NEOPLASTIC DISEASES OCCURRING AMONG MICE SUBJECTED TO GENERAL IRRADIATION WITH X-RAYS 1

n. OVARIAN TUMORS AND ASSOCIATED LESIONS

J. FURTH AND J. S. BUTTERWORTH (From the Department of Pathology, Cornell University Medical College, New York)

Mice were subjected to single or repeated large doses of x-rays, 200 to 450 r, as described in the preceding paper (p. 54). Irradiation increased the incidence of myelosis 8 times, of lymphomatosis from 2 to 7 times, while ovarian tumors were approximately 15 times more frequent among the irradi­ ated mice than among their unirradiated siblings (1). The present communication describes the morphological characteristics of the ovarian tumors found among both x-rayed mice and controls and some associated changes. An attempt is made to correlate the histologic characters of the ovarian tumors with hormonal activity, and with the presence or absence of breast tumors. The material is in some respects incomplete. The discovery of the tumors was accidental, and the majority of the animals were discarded without pre­ serving tissues of the endocrine organs. Indeed, the possibility that the ovarian tumors were part of an endocrine disturbance caused by exposure of the entire body to massive doses of x-rays was not recognized until the study was almost completed. We have encountered many findings that are difficult to interpret, but it seemed desirable to present our observations, leaving the solution of these problems to future investigations. The literature abounds in descriptions of the changes which immediately follow exposure to x-rays but nothing is known of the late effects of irradiation on endocrine organs such as the adrenals and hypophysis. It will be the purpose of future investigations to fill this gap in our knowledge.

LITERATURE Effect of X-rays on the : The histologic changes in exposed to sterilizing doses of x-rays have been fully described in a series of articles by Brambell, Parkes and Fielding (2-7). Reference to the earlier literature may be found in their communications and in the reviews by Regaud and Lacassagne (8), Allen (9), and Zondek (10). Brambell, Parkes and Fielding (2) found that in mice exposed to steriliz­ ing doses of x-rays at weaning time (three weeks old), all the oocytes degen­ erated completely. The cells of the and the theca in­ terna subsequently underwent a similar change. In some cases the follicles

1 This investigation has been supported by a grant from the Lady Tata Memorial Trust and by a Fund for the Study of Leukemia. 66 NEOPLASTIc DISEASES AMONG MICE SUBJECTED TO X-RAYS 67 did not degenerate, but grew and formed corpora lutea atretica. These per­ sisted but had no effect on the vaginal cycle. Proliferating cords of cells of germinal penetrated into the medullary region. They formed the major part of the irradiated gonad and were probably the chief source of estrin. A second proliferation of cords from the germinal epithelium oc­ curred, producing anovular follicles. These cords were shown to have no influence on the . Irradiation of non-parous adult mice produced similar changes, but no post-irradiation proliferation from the germinal epithelium was observed (4). The elements already forming the cortex at the time of irradiation persisted. The sterilized ovary consisted of follicles and interfollicular tissue originally derived from the embryonic germinal epithelium. Brambell and his associates believe that these elements produce estrin in the normal ovary and continue to do so without interruption after irradiation. Anovular follicles were formed from the smaller follicles by degeneration of the oocytes and growth of the membrana granulosa cells. These were identical in every respect with the cords of the second proliferation in the animals x-rayed before . The latter, however, developed directly from the germinal epithelium after the follicles had disappeared, and contained no oocytes. The degenerative changes in the of mice irradiated during pregnancy (3) or lactation proceeded more slowly than in normal ovaries and old corpora lutea became in most instances permanent components of ovaries sterilized by x-rays. The salient observations of these investigators have been amply confirmed (9, 10). X-rays when applied to the ovary in amounts sufficient to destroy all ova do not bring about cessation of estrin production, as evidenced by persistence of the estrus cycle. The smallest dose of x-rays that brings about sterilization of the ovary is 200 r in man and 54 r in mice (Schugt, 11). The cycle is present in the mouse even after irradiation with 400 r (10). The amount of estrin excreted in the urine of guinea-pigs with irradiated ovaries is, however, less than normal and the cycles are irregular, with prolonged pe­ riods of estrus, and several months after irradiation the cycle may be alto­ gether absent. Development of mammary glands occurs (Schmidt, 12). Ovarian Tumors in Mice: Primary tumors of the ovary in mice are un­ common. Only two cases have been reported since the elaboration of a clas­ sification of ovarian tumors based upon endocrine function. Schiller (13) irradiated mice with 180 r and subsequently injected them two or three times weekly with of the anterior lobe of the hypophysis. In one of these mice he found, several months after irradiation, bilateral ovarian tumors which he believed were the result of the stimulating effect of hypophyseal hormones on the granulosa cells of the ovary. Apparently he did not investigate the possibility that irradiation alone may be sufficient to produce such growth. While the present paper was in preparation, Gardner, Strong and Smith (14) described a chromophobe adenoma of the hypophysis, granulosa-cell tumors of both ovaries, and adenocarcinomata of the breast in an unirradiated mouse twenty-three months old. ]obling (15) reported the first ovarian tumor in a mouse, which had a sar­ coma of the chest wall and papillary cystadenomata of both ovaries. Later 68 J. FURTH AND J. S. BUTTERWORTH

Jobling (16) described a second case in which the left ovary alone was en­ larged. Microscopically there were in this growth many cysts lined by high columnar epithelium without cilia and separated by tubules. Among the tubules there were smooth muscle fibers with many mitotic figures. The more solid parts had the appearance of a leiomyoma. Tyzzer( 17) reported four cases of primary solid papillary cystadenomata of the ovary, of which two were bilateral. These tumors all occurred in mice over twenty-four months of age. They were essentially solid, with con­ spicuous papillary ingrowths, and showed only a slight tendency to form cysts. Tyzzer says that the tumor cells resembled those of the peritoneal . Haaland (18) reported two ovarian tumors occurring among 288 mice. One of these was possibly a metastatic adenocarcinoma, the other a solid folliculoma similar to that of our mouse Rr 288 (see p. 74). Slye, Holmes and Wells (19) described 44 cases of primary ovarian tu­ mors occurring among 22,000 mice, an incidence of 0.2 per cent. Simple cysts of the ovary were also encountered but were not included with the tumors. Thirty-eight of the 44 cases were designated as benign solid papillary adeno­ mata and in one-half the tumors were bilateral. One tumor was a solid tera­ toma and one a papillary cystadenoma. The remaining 4 tumors were re­ garded as primary malignant tumors of the ovary. Of the 40 mice with benign ovarian tumors, 14 had one or more tumors of the mammary gland and 3 had adenoma of the lung. The tumors varied from 3 to 10 mm. in diameter', were white and firm, and often showed a lobulated surface. Microscopically they were composed mainly of two types of cells in varying proportions; low cuboidal epithelial cells and spindle-shaped cells like those of the ovarian stroma. The cuboidal cells formed tubules, solid plugs, or alveolar masses. Blood vessels were scanty and mitoses were rarely seen. About the tumor there was in most cases compressed ovarian tissue in which ova and follicles were rarely present. The authors believed that histogenetically the benign tumors were similar growths. Apparently the cells composing the tumors grew under great pressure, which did not allow the same degree of differentia­ tion in all cases. The four tumors that were considered to be primary malig­ nant growths of the ovary were unilateral, and in two metastases were found. Three cases were associated with primary carcinomas of the breast and the fourth case with papillary adenoma of the lung. Mitotic figures were seen in all of these cases. The authors believe that the tumors of the ovary, testicle, and adrenal which they have studied "represent reversions to the primitive embryonal tissues of the urogenital anlage, and are probably best designated by Adami's term, mesothelioma." Figure 1 of Slye, Holmes and Wells, designated as solid tubular adenoma, is like our tubular adenomata with abundant spindle-shaped stroma cells. Their Figs. 9 and 10, designated as malignant epithelial tumors of the ovary, appear to resemble granulosa-cell growths, and the latter may be taken to illustrate solid granulosa-ceIl tumors. Both of these tumors were associated with carcinoma of the breast. A fibrosarcoma of the ovary of a mouse was described by Heidenhain (20). NEOPLASTIC DISEASES AMONG MICE SUBJECTED TO X-RAYS 69

MATERIAL AND METHODS The material studied was obtained from 68 mice with ovarian tumors, of which 57 had been irradiated. Most of these mice belonged to the series described in the preceding communication (1). The rest were found in the course of more recent observations. Irradiated mice with no ovarian tumors were studied for the purpose of investigating the changes that precede the development of ovarian tumors. The mice were irradiated in groups of 15 to 25, the factors being as follows: 190 kv., 30 ma., 50 em. distance, 0.5 mm, Cu + 1 mm. Al filtration. The amount of x-rays that each mouse received is stated in the individual protocols. The tissues were fixed in Zenker-forrnol or formol-bromide solu­ tions. In a few instances the sections were taken from tissues kept in Kaiser­ ling's fixative. The sections were stained with hematoxylin and eosin. Mal­ lory's aniline blue was used for the demonstration of the different cell types in the hypophysis.

CLASSIFICATION AND NOMENCLATURE The classification of ovarian tumors offers considerable difficulty. Tu­ mors are usually divided into epithelial and connective-tissue growths, but it is well known that many ovarian tumors have the appearance of both. Fur­ thermore, it is impossible to predict with certainty from the morphological appearance whether the tumors are benign or malignant. None of the tumors studied by us gave rise to metastases, although histologically several of them were similar to those described in the literature as malignant. Endocrine activity is now given eminent position in classifying ovarian growths, but in many cases this cannot be determined from the morphological appearance of the tumor alone. In the current literature the solid special ovarian tumors are classified as granulosa-cell tumors, arrhenoblastomata, disgerminomata, and Brenner tumors. The tumors which are morphologically similar or histogenetically related to the cells of the ovarian follicles will be designated granulosa-cell tumors. The cells usually appear in cords or clusters (Fig. 10) separated by variable amounts of . Brambell, Parkes and Fielding have noted that in x-rayed ovaries the sharp outline of the follicles may be absent and there may be transitional forms between the typical follicular and interfollicular (interstitial) cells. This is so conspicuous in some of the ovarian tumors that they are diagnosed sometimes as carcinoma and sometimes as sarcoma (see Fig. 21). The luteomata may be regarded as a variety of the granulosa-cell tumors. By " clear" cells we mean cells, almost certainly derivatives of lutein cells, that have large amounts of pale-staining, often vacuolated cytoplasm, prob­ ably containing large amounts of fat (Figs. 2, 6). Pigment cells resemble clear cells but their nuclei are smaller and more compact and their cytoplasm appears pale brown. The pigment is probably of hematogenous origin. These cells are often present in the ovaries of irradiated mice. 70 J. FURTH AND J. S. BUTTERWORTH

EXPERIMENTAL: I. OVARIAN TUMORS OCCURRING AMONG IRRADIATED MICE Table I shows the number of x-rayed mice with solid ovarian tumors that have been studied. The figures in parentheses indicate the number of ovarian growths that were associated with breast tumors. The granulosa-cell tumors, including the luteomata, are believed to be -producing, while the tubular adenomata appear to be devoid of endocrine secretion.

T ABLE I: Survey of the Ovarian Tumors in Irradiated Mice and Their A ssociation with Breast Tumors

Stock of mice Ovarian growth Total A R S ------Granulosa-eel! tumors including luteoma, presumably hormone-prod uri ng Small (3 mrn, or less) 4 (I) 1 (0) 1 (0) 6(1) Medium or large (over 3 mm.) 27 (10) 8 (0) 1 (1) 36 (II)

Tubular adenomata and miscellaneous types, presurn- ably not hormone-producing SmaI! (3 rnm. or less) 5 (0) 1 (0) 3 (0) 9 (0) Medium or large (over 3 mm.) 2 (0) 3 (0) 1 (0) 6 (0)

TOTAL 38 (11) 13 (0) 6 (I) 57 (12)

As shown in Table I, the majority of ovarian tumors were found in mice of Stock A. The incidence of both breast and ovarian tumors was greater in this stock than in Stocks Rand S (see Table II and the preceding paper). The majority of the tumors were composed of cells to which hormone produc­ tion might be ascribed. These will now be described.

Tumors Among the X-rayed Mice with Euidcnce of Hormonal Activity Cystic endometrial hyperplasia can be produced by long continued ad­ ministration of estrin and its presence may be regarded as suggestive evidence of continued hormonal secretion by the ovaries. This change was most con­ spicuous in three mice with luteomata in which endometrial tissue penetrated the muscularis and in one case became established on the serous coat of the . These alterations may be regarded as similar to those termed endometriosis.

Mouse Rr 294: Luteoma of the Ovary, Associated with Cystic Endometrial Hyperplasia and Mucous Transformation of : This mouse was exposed to a single dose of 400 r at forty days of age and died at twenty-seven months. Gross examina­ tion (Fig. 1) showed a yellow tumor measuring 2.0 X 1.3 X 1.0 em. replacing the right ovary, thickening of the uterine horns, and moderate enlargement of the liver and spleen. Micro­ scopic examination showed the ovarian tumor to be composed almost entirely of cells like those of the normal corpus luteum (Figs. 3-5). Fig. 4 shows a mitotic figure in a lutein cell of this growth. The enlargement of the uterine horns was caused by cystic hyperplasia of the (secretory phase) similar to that shown in Fig. 7. This finding in an irradiated mouse doubtless indicates excess production of ovarian hormones. The vaginal epithelium was lined by -producing cells, suggesting abnormal production of the corpus FIG. 1. LUTEOMA OF OVARY WITH ENLARGEMENT 01' UTERINE HORNS. RR 294: IRRADIATED. XU FIG. 2. TRANSITIONAL FORMS BETWEEN LUTEIN CELLS WITH PINK-STAINED CYTOPL,ISM AND THOSE WITH PALE, ALMOST COLORLESS VACUOLATED CYTOPLASM (CLEAR CELLS) IN LUTEOMA OF IRRADIATED MOUSE AR 273. X 240 FIG. 3. MICROSCOPIC ApPEARANCE OF LUTEOMA OF RR 294 (FIG. n, X 190 FIG. 4. MITOTIC FIGURE IN LUTEOMA OF RR 294. X 380 FIG. 5. HIGHER MAGNIFICATION OF LUTEIN CELLS OF GROWTH }'ROM RR 294. X 575 (These and other sections stained with hematoxylin and eosin. Magnifications approximate.) 71 72 J. FURTH AND J. S. BUTTERWORTH luteum hormone. There was profound atrophy of the adrenal cortex, the greater part of which was replaced by spindle-shaped connective-tissue cells. The hypophysis showed no conspicuous changes. Mouse Alta 12: Luteoma of the Ovary, Endometriosis, Adenocarcinoma of the Breast: This mouse was sixty-one days old when irradiated with 400 r. It died at the age of seven­ teen months. Each ovary was replaced by a growth measuring approximately 0.6 cm. in greatest diameter. Microscopically the growths were identical and consisted largely of lutein cells like those shown in Figs. 3-5. A smaller part was occupied by clear polyhedral cells (Fig. 6), and there were transitional forms between these two types of cells similar to those shown in Fig. 2. There were areas in which spindle and polygonal cells were pre­ dominant, among which tissue mast cells were numerous (Fig. 8). The uterine horns wen lengthened, tortuous, and greatly thickened. Microscopic examination (Figs. 7 and 9) showed cystic hyperplasia of the endometrium, penetration of the muscularis by endometrial tissue forming nodular prominences covered by the thin serosa of the uterine horn, and papillomatous growths on the serosa. The endometrium was in the secretory phase and the vaginal epithelium had a surface layer of mucus-producing cells. The greater part of the adrenal cortex was replaced by spindle-shaped and polygonal interstitial cells. The hy­ pophysis showed no noteworthy change. The mouse had adenocarcinoma of the breast with metastasis to the lungs. Mouse Aha 13: Luteoma of the Ovary, Adenocarcinoma of the Breast: This mouse, a sister of the mouse described above, was irradiated with 400 r at the age of sixty-one days. and died at the age of fifteen months with carcinoma of the breast. The ovary measured approximately 0.5 ern. across. Microscopic examination showed a luteoma similar to that of Aha 12. The endocrine organs were not preserved for study. Smaller ovarian growths, composed largely of lutein cells, were found in 7 additional mice; the histories of 3 will be briefly given.

Mouse Rra 20: This mouse was irradiated at the age of seventy-five days with 400 r, and died at the age of twenty-eight months. The left ovary measured 0.8 em. Its bulk was composed of clear cells separated into alveolar structures by spindle-shaped stroma cells. It contained a circumscribed area of lutein cells, an area of granulosa cells, and dilated ducts lined by cuboidal epithelium. The growth could be palpated during life. It was removed aseptically. Part of it was cut up into small particles and injected into 8 mice, 4 subcu­ taneously and 4 intraperitoneally. There was 110 growth at the sites of injection. The effect on the vaginal cycle was not studied. Mouse Am 143: This mouse was irradiated at the age of sixty-four days with 450 r, and died at the age of seventeen months. One ovary was 0.5 cm. in greatest diameter and consisted largely of lutein and pigment cells with transitional forms between these types. The smaller part of the growth was occupied by remnants of follicles and spindle-shaped cells, occasionally in mitotic division. The second ovary of this mouse presented the char­ acteristic changes of x-ray injury, namely, overgrowth of spindle-shaped stroma cells among numerous pigment and clear cells, anovular follicles and down-growth of germinal epi­ thelium whose cells resembled those of the stroma. Mouse Ar 273: This mouse was irradiated at the age of thirty-six days with 200 rand at sixty-seven days with 250 r, It died at fifteen months of age. The ovaries measured ap­ proximately OAcm. each and were composed almost entirely of lutein cells. clear cells and transitional forms (Fig. 2). Discussion of Luteomata: Luteoma is a rare type of ovarian tumor in women and to our knowledge has not been described in animals. It is com­ monly regarded as a variety of solid granulosa-cell tumor in which the granu­ losa cells have become luteinized. The mice with luteomata were irradiated at from forty to seventy-five days of age. They may have had corpora lutea at the time of irradiation, but fol­ lowing- exposure to x-rays corpora lutea slowly degenerate (Brambell, Parkes FIG. 6. CLEAR LUTEIN CELLS IN OVARIAN GROWTH OF MOUSE AHA 12, IRRADIATED. X 480 FIG. 7. CYSTIC ENDOMETRIAL HYPERPLASIA AND ENDOMETRIOSIS IN AHA 12. X 18 FIG. 8. PART OF OVARIAN GROWTH OF AHA 12, COMPOSED OF SPINDLE-SHAPED AND POLYGONAL CELLS AND NUMEROUS DARK-STAINED TISSUE MAST CELLS. X 480 FIG. 9. HIGHER MAGNIFICATION OF AREA MARKED IN FIG. 7, SHOWING PE:-IETRATION OF ENDO­ METRIAL TISSUE INTO TIlE MUSCULARIS OF THE UTERINE HORN. X 9S

7.~ 74 J. FURTH AND J. S. BUTTERWORTH

and Fielding). Moreover, normal corpora lutea are circumscribed, while the lutein cells of the luteomata have no well defined boundaries. According to the literature of the subject, lutein cells do not exhibit evi­ dence of multiplication, and it is supposed, therefore, that in the luteomata they are derived from granulosa cells. Mitotic figures may be found in the luteomata (Fig. 4), although they are scarce. Nevertheless, their presence indicates that lutein cells of luteomata may reproduce themselves and are not necessarily derived from granulosa cells. The literature of human luteomata has been reviewed recently by Wills and Romano (21), who presented a typical case of their own, in which there was amenorrhea of nine months, followed by normal menstruation twenty-six days after the removal of the tumor. Microscopically, the growth was com­ posed largely of cells with eccentrically placed, deeply staining nuclei and abundant, finely granular and vacuolated cytoplasm staining freely with eosin. The microscopic appearance of the tumor, as shown in Fig. 2 of Wills and Romano, is very similar to that of our luteomata with clear cells, illustrated in Fig. 6. They state that in some areas of the tumor the constituent cells look more like granulosa cells than lutein cells. Similar transitional forms are present in our growths.

Granulosa-cell Tumors Associated with Hyperplasia of the Anterior Lobe of the Hypophysis Several investigators have observed recently that long-continued injections of estrin produce hyperplasia of the anterior lobe of the hypophysis (22, 23, 24). Since this condition was found only among the mice with granulosa-cell tumors, it may be supposed that the ovarian tumors that were associated with hyperplasia of the anterior lobe of the hypophysis produced large amounts of estrin over a long period.

Mouse Rr 288: Granulosa-cell Tumor of the Ovary, Hyperplasia of Endometrium and Anterior Lobe of the Hypophysis: This mouse was forty-two days old when exposed to 400 r and died at the age of twenty-three months and a half. The right ovary was replaced by a yellow-gray growth measuring 0.8 X 0.6 X 0.6 em. The greater part of the growth con­ sisted of nests of cell? like those of the ovarian follicles, surrounded by spindle-shaped cells (Fig. 10). There were vast numbers of pigment cells at the periphery of the growth. The tumor evidently produced estrin, for there was moderate cystic hyperplasia of the endo­ metrium (proliferating phase). The vaginal epithelium was covered with flat nucleated epithelial cells. The anterior lobe of the hypophysis was greatly enlarged. The greater part of it was occupied by a circumscribed area of cells that were somewhat larger than normal and contained scanty, seemingly chromophobe cytoplasm. At the periphery of this nodular hyperplastic area there were normal cells of the anterior lobe. In the hyperplastic area there were many large sinusoidal spaces filled with erythrocytes, about which there were few, if any, endothelial cells, and it was difficult to distinguish between sinusoidal vessels and extravasations of blood. This lesion appeared to be similar to that found in mice that were injected with estrin over long periods of time. Mouse Rm 55: Granulosa-cell Tumor of the Ovary, Hyperplasia of the Anterior Lobe of the Hypophysis: This mouse had several litters and was nine months of age when ir­ radiated with 400 r, It died at the age of thirty-two months. At autopsy there was evi­ dence of post-mortem decomposition. The left ovary was replaced by a grayish-white growth 1.5 X 1.0 X 0.7 em. in size. Microscopically, a large part of the tumor con­ sisted of degenerated tissue that could not be identified: another large part showed nu- FIG. 10. CELL NESTS LIKE THOSE OF NORMAL FOLLICLES SURROUNDED BY CONNECTlVE-TlSSut: CELLS IN OVARIAN GROWTH OF MOeSE RR 288, IRRADIATED. X 58 Fro, 11. Low MAGNIFICATION OF OvARIAN GROWTH OF UNIRRADIATED MOUSE Arc 12 In the central part of the growth are numerous anovular follicles with cavity formation; in the lower part clear cells and yellow cells. The rest of the growth is occupied by anovular follicles and youthful spindle-shaped and polygonal stroma cells. X 30. FIG. 12. AREA OF ABOVE TUMOR (Ale 12), X 430, SHOWING A!\iOVULAR FOLI.ICULAR GROWTHS FIGS. 13 A!\D 14. MITOTIC FIGURES IN ANOVULAR FOLLICLES IN OVARIAN GROWTH OF IRRADIATED MOUSE AF 597. X 430 75 76 J. FURTH AND J. S. BUTTERWORTH merous areas of granulosa cells, mainly in follicular arrangement, and areas of clear cells. The uterine horns were greatly thickened. The vaginal epithelium was only slightly thick­ ened, but was cornified. The adrenal cortex was thin and showed extensive peripheral re­ placement by fibroblast-like cells. Between the cortex and medulla was a wide zone of large cells with vacuolated brown-stained cytoplasm similar to those described by Cramer and Horning (22) in mice receiving long continued administration of estrin. The anterior lobe of the hypophysis was greatly enlarged. filled with cavernous vessels and extravasations of blood. Mouse A¢f 199.' Luteoma of the Ovary, Cystic Hyperplasia of the Endometrium, Ade­ noma of the Hypophysis, and Carcinoma of tile Breast.' This mouse was sixty days old when irradiated with 400 r and was killed at the age of twenty-one and a half months. At autopsy the breast tumor (adenocarcinoma) measured 2.5 X 2.5 X 1.5 em. The right ovary was re­ placed by a growth approximately 0.6 em. in its longest diameter, while the left ovary was not enlarged. Microscopically the ovarian growth consisted largely of typical lutein cells. clear cells, and transitional forms. Among the variants of lutein cells were numerous cysts lined by flat or low columnar epithelium and ducts lined by cuboidal epithelium. There was cystic hyperplasia of the endometrium. The vaginal epithelium was covered by mucus­ producing cells. The medulla and cortex of the adrenal were separated by a wide zone of large, vacuolated, brown-stained cells, many of which had pyknotic nuclei, but the cortex and medulla appeared normal. The anterior lobe of the hypophysis was large and con­ tained a nodular area approximately 0.5 mm. across, consisting of seemingly chromophobe cells somewhat larger than normal with abundant cytoplasm and containing numerous cavernous spaces filled with blood cells.

The Relation of Ovarian Tumors to Those of the Breast The occurrence of breast tumors in mice indicates the production of ovarian hormones in the host preceding the development of the tumor (Loeb, 25; Cori, 26; Burrows, 27). The hormones, however, produce the tumors only in susceptible mice (27). Table I shows that the incidence of breast carcinoma was very high among the mice that had hormone-producing ovarian tumors, notably among mice of Stock A, in which cancer of the breast was more frequent than among the mice of the other stocks. Table II is of further interest in this connection. The figures in Table II show that breast tumors were very frequent among mice having ovarian tumors of the granulosa-cell type, but that they did not occur among mice having tubular adenomata. In a more recent series one case of tubular adenoma was found to be associated with adenocarcinoma of the breast, but in this instance there were also present numerous lutein cells in the ovarian tumor. Our records show the occurrence of 10 breast tumors among 374 irradiated female mice having no ovarian tumors (2.7 per cent). These will be dis­ cussed in the subsequent section. Data presented in the preceding communication (1) show that the stocks in which breast carcinoma is most common have also the highest incidence of spontaneous ovarian tumors among their irradiated members. These observa­ tions suggest that there are constitutional differences in the ability to respond to cancerogenic stimuli in the ovaries of these stocks, as well as in their mam­ mary glands. The occurrence of two breast tumors in mice with lutein cell growths has already been described. Five additional mice with ovarian and breast tumors were irradiated at thirty-seven, thirty-nine, sixty, sixty-seven, and seventy- NEOPLASTIC DISEASES AMONG MICE SUBJECTED TO X-RAYS 77

four days of age with 400 r and died at twenty, twenty-one, twenty-two, thir­ teen, and fourteen months, respectively. The conspicuous cell types in these ovarian growths of mice with breast tumors are illustrated in Figs. 21 and 24. They are polyhedral and spindle-shaped with continuous transitional forms between the two. The vaginal epithelium was examined in 3 of the mice and appeared to be in the resting stage; the uterine horns appeared normal in 2 of the mice examined. The hypophyses of 2 mice were sectioned and appeared normal. The adrenal of only one mouse (A¢f 196) was examined and showed degenerative changes (see below).

TABLE II: Incidence oj Breast aud Ovarian Tumors

Incidence of tumors Mice Breast Ovary Per cent Per cent Stock A Not irradiated 17.3 1.8 Irradiated (total) 9.7 27.9 Per cent of breast tumors in 374 irradiated mice 2.7 with no ovarian tumors Per cent of breast tumors in 57 irradiated mice 21.0 with ovarian tumors Per cent of breast tumors in 42 mice with 28.6 granulosa-cell tumors of the ovary Per cent of breast tumors in 15 mice predomi­ 0 nantly with tubular adenoma of the ovary

Stock R Unirradiated 6.1 1.2 Irradiated .3.7 18.5

Stock S Unirradiated 6.8 0 Irradiated 0 4.1

It is noteworthy that mice Ajiif 196 and 199 were sisters, received the same dose of radiation (400 r) on the same day, at sixty days of age, and died at approximately the same age, twenty-two months, the former with a solid granulosa-cell tumor and the latter with a growth composed largely of lutein cells. Both had carcinoma of the breast and degenerative changes in the adrenal cortex. Mouse A¢f 199 has been described above. The following is the history, in brief, of Mouse Ajiif 196.

Mouse Ailf 196: Solid Granulosa-cell Tumor of the Ovary: This mouse was irradiated with 400 r at the age of sixty days and died at the age of twenty-two months. The left ovary was replaced by a gray, moderately firm, solid tumor spotted with hemorrhage, and measuring 2.5 X 2.0 X 2.0 em. (Fig. 19). The right ovary was yellow and measured 0.4 em. in its longest diameter. The cells of the larger tumor (Figs. 20, 21) were round or spindle­ shaped and fairly uniform in size and shape. Intercellular substance was very scanty and in most parts the tumor appeared to be almost devoid of capillaries. A part of the tumor, on the contrary, contained numerous large sinusoidal spaces lined by single layers of en­ dothelium and containing blood cells. In another part of the tumor there was a large old hematoma in the process of organization. There were in the tumor numerous areas of clear 78 .T. FURTH AND J. S. BUTTERWORTH cells. The vaginal mucosa was thin, and the uterine horn was likewise in the resting stage. The hypophysis and the thyroid showed no conspicuous alterations. The adrenal showed mild subcapsular fibrosis of the cortex, and in the intermediate zone there were numerous pigment cells.

Breast Tumors in Irradiated Mice Unassociated with Ovarian Growth Approximately 2.7 per cent of irradiated mice showed on routine post­ mortem examination tumors apparently of the breast gland with no conspicu­ ous enlargement of the ovaries. All these autopsies had been made before the relationship of ovarian tumors to those of the breast was discovered, and no special attention was given to the sex organs. Fortunately 3 of these mice had been preserved in Kaiserling's solution and examination was made of sections taken from tissues kept in the preservative during approximately two years. It is noteworthy that these mice died at the age of from ten to twelve months, before ovarian tumors make their appearance in our stocks of mice.

Mouse Aqc 17: This mouse received 200 r at the age of thirty-six days, followed one month later by another general irradiation with 250 r, The breast tumor (adenocarcinoma) measured 1 em, in its greatest diameter when the mouse was nine and a half months of age. The ovary was small; follicles and ova were not seen in the sections, and in general the microscopic picture was that of an ovary severely damaged by irradiation. The greater part of the organ was composed of clear and yellow cells; spindle-shaped stroma cells were predominant at the periphery, and among them there were tubules like those that arise from down-growth of the germinal epithelium. This ovary may have had enough endocrine ac­ tivity to produce the breast tumor. The endometrium appeared normal, with focal areas of hyperplasia showing numerous mitoses. The state of the endometrium likewise suggested ovarian hormonal activity. The adrenal and hypophysis showed no conspicuous change. Mouse Ar 252: This mouse received 300 r at the age of forty-three days, followed by two similar doses at monthly intervals. The breast tumor (likewise adenocarcinoma) meas­ ured 0.8 X 0.8 X 0.6 em. when the mouse was one year old. It grew rapidly, reaching the size of 3.0 X 2.5 X 2.0 em. twelve days later, when the mouse was killed. There were metastases to the lung. Histologically, the ovaries presented the same appearance as those of the mouse just described. Mouse Aqc 20: This animal received 200 and 250 r at the age of thirty-five and seventy-two days, respectively. A firm tumor approximately 0.8 em. across made its ap­ pearance in the subcutaneous tissue of the left groin when the mouse was nine and a half months of age. At autopsy, two weeks later, the tumor (adenocarcinoma) measured 2 em. in the greatest diameter, and was ulcerated. The ovaries were similar to those described above, but at the periphery there were numerous tubules, evidently resulting from down­ growth of the germinal epithelium. These ovaries may be regarded as the site of early tubular adenomata. The cortex and medulla of the adrenal were separated by a wide zone of large polygonal cells with pale brown vacuolated cytoplasm.

The study of these 3 mice indicates that sterilization by irradiation at the age of from thirty-five to forty-three days did not prevent the develop­ ment of carcinoma of the breast. The ovaries injured by x-rays apparently produce estrin in amounts sufficient for development of these tumors. It is also possible, however, that the amount of estrin produced before irradiation was sufficient to account for the appearance of carcinoma of the breast (d. Cori, 26), and that irradiation at an earlier age might further diminish or al­ together prevent the development of breast tumors. FIG. 15. A SOLID PART OF THE OVARIAN GROWTH OF IRRADIATED MOUSE AlB 45, SHOWING A MITOTIC FIGURE NEAR THE CENTER OF THE PICTURE. X 450 FIG. 16. NESTS OF PALE-STAINED EPITHELIAL CELLS SURROUNDED BY ABUNDANT SPINDLE-SHAPED STROMA, AlB 45. X 200 FIGS. 17 AND 18. ADENOMATOUS GROWTH IN OVARY OF MOUSE SGB 26. X 200 AND X 450 The higher magnification (Fig. 18) shows the tendency to form papillary projections into the lumen of the gland-like structures. 79 80 J. FURTH AND J. S. BUTTERWORTH

Granulosa-cell Tumors of the Ovary Unassociated with Breast Tumors The occurrence of ovarian tumors without breast tumors is in itself insuffi­ cient evidence for the statement that the tumors did not produce estrin, for breast tumors can be produced by estrin only in genetically susceptible mice by large doses of the hormone administered over long periods of time. The and examined in 3 of these mice likewise failed to give evidence of excessive hormone production by the tumor. It is uncertain whether the cycle persisted until death. Morphologically, these tumors were similar to those that gave evidence of excessive hormone production. Table III gives some significant data on 12 mice with ovarian tumors, presumably of the granulosa-cell type, which were unassociated with breast tumors. The tumors measuring 0.3 em. or less are described as small, those measuring from 0.3 to 0.6 em. as medium, and those measuring more than 0.6 em. as large.

TABLE III: Data on Mice with Granulosa-Cell Tumors Unassociated with Breast Tumors ...

Age at Dose Age Ovarian growth Mouse irradia- of at number tion x-rays death (days) (r) (months) Size Cell types

Af 597 37 200 14 Large Solid, granulosa cells, with follicle- like struc- 68 400 tures Af 732 36 300 14! Medium Granulosa cells with tubules 190 400 Aq 114 38 400 22 Large Solid, granulosa and dear cells Ai 147 32 400 20 Small Granulosa cells wit h few yellow cells Aib 32 31 300 16 Medium Granulosa cells: decomposed 62 300 92 300 Aib 44 37 200 17 Large Solid, granulosa cells, few follicle-like st ruc- 68 400 tures and few tubules Akd 18 32 200 17 Medium Solid, granulosa cells with tubular adenoma 64 250 Ara 33 61 400 19 Medium Granulosa cells and a few tubules Ara 34 61 400 20 Large Solid, granulosa cells; one cyst Rr 267 40 400 17 Medium Granulosa cells with tubular adenoma Sxa 38 35 200* 14 Small Solid, granulosa cells 5gb 26 65 400 19 Small Atypical adenoma Rxc 31 24 200 20 Medium Atypical adenoma 60 400 Afc 5 37 400 24 Large Solid, granulosa-cell tumor, with interwoven tubular adenoma

* Seven times at approximately monthly intervals. The majority of tumors grouped in this section were composed of granu­ losa cells such as are illustrated in Figs. 20 to 22, and 24. In most of them there were also small areas of clear or yellow cells and in many there were scattered among the solid granulosa cells tubules, lined by cuboidal epithelium, and small cysts. A precise quantitative description of the cellular composition of these tumors cannot be given because of the variability of cell types in dif­ ferent tumors, and in different parts of the same tumor, and because only one or a few sections were made of each tumor. The sections show, however, that FIGS. 19-21. SOLID GRANULOSA-CELL TUMOR OF OVARY, MOUSE A¢F 196, IRRADIATED The microscopic pictures, X 45 and X 400, show the dense masses of polygonal cells forming this growth. FIG. 22. A MITOTIC FIGURE IN AN AREA OF OVARIAN GROWTH OF IRRADIATED MOUSE AKD 18, COMPOSED OF CELLS SIMILAR TO THOSE IN FIG. 21. X 360 FIGS. 23-24. SOLID BILATERAL OVARIAN GROWTH WITH THICKENING OF THE UTERUS, MOUSE AFA 12, UNIRRADIATED The microscopic picture, X 135, shows clefts or ducts lined by cuboidal cells and separated by abundant, densely packed polygonal cells and transitional forms.

81 82 J. FURTH AND J. S. BUTTERWORTH all of these tumors contained cells that supposedly produced estrin. Of these growths the following are illustrated. Mouse AI 597: Granulosa-cell Tumor 'witll Follicle-like Structures: The ovary was re­ placed by a tumor measuring about 0.7 em. across, composed almost entirely of nests of follicles surrounded by theca-like connective-tissue cells. Some of the follicles were small, consisting of about ten cells, but others were very large, consisting of hundreds of cells. Many mitotic figures were seen in the cells of the follicles (Figs. 13 and 14). Some of the follicles were sharply outlined like normal follicles, while others were ill defined. There was great variability in the size and shape of the cell nuclei and cytoplasm. The endocrine organs were not examined. Mouse Sgb 26: Atypical Adenomatous Grounh: This mouse was fifty-eight days old at the time of irradiation with 400 r and died at the age of nineteen months. A section of one ovary, which measured 0.4 cm., showed it to be replaced by an adenomatous growth (Figs. 17 and 18). The gland-like structures were sharply circumscribed, surrounded by flat stroma cells like those of the , The epithelial cells of the adenoma varied greatly in size and shape; some were polygonal or cuboidal and others cylindrical. Some stained deeply with hematoxylin and eosin; others were pale. They formed polypoid prominences protruding into the lumina of the gland-like structures. Mouse Rxc 31: Atypical Adenomatous Growth: The cells which formed this growth (Figs. 29 and 30) were small and uniform in size and shape. The general appearance of the tumor was suggestive of its follicular nature and it showed a great resemblance to the tumor of Rr 292 (Fig. 31), in which follicle-like structures were present. Typical follicles, how­ ever, were scant in this growth, the bulk of which was made up of ducts with numerous in­ foldings suggestive of papillary adenoma (Fig. 30). Mouse AI 732: Granulosa-cell Tumor with Gland-like Inclusions: One ovary (Fig. 36) measured approximately 0.4 em. in its longest diameter, and consisted mainly of spindle­ shaped granulosa cells containing numerous tubules lined by cuboidal epithelium, cystically dilated (Figs. 35-37). The other ovary showed an early ovarian growth of similar type. The uterine horns appeared to be in the resting stage. Except for slight cortical fibrosis the adrenal showed no conspicuous change. The hypophysis seemed normal; it was composed largely of chromophobe cells. Mouse Akd 18: Early Solid Granulosa-cell Tumor with Tubular Adenoma: This early ovarian growth measured 0.5 em. in longest diameter. The greater part of the growth con­ sisted of nests of solid granulosa cells similar to those illustrated in Fig. 21, a smaller part of tubular adenoma. Fig. 22 shows a mitotic figure in a granulosa cell. Mouse Alc 5: Solid Granulosa-cell Tumor with Interwoven Tubular Adenoma: The growth was bilateral. The tumor on the right side measured 1.0 X 0.6 X 0.4 em.; that on the left side 0.5 X 0.4 X 0.3 em. They were predominantly granulosa-cell growths similar to that illustrated in Fig. 24. Scattered in the solid masses of polygonal cells were a few ducts lined by cuboidal cells that, aside from their shape, were very similar to those be­ tween the ducts. Transitional forms were numerous. In some areas these cells formed numerous follicular structures similar to those shown in Fig. 11. In parts of the tumor, however, there was conspicuous formation of ducts (Fig. 33), many of which were dilated, containing pale-stained granular matter (Fig. 34), and stood out sharply against the granulosa-cell matrix. Nevertheless, there seemed to be transitional forms between the lining cells of the tubules and the interstitial cells. Both ovaries showed essentially the same structures. The vaginal epithelium was in the resting stage.

Tubular Adenoma The development of epithelial tubes in the x-rayed ovaries of 3 mice was observed by Brambell, Parkes and Fielding (2), who described these as anomalous structures found only in irradiated ovaries. The tubes "ramify deep into the gonad and open on its surface (Plate 2, Fig. 4, and Plate 5, Fig. 1 [of their article]). The lumen of these is clear and the epithelial lining is FIGs. 25 AND 26. TtTBULAR AND CYSTIC OVARIAN ADENOMA, MOUSE AKA 72, IRRADIATED. X 25 AND X 400 FIG. 27. EXTENSIVE MYELOID INFILTRATION IN LIVER OF IRRADIATED MOUSE RR 287 There are large numbers of myeloid cells in the blood vessels. X 80. FIG. 28. TUBULAR ADENOMA OF OVARY OF RR 287, INFILTRATED BY IMMATURE MYELOID CELLS There is a conspicuous leukemic infiltration about the capsule. X 80.

83 84 J. FURTH AND J. S. BUTTERWORTH

cubical and resembles the germinal epithelium with which it is continuous. Surrounding the epithelium is a thin layer of connective tissue resembling and continuous with the tunica albuginea. It would appear that they are formed by invaginations of the germinal epithelium carrying with them the surround­ ing layer of tunica albuginea cells to form a sheath." In studying the histogenesis of tubular adenoma we arrived at a similar conclusion regarding the origin of such tubules found in the ovaries of many of our x-rayed mice. Our material shows that the proliferation of these tubules may take on tumor-like proportions. The 3 mice of Brambell, Parkes, and Fielding, in which such epithelial canals were found, were irradiated when three weeks old and were examined 195, 98, and 98 days respectively after irradiation. These investigators did not find epithelial canals in the irradi­ ated non-parous adult mice or in normal mice. All our mice that had ovarian tumors composed of such canals were irradiated, but many of them were young adults at the time of irradiation. After approximately one year almost the entire ovary of several x-rayed mice was found to be composed of tubules, many of which were continuous with the germinal epithelium, the lumen open­ ing in the capsular space of a normal ovary. The size of the early tubular adenomata did not exceed that of a normal ovary. Indeed, it is highly prob­ able that many of them were overlooked. Irradiated mouse Rxc 35 died at the age of eleven months with mediastinal lymphomatosis and its ovaries ap­ peared normal on gross examination. An early tubular adenoma of this mouse was discovered only when sections were taken from the Kaiserling-pre­ served specimen for the purpose of establishing the changes that precede the development of ovarian tumors occurring in x-rayed mice. None of these tu­ mors was associated with lesions that could be attributed to excess of ovarian hormones. Neither did Brambell, Parkes and Fielding suggest that these epithelial cells were hormone-producing. Table IV shows some salient data on mice with tumors of this type. Here, as before, the tumors measuring 0.3 em. or less are described as small, those measuring from 0.3 to 0.6 em. as medium, and those measuring more than 0.6 em. as large. The following is a brief history of a mouse with typical tubular adenoma. Mouse Aka 72: The right ovary of this seventeen-months-old mouse was replaced by a growth that measured 0.6 em. across, and consisted of tubules lined by cuboidal epithelium (Figs. 25 and 26). Some of them were collapsed, others showed cystic dilatation. Variable amounts of flat spindle-shaped stroma cells separated the tubules and there were no tran­ sitional forms between the epithelial and the stroma cells. As shown in Table IV, only two tubular adenomata exceeded 0.6 em. in longest diameter, and most of them were small. The uterine horns of 5 mice were examined. In 3 they were normal or hypoplastic and in 2 hyperplastic, but in both of these latter mice granulosa or lutein cells were abundant. One of the latter mice (Afd 81) was the only one of this series to have carcinoma of the breast: Mouse Afd 81: This irradiated mouse came under observation during the preparation of this manuscript. It developed adenocarcinoma of the breast at the age of thirteen months. Both ovaries were normal in size, and the uterine horns were thickened, tortuous, and congested. Microscopic examination showed essentially the same change in both ovaries. The greater (peripheral) part of the organ was replaced by a tubular adenomatous FIGs. 29 AND 30. ADENOMA OF OVARY OF MOUSE Rxc 31, IRRADIATED The cells are densely packed, in some parts forming structures resembling follicles, but in the greater part arranged as in papillary adenomata. X 3S and X 100. FIG.31. WALL OF A CYST IN OVARIAN GROWTH OF IRRADIATED MOUSE RR 292, SHOWING PAPIL- LARY PROJECTIONS FORl\1ED BY CELLS LIKE THOSE OF OVARIAN FOLLICLES Deeper in the stroma are gland and follicle-like structures composed of similar cells. X 100. FIG. 32. GROWTH IN MOUSE AKA 308, IRRADIATED Part of the growth consists of solid adenoma like that in Fig. 29, part of numerous cysts. X 20.

85 86 J. FURTH AND J. S. BUTTERWORTH

TABLE IV: Data on Mice with Ovarian Growths Composed Mainly of Tubular Adenomata

Age at Dose Age Ovarian growth irradia- of at Mouse Notes number tion x-rays death (days) (r) (months) Size Cell Types

Af 603 33 200 17 Small Early tubular Uterus, hypophysis 63 adenoma normal; cortical fibrosis of adrenal Afd 76 42 450 16 Medium Tubular adenoma with few granulosa cells Afd 81 67 450 13 Small Same as above Moderate endometrial hyperplasia: adeno- carcinoma of breast Aib 1 165 400 20 Small Early tubular Papillary adenoma of adenoma lung Aka 59 63 400 17 Small Tubular adenoma Adenoma of liver with many spindle- shaped stroma cells Aka 71 65 400 19 Small Tubular adenoma with spindle-shaped stroma cells Aka 72 65 400 17 Medium Tubular adenoma Aka 85 69 400 18 Small Tubular adenoma Slight endometrial hy- with granulosa cells perplasia Aka 308 60 450 22 Large Tubular adenoma Uterus, vagina, hy- with solid granu- pophysisand thyroid losa-cell areas normal Akd 17 32 200 12 Small Tubular adenoma Uterus and vagina 63 250 normal Rrnb 6 71 400 28 Large Tubular adenoma Adenoma of lung with much stroma Rmb 11 60 400 22 Medium Tubular adenoma with multiple serous cysts Rr 287 42 400 21 Medium Tubular adenoma, in- Myelosis filtrated with myel- oid cells Sgc 26 64 400 14 Small Early tubular adenoma Sib 65 58 400 18 Small Tubular adenoma with papillary pro- jections

growth and in the central part there were numerous pink-stained lutein cells. There was cystic hyperplasia of the endometrium (secretory phase). It is noteworthy that this mouse was mature, sixty-seven days of age, when exposed to x-rays. In the series upon which the incidence of ovarian tumors among x-rayed mice was determined (1) such mice as Afd 81 were included among those with no ovarian tumors, because the ovaries were not enlarged, and no sections were taken. As shown in Table IV, one mouse CRr 287) with tubular adenoma also had myeloid leukemia. Fig. 28 illustrates the ovarian growth of this mouse. There is a conspicuous leukemic infiltration of the growth, the ovarian capsule. and pericapsular fatty tissue. The uterine horns were likewise infiltrated. Fig. 27 shows a section of the liver of this mouse with extensive invasion of the FIGs. 33 AND 34. ADE~OMA OF IRRADIATED MOUSE AFC 5, WITH ABUNDANT STROMA CELLS RE­ SEMBLING THOSE THAT LINE THE GLAND-LIKE STRUCTURES. X 280 FIGs. 35-37. OVARIAN GROWTH OF IRRADIATED MOUSE AF 732 The greatest part of the growth is solid, composed of polygonal and spindle-shaped cells in which are embedded many -like structures with cystic dilation. X 75, X 25, X 400.

87 88 J. FURTH AND J. S. BUTTERWORTH

portal area and blood vessels by immature myeloid cells. The ovarian growth of mouse Aib 1 that also had lymphoid leukemia showed similar infiltrations of lymphoid cells. Most of these mice were discarded after post-mortem examination and no sections of the endocrine organs were made. The adrenal of only one mouse (Afd 76) was sectioned and showed, except for a slight outer cortical fibrosis and congestion of the medulla, no noteworthy change. The uterine horn of this mouse was not hyperplastic and the hypophysis showed no conspicuous alterations. The hypophysis of only one additional mouse with tubular adenoma was examined and was likewise normal (Aka 308). Mouse Aka 308: Tubular Adenoma with Multiple Cysts: The right ovarian growth measured 0.5 em. in largest diameter. Part of it was composed of serous cysts lined by flat or cuboidal epithelium and another part by densely packed cuboidal epithelial cells with few ducts (Fig. 32). The left ovary measured 1.5 X 1.3 X 1.0 em. Microscopically the greater part of it was disintegrated and unsuitable for study. Part of it was occupied by a hematoma and another part by cysts. The uterus, vagina, and hypophysis were normal.

Mixed Tumors in Irradiated Mice The histologic structure of several small ovarian growths was very com­ plex. This may be explained by the complexity of the changes initiated by the x-rays, including proliferation of follicular and interfollicular tissue, for­ mation of epithelial tubules by the germinal epithelium, and luteinization of follicular epithelium. Several of these changes proceed simultaneously in the same ovary, and it is evident from the description of the tumors already pre­ sented that their structure is often not uniform. The following are some salient data on a mouse with an ovarian tumor of varied structure: Mouse Aib 49: This mouse received 200 r at the age of thirty-eight days and 400 r at the age of sixty-eight days. It died at the age of sixteen months. The ovarian growth measured approximately 0.5 em. and was composed of solid granulosa cells, with few tubules, nodular growths of follicular cells, tubular adenomatous growth, serous cysts lined by cuboidal epithelium and an area of cavernous hemangioma.

Miscellaneous Ovarian Tumors in Irradiated Mice Mouse Sic 16: Fibroma-like Ovarian Growth with Adenoma: This mouse was irradiated with 200 r at the age of thirty-six days and 400 r at sixty-eight days, and died at seventeen months. The lung contained a papillary adenoma 0.2 em. in diameter. The right ovary measured 0.7 em. in its greatest diameter (Figs. 38-40). A small peripheral area was com­ posed of ducts and tubules (middle upper part of Fig. 38). The remainder of the growth was solid and composed of spindle-shaped cells like fibroblasts and small cells with round or oval nuclei and abundant pale-staining cytoplasm. Ovarian tumors of this type have been described in women as "thecoma" (d. 28). The left ovary contained a large cyst and was not examined microscopically. Mouse Rr 292: Papillary Cystic Adenoma: This case illustrates the difficulty of dis­ tinguishing between papillary adenomatous and follicular growth. The mouse was irradiated at the age of forty days with 400 r and died at the age of twenty-three months. One ovary was 0.5 em. in its greatest diameter, while the other was normal in size. The greater part of the enlarged ovary consisted of clusters and strands of epithelial-like cells separated by abundant spindle-shaped stroma. Attached to the ovary was a serous cyst into which grew papillary projections of epithelial cells that morphologically closely resembled those of fol­ licles (Fig. 31). In the wall of the epithelial cyst was a nodular area, shown in Fig. 31, re­ sembling an anovular follicle. FIGs. 38-40. OVARIAN GROWTH OF IRRADIATED MOUSE SIC 16 The bulk of the growth consists of connective-tissue cells that are possibly derivatives of, or related to, theca cells. The upper part of Fig. 38 (X 22) shows an area with tubular adenomatous growth. At the right of this is a long epithelial canal communicating with the subcapsular space. Figs. 39 and 40 show higher magnifications (X 80 and X 400) of the fibromatous (or thecoma­ like) structure.

89 90 J. FURTH AND J. S. BUTTERWORTH

II. OVARIAN TUMORS AMONG UNIRRADIATED MICE Ovarian tumors were found in 11 unirradiated mice. All but one were of the granulosa-cell type. Six occurred in Stock A, of which 2 were of small and 4 of medium size. Two mice with ovarian tumors of medium size had carcinoma of the breast; the rest of these 11 mice had none. Three tumors were found in Stock R; none was found in Stock S. In unirradiated mice there was only one tumor, a papillary adenoma, which was of a type not associated with endocrine secretion. Mouse Rxa 4: This mouse died at the age of eighteen months. The ovarian tumor was spherical and measured 0.7 em. across. Microscopic examination showed it to be a papillary cystadenoma. The cysts were lined by columnar epithelium and contained serous fluid. The mouse also had a small (0.2 em.) adenoma of the lung. No tubular adenoma was found among the unirradiated mice, nor has this type of tumor been hitherto reported in mice. The tumors presumably with endocrine secretion were of the same type as those occurring among the x-rayed mice. One was a luteoma associated with similar endocrine disturbances as the luteomata found in irradiated mice. The remaining tumors were either solid granulosa-cell growths or of a mixed structure, composed of granulosa cells, typical lutein cells, and" clear" cells. It is noteworthy that the sections did not show normal ovarian tissue in as­ sociation with any of these granulosa-cell tumors. The following are some data on unirradiated mice with ovarian tumors: Mouse Afa 12: Solid Granulosa-cell Tumors and Adenocarcinoma of the Breast: This mouse died at the age of twenty-two months. Six weeks before death glandular tumors were felt on the right side of the chest and neck. Fig. 23 shows the bilateral ovarian growths of this animal and the greatly thickened uterine horns. Microscopically (Fig. 24) the bulk of the ovarian tumors consisted of spindle-shaped and polygonal cells and transitional forms between the two. There were cleft- or duct-like spaces, lined with cuboidal cells, scattered throughout the growth, but the numerous transitional forms between these cells and those of the intervening stroma made it evident that all the cells were variants of the same cell type. There was cystic hyperplasia of the endometrium (mainly proliferating phase). The vaginal epithelium was in the resting stage. The hypophysis showed no noteworthy change, and there was only very slight subcapsular fibrosis of the adrenal cortex. Mouse Ax 100: Granulosa-cell and Lutein-cell Tumor of the Ovary: This mouse died at the age of twenty-seven months. It had an ulcerated tumor on the right side, infiltrating the skin over an area of approximately 3 X 3 em. On the left side was a similar but smaller tumor invading the skin. Microscopically both tumors were lymphosarcomata. The blood­ forming organs were normal. The right ovary was replaced by a tumor measuring 1 X 0.5 X 0.5 em., and was composed mainly of granulosa cells; the left ovary was replaced by a growth approximately 0.6 em. across, and was composed mainly of lutein cells. There were infiltrations of lymphoid cells in and about the tumor. There was mild cystic hyperplasia of the uterine horns (secretory phase) and mucous transformation of the vaginal epithelium. There was atrophy of the adrenal cortex, and the subcortical area was replaced by spindle­ shaped cells. The hypophysis showed no noteworthy change. Mouse Rr 316: Cystic Adenoma of the Ovary witJl Luteinized Stroma: This thirty­ months-old mouse died during ether anesthesia for attempted removal of the ovarian growth that could be palpated during life. The growth was spherical, measuring 0.4 em. across, and was composed of greatly dilated tubules lined with flat to columnar epithelium, among which were collections of clear cells. Masses of clear cells were found at the periphery of the tumor and formed the bulk of the growth. There was cystic hyperplasia of the endo­ metrium (proliferating phase). NEOPLASTIC DISEASES AMONG MICE SUBJECTED TO X-RAYS 91

Mouse A] 366: Atypical Ovarian Growth and Adenoma oj the Breast: This mouse died at the age of twenty-four months. In the subcutaneous tissue on the left side of the neck was a sharply circumscribed glandular growth approximately 1 em. across. The right ovary measured 0.5 crn., the left 0.3 em. Microscopic examination of the right ovary showed complete replacement by epithelial cells which exhibited great variability in size and shape. The majority were polygonal; many were cuboidal and formed duct-like struc­ tures. Transitional forms between cuboidal, polygonal and spindle-shaped forms were nu­ merous. Characteristic of the growth were focal masses of epithelial cells which either ap· peared to be surrounded by a clear area or were attached to the surrounding tissue, each by a small pedicle. Part of the tumor was similar to that shown in Fig. 18. Mouse Aic 14: Granulosa-cell Tumor Unassociated with Breast Tumor: The greater part of the ovarian tumor of this mouse, dying at the age of twenty-six months, was indis­ tinguishable from that of Afa 12 (Fig. 24). It was likewise bilateral, but somewhat smaller, each growth measuring approximately 0.5 em. in the greatest diameter. At the periphery of the growth proliferation of granulosa cells occurred in follicular arrangement. A similar but somewhat smaller unilateral ovarian growth was found in mouse Aib 24. Mouse Aic 12: Follicular Granulosa-cell Grouitli oj the Ovary: While Aic 14 had an ovarian tumor, as just described, consisting of spindle-shaped and cuboidal cells with only a small peripheral area of follicular growth, its sister, Aic 12, dying at twenty-seven months, had on the right side an ovarian growth measuring 0.6 em., a large part of which showed follicular growth (Fig. 11, 12). Another part was composed of clear cells. The left ovary measured 0.3 em. in its greatest diameter and was composed of a cellular spindle-shaped stroma in which there were embedded nests of pale-stained epithelial cells which were polygonal. columnar or cylindrical; many had a gland-like arrangement and there were nu­ merous glomerulus-like clumps of epithelial cells. The microscopic appearance of the glandular parts of the growth were like those illustrated in Figs. 17 and 18, and the solid parts like that shown in Fig. 15. Mouse Rrc 2: Folliculoma oj the Ovary: This mouse died at the age of twenty-six months. The ovarian growth. measuring 0.5 em. across, consisted almost entirely of nests of follicles like that shown in Fig. 10, but there was more abundant spindle-cell stroma among the follicles. In several of them there was a pink-stained liquid, probably follicular liquid, which was absent in the growth shown in Fig. 10. The hormonal activity of the growth in mouse Rrc 2 was indicated by cystic endometrial hyperplasia (proliferative phase), This mouse also had an osteoma attached to the left scapula.

In none of these ovarian tumors save the papillary adenoma was normal ovarian tissue found in the available sections, and anatomical evidence that the mice were not irradiated is wanting. It is possible, however, that serial sec­ tions might have revealed normal ovarian tissue.

DISCUSSION Histogenesis: The histogenesis of the special tumors of the ovary is un­ settled, as is that of the more important histologic elements of this organ. The almost universally accepted view that the epithelial cells of the follicles are de­ rived from the germinal epithelium has been challenged recently by Fischel (29), who maintains that these cells originate from the ovarian mesenchyme and that the germinal epithelium acts as a mere cover to the ovary, contribut­ ing none of its structures. Studies by BrambeIl, Parkes and Fielding (2) on the regenerative changes that follow x-ray injuries have shown, on the contrary, that in mice irradiated before puberty the germinal epithelium proliferates, producing cords of cells and anovular follicles. These cells may become luteinized. But in mice ir­ radiated when mature there are no signs of proliferation from the germinal 92 J. ~'URTH AND J. S. BUTTERWORTH epithelium, and the tissues of the sterilized adult ovary are derived from the interfollicular elements and from the disorganized follicles. Histogenesis of the human ovarian tumors is almost universally explained by reference to anomalies in embryological development. According to the widely accepted view proposed by Robert Meyer (30), the granulosa-cell tumors are derived from embryonal rests of these cells (Granulosaballen). The experiments with x-rays do not exclude this possibility, although they indi­ cate that these tumors may originate from remnants of disorganized follicles or from secondary down-growths of the germinal epithelium. In this connec­ tion the presence of numerous mitotic figures in the follicular epithelial cells of x-rayed ovaries and the observations of Brambell, Parkes and Fielding al­ ready cited are significant. While the study of our granulosa-cell tumors does not give conclusive evidence concerning their histogenesis, the tubular ade­ nomata can definitely be traced to down-growths of the germinal epithelium. In view of these observations, the opinion that the follicular epithelium is a satellite tissue, depending for its existence upon the ova, and consequently not capable of giving rise to tumors, requires revision. Post-natal Ovogenesis: The view that all ova in the mammal are formed before birth is almost universally accepted. Waldeyer has estimated the life of many human ova at fifty years. Several workers, however, have suggested that the ova of adult life are new formations, produced rhythmically, and Evans and Swezy (31) believe that they may have a shorter life span than any cells other than blood cells. In x-rayed ovaries there is proliferation of germinal and follicular epithelium and of the cells of the interfollicular tissue as well, but we have never observed any process suggestive of ovogenesis. Proliferation of follicular cells does not cease after irradiation with x-rays, yet ova are not formed. This observation may be interpreted in favor of the older concept that ova are not formed in the adult ovary. Hormonal Aspects: The Hypophysis: Some of the observations here de­ scribed are in harmony with the recent experimental results of Cramer and Horning (22), Zondek (24), and McEuen, Selye and Collip (23), who found that long continued administration of estrin produced hemorrhagic chromo­ phobe adenoma of the hypophysis. In the animals treated with estrin loss of weight may occur without adenoma of the hypophysis, due to a diminished output of the growth-promoting hormone of this gland (Zondek). Whether the cachectic condition of x-rayed mice is due to hypophyseal dysfunction re­ quires further study. The observations showing that the hypophysis itself is x-ray resistant (Regaud and Lacassagne, 8) lend further support to the view that the hypophyseal disturbance is secondary to the ovarian growth. Never­ theless, our mice were irradiated over the entire body and the direct effect of the x-rays upon the hypophysis must be considered. The reports in the litera­ ture on the effect of x-rays on the hypophysis are scanty and are concerned only with the early effects of the irradiation. The older studies are reviewed by Biedl and Borak (32). The x-rays are believed to injure only the glandu­ lar part of the organ. Degeneration and atrophy occur mainly in the eosino­ phils, but there is later a relative increase of these ceUs and decrease of both chromophobe and basophile cells. If the hypophysis of a young animal is irradiated retardation of growth occurs. There is a secondary decrease in the NEOPLASTIC DISEASES AMONG MICE SUBJECTED TO X-RAYS 93

size of the ovaries with degenerative changes in the follicles and atrophy of the uterus. Adrenal: In several mice with ovarian tumors there was atrophy of the cortex with subcapsular fibrosis and accumulation of pigment cells in the in­ termediate zone. So far as we know, the occurrence of similar lesions in nor­ mal mice has not been observed, but they have been described and illustrated in mice that received large doses of estrin over long periods of time (Cramer and Horning, 22; McEuen, Selye and Collip, 23). The question whether the lesions found by us are specific and whether they are caused directly by x-rays or indirectly by other endocrine disturbances remains to be investigated. According to the review of Biedl and Borak (32) the cortex of the adrenal is much more susceptible to x-rays than the medulla. The stratum fascicu­ latum and reticulare are chiefly affected. In the cells of the latter zone there is conspicuous accumulation of pigment. Severe cachexia follows injury to the cortex by irradiation. These scanty data do not permit any conclusion concerning the late changes which may take place in x-rayed adrenals. The Question of Malignancy: Most if not all of the ovarian tumors here described appear benign. The human granulosa-cell tumors, on the contrary, are often regarded as malignant. Local recurrence has often been noted in granulosa-cell tumors, but this is not proof of malignancy, because it is. pos­ sible that part of the growth was left behind. Peritoneal metastasis, noted in a few cases, is not conclusive evidence of malignancy, and, indeed, many work­ ers regard the majority of granulosa-cell tumors as benign (d. Bland and Goldstein, 33). Similarity of Human Ovarian Tumors to Ovarian Tumors in Mice: The similarity of human granulosa-cell tumors to those of mice becomes evident on comparison of the microscopic appearance of these tumors. Arrhenoblastoma was not diagnosed in this series, but it is not unlikely that some of the tumors were of this type. From the literature it seems that masculinization is the chief basis for the diagnosis of arrhenoblastoma, as the histologic picture varies greatly. Microscopically some of the human arrheno­ blastomata observed appeared to be of granulosa-cell type. Masculinization in mice to the extent found in women would probably have been overlooked by us. In order to establish if some mouse tumors are arrhenoblastomata a hormonal assay of the growth is desirable. Brambell, Parkes and Fielding (2) have recognized the morphological re­ semblance of the epithelial cords arising in x-rayed mice by down-growth of germ epithelium to the spermatic tubules, but the true nature of these struc­ tures is still unknown (pp. 53-54 of their article). The tubular adenoma of mice described by us results from the down-growth of germinal epithelium and, unlike "adenoma tubulare testiculare" of Pick, does not appear to be a variety of arrhenoblastoma. Tumors with large round cells characteristic of dysgerminoma (seminoma) were not found in this series, and they have thus far not been described in mice. A study of the literature suggests 'that these tumors are truly em­ bryonal, since many of them occur in hermaphrodites and many are manifest during early childhood. In contrast to the granulosa-cell tumors they are highly malignant. 94 J. FURTH AND J. S. BUTTERWORTH

It is uncertain if any of the ovarian growths of mice described are related to the Brenner tumors. Typical Brenner tumors (cf. Bland and Goldstein, 33) with follicles and cysts containing mucoid material separated by abundant fibrous stroma were not found, but in several of our granulosa-cell tumors there was a hollowing out of the center of the follicle-like structures and for­ mation of epithelial cells that resembled somewhat the mucoid cells of the Brenner tumors.

SUMMARY AND CONCLUSIONS The destructive changes in the ovaries of mice which immediately follow irradiation with x-rays are followed by slowly progressive proliferative changes resulting after from one to two years in the formation of growths with the character of neoplasms. All of the solid ovarian tumors that were found oc­ curred in senile mice. Tubular adenomata arise from down-growth of germinal epithelium in the form of epithelial canals. There is no evidence that these growths have endo­ crine function. Proliferation of follicular epithelial and interfollicular spindle-shaped cells, both of which are probably derivatives of the germinal epithelium, give rise to granulosa-cell tumors. These tumors may be composed of spindle-shaped cells like those of the ovarian stroma, or of epithelial cells like those of ovarian follicles, or of luteinized epithelial cells, or of several different morphological variants of the granulosa cells. In many of the mice with granulosa-cell tumors excessive endocrine func­ tion is indicated by extensive cystic glandular hyperplasia of the endometrium, and in a few mice by hyperplasia of the anterior lobe of the hypophysis. The incidence of breast tumors among mice with granulosa-cell tumors is greater than among unirradiated females of the same stock, but it is less among irradiated mice with no ovarian tumor or with tubular adenoma of the ovary. Sterilization of the ovaries, however, does not cause cessation of estrin pro­ duction and does not altogether prevent the formation of breast tumors.

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