Experimental Carcinoma of the Cervix: A Comparative

Attempts to produce carcinoma of the uterinecervix experimentally began shortly after the discovery of the carcinogenic properties of crude tar.The earliest experiments consisted of the intravaginal application of crude tar by injection or implantation in the vaginal vault, cervix, and loweruterine segment. Although squamous metaplasiawas frequently produced, invasive carcinoma developed in only a few cases (3, 5, 8, @5).The tumors were slow-growing, failed to metastasize, andoften regressed even during continued applicationof tar.

With the stimulatory effect of estrogenic hormones upon the growth and development of female genital tissues an established fact, studies onthe possible carcinogenic effect •of various estrogens were reported by several workers (15—17, @8).A small number of lesions closely resembling earlyepidermoid carcinoma were observed in the cervices of monkeys that had received estrone parenterally and whose cervices had been traumatized(@1). It is interesting to note that the epithelialmetaplasia produced with estrone alone regressedfollowing discontinuation of the hormone treatment; however, some of the animals which received estrogen together with trauma to the cervixdeveloped invasive carcinomas. A small number ofcervical carcinomas were also observed in mice

whose neck and back had been painted with1,@,5,6-dibenzanthracene while they simultaneously received injections of estrone. The results of thisexperiment would seem to indicate that estrogenichormones possess some co-carcinogenic effect bysensitizing the uterine cervix to the carcinogenicsubstance even though the latter was applied at adistant site (@4, @5).However, owing to the smallnumber of animals which developed malignant lesions, it is difficult to draw any valid conclusionsfrom these experiments. The prolonged subcutane

S T1'@ study was aided by an institutional grant from the

American Cancer Society.Presented in part at the Inter-Society Cytology Council,

Boston, November 18, 1954.

Received for publication February 12, 1955.

ous administration of estrogens for periods of over

1 year produced cervical carcinomas in variousstrains of mice ; one of these metastasized widely(10). However, the percentage of tumor yield waslow, and the mortality of the mice from mammarycarcinomas and pyometra was high. This experiment was further complicated by the fact that thedevelopment of mammary carcinomas occurredlong before the appearance of uterine carcinomas.This often necessitated surgical removal of themammary lesions to prevent death of the mice.

Experiments on the possible etiologic role of

human smegma in cervical cancer were reportedby Fishman et al. (7) ; no tumors in mice werefound even after 16 months of intravaginal painting with smegma.

Purified carcinogenic substances applied directly to the uterine cervix resulted in a considerably higher yield of epidermoid carcinomas thanoccurred following either the application of crudetar or the injection of estrogens. The epithelium ofthe vaginal vault and cervix of the mouse responded to carcinogenic hydrocarbons in a mannersimilar to the epitheliuxn of the skin, although thetumor yield from the vagina and cervix was significantly lower. This may have been owing to thefact that application of carcinogenic substances tothe uterine cervix was technically more difficult, orthat the-cervical and endometrial secretion dilutedor removed the applied carcinogen so rapidly thatit became much less effective. Von Haam andMenzies (14) were able to produce carcinoma ofthe cervix in a high percentage of CSH mice by intravaginal painting with 3,4-benzpyrene by meansof a wire loop applicator which permitted accurateplacement of the carcinogen to the uterine cervix

and vaginal fornices. Murphy (@0)solved the problem ofaccurate placement by inserting a string saturated with methylcholanthrene into the cervicalcanal under direct visualization and was also suecessful in producing a considerable number of cervical carcinomas. The significance of these resultsis greatly increased by the fact that spontaneousuterine tumors in mice are extremely rare, num

449

Cytologic and Histologic Study*

E. VONHAAM ANDD. G. SCARPELLI

(Department of Pathology, Ohio Slate University, Columbns, Ohio)

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450 Cancer Research

bering less than 0.06 per cent according to reportsby Slye, Holmes, and Wells (@7). Reports on theexperimental production of carcinomas of theuterine cervix are summarized in Table 1.

Von Haam and Menzies were able to demonstrate malignant cells in the vaginal smears oflaboratory animals some time before the gross appearance of tumors and pointed out the great advantages of this procedure for studies in exfoliativecytology. Because decreased cellular adhesivenessis present in neoplastic tissues, the rate of cellular

exfoliation is increased, and the vagina becomes a

“cellsac―providing a steady supply of malignant

cells. Since it was felt that this method would lenditself well to a comparative study of the histologicand cytologic changes which occur during the development of the neoplastic process, the presentseries of experiments was undertaken.

MATERIALS AND METHODS

One hundred and ten C3H mice, 2-3 months of age, wereused for the experiments; 80 animals were painted intravaginally twice weekly with a 1 per cent solution of 3,4-benzpyrene in acetone. Thirty animals, which served as controls,were painted intravaginally twice weekly with acetone. Painting was accomplished by means of cotton-tipped bacteriologicwire loops which were bent slightly to fit the curve of the

TABLE 1

SUMMARYOFEXPERIMENTALPRODUCTIONOFCARCINOMASOFTHE UTERINECERVIXNo.. species and strain, of experimental

animals

White rat, No.?

White mice, No.?

50 White rats

9 Macacm rhesusmonkeys

White mice, No.?

40 mice

1 mouse“OldBuffalo―27 mice

75 mice

234 mice—A,C57, D,Cs!!, CBA, new andoldBuffalo

100mice, CSH

324 mice—A, C57, D,new and old Buffalo,CSH, CBA

1939 (7) 134 mice—CSH,CBA,A, C121, C57, N,Jx,F

170 white mice

44mice—C57,

10 mice—A20mice-A16 mice—CBA, C57,

AXA35 mice—CSH

BALB mice, No.?

80 mice—CSH

1953 (16) 74 mice—A,C57

Mode ofapplica.

tion

I.V., skin“V.“V.S.C.

Induction

time(wks.)

24

No.tu'

mors

1Carcinogen

Crude tar

Crude tar in xylol

Crude tar

Estrone and cervical trauma

Crude tar

Estrone

Estrone1,2,5,6-dibenzan

thracene, Estrone

1,2,5,6-dibenzanthracene, Estrone

Estrone

Estradiol and Estrone bensoate

Estrone benzoate

Estrogens and Teatosterone propionate

CoaltarFolliculol

Estradiol benzoate

Estrogens and androgen

Estradiol sensitizedcollodion particles

3,4-Benspyrene

Methylcholan- IC.threne-saturatedstring

RemarksPrecancer

Epidermoid carcinoma

40 1 Epidermoid carcinoma13 3 Precancer

Early epiderm. ca.

1 Epidermoid carcinomaI Epidermoid carcinoma1 Carcinoma-likelesions3 Epidermoid carcinoma

Epidermoid carcinoma

106 26 Precancer

38—52 19 1 metas. epiderm. ca.18 early epiderm. ca.

4-80 0 Metaplasia

55 26 S epidermoid23 precancer

30-35 2 Epidermoid carcinoma

52 25 PrecancerEpidermoid carcinoma

28-56 10 Epidermoid carcinoma

10—28 9 PrecancerEpidermoid carcinoma

29 13 PrecancerEpidermoid carcinoma

64 38 PrecancerEpidermoid carcinoma

16-40 31 Carcinoma in situInvasive epiderm. ca.

30-40 27 Invasive epidermoidcarcinoma

Year andreference

1926 (25)

1929 (8)

1982 (8)1988 (17)

1985 (5)1986 (12)

1986 (13)

1986 (20)

1987 (21)

1988 (24)

1938 (6)

1938 (14)

1.V. 22S.C. 11-29

S.C. 104

Skin-painted 2@

Skin-painted 26—40

1940 (15)

1941 (1)

1942(7)

1944 (4)

1948 (18)

1953 (26)

1958 (10)

8,4-BenzpyreneHuman smegma

Estradiol bensoate

S.C.

S.C.

S.C.

S.C.

Tar I.V.S.C.

S.C.

“V.“V.S.C.

S.C.

S.C.

“V.

S.C.—subcutaneous. I.V. —intravaginal. I.C. —intra@rvical.

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VON HAt@ @u@mSCARPELLI—Experim.enial Carcinoma of Cervix 451

genital canaL The cotton-tipped wire loops were carefullyplaced in the vagina, which was stretched open by dorsalfiexion of the tail, and inserted until firm resistance was felt;the cervix was then painted with several rotary motions andthe wire ioop removed. The animals were kept in groups offive in an air-conditioned room and fed Purina Dog Checkerssupplemented by daily addition of fresh bread and carrots.

Smears were prepared previous to each painting period byinjecting a small quantity of saline into the vagina followedby immediate aspiration. The smears were fixed immediatelyand stained by the method of Papanicolaou (23). In addition,smears were prepared for the following cytochemical reactions: Feulgen's reaction for the determination of deoxyribonucleic acid (5); methyl green-pyronine stain of Brachet forthe determination of ribonucleic and deoxyribonucleic acid (2);the PAS reaction of McManus for the detection of glycogen(19); Gomori's reaction for the detection of alkaline phosphatase (11); and Gomori's reaction for the demonstration ofphosphamidase (12). Smears for cytochemical studies werefixed in an alcohol-ether mixture except for those used for thephosphamidase and alkaline phosphatase reactions, whichwere fixed in ice-cold acetone. The standard histochemical procedures were modified for application to vaginal smears as follows: Smears prepared for the Feulgen reaction were subjectedto N HC1 hydrolysis at 6V C. for 12 minutes. The substrateused for the phosphamidase reaction was a 0.1 M solution of theammoniuni salt of p-chlorophenyldiamidophosphoric acidcommercially available from Bayer-Leverkusen, Germany.The reaction was modified by shortening the incubation time to18 hours. Rinsing with distilled water and acetate buffer waslimited to 14 minutes. The above-cited cytochemical reactionswere performed once weekly. Smears stained with the Papanieolaou method were examined for the degree of estrus, thepresence of leukocytes and erythrocytes, and the appearanceof atypical and malignant epithelial cells.

As soon as abnormal cells appeared in sufficient numberto be considered diagnostically significant, the animals weresacrificed, the uterus and vagina removed, and serial sectionsprepared for microscopic examination. Seven groups of animale were sacrificed during a period from 12 to 40 weeks afterthe beginning of the experiment; this permitted a comparisonbetween early and late cytologic and histologic changes produced by the carcinogen. By means of differential counts,quantitative “cytograms―were constructed; although theydo not reveal the complete estrous cycles of the animals, theypermitted us to follow the cytologic pattern observed duringthe period of experimental carcinogenesis.

RESULTS

On the basis of our histopathologic studies,three distinct lesions could be differentiated : (a)dysplasia of the epithelium of the cervical andvaginal mucosa; (b) noninvasive carcinoma (car

cinoma in situ) ; and (c) invasive carcinoma. Epithelial dysplasia was found in six animals sacrificedat an early stage of the experiment, noninvasivecarcinoma in @1animals, and invasive carcinoma

in fifteen animals (Table @).In fourteen animalsthe pathological changes were restricted to theuterine cervix (Fig. 1), in ten animals the lesionwas present in the vaginal wall (Fig. @),and ineighteen animals it was impossible to determinewhether the primary site was the uterine cervix orthe vaginal mucosa (Fig. 8). Cervical dysplasia ap

peared during a period of 1@—@8weeks after thepainting had been started, carcinoma in situ during a period of 16—40weeks, invasive carcinomaduring a period of p21—40weeks. None of the control animals painted with acetone developed anygross or microscopic lesions of carcinoma in vitu orinvasive carcinoma, although evidence of vaginitiswith some epithelial dysplasia could be demonstrated in some.

Our cytologic studies revealed definite nuclearand cytoplasmic changes in the exfoliated cellsduring the development of cervical dysplasia andcarcinoma. Certain cellular changes preceded by aconsiderable time the appearance of neoplasmsand therefore might be classified as precancerouschanges. Other changes were demonstrable only inthe presence of either carcinoma in situ or invasivecancer and therefore were considered as changestypical of malignant cells.

TABLE 2

INCIDENCE, TYPE, AND SITE OF LEsIONs PRODUCED BY

THE INTRAVAGINAL APPLICATION

OF 3,4-BENZPYRENE

SITEVaCerTOTALginavizBothNO.1326777212491510141842

Tyraor

Epithelial dysplasiaCarcinoma in situInvasive carcinoma

Total:

Watts

12-2816—4021—40

Epithelial dy8plasia.—The histological conceptof cervical dysplasia as defined by Reagan andMoore (@6) consists of a disturbance in the regulardifferentiation and keratinization process of thebasal-cell layer with considerable “disorderly―arrangements of the cells but without evidence of increased proliferation. In addition, we observedsome loss in the polarity of basal cells, and cellsundergoing necrosis became intensely acidophilicand attracted small intra-epithelial accumulationsof leukocytes (micro-abscesses) (Fig. 4). Cells exfoliated from this lesion had pale, large or multiplenuclei, cytoplasmic granules, vacuoles, and precocious cornification (Figs. 5—7). These cells wereconsidered “atypical―and were not present duringan ordinary inflammatory process. They were alsopresent in animals with invasive carcinoma butwere not considered malignant.

Carcinoma in situ.—Carcinoma in situ, or intraepithelial carcinoma, showed a marked proliferation of basal and parabasal cells within widenedepithelial boundaries but within intact basal membranes (Fig. 8) . The nuclei of these cells were enlarged and hyperchromatic, there was an increasednumber of mitotic figures, and one had a definite

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45@ Cancer Research

impression of “cellcrowding― (Fig. 9). Because ofthe latter phenomenon, the atypical appearance ofindividual cells was sometimes not so impressive inthe histologic preparation as in epithelial dysplasia. Cells exfoliated from this lesion were characterized by large nuclei with definite change in thenuclear-plásmatic ratio, having a coarse chromatinpattern, multiple and atypical nucleoli, and in

observed in our animals was mostly of the well differentiated squamous-cell type (Fig. 10). The islands of cancer cells showed numerous pearlybodies and were usually surrounded by a very severe inflammatory reaction. Only in a small groupof animals did we obtain an undifferentiated,highly anaplastic type of carcinoma with smallpyknotic cells and no evidence of keratinization

TABLE 8

CYTOLOGICAL-HISTOLOGICAL CORRELATION

RANGE OF DISTRIBUTION

ANIMALS WITH LESIONS OF NORMAL EPITH. CELLS RANGE OF RANGE OF

Watt Carci. Invasive Corni!. ATYPICAL MALIGNABI'OF Cervi- Dyspla- Hyper- noma carci. Basal cells Lutein czu.a czi@xs

WEE. citia sia plasia in situ noma cells (per cent) cells (per cent) (per cent)

12 2 70—75 20—25 5 4—9 01 65 30 5 4 0

2 85—40 25—SO 80-45 6—7 014 1 30 40 80 5 0

4 10—70 15—80 10-70 6—16 0

16 5 40—90 5—SO 5—80 12—22 8-618 8 65—75 15—SO 5—10 8—17 2—420 4 65-85 10—SO 5-15 11—52 3—17

1 75 15 10 43 1222 4 60-65 15—80 5—20 12—88 4-924 1 60 20 20 10 0

6 25—80 15-30 5—45 16—37 2—il2 85-95 5—iS 0 88—64 26—43

40 1 75 15 5 5 15ii 60—95 5—25 0—15 26—46 3—58

some cases quite bizarre shapes (Figs. 1@—15).The (Fig. 11). Exfoliated cells from this lesion were notcytoplasmic changes were usually similar to those much different from those found in animals withfound in cells with epithelial dysplasia, but the intra-epithelial carcinoma, with the exception that

they appeared in larger numbers. This seemed to_1@ Bosol cells us added proof, if such is still needed, that intra(I)

-J@ Cornifled cells epithelial carcinoma should be considered a malig

@ 80 Luteinized cells nant and not a premalignant lesion.@ Atypical cells60 Malignant cells@ Table 3 represents a correlation between the

, histologic type of lesion and the cytologic findings40 in vaginal smears immediately before the animals

were sacrificed. The number of atypical cells in the@ 20vaginal smears varied from 4 to @6per cent in ani

@ 0- ____ ____ ____ ____ mals with epithelial dysplasia, from 8 to 38 perCervicitis Epitheliol Carcinoma lnvoslve

cent in animals with carcinoma in situ, and fromdysplosia in situ carcinoma@6to 64 per cent in animals with invasive car

TYPE OF LESION crnoma. Definitely malignant cells, which wereabsent in all but one of our cases of epithelial

CHART 1.—The different average distribution of normal, dysplasia, were found in 3—17 per cent of our cases

atypical, and malignant epithelial cells in the four types of of carcinoma in situ and in 3—53 per cent of ourhistopathological lesions. These are based on vaginal smearstaken immediately before the animals were sacrificed.@ of invasive carcinoma. The fact that one ani

S Fifteen per cent malignant cells'were found in one animal ma! (NO. 55) with histologically invasive carcino

with epithellal dysplasia. ma exfoliated as few as S per cent recognizablymalignant cells seems interesting in view of the re

process of atypical keratinization was found much ported false negative smears in cases of humanmore frequently, and the appearance of small cells cervical carcinoma. Chart 1 shows the average diswith a deep orange color of the cytoplasm was tribution of normal, atypical, and malignant epiquite characteristic. the!ial cells in the vaginal smears according to the

Invasive carctnoma.—The invasive carcinoma types of histologic lesions and clearly demonstrates

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VON HAAM AND SC@u@Pi@u—Experimental Carcinoma of Cervix 453

the sharp cytologic difference between epithelialdysplasia and carcinoma in situ.

On the basis of our weekly differential counts,other interesting differences were found to existbetween dysplasia, carcinoma in situ, and invasivecarcinoma. Smears from animals with dysplasia

CHART 2.—Cytogram of experimental animal No. 70 sacri

ficed after 40 weeks. Note the change of the cyclic cellular pat

and carcinoma in situ showed no significant changesin the normal epithelial cell population, while inanimals with advanced invasive tumors a markedincrease in basal cells existed. This is shown inChart @,which represents the cytogram of animal70, which was sacrificed 40 week@ after the beginning of the experiment with a large fungating carcinoma of the cervix; malignant cells had beendemonstrated constantly for the last 1@ weeks.The cyclic nature of the cellular pattern was markedly altered at about the @4thweek, and luteinizedcells were almost absent for the last 10 weeks.

In most of the animals with epithelial dysplasiaor noninvasive carcinoma of the cervix, the cydie cellular pattern was only slightly disturbed,and luteinized cells could be found in considerablenumbers at set intervals. In animals with invasivecarcinoma, keratinized and luteinized cells decreased sharply, and basal cells of the parabasaland intermediate types appeared to predominate.

Red blood cells were generally absent during thestages of dysplasia and noninvasive carcinoma.They were always a prominent feature in animalswith invasive carcinoma.

The results of the various cytochemical reactions are presented in Table 4. Deoxyribonucleic

PRECOC. COIN. CELL$

tern after malignant cells appear in larger numbers (at aboutthe80thweek).

acid seemed increased in large and atypical cells,but this increase was not uniform and never sopronounced as in the leukocytes of the exudate.

TABLE 4

SUMMARY OF CYTOCHEMICAL STUDIES

CYTOGRAM OF ANIMAL 700I

I0

80 HIST0PATH0L0GY@ADVANCED CARCINOMAOP CERVIX

60

40

20

0 481216202428 32 36 40

WEEKS

Reactionor stainSubstance tested forAtypical cellsMalignantcellsFeulgen

Methyl greenpyronine

PASPhosphamidase

reaction ofGomoriDNA

RNADNAGlycogenPhosphamidaseIncreased

IncreasedIncreasedNo changeIncreasedhcreased

Increased.IncreasedNo changeMarkedly

increasedPhosphatase

reactionofAlkalineEquivocalEquivocal.Gomori

(Figs. @0, @1).Ribonucleic acid was also increasedin the cytoplasm and the nucleoli of malignantcells. However, the staining technic With methylgreen-pyronine proved difficult, and reliable results

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454 Cancer Research

could not be obtained. The glycogen content of themalignant cells seemed not to be increased as compared with normal cells, nor was the alkaline phosphatase reaction significantly different. The phosphamidase reaction was strongly positive in manyatypical and malignant cells, but again the resultswere not consistent. In some instances the reactionwas present in the cytoplasm (Fig. %2), while inother malignant cells the reaction was more pronounced in the nucleus (Fig. @3)or was entirelyabsent (Fig. @24).The reaction depended greatly onthe technic employed and for this reason demanded a very exact standardization of the technical method. While in our experience cells from normal animals did not give a positive reaction, wefeel that only a comparatively small number ofexfoliated malignant cells were phosphamidasepositive.

DISCUSSION

The pathogenesis of 3,4-benzpyrene-inducedcervical and vaginal carcinoma in CSH miceseemed to be characterized by definite cytologicand histologic changes. The development of carcinoma appeared to progress from epithelial dysplasia to carcinoma in situ and finally to invasivecarcinoma. Qualitative and quantitative changesin the cell pattern exhibited by exfoliated cellsparalleled the histologic changes during the development of neoplasia. These cytologic changes canbe classified into the groups of precancerous andmalignant changes. Precancerous changes, withthe exception of precocious cornification, precededthe appearance of neoplasia by a considerable penod of time and were also found occasionally incells from control animals painted with acetonealone. The impairment of normal growth and maturation of vaginal and cervical epithelium and theappearance of precociously cornified basal cellssuggested to us some close relationship. The malignant changes were primarily characterized by alteration of the nucleus, although variations in sizeand shape of cells were most marked in animalswith invasive carcinoma. Nuclear pyknosis, precociously cornified basal cells, leukocytes, and red

FIG. I .—Mouse uterus with advanced carcinoma of thecervix. The distended urinary bladder is secondary to urethralobstruction. X2.

FIG. 2.—Mouse uterus with a carcinoma of the vaginal wall.xS.

Fia. 8.—C8Hmouse with an extensive cervical carcinomafilling the entire pelvis.

FIG. 4.—Cervical dysplasia. There is a disorderly arrangement of both basal and parabasal cells with intra-epithelialmicro-abscesses. X585.

blood cells were found so constantly in animalswith invasive or noninvasive carcinoma that theycould be spoken of as representing a “malignantpattern,― although none of these changes by itselfwas diagnostic of a malignant process. This observation is identical to one made by von Haam (13)during the examination of a large number ofvaginal smears in women.

It is interesting that the normal cyclic cellularpattern of the animals continued during the firsttwo stages of neoplastic development and was onlydisturbed in animals with invasive carcinoma.This must be interpreted as an effect of the diseaseupon the hormonal health of the animals and notas a failure of the remaining vaginal epithelium torespond to estrogens. Epithelial dysplasia preceded the appearance of noninvasive carcinoma bya considerable length of time. The fact that it wasonly very occasionally observed in our control animals would suggest that it may belong to thegroup of precancerous lesions, although additionalexperiments are needed to support this theory.

Our cytochemical studies were disappointing inthat no clear-cut difference among normal, atypical, and malignant cells could be established.Many obviously malignant cells gave a strong reaction for ribonucleic and deoxyribonucleic acidand were phosphamidase-positive. The increase inthe deoxyribonucleic acid content of malignant nuclei is probably related to the polyploidy so characteristic of cancer cells. However, it was also feltthat many malignant cells did not give these reactions, and for this reason we cannot consider anyof these reactions as significant or possessing muchdiagnostic value. We feel, however, that the equiv

ocal results we have obtained may be attributed inpart at least to the length of time the exfoliatedcells had been in the vaginal pool. The importanceof this factor becomes quite evident when one considers that all intracellular substances are subjectto denaturation. Therefore the varying intensitiesof reaction seen in cells of the same type may be areflection of cellular hypoxia, malnutrition, senescence, trauma, or death following exfoliation.More definitive investigations with regard to cyto

Fia. 5.—Cervical dysplasia. Vaginal smear. Parabasal cellwith two nuclei. X 1200.

Fia. 6.—Cervical dysplasia. Vaginal smear. Basal cell contaming cytoplasmic granules. X 1250.

FIG. 7.—Cervical dysplasia. Vaginal smear. Basal cell contaming cytoplasmic vacuoles. X 1250.

FIG. 8.—Carcinoma in situ, A proliferation of intenselybasophiic cells can be noted within an intact basement membrane. X125.

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Fic. 9.—Carcinon@a 111 situ. Iligh power photoinicrographsho@ving hyperchromat ism, niitotie figures and marked cellularcrowding. X475.

FI( . I O.—%Velldifferentiated epi(lermoid carcinoma of the(erv@x. Marked keratiuization with pearl formation is present.XU.5.

Fin. I I .—Anaplastic epidermoid carcinoma of the cervix.‘[hislesion is conlpose(l of pleornorphic cells arranged in (leilsesheets with no evidence of keratinization. X 1@5.

Fin l@&—Vaginal smear. Malignant basal cells showinghypcrchroinatism, iiiarked variation in size and indistinct cytoplasniic borders. X 1@5O.

Fin . 13 .—Vaginal smear. \-lalignant basal cells showingmacro:iucleoli. X l@5O.

Fin. 14.—Vaginal smear. Malignant basal cells exhibitinghypcrchroinatisin, pleornorphism, macronucleoli, perinuclea rchroinatin condensation, afl(l mitosis. )< 1@5O.

Fio 15.—Vaginal sniear. \Iultinucleated malignant basal((IIX 1@5()

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FIG. 16.—Vaginal smear. Malignant cell of the fiber celltype. X875.

FIG. 17.—\aginal smear. Malignant cell of the tadpole cell

type. X1250.FIG. 18.—Vaginal smear. Cluster of elongated malignant

celLs (fiber cells). X550.FIG. 19.—Vaginal smear. Malignant basal cells exhibiting

marked pyknosis (anaplastic carcinoma). X 1250.FIG. 20.—Feulgen reaction. Normal cervical epitheliuni.

X675.FIG. ‘@L—Feulgen reaction. Epidermoid carcinoma of the

cervix. Distinct increase in nuclear size and nuclear content ofdeoxyribonucleic acid. X675.

FIG. @.—Phosphamidase reaction. Vaginal smear. Malignant basal cell exhibiting a strong cytoplasmic and no nuclearreaction.X1250.

FIG. 25.—Phosphamidase reaction. Vaginal smear. Malignant 1)asal cell showing a moderately positive cytoplasmic andstrongly positive nuclear reaction. X 1250.

FIG. @24.—Phosphamidase reaction. Vaginal smear. Multinucleated malignant basal cell showing a negative reaction incytoplasm and nucleus. X 1250.

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VON HAAM AND SCARPELLI—Experimental Carcinoma of Cervix 455

chemical characteristics of malignant cells are nowunder way in our laboratory.

SUMMARY

1. The results of our experimental study mdicate that the pathogenesis of 3,4-benzpyrene-induced carcinoma of the cervix and vagina in CSHmice is characterized by four definite stages : acuteand chronic inflammation, epithelial dysplasia,noninvasive carcinoma, and invasive carcinoma.

@2.On the basis of cytologic and histologic studyit was established that a definite correlation existed between the histopathology and the cells cxfoliated during these stages.

3. Atypical and malignant cells could be identifled and distinguished among the exfoliated cellsduring the development of the neoplastic process.No differentiation could be made on the basis ofexfoliated cells between noninvasive and invasivecarcinoma.

4. The cyclic pattern of estrus in animals withdysplasia and carcinoma in situ was similar to thatfound in normal animals. It was definitely alteredin animals with invasive carcinoma.

5. Cytochemical studies showed an increase in

deoxyribonucleic acid and ribonucleic acid and apositive phosphamidase reaction in many but notall exfoliated malignant cells.

REFERENCES

1. ALLEN, E., and G@qitDwxit,W. U. Cancer of the Cervix ofthe Uterus in Hybrid Mice Following Long-continuedAdministration of Estrogen. Cancer Research, 1:856-66, 1941.

2. Bit.@cun@r,J. Nucleic Acids in the Cell and Embryo. Symp.Soc. Exper. Biol., 1:207—24, 1947.

8. Czou, R. Tumori sperimentali da catrame dell'utero eloro sviluppo in rapporto alla funzione ovarica. Boll. eatti d. r. Accad. med. di Roma, 55:291—96, 1929.

4. CROSSEN,R. L, and Lonn, L. Effect of Long-continuedAdministration of an Estrogen on the Sex Organs of MiceWhich Have Passed the Reproductive Period. Arch. Path.,37:808—12, 1944.

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