gain access to the bronchial vascu- 
lar bed the pulmonary vein to the affected lung was ligated near its 
entra.p-cc to the heart. Diodrast injection of these preparations seemed 
to visualize the bronchial vascular bod. However, nitrogen mustard inject- 
ions failed to, produce specific burning of tht bronchial mucosa, and 
further studies on the collateral vessels which hrvo developed after pulmo- 
nary vein ligation have revealed that these are actually conn.:^ctions 
between pulnonary and systemic veins which sidetrack the bronchial vascular 
bed. The most "pronounced local effect on Imig tissue as compared with a 
general body effect mcasirred by bono marrow examinations, occurs immediately 
after ligation of tho pulmonar'/ veins. Injection of pulmonary v^ins with 
nitrogen mustard occasionally produced >:,nough daiiiage to cause local necrosis 
of lung tissue. 

Investigations have now beguai on utilizjjig th^ thoracic aortr itself 
as an access vessel. If the aorta were temporarily obstructed at the 
level of tho diapliragm and ^ quantity of chemotherapeutic agent intro- . 
duced into the thoracic aorta it would immediately be carried to tlie 
bronchial circulation, chest wall, mediastinal l^niiph nodes and other 
structui\,s usually involved by locally advanced bronchogenic carcinoma. 
Methods of accomplishing this at open thoracotomy and h-y aorta catheteri- 
zation in the intact anlnial are being studied. 

Significance to Cancer Research : 

Continuing increase in the incidence and death rate due to lung 
cancer demands maxlfial efforts tj control the disease. Tho primary or 
basic science approach involves efforts to determ.ine the biologic nature 
and environmental relationships of lung cancer. Wliile of great hope, 
this research has yet to produce applicable results. A second phase is 
seen in programs aimed at the discovery and treatm.ent of lung cancer in 



PROJECT REPORT FORM (Cont'd) 



PAGE 3 



NG 1-756 
SERia NO. 



Significance to Cancer Rcsoarch : (Continued) 

its earlibSt stages. This attack has succeeded in curing up to $% of 
all patients seen hj extirpative surgery, the only curative treatment 
now known. The third phase of lung cancer research involves improvement 
of treatment of the disease when it is past its earliest stages, and the 
large number of well-advanced lung cancer pati^-nts now seen provides an 
urgent motivation for the laboratory inve3tigat6r to search for an 
approach such as described in this project. 

Proposed course of projoct ; 

Continued studies will be made to determine the most advantageous 
vascul-^r route of drug administration in bronchogenic crrcinoma, The 
project will provide a continuing opportxmity to evaluate changes in 
a catheterization technique to be employed in humrns. After deciding 
on a standard technique we would like to use that technique for toxicity 
studies in dogs before using new drugs in hum-^ns by the regional vascular 
approach. 

11. 



BU£)(JST AcTtVlTV: 



RESKIRCH /17 ADHINISTR'_TIOIJ £7 

REVEilJ & APPROVAL r? TUGHI^IICAL ..SSISTAMCE /~7 



NO ENTRIES FOR ITEIiS 12, 1.3, lii, 15' & 16. 



PROJECT REPORT F0RI4 PAGE 1 



1. National Cancer Institute 2. Surgery Branch 

" toJS'Tll'UTE UiBORjlTORY OR BR-'iNCH 



3. h. 5.- NG 1-757(0 

SECTION OR Service location (1F other TH^M B^THkSM) SERIAL m. 



6. 


Prevention of Operative Site Recurrence by Improvement of Biopsy 
Wound Closure Technique 




PUoJECi 'TITLE 


7. 


Dr. John H. Waite 




PRlNdlpAL 'lMVESTia\TOR(S; 


8. 


Dr. Robert R. Smith - Dr. Albert Hilberg - Dr. Horace Herbsman 




OTl-lEK iWVEtJTi&iTORS ' 



9, PROJECT DESCRIPTION 

Objectives ; 

,The. purpose, of this project has been to gather some experimental 
data concerning the validity of biopsy wound closure techniques, to 
determine if any currently used techniques are satisfactory, and to 
develop, if necessary, improved techniques for management of biopsy 
wound closure. 

Ifethods employed : 

Patients with apparently primary operable carcinoma of the breast 
are prepared for surgery. A routine local incision is made, and the sus- 
pected tumor excised, examined and submitted for frozen section histodiag- 
nosis. The cut surface of the excised tumor is carefully washed to procure 
positive c,ytologic material for comparison with later skin surface washings. 
Cases with positive frozen section are suitable for study. 

The local wound is closed by one of the standard methods vinder 
study, and the skin area completely cleansed and redraped for radical 
mastectomy. At this point, the closure is carefully examined for gross 
escape of serous or bloody fluid. Any of this serous or bloody fluid 
which does escape is carefully aspirated for cytologic bxamination, and 
in addition, washings are taki^n from the margins of the closed biopsy 
wound or any cover which may be fixed over it for cytologic examination 
to determine whether cancer cell escape has occurred with the method 
under evaluation. 



PROJECT REPORT FORII (Cont'd) PACE 2 



NGI-757(C) 

bKKL'iL i\IU, ....•• 

Methods employed ; (ContJJiued) 

The present' s-cope- of ■ the project is limited to the following 
closure methods: 

1. Closure with a tight interlocking continuous suture. 

2. Closure ^^rith a tight Interlocking continuous suture followed by ?. 
plasticized film, 

3. Suture closure with a sheet of rubber submitted to the surrounding 
skin. 



Patient material: 



No. Average Stay Days 



:\djTiissions: Adiilt males 2 lb 

Adult females . ■, '^ ...... . „, ^^ 

Outpatient: Number of patients 10 
Number of visits IjO 

Major findings : 

Five breast carcinomas and one chondrosarcoma have been carefiilly 
studied according to the methods outlined. That the opportunity exists 
for wound seeding, to occur from the biopsy site appears probable from 
the following data: 

1, Three of these six patients yielded cr.ncer cells from the skin sur- 
face, using cytologic detection techniques. 

2. In all six patients, oozing of serous fnd bloody material through 
the closed biopsy wound occurred. This occurred despite a tight closure, 
and it was noted that the bleeding usually occurred from the suture needle 
puncture site. It was reasoned that the blood escaping from the biopsy 
site might easily contain a suspension of tumor cells. 

Closure of the biopsy site with a continuous tightly interlocking 
suture was unsuccessful in sealing the v7ou:id as was Aeroplast (an aero- 
solized plastic spray which dries rapidly to form a film) which was 
applied in all of the five breast ccrcinona patients. Oozing from the 
closed biopsy wound occurred, lifting the dried plastic film from the 
skin surface in a few seconds' time. 

The four patients who were found to have benign tumors on biopsy 
contributed to the program by yielding cytologic control specimens. 2 cc. 
of a dye placed within the depths of one of these biopsy woiinds appeared ■ 
immediately on the surface of the skin through the suture needle puncture 
sites, imitating, we th^nk, the case with which a tumor suspension could 
reach the skin surface. 



PROJECT REPORT FORM (Cont'd) 



PAGE 3 



NCI-757(C ) 

9. PROJECT DESCRIPTION (Continued) 

Significance to Cancer Research ; 

Wound recurrence following biopsy and local surgery is not uncommon. 
In treating one of the most common cancers, that of the breast, leading 
clinics admit to a 12^ local recurrence rate. The two most likely factors 
are inadequate surgical margins and spillage of cells at the time of 
biopsy. Exfoliated cells have been recovered from skin surfaces by the 
principal investigator even after more than usually careful attempts at 
biopsy site closure and sealing off, using conventional techniques. A 
simple method which would guarantee 100% riddance of skin surface tumor 
cells would be of immediate value to all surgeons in treatment of many 
common malignancies. 

Proposed course of project ; 

Thus far none of the techniques in current practice for biopsy 
site closure is even theoretically capable of preventing escape of a 
tumor cell suspension from the closed biopsy wound. We will evaluate 
other methods in common use. In the meantime we will be trying to 
develop a foolproof technique under the animal project no, 758. We 
will continue to get washings from the operative wound site to correlate 
positivity of skin surfrice washings vjith the radical operative wound 
washings, and we will continue to follow these patients clinically in 
order to correlate tumor recurrences in the operative site wi.th the 
positivity of skin washing cytolog.-. 



11. 



BUDGET ACTr\fITY: 



RESEARCH /x7 ADMINISTR/iTION £7 

REVIEW & APPROVAL /~~/ TECHNICAL ASSISTANCE /V 



NO ENTRIES FCR ITEMS 12, 1.3, lii, iS & l6. 



PROJECT REPORT FORil PAGE 1 



1. National Cancer Institute 2. Surgery Branch 

BISTtt'tJfE L^SOPulTORY OR BRaMCH 



3. Ii-. 5. NC I-7$8 

SECTIOM OH Si^RVKE LOCATION' (IP OTflER TJ14N BETHEiifiA) 'SERI'.L NO. 

Prevention of Operative Site Turior Recurrence by Improvei-nent of 
6. Biopsy Wound Closure Techniques: Laboratory Research 

PROJECT TITLE 



7. Dr. John H, Waite 

PRINCIPAL raVESTIQ\TOR(Sj 



Dr. Horace Herbsman - Mr. Richard V. Eck 
OTHER INVESTiaiTORS ' "" 



9. PROJECT DESCRIPTION 

Objectives ; 

The purpose of this project is to evaluate methods vrhich have been 
proposed for biopsy wound closure by laboratory techniques on the experi- 
mental andjiial. A closiire method is sought which would prevent the escape 
of tumor cells fropi the skin surface into the radical surgery wounds 
after preliminary biopsy. If currently used techniques are fo-und to be 
unsatisfactory, new techniques will be developed. 

Methods employed : 

■ For experimental convenience, a blue dye is used to imitate the 
turaor cell suspension which must be retained within the biopsj'" wound 
in order to prevent radical surgical wound contamination. Using rabbits 
as experimental animals we have made standard biopsy wounds, closed them 
with continuous interlocking sutures, and injected dye into the biopsy 
wound cavity, to determine the quantity and pressure of fluid retained 
by the wound closure being tested, before leakage occurs onto the skin 
surface. 

Thus far three methods of closure have been evaluated; 

1. Closure with a continuous interlocking^ suture as described. 

2. Closure with a continuous interlocking suture overlaid with a plastic 
film sprayed onto the skin surface. 

3. Closure using Raney sur:?ical skin clips. 



PROJECT REPORT FORI-I (Cont'd) PAGE 2 



MCI-7g8 : 

SKdiAj MO. 



PROJECT DESCRrPTrON-tC-ontiritlca')- '■-- ' ." . ■ __ ' ' . •' 

Mr. j or . f indings : 

• ' 1. : After clasiire' of • the" standard biopsy, wound with a continuous 
interlocking suture only, 1-2 cc. of dye injected under 2 cm. of water 
pressure appeared. JJTimediately on the^ surface of the closed biopsy wound. 
The appearance. of this dye was not at the incised skin margin, but always 
at a suture needle puncture site/- the same-., locus from which bloody oozing 
was invariably noted after the clinical closure of biopsy wounds in huraans. 
Apparently the skin defect created by a suture needle puncture should be 
regarded as continuous with the incision into the underlying tun or, ^ and 
a likely BO'urce f 'or" turror cell "escape. v 

2, Sealing of the closed biopsy wound with Aeroplast (an aerosolized 
plastic spray. which dries rapidly to, fcrpi a film), permitted about three 
times as mueh dye to 'be in^edted '"under a. slightly higher- pressure. However, 
if only sli'-;ht manipulation of the closed biopsy wound was performed, dye 
immediately burst from beneath this very fragile seal, easily dissecting 
the plastic seal off of the surface of the skin. Thus, this type of a seal, 
including the older collodion seal, would seem to add little protection 

to the biopsy site. 

3. Application ofRaney clips to corpt skin margins resulted in an 
extremely efficient closure -permitting 'Ux injection of about ^0 cc, of 
dye under about lu cm. of vrater pressuro, before appearance of the dye at 
the openings of nipples or sweat glands. 

Significance to Cancer Resegrch ; - 

The intact skin overlying a s-ft part cancer must usually be incised 
to obtain a biopsy, rupturing the envelope ox normal tissue which theoreti- 
cally should be removed intact with its indwelling m.alignancy.- The pre- 
vention of escape of malign?nt cells through this incision constrbutes one 
of the oldeat problems in Cc-^ncer surgery.. Many methods of closure of 
biopsy wounds have been recommended e-np^jr^icrlly. It is hoped that utili- 
zing the testing methods described, we will be ablo. to recommend or 
develop -^ standard tiethod for biopsy wound closure backed by experimental . 
data. 

Proposed course of project ; 

The most important untried method for wound closure is a method which 
has been recommended using tho cementing of a sheet of rubber over the 
closed biopsy wound. This technique will be tested, and further experi- 
ments will be condu:;ted on the Ranejr clip method of closure. 



PROJECT RE^PORT FORM (Cont'd) ' PAGE 3 



10. NC 1-758 

SERL'-L NO. 



11. 

BUDO^T ACTIVITY: 



RE,SEi;RCH frf ADME'JISTRATION £7 

REVIff."! & APPROVAL /"/ TECHI-Iia.L ASSISTANCE /~J 



NO ENTRIES FOR ITEMS 12, 13, lU, l5 & l6. 



PROJECT REPORT FORM PAGE 1 



1. LTa tional C ancer Institute 2, Surg ery Branch 

INSTITUTE L.B01i;T0RY OR BR.'i!CH 



3. h. ' 5. NCI-759 

SECTION OR SERVICE LOi^TiO^J (11? i^lM Tii.H BIDTHSSD..) SERL'.L NO. 



6. Ttchnic of Hypophysectoiiiy in the Guinea Pif 
prO^CT TiTi£ ^ ' 



7. Dr. Lt,ster M. Cramer 

"p^^inTpXTFTESTmr^rrsT 



0. Dr. Eli Nadel 

OTHER BIVESTIG'.TORS 



9. PROJE.CT DESCRIPTIO'^- 

Objectives ; 

To develop and perfect a technic for hypoph^z-sectomizing the guinea pig. 

Methods employed ; 

Guinea pig hypophyseal fossae were appruached trans cervic ally, 
transcranially, and transbuccally and the pituitarj- gland renoved by 
suction. Animals m:>intained postoporativel3^ on antibiotics, clyses, 
and steroid. 

Major findings : 

No complete hypophysectomies were accomplished, the Injaiting factor 
being the extension of anterior pituitarj^ colls up around the stalk to 
the hj'pothalamus. ^ 

It is felt that radioactive substances, such as yttrium., implanted 
into tht, pituitarj" fossa will offer a better chance to perform a com- 
plete hjTDophysectomjr. Neither investigatur is authorized to use radio- 
active substances, and the project is arosently shelved until an interested 
personage with the necessary radioactivity clearance can be included in 
the study. 



PROJECT REPORT FORl-I (Cont'd) PAGE 2 

NCI-759 • ■ 

&mt.1 V.IO. 

PROJECT DESCRIPTION (Continued) 

Significance to Cancer Research : 

Continued developments in the f ic^ld of endocrinology demonstrate 
the Mportance O'f pituitarj^ hormones- upon cf-ncer •■ tissue * If "complete, 
removal of the gland could be easily done in rjuinea pigs, a very useful 
tool xsrould be available to further modifj'- this control. 



Proposed course of proJL:Ct ;' ■ 

Pending the availability of radioactive j'ttrium, no further work 
is planned on this project. 



11. 



rese:.rch /T/ ;.D'ii'asTR.TioN A7 

REVIEW ic IJpyRLNLL H TECffi'lICL ASSISL^NCE /"T" 



NO ENTRIES FOR ITEiMS 12, 1.3, lU, 15 "x l6. 



PROJECT REPORT FGIG' ' ' P;.GE 1 
1. National Ca ncer Institute 2. Surgery Brnnch 

-TH^TTTITTE li^BCii-xTORY OR br;.itch 

3. Ii. . S. NCI 760 

SECTION OR Sfiftv'iCE LOCATION {W OTIffiR TH.UI 3ETIDLSDA ) SERIAL NO. 

Th>.. Studjr of the Effects of Beta Nn.phthylr.mint and U-Aminodiphcnol on 
Qastrointestinal -'.nd .Bladder ;''ucosa by Direct Contact in Dogs. 

a. DevelopCTont of a Surgical Tuclinique for In Jivo Study of 
Drugs in the GastrointestLnal and the Isolated Urinarj^ Bladder. 

b. A Studj^ of the Techniques for Handling the Genitourinary Tract 
6, after Cy stec tomy. ; ' 

PRD'JECT TI1*Le ~ 



7. Dr. Donald Cole - Dr. Willian Hueper 
PRlNdlR.L INViSTia-TOR(S; 



■oTl-M INVEST IG-.TORS ^^ 

9. PROJECT DESCRIPTION 

Objectives ; 

A study of the carcinof^enic potentialities of certain aromatic "nines 
and azo compound yt^llow ;b. These comp>^unds along with yellox^ ,..B and 
yellow OB are involved in food dycs in comnion use. This iS the problem 
of Dr. William Hueper of tlie Environmuntal Cancer Section of the National 
Cancer Institute that lie will report in detail. 

Ifcthods em-ployed : 

An acute experiment was devised in order to study the naetabolism 
of the djes in an isolated segment of the gastrointestinal tract for the 
length of. time it may bq present duriiig norraal digestion. In., effect, 
in vivo test tubes were created by isolating the gastrointestinal tract 
of dogs tliroughout its length from the stomach to tht, rectum with no 
reconstruction of the ana tony. In another group of chronic experiments, 
in vivo test tube bladders and segments of gastrointestinal tract, stomach 
To colon, were isolated with reconstruction of the anatomy for what was 
believed to be the best fiuictional results. A total of 19 acute experi- 
ments were performed on the gastrointestinal tract. The dogs were prepared 
preoperatively so that their gastrointestinal tract was clean. The organ 
was satisfactorily isolated at the time of surgery, bejjig caroful to main- 
tain the integrity of the blood supply. Tlie drug under study was then 
placed into the isolated loop and speciraens obtaiiied from the isolated loop 
at various intervals. In the chronic 'experiments 10 mg. of yellow AB were 
fed in meat balls to the dogs. After tliree weeks they were sacrificed and 
autopsied to be examJ-ned for metabolic analysis and pathologic changes. 



PROJECT REPORT FORli (Cont'd) Ri( 



NCI-760 ' 

Methods employed ; (Continuod) 

Two dops hr-d pouches formed with implantation of ^ grsra of yellow ;.B and 
two-. others had jejunal loops and colon loops prep.ared and the drug placed 
into the isolated segments ia a variety of circumstances. Multiple pro- 
cedures were carried out to ascertain the best method for diverting the 
urinary stream. to obtain an isolated urinary bladder and still maintain 
life. The drug- was placed thro-ugh the dome of .the empty bladder which 
was then closed with silk sutures. The urethral ope:ning/was_ then closed 
in'a similrr manner;-"^ '■'""■ " ' ""'-■■ ''~ ■""•"' "" "' 

Major findings ; 

"■ The result of the' cxperinient -is "not complete as yet. The dyes were 
not left in place long enough to produce any histologic changes, .'.s for 
the chemical degradation products for;ned by the compounds^ definitive 
results are not as yet available. ..'.s in the similar instance of using 
humans, the placement of ±h^ ureters in the colon or on the skin produced' 
a hydroneplirosis and hydroureter with ascending urinary infection which 
made it impossible to maintain adequate kidney function. The placement 
of the carcinogenic agent in the bladder with the urinary stream diverted 
has not boon completed as yet. 

Significance to Cancer Research ; 

A more thorough understanding of the possible carcinogenic effect 
of certain dyes, would possibly l^^nd tu - useful prophyl^actic measure in 
the prevention of c?,ncer.'. • . 

Proposed course of project : 

Sec report of Dr. William Huepor re '■.warding future course of study. • 



11. _____ 

BUDarT ACTIVITY: ^ ~ 

, rese,'-RCh /tj ;DniNiSTR.\Tioi-j /y 

■ REVE^J i ;.PPRaV.-.L £7 Tt.CHNIC:.L ..S3IST;,.NCE £7 . 
NO ENTRIES FOR ITEI-IS 12, 13, lij., i$ :k 16. 



PROJECT REPORT FORM PAGE 1 



1. Np.tional Cancer Institute 2. Surgery Branch 

IMSTITUTE L;Sta.'.TOnT OR BRAMCH ~~~~ 

Memorial Hospital & 
3. li. Sloan-Kettcring Institute i; NCl-761- 

"S^CTIO-'.^ OR SERVICE LOa:.TiOM (IF G^nm T^-N MTHESC;.) gERnL l^Ol 



6, Blood-Pressure Sorvo-Reg-ulator 
PROJECT "TITLE 



7. Dr. Jolin H. Wait e . 

PRINC 1P;.L IN\7ESTli.-TCR( t ) 



8. John Laughlin - Naorai Sager - Williara Poppall - William Rowland 

othf:r JiivEST la'. tors ' ' 



9. PROJECT DESCRIPTION 

Objectives ; , _. . . 

To construct a machine for automatic regulation of blood pressure in 
shock viuc to vasodilation^ and in hypotensive anosthtsia. 

Methods employed ; 

The mean arterial blood pressui'-e is measured directly with a trans- •.■ . 
ducing manometer. Appropriate vasopressor (norepinephrine), or vaso- 
depressor (arphonad) solution- is c7:drn;inistered continuously intravenously 
by a variable spe^^d' infusion pump. The pump speed is regulated by a 
signal generated by a servo-loop. The servo-loop signal measures the 
difference between the blood pressure measured, and the blood pressure 
clinically desirable for the patient being treated. T: e therapist thus 
needs o^nly to set the control panel for the desired blood pressure, and 
the auto-regulated machinery administers blood pressure regulating 
substance as needed. 

T'ajor findings ; 

Construction of the blood-pressure servo-regulator apparatus has been 
completed. The apparatus has been tested on a series of experimental 
animals (dogs) in the Physiological Laboratories of the Sloan-Kettering 
Institute. Necessarj'- refinements in design and construction have boon 
made r'nd completed. The apparatus has now been moved to the recoverjr 
room of the Memorial Hospital. The participation by the principal investi- 
gator has been on a consultative basis d^oring the calendar year, 1955. 



PROJECT REPORT FORiM (Cont'd) PAGE 2 



NCI-761 
aERr.L WO. 



PROJECT DESCRIPTION (Continued) 

Sit^nificance to Cancer Research ; 

The raochine, if successful, will be an aid in maintaining blood 
pressure in patients with neurogenic shock due to radical surgery or 
drug reactions. It will also provide a method for controlled lowering 
of blood pressure (hypotensive anesthesia) in patients undergoing 
radical surgerj'- for cancer. 

Proposed course of project ; 

The apparatus will be evaluated in controlling the blood pressure 
of the next six patients requiring vasopressor agent administration to 
maintain blood pressure at the Menorial Center. 



11. 

BuB'GET'ACTWir^ — ' ' — ~ 

RESEL\RCH /^ /J^IIEIISTR-.TION £7 

REVIEI'-! & AP?ROW.L /~7 TECrffiia.L .ASSISTANCE A7 



12. Sloan-Kettering Institute - I''crr:orial Hospital, New York City 

COOPEUVriMG JtilTS OP THii PuBlIC HL' LT:rS-^R\/iCh, OR aH'Ml ORa.MIZATlOWS, 
. PROVIDING FJiiDS, F CILITES, OR PERSOlll^EL FOR THIS PROJECT IN EITHER 
1956 or 1957. 

WO ENTRIES FOR ITE.iS 1.3, Hi, 15 & I6. 



PROJECT REPORT FCMl R^iQE 1 



National Cancer Institute 2. S'urger^r Branch 

ii. 5. :ici-762(c) 

Blood Volurr.e, plasna Volume, Red Cell Mass and Total Circulating Plasna 
Protein and Electrolytes in Cancer Patients D"ar:ng preoperative Bowel 

Preparation and the Period after Rad i cal S^Jirger- .^. 

"PROJECT IITLE '' 



7. Dr. Rcoert ;.. >^ilch - Dr. Jesse L. Steirifeld 
PRi-xP/lL llfV£STI3i^TcR(Sj 



\. Surgical Staff, :'.C.: 



9. FRO.^CT DESCRIPTIMf 

Objectives ; 

Although considerable data have been presented from several Isbora- 
toriss concerning the netabolic responses to surgery and other forms of 
tra^'Pia, little attention has been directed to nietabolic changes in patients 
undergoing various preparatory- procedures during the imnediat-e preopera- 
tive period, the operative and immediate postoperative oeriod. This is 
particulsjrly true of female patients undergoing extensive preparation of 
the bowel for pelvic surgery prior to contemplated exenterative procedures. 
The objectives of the present study are to determine the above parameters 
seris.lly in patients undergoing "vigorous" as opposed to "mild" prepara- 
tion of the gastrointestinal tract for both pelvic and extra-abdom.inal 
surgical proced'jres. 

''ethods employed: 

.Injection of known small amounts (approxiniately 10 microcuries) of 
1 ■'- labelled human series albi-r-iin and measurement of its dilution at 
15 and 30 minutes after injection in both whole blood and plasma. Coi;5)led 
with serial determinations of xhe plasma concentration of red cells (hemato- 
crit), plasm.a protein, and senum electrolytes, it will be possible to cal- 
culate the total amounts of these various substances v/hich are circulating 
in the plasma and the effect of bowel oreparation of thiem from calculated 
data on red cell mass, plasma volume and blood volume. 

pa~ient material; 

-ati&nts undergoing surgery by the N.C.I. Surger;.'- Branch /rill be 
stu'Jied. Daily EKG's and if possible, ZSG' s will be taken. I'o patients 
admitted during this year for this project. 



PROJECT REPORT FORM (Cont'd) PAGE 2 



MCI-762(C) 
SERIilL KO. 



9. PROJECT DESCRIPTION (Continiaed) 

Major findings ; 

None to date. 

Significance to Cancer Research ; 

In order to improve our methods of treating cancer j mere precise 
information is necessary concerning the metabolic status of cancer 
patients coming to surgery. This project should help the surgeon pre- 
pare patients for major surgery. 

Proposed courae of project ; 

During the next year it is planned to study patients undergoing 
radical pelvic surgery, both during the preoperative and immediate post- 
operative periods. It is anticipated that approximately -2ii-l 8 patients 
will receive such therapjr and be available for such studies. 

11, 

"DUDGET ACTITTTyI 

. RESK'iRCH /p ADMINISTRATION £7 

REVIEV. k AP ROVi\L/y TECHNICAL AbSISTASCE £7 

NO EFTRIEb FOR ITE. S 12, 13, ih, l5 & l6. 



PAGE 1 



PROJECT REPORT FORM 



1. N"tional Can cer Institute 2. Surgery Branch 

-EISTTTUTE ■ UBOPu-TORY OR 3R.U-ICH 

3. h. ^- NGI- 76.3 

SECTION OR SERVICE LOCATIOW (i^' OTHER TR-.N BMHesdA) SERia 'No. 



6. The Effect of Prednisone (Metacorten) Upon the Healing of Wounds 
PROJECT TITLE ' 



7. Dr,- Lester I'l. Cramer 

PRINCIPAL IHVESTI(i-.TOR( S ; 



• "OTHER I^]VESl'I(i;'roRS 

9. PROJECT DESCRIPTION ' • ' ■ . 

Objectives ; 

Because of its greater anti-inflammator;^'- action and its lesser effect 
upon ion netabolisiTi, prednisone is rapidly replacing compounds E and F in 
a large segment of clinical medicine, especially in the management of 
rheumatoid ?rthritis, chronic pulmonary asthma, and many ocular and derma- 
tologic conditions. ■ . 

The inhibitor^,r effect upon wound healing of compouiids L and F is well 
docuTiiented. It becomes Imperative to know if this- new -.Gompound will have 
greater wound healing inhibitory effect as night be expected from its 
greater effect upon inflajumation. 

Methods employed ; ' ■ . 

280 C57BL nice were divided into two groups of li|0 each, and then 
each of these subdivided into lli, groups of 10 each. One-half of all 
the animals were given prednisone .25 mgm/20' gm* daily for 5 days before 
wounding, the other one-hnlf served as pair -fed controls. 

The wound consisted of a 2 cm. incision in the anterior wall of 
the distal stomach. 5-0 nylon thread was used to close the wound in 
linear fashion, used a continuous horizontal mattress suture for serosal 
coaption. 



P;.GE 2 



PROJECT REPORT FCRM (Cont'd) 



NCI-763 

SERni wo. 



Methods employed ; (Continued) 

Prednisone was given daily postoperatively, and all animals were 
weighed daily. A. group from .each of the -prednisone and control groups' 
iras sacrificed on each das'- for fourteen days, and the wounds studied in 
two ways: (1) Strength of wound as determined by introduction of con- 
pressed air until the suture line bursts. (2) Histologic character. 

Major findings ; 

_'.ll of the technical aspects of the work have been completed. 
I. Bursting strength ..re suits. 

a. Curve has been established for the healing wound in 

thu mouse stomach which is comparable to the accepted curve for a similar 
xiTound in the rat stomach. 

b. The curve increment of .healing strength in the mouse' ■ 
treated with prednisone is ox the same shape as the normal curve, but 
is very slightly lower. Limited studies using cortisone showed that 
healing strength was about the same at i^iid points on the curve as that 
found with prednisone. 

II. Histology' results. 

Not available, slides are being processed. 

Significance to Cancer Research ; 

The use of new steroids as a palliative agent in treatment of cancer 
produces serious problems in tlie surgical management of patients, especi- 
ally, if it could be shoxm that these drugs do inhibit wound healing. 

Proposed course of project ; 

Until complete analysis of the material already at hand has been 
performed, it remains impossible to definitely state whether this drug 
has any effect on wound healing. It may be necessarj- to run a few more 
anLmals to further determine this point. 



11. • 

BuDGE'T'AOTWITt: ^ ^ . — _ 

RESK-.RCH' /T/ ;.DMnJISTR-.TION /7 

REVEi'j St :.?mm:L fl technical ;.ssistance f~f 



NO Ei'ITRIES FOR ITEMS 12, 1.3, lIi, l5 h. l6. 



PAGE 1 
PROJECT REPORT FORM 



1. i\Tg.tiong.l Cane or Institute 2. Surger:: Branch 

INSTITUTE- L:.BOR.iTbR^ OR BRjiCH 



3. k. . . 5. NCI-76I. 

SSCTIOK CR SKRVIOE LOdJl ' lOM (1^ ' OTtli^R m.i'] jJE'i'tiESUA ) SUSl'L NO. 

6. The Transplantation uf "Conditioned" Endocrine Tissue 

PROJECT TITLE 



7. Dr. Lester K. Cra-ier 



8. Dr. Williajii Schatten - Dr. Williain rlohler - Dr. Jesse Steinfeld. 
OCiiER IJI^SI^I'Q'.TORS 



9. PROJECT DESCRIPTION 

Objectives : 

It has been shox>ni, but not conclusivelyj th?t the imraune response 
to hoLiografted tissue may bo abrogated bj' riodifying the antigenicity 
of the donor tissue. 

Wo plan to use a tissue cvlt-or^ :p.ethL>d of modifying neonatal rabbit 
thyroid glands, ?nd then trans nl-^nting then to hjrpothyroid recipients. 

Methods employed : 

Thyroidectorry- is periorined on rabbits less than iiB hours old, and 
explants of these thjToids grown in media consisting of Eagle's nutrients 
and serum of the recipient rabbit. Thyrotropic hormone is placed in one- 
half of the cultures, and all of the culture have histologic section and 
I --^ uptake performed on a representative sample. 

After a' suitable lengtli of time in tissue culture, the explsnts are 
placed subcutaneously in the anterior abdominal x\rall of a rabbit which 
has been thjToidectomized two weeks previously. Thyrotrophic hormone is 
cdministered two dajrs prior to and ten days after the homografting, 

1.31 
I ■ uptakes will be determined over the graft bi-weekly for a three 

month period at which time grafts which are not functioning will be sacri- 
ficed. Functioning grafts will be left intact and I-'--^-'- uptake determined 
monthly for at least six more m.ontlis at x-^hich time histologic examination 
will be performed. 



Pi.GE 2 



PROJECT REPORT FORii (Cont'd) 



MCI-761i 



Methods emplo;^ed ; (Continued) 

It is .contenplated using the transparent chajnber for some of the 
transplants when function has been demonstrated '. Controls are liver 
treated in the same fashion. 

Major findings ; 

Presently we are working on technics for ,>ving TSH in vitro , for 
counting I'^-^-'- in vitro , and determining optirnui-;i tiiTiing for the condition- 
ing. Tliree gr'afts have been in recipients for two weeks. Two of these 
have demonstrated no function, the third- one has picked "up 6;^ of the I • , 
but the control transplnit of liver tissue has also picked up 6/j of the 

Significance to Cancer Research : • ■" 

In the treatment of cancer of endocrine glands it is often necessary 
to remove the entire gland. This necessitates that the patient continue 
taking substitution drug treatment for the remainder of life. If a method 
of producing a functioning homologous graft could be developed, possibly 
using tissue cultured grr'fts, considerable benefit to clinical management 
would be obtained. 

Proposed course of project ; 

It is planned to- continue th^^ experiment as outlin>^d above. If 
it can be shown that these grafts grow r-nd function, one m.ight consider 
the possibility of attempting the sam^ procedure in humans. It is 
doubtfxil if during the present year, progress on this experiment will 
develop to the point that would allow such a study. 



11. 



BUDGET ACTIVIiT: 



RESE'.RCH (V ;.dministr,;tion n 

REVIEla' 2t ..PPR0V..L £7 TECHNICAL ASS1ST..NCE /7 



MO ENTRIES FOR ITEMS 12, 1.3, H-l, l5 & l6. 



PROJECT REPORT FORM 



1» National Cancer Institute 2, Surgery Branch 



INSTITUTE LABORATORY OR BRANCH 



A. _ 5. NC 1-765 



SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NC . "" 

&. Effect of Carcinogenic Agents using a Bronchial.. Pouch Preparation in tho t r 
PROJECT TITLE 



Dr. John H. Waite 
PRINCIPAL INVESTIGATOR(S) 



Dr. W, C. Huepsr and Dr. William Banfield 



OTHER INVESTIGATORS 

?. PROJECT DESCRIPTION 
Objectives.: 

(1) To introduce and utilize the blind pouch technique into broncho- 
genic carcinoma carcinogenesis study. Results may be added to current 
evidence concerning the etiology of human lung cancer. 

(2) To provide a satisfactory large animal tumor for treatment 
studies. 

M ethods Employed ; 

As performed at present the pouch is fashioned from the left lowe-^- 
lobar bronchus. A left lower lobectomy is first performed, amputating- 
the bronchus more distal than usual. The lower lobe bronchus is then 
re-amputated at a higher level, severing the cartilagenous ring and endo- 
bronchial structures, but leaving the peribronchial structures intact as 
a vascular pedicle. The left main bronchus is sutured as are both ends 
of the pouch, forming in effect a pseudocyst. The end of a fine poly- 
ethylene catheter is implanted into the lumen of this pouch, and the othe 
end of the catheter is exteriorized through the chest wall where it is 
buried beneath the skin, available for injection purposes. 

Suspected carcinogenic agents will be injected into the bronchial 
pouch via the polyethylene catheter at regular intervals. Suitable coritr:'.- 
where the v«hicle only is injected, will be included. Serial roentgen- 
ograms will De taken to detect and follow the progress of any left hilar 
radiodensity which may develop. The animals will be followed for four- t-: 
five years, after which time they will be sacrificed. It is proposed to 



PAGE 2 
SERIAL NO. 

Methods Employed ; (Continued) 

study the following agents initially: 3,4 benzpyrene, tobacco tar, and 
internal combustion engine eschaust concentrate. 

If sufficient animals and facilities are available, rates of uptake isf 
radio-labelled samples of these compounds may be studied. Further e^cperijiier . . 
will be conducted to attempt production of a bronchial, pouch or bronchostomc 
continuous with the skin surface, for carcinogenesis studies. 

Ma.ior Findings; ■ 

In a pilot group of 3 dogs that were studied for technique development 
only, all three dogs survived the procedure and in all three a satisfactory 
pouch preparation was found at sacrifice two weeks later. Pathologically, 
the pouch appeared to be identical to a pseudocyst lined by healthy bronchial 
mucosa and filled with a non-infected gelatinous material. 

S ignificance to Cancer Research ; ■•■•■.■;:-•-■.,,:, 

To date the majority of evidence purporting to bear on the genesis cf 
human bronchogenic carcinoma is adduced from the ability- of .a suspected 
carcinogenic agent to produce a tumor when painted on the skin of ' a~. rabbj,t . 
or mouse . In interpreting the results of these experiments , not only must • 
the heterogenecity of the e3q)erimental animal with respect to humans be co'f^ 
sidered, but dissimilarity between epidermis and bronchial epithelium plays 
an important role. Exposing the experimental animal to an atmosphere ladon; 
with the suspected carcinogenic agent has almost always been unsuccessfur: 
in producing tumors. This has lead to the conclusion by some investigators? 
that the agents are not respiratory carcinogens and by others -that the 
animals respiratory defense (the cough, the dilution of the carcinogenic 
agent in respiratory secretions, and the action of cilia) are so f f ficier!'.. 
as to make exposure to the carcinogenic agent negligible using this tecliXLlqac 
There is much logic to support the second hypothesis, and a good method to 
provide contact with a strong concentration of the suspected agent over a prc= 
longed period of time and against which contact the animal may not have an 
effective defensive mechanism might be of some value in settling this point. 

We feel that a study on the uptake of radioactive benzpyrine from our 
pouch preparation alone might yield sufficient data to justify the project. 

11. BUDGET ACTIVITY; ^ '.. 

RESEARCH ^O ADMINISTRATION: /~~7 
REVIEW & APPROVAL A7 TECHNICAL ASSISTANCE /^/ 



(No Entries for Items 12, 13, 15 & 16.) 



PAGE 1 
PROJECT REPORT FORII 

1. T\Tacional Cancer Institute 2. Surgery Branch 

IMSTlTU'i'!^ WiaOiiVl'ORY OR BIlVJCH 



3. h. 5. MC 1-7 66(C ) 

SECTIO^J OR SERVICE lOOATIOW (IF oThER fmi] feEthESDA) SERL'.L NO. 



6. C crab ination Therapy of Esophageal Carcinoma. 
PROJECT TITLE " 

7. Dr. John H. Waite - Dr. J. Robert Andrews 



pRTNCmL i^;\/EtiTlL^iTuR(t.)' 
8. Dr. Andrew G. Morrow 



OTHER IlI^TESTiaiTORS 

9. PROJECT DESCRIPTION 
Objectives ; 

This project will attempt to develop a satisfactor;;'- method for 
treatment of squamous carcinom.a '-..f the esophagus. Iraprovement of ■ 
operability, resectability, and cure rate, will be sought by a pro- 
gram of preoperative rotational supervoltage radiation, A byproduct 
of the studj'- will be the availability of the recently irradiated esopha- 
gus for intensive pathologic study to evaluate the effects of rotational 
supervoltage on this tumor, B}/ use of tube feedings during the entire 
period of preoperative radiation an attempt will be made .to improve 
caloric intake and reduce the incidence of hemorrhage and perforation 
during the course of therapy. 

Methods employed ; 

(1) Complete workup 'to determine the extent of tumor. Patients with 
metastatic carcinoma or with. regional Invasion of a vital structxjre, 
such as heart or. lung, will not be suitable. 

(2) Insert plastic Levin tube at esophagoscopy to provide maintenance 
and improvement of nutrition. Consider insertion of a nuchal esopha- 
gostomy tube if patient develops hypopharyngeal irritation due to the 
indwelling Levin tube. 

(3) Radiation plan of study bj' Dr. Andrex^^s as coinvestigator. In 
general, he plans to give external two million volt therapy by rotation 
teclinique, beginning with low doses and increasing gradually to a total 
of 6,000 to 7,000 "r" axial dose over a period of six to seven weeks. 
If difficulty in achieving a satisfactory vertical width of port is 
encountered, the possibility of delivering supplemental gamma rays from 
an available intra-luminal source will be considered. The presence of 
the indwelling Levin tube would be ideal for this technique. 



PROJECT REPORT FORM (Cont'd) P^'^^E 2 



NC 1-7. 66(C). 

SERL\L No. 



Methods employed ; (Continued) 

ii. Surgery (with Dr. Morrow) about four weeks after completion of 
radiation. If resectable, total esophagectomy with dissection of the ^ ■ 
regional nodes will be performed with iiranediate or delayed reconstruction 
of gastrointestinal continuity. 

Evaluation criteria will include (l) changes in tumor size by serial 
roentgenography during radiation, (2) extent of radiation. reaction- and 
tumor fixation at the time of surgery, (3) extent of twaor and lymph node^ 
metastases by pathological examination of surgical speciiaen, and {h) clini- 
cal course oa patient after treatment. 

Patient Material ; ■ " ' 

Patients studied during the calendar year 1955 are reported by 
Dr. R. R, Smith under Project No. 751(C), "Evaluation of Radical Surgery 
and Development of New Surgical Techniques as a Therapeutic Method of 
Palliating or Definitive Therapy of Advanced Cancer." This included the 
initial 3 patients studied, for an average of Ul hospital days and one 
outpatient visit each. ... 

Major findings ; 

Of the 3 patients studied,, 1 patient was unsuitable for the program 
because of extreme senile debility, and 2 p?tients were suitable. These 
patients both satisfactorily completed a course of rotational 2-raillion 
volt therapy, and came to surgery one month after completion of therapy. 
Maintenance of alimentation by tube feedings was very successful in both 
patients and in itself maj prove to be a significant contribution to the 
treatment of this lesion. Of the 2 patients brought to surgery, one proved 
incurable by virtue of abdominal metastases found at the time of prelimi*- 
nary laparotomy. 

An esophagectomy was performed in one patient. Resection of the lesion 
was not hindered in this one instance by preliminarjr radiation, on the 
contrarjr it is felt that the prelirainrry radiation probably improved the 
resectr^bility of the lesion which wps in the region of ti:-e r;rch of the 
aorta. This patient expired postoperatively due to a culmination of tempo- 
rary hemorrhagic shock during surgery, poor cardiac action, nnd pulmonary 
edema postoperatively. An operative mortality of about l5^ is expected 
for this tjTpe of procedure but will be acceptable because of the certain 
early fatality in esophageal carcinoma treated by current methods. 



PROJSCT REPCRT FORM (Cont'd) R'.GE 3 



MCI-766(C) 
SERIAL rIO. 



PROJECT DESCRIPTION (Continued) 

Significance to Cancer Research ; 

Carcinoma o^f the esophagus is the fifth highest in frequency among 
internal cancers in ndiilt males. This frequency and the poor results 
available from present methods of treatment make it an ideal subject for 
cancer research. Adequate siirgery and adequate radiation each fails in 
its own way to control more than 2% of the entire group of patients 
presenting themselves with esophageal carcinom.a. The chief advantage 
of supervoltage rotational therapy x-ray is appearing to be better immediate 
palliation, although the incidence of eventual recurrence and death probably 
will not be much lower. Combination of surgery and supervoltage rotational 
therapy is being tried at several centers^ utilizing surgery as the pri- 
mary treatment and roentgen therapjr as a "mop up" treatmt^nt. For the 
reasons already enumerated, the principal investigator feels that the 
initial use of radiation may provide superior results. 

Pr oposed course of project ; 

Sec "Methods employed" above. The availability of material will 
make this a slowly developing project.. 

11. _^ 

BUDGET ;.CTlVriY: 

RESE..RCH fTJ -:.DimiISTK-.TION [J 

REVL.V^' k EPPRO'vV.L /T" TECHMCAL ;.SSIST..MCE /~7 

NO ENTRIES FOR ITEI'IS 12, 13, lU, l5 ?c 16. 









1 . Ifc^-— =- ^=--=~ 



".^.il ^'---.---zi isscoisted with 



5. :ri-- 









.-■C Z^T'. --■ 






.^agec"^ "x i=_e per- 






PnDJECT REPORT FORII (Cont'd) 
NCI-767(C) P^^^ 2 

SERIAL NO. 



9. PROJECT DESCRIPTION (Continued) 

Method s, emp loyed ; 

Temperatures of the patient from various- body suxfaces v;ill be 
recorded simultaneously with a multi-channel thermocouple which will 
also I'ecord the prevailing room temperature. Various physiologic 
determinations on the patient will be obtained using standard methods, 

Patient _ Ija terj-.al ; " 

liaterial for this project \irill be obtaiiied during operations, on 
patients ad-nitted for other projects. 

Ha .lor Findings t 

None. Not started yet, ■ r ■ 

Siftmfican ce to Cancer Res earch; 



The information to be derived, from this study will be used to help 
render major surgical procedures for cancer more successful. 

Propose d Coijrse pf^Pro;ie_ct; 

During the coming year many of the patients undergoing major surgery 
for cancer will be studied diuring their" surgical proced-ures. Constant 
body temperatures and other physiologic data will be determined to be 
correlated with the procedure being done," the room temperature and 
htmiidity, and the other contributing factors. ■ ■ ■ 



PROJECT REPORT FORM (Cont'd) 

PAGE 3 



10. _2Mcl 



SERIAL NO. 
11, _, .„ 



BUDGET AGT.IVJTY: 

RESEARCH ^ ADFETISTRATION [J 

REVIEW & APPROVE! L [J TF.CIMICAL ASSISTANCE O 

12. N N_E .^ . _-_____^ .___ 



C00PERATI:mG units of riC PIjTELIG IjEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT. 



13, N N r 



IF THIS PROJECT PEEIQIES, COliPLEIENTS, OR PARALLELS PvESEARCH DONE ELSEl-fflERE 
IN THE PUPLin HEALTH SERVICE (WITHDUT INTERCHANGE OF PERSONNEL, FACILITIES OR 
FUNDS), IDENTITY S'JGH RESEARCH. 



15. NONE 



PUBLICATIONS OTHER THAN ABSTPulCTS FRDII THIS PROJECT DURING CALENDAR YEAR 1955 



16, N ONE 



HDNDRS AJ^E) AWATxDS TO PERSOMEL REL/ITING TO THIS PHDJECT DURING CALENDAR 
YEAR 1955. 



p:iOJECT REPORT FORM ■ PAGIl 1 
1. National Cancer Institute 2, Siirgery Branch 



fiTSTlruTfi LlBOiIvtcry or branch 



3. U. " 5. yci-7 63 



6. Studv of the Role of the Pronerdin System in Natural IiTiinunit3?- 

PROJECT 'knam ' 



7. Dr. TJiiiiain E. Schatten 
PRINCIPAL ll<i\i^^TiCiU'ali(S) 



Dr. Lester I'l. Cramer - Dr. Horace Herbsnan - Dr. Robert R. Smith 
OTHEIR lN\/hS'i'iaii'i'okS ' ' 



9. PROJECT DESCRIPTION 

Objectives ; • , ■ ' 

To evaluate the effect of altering the serum properdin level in an 
attempt to abrogate the immune mechanism that plays a role in the neutrali- 
zation of viruses and in the prevention of succescful homo- and hetero- 
transplantation of tumor tissue. 

Methods employed : 

One stud" that would indicate whether or not zjrmosan, a carbohydrate 
that combines with the serum globulinj properdin, is effective in alter- 
ing host resistance would be to ascertain whether or not z;;Tnos3n is effect- 
ive in the heterotransplantation of tumor tissue. If zjTdosan were effective, 
it would strongly indicate that properdin pla3''s a role in the natural resist- 
ance of host anuials, since zymosan has a specific action on properdin. 
This study will be carried out by adrainistering zymossa intravenously to 
rats in different doses at different rntervals of time prior to and aftar- 
inoculation of rats with tujnors. The S-91 melanoma of mice will be used 
for heterotransplantation in this study of the effects of altering properdin 
levels in rats. If it is proved that z^nnosan is effective in successful 
heterotransplantation, then transplantation of tumors from patients to rats 
will be studied, Tuinors will be transplanted ijito groups of rats tliat have 
been pre-treated with zymosan .-'nd with zjrmosan and cortisone. Tumors that 
survive transplantation and grov; will be periodically transplanted to 
other rats, some pre-treated, and others not treated. This will be ?n 
attempt to adapt a transplantable tumor strain. Tumor groirrth will be mea- 
sured directly following subcutaneous inoculation and b weight of the 
tumor following ijitraperitoneal inoculation. 



PROJECT REPORT FORM (Cont't) PAGE 2 



HC 1-768 
SERML NO. 

Methods employed ; (Continued) 

If heterotransplantation of tumors can be effected successfully, the 
oncolytic effects of viruses on tumors will be studied by injection of 
viruses into the tumors. .Following vjxus injection, siilocutaneous tumors, 
will be measured and, biopsies. -taken periodically for histologicsl studies. 
The effects of different viruses, on a tumor, as well ?s the role of zymo- 
san, cortisone, and other agents in enhancing virus effects will be studied. 
Also, this would provide a method of increasing' the virulence of a virus 
by passing the virus thru a patient's tumor to "train" it against' the 
tumor before adrninstering the virus to a patient. 

Major findings; ' , . 



This project is in its preliminary phase. However, it is believed 
that there was a definite difference in the size of heterotransplanted 
tumors in rats that were treated with zymosan prior to tumor inocula- 
tion when compared to. rats, .that were- not treated. \ 

Significance to Cancer Research ; 

If huma-n tumors could be heterotransplanted into other animals 
successfully, this would provide a method for studying individual turiiors 
of patients in laboratory'- animals. This would be a method of testing 
many chcmotherapeutic agents in laboratory animals. As mentioned above, 
this would also provide a method, for the evaluation of the oncolytic 
effects of viruses on tumors snd perhaps this would be a method of con- 
ditioning a virus to become more virulent with regards to its effect on 
a particular tumor. If zjmiosan were effective in altering host resist- 
ance so that a tumor could be transplanted, it might also be used to 
alter the immune mechanism that prevents continued propagation of a virus 
following its administration. This antibody response following virus 
administration is one of the major probltns that now presents itself in ■ 
the- virus treatment of carcinoma.. 

Proposed course of project : . . 

Heterotransplantation of tumors will be performed in a sufficient 
number of rats that are pre-treated with zymosan and with zymosan and 
cortisont to determine the effectiveness ,. of this treatment in hetero- 
transplantation. If •z-ymos'.'n does promote successful heterotransplanta- 
tion, then further studies wilt be carried out as outlined above. 



PiiOJECT REPORT F0RI4 (Cont'd) PAGE 3 



10. NCI- 76 3 

SERilL iv'O. 



11. 



Budget acthity: 

RESEARC:i /x7 ADMINISTR^ITION /T* 

RE/IEiJ 1 A??R0VA.L,/7 TECHNICAL ASSISTANCE /7 



NO ENTRIES FOR ITEJ4S 12, 13, lli, 13' ?: 16. 



PROJECT REPORT FOM PAGE 1 



1. N^.tional Cancer Instit ute 2. Surgerj;- Branch 

liSTITUTE LiillftlTORY OR iTUvAQU 



3. it. 5. MCI-770 

ggc T ION OR 5^\llC& LOCATTO?! (1^' CTH^R T'^J' br'j^HESDA ) SERI/.L NC. 

Study of the Effect of Growth Hormone on Cprtilage 'Transplants and 
6. of the Sxxrvival and Gro wth of These Transplants 

projj,ct title ' ' 



7. Dr. William 2. Schatten 



8. Dr. Lester '.' . Cramer - Dr. Horace Herbsman - Dr. Delbert I'l. Ber^^enstal 
OTHER ItTVESTlG.\TORS 



9, PROJECT DESCRIPTION 

Objectives : 

To develop a method of stirirolation of growth of cartilage transplants 
and to stud"- the survival ard rrox^h of homotransplants and heterotrans- 
plants of cartilage. 

Ile'thods employed ; 

Hypopbysectomized rats are used iri this study because they are sensi- 
tive to small amounts of growth hormone and becc?use non-treated hypophy- 
sectomized animals are oetter controls than normal rats in this experiment. 
Three groups of rats are being studied. Autografts of xiphoid and costal 
cartilages are transplanted in one :;,roup, homografts in another, and 
heterografts from rabbits in the third group. The cartilage grafts are 
transplanted into the axilla and abdominal wall. Rats in each group are 
sacrificed at weekly intervals. On the second and third days prior to 
sacrifice, S-35 is injected intraperitoneally. After the animals are 
sacrificed the cartilage grafts and the aniraal's own costal and tibial 
cartilages are removed f r stLidj"". The prima r-y method of studying acti- 
vity of the cartilage is by deternining the S-35 uptake of the' cartilage 
as expressed in counts per 100 mg. of dry cartilage per minute. The 
transplants are com.pared with animal cartilage that was not transplanted 
and also with the transplants of p control animal that did not receive 
growtli horm.one. The growth of cartilage transplants is also evaluated 
by means of comparisons of measurements and wet weights of the cartilages 
taken at the time of transolantation and at the tiime of sacrifice. 



PROJECT RjiPORT FORM (Cont'd) KiGJi 2 



NCI-770 



9. PROJECT DESCRIPTIOW (Continued) 
Major findings ; 

The project is in oh? preliminary phasa, 1% is believed that the 
data that has been accimulated up until the present time shows that 
autografts of cartilage can be stimulated by gr^/'th hor'none. Homo- 
grafts and hetfcr.graf-c:: of cartilage can be stitnUlat^u to a lesser extent 
by growth hormone. In addition, het.erografts of cartridge inL anim-^ls 
treated with growth honnone do not take up as nuch J-3$ as hett-rografts 
in non-trur.ted hyp jphysector.iA.:ad anmals. This finding is, of course, 
of great interest and is bcir^- more fully investigated, 

Signif icanr'e to Cancer Research ; 

It is go'-.^rill" csrecd that honologous cartilage grafts surx'ive 
transplantaxj-on ior vaxying periods of time. The exact reasons that 
cartilage grafts are not affected oj an immune response as are alinost 
all other tissues, is not known. It is also not known, how well homo- 
grafts of cartilage do survive. It is difficult to perform man;'- studies 
that might give this answer bocause of the low mutabolic activit;^ of 
cartilage. It is believed that the uptake of S-35 by a cartilage graft 
will be a critical test of "letabolic survival. It has been shoi-m that, 
after dialysis of cartll-^ge rc' [j.8 hours following removal of a speci- 
men from an anm'.l tha-i; h.s received S-35, 9^ of the sulfur is bound 
in ■ n organic couipound. Srlp>^'^.te is takc;n up by cai-tilage cells where 
it is probably attach.-.d to the mucopolysaccharide molocule and thereafter 
appears in the ground substance. It is hoped that this study will indi- 
cate some of the reasons that honografts of cartilage svirvive transplanta- 
tion. 

It is also the purpc-se of this experiment to determine if cartilage 
grafts, including autografts, homografts and heturografts, can be stimu- 
lated by growth hormone following transplantation. If this could be 
achieved, it would have clinical application in various reconstructive 
procedures in which cartilage grafts are used. 

Proposed course of project ; 

Three groups of hypophysectomized rats will be used for studying 
autografts, homografts, and hettrografts of cartilage. There will be iiO 
rats in each group, 20 servijig as controls, and the other 20 reciving 
growth hormone following transplantation. Following transplantation 
of all ijO ratsj 10 rats, 5 cuntrols and 5 treated, will be sacrificed each 
week for k weeks. S-35 will be given to the rats prior to sacrifice as 
outlined above. Growth of the transplants will be studied as outlined 
above. If autografts can be stimiilated to grow by the use of growth 
hormone in hypophysectomized rats, normal rats and other anixiial species 
will then be used in an attempt to sec if growth hormone will stimulate 
the growth of cartilage transplants in those animals. Also, cartilage 



PROJECT RiLPCRT FOWi (Cont'd) PAGE .3 



NCI-770 



Proposed courso of project ; (Continued) 

transplants will be performed and period of time allowed to lapse prior 
to adjiiinistration of growth hormone. This will be done in a separate 
group of aniraals. 

The fact that heterografts of cartilage have ben found to take up 
less S-35 in hyp ophyse eternized animals that receive growth hon-aone than 
in h^'-pophysectomized animals that are not treated indicates that hj'po- 
physectomized aniraals may not be able to react to foreign tissue with 
an immune response. If this finding that Wc?.s present in the first few 
experiments that were done is corroborated by our further exper jjiients 
it would be indicated to heteroti-'ansplr'nt tumors to hypophysectomized 
rats in an attempt to sec if the tumors would not be affected hy host 
resistance and would therefore progress at their own rate of growth. 
If this were true, then such turners could be passed one or more times 
in hypophysectomized rats and t!ie tujnors night become adapted to the 
new hosts. They might then be transplanted to normal rats and the tumor 
developed as a transplantable tumor. 



11. 

BUDGET :XTI'/rrY: 



RESK\RCH /x7 ADMINISTRATION £7 

REVIEW k APPROVAL /T" TECHNICAL ASSISTANCE /~y 



NO ENTRIES FOR ITEIIS 12, 13, lii, 1$ ?c l6. 



PROJECT REPORT F0RI4 PAGE 1 



1. Nrtional Cancer Institute 2. Surgery Branch 

INSTITITI'E LVBOliLTORV Uk 'dk'MA 



3. h. 5. NC 1-771 

SfeCTlor OR SERVICE LOCATlOiJ (IF OTl-i^R TiUW BETH3SDA) sERi'.L NO. 



6. Experimental Reconstruction of the Extra-hepatic Biliary S3^stera 
PROJECT TITLE 



7. Dr. IJilliam E. Schatten 

Pki!101P.'.L i*JVESTiti.TcR(SJ 



8. Dr. Lester 11. Cramer - Dr. Horace Herbsman - Dr. Robert R. Smith 
dTHER IMVESTICATORS : ' [ — ' 



9. PROJECT DESCRIPTION " 

Objectives ; 

To develop a successful method of reprir of commoii bile duct defects. 

Methods employed ; 

Adult mongrel dogs are selected as experimental animals. A chole- 
cystectomy and ligation of the comi-aon bile duct is performed in ordtr. 
to closely simulate conditions encountered, clinically. Tiiree daj^s 
following this operation, a second operation is performed. At that time, 
a split-thickness .graft of skin is taken from the -bdominal wall. The 
common duct region is exposed and a portion of comiiion duct proximal and 
distal to the area of previous ligation is excised in order to create a 
defect that is at least 2| cm, in length. The skin graft is then used 
to form a tube over a rubber T-tube, and the T-tube is then introduced 
into both ends of the common duct and an accurate anastomosis between 
the comm.on duct and skin graft is performed. After the graft is sutured 
in place, a tab of omentum is placed in the region of the graft. The 
.end of the T-tube is tied off, brought out through a stab wound in the 
right upper qi:ia.drant, and then placed in a subcutaneous pocket in the 
musculature of the right upper quadrant of the abdomen. The skin graft 
is constructed into a tube in the direction of Langer's lines in one-half 
of the dogs and is constructed so that the long axis runs perpendicular 
to Langer's lines in the remainder of the dogs. The T-tube will be 
allowed to remain in plrcc for tliree months in one-half of the dogs and 
for six months in the remainder. Thu dogs will be observed for three 
to six: months following removal of the T-tubes in order to observe them 
for evidences of bile duct stricture formation. The dogs will then be 
sacrificed and gross and histological studies of the coMnon ducts will 
be performed at the time of autopsy. 



PROJECT RiiPORT FOffli (Cont'd) R/iGE 2 



NCI-771 
SER1/.L NO. 



9. PROJECT DESCRIPTION (Continued) 

Major findings ; , . ' 

At the present time the above procedure h?.s been carried out success- 
fully in ik dogs. The procedure has been performed in three other dogs 
and these do-js have died at varying interv^.ls postoperatively due, to' 
leakage of bile- in the region of the' graft. Technical points have been 
learned from the loss of these three dogs and none. of the last nine dogs 
has been lost because of bile leakage. 

Significance to Cancer Research ; 

The repair of common bile duct defects is one of the most difficult 
problems in surgery today. This is i^videnced by the multitude of experi- 
mental and operative procedures that havJ been employed. There is general 
agreement that it is advantage. ous to preserve the sphincter of Oddi in 
reconstruction of the biliary tract. For this reason it -would be more 
preferable to repair common bile ducts by means of a successful. tr.':'ns- 
plantation of tissue to bridge existing defects than to effect repair 
by means of Roux-en-Y procedures tiiat are advocrted today. The proposed 
method of reconstruction of the common bile duct will alloinr preservation 
of the spincter of Oddi and therefore will be of valuu as a now surgical 
technique. If successf iJ., this method will be recommended to surgeons 
for clinical application. 

Proposed co-urse of project : 

This m.ethod of common bile duct reconstruction will be performed 
in a number of dogs so that a total of 20 will have survived the procedure 
and cm then be observed for evidences of stricture formation over the 
proposed period of tine. 



11. 

BUDGET ACTIVITY: 



RESiL.\RCH' /T/ ADiIIi\IISlTR;iTIOM £7 

REVIEM & APPROVAL 7^ TECHNICAL ASSISTANCE /~7 



NO ENTRIES FOR ITEl"iS 12, 1.3, lU, l5 'i 16. 



PAGE 1 
PROJECT REPORT FORl^l 

1. N. C. I. _^ 2, Endocrinology Branch 



INSTITUTE LABORATORY OR BRANCH 

3, Endocrinology Service 4-. 5 . NCI-800C 

SECTION OR SERVICE LOCATION (IF OTIiER THAN BETHESDA) SERIAL NO. 

6, Endocrine Aspects of the Progression and Therapy of Cancer of the 

PROJECT TITLE 

Breast in Women and Men 



7. Roy Hertz, Delbert !:. Bergenstal. and M. C. Li 
PRINCIPAL It'TVESTIGATCR(S) 



8, James A. Pittman. Harold Altman (M. G. Sherer and A, Breslow up to 

OTHER INVESTIGATORS 7/1/55) 

9. PROJECT DESCRIPTION - • 

Objective s ; 

1, To extend and intensify detailed clinical observation of the 
natural course of cancer of the breast in men and women in order to 
better appraise the effectiveness of various methods of therapy and 
learn more concerning the etiology and pathogenesis of the disease. 

2, To improve upon existing forms of hormonal therapy for pallia- 
tion of advanced breast cancer and to elucidate the mechanisms involved. 

I'fethods employed ; 

1. The acceptance of complete clinical responsibility for the 
further management of referred patients with proven diagnosis of 
cancer of the breast and the application to their problems of all 
available modes of therapy, 

2. The exploration of new modes of hormonal therapy for palliation. 

3. The detailed analysis of endocrine and metabolic factors operat- 
ing in relation to the genesis and course of the disease in these pa- 
tients up to the tiiri.e of their demise. 

A^ Careful appraisal of pathological specijnens obtained at biopsy 
or autopsy in collaboration with Poithology staff. 

Patient material ; 

No. Average Stay Days 
Admissions: Adult females 4-3 43 

Outpatient: "Tumber of patients LA 
^^umber of visits 272 



PAGE 2 
PROJECT REPOP.T FOmi (Cont'd) 



NCI-.800(C) 

SmiAL NO. 



m^oT findings - 12/l/5^-12/l/55 ; 

.1. The momber of ...cases of breast cancer which have come under our 
observation now eqioals 213, These patients have been studied with 
varying degrees of intensity but they all have contributed to the 
general clinical background and teaching functions of the group. This 
activity continues to provide constant liaison vxith local physicians 
and medical institutions for additional patient referral to the 
Clinical Center. 

2, The necessary clinical precautions to be taken in the adminis- 
tration of massive parenteral estrogen therapy in patients with breast 
cancer and now rather completely determined, thus making this form of 
therapy an entirely feasible procedure in this and other clinics. In 
the face of the hi^ily variable clinical conditions present in patients 
with advanced breast cancer, it seems increasingly unfeasible to . 
attempt to determine definitively whether this form of therapy presents 
any distinct clinical advantages over more conservative forms of es- 
trogenization. Nevertheless, our own clinical impression at this time 
is that in certain selected cases massive parenteral estrogen adminis- 
tration presents certain advantages for prompt and intensive hormonal 
therapy. 

3, Additional data on estrogen withdrawal bleeding in elderly 
patients with advanced breast cancer indicate an incidence of appro- 
ximately 15 percent. These obser^/ations confirm the marked difference 
in endometrial response in senile women from the uniformly positive 
response seen in women under 4-0 years of age. This difference in 
response suggests that an additional factor other than estrogen is 
essential for the activation of the hujuan endometrium, 

4-. The failure to encounter a single case of endoro.etrial carcinoma 
among all of our breast cancer patients treated with prolonged and 
intensive estrogen confirms the lovj level of carcinogenicity which 
estrogens have for the endometrium in patients over 4-0, 

5, In-".tial studios on the use of massive intravenous androgen 
therapj- hf.ve proven discouraging. Local and systemic tolerance is 
not gor.d and lie have failed to detect any material blood level of i 
circulating androgen by bioassay even after as much as 750 mgm-, tes- 
tosterone propionate given intravenously. Moreover, the mechanical 
problem of rendering such androgen solutions free of particulate 
material has thus far proven insurmountable. Accordingly, our 
clinical efforts in the development of massive androgen therapy in 
breast cancer have been momentarily suspended. 

6, Techniques for complete balance studies of breast cancer 
patients have been developed. These patients can now be followed 
with respect to balance of sodium, potassium, calcium, nitrogen. 



MCI-500(C) 

SERIAL MO. 



PAGE 3 
PROJECT REPORT F0RI4 (Cont'd) 



Ma.jor findings (cont'd.) 

phosphorous and creatinine. 

These techniques have thus far permitted a -coaplste characteriza- 
tion of the metabolic action of the newer corticoids, suggesting a 
greater field of usefulness for these compounds in the practical 
palliation of breast cancer patients. One of the more immediately 
demonstrable effects is a rapid correction of hypercalcemia, a fre- 
quently fats-l complication in patients with breast cancer with ex- 
tensive osseous metastases, 

7. The aforementioned balance techniques have provided more re- 
liable quantitative data on the B-complex balance in patients under- 
going protein catabolic effects of exogenous corticoids. Although 
excretion of biotin, riboflavin, pyridoxin and folacin is unaltered 
during corticoid-induced catabolism there is a distinct and repro- 
duceable loss of pantothenate. Thus, the participation of this 
dietary trace factor In the steroid catabolic effect is suggested, - 
In addition, these data extend earlier observations indicating a 
retention of pantothenate during androgen induced anabolic states. 

8. Cytological studies of the vaginal smears of patients treated 
with estrogen, androgen, corticoids and progesterone have been con- 
tinued. No evidence of metaplastic changes have been noted, 

9. A new steroid, 17 ethyl 19 nor testosterone which is knoi-m 
to be highly anabolic has been p^.rtially evaluated for its relative 

' androgenicity in a series of six breast cancer patients. The compound 
thus far seems to present no marked advantage over other androgens 
available, 

10, A study of the effects of potent pituitary follicle-stimulating 
hormone preparations on the himian ovary has been conducted in six 
patients Just prior to surgical o-variectomy for palliation of advanced 
breast cancer. Marked follicular stimulation was observed in 4 cases 
and no response in 2. No detectable rise in serum or urinary estrogen 
accompanied these effects and no detectable estrogen was found in the 
ovai'r-ian vein blood obtained at the time of ova.riectomy. Thus the 
tro;-ihic response of the human ovary is not accompaniad by. the readily 
detectable changes in blood and urinary steroid level seen when the 
human adrenal is activa.ted by ACTH. 

11, Attempts to modify adrenal cortical function -in 'breast cancer 
patients by Amphenone administration ho.ve led to a more compl:;te 
appreciation of the clinical toxicity of this compound and its con- 
sequent limitations as an Inliibitor of adrenal cortical function in 
man. Nevertheless, this information has proven usefiil in application 
of this agent to other endocrinological problems to be described under 
project 803, 



PAGE 4 
■PROJECT REPORT FORI'! (Cont'd) 
NCI>>^00(C) 
SERIAL NO. 



Slgnificange to cancer research ; 

The direct pertinence of the foregoing studies to the grave 
problem of advanced or persistent breast cancer is too obvious to 
require discussion. 

Proposed course of pro.i'ect ; 

These comprehensive clinical, endocrinological and metabolic 
studies in breast cancer patients are to be extended essentially 
along the lines already described above. A nev7 departure vjill be 
the evaluation of the place of hypophysectomy in the management of 
breast cancer, a study to be carried out in cooperation with the 
neurosiirgical staff of N,I.N,D.B. In addition, studies of the 
effect of radiothyroidectomy on the clinical course of breast cancer 
will be undertaken. 



11.. 



BUDGET ACTIVITY: 

RESEARCH [J ADl'IINISTRATION [J 

REVIEVJ & AFPPOVAL [J TECIffilGAL ASSISTAI'CS [J 
NO ENTRIES FOR ITEl^iS 12 & 13 



15.. 



PUBIJCATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR 
YEAR 1955: 

1, The effect of meticorten and meticortelone on thyroid 
function, by M, G, Sherer and B, N. Siefring (J. Clin, Endo- 
crinol. & Me tab, ) In press. 

2. Amphenone: Toxicity and effects on adrenal and thyroid 
function in man, by R. Hertz, J. Pittman and M. Graff (J, 
Clin. Endocrinol. & Me tab, ) In press. 



16.. 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1955: 

1, Dr. D, M, Bergenstal was invited to attend the International 
Rheumatology Congress, Rio de Janeiro, Brazil, August 1955. 

2, Dr. R, Hertz V7as invited to participate in the meetings of the 
Societe d'Endocrinologie d'Haiti, Port-au-'Prince , Haiti, Dec. 
1955 (unable to attend). 



PAGE 5 
PROJECT REPORT FOPJI (Cont'd.) 
NCI-800(C) 
SERIAL NO. 

16. HONORS AND AIJARDS (Cont'd.) 

3, Dr. R. Hertz is Invited to preside over forthconing session 
of the Endocrine Society, Jime 1956. 

A, Dr. R. Hertz was reappointed Chairman of Endocrinology Panel 
and Member of Executive Coromittee on Growth, National Research 
Council. 



PAGE 1 
PROJECT REPORT FORM 

1. Kf.C.I. 2. Endocrinology Branch 



INSTITUTE LABOPJITORY OR BPiillCH 

3, Endocrinology Service A. HGI-3Q1(C) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHSSDA) SERIAL NO. 

6. Endocrine Aspects of Cancer of the Prostato in M-^.n 

PROJECT TITLE 

7. Ro7A Hertz, Deroert M. Borgonstal. M. C, Li, and Lois F» Hallnan 

PRINCIPAL INVESTIGATOR (S) 

8. James A. Pittman and liarold Altman 

OTHER BTVESTIGATORS 

9. PROJECT DESCRIPTION - 

Objectives ; 1. To improve non-surgical therapy of cancer of the prostate 

2, To increase understanding of endocrine factors involved :' 
genesis and course of prostate cancer. 

3, To gain basic knowledge of the metabolic action of cer- 
tain steroids in man, 

ITethods employed ; Detailed clinical and metabolic observations of 

patients with advanced prostatic carcinoma who are 
no longer amenable to accepted forms of clinical management. 

Patient material ; 

No, Average Stay Days 
Admissions: Adult males 4 51 

Outpatient: I^Iumber of patients 2 
Number of visits 4- 

Ma.jor findings ; 1. The administration of chorionic gonadotropin to 3 
patients with prostatic carcinoma just prior to or- 
chiectomy has shovm that such gonadal activation even in the senile 
man will aggravate the disease as manifested by exacerbation of pain 
and elevation of acid phosphatase. The expected rise in urinary ex- 
cretion of biologically active estrogen was not detected. The spermatic 
vein blood obtained at the tim.e of the orchiectomy also contained no 
detectable androgen or estrogen. Thus, although it is apparent on 
histological and clinical grotmds that the senile human testis can be 
activated by chorionic gonadotropin, this trophic action on the testis 
is not reflected in detects^ble increments in steroidogenesis as is the 
case in the activation of the human adrenal by ACTH, 

2, Further studies of the mechanism of the elevation of 
serum acid phosphatase in prostatic cancer patients have yielded no new 
data of value. Thus we have failed in our attempts to transmit this 
activity to human fibroblasts in tissue culture. This was attempted 



PAGE 2 
PROJECT REPORT FORM (Cont'd) 
KC 1-801(0) 
SERIAL NO. 

I-ia.ior findings (cont'd.) - „.",.,.• 

on the theory that this enzymatic activity may possibly be associated 
■ v;ith a virus-like agent v;ho^se. titre rises in advanced . prostate cancer 
patients. Initial impressions that this elevation of enzymatic acti- 
vity represents the failure of an iriiibitory agent in normal serum 
have failed of confirmation. 

Significance to cancer research ; The high frequency of prostatic car- 

.,- -. cinoma in man and. initial inroads 
upon it by hormonal therapy indicate the pertinence of this study to 
cancer research. 

Proposed course of pro.i'ect : Additional studies of the clinical and 

endocrinological factors involved in the 
extension and growth of advanced prostatic cancer will be undertaken. 
I'lajor emphs.sis will be placed upon the evaluation of several atypical 
estrogenic compounds and corticoidal steroids in the therapy of pros- 
tatic carcinoma. 

11 . : 

BLTDGET ACTIVITY: 

RESEARCH . U ADi-IEICESTEiATION £J 

REVIEI7 & APPROVAL /^ 1201211011. ASSISTANCE /7 



NO ENTRIES FOR ITE^S 12, 13, 15 & 16. 



PAGE 1 
PROJECT REPORT FORM 

1. N. C. I. 2. Endocrinology Branch - 



INSTITUTE LABOFt/iTORY OR BRANCH 

3. Endocrinology Service K. , 5. NCI-.8Q2(C 

SECTION OR SERVICE LOCATION (IF OTHJl THAN BETHESDA) SERI/iL NO. 

6 . Preoperative Hormonal Therapy of Cancer of Cervix 

PROJECT TITLE 

7. Roy Hertz, John H. Walte, and Robert Smith 

PRINCIPAL IWESTIGATOR(S) 

8. James A, Plttman and Harold Altman 

OTHER INVESTIG/\TORS 

9. PROJECT DESCRIPTION ' 

Ob.jectlves ! 

To determine the value of steroid therapy as a palliative measure 
in advanced cancer of the cervix. 

Methods employed ; 

Patients who are considered amenable to definitive surgical therapy 
are pre-treated for 4- to 6 weeks with massive doses of various steroid 
hormones singly and in combination and then are operated upon. Their 
progress is evaluated by frequent pelvic exaralnatlon, serial biopsies 
and smears and by histopathological study of the surgical specimens 
removed. Pyelograms ajid barium enema ta are also employed to eva.luate 
changes in the pelvic viscera. The amount of vo.ginal bleeding is ■ 
checked from day to day. The patient's general clinical status v/ith 
respect to v;elght, appetite, blood status, and pain are also closely 
observed. 

Patient material ; 

No. Average Stay Days 
Admissions: Adult females 3 10 

Outpatient; Number of patients 3 
Number of visits 12 

Ma.jor findings ; 

No extension of our findings beyond last year's report has been 
made due to the justified preocupation of our stirgical staff with 
the virus approach. 



NCI-g02(C) 

SERi;.L NO. 



PAGE 2 
PROJECT REPORT FORM (Cont'd.) 



Significanco to cancer resoaroh ; 

Palliation of advanced corvical carcinomas urgently needed in 
view of the low over-all 5 year survival rate of patients with this 
disease {UO-^Ofo), ... 

Proposed course of pro.ject ; 

The further course of these studies will depend upon the interests 
of the surgical staff Xirhich is at the moment justifiably concentrated 
on the virus approach to cervical cancer, • • 



11. 



BUDGET ACTIVITY: 

RESEARCH ^J ADMNISTR/.TION ■ /^ 

REVIEt'J & APPROVAL /7 T3CMICAL ASSISTANCE . U 



12. 



COOPJ?a"-Tr:G TIIIITS OF TH3 PUBLIC HE/.LTH SERVICE, OR OTHER ORGANIZ/.TIONS , 
P^,OVIDirG FU^IDS, FACILITIES,. OR PERSOMEL FOR THIS PROJECT IN EITHER 
1956 or 1957: 

Surgery Branch, NCI - Dr. R. Sraith and staff. 



NO ENTRIES FOR ITEFS 13, 15 & 16. 



PAGE 1 

PROJECT REPORT FOPuM 

1. N. C. I. 2. Endocrinology Branch 

INSTITUTE L/iBOPJ.TORY OR BRA-NCH 

3. Endocrinolo^ Service /^. 5. NCI-803(C ) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. The Nature of Hormone -Producing Tumors of Pltultar:;-, Adrenal, Ovary. 

PROJECT TITLE 

Testis, Pancreas, Parathyroid and Chorion; Abnorma- 
lities of Somatic Development and Growth 

7. Roy Hertz, D. li, Bergenstal. M. G. LI. Sally B, Fand and li. M. Graff 

PRINCIPAL INVESTIGATORCS ) 

S . James A. Plttman, Harold /Jtman. I'. W. Tullner and Donald Spencer 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Ob.jectlves ; 

1. To learn more about the basic metabolic phenomena Involved 
in the process whereby neoplasms In hormone-producing organs produce 
highly excessive sjnounts of hormone, resulting in such syndromes as 
Gushing 's Disease, Acromegaly, Hyperparathyroidism, Hype radrenal ism, 
and slinilar Gndocrlnopathies. 

2. To maintain in tissue culture hormone-producing tissues and to 
ascertain the factors involved in quantitative hormone production in 
vitro, 

3. To assess the capacity of new drugs to alter the, course and 
hormonal activity of hormone-producing tumors. 

Methods employed ; ' . 

Hormone producing tiimors" from patients with one of the above-named 
syndromes are obtained at surgery and carried in tissue culture. The 
supernatant fluid from the cultures are tested for hormonal activity 
by appropriate bioassay methods and the quantity recovered compared 
with the content of a piece of tissue exactly equivalent to that ori- 
ginally placed in culture. 

Patients presenting such sj'ndrom-es are completely characterized 
endocrinologlcally and metabolically before and after therapy in order 
to ascertain the relative effects of \'arlous forras of therapy. 



NCI~803(G) 

SERIAL NO. 



PROJECT P^EPORT FORl-I (Cont'd). 



PAGE 2 



Patient matorial; 



Admissions! 



Outpatient: 





No, 


Average Sta 


y Days 


Adult males 


6 


11 




Adult females 


16 


33 




Children males 


k 


11 




Children female s .. 


k 


9 




Number of patients 


": :■ 72 






Number of visits 


2S9 







I-h.l'or findings ; - ..„.. ,i :,, • 

1. Two additional patients with proven chorio carcinoma have been 
■shown to- lack the histidinuria.seen, in norr.ial pregnancy, 

2. A patient v/ith proven choriocarcinoma has exhibited a marked 
suppression of urinary gonadotropin secretion while on ojaethopterin 
therapy. This indicates a specific interference with the nornr.\l 
metabolisn of this hormone-producing tumor, 

3. Attempts to transplant huiian hormone -producing tvimor tissue 
to cortisone-treated rats have failed in the case of 1 islet cell 
tum.gr, 2 adrenal adenorxata, 1 parathyroid' adenoma and 1 simple 
follicu3.ar cyst, 

Lr. An attempt to grow one islet cell adenoma in tissue culture 
has. failed and one additional islet cell tuxaor is surviving in tissue 
ciilture 2 weeks after oxplantation. Its further course and possible 
in vitro production of insulin remain to bo evaluated, 

5, Two adrenal carcinomr. patients have been studied directly by 
us and two in collaboration with outside- investigators at other hos- 
pitals. All 4- have shown a decisive drop in urinary corticoid ex- 
cretion under Amphenone administration. This demonstrates that 
Am.phenone will markedly depress the activity of this hormone-producing 
tumor. The duration of those studies was too limited by clinical 
circuristances to permit any evaluation of the effect of Am.phenone 

on the course of the m.alignancy, 

6, In one patient with advanced metastatic adonocarcinomxi of 
undetermined origin the administration of Germanin (Bayer 205) has 
led to an extensive necrosis of the adrenal cortex similar to that 
observed experimentally and in prior clinical studies in patients 
with pemphigus. This patient also showed clinical and biochom-ical 
evidence of adrena.l insufficiency 

7, lihdocrinological analysis has porrrdtted the differentiation 

of constitutional sexual precocity from that due to horm.one -producing 
hyperplasia or tumor in 5 additional children. 



PAGE 3 
- PROJECT PilPORT FORiM (Cont'd.) 
NCI~803(C) 
SSRL'J. NO. 

Ma..ior findings (cont'd.) 

8, Cytological nethods for the chronosonr.1 detorr-iinr.tion of the 
truG sonatic sex of patients with adrenal hyperplasia or abnomo.l 
gonadal development has facilitated the endocrinological evaluation 
of 10 problen cases, 

9. Further observn.tions in acronegaly have been seriously limited 
by paucity of case iiaterial. Nevertheless, in 2 instances appropriate 
specimens for growth hormone studies have been obtained, 

: 10, Conplete endocrinological stiid;'- of a patient with Lawronce- 
Moon-Biedl syndrone and of another in.th. Sjorgen's syndrone revealed 
no distinct endocrinopathic pattern in those cases, 

11, A new progestational conpound, 19-nor ethisterone previously 
found by us to be 5 tines as active as oral progesterone in rabbits 
and nonkeys has been assayed in 5 patients for oi^l progestational 
potency, Endonetrial biopsy showed that this conpound has the sane 
enhanced potency in the hur:ian as was shovm. oxperirientally. It is 
now available in anounts sufficient to pemit its trial in the therapy 
of breast cancer, , 

Significance to cancer research ; 

Further understanding of the conditions pernitting. the excessive 
horraonal production of tur.iors of endocrine origin is basic to otir 
understanding of the netabolisr'. of neoplastic tissue generally. In 
addition, further analysis of abnoma.lities in'sona.tic growth and 
developnent should provide data pertinent to the problens of tissue 
groTATth generally, 

■ . Proposed course of pro.ject ; 

Further elaboration of these studies with najor enphasis on bio- 
chenical and phamacological control of ho mono -producing tunors and 
anonalous tissue growth is projected. 



11. 



BUDGET ACTIVITY: 

RESEARCH /^ ADMNISTR/iTION /^ 

REVIEl'J & APPROVAL £J TECHNICAL ASSISTANCE £J 

12. 

COOPER/iTING mJLTS OF THE PUBLIC HEi'iLTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUl^JDS, FACILITIES, OR PERSONFEL FOR TlilS PROJECT IN EITHER 1956 
or 1957: 

Dr, Earlo's Tissue Culture Unit collaborates in this , 
pro gran. 



PAGE 4. 
PROJECT REPORT FOPJl (Cont'd. ) 
HCI-803(C) 

SERI/i NO. 

NO ENTRY FOR ITEI4 13, 

15. . . 

PUBLICATIONS OTHER TFi.N AB3TR/.CTS FROM THIS PROJECT DURING CALENDAR 

YEilR 1955: 

1. Green, Stanley, Evans, J. M. and H^rtz, R., Acyclic nenstrual 
■ bleeding associated vdth erythrocytosis, J, Clin, . Endocrinol. E: 

Metab. 15*. 199, 1955. 

2. Hertz, R., Tullner, U. H., Schricker, J. A,, Dhyse, F, G. , and 
Hallxian, L, F., Studies on Anphenone and related conpounds. Recent 
Progress in Homone Research, Vol. 11, pp. 119-14-7, 1955. 

3. Hertz, R,, l-'aite, J. H. , and Thonas, L., The progestational 
effectiveness of 19-nor-ethisterone by oral route in wonen, J, 
Clin. Endocrinol. & Metab. (In press), 

U* Thorn, G. V. , , Renold, A. E., Goldfien, A,, Nelson, D. H. , Reddy, 
¥. J,, and Hertz, R,, Inhibition, of corticosteroid secretion by 
Anphenone in a patient with adrenal cortical carcinona, New Eng. 
J, Med, (In press), 

5. Hertz, R., Pittnian, J. A., and Graff , M* M< , Anphenone: Toxicity 
and effects on adrenal. and thyroid function in nan, J, Clin. Endo- 
crinol. & Metab, (In press). 



^6. 



■HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALEIID/Jl 

YE/'iR 1955: 

Translation of paper entitled "Studies on Anphenone and 
Related Conpounds" into Spanish and published in "Revista Argentina 
de Endocrinologia y Metabolisno, Btienos- Aires,. Argentina, 1955, 



PAGE 1 
PROJECT P.EPOP.T FORM 

1, National Cancer Institute 2, Endocrinology Branch 



INSTITUTE LABORATORY OR BRANCH 

3, Endocrinology Service k» _«________________, 5. NCI-804.(C) 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA SERIAL NO. 

6, Biotin and the Avidin-Biotin Complex in Relation to Normal and Neoplastic 

PROJECT TITLE 

Tissue Growth 

7. Roy Hertz. Frederick G. Dhyse and William U« Tullner 

PRINCIPAL INVESTIGATOR(S ) '. ' '. '■ 

8, Dr. Lloyd Law . 

OTHER BIVESTIGATORS 

9. PROJECT DESCRIPTION A) Objectives ; 1, To ascertain the role of biotin, a 
dietary trace factor, in the growth of normal tissue' and in the development of 
neoplasms in animals and man, 

2. To learn the normal metabolism, of biotin in man and to produce biotin de- 
ficiency in cancer patients administering lo^^r biotin diets plus concentrates of 
avidin prepared from egg-white, 

3. To develop methods for the cheap and practical preparation ox large amoTints 
of avidin for therapeutic trial in selected cancer cases. i 

B) Methods ; 1. Standard methods of protein fractionation of egg white are 
employed to prepare potent avidin concentrates which are assayed for growth 
suppressing effect on a biotin requiring yeast organism, 

2, Biotin deficiency is produced in mice by feeding egg white and avidin 
concentrates and the effect of this deficiency on such standardized tumors as 
transplantable leukemia is determined in collaboration v/ith Dr. Lloyd Law. 

3. Patients are carried on diets of knovm and constant biotin content and the 
urinarj"- biotin excretion is followed microbiologically. The effects of egg white 
feeding and avidin feeding are observed upon the patient's clinical nutritional 
status and upon the urinary excretion of biotin, 

C) Ma.jor Findings ; 1. An excellent, economical method of preparing potent 
concentrates of avidin has been developed and a stock pile of this material for 
clinical and experimental use has been prepared, 

A large industrial laboratory with ready access to abundant supplies of fresh 
egg-white has adapted this method to mass scale and is now providing our first 
adequate supplies of avidin for extended clinical trial. 



PAGE 1 
PROJECT PaiPORT FORM 
1, N.C.I. . 2, Endocrinology Branch 



INSTITUTE LABOR/^/rORY OR BR/.NCH 



3, , 4. - 5. NCI-805C 

SECTIOLI OR SERVICE LOCATION (IF OTIiSR THAN BSTHESDA SERLfi NO. 



6. Effect of Steroid Therapy on Metabolism of B~Complex Factors in Cancer 
PROJECT TITLE 

Patients 



7. Roy Hertz and Frederick G. Dhyso 



PRINCIPAL INVESTIGAIOR(S) 
8, Phillip Rayford 



OTHER IMESTIG/^.TORS 



9. PROJECT DESCRIPTION - ■ 

Objectives ; To determine the effect of protein anabolic action of 

steroid therapy in cancer patients upon the metabolism 
of B-oonplox factors oonaonod in tho diet. 

To correlate the foregoing with specific therapeutic 
effects obtained. 

Methods employed ; Patients are maintained on a fixed diet and their 
dietary intake determined by a balance procedure. 
Continuous urine collection provides specimens for determining urinary 
excretion of nitrogen, biotin, riboflavin, niacin, folocin, and panto- 
thenate (See project SOOC). 

Patient material ; 

No, Average Stay Days 

Admissions: Adult fern-ales 2 90 

Outpatients: None 

Proposed course of project ; It is proposed to develop and extend those 

studies as a new project largely by conti- 
nuing studies already in progress. Observa,tions would be made in suit- 
able patients with andro gen-induced protein-anabolism and in cortisone- 
induced protein-catabolic states. In this manner the relationship of 
B-complex balance to nitrogen balance may be determined vjith a view to 
clarifying the mechanism of action of those steroids on protein meta- 
bolism generally. 



PAGE 2 
NCI-805C PROJECT REPORT FORII (Cont'd) 

SERTixL NO. 

Proposed course of project : (cont'd.) 

The paired tray technique has been replaced by full 
metabolic study. This more exact technique will provide necessary 
control data regarding the differences between androgen- induced 
nitrogen retention and simple retention folloi.dng increased intake. 



11. 



12. 



BUDGET ACTIVITY: , 

RESEARCH fH ADMINISTPiiTION / 7 

REVIEW & APPROVAL /"~7 T JCHIIIC/iL ASSISTALICE /~7 



COOPER/'.TING UfllTS OF THE PUBLIC HE/lLTII SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONKSIL FOR THIS PROJECT IN EITHER 
1956 or 1957: 

Dr. Donald Uatkin, General Medicine Branch, is collaborat- 
ing in completing the studies on patients on the complete balance 
regime. 



NO ENTRIES FOR ITEMS 13,..15-& 16. 



PAGE 1 
PROJECT REPORT FORM 



1. N. C. I. 2. Endocrinology Branch 

INSTITUTE LABORATORY OR BRANCH 

3. Endocrinology Service 4-. 5. NGI~806(C) 

SECTION OR SERVICE LOCATION (IF OTHER. TMN BETHESDA) SERIAL NO. 

6. Endocrine Characterization of Females with Cancer of the Larynx 

PR.OJECT TITLE 



7« Roy Hertz. Morris M, Graff and Lois F. Hallman 

PRINCIPAL I?]VESTIGATOR(S) 

3. James Pittraan and Harold Altinan 

OTIiER BTVESTIQATORS 

9. PROJECT DESCRIPTION 

Objectives ; 1. To determine the endocrinological characteristics of the 
relatively rare female patient with cancer of the larynx in 
order to assess the role of endocrine factors in the pathogenesis of this 
disease. 

Methods employed : V/omen with proven cancer of the larynx will be hospi- 
talized and a complete endocrine evaluation made by: 
l) recording a complete pubertal, menstrual, and reproductive history 
and any other pertinent endocrine facts regarding the patient and her 
famUyi 2) complete physical appraisal of endocrine make-up with emphasis 
on obesity, masculinity, size of clitoris, hirsutiarn, voice pitch, acne, 
baldness, vaginal smear and other endocrine factors; 3) determination of 
urinary cortlcoids, estrogens, ketosteroids, and gonadotropins; also 
glucose tolerance, B.M.R., protein bound iodine, eosinophlle response 
test and other measures of endocrine functions. 

These data would be collected in order to determine v/hether there 
is any evidence of gonadal or adrenal androgenlzation of the female who 
gets cancer of the larym: as compared with normal females of the same 
age group. 

Ifa.ior findings : Preliminary superficial clinical observation of 2 

females with cancer of larynx has revealed no striking 
difference. 

Significance to Cancer research : Literature review indicates that the 

sex ratio for cancer of the larynx 
is variously estimated as between 16 to 1 and 200 to 1. In any case 
it is clear that females are relatively rare among patients vrith laryn- 
geal cancer. 



PAGE 2 
PROJECT REPORT FORl^i (Cont'd.) 



NCI-306(C) 
SERIAL NO. 



Sip inificance to cancer resea rch (cont'd.) 

The larynx is a secondary sex organ in that its pubertal 
differentiation is homonally dependent. Moreover, androf;en ad- 
Snistration readily alters' the larynx in the ^man female but no 
case of cancer of the larynx in the htrnian female has yet been re- 
ported following androgen administration. 

ProposedcourseoL.E£OJect: This project is deferred pending 
rroposea cou , , ti_-.J additional clinical facilities for 

the admission of appropriate patients. 



11. 



BUDGET ACTI\n:TY; 

RESEARCH • O ADMINISTR/iTION. ■ O 

REVIEW h APPROVAL O TECmilGAL ASSISTANCE O 



NO ENTP.ISS FOR ITS!1S 12, 13, 15 & 16. 



PAGE 1 
PROJECT REPORT FORM 
1, N,G,I, 2, Endocrinolo^ Branch 



INSTITUTE LABOPivTORY OR BRANCH . 

3, Ilhdocrinology Service K, 5. NCI-.807(C ) 

SECTION OR SERVICE LXATION (IF OTHER Tli/^.N BETHESDA) SERI/.L NO. 

6, Methods of Steroid Analysis in Urine and Blood from Cancer Patlsnts 

PROJECT TITLE 

7. Morris M. Graff 

PRINCIPAL INVESTIGATOR(S) 

8, Silas Jackson, Foster Burnett and Willie Logan 

OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION - 

Objectives: To develop and iroprove the chonical nethodology for deter- 
mination of urinary corticoids and ketosteroids and blood 
corticoids, estrogens, and androgens. 

Methods employed ; Classical methods of reagent and enzymatic hydrolysis, 
chromatographic adsorption and elution, paper chroma- 
tography, and spectrophotometric determination of specifically developed 
chrom.ogens. Chemical methods to be correlated with equivalent bioassays 
available under Project SOo, 

Major findings ; Methods for urinary ketosteroids and corticoids have 

been developed and perfected and are now adaptable for 
clinical studies. 

Blood corticoid methods have bejn investigated and an 
improved method devised which is now available for clinical research 
studies involving assossm^ent of adrenal corticoid fvmction. 

Progress in the developm.ent of a clinically useful 
method for estrogen determination in body fluids has been very liraited. 
However, certaiji technical difficulties have been overcome. 

Significance to cancer research ; These methods are basic to the endo- 
crinological study of patients with 
cancer of the breast, prostate, cervix, and larynx. 

Proposed course of project : The further extension of methodological 

investigation is proposed especial3y for 
blood estrogen and iirinary estrogen by fluorom.etrie methods. Extended 
investigation of the use of the Anthrone reagent In steroid analysis 
is planned. 

Methods for the fractionation of alpha 
and beta ketosteroids and for pregnandiol detemlnrutlon are to be de- 
veloped in relation to further endocrine characterization of patients 



PAGE 2 
PROJECT ■ REPORT FORM ( Cont ' d ) . 
NCI--807(C) 

SERI/iL NO. 

Proposed course of project (cont'd.) 
with timors of adrenal, testis, or ovary. 

11,5 . , 

BUDGET ACTIVITY: 

P.ESSARCH /y ADMNISTRATION /7 

. REVIEl«J & APP?.OV/iL jT/ TECHNICAL ASSISTANCE ^ 



NO ENTRIES FOR ITEl-K 12, 13, 14-, & 16, 



■ . PAGE 1 

.- _ , ■ ■.PROJECT. REPORT FORM 

1. N. C. I. 2. Endocrinology/ Branch ■- 



mSTITUTE LABORATORY OR BRANCH 

3. Endocrinology Service ^ U. 5 .NCI-??0SC 

SECTION OR SERVICE ;• LOCATION (IF OTHER, THAN BETHESDA) SERIAL NO, 

6, Endocrine Factors Governing Hormone-Induced Tissue Growth and Ttrnior 

PROJECT TITLE 

Formation in Animals; Bioassay of Blood and Urine 
from Cancer Patients 

7. Roy Hertz, Will3-an W. Tullner. Donald Spencer, D. M. Bergenstal 

PRINCIPAL INVESTIGATOR(S) 

B. Sally B. Fand and Morris M. Graff 

• OTHER im/ESTIGATORS 

.9. PROJECT DESCRIPTION 

Objectives ; 

^1. To gain basic information concerning endocrinological and nu- 
tritional factors governing homonally conditioned growth and involu- 
tion in organs such as the uterus, breast and prostate which are the 
frequent seat of hormone-sensitive neoplasms. 

2, To develop and improve bioassay methods for the quantitation 
of hormones in the blood and urine of patients with cancer who are 

. either receiving various forms of hormonal therapy or who present un- 
usual hormonal patterns, 

3. To determine the biological properties of steroid and related 
compounds in terras of their potential usefulness in cancer therapy. 

Methods employed ; 

Standard methods of biological study are employed. These include; 
' l) the maintenance of animals of various species on specified hormonal 
or nutritional regimens j 2) daily observations of physical and beha- 
vioral changes in such animals; 3) complete autopsy of test animals 
\i±th detailed weighing and chemical analysis and histochemical study 
of specific organs; J+) surgical alteration of the endocrine status 
of animals by gonadectomy, adrenalectomy, hypophysectomy, thyroidectomy, 
parabiosis, hysterectomy and other operative procedures. 

I-fejor findings; 

These are detailed in the publications listed under Item 15. They 
may be briefly enumerated as follows; 



PAGE 2 



PROJECT REPORT FOR^l (Cont'd.) 

NCI~808(C) 

SERIAL NO, ■,,,..:.::...-■' . ... -, ,... . ■:.... 



Majior fln riin prs (cont'd. l 

1 . The -biolosical properties of Amphenone- in relation ^to adrenal 

S?.ersSpr.rsSS trrls%rt.flXl :L.^ to e.c^nou. corticoia 
is readily demonstrated. 

A .series of analogues of Amphenone have been synthesized and 
studied ?hesf i^lX alpha, beta, dmethyl-4 V stilbenediarixne, 
?hf dS^th^fa^ino ^erivati^e ^f Mphenone,^he o.ine ^er-t.ve f^ 

s:-'.iSoS:ar^-tXrs^^^^^ ~- 

If,, Axes are bein^^ made with respect to relative toxicity ana c^nxrax 
nervous sjstem dlp^ssant effect. Twenty-five related compounds have 
Serfou^d to be inert, indicating a relatively l^g^f^^ °^ ^P^ 
cificity for these interesting endocrinological eficcts. 

Extended work with these and related substances, j;j2/'sScinc 
Gemanin, is projected with a view to deriving a non-toxic, specific 
Inhibitor of adrenal-cortical function. 

Chromatographic analysis of the steroid content of ^^e a^enal 
vein blood of the Amphenone-treated anir.ials is also to be carried out. 

2 Fui'ther studies on potent anti-gonadotrophic sera have l^d to 
some de Jee of purification of the active inlaibitory material These 
ef?orts^re to be extended by means of specific immunochemical ad- 
sorption and elution techniques now being developed. 

3. Additional steroids have been screened for P°^?j:^^\J!;^^°°^" 
tion of pituitarj^ depressant action from other biological effects. 
The studies on ?he biological properties of allopregnane, 2l-ol-3, 
S dione and Lichstein's compound S have been completed and reported. 
Ssays of ?he new corticoids, metacortandrosin and metacortandrolone 
have indicated no material dissocio.tion of pituitary depressant 
action and enhanced corticoid action. 

U. Extension of studies on the local catabolic function involved 
in the involution of hormone sensitive organs. fpllovanggonadectomy 
SbstanSIte the complete quantit-ative and chronologicar independence 
S this process from general catabolic effects on the body as a whole 

5. Substantial progress has been made on a sensitive and highly 
specific assay for luteinizing hormone, A normally cycling lemaie 



PAGE 3 
:PROJECT REPORT FORJ'I (Cont'd.) 



NCI-808(C) 

SERIAL MO. 



Ma.ior findings .(cont'd. ) 

rat is followed by vaginal smears until she is found to be in the 
pre-ovulatory or pre-estrus phase which is characterized ty the 
appears-nce of very large numbers of large rotuided, nucleated epi- 
thelial cells in the vaginal smears. Our previous studies had shovm 
that hypophysectoray at this point will abort the succeeding ovula- 
tion. Since this ovulation depends entirely on luteinizing hormone, 
a sensitive and specific assay is- based on the restoration of ovula- 
tion by injection of such hormone preparations. It is hoped that 
this will permit us to assay for this hormone in the blood and urine 
of cancer patients under steroid regimens, 

6. A strain of guinea pigs has been identified which has a large 
number of hypogonadal males. These animals have normal mating beha- 
vior but have complete aspermia and extremely atrcphic testes, A 
complete endocrine, behavioral, and anatomical stu.dy of these animall.s 
is being undertaken in collaboration with Dr. V, C. Yovmg, Prof, of 
Anatomy at the University of Kansas. 

7. The hormonal factors involved in the post-natal differentiation 
of the ovary in the rat and rabbit have been investigated, A post- 
natal period of 10 days in the rat and of 10 weeks in the rabbit 

has been characterized as a period during which the ovary is completely 
insensitive to pituitary gonadotropin stiiaulation. During this period 
the ovary undergoes the first delineation cf normal follicular 
structure, the final step being that of antmmi formation. It is 
only such antrum-containing follicles that respond to gonadotropin 
administration. Moreover, the ovary of a new-born rat grafted to 
its mother's ki'dney vn.ll undergo normal, post-natal differentiation 
even when the mother had been completely hjrpophysectortized. Further 
studies on the possible role of the hypothalamus in this process 
are under vjay, 

8. The overlap between progestational and corticoid action of 
certain steroids is well exemplified by the fact that desoxycorti- 
costerone is about 10^^ as active as progesterone for progestational 
effect. 1'fe nox-; find that whereas, hydrocortisone lacks progesta- 
tional activity, the introduction of the fluorene atom in the 9 
position imparts not only increased corticoid potency but also renders 
the compound progestationally active at about 5/' the activity of 
progesterone itself in both the monkey and rabbit. Similarly the 
fluorination of 11 beta hydroxyprogesterone imports progestational 
activity to this compound .at a level equ?.l to 10 percent the activity 
of progesterone. These observr.tions indicate that fluorination has 
implications not only for corticoid action but also for gonadal 
steroid properties. Moreover, the availability of com.pounds having 
combined corticoid and progestational action provides, an £ipporttmity 



NCI-808(C) 

SERIAL NO. 



PAGE U 
PROJECT REPORT FORM (Cont'd.) 



Vh.^ov findings (cont'd. ) 

for study of a compound which vdll proliferate the uterine epithelial 
"structures at the same time that ' stromal resistance is depressed. 
Long-term studies are in progress to determine what the final re- 
sult of such tissue changes may lead to in the way of architectural 
derangement possibly leading ultimately to m.alignancy, 

9. Ve have previously reported that a five-fold enhancement of 
progestational activity in the rabbit is effected by demethylation 
of progesterone at the 19 position, yielding 19-nor-progesterone, 
Similarly, 19 nor-ethisterone was shown to have the same increased 
progestational action in the rabbit when orally administered. These 
studies have now be on extended to the monkey, employing the inhibi- 
tion of estrogen-withdrawal bleeding and the production of a secretory 
endometrium. Both compounds show comparably enhanced effectiveness 

in the monkey. These studies have led to siir.ilar observations in 
the human (see project 803, item 9 Cll), Thus the quantitative 
ratios are similar in rabbit, monkey, and human. 

10. Recent studies on the effect of hyp":ph3r<5eotomy on breast 
cancer have implicated the pituitary grov/th ho:tT,iona as a direct 
trophic influence on breast cancer. It becomes iciperative to be 
iable to measure the level of growth hormone in human blood or urine. 
The rat's tibial cartilage and costal .cartilage have been shox-m to 
respond to administered grov/th hormone by increased uptake of radio- 
active su].fur. Preliminary studies suggest that this may provide 

a sufficiently quantitative and sensitive index of the action of 
grovrbh hormone to permit its assay at much smaller levels than 
current histological methods permit. 

11. Detailed histochemical analysis of the reaction of hormone 
■"'sensitive tissues and tumors to altered hormonal states have been 

initiated. Examples are: (a) the relationship of initial fat 
accumulation to the post-natal differentiation of the ovary; (b) 
the relationship of carbonic-anhydase to the progestational response 
of the rabbit and monkey uterus j (c) the histochemical characteriza- 
tion of the lipoidal substance present in the adrenal of the Amphenone- 
■ treated rat, and (d) the relationship between the functional state 
of the human pituitary and specific enzyma.tic content as demonstrated 
histochemically. 

12. The -role of dietary trace factors in hormonal response has 
been stvidied in particular relationship to the activation of the 
adrenal gland of the hypophysectom.ized dog by exogenous ACTH, It 
has been dem.onstrated that the pantothenate, riboflavin, biotln, 
folacin, pyrldoxine and ascorbic acid content of the adrenal gland 
remains unchanged vrhen the gland is at complete rest as manifested 
by low cortlcoid content of the adrenal vein effluent or after 



PAGE 5 
PROJECT REPORT FORM (Cont'd.) 



WCI-80B(C) 
SERIAL NO. 



Ma.jor findings (cont'd.) 

maximtim activation by exogenous ACTH leading to enonr.ous elevn.tion 
• • * in corticoid- content of the.adjrenal vgin blood. Moreover, the 
adrenal vein blood in either state . shows no change in -its content 
of these trace factors despite the very marked rise in corticoid 
content. Thus it appears that these trace factors are not quanti- 
tatively critical for this type of iimnediate hormonr-l response to 
ACTH. 

Proposed course of pro.ject ! 

It is expected to continue and extend the foregoing studies 
along the lines already indicated. 

11. . 

BUDGET ACTIVITY: 

RESEARCH /~/ ADiINISTR<1TI0N £J 

REVIS'J & APPROVAL /^ TECHI'IICAL ASSISTANCE jTJ 

NO ENTRIES FOR ITEI^E 12 & 13 

15. 

PUBLICATIONS OTHER THAN ABSTR/iCTS FROM THIS PROJECT DURING CALEITDAR 
YE/iR 1955: 

1. The effect of 17-alpha-hydroxy-ll-desoxycorticosterone on estrogen- 
stimulated chick oviduct grovrth, by W. V. . Tullner and R, Hertz. 

(Endocrinology) (In press). 

2. Progestatf-onal activity of the halogenated corticosteroids and 
related compounds in the rabbit and monkey, by R, Hertz, and 
W. W. Tullner (Proc. Soc. Exper, Biol. &. Med.) (In press). 

3. Amphenone inhibition of adrenal corticosteroid output in the hypo- 
physectomized dog, by U. W. Tul^lnor, M. M. Graff and R. Hertz 
(Endocrinology). (In press). 

4-. High progestational activity of nor-ethisterone and norprogesterone 
in the m.onkey, by R. Hertz and W. W. Tullner. (Endocrinology). 
(In press). 



NCI-808(C) 
SSRI/iL NO. 



PAGE 6 
PROJECT REPORT FORl'I (Cont'd. ) 



15. Publications (Cont'd.) 

5, Vitfunin content of dog adrenals and adrenal vein blood before 
and after ,ACTH, by R. Hertz, W. W. T-ullner. M. Graff and J. A. 
Schricker. (Proc, Soc, Exper. Biol, 8: Med.; (In press), 

6. Studies on Amphenone and related conpoiinds, by R. Hertz, 11. W. 
Tullner, J. A. Schrieker, F. G. Dhyse, and L. F, Hallnan. 
(Recent Progress in Hornone Research, Vol. 11, pp. 119-14-7, 
1955). 

16. ^ \ \^ 

' HONORS AND AWARDS TO PER.'JOBIiilL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1955: 

, Mr, William W, Tullner uas admitted to the Graduate Council of 
George Washington University as a candidate for the degree of 
Doctor of Philosophy in Physiology to be conferred in June 1956, 



PAGE 1 
PROJECT REPORT FORM 
1. N. C, 1, 2. Endocrlnolo^ Branch 



II'ISTITUTE LABORATORY OR BRANCH 

3. Endocrinology Service U. 5> NCI-809(c; 

SECTION OR SERVICE LOCATION (IF OTHi^. THAN BETHESDA) SERIAL NO. 

6. The Preparation of Crude Natxiral Extracts of Hormonal Activity from Ovary, 
PROJECT TITLE 

Testis, Adrenal and Pituitary and their Biological and 
Clinical Characterization. 



7. To be recruited. 



PRINCIPAL INVESTIGATOR(S ) 



OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives ! To prepare for biological and clinical assay crude 

preparations of hormonally active tissues in order to 
determine their effect upon tumors in hormone -sensitive organs. We 
know the crystalline steroids now so widely used represent only 
partial elements of the over-all activity of the various endocrine 
organs and that even optimum mixtures of them do not reproduce the 
characteristic effect of the total internal secretion of the endocrine 
gland itself. Such effects maj be more profound and more desirable 
than those obtained by the separate synthetic cr;'"sta.lline factors. 

Methods employed : 1. Standard chemical methods of total lipid and 

protein extraction of freshly prepared tissues to 
be collected at the slaughter house and processed immediately, 

2, The application of bioassay techniques to such preparations to 
determine their endocrine effectiveness and their toxicity. 

3, Clinical trial of non-toxic, active materials in suitable 
patients with endocrine deficiency or cancer of breast, prostate or 
cervix, 

NO ENTRIES FOR ITEI^IS 11, 12, 13, 15 & 16. 

N.B, This project has not been activated due to failure to 
recruit properly qualified professional personnel. 



PAGE 1 

PROJECT PEPORT FORM 



1. N. C. I, «__>_ 2. Endocrinology Branch 



INSTITUTE LABOPJITORY OR BRANCH 

3, EndocrJbiolQgv Service L. _ , , , 5,NCI-8lO(C) 

SECTION OR S^VICE LOCATION (IF OTHER TIIA.N BETffiISM} SERIAL NO. 



6. Studies in Thyrnid Function 
■ PROJECT TITLE 



7 KG Sher^'f 
'principal xlIVESTIGATOR(S) 

8 H-a -r^-Ld Altman. Betty N^ Siefring and RiuL liarks 



■q^FTER INVS.STIQATORS 

,, PROJECT DESCRIPTION - A) The hepatic clearance of plasma protein bound 

iodine in ms.n. 

Objective: To determine, in Ernn, the role of the liver in the excretion, 

conjugation, and utilization of thyroid hormone. Results 
obtained are to be compared with experimental information available 
from similar work done in animals (rat, mouse, cat, dog). 

Methods employed ; Hepatic vein catheterization in man; serum or plasma 

protein bound iodine determinations, hepatic blood 
flow measurements employing the brom-sulfaphthalein technique, 

Mia .1 or findings ; The major findings of this project during the past 

year have been the demonstration of a significantly 
lower plasma protein bound iodine concentration in blood from the 
hepatic veins of hvimans as compared to the concentration of plasma 
protein bound iodine in the peripher8.1 veins or femoral artery blood 
of the same subject. This enables one to calculate the amount of 
protein bound iodine removed by the liver and gives a measure of the 
entero-hepatic circulation of thyroxine; the first such information 
available in man. 

Proposed course ; See notation atta.ched to page 1, 

B) The effects of varioiis steroids on thyroid function and the 
metabolic fate of thyroxine. 

The effect of estrogens, androgens and corticoids on thyroid 
function and the metabolic fate of thyroxine. 

Methods employed ; Radioiodine 24- hour tracer uptakes; thyroid gland 

hormone secretion rates; paper chromatography of 
protein bound radioiodine com-pounds, serum protein bound iodine de- 
terminations; disappearance rate measurements of blood radiothyroxinej 
serum protein thyroxine binding capacities. 



PAGE 2 
PROJECT REPORT FORM (Cont'd.) 



NCI-810(C) 
SERIAL NO. 



PROJECT DESCRIPTION (Cont'd.) 

Ma.lor findings ; a) Estrogens cause a significant rif5e in serum protein 

bound iodine without a concomitant rise in 24- hour 
tracer radioiodine uptake of the thyroid gland; b) secretion rate: 
in 2 patients, so far, one has.. had no change in secretion rate with 
estrogen administration; one lias had slowing of secretion rate with 
estrogen administration; c) testosterone causes a mp.rked fall in 
serum protein bound iodine and. .24. hour radioiodine tracer uptake by 
the thyroid gland; d) Metacortandrocin and Metacortandrolone, cor- 
ticoids similar to hydrocortisone, cause a marked fall in uptake 
and senmi protein bound iodine; g) jxiper partition chromato.graphy has 
failed to reveal any qualitative change in the iodinated compounds 
released by the thyroid under the influence of the steroids mentioned 
o.bove , 

Proposed course ; It is proposed, during the next calendar year to 

extend these studies to a greater number of cases 
and to employ radioactive iodine labelled thyroxine for tissue uti- 
lization studies during control and steroid administration periods 
in the same patient, 

C) Ejctra thyroidal effects of thyroid stir.iulating hormone; T3H effect 
on thyroxine to triiodo-thyronine conversion: 

Ifethods employed ; Measurements of serum or plasma protein bound iodine, 

rate of radiothyroxine utilization in vivo by scin- 
tillation gamiTia counting of blood protein fractions in sequentia.l 
fashion. 

Ma.-]' or findings : One case studied so far over 2 week period. No change 
in thyroxine utilization rate was found. 

Proposed course ; ictension of present study to several patients includ- 
ing radiothyroxine utilization studies. 

D) In vitro conversion of thyroxine to triiodo-thyronine by surviving 
tissvie slices and homogenates, and the factors influencing this 
interconversion. 

Objectives ; To confjjrm and extend the work of Albright and Larsen vrho 
demonstrated in vitro conversion of thjrroxine to triiodo- 
thj'ronine by rat kidney slices. To isolate the enzyme systems respon- 
sible for the deiodination involved and to ascertain the factors in- 
fluencing this enzyme's action. 

Methods employed ; Surviving tissue slices, tissue cultures and homo- 
genates incubated with radioiodine labelled 1- 
thyroxine. The products of these incubations are then analyzed by 
paper partition chromatography. 



PAGE 3 
PROJECT P.EPORT FOM (Cont'd.) 



ECI-810(C) 

SERIAL NO. 



PROJECT DESCRIPTION (Cont'd.) 

Major findings ; No conversion to triiodo-thyronine by mouse liver 

tissue cu3.tures5 slight appearance of triiodo 
thyronine after incubation with monkey surviving kidney slices and 
homo ge nates in phosphate buffer. 

Proposed course ; This project was discontinued due to separation 
from the Service of the principal investigator. 
No additional progress is reported at this time. 

Significance to cancer research ; Further elucidation of our know- 
ledge of the m.echanism of action 
of the steroid hormones on peripheral tissues may be expected to 
help explain som.e of the favorable and unfavorable effects on 
cancer of the breast, prostate and uterus. 



11. 



BUDGET ACTIVITY: 

RESEARCH /^ ADMINISTRATION /~/ 

PJCVIEVJ & APPROVAL /^ TECHNICAL ASSISTANCE /^ 



12.. 



COOPERATING UNITS, 0? THE PUELIC I1E/.LTH SERVICE, OR OTItR ORGANIZATIONS, 
PROVIDING FUtlDS, FACILITIES, OR PERSOMJEL FOR THIS PROJECT IN EITHER 
1956 or 1957: 

Dr. Pb.ul Marks, tJIAMD cooperated in this project. 



15.. 



PUBLICATIONS OTHER TH/iN ABSTR/iCTS FROM THIS PROJECT DURING CALEIvTDAR 

YEAR 1955; 

The effect of meticorten and meticortelone on thyroid 
function, by M. G. Sherer and B, N, Siefring, J. Clin, 
Endocrinol. & Me tab, (In press). 



NO ENTRIES FOR ITEt-IS 13 and I6. 



PAGE 1 
' PROJECT REPORT FORM 

1. H. C. I. 2. Endocrinolopy Branch 



INSTITUTE LABOR.'iTORY OR BRANCH 

3 ♦ Endo crinolo gy ^e rvice 4-. ^ 5. IICI-3ll(C ^ 

SECTION OR SERVICE , LOCATION (IF OTHER Ta'.N BETIiSSDA ) SERIAL NO. 

6. Studies on Thyroid Adenomata and Related Problems in Pituitary-Thyroid- 
PROJECT TITLE 

HjTothalrjnic Relationships 

7. Monte A. Greer 

PRINCIPAL INVESTIGATOR (S) ' 

8. Leslie J. Do Groot, E].eanor Siperatein, Howard En-fin and Robert . Scow 
OTHEP. IIWSSTIGATORS • 

9. PROJECT DESCRIPTION - Correlation of histologic morphology and metabolism 

of simple goiter with its response to thyroid therapy; 

Objectives: To determine whether theoo is any correlation between the 

microscopic appearance and metabolic activity of various 
types of simple goiter tri.th their diminution in size v;hen desiccated 
thyro'id therapy is administered. 

■ Methods employed ; Studies of the metabolic activity of simple goiter 

are rxido with 1-131 tracer studies. A vredge biopsy 
is then made of the gland and 3 weeks later administratien of 3 grains 
daily of desiccated thyroid begun. At the end of a 4--6 month period 
a final biopsy is performed. In the case of single nodules, the entire 
nodule is removed at the end of the therapy period. 

Patient material: 



Admissions: Adult males 

Adult females 


No, 

u 

7 


Average Stay Days 
15 
29 


Outpatient: Number of patients 
Number of visits 


15 
77 





I'la.ior findings ; During the past year, only U such patients have been 

studied. These included cases of simple goiter, chronic 
thyroiditis, and single thyroid adenomas. All goiters thus far studied 
have experienced considerable diminution 3ji size. 

Additional findings of note are indicated by the titles of publica- 
tions listed below under Item. 15. 

Proposed course ; A continuation of the study as outlined above is 
planned for the coming year. 



PAGE 2 
PROJECT REPORT FOM (Cont'd.) 



NCI-811(C) 

smuij NO. 



PROJECT DESCRIPTION (CONT'D.) 

B) Isolation; and identification-of precursor of naturally occurring 
antithyroid compound, 1-5 vihylthioo3cazolidone. 

Objectives ; To isolate, crystallize, and identify the structure of 

the precursor of the only known naturally occurring anti- 
thjToid substance, venylthiooxazolidone. The procursor may be of a 
type that is more widespread among various plant foods than is now 
believed. 

Methods employed ; Brassica seed extm.cts are fractionated by various 
procedures, including column chromatography, after- 
first heat denaturing the thioglycosidase which specifically hydro- 
lyzes the precursor. Assay of the fractions is greatly facilitated 
by following the UV abortion spectrum. The precursor has a specific 
peak absorption at 2270 A°. Crystallisation and identification of 
the pure compound are by standard chemical and microchemical procedures. 

Major findings ; During the past year, the precursor has been purified 
and crystallized. It has been found to be a thiogly- 
coslde having a molecular weight of about 1000, m.p. 130°C. Both 
sodiim, potassium, and 304. — have been identified in the molecule. 
Tlie glycoije has been tentatively identified as a fructose polymer. 

Proposed course ; It is planned to continue with degradation experiments 

to \rork out the structure of the compoiind and to de- 
termine T:^ animals and human assay whether any biologic system not 
containing the specific thioglycosidase foiond in Brassica members is 
capable of liberating thiooxazolldone from it. 

C) Stim-ulation of the central nervous system, of rats by radiofrequency 
transmission. 

Objectives ; To devise apparatus capable of stimulating the central 
nervous system, particularly the hypotlialamus c of rats. 
The animals are to be completely unrestrained and without any external 
connections to the pulse generating equipment. It is hoped to produce 
hyperf unction of the pituitary by stimulation of those areas vrhich, 
when destroyed by electrolytic lesions, result in decreased hypophysial 
function. In this way, considerable information may be gained by neuro- 
endocrine interrelationships. 

Methods employed ; A technique has been devised for fixing radio receivers 

to the skull of rats, vdth stimulating electrodes buried 
according to calculation in various parts of the hypothalamus, A ratio 
sending and receiving apparatus has been devised to carry variable 
pulses to the hypothalamic electrodes. The receiver in the rat is 
entirely subcutaneous. It is possible to stimulate 36 or more rats at 



PAGE 3 

PROJECT REPORT FORM (CONT'D). 



NCI-8ll(C) 

SERIAL NO. 



PROJECT DESCRIPTION (Cont'd.) 

one time and with variable intensities and frequencies. 

Changes in hypophysial function are measured by gross and cyto- 
logic changes in the pituitary and target endocrine glands and by 
noting alterations in the functional activity of the target glands. 

Concomitantly observations are made of the behavioral changes 
■produced in the animals by stimulat5.on. 

I>fei.1or findings ; During the past year, the major emphasis lias been 

on developing equipment and techniques to a satis- 
factory level. It has been discovered that stimulation of the hypo- 
thalamic area lateral and Just posterior to the paraventricular nuclei 
will induce narked drirJcing behavior in the rats. Stimulation slightly 
posterior to this area will induce marked eating behavior. 

Proposed cour se t The major emphasis during the coming year will be 

devoted to vrorking out the "bugs" in the stimulation 
system and develop it to the point where' stimulation can be continued 
for several weeks without any appreciable loss of response in the 
stimulated animal, 

D) Study of the structure and function of heterotopic compared with 
normal pituitaries. 

Objective s: To determine the alterations in structure and function 
produced in the pituitary gland by transplanting 'it to 
an abnormal site and to determine what factors are involved in the 
changes thus produced. 

Methods employed ; Pittiitaries from new born mice are transplanted into 

the eye or kidney of genetically homogeneous mature 
mice. The hosts are hjrpophyse eternized one week later. Three v/eeks 
following hypophysectomy the function of the heterotopic pituitaries 
is measured by following changes in body growth and changes in the 
metabolism and structure of the target endocrine glands. Pituitary 
cytology is studied by the various advanced techniques now available. 

Major findings ; 1, Heterotopic pituitaries are able to maintain thy- 
roidal 1-131 metabolism at a level approximately equal 
to that in intact animals. They are not able to m.aintain thyroid weight 
or the weight of other target endocrine organs significantly above 
that of hypophysectomized animals. Raising or lowering the level of 
circulating thyroxin yxU. cause marked changes in the secretion of the 
pituitary hormone controllijig thyroidal 1-131 metabolism in these animals 
without having any significant effect on thyroid v/eight. 



PAGE U 
PROJECT REPORT FORI I (Cont'd.) 



NCI-311(C) 
SaiLIL NO. 



PROJECT DESCRIPTIOII (Cont'd.) 

2. Glycogen has been demonstrated in the pituitary for the 
first tine. This is present particularly in the younger glands, and 
in highest concentration in the pars tuberalis. 

3, The differences in cytolog;^ of heterotopic compared to in aitu 
pituitaries has been compared for va.rious ages. One of the most 
striking differences is the narked drn.inution in chrom.ophiles in the 
heterotopic glands. There is also a narked hypertrophy of the pars 
intermedia comparable to tkat seen following hypothalamic lesions. 

Proposed course ; The major effort during the coming year vdJ-1 be de- 
voted to a study of whether function of the heterotopic 
pituitaries can be increased by transplanting -vT^-rious p-^rts of the 
central nervous system, particularly the hypothalrius , in direct 
connection with the pitui.tary transplants. A system has already been 
devised for making transplants of considerable size to the kidney of 
adu!l.t mice. 

It will also be detexmined whether the heterotopic pituitaries 
secrete the substance necessar:/ for ovarian and uterine response to 
chorionic gonadotropin, A further study vri.ll invoj.ve whether they 
can concentrate isotopically labelled triiodothyronine to as marked 
an extent as do normal glands, 

E) The effect of hypothalamic lesions on pituitary function. 

Objectives ; To deteiT'.ine what effect electrolytic lesions in various 

parts of the h;7pothalarus have upon the regulation and 
secretion of the various hormones produced by the pituitary gland. 

Methods employed ; Electrolytic lesions are made iji the hypothalamus 

of the rats by means of a specially designed stereo- 
taxic instrument. Following a period of 1-3 weeks for reoovarj'', 
studios are made of the ability/ of the pituitary to secrete the 
various trophic hormones. These deterriinations are made by studying 
the alterations of structure and function produced in the target endo- 
crine glands by v^.rious stimuli. For exar.iple , thyrotrophin secretion 
is studied by the change produced in thyroid size and. 1-131 metabolism 
by chronic administration of propylthiouracil, 

Ilaj or. findings ; During the past year it has been found that the location 
of the hypothalamic area controlling thyrotropin secre- 
tion in the rat lies in the midline between the paraventricular nuclei 
and the media.n emdnence. Destruction of all or part of this area will 
markedly interfere with thyrotrophin secretion. 



PAGE 5 
PROJECT REPORT F0R14 (Cont'd.) 



NCI-811(C) 

SERIAL HO. 



PROJECT DESCRIPTION (Cont'd.) 

It has also been found that although lesions in the above area 
will prevent the goitrogenic response to Anphenone administration, 
they ijill not prevent the usual conconitant adrenal hypertrophy. 
This provides further evidence that the hypothalamic areas cohtrolling 
the various pituitary fxinctions are discrete. 

Proposed course ; It is planned to make a study of the location 'of the 
hypo thai pjnic area responsible for maintaining normal 
growth of the pars intermedia of the hjixiphysis. , Material from over 
500 rats with hypothalrjaic lesions has already been assembled and the 
remaining requirement is to determine hc!'; far. from the hj^Dophysial 
stalk it is liecesaary to place lesions i:i. order to avoid the gross 
and bizarre hypertrophy , of the pars interriiedia. xrhich occvirs with le- 
sions close to the stalk, 

■ It is also planned- to investigate the area controlling ACTH 
secretion r.ore thoroughly and to see if it is possible to place 
lesions that will interfere with ACTH secretion as much as previous 
lesions have been intarfered with thyrotropin secretion, 

F) Study of the mechanism of action of stable iodine in thyrotoxicosis. 

Objectives ; To determine the mechanism, of action of stable iodine 

. in .ameliorating thyrotoxicosis and, secondarily, to --.ga'in 
some insight into the pathogenesis of thyrotoxicosis. 

Methods employed : Thjrro toxic and euthyroid subjects are given a 100 

rdcrocurie tracer dose of I~131. Twenty-four hours 
later the administration of 3O-6O ng, tapr.zole every 8 houi-s is begun to 
prevent reaccumuJIation in the thyroid of 1-131 broken doim from endo- 
genously labelled thyroxin. In vivo counting over the thyroid is ma.de 
by means of an. extema.lly placed scintillation counter. The thyroidal 
secretion rate can thus be deterr.iined by plotting on serdlog paper. 

Iflhen the rate has been established, 300 ng. per dr.j of Nal are 
added 'to the regimen. When the maximum effect from iodine has been 
obtained, 10-100 units of thyrotrophin are given daily for a period 
of several days. 

In the case of the control euth;!/roid subjects, thyrotrophin was 
frequentD.y given before iodine adm.inistratibn was begun. 

Major findings ; It has been found that iodine interferes with the 

release of hormone from, the thyroid gland in thyro- 
toxic subjects but has little, if any, effect in euthyroid subjects. 
It has also been foiond that iodine does not interfere with the stimu- 
lation of euthyroid glands by exogenous thyrotropin. 



PAGE 6 
PROJECT REPORT FORM (Cont'd.) 



NCI-811(C) 
SERIAL NO. 



PROJECT DESCRIPTION (Cont'd.) ' 

"\_ These results suggest thnt iodine nets in hyperthyroidisn by- 
inhibiting the release of thyrotrophin fron the pituitary. There- 
fore, hyperthyroidism nust be a supro.thyroidal disturbance and not 
solely a disease of the thyroid gland. .' 

Proposed course ; The project is teminr.ted due to the separation 
of Dr. Monte A, Greer, 

Significance to cancer research ; Studies of thyroid adononata will 

have a direct bearing on the problein 
of the pathogenesis of thyroid cri.ncer. In addition, further know- 
ledge of the pituitary''- thyroid interrelationship vjill provide potential 
means of controlling abnormal thyroid grovrbh. 



11.. 



BUDGET. ACTIVITY: 

RESEARCH £7 ADMINISTRATION £7 

REVIEW & APPROVAL £7 TECHNICAL ASSISTANCE /~/ 

12. 

COOPEPATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTIiER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES ^ OR PERSONI^IEL FOR THIS PROJECT IN EITHER 
1956 or 1957; 

Dr. Robert 0. Scow, NIAMD, collaborated. 

NO ENTRY FOR ITE14 13. . 

15. ., 

PUBLICATIONS OTHER THAN ABSTR/iGTS FROM THIS PROJECT DURING CALEIMDAR YEAR 
1955; 

1. Denonstration of thyroidal response to exogenous thyrotropin 
in irats' with anterior hypotlmlanic lesions, by M, A. Greer 
Endocrinology (In press). 

2. Observations on the norphology and histo'chenistry of the 
mouse pituitr.ry inplanted in the anterior eye chamber, by 
E. R. Siperstein and M. A, Greer, J. Nat, Cancer Inst, 
(In preparation), 

3. Effect on the thyroid gland of experimental alteration of the 
level of circulating thyroxine in mice with heterotopic 
pituitaries, by R, 0, Scow and M. A. Greer, Endocrinology; 56: 
590, 1955. 



PAGE 7 
PROJECT RSPOHT FOP^i (Cont'd.) 



ITCI-ai(C) 
SERI/iL NO. 



15. FUBLICATIOITS (Cont'd.) 

4.. Rlstocheriical denonstration of glycogen in the mouse 
pituitary, by Eleanor R. Siperstein, Proc. Soc. Sxper, 
Biol. ^ Med. 8S: 296, 1955. 

5, Suggestive evidence of a prinary"drirJ<:ing center" in 

hypothalamus of the rat, by M. A. Greer, Proc. Soc. Exper. 
Biol. & Med. 89:59, 1955. 

6. A modified Krieg stereotaxic instioment for producing intra- 

cranial lesions in the rat, by Monte A. Greer, Proc. Soc, 
Exper. Biol. & Med. 89: A^O, 1955. 



NO EI'ITRY FOR ITEl-I I6. 



PAGE I 
PROJECT REPORT FORM 



^ • National Cancer Institute 2 . Patholo g ic Anat o my Branch 

INSTITUTE LABORATORY OR BRANCH 

^ • Surgical Pathology & Post-Mortem Serv ice 4 . 5 ■ NCI—S^ 

SECTION OR SERVICE ' LOCATION SERIAL NO. 

6. No specific research projects in this Branch. However, the diagnostic 
service is designed to aid the investigative efforts of the N.I.H. staff 
responsible for patient care. In many instances, investigative study of 

pat holcgical t issues is of great im portanc e to clinical research. 

PROJECT TITLE . ~ - - . 



PRINCIPAL INVESTIGATOR (S) 



Dr. Harold L. Stewart, Chief, Pathologic Anatomy Branch 

Dr. Louis B. Thomas, Head, Surgical Pathology & Post-Mortem Service 

Dr. Albert W. Hilberg, Head, Cytodiagnosis Service 

Dr. John H. Edgcouib, Pathologic Anatomy Branch 

Dr. Alan S. Rabson, Resident, Pathologic Anatomy Branch 

Dr. Richard L.. Swarm, Laboratory of Pathology, N.C.I. 

Dr. Clyde Dawe ■ " " 

Dr. Eli Nadel " " 

Dr. Joseph Leighton " n 

Dr. William Banfield " " 

Dr. C.Harold Steffee. " " 

Dr. Edwin Lerner, Laboratory of Pathology and Histochemistry, N.I.A.M.D. 

Dr. Leon Sokoloff " " '' " 

Dr. Harold Stanley, Oral Pathologist, N.I.D.R. 



* 



These pathologists spend full time in the service and diagnostic 
functions of the Pathologic Anatomy Department, Clinical Center 
(Pathologic Ajiatomy Branch, N.C.I.) 

These associate pathologists spend only part time in the activities 
of the Pathologic Anatomy Department, principally in the performance 
of postmortem examinations. 



OTHER INVESTIGATORS 



SERIAL NO. NCI -85^ PAGE II 

PROJECT REPORT FORM (Cont'd) 

9. PROJECT DESCRIPTION: 

No specific research projects. The services and diagnostic functions 
of the Department during 1955 have included: 

a. 148 autopsy examinations. 

The average number of postmortems performed by each pathologist 
was about 15; the range was from 21 to 8 with the smaller number being 
performed by those who were here less than one year. 

b. 1778 surgical pathology accessions. 

Most of these were worked up by Doctors Thomas, Edgcomb/ Swann, 
Rabson, Sokoloff and Stanley. 

The objectives of the Surgical Pathology and Post -Mortem Service in the 
Clinical Center are twofold: first^ to furnish a diagnostic service in 
autopsy and surgically rei\ioved tissues, and second, to aid from a morpho- 
logical s'tatidpoint the various clinical research problems which are under 
study in the Clinical Center. 

The methods used are those standard methods which are used in the 
description of organs and tissues, the fixing aiid sectioning of this material 
and the preparation of histological slides. A wide variety of special 
staining procedures are used in the Histopathology Laboratory of the 
Department . The appended chart shows the December monthly report of the 
material studied in this Department and includes in the right hand column 
totals for the accessions during the calendar year of surgical specimens 
and autopsies performed. Note that the autopsy rate has doubled that of 
1954 and the number of surgical specimens has increased by over 50 per cent. 

In addition to the diagnostic services, there are several other functions 
of the Department which have continued and expanded during the past year. At 
present there are fifty scientists in the various laboratories at N.I.H. who 
have requested particular types of tissue from surgical and postmortem 
specimens examined in this Department. On many occasions it is possible to 
furnish these investigators with fresh human material.;. .: -. 

The staff of the Department continues to take active part at numerous 
Clinical Center staff meetings, and in addition, routinely conducts four 
Departmental meetings weekly. These are the Brain Cutting Conference on 
Monday, the Autopsy Conference on Tuesday, the Joint Pathology Staff Con- 
ference on Wednesday and the Surgical Pathology Conference on Friday. 



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PAGE III 



PROJECT REPORT FORM (Cont'd) 



10. NCL.S52— 



SERIAL NO. 



II. 



BUDGET ACT^'VITY: 



RESEARCH' /_/ 
REVIEW & APPROVAL /~7 



1 2 . None 



ADMINISTRATION /_/ 
TECHNICAL ASSISTANCE /~7 



COOPERATING UtillTS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 or i9i7 



13 . None 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELS^T^'HERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR 14, 15, or 16, 



PAGE I 
PROJECT REPORT FORM 



I . National Cancer Institute 2 . Pathologic Anatomy Branch 

INSTITUTE LABORATORY OR BRANCH 

3 . Cytodiagnosis Service : : A i -rJ.: "' ^ 5 . NCI 851 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO. 

6. Exfoliative cytology applied to diagnostic and research problems in the 

Clinical Center, N.I.H. . ^ ..__ 

PROJECT TITLE 



7. Albert W. Hilberg 



PRINCIPAL INVESTIGATOR (S) 



8. None 



OTHER INVESTIGATORS 



9, PROJECT DESCRIPTION: 

Project: Application of Exfoliative Cytology to Human Diagnostic and 
Research Problems. 

Objectives: To provide the service of exfoliative cytology for the diagnosis 

of malignant tumors of various sites in the body, notably the 
cervix uteri, lungs, stomach, and body cavities. Evaluation of effects of 
various types of therapy for cancer such as surgery, virus therapy, and 
endocrine therapy. To evaluate effects of hormones in non -cancer patients 
studied for endocrine balances. Further objectives involve attempts to 
better evaluate the cytological criteria of malignancy. 

Methods Employed: Routine Papanicolaou staining and careful cytological 

4 screening procedures together with concurrent tissue and 
clinical evaluation. Additional methods involve special fixation preparation 
and staining techniques as developed in histochemical procedures and as 
needed for the precise handling of specimens of various kinds. 

Patient Material: See appended chart. 

Major Findings: The correlation of recurrent cancer at the site of surgical 

procedure and the presence of malignant cells in washings 
from this site has been made. Several previously undetected cancers of the 
cervix were found by the methods of exfoliative cytology. The presence or 
absence of estrogenic activity in prepubertal girls or in cases of amenorrhea 
was determined in many clinical cases. Evaluation of menstrual cycles. 
Argyrophil secretion patterns indicating estrogenic activity of a notable 
degree past the menopause was noted in cases of carcinoma of the cervix uteri 
and evaluation of this continues. 



SERIAL NO. NCI 851 PAGE 11 

PROJECT ILEPORT FORM (Cont'd) 

PROJECT DESCRIPTION (Cont'd): 

Significance to Cancer Researdh; '.T^is project is a continuing 'demdnStration 

of the value of exfoliative cytology as a 
diagnostic tool for the clinician in the attempt to detect early cancer^ 

The suirgical wound washing cbtitiriues to show distinct and valuable 
correlation of residual tumor cells and tumor recurrence in surgical sites. 

Exfoliative cytology provides an excellent ! adjunct to tissue evaluation 
of the effects of various forms of therapy on tumors wheri it is 'riot possible 
or practical to remove tissue for biopsy or In conjunction with biopsy study. 

Proposed Course of Project: The continuation of diagnostic service to all 

Institutes in t.he Cllttical Center'.' 'Continued 
collaboration in evaluation of various clinical research projects such as 
virus therapy of cancer, hormone balance, and wound washings. Further 
development and refinement of special preparation, fixation and stashing 
techniques as applied to cytologic material. Expansion of activities is 
planned to meet increased demands for diagnostic service and to further 
assistance in clinical research. 









.sbsihi 'iuox .'i-K-- 'it h ftnoffi}- 



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PAGE m 



PROJECT REPORT FORM (Cont'd) 



10. NCI 851 



SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH M 
REVIEW & APPROVAL l~l 



ADMINISTRATION /_/ 
TECHNICAL ASSISTANCE l~l 



12 None to my knowledge 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, 
PROVIDING FUNDS , FACILITIES , OR PERSONNEL FOR THIS PROJECT IN EITHER 
1956 or 1957 



13. None to my knowledge 



'if THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, 
FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 



PAGE IV 
PROJECT REPORT FORM (Cont'd) 



14. NCI 851 



SERIAL NO. 



15 . '-^ ■ : ■ -::r 

PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PR5(ijECT DURING CALENDAR YEAR 
1955 '' ■ ■ ■■ • 

Cancer Cell Seeding of Operative Wounds, Smith, Robert R. and Hilberg, 
Albert W., J. of Nat. Cancer Inst., Dec. 1955. 

A Ndw Inexpensive Marking Deyice for Slides, belVeccKio, P.R., 

Ziegler, E., and Hilberg, A.W. , J. of Nat. Cancer Inst. 

(In Press) , 



•v:.. HONORS AND AWARDS TO PERSONNEL RELATING tO THIS PROJECT DURING CALENDAR 

YEAR 1955 







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PAGE 1 
PROJECT REPORT 



. National Cancer Institute 2» - Laboe^oiy of Pnysiolcgy 

INSTITUTE tAW^rtJ^-Y OR BEA>*CH 



U Cancer Physiology Section 4. -- :.. j^-'- 

SECTION OR SERVICE LOCATION tif other tiun Betoesda) SERIAL Na 



5. The "appetitiB factor^ in Walker 25g 
PROJECT TITLE 



'. Julius White 

„ .PRINCIPAL INVESTIGATORiS^ 



3. 

OTHER INVESTIGATORS 



9, PROJECT DESCRIPTION 

Ob|edtive8;''1*6' continue Iftd stoidlies qf the growtij-stimulating factor present 
in Walker carcinoma 256. 

Methods Rn\ploye<fe Fractu- -.:..- ^ of th« Walker 256 witix the purpose of 
isolating the growth factor. 

Major Findings ; 

1. Verification of the previous obMrvaHMS ol 0r. C» B^ MldM' 
that supplenientijig the t^asal diet of animal kMUteg )ayqe» 
plantable tumors at the point where the ml—iBt llSt Vi^ 
Intake, results in a resumption of tooti Mate wd ffowth increase. 

2* Extraction of lyophiUised tumor with iteft|»| tlfcuf 4M8 BOk remove 
the factor. 

3, The protein fraction of the tumor tissue e'er :^ .' :? ': " tiie ^SP^tiUto 
factor (preliminary observation). 

4. Pair (e«<ttng tixpMrtmMili;^ cv> ,v. ^ ^vw^ > ; = .^ . young r«ls 
Ingesting a 20^ casein and a 20% tumor vsource of tuirvgenj iiet, show 
Identical growth curves. 



PAGE 2 



SERIAL NO. ■nj-x.,..i. .>... 



5. Animals ingesting the ^0% tumor diet ad libitum siiow ain ippfoxi- ' 
mately 25 per cent higher growth increment than do litter mate animals 
ingesting the 20% casein diet ad libitum . 

: y ■''•■'; 6.: ■ ^'Aniittkls, ingesting the 20% casein- diet exci«^it^&^i^rii|-'^^ '' 

nitrogen as do the pair fed 20% tumor diet. ' ' ' ' ' ^' 

7, Urea nitrogen excretion of the tumor fed ^nimals jls approxim,a,tely.. ^ 
half of that excreted by the casein fed animals. ' ■^''^■':;^j. i-y^'i^ 

8, Allantoin excretion from rats ingesting the casein diet is approxi- 
mately half of that excreted by the rats ingesting the tumor diet;, 

9. Animals fed the 20% casein diet and bearing Walker 256 transplanted 
tumors show emaciation and die sooner than do litter mate rats 
ingesting the tumor diet. 

10. Animals ingesting 20% tumor diet and bearing a Walker 256 trans- 
planted tumor grow massive tumors (50 per cent of total tumor and 
body weight) without loss of appetite or loss in weight. 

Significance to Cancer .fieseaypti . There is, undoubtedly, a f actor jpresent in 
Walker carcinoma 256 which stimulates growth, spares hit;p(>geh and 
has a high "food efficiency" value. 

Proposed dourse of project : To continue in seaifch o! tne^ active principle ■ 
in tumor tissue which is responsible for this "appetite factor, " 

•■'J?;-'.' ' \ '■' - , ■ '.„ ^ ■'■, \0 i^lb hi'dui_^(>i '^iv • ■^'' ■■:■ ; ;; ' 



12, 



900 PAGE 3 

SERIAL NO, 



BUDGET ACTIVITY: 

RESEARCH fT ] ADMINISTRATION [ZJ 

REVIEW AND APPROVAL /~7 TECHNICAL ASSISTANCE ri 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER 
ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN EITHER 1956 or 1957, 

None, 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS 
RESEARCH DONE ELSEWilERE IN THE PUBLIC HEALTH SERVICE 
(WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUNDS), 
IDENTIFY SUCH RESEARCH: 

None. 



NO ENTRIES FOR ITEMS 14, 15 and 16. 



■orrASTaiv^i 



T^m 



'143. Ov4 



PAGE 1 
PiElOJECT REPORT J>i^i 



1. National Canter Iftstitate! : ; >^yA L abor atory of Physiology 

institute:, •■ ^ •'■■■■■■■■ :.'■..,; .-^i.^ ., .,. "^BaR/lTdRX ok brangh 



3. Cancer Physiology Section 4« v^ ■■ " ; . ... ; ' 5> '9(31 ' 

SECTION OR SERVICE LOCATION (if other than Bethesda) ' SERIAL NO. 



6. Probable Sources of Available Nitrogen for Tmior Growth 
PROJECT TITLE • . :. . ; ~~^. 



7. Julius White 

PRINCIPAL INVESTIGA'rOR(S) 



8. 

OTHER INVESTIGATORS 



9. PROJECT DrilSCRIPTION 

Objectives ; To determine the availability of nitrogen for the rapidly growing 
transplanted Walker carcinoma 250. 

Methods Employed ; Young, growing, as well as adult, rats were placed on a 
20% casein diet of known composition. The Walker carcinoma 256 was 
implanted subcutaneously and, at the same time, N*^ glycine was in- 
corporated into the diet. At various intervals the animals were sacri- 
ficed and the total nitrogen balance was determined. 

Major Findings; 

1, The uptake of N^ glycine by the transplanted tumor was equivalent 
to that taken up by the host. This occurred whether the isotope was fed 
for 7 days or 14 days and followed by a 7-day period on the basal diet 
or sacrificed immediately after the cessation of isotope feeding, 

2, In all cases the tumor reached a maximum size of 8 grams. It 
appears that tumors in their early stages of growth can utilize available 
nitrogen at the same rate as the host. 



;.901; PAGE 2 

iERlAL NO. i-^lO'lMi TO'M&V) 

Signif icance to Cancer Research ! To obtain a better understanding of the 
tumdr-libst relationship. A.t what stage of tumor growth does the 
metabolic insult appear in the host? Is the nitrogen present in tuinor 
tissue available under some circumstances for growth of the host? 
ii'^re the "building blocks" used by the tumor similar to those used by 
.<5^ivJ#ililie'-host?'^' :■■ :ii^:i limsoMM'i' ' •■ /.■-v/ua*. -^ ; 

.'.->,,.■,?',,>.'■' ;■■-.■ ■ . . ■ '• 'i \ \ ■ *\ ■ ' ■ - . ' . • 

■ ' - *'''#- '.I ■■■, . ' ■ : i , , 

Pr oposed course of project ; To make a similar study of nitrogen utilization 

in tumor -bearing animals in which the tumor has reached a growth 

increment where derinand of energy exceeds energy intake* To make , 
serial killings and determine if a transition takes place, where it takes 
place, and the nature of such an event. 



••ta^iVJii >idhi' 



IfOTTq^ 



'' ■''■"' '''•■■"■■ '■'■■■ ■ Jy9n!ttf^fJ:JW^siw-^or>te^';<i'no5ib-i5^ bf^a. ft-s^n. 



od)9 i'/! 







lOi 901 PAGE 3 

SERIAL NO. T-1 



U, 

: O BUDGET ACTIVITY: 



L2. 



13. 



RESEARCH /X7 ADMINISTRATION [ZJ 

REVIEW AND APPROVAL /~7 TECHNICAL ASSISTANCE fl 



COOPSRATIHO UNITS OF THE PUBLIC HEALTESSRVICE, OR OTHER 
ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN EITHER 1956 or 1957. 

None, 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, CR PARALLELS 
RESEARCH DONE 3LSEWHERE IN THE PUBLIC HEALTH SERVICE 
(WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUNDS), 
IDENTIFY SUCH RESEARCH: 

None. 



M 



14, 901 . _ .-lOPAGE^; 

SERIAL NO. ■■ryy.rii-^/u: . 



X5. . , ..____.-, - ' 

I^IJBLICATIONS other tHAli AfiStftACtS t'ROM tHISFROJECT DURING 
CALENDAR YEAR 1955 



None. 






16. _^^ ' __ 

ttdl^ORS AJiD AWARbS To t>ERSONNEL ft£LATiNG To tHiS ^ftOjECf 
DURING CALENDAR YEAR 1955: 

Elected to membership "Who's Who" In Chemistry. 



PAGE 1 
PROJECT REPORT 



1, National Cancer Institute 2, Laboratory of Physiology 

INSTITUTE """ LABORATORY OR BRANCH 



3. Cancer Physiology Section 4. 5. 902 

SECTION OR SERVICE LOCATION (if other than Bethesda) SERIAL NO, 



6. The Effects of X-irradiation on the Nucleic and Protein Synthesis of the Small 

Bowel Epithelium of the Rat as Related to Cell Division 

|»ROJECT TITLE ~" ^~™~ '' '- 

7. Julius White ' - . ,' . 

PRINCIPAL !NVESTIGATOR(S) '^. 

8. Dr. R. Bland Williams, Naval Medical Research Institute; 

Dr. James C. Reid and Jane N, Toal. ■ . : 

OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION 

Objectives ; To correlate changes in nucleic acid and protein synthesis with 
histopathological changes in the small bowel epithelium produced by 
x-irradiation. 

Methods Employed ; Rats are exposed to total body x-irradiation of varying 
concentration (from threshold to lethal dose) after receiving a known 
quantity of N^° labeled glycine, Histopathological changes in the small 
bowel with respect to mitotic arrest, acute cell destruction, prolongation 
of interphase, overshooting in recovery and incomplete recovery are 
made. Changes in protein and nucleic acid synthesis are determined to 
parallel the histopathological studies. 

Major Findings ; It was previously demonstrated that with an arrest in cell 
division there was always associated an arrest in DNA synthesis but no 
depression in protein synthesis. This occurred if the x-irradiation 
exposure was 450 to 2, 000 r and the synthesis studied 0-5 hours after 
exposure. But if examined 68-73 hours after exposure (450 r) there is 
a threefold increase in protein synthesis and an eightfold increase in 
DNA synthesis. This increased synthesis has been associated with the 
cells which were in interphase and are now dividing. 



902 .''HQ^i#l%l!iOft^''' PAGE 2 

SERIAL NO. 



In the preseht ferries, the tdkl dose of x«ir radiation was fractionated in 
which the tiss'ie was allowed ta recover before repeating the initial dose 
and to determine the effect of keeping the total dose and total time con- 
stant/ bi>t varyiag th^ size of |hoip4ivi±aal dbs^is.. Divided doses were 
; mdre ek'h'cti%'thkti k single dos 3 1'^ enhancing recovery (prolongation of 
interphase) or preventing destn.ction of crypts. Protein and DNA 
^^ synther^EJ paralled Uiese histopjithplogical findings. Exposure to 600 r 
"mB iolloweci by a slmiiir^^^^d^^^ later repeated the' delay in division, 

but when givsn 12 hours altt^r the ifiitiai irradiation, there results a 
severe destruction of large numbers of crypts, with a marked arrest in 
DNA sjmthesis* Nine hundred r were given over a 12-hour period or 
225 r every four hours; as 450 r followed 12 hours later by 450 r; and 
as 700 r follcwed 12 hours later by ^00 r, respectively. The degreie of 
recovery of DHA synthesis was depenient Upbn the size of the individual 
doses. DMA synthesis (recovery) wae greatest following 4 doses of 
225 r and least after ,700 r followed ^2 hours later by 200 r. 



Signific ance to Ca n^^er Research; To get a better TihdQrstanding of the hlsto- 
pathokig?cai aid chamicai changes which take place in rapidly pro- 
liferating tissue (epithelial lining of the small bowel) following 
x-irradiation, which may be useful in tumor therapy by x-irradiatlon 
and/or chemotherapeutic agents. 

Propbs^d bd^ii'se of prdject ; To rbuhdoff the program reported above and to' 
present the data obtained for' publication. 



■'■;(■;, .1. '■y<{'J 



«:^,'d3'i% ]^.riJfc9.S,i:;'^ '. • '■ /latifOi*^'^ *:• 






10. 902 

SERIAL NO. 



PAGE 3 



U. 



BUDGET riCTIVITY: 

RESEARCH [YJ ADMINISTRATION [^ 

REVIEW AND APPROVAL [^ TECHNICAL ASSISTANCE [^ 



12. 



COOPERATING UNITS OF THE PUBLIC HEi^XTH SERVICE, OR OTHER 
ORGnNISiViIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN EITHER 1956 or 1957. 

Naval Medical Research Institute, National Naval Medical Center, 
Ee:hesda, Maryland, 



13. 



IF THIS FRO JS C.r RESEMBLES, COMPLEMENTS, OR PARALLELS 
RESEARCH DO^IS ELSEWHERE IN THE PUBLIC HEALTH SERVICE 
(WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUNDS), 
IDENTIFY SUCH RESEARCH: 



NO ENTRIES FOR ITEMS 14, 15 and 16. 



fei •U'7-i'\''i 






MmihVk ,sit^Ua-.;i 



4 i. k^|U >\ 'i; 



M: him U ,1-li SM-.'iIl H^m 8^HTVi;-t •')j^f; 



PAGE 1 
PROJECT REPORT 



1. National Cancer Institute 2. Laboratory of Physiology 

INSTITUTE , LABORATORY OR BRANCH 



3. Cancer Physiology Section ,4» . 5 . 903 

SECTION OR SERVICE LOCATION (if other than Bethesda) SERIAL NO. 

■ ■ ■ ' a^ts? 

6, To determine factor(s) which produces cirrhosis of the liver in rats ingesting 

a low protein diet and exposed to 450 -500 r total body x-irradiatlon. 

PROJECT TITLE ~ 



7, Julius White 

PRINCIPAL INVESTIGATOR(S) 



OTHER INVESTIGATORS 



PROJECT DESCRIPTION 

Objectives : To determine whether transmethylating agents or lipotropic 

substances, when added to the low casein diet, will have any effect on 
the production of cirrhosis of the liver produced by whole body 
x-irradiation. 

Methods Employed ; Rats were fed one of four diets differing from each other 
only in the amount of choline, methionine or both. Four diets were 
employed! (a) 6% casein diet, (b) 6% casein supplemented with 0, 5% 
methionine, (c) 0. 5% methionine and 0. 5% choline, and (d) 0, 5% choline. 
All animals received 450-500 r total body x-irradiation. 

Major Findings : As reported in the last annual report, an incidence of 

approximately 50 per cent of cirrhosis of the liver was observed in the 
animals in group (a). No cirrhosis of the liver was observed in the 
other three groups of animals. Since methionine and choline are both 
intimately involved in transmethylation, it may be that the utilization 
of the low concentration of methyl donors present in the diet of animals 
in group (a) is influenced by exposure to x-irradiation and ample 
quantities of methionine or choline counteracts the irradiation effect. 



903 "' PAGE 2 

SERIAL NO. 



Significance to Cancer Research ; In no case was carcinonia 6t the liver 
produced. Yet, the fact that cirrhosis of the liver appeared in 50 
per cent of the animals on the unsupplemented diet suggests that the 
nutritional state of the animal can influence the effect of total body 
x-irradiation on the liver and; perhaps, other tissueis. 



Proposed course of project: None. 



10. 9Q3 PAGE 3 

SERIAL NO. 



U. • •' • ' 

BUDGET ACTIVITY: 

RESEARCH (T] ADMINISTRATION /~7 

REVIEW AND APPROVAL [~J TECHNICAL ASSISTANCE [^ 



12, ■ ' - ■• ' • • 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER 
ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR 
THIS PROJECT IN EITHER 1956 or 1957. 

None. 



13. ^ 

IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS 



RESE/iRCH DONE ELSEWHERE IN THE PUBLIC HEALTH SERVICE 
(WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUNDS), 
IDENTIFY SUCH RESEARCH. 

None, 



14» 903 ■ v<-::i^\iAlPAOE 4 

SERIAL NO. 



15 
1 PUBLICATIONS 6THER TH AN ABSTRACTS FROM THIS PROJECT UUKIINU 
^ CALENDAR YEAR 19551 'v;iad ■ 

\ V mks White, Charles C. Cohgdon, PhUip W. Davtd, and/ Mona S. 

Ally: Cirrhosis of the liver of rats following total body 
x-lrradiation. J. Nat. Cancer Inst. 15:X165-1163, 1955. 



..1. 1 






' HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT 



DURING CALENDAR YEAR 1955; 
None. 



PAGE 1 
PROJECT REPORT 



1, National Cancer Institute 2, Laboratory of Physiology 

INSTITUTE [ LABORATORY OR BRANCH 

' :, Departmeiit of Biology, Princeton 

3. Office of the Chief 4, University. Princeton, N. J. 5. 904 



SECTION OR SERVICE LOCATION (if other than Bethesda) SERIAL NO. 

6, Study of biological Effects of Ultraviolet Irradiation, ■ 

PROJECT TITLE 

7. Harold F. Blum 

PRINCIPAHNyESTIGATOR(S) ~"~" " " 



8, Drs, Elmer G, Butler, J. J, Chang, R. C, Mawe, S. E. Schmidt, and 
A, K, Parpart, of Princeton University; and Dr. J. W, Green of Rutgers 

University 

OTHER INVESTIGATORS '"" ~~ 



9. PROJECT DESCRIPTION 

Objectives ; to study the biological effects of ultraviolet irradiation with the 
ultimate aim of better understanding of the carcinogenic action of that 
agent, these studies aim to combine an experimental and theoretical 
approach. 

Methods Employed; The approach is a combined theoretical and experimental 
one. Using data available from our earlier work on the induction of 
cancer by ultraviolet light, a mathematical model has been constructed 
which explains certain aspects. Realizing that any such model can only 
" have meaning if related to real biological phenomena, experimental 
studies of various effects of ultraviolet radiation on living systems have 
been undertaken. Our model would indicate that the mechanism of cell 
division and its control are of particular importance in this problem, 
and hence the effect of ultraviolet radiation on this phenomenon has 
; been a principal preoccupation. Our studies haye extended into related 
. fields as this has seemed indicated. 



Q04 .,^,., PAGE 2 

SERIAL NO. 

Major Findings ; 

^* Studies of regenerationi after ultraviolet radiation In the larvae of 
the urodele anrtphibian . (la coUabora'don with Professor Elmer G» 

\i:. Butlei'j with the assistance of J* J. • Change R« p. Mawe andS, E, 
Schmidt,) Ws have been concerned with qusstions of dosage, and 
relationships to photorecovery, as a necesoary matter of background, 
but oitr mo.fit intsrestii^ study has; been a rather extensive, comparison 
of regcensratlon cf irradiated and normal limbo after amputation, A 
rather complex situation is discovered by these experiments: The rate 
of regeneration is reduced in the irradiated limb; the degree of re- 
duction varies with the time at which the amputation is performed, and 
doea not follow the course of regression, Photorecovery is observed^ 
illumination for two days after the irradiation greatly Increasing the 
regenerative capacity toward normal, and reducing the development 

; of abnprmalities to a negligible number,. Tiiere .seems -to be no close 
correlation bei;v/9en regressionof the limb, regeneration of the limb 
or the incidence of abnormalities in the regenerate} our experiments 
clearly separating thes6 different aspects, apparently for theiirst 
time, A manuscript describing these experiments has been prepared 
for publication in the Journal of Cellular and Comparative Physiology 
and will be subm.itted for approval in the near future, 

^ : II, . Effects of uLtra^violet radiation gn mouse skin , (Begun in coUab- 
■ oration with Drr R;, C, iVIawe with the assistance of Mrs, F, 
CorA,3tance; ) In these STadies it is planned to follow, the changes in 
mouf^Q skin resulting from ultraviolet radiation, with- particular 
atte:).tIon to the course oi hyperplasia and its regression. Analysis of 
our earlier studies on earcincgenesis by ultraviolet radiation indicate 
that th-ase^ events may have important bearing on that process, A 
method for reproducible, quantitative dosing of mouse, ears has been 
.worked ov-fc and some hintological stadias have been made. More 
precise hietological methods are naoded, and these are at present 
being worked out by Mr, Vincent Gregg, . technician to Professor 
■-: . Butier, , '^ ■!'■'-.: :- ^ ■ I i- .- ■■■■'. ... . .:■;..■.'.■ 

m. studies of! henroU'sis by ultra violet radiatio n. The question of 
■r..- I lysis by this agent waS' reopened bFo^ stu;die&"on cytolysis of 

Arbacia eggs, carried out at, the Marine Biological I^abpratory, Woods 
Hole, in the summer cf 1952. (Blum, Cook and Loos, J, Gen, Physiol,, 
37j 313, 1954.) Subsequently, John S. Cook undertook studies of 
ultraviolet hemolysis as his thesis for the Ph, D, degree, under my 
supervision. In an extensive paper, soon to be published in the 
J. C. C, P. , Dr, Cook lias shown two new things of particular interest: 



904 PAGE 3 

SERIAL NO. 



(1) that hemolysis by this agent is a second order process, and 

(2) that ultraviolet radiation of short, wavelengths (0,1860jli to 0.2500|i) 
is much the iiiost effective part of thfe mercury arc spectrum for 
producing the hemolysis. Dr. Cook is now in Bern, Switzerland, on a 
Public Health Fellowship, studying effects of ultraviolet radiation on 
nerve with Prof esjsor Alexander von Miirklt. 

Dr. Cook and I began together a study of the O, dependence for ultra- 
violet hemolysis, whi(± was suggested by his studies, and had just 
turned up some unexpected things, when he left* I am just resuming 
these studies, after the Construction of more adequate apparatus. It 
does not seem wise to report our results to date, because they need 
repetition and expansion, but at the present moment they seem rather 
fundamental, and may perhaps bear on the (to my mind) confused 
picture of O^ dependence in effects of ionizing radiations, 

IV. Studies of photosensitized erythrocytes . (In collaboration with 
Professor J. W. Green, Rutgers University, and Professor A. K. 
Parpart, Princeton University. ) Studies on permeability of 
erythrocytes photosensitized with rose bengal and exposed to visible 
light, have shown that this treatment results in increased permeability 
to K"^ and Na"*" ions, but does not alter the metabolism of glucose by 
these cells. These studies are being continued. An abstract is being 
published in the Journal of Cellular and Comparative Physiology. 

V, Polyspermia in the Arbacia egg . An exploratory study involving 
quantitative determinations of polyspermia in the Arbacia egg was 
carried out with Dr. Murray Eden at the Marine Biological Laboratory, 
Woods Hole, in the summer of 1954. The analysis of the extensive 
data we obtained has only now been completed in Dr. Eden's labora- 
tory. It is hoped to prepare this material for publication in the near 
future. 

Plans for work at the Marine Biological Laboratory, Woods Hole, 
during the past summer, were upset by my contracting whooping 
cough at a crucial time. This prevented any extensive experimental 
work, so the summer was spent chiefly in reading, writing, and 
working up with Professor Butler the results of our investigations on 
amphibia, carried out at Princeton during the spring. 

Significance to Cancer Research ! To throw light on the relationship of 
ultraviolet exposure to carcinogenesis. 



904 PAGE 4 

SERIAL NO. 

PfoposBd course of projfect : Experiments designed to explore further the 
. effects of ultraviolet radiation ©n rejgeneratiori are. now' under way. 

. Resumption of study of the Oj dependence for ultraviolet hemolysis 
using more ade(3[uate apparatus. 

Continuation of studies of photosensitized erythrocytes, 

the synthiBtic approach to the problem of caVcinbgiBnesis by ultra- 
violet radiation will continue^ ' 



Silfiqa '.^iii'^ritiift m 



10, 904 PAGE 5 

SERIAL NO. 



11. 

:'u BUDGET ACTIVITY; 



RESEARCH /Xj ADMINISTRATION /~7 

J;a.^.;^; jREVIEW^AND APPROVAL /C7 TECHNICAL ASSISTANCE fZ/ 



li. ^^ _ • _■ -■-■ — - • ■ ■ ■ .-■ 

- COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER 
ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR 
THIS PROJECT IN EITHER 1&56 or 1957. 

Department of Biology, Princeton University, 



IF THIS- PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS 
RESEARCH DONE ELSEWHERE IN THE PUBLIC HEALTH SERVICE 
^' (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUNDS), 
IDENTIFY SUCH RESEARCH: 

: •::;•■; ^•:- None, ' "■'''^•''' '•'•^■' • " -^iio^'-^ntm'^ ^ > v^^ 

• ';;•:?:;■, ■ •■..•; .v.. . . ■ ■•.V'..: ;IA ^ . . •• '. - ■ ^ '• . ^'1 ftt- ^ ^'i ■ 






Uh^iJA W i,V' 



14, 904 PAGE 6 

SERIAL NO. ' 



15. ;- 

PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
CALENDAR YEAR 1955: 

Butler, E.G.,. and Blum, H. F. : Regenerative growth in the urodele 
foriBiinib fdiiowing ultravlQlet radiation. J, Nat. Cancer Inst. 15: 
, . . a77-e89, 1955„ , ;., . . 

Blum, H. F.: Sunburn. In Radiation Biology (HoUaender, A., ed.). 
New York; McGraw-lUri, 1955, Chapter 15, pp. 487-528. 

BIum> Hi F.J Ultrd violet ■Miakoft' and 'Cancer, Ig Radiatibft Biology 
■ (HoUaender, A., e4 )/ New Yorkj McO^ 1956, ''^ 

Chapter 14, pp. 529-559!* -/ ^' ■ • ' ' ^^ 

Blum, H. F.: 'Siinlight as an environmental faictor in Cancer of the skin. 
Military Medicine 117: 202-208, 1955. 

Blum, H. F,: Timers Arrow and Evolution. 2nd Edition. Princeton 
University press, 1955, Princeton. 

- Blum, Hii Fi I i Perspectii^efe in Evotation. Ameirican' Scientist 43i 
•; ■> ■^■■■595-610,. 1955.^-^'-^ hI -■ hii.rr-i-'-v ,^.:.y:i ■'. ■ .fji' - ^^. a:-:::.x 

Papers describing work carried out under ;my super Visions i 

Zimskin, P. D, , aEid Schisgall, R, M.: Photorecovery from 
ultraviolet-induced pigmentation changes in anuran larvae. 
J. Cell. Comp. Physiol, 45: 167-176, 1955. 

16. 

HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 1955. 

1. I gave the initiation lecture to the Sigma Xi chapter of the 
University of Vermont, Burlington, on April 26, The title was 
Perspectives in Evolution . 

2. I took part in a symposium on Biogenesis together with 
H, C, Urey, Philip Abelson, Sidney Fox and George Wald, 
as part of the Centennial Celebration of the Polytechnical 
Institute of Brooklyn on May 6. My position in the symposium 
was that of summarizer. 



904 PAGE 7 

SERIAL NO. 



3. I presented a paper in a symposium at the Dedication of the 
Armed Forces Institute of Pathology, Walter Reed Medical Center, 
Washington, D. C. , on May 27» The title was Sunlight as an 
Environmental Factor in Cancer of the Skin. 



PAGE 1 
PROJECT REPORT 



1. National Cancer Institute 2, Laboratory of Physiology 

INSTITUTE. ; ';■' ■'■"^r.'--- LABORATORY PR BRANCH 



3. Cancer Physiology Section 4, • 5» 905 

SECTION OR SE;RVICE ■ LOCATION (if other than Bethesda) SERIAL NO 

C Iodide Concentra:ting.]VJfechanism in the Thyroid Gland . 

PROJECT TITLE . j~— ^- , ■ • .■ , , — 



S. H. Wollman 

PRINCIPAL INVESTIGATOR(S) 



Dr. R, O, Scow of NIAM D 
OTHER INVESTIGATORS^ 



PROJECT DESCRIPTION 

Objectives ! To learn about the mechanism by which the thyroid gland main- 
tains a concentration of radioiodide elevated above that in the blood and 
about the way the thyroid radioiodide and iodide concentrations influence 
the uptake of radioiodine. 

Methods Employed; Kinetic studies will be made of the ratio of radioiodide 
concentrations in the thyroid gland and serum and factors which control 
it. 

Major Findings ; A study was made of the inhibition of the iodide concen- 
trating mechanism by thiocyanate. The dependence of the ratio of the 
radioiodide concentrations in thyroid gland and serum on the concen- 
trations of iodide and thiocyanate in the same animal was examined. 
The observed data can be fit quantitatively by two qualitatively 
different models, an adsorption model and an active transport model. 
In these models iodide complexes with a substance in the thyroid, one 
type of site is involved with no interaction between sites; thiocyanate 
also complexes with the same sites and competes with iodide for the 
sites. The affinity of the site for iodide is twice as great as for 
thiocyanate. The affinity for thyroid sites is more than an order of 
magnitude greater than that for serum albumin sites. With the 



905 rVvvyr.':.. .^ ■;:-,- ; PAGE 2 

SERIAL NO. 



- observed constantsy the adsorption model requires thiocyanate Ije. 
concentrated^ That it is not concentrated in rat and sheep thyroids , 
suggests that the iodide concentrating mechanism Is not an adsorption 
but an active transport process. , :.:;, 

Significance to Cancer Research : Transplantable tumors of the thyroid gland 
in mice appear to have a marked impairment in or loss of the iodide 
concentrating' mechanism. This loss appears directly .responsibl^i for 
the loss of ability of certain lines of thyroid tumors to accumulate 
radioiodine. The above experimental results are directeid largely to 
provide an understanding of the iodide concentrating mechanism and 
the factors controlling it, ' . 

Proposed course of project : Further kinetic studies on the iodide cohcen- 
trating mechanism will be undertaken. In addition, a more direct 
attack will be made on the problem of deternalning the factors w|iich 
most seriously limit the accumulation of radioiodine. 



OioTfM'J" ■ ■ " . •■• •"" • ;, . ■,, , 5 ■•.■>«-^if?:;;-^'*..Si'':-i«Pj 



y 0h 



10. 905 PAGE 3 

SERIAL NO. 



11. 

BUDGET ACTIVITY; 

.V RESEARCH flT/ ADMINISTRATION / 7 

REVIEW AND APPROVAL /~7 TECHNICAL ASSISTANCE/ 7 



12. ___■ 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER 
ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR 
THIS PROJECT IN EITHER 1956 or 1957, , 



NIAMD, Dr. R. O. Scow 



13. 

IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS 
RESEARCH DONE ELSEWHERE IN THE PUBLIC HEALTH SERVICE 
(WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUNDS), 
IDENTIFY SUCH RESEARCH! 



This project may complement research in the Clinical 
Endocrinology Branch, NIAMD. 



14. 905 PAGE 4 

SERIAL NO. 



15. , ^ .... ■ . ■ 

PUBLICATIONS OTtlER TfiAN ABSTRACTS FROM TdlS PROJECT DURING 
CALENDAR YEAR 1955; 

S, H. WoUman and R. O. Scow; . Effect of various goitrogens and of 
dose of ptopylthiouracU bn thiB ratio of radibiodlde con- 
centrations in the thyroid gland and serum in mice. 
Endocrinology 56; 448-454, 1955. 

. ; . S. IL Wolirfidii .arid R, Q, ScQ^';, Effect of propylthiouracil oh the 

' ratio of the radioibdi^/s 6pA<?^".trati^^ 

serum in normal ahti hypbphysectomlzed rats. 

Endocrinology 56; 445-447, 1955. 



16. 



HONORS AND AWARDS TO'PERSONNEL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 1955. 



Elected tb the American Physiological Society. 



PAGE 1 
PROJECT REPORT 



1. National Cancer Inatitute 2. Laboratory of Physiology 

INSTITUTE LABORATORY OR BRANCH 



3. Cancer Physiology Section 4. . 5. 906 

SECTION OR SERVICE LOCATION (if other than Bethesda) SERIAL NO 

131 
6. Transplantable Thyroid Tumors: Production and I Metabolism, 

PROJECT TITLE 



7. S. H. Wollman . ; ;;vn4 



■JuaXm" IITt^o ■ aTlwjT)>ii ^ 



PRINCIPAL INVEStlGATOR(S),::S3^^o.l--:'J--^:^' ''^I'.-J'V^ ll'^t^:^ \:/^ 



8. Dr. R. 0. Scow of NIAMD 
OTHER INVESTIGATORS 






9. PROJECT DESCRIPTION • f^^.mTHiPrf^^ 



Objectives : '^?^^^i 



^a) To produce transplantable thyroid tumors in the rat and the mouse^ 
(b) To compare iodine metabolism in thyroid tumors and normal 
thyroid. 

Methods Employed: 

(a) Thyroid tumors are being produced by chronic feeding of thiouracil. 

(b) Iodine metabolism was studied with the use of radioactive iodine. 

Major Findings; 

(a) We now have transplantable thyroid tumors in both mice and rats. 

These tumors will grow in hosts fed goitrogen free diets. 

(b) No new results. 

Significance to Cancer Research : Tumor material will now be available for 
radioiodine studies in two experimental species. 

Proposed course of project ; Studies of the radioiodine metabolism of thyroid 
tumors in the rat will be undertaken this year. 



jQ QQg T^cmam yD:;!&c.vR page 2 

• ' SERIAL NO. 






BUDGET AC'TIVITV: 

...|^...RlSEAI^Ca.-.^^.,-._.,-.^-^. m . ADMINISTRATK)Nyo.^ :...... O^ 

REVIEW AND APPROVAL [J TECHNICAL ASSISTANCE £7 

-—'—-■— — — - -- " '"'liSlWTJMm'i 

12. 



COOPERATING UNITS OF TH £ PUBLIC HEALTH SERVICE, OR OTHER 
SrOANI^SI^S, PROVmiNG Ji;i^, FA^^^ 



THIS PROJECT IN EITHER 1956 or 1957 
Dr. R. O. Scow, NIAMD 






RESEARCH DONE ELSEWHERE IN THE PUBLIC HEALTH SE^VICE^ 
(WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR F^pS)^. 
IDENTIFY SUCH RESEARCH: 

U^m flhWjaiprojeet may p^s^wiW^ .?;f Sif arch iP; tlie; l^appr^xqi^ |0i . ; 

Biochemistry, NCI, ,;i.;ii; ,,!) 



14. 906 

SERIAL NO, 



PAGE 3 



15. 



Place following wording on front 



""'^^' THIS PROJECT DURING 



of thyroid lobes, 
Growth and I^^^ 



7, 1955. 



...ijonSj IN- 



ACTIVITIES 
OF HEALIH 



STIKTE 






G TO THIS PROJECT 



NATIONAL INSTimrES OF HEALIH 
PUBLIC HEALTH SERVICE 
U. S. DEPARIMEKT OF HEALffi, EDUCATION, AND WELFABE 



, „ "' ' ^^a "TSCB^-rflJ tDM^Cm'i PAGE 2 

lu. yuo 

SERIAL NO. 

u. "• '•■ • • •- • • • •••'•• — 

BUDGET A'JTIVITY: 
' ^''''" REVIEW AND APP^VALO ASSISTANCE fj 

i t' i 

■■^•^"^^'"'"^' ^'"- ■ ■• ^- -— --^ — ~ - '^swf''^M:ms 



^^* - rnnpyY<ATING UNITS OF TH£ PUBLIC HEALTH SERVICE, OR OTHER 



li 



THIS PROJECT IN EITHER 1956 or 1957. 
Dr. R. O. Scow, NIAMD 






I 



13. 



IF THIS PROJECT' KESEMiiLfi^, ^"MPLKMi;OT^^;J^KALljEI|.^,, , 
RESEARCH DONE ELSEWHERE IN THE PUBLIC HEALTH SERVICE 
fw™T mTERCHANGE OF PERSONNEL, FACILITIES OR.,,^WpS), 
IDENTIFY SUCH RESEARCH: 

i^;^^. r1?tots(ipr^e6tirnay ij^S^w^^i^e^airph ln:the;%a^qr^qryiO^,:; 
Biochemistry, NCI. ,;;;-: ,;,ii 

jiis-i"?. hiii;, ^M\>->i i\i*'xi !,d 'y^tiotviiiKi sua-- ,^r!.s •v'f:7?,?«t-;i!K.i^fj.i? rtij<.;i %'mt 3W In^) 



L4, 906 PAGE 3 

SERIAL NO. 



15. 

PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 



CALENDAR YEAR 1955 

S. H. Wollman and R, O. Scow: Comparison of thyroid lobes, 
autotransplanted and in situ, in the rat: Growth and I^^^ 
uptake, J, Nat. Cancer Inst. 15: 943-947, 1955. 



16. 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 1955. 



None 



, k 






PAGE 1 
PROJECT REPORT 



, National Cancer Institute 2, Laboratory of Physiology 

INSTITUTE " LABORATORY OR BRANCH 



I. Cancer Physiology Section ; 4^ 5. 907 

SECTION OR SERVICE LOCATION (if other than Bethesda) SERIAL NO. 

131 
J. Autoradiographic Localization of I in Normal and Neoplastic Thyroid Tissue 

PROJECT TITLE 



K S. H. WoUman 

PRINCIPAL INVESTIGATOR{S) 



J, Mr. 1. Wodinsky, Laboratory of Chemical Pharmacology, NCI 
OTHER INVESTIGATORS 



). PROJECT DESCRIPTION 

Objectives : , ; ^ 

(a) To locate the sites of high concentration of radioiodide in the 

thyroid gland, 

(b) To locate the site at which the binding of radioiodine occurs in the 

thyroid gland, and to determine changes in the localization of 
the I^^^ after binding. 

Methods Employed ; High resolution autoradiography. 

Major Findings ; No new results this year. 

Significance to Cancer Research ; Localization of I^^^ by autoradiographic. «, 
techniques is Important in understanding of mechanism of uptake of I 
by thyroid gland and thyroid tumors. 

Proposed course of project; To be continued. 



■10- 907 . r^mm xo-iLOH^ page 2 

SERIAL NO. 



BUDGET ACTIVITY: 

RESEARCH /S7 ADMINISTRATION O 

REVIEW AND APPROVAL £[7 TECHNICAL ASSISTANCE /~7 



■12. ' '-■'.- ■ '• I } -^ 

COOPERATtNti UtJlTS Ot" The Pt)6LlC HEALtH SfiftVlCfi, OR OTHER 



ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR 
THIS PROJECT IN EITHER 1956 or 1957, 

Laboratory of Chemical Pharmacology, NCI, 



J3. ^ 

If THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS , 
RESEARCH DONE ELSEWHERE IN THE PUBLIC HEALTH SERVICE 
(WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUJpS), ; 
IDENTIFY SUCH RESEARCH: 

None •" ' ... - j..f;^,..; ctt-'i-j^iii 



J ;!;■*. !u'.'.-..i ':>••>. ';-;.; .^itaj,Oi.. .' ' ' .' -,' V'iH. 



14. 907 PAGE 3 

SERIAL N0» 



15. 

PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS I^ROJECT DURING 



CALENDAR YEAR 1955 



S, H. Wollman and I. Wodinsky: Localization of protein-bound I ^ 
in the thyroid gland of the mouse. Endocrinology 56: 
9-20, 1955. 



16. 



HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 1955. 



None 



PAGE 1 
PROJECT REPORT 



I. National Cancer Institute 2. Laboratory of Physiology 

INSTITUTE LABORATORY OR BRANCH 



. Cancer Physiology Section 4. . ' - ,■:,.-: -., 5. 908 

SECTION OR SERVICE LOCATION (if other than Bethesda) SERML NO. 



5. Theoretical Analysis of Carcinogenesis Based on the Mutation Hypothesis 
PROJECT TITLE ~ 



I. S. H. Wollman . . ■; ■;. ■ 

PRINCIPAL INVESTIGATOR(S) 



OTHER INVESTIGATORS 

). 'PROJECT DEeJCRIPTION> '. ;' ■/;;:•: : : : V 'ri- . f ; ; 

Objectives ; ^ To aid in the test df a- possible mechanism; of qar-cinogeneais. 

Methods Employed ; A simple quantitative model was designed. Predictions 
of the model were compared with dose-respons.e data in experimental 
carcinogenesis. 

Major Findings ; A model was designed which avoided assumptions about the 
growth rates of tumors, and instead involved total incidence of tumors 
of a particular type. Tests of the analysis do not appear possible at 
present because of the absence of suitable data. The suitability of most 
data is impaired by complications which are a consequence of the 
methods of administering carcinogens, by difficulties in making esti- 
mates of the number of tumors produced, and by dose -dependent lethal 
effects and dose -dependent indirect effects of carcinogens. 

Significance to Cancer Research ; Emphasizes need for further experiments 
avoiding interpretive difficulties recognized in the present analysis of 
available data. 

Proposed course of project ; This project has been completed. 



10, 908 :.•:;;:..- PAGE 2 

SERI/vL NO. 



li; • - " ' ' -■ ■-- "■■■ ■ •■.:/. 

BUDGET ACTIVITY: 

V V . RESEARCH /Tk/ ADMINISTRATION / 7 

REVIEW AND APPROVAL / 7 TECHNICAL ASSISTANCE/ 7 



12. ■ ■ - ■ ' • ./• 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER 
ORGANIZATIONS, PROVIDING FUNDS, F;.CILITIES, OR PERSONNEL FOR 
THIS PROJECT IN EITHER 1956 or 1957. 

None 



13. 

IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS 
RESEARCH DONE ELSEWHERE IN THE PUBLIC HEALTH SERVICE 
(WITHOUT INTEjR CHANGE OF PERSONNEL, FACILITIES OR FUNDS), 

... IDENTIFY SUCH RESEARCH: 



14. 908 PAGE 3 

SERIAL NO. 



15* 

PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 



CALENDAR YEAR 1955 



WoUman, S* H,; Comments on the analysis of dose-response 
data in experimental carcinogenesis. J. Nat. Cancer 
Inst. 16: 195-204, 1955. 



16. ^ 

HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 1955 

None 



>Vi\\ 



PAGE 1 
PROJECT REPORT _ v,> 

1, National Cancer Institute 2, Laboratory of Physiology 
... . INSTITUTE LABORATORY OR BRANCH 

5, Cancer Physlology''Section h» 5, 909 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) SERIAL NO 

6, Metabolic Fate of the Histldlne Side Chain 

.PROJECT TITLE ... . . " __ [^ I ^ 

7. James C . Reid ■ . ■"'.'..'" . 

PRINCIPAL INVESTIGATOR (S) ' "' '■ ■ ' ' 

8. Florence K, Millar :■■■-■': 

OTHER INVESTIGATORS 

9» PROJECT DESCRIPTION 

Objectives : The purpose of this pToject is Ibo acquire more information on the 
process by which histldlne is used f pi!'. th6 .synthesis, of nucleic acldt 

Methods Employed : Radioactive carbon is beitig used as a tracer to study the 
problem. 

Major Findings : Administration of DL-histidine labeled in the side chain with 
radioactive carbon leads to the formation of labeled aspartic acid, gluta- 
mic acid, alanine and threonine. In addition ^ nucleotide fractions 
derived from ribonucleic acid are highly radioactive. Before these 
results can be confirmed and interpreted it is necessary to resolve the 
histldlne so that the L-form alone can be given* A resolution technique 
has been worked out. 

Significance to Cancer Research ; The significance of these findings to cancer 
research is that they add to our knowledge of the metabolism of protein 
and nucleic acid. Both of these substances are important for the growth 
of cancer. 

Proposed course of the project ; During the coming year the labeled DL- 

histldine will be resolved and the L-form will be used to confirm and 
extend the findings made to date. In particular , chemical degradation 
of labeled metabolites will be carried out to gain infoimatlon on the 
probable mechanisms of the transformations. 



10' 909 



SERIAL NO . 



11. 



l^vicJ^I'jr 






PAGE 2 



BUDGET ACTIVITY: 



RESEARCH.. .^ 
REVIEW & APPROVAL £J 



ADMINISTRATION /~J 
TECHNICAL ASSISTANCE £J 



12. 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 195^ or 1957. 



'^ ^'■IbjiimiAQim^vii 



None 






15. 



IF THIS PROJECT RESEMBLES , COMPLEMENTS , OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDENTIFY SUCH RESEARCH: 



None 



111 • .an 



HO ENTRIES FOR ITEt© ih, 15 and l6« 



ifixl 



Jllw e- 
^ aiiS 'r- 



PAGE 1 
PROJECT REPORT 



.. National Cancer Institu te 2. Laboratory of Physiology 

INSTITUTE LABORATORY OR BRANCH 

5. Cancer Physiology Section h. __^ ^. 910 

SECTION OR SERVICE ' LOCATION (IF OTHER THAN BETHESDAJ SERIAL NO, 

). Tumor Autora d iography. ^ ,__ 

PROJECT TITi£ 



^ James C. Reid 

PRINCIPAL INVESTIGATOR (S) 



5. Julius White 



OTHER INVESTIGATORS 
). PROJECT DESCRIPTION 



Objectives : It is desired to obtain information on the circulation pattern in 
tumors and the stability of tumor protein. 

Methods Employed : Animals bearing tumors are dosed with radioactive amino 
acids and J after various periods of time^ the tumors are removed and 
sliced. The slices are placed in contact with photographic film. 
After development^ the film bears an image vrtiich shows the distribution 
of radioactivity through the slice. 

Major Findings : When radioactive glycine is administered to rats bearing 

the Walker 256 tumor; isotope is found in the tumor tissue within one hour 
but cannot be demonstrated in 10 minutes by the methods employed. 
Radioactivity is found in substantial amount as late as 11 days after 
dosage. Necrotic areas present in the tumor when the glycine is admin- 
istered do not become labeled. Conversely ^ if an area becomes labeled 
and subsequently becomes necrotic the fixed isotope is not lost. The 
inertness of the protein metabolism of necrotic tissue is thus established. 
It is also desired to obtain information on the possibility of gradients 
within the viable tumor tissue. This is the major aspect of the in- 
vestigation and bears not only on the circulation pattern but on the 
question of the degree to which tumor protein is in labile equilibrium 
with host protein. No certain conclusions can be drawn on this point 
yet. 



i 



,10 

ERIAL NO. 



PAGE 2 



Significance to Cancer Research: The question of the relationship between 
the nutrition of a tumor and that of its host is an important one in 
understanding the uncontrolled growth of tumors. These experiments 
represent another mode of approach to this question. The information 
gained is also of interest in connection with the question of how 
necrosis affects the over-all metabolism of a tumor. This problem is 
a troublesome one for investigation of tumor chemistry. 

Proposed course of project : The next step in the investigation will be to 
check the results obtained with glycine by repeating the experiments 
with labeled lysine. Glycine may not be the most suitable amino acid 
for this work because it undergoes extensive biochemical side reaction 
in addition to protein formation. It is then planned to extend the 
experiments to other tumors and; if it seems necessary > to other amino 
acids . 



1<^' 910 PAGE 5 

SERIAL NO, 



11. 



BUDGET ACTIVITY: 

RESEARCH jT} ADMINISTRATION [J 

REVIEW AND APPROVAL [J TECHNICAL ASSISTANCE [J 



12. 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER I956 or 1957. 



None 



13. 



IF THIS PROJECT RESEMBLES, COMPLEIvIENTS , OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDENTIFY SUCH RESEARCH: 



None 



NO ENTRIES FOR ITEMS 1^+, 15 and I6. 



PAGE 1 
PROJECT REPORT 



1. National Cancer Institute 2. Laboratory of Physiology 

INSTITUTE LABORATORY OR BRANCH 

5, Energy Metabolism Section k-. 5' 911 

SECTION OR SERVICE ' LOCATION (IF OTHER THAN BET^HESDA) SERIAL NO, 



6. Energy Metabolism Project 



PROJECT TITLE 




7. A. W. Pratt W. C. White and F. K. Putney 




PRINCIPAL INVESTIGAl'OR(S) 




8. 





OTHER INVESTIGATORS 

9. PROJECT DESCRIPTION 

Objectives : The overall objective of this program remains the continuous, ac- 
curate measure of the energy metabolism of small experimental animals 
under different experimental conditions for prolonged experimental 
periods. 

As the program progresses, certain immediate or relatively short term 
objectives may be examined in relation to accomplishment. The immediate 
objective of this reporting period relates to the performance of the 
total proced\ires and specifically the instrumentation^ in the continuous 
routine study of tumor-bearing animals. 

Me t h ods Employed : Not applicable. 

Major Findings : 

a) Instrumentation 

At the end of the last reporting period, it was considered that the 
short term studies conducted on the energy exchanges of rodent experi- 
mental animals indicated the need of further instrumentation on the 
recording -measuring equipment if continuous, accurate measureswere to be 
accomplished. To this end, modified computer techniques were adapted to 
the instrument in the fom of an analog to digital converter measuring 
and recording system. The new device (teleducer-telecomputer) was es- 
sentially a multi -channel system which would allow continuous measure 
and recording of all the desired parameters, i.e. Os output, COa output, 



911 PAGE 2 

SERIAL NO. 



radiation chamber, cage and room temperature comparisons, etc. In addi- 
tion, we are afforded the advantage of being able to convert the total 
system to a fully automatic processing of the total data should such be 
indicated. It is apparent that the addition of this new component 
solved the problem of continuous, accurate measure of low value d.c. 
potentials over prolonged experimental periods while affording a com- 
plete printed tabulation of all data. The new instrument substantially 
reduced the number of technician man-hours needed for overall data 
handling. 

As with any electromechanical device of this complexity the expected 
electronic, difficulties presented themselves once the instrument was in 
continuous operation. While they led to extensive "down-time" in the 
early application, the development of "by-pass circuitry" by our group 
has made the instrument into a dependable, accurate component in our 
application. 

In summary, it would appear that the study of four individual animals 
for an average continuous experimental period of 2.5 months each indi- 
cate that the new instruments will accurately and continuously measure 
the energy exchanges of the rodent experimental animal. In addition, 
three animals have been studied in the Hays instrument. While this in- 
strument has perfonned satisfactorily, it is apparent that only day to 
day monitoring can be achieved with this instrument. 

b) Animal Findings 

The animals under study were Osborne -Mendel males of an inbred strain. 
The dietary regime was 20^0 casein with added vitamin supplements and in- 
organic salts . The experimentaJ. period consisted for each animal for 
approximately JO to 80 days. The experimental period was divided into a 
control period, induction period and a tumor period. During the control 
period observations were made on the normal animal and the energy intake} 
energy expenditure was determined in the usual fashion. When it was cer- 
tain that the control period was adequate in length of time to reflect 
the metabolic behavior of the animal, a Walker 256 transplant was ad- 
ministered by sterile trocar and the observations on the animal con- 
tinued. The induction period spanned the time between transplant and 
the point when the tumor was first palpable in the animal. After the 
tumor was palpable the tumor period was initiated and ended when the 
animal was sacrificed. Maximum growth of the tumor was very carefully 
regulated in that no animal was allowed to grow a tumor larger than l8^ 
of the total host-tumor vreight. 

The general findings which are discussed below reflect the average be- 
havior of tumor-bearing hosts studied under the conditions mentioned 
above. Following tvmor transplant the oxygen consumption as expressed 
in liters per day abruptly rises to a level significantly above that 



911 PAGE 5 

SERIAL NO. 

observed in the control period , reaching a maximum on the third or fourth 
day. The maximiam is followed by a decrease which; at the time of appear- 
ance of tumor, is at a new level intermediate between the highest level ob- 
served in the control period and the highest level observed in the in- 
duction period. Carbon dioxide production roughly parallels the oxygen 
consumption curve. In the tumor period, the oxygen consumption level 
gradually, but steadily, rises as the tumor contributes a greater and 
greater percentage to the total host-tumor weight. It has been our 
general observation that the carbon dioxide production in the tumor 
period tends to decrease during the tumor growth period with the net 
result of a fall in R. Q. and a significant fall in the non -protein R. Q. 
It is not, at the moment, possible to interpret this observation in the 
sense of a real finding. It isi not known whether the fall in non-protein 
R. Q. represents solely an increased metabolic usage of fat or whether, 
in addition to the increased usage of fat, there is a superimposed 
retention of carbon dioxide. With the fall in the value of the non- 
protein R. Q., the energy equivalent of calories per liter of oxygen 
consumed falls correspondingly, with the net result that the increase 
in oxygen is not a true reflection of the observed increase in energy 
expenditure as a function of tumor growth. We have always observed a 
significant slope in the increase of oxygen consumption throughout the 
induction and tumor periods. However, only in the induction period does 
an abrupt rise in energy expenditure coincide with the abrupt rise in 
oxygen utilization. In the tumor period, the fall in carbon dioxide 
produced to affect a more gradual or gentle slope in energy expenditure. 

Energy intake : The energy intake of the animal during the control period 
is always substantially in excess of the energy expenditure and the net 
change in body weight can be grossly correlated with the energy retained. 
In the induction period the difference between the energy intake and the 
energy expenditure is substantially reduced, but the energy intake remains 
in excess of the expenditure. In animals growing a tumor, which at max- 
imum is equivalent to approximately 15lo of body weight, it has been noted 
that the energy intake gradually reduces and this effect can be seen almost 
from the beginning of the tumor period. 

Energy expenditure : Ccsnparison of the energy expenditure of the animal 
in the induction period to the control period shows that, following 
transplant of the tumor, an abrupt and significant increase in energy 
expenditure occurs. The maximiim of this expenditure is observed on the 
third or fourth day following the transplant of the tvimor. The energy 
expenditure then declines, reaching levels at the beginning of the 
tumor phase which is intermediate between the highest value observed for 
the control period and the highest value observed during the induction per- 
iod. As the animal experiences progressive tumor growth, the level of 
energy expenditure graduaU.y increases. As the energy intake decreases 
and the energy expenditure increases, the animal may achieve a condition 
of isocaloric balance. It has been our obsei'vation that the contribu- 
tion of protein to the energy expenditure is roughly constant throughout 



911 PAGE k 

SERIAL WO. 



the control period^ induction period and for most of the tumor period. 
As the non -protein energy contribution to the energy expended becomes 
equal to, or larger than, the non -protein energy intake, the slope of 
the protein .energy expended begins to gradually increase. From our 
observations, it is clear that the excess energy expenditure observed 
in the induction period is entirely contributed to by non-protein 
materialB. In addition, the increased expenditure during the tumor 
period appears to be primarily non -protein in nature. The net result 
being that isocaloric balance between the non-protein energy intake 
and the non-protein energy expenditure is achieved very early after 
gross tumor grov/th is, apparent. , Use of protein for energy needs of the 
animal is sj^ared until a negative balance in the non -protein energy oc- 
curs. It has been our observation that the weight change of the tumor - 
bearing animal progressively rises as long as the protein balance is 
positive, even in the face of an isocaloric balance or perhaps a slightly 
negative balance; of the non-protein energy. Pi-esvunably, this is due to 
the retention of new protein body weight in the form of tumor, and, 
in addition, an extensive hydration of. the animal body as the tumor 
process progresses. ,, ; ' . 

Certain gross correlations may be made by comparing the energy expen- 
diture with other parameters. Weight change, in; the non-tumor periods re- 
flects the retention of prptein and non -protein energy in addition to 
■ water. The weight change during the tinior period is primarily dependent 
upon the protein energy balance and the degree of hydration. A high de- 
gree of protein sparing, occurs in the sense that a significant amount of 
protein will be retained and dep'osited as tumor, while the non -protein 
energy contributes almost entirely to the energy demands of the animal. 
The energy intake does not. maintain pace vn'.th the increase in- energy ex- 
penditure observed in the. induction and tumor periods. On the contrary, 
the anorexia substantially red.uces the animal's energy intake. 

Significance to Cancer Research : Study of the effects on the energy metabolism 
of a host animal, follov/ing tumor implant and during subsequent tumor 
■ growth, promises to yield significant information bearing on host-tumor 
relationships. The development of these new techniques for these studies 
should allow an accumulation of whole animal data which, together with 
the advances iri intermediate metabolism, should increase our understand- 
ing of the pa,thological physiology of the. tumor process. 

Proposed course of project; We believe vre Jiave been successful in accomplishing 
an accurate measure of energy expenditure .and energy XJ^ take and, thus, the 
energy retained. Our next immediate objective will be the, distribution of 
retained energy into "fat-free, body vreight" and "fat body weight" for 
correlation with observed weight gain. , 

In addition,- comparative studies on the body composition of the observed 
animals at the time of sacrifice are being, undertaken. 



911 PAGE 5 

SERIAL NO. 



The need of a direct calorimetric measure of the heat output of our 
experimental animals during these prolonged experimental periods is neces- 
sary if we are to ccsupare and interpret the animal's metabolic behavior. 
To that end we are beginning pilot construction on a direct calorimeter 
which will utilize the adiabatic principle. 



10. 911 . PAGE 6 

SERIAL NO. 



11. 



BUDGET ACTIVITY: 

RESEARCH /xj ADMINISTRATION [j 
REVIEW & APPROVAI. O TECHNICAL ASSISTANCE [J 



12. 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUl^lDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER I956 or 1957. 



None 



15. ^ 

IF THIS PROJECT RESElvBLES, COMPLEMEINTS , OR PARALLELS RESEARCH DONE ELSEl-JHERE 



IN TI-IE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDENTIFY SUCH PvESEARCH: 



None 



NO ENTRIES FOR ITEMS 14, I5 and I6. 



PAGE 1 
PROJECT REPORT 

1. National Cancer Institute 2. Laboratory of Physiology 

INSTITUTE ' — — . LABORATORY OR BRANCH 

5» Energy Metabolism Section k, S 912 

SECTION OR SERVICE lOCATION (IF OTHER THAN BETHESDA} SERIAL NO . 

assiO'Lq:-: 

6, Mass Spectrometer' Frggram 

PROJECT TITl¥~ ~~' "" ' ' '. '' 

7. A. W. Pratt and Bernard E. Burr . ■ " 

PRINCIPAL INVESTIGATOR (S) 

8. ^■_ - ■ ■' ■- ■. ■■ . • . .' : 

OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION 

Objectives ; The instrument is in continuous routine use for calibration and 
monitoring of the energy metabolism instruments. Secondly, studies on 
diffusion rates of gases obtained from biological sources continues. 
This study is particularly concerned with the deviations that exist in 
the diffusion rate of these gases from that predicted by Graham's Law. 
Thirdly, pilot experimentation has been tried out relating the rate eind 
kind of gases which are released from pyrolyzed proteins. 

Methods Employed ; Mass spectrometric techniques. 

Major Findings ; The primary use of the mass spectrometer continues as a 

basic reference instrument for gas analysis of our energy metabolism in- 
struments. The mass spectrometer allows frequent and accurate standardi- 
zation of our analytical respirometers and is primarily responsible for 
the present accuracy and the continuous performance of cur new analytical 
respirometers. To completely define the accuracy of our energy metabolism 
measure, it is necessary that an extensive performance catalog of the 
mass spectrometer exist. Compilation of these data will occupy most of 
the mass spectrometer time for an extended period. 

Studies on diffusion rate of gases is progressing very slowly. The immedi- 
ate problem at hand deals with the deviations that are observed in the 
diffusion rate of these gases from Graham's Law. We are confronted here 
with fundamental problems in gas behavior and vrhile we have made consider- 
able effort in relation to this problem we have been unable to resolve the 
questions related to the fundamental behavior. 



PAGE 2 

912 .■r.)>H,;. .,..;. 

SERIAL NO. 

The studies from the pyrolysis" products of P^^^^^^'^f ,J^f .J^^ i^""" 
Plate to he discussed at this moment. It is apparent that the study of 
?he diffusion rates and the study of pyrolysis products are long-temed 
^ phJsicSche^ci. problems which we feel will extend the use^af the mass 
spectrometer but which at the same -time will he resolved slowly, • , 

Significance to Cancer Research ; It is hoped hy ^J/P^J^f ^J^J^?J;'_f the 
- hiiirihj^claliSt;i?r^?rian utilize the considerable potential of thp 
mass spectrometer in understanding biological functxon and ff-^^^^ 
composition of important proteins. The P^?P°s^\?°^^^\°^,^f .H^'f 
will be to continue the pilot experimentation until such time as posi- 
tive findings direct the development of the program, 

Pro-DOsed course of project: It is apparent that the study of the diffusion 
^ rates Slthe study of pyrolysis products are long-termed physical chem- 

ic2 pSblems which we ?eel will extend the use of the mass spectrometer 

but which at the same time will be resolved slowly. 







-:cf- 


tQ&m^ ■ 



10. 912 



SERIAL NO. 



PAGE 3 



11. 



BUDGET ACTIVITY: 



RESEARCH /T/ 
REVIEl'J & APPROVAL [J 



ADMINISTRATION [J 
TECHNICAL ASSISTANCE [J 



12. 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHEIR ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER I956 or 195? 



None 



13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEM.TH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDENTIFY SUCH RESEARCH: 



None 



NO ENTRIES FOR ITEMS lU, 15 and I6, 



PAGE 1 
PROJECT REPORT 



1. N ational Cancer Institute 2. Laboratory of Physiology 

INSTITUTE LABORATORY OR BRAJMCH 

5. Energy Metabolism Section k. 5, 913 

SECTION OR SERVICE LOCATION "(iFOTHER THAN BETHESDA)" SERIAL NO, 

Determination of the radiochemical mechanism of L-raalate decomposition 

6. in oxygenated, aqueous solutions. 

PROJECT TITLE 



T» A. W. Pratt and Franc es K, Putney 

pmciPAL investigatdrTsI 



OTHER INVESTIGATORS 



9. PK)JECT DESCRIPTION 

Objective s : To determine the radiochemical mechanism of L-malate decomposi- 
tion in oxygenated;, aqueous solutions. 

Methods Employ ed: Chemical analysis by fluorometry, chromatography^ and 
absorption spectroscopy. 

Major Findings : It has been i-eported that the level of oxygen tension in the 
ambient air and thus , in the blood of an experimental animal exerts a 
significaoit effect on the per cent survival ratio and survival time of 
irradiated animals. One approach to the understanding of this system 
is to study the radiochemical decomposition of important biological 
compounds in oxygenated, aqueous solution. Studies of x-irradiated 
malate solutions revealed that 6% of the decomposed malate was con- 
verted to oxalacetate and the remainder to a-keto, P-hydroxy succinate 
in the presence of oxygen. Neither of the compounds are formed in non- 
oxygenated solutions. The oxygen effect in the radical reaction is ap- 
parent from these observations. The full data including the probable 
radiochemical mechanisms of these reactions, which is now to be sub- 
mitted for publication; clearly indicates the importance of molecular 
oxygen in determining decomposition reactions following exposure to 
ionizing radiations. 

Significan ce to Cancer Research : To aid in the understanding of the biologi- 
cal effect of ionizing radiation. 

Proposed course of project : Completed . 



10. £1^ PAGE 2 

SERIAL NO . 



11. . 

BUDGET ACTIVITY: 

RESEARCH /x/ ADMINISTRATION [J 

REVIEW MD APPROVAL [J TECHNICAL ASSISTMCE [J 



12. 

COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE , OR OTHER ORGANIZATIONS , PRO - 
VIDING FUNDS. FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 195^ or 1957. 



None 



13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS; OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC m^}FYi SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FWros), IDELWIi'.- SUCH RESEARCH. 



None 



ENTRIES FOR ITEMS \\ „ 15 and l6. 



PAGE 1 
PROJECT REPORT 



1. National Cancer Institute 2. Laboratory of Physiology 

INSTITUTE LABORATORY OR BRANCH 



3. Physical Biology Section 4. ' ' 5, 914 

SECTION OR SERVICE LOCATION (if other than Bethesda) SERIAL NO, 



6, In Vivo Measurement of Tumor pH With the Capillary Glass Electrode, 
PROJECT TITLE 



7. H, Kahler 

PRINCIPAL INVESTIGaTOR(S) 



8. B. Haines 



OTHER INVESTIGATORS 



9. PROJECT DESCRIPTION 



Objectives ; The object of this project is to determine the in vivo pH of 
tumors under different experim.ental conditions. 

Methods Employed : Following anesthesia, three fine-tipped capillary 
electrodes were inserted into each tumor in selected sites, one 
electrode was inserted into normal leg muscle, and the pH was 
recorded by automatic equipment over a period of 18 hours. Fine 
thermocouples were also inserted into selected sites such as skin, 
tumor, muscle and rectum, and the temperatures were recorded on 
another instrument throughout the experiment, After an initial 
period, an injection of one of numerous sugars was made. 

Major Fi pd'r.g3?. The aiid reaction of tumors follo'^^Ing glucose administra- 
tion if'S2m3 io be quite general; all 14 rat tumors studxed chow this 
effect 

In a gh'en ";umor, the pH varies with location within the tumor, 

SignifAca nc? to C-m?^v Research ; The pH of a tissue is one of its basic 
pzc-jc^tiGf. ana whore this pH deviates from tj.^. normal tissue value 
ths celxHed etudy of these changes is essential to an understanding 
of tumor chemical processes. 



914 r ; ; '■. ' i PAGE 2 

SERIAL NO, 



Proposed course of project ; The variation of pH with position in a tumor 
suggests it may be a function of the proximity to blood vessels in 
addition to the proximity to the surrounding normal tissue. This 
remains to be seen. 

An analysis of tumor temperatures for information on energy 
characteristics of tumors. 



10. 



11. 



914 

SERIAL NO. 



PAGE 3 



BUDGET ACTIVITY: ' ~ ~ 

RESEARCH /T7 

REVIEW AND APPROVAL . /^ 



ADMINISTRATION r~7 

TECHNICAL ASSISTANCE /"~7 



12. 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER 
ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN EITHER 1956 or 1957 

None 



13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS 
RESEARCH DONE ELSEWHERE IN THE PUBLIC HEALTH SERVICE 
(WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUNDS), 
IDENTIFY SUCH RESEARCH: 

None 



14/ 914 PAGE 4 

SlmAL NO. ' 



15. 



PUBLICATIONS OTHER f HAN"ABSTlAgTS FROM THIS PROJECT DUKlJsiu 
CALENDAR YEAR 1955: 

M. Eden, B. Haines and H. Kahler: The pH of rat tumors 
measured^ vivo. J. Nat. Cancer Inst. 16t 541-556,, 
1955. 



16. 



HONORS AND AWARDS TO 'PEl'SONNEirRELATING TO THIS PROJECT 
DURING CALENDAR YEAR 1955. 

None 



PAGE 1 
PROJECT REPORT 



1. National Cancer Institute 2, Laboratory of Physiology 

INSTITUTE LA BORATORy. qk BfeANCH ~~~ 

-"i'X:H,iQ'i. xy ■ ■ ■■■'■■ 

3. Physical Biology S6ctiori 4. ■■ '' ^^^ '--■<■ ■ ^i '' 915 

S E CTIO N OR SE RV ICE LOCATION (if other than Bethesda) SERIAL NO. 

6, Physical Characterization of Cellular Bodies; Ultracentrifugal Study of 

Desoxyribonucleic Acid (DNA) in Bivalent Ion Solutions, 

PROJECT TITLE ~~~~ ~" ■ -■ ■ ■;■ :. .-. ■. ' ~T~, 



H> Kahler 

PRINCIPAL INVESTIGATOR(S) 



J, Shack and B. J. Lloyd 
OTHER INVESTIGAT(5R§" 



PROJECt DESCRIPTION '^ .; 

Objectives ; To compare the sedimentation characteristics Of DNA in bivalent 
and monovalent ion solutions. 

Methods Employed; Solutions of different DNA preparations from normal and 
tumor tissue were centrifuged in various monovalent and bivalent ion 
solvents. The optical photographs were analyzed for sedimentation 
velocities and boundary spreading. 

Major Findings: DNA has a higher sedimentation velocity and greater 
boundary spreading in bivalent ion solutions than in monovalent ion 
solutions. 

Significance to Cancer Research ; The properties of DNA are of significance 
to genetics and, hence, to cancer. It is important to know not only the 
properties in monovalent solutions but also in solutions containing 
calcium and magnesium. 

Proposed course of project ; Continuation of DNA study, utilizing light- 
scattering, ultracentrifugation, electrophoresis, and electron 
microscopy. 

Continuation of virus part of project by studying morphology of tumor- 
inducing viruses sliced with a microtome, and sectioned viral tissue. 



10- 915 TAm .in TD5i5 ■ : , PAGE 2 

SERIAL NO. 





BUDGET ACTIVITY: 










,1:; RESEARCH 


/x/ 


ADMINISTRATION. , .^, .. 


/ / 




REVIEW AND A PPROVA L 


n 


TECHNICAL ASSISTANCE 


■/ / 


12. 


>; Vu:4&rs-v/li!.- ■:■; . ■,.:■, 




: : lM>yKtfi^O .k'M'.?';" 






COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER 
ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN EITHER 1956 or 1957 




None 




:-■•■_■■■ • .-..J v-/,i '. ,r ■■ . '■■■ 





13. 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS 
RESEARCH DONE ELSEWHERE IN THE PUBUC HEALTH SERVICE i ^ 
(WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUNDS), 
IDENTIFY SUCH RESEARCH 



None 



^iMii^iQ ux B;jin£'-' 



14. 915 PAGE 3 

SERIAL NO. 



15. 

PUBLICATIONS OTHER TiiAN ABSTRACTS FROM THIS PROJECT DURING 
CALENDAR YEAR 1955 

B. J, Lloyd and H. Kahler: Electron microscopy of the virus 
of rabbit fibroma, J, Nat, Cancer List, 15: 991-999, 
1955. 



16. 

HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT 



DURING CALENDAR YEAR 1955 
None 



PAGE 1 
PROJECT REPORT 

1, National Cancer Institute 2. Laboratory of Physiology 

" INSTITUTE LABORATORY OR BRANCH 

5. Physical Biology Section , k, ' ^-> ■'5> 9l6 

SECTION OR SERVICE lOCATION (IF OTHER THAN BETHESDA; SERIAL NO 

Physical Chemical Characterization and Comparison of Nucleic Aoids .and 

6. Nucleoproteins of Normal and Neoplastic Tissues, ->... v,. .,.. . ,. ;..o.,.. 

PROJECT TITLE ■ ,^ ii c:. .-.1 besB'isq:-^ Cnll 

■;..oo "jo aoiaJotsqo?!.,::, 

y.r Joseph Shack j,.. ^ . ., bt^s ncxi-a-^-dxTss u;- 

.: £, PRINCIPAL INVESTIGATOR (S} ' i;.i:j^^>:i , v.:^--.i.vV,amo-.!^uo} • 

OTHER INVESTIGATORS , ,;j::^ o<yn Xii>;iu-/ >;.n.t t>'iJ- i;: y^-5.- i.. e.i sxJocfeJv 

9. PROJECT DESCRIPTION 

Objectives ; The olpjective of this project is to isolate in a native form the 
nucleic acids and nucleoproteins of various normal and malignant tissues, 
to develop and apply appropriate methods of fractionating and characteriz- 
ing them as chemical entities ^ and to discover what differences, if any, 
there are between normal and malignant tissues with respect to these 
components . 

Methods Employed : During the last calendar year modified deoxyribonucleates 
(dNA) were prepared from the DKA of calf thymus and strain A mouse 
lymphoma L#l by exposure to heat, acid, alkali and low salt concentra- 
tions. The ultraviolet absorption and viscosity behavior of both the 
original and the modified DNA were investigated over a range of tempera- 
tures, pH values and salt concentrations. 

Major Findings ; The ultraviolet absorption of native DNA is the same over a 
wide range of temperatures and. salt concentrations. The modified DNA's 
all show marked variation over the same range of conditions. These 
findings suggest that the native nucleate has a high degree of structural 
rigidity which is lost on even mild denaturation . The results afford 
some new simple criteria for recognition of the native state of DNA. 

Significance to Cancer Research : According to current theories nucleic acids 
play a role in genetic processes, in bacterial transformations, in pro- 
tein synthesis, in virus reproduction and in the action of radiation and 
of some carcinogens and chemotherapeutic substances. The importance of 
acquiring knowledge of the nature of the nucleic acids and nucleoproteins 



9l6 PAGE 2 

SERIAL NO . 



of malignant tissues and of differences that may exist between normal 
and malignant cells is indicated by these facts. 

Proposed course of project : Recent evidence shows that the total nucleic acid 
of a cell consists of a large number of different chemical individuals. 
Elucidation of differences among tissues must be concerned with study of .■ 
the separated fractions as well as with the total nucleic acid. It is 
planned to carry out such fractionations on the nucleic acids and 
nucleoproteins of certain normal and malignant tissues. During the past 
year exploration and evaluation of some of the methods to be used in this 
work (chromatography, fractional precipitation and extraction) have been 
carried out. It is also planned to carry out such fractionations on the 
nucleic acids of the tissues of normal and tumor bearing animals that 
have received isotoplc tracers in order to gain information regarding 
the metabolic activity of the individual fractions or components as well 
as of the total nucleic acid. 






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10' 9X6 PAGE 5 

SERIAL NO . 



11. 



BUDGET ACTIVITY: 

RESEARCH fTf ADMINISTRATION [J 

REVIEW 8s APPROVAL [J TECHNICAL ASSISTANCE [J 



12, 



COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE , OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS; FACILITIES; OR PERSONNEL FOR THIS PROJECT IN EITHER 195^ or 1957. 



None 



15. 



IF TEES PROJECT RESElyiBELS . COMPLEMENTS; OR PARALLELS RESEARCH DONE ELSEV/HERE 
IN THE PUBLIC HEALTH SERVICE (V/ITHOUT INTERCHANGE OF PERSONNEL; FACILITIES OR 
FUNDS); IDMTIFY SUCH RESEARCH: 



None 



NO ENTRIES FOR ITEMS 1^^, 15 and l6. 



VfGZ 1 

Cs-T^iiv^: . : -i - . /■.■.-.--■'' A xblB -project i£ to deteiadiie ttos oatore^ gjoaatltsr 
i.- : ^'.;=:- --; -^ .^ i'^'.c si'z^CDaeleasee of laallgnattt ana noiMal tlasties 
5-1 ,: .;. :L. .-: 1^ .'^, psrtlealarly of ISaoee tissoee viioee 

'.■:--. :.-.'tz 5rT-cv^i : I ;-. ". r ^ -. -. '. '.r -.-'.-..-:: '_--'! pr€psratlOB6 of ■body flxiids suit- 
iil^ f-r 5.:-:-i.-. i::i i. : -. .zTBatic actlcQ OQ pure 23ticlelc 



10. 91? PAGE 2 

SERIAL WO . 



11. 

BUDGET ACTIVITY: 

RESEARCH [TJ , ADMINISTRATION [J 

REVIEW & APPROVAL [J TECHNICAL ASSISTA^ICE [J 

12. \ " 

• COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PRO- 
VIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN EITHER 195^ or 1957 



None 



15. 



IF THIS PROJECT RESEi^LES,, COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEWHERE 
IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACILITIES 
OR FUNDS), IDE]\ITIFY SUCH RESEAJ-'vCH: 



None 



ENTRIES FOR ITEMS ll+, 15 and l6. 



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