Spontaneous Malignant Mixed Tumors of the Rat, and the Successful Transplantation and Separation of Both Components from a Mammary Tumor Wilhelmina F

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Spontaneous Malignant Mixed Tumors of the Rat, and the Successful Transplantation and Separation of both Components from a Mammary Tumor Wilhelmina F. Dunning, Ph.D., Maynie R. Curtis, Ph.D.,* and Mark E. Maun, M.D.

(Department o/ Pathology, Wayne University College o/ Medicine in co-operation with the Detroit Institute o/ Cancer Research, Detroit 26, Michigan)

(Received for publication June 1, 1945)

Malignant mixed tumors in man are relatively rare, comas and 1 a mixed tumor. Loeb (14) reported the but their occurrence has occasioned considerable dis- transplantation of an adenocarcinoma of the sub- cussion of their probable histogenesis. In 1937, Raso maxillary gland of a mouse, which developed into (16) reported an osteoid chondrosarcoma of the breast mixed tumors and spindle cell sarcomas in the first and collected from the literature, dating from 1835, transplanted generation. A transplantable mixed tumor 74 mammary tumors containing bone and cartilage, of the fowl was reported by Foulds (10). The primary but only 5 were considered to be true mixed tumors. growth, which appeared to be a carcinoma of the ovi- Saphir and Vass (17), in 1938, reviewed 153 cases of duct, was transplanted for 12 generations with varying "carcinosarcoma," excluding any tumors containing success into the pectoral muscles of fowls of the same large amounts of cartilage and bone that might be in- and different breeds. Bone and cartilage were found terpreted as teratomas. They concluded from the mor- in growths of the first and succeeding generations of phology of these neoplasms that in all but 3 or 4 cases transplantation. No metastases were recorded, and a dual nature was very questionable. The majority Foulds concluded that only the epithelial component they interpreted as primary carcinomas, evaluating the survived from generation to generation, the stroma sarcomatous portion as composed of carcinoma cells being formed anew by the host's connective tissue cells showing transitional features, or marked anap|asia, or under the influence of the neoplastic epithelial paren- as a lymphocytic reaction due to chronic inflammation. chyma. Other cases they believed to be sarcomas invading meta- The frequency with which sarcomatous transforma- plastic epithelial structures. tion of the stroma of transplanted mammary carci- Budd and Breslin (2) reported a malignant mixed nomas has been observed by Ludford and Barlow (15) tumor of the chest wall, which Ewing considered a in highly inbred stocks of mice and in studies of these primary mammary carcinoma with bone deposits added growths in vitro has been attributed by these authors by a curious type of metaplasia. Mallory diagnosed it to either of two factors; the considerable stimulating as a carcinoma and an osteogenic sarcoma, one invad- action of the carcinoma cells on the fibroblasts, or per- ing the other, and Masson believed in this case that sistence of the stromal cells through repeated trans- he could follow a direct transformation of the epithe- plantations due to homozygosity of the cells of the lioma into osteosarcoma. graft and of the new host. In each case there was a Allen (1) described 4 mixed tumors of the mam- progressive increase in quantity and cellularity of the mary glands of dogs, and 1 of the human breast. In stroma, which preceded the appearance of the sarcoma 3 of the canine tumors he believed the cartilage was as had earlier been observed by numerous investigators derived directly from adult epithelium. This view was including Ehrlich and Apolant (8), Haaland (11), endorsed by Ewing (9). In contrast, Harvey, Dawson, and Lewin (13); by the latter also in the case of a and Innes (12) and Sheldon (18) believed the cartilage transplanted adenocarcinoma in a rat. In most cases and bone of mixed tumors of the salivary glands arise the authors excluded the probability that these growths by metaplasia from the connective tissue stroma. could have been mixed tumors from their inception, In the mouse, Slye, Holmes, and Wells (19) re- since they had been under observation for from 5 to ported 12 malignant neoplasms of the thyroid gland as many as 67 generations before the sarcomatous of which 4 were of dual origin, 3 being carcinosar- element was identified. In one case (8) the appear- * Fellow of the Detroit Branch of the Field Army of the ance of a mixed tumor persisted for 20 generations, American Cancer Society. but more commonly the sarcomatous portion overgrew 644

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1945 American Association for Cancer Research. Dunning et al.--Spontaneous Malignant Mixed Tumors 645 the carcinomatous within a generation or two after its SPONTANEOUS MIXED ']'UMORS OF RATS recognition and ultimately supplanted it entirely. Ehr- A study of the spontaneous mixed tumors that oc- lich (7) and Haaland (11) each hastened this separa- curred in the rats of our colony offers pertinent data re- tion process by experimental measures. The former garding the nature of these growths. Metastases fre- used heat or cold to destroy or selectively weaken the quently occurred and were often a direct factor in carcinomatous portion, and the latter inoculated the the death of the hosts; examination of these metastatic mixed tumor into mice experimentally immunized to growths established beyond a doubt the malignant mouse carcinoma cells; in both instances some pure sarcomas resulted, whereas the control series continued character of one or more of the tissues observed in the as mixed tumors. primary tumor. Mixed fibroepithelial neoplasms occur spontaneously Among the spontaneous neoplasms 27 mixed tumors (4), and can be readily induced with comparative of the mammary gland, uterus, and face were observed frequency in the rat; often both components appear (Table I). In the mammary gland 41 malignant to be malignant. In 1928 Bullock and Curtis (3) rc- growths were found; 24 were mesodermal, 12 epi- ported a Cysticercus tumor of the rat's liver classi- thelial, and 5 were mixed tumors. The sarcomatous fied as a carcino-osteochondrosarcoma. Peritoneal portions were mixed with portions of adenocarcinoma metastases were abundant, and microscopic examina- in 3 instances, while 2 tumors had these elements plus tion revealed scattered islands of osteoid tissue and portions of squamous cell carcinoma. Only one of

TABLE I: TilE NUMBER AND PERCENTAGE OF SPONTANEOUS TUMORS AT 3 SITES THAT WERE OF EACH MORPHOLOGICAL PATTERN

Mixed tumors r Sal'c~ "~ and adeno- Classification Sarcoma Sarcoma carcinoma ~'- -~ and and and r Fibro- Adcno- Sq: cell Adeno- adeno- sq. cell sq. cell Location sarcoma carcinoma carcinoma acanthoma carcinoma carcinoma carcinoma Total Breast No. 24 9 1 2 3 0 2 41 % 59 22 2 5 7 5 100 Uterus No. 68 12 24 2 3 8 1 118 % 58 10 20 2 2 7 1 100 Face No. 21 0 57 1 0 10 0 89 % 24 64 1 11 100 Total 113 21 82 5 6 18 3 248

cartilage in the diaphragm and neoplastic epithelial these metastasized, with the production of extensive cells within the lumina of the vessels in the uninvolved areas of adenocarcinoma in the lungs. One of the 2 part of the cyst wall. This tumor was one of 3, in that contained both adenocarcinoma and squamous a series of 2,100 induced mesothelial tumors, that cell carcinoma (R2572) was used in the transplanta- contained neoplastic epithelium. In 1936 Dunning, tion studies described here. Curtis, and Bullock (5) reported a total of 9 mixed The uterus was the site of the largest number of tumors in a series of about 900 benzpyrene and dibenz- mixed tumors in these rats. Of a total of 118 malig- anthracene neoplasms of the rat. In this article were nant neoplasms in this organ, 68 were sarcomas, 38 included 25 mixed tumors of the mouse in a series of were carcinomas, and 12 were mixed tumors. Most some 500 benzpyrene tumors. Later, in 1940 (6), 17 of the latter were composed of squamous cell carci- mixed tumors were discovered among approximately noma mixed with mesodermal elements. Metastases 900 subcutaneous methylcholanthrene neoplasms. The were found in the peritoneum and abdominal viscera epithelial component of the tumors previously men- in 6 cases, with both components of the tumors present tioned was of mammary origin and probably resulted in 5. from direct local action of the carcinogen on the Another frequent site of malignant mixed tumors in glandular tissue or the areas of metaplastic squamous the rat is the skin of the face near the orbit. Here epithelium frequently observed in the mammary ducts 89 malignant neoplasms were found; 57 were squamous adjacent to foci of injected carcinogen. Metastases cell carcinoma, 1 contained squamous and glandular were scanty and contribute little evidence for the epithelium, 21 were fibrosarcomas, and 10 were mixed malignancy of either component, since the hosts were tumors made up of sarcoma and squamous cell carci- sacrificed soon after the tumors were observed. noma. Metastases were found from only 3 of these

10 mixed tumors. In 1 case the secondary growths that segregation was taking place. The squamous were composed of pure squamous cell carcinoma, in cell component was not observed after the second another of pure'fibrosarcoma, and in the third both generation, and the sarcomatous and bony component components were present. was found in only about half of the growths. It seemed From examination of these data it seems probable feasible, therefore, to attempt to separate the epithelial that in the rat malignant mixed neoplasms composed of and connective tissue elements. Table II shows by 2 independent neoplastic components growing as a generation the history and classification of the trans- single tumor occur at sites where growths composed of planted fragments. each component are frequent. Either or both of the components may metastasize. In all, 27 mixed tumors TRANSPLANTATION STUDIES have been observed, with metastases occurring in 10, Procedure.--In preparation for the transplantation of which 6 showed both malignant mesodermal and of the fifth generation 9 grafts, each weighing about malignant epithelial components. In order to evaluate 10 mgm., were cut from one of the mixed tumors further the nature of a mixed tumor the following (Fig. 7) of the fourth generation. Each graft was transplantation experiments were undertaken. cut in half and one half was again divided and implanted on the right and left sides of each of 9 rats. EXPERIMENTAL PROCEDURE The other half was fixed in Zenker's fluid for serial TRANSPLANTATION HISTORY OF MIXED TU~OI~ R2572 sectioning. Animals.--The rats used in the transplantation The tumor selected for transplantation studies was studies were all of the same inbred strain, A• R2572. This was a soft, mottled, encapsulated mass in Line 9935. the subcutaneous tissues of the left groin. It had Histologic features o/ transplanted tumors.--Ex- grown rapidly, was free from the skin and underlying amination of sections of the 9 tumor fragments revealed muscle, and measured 4.5 • 4.0 • 2.2 cm. when excised 5 to be pure adenocarcinoma; 3 were a mixture of for transplantation. A diagnosis of papillary cyst adeno- adenocarcinoma and fibrosarcoma, and 1 contained carcinoma was made from a frozen section, which was areas of cartilage in addition. The host into which the later confirmed from a paraffin section (Fig. 1). other half of the tumor fragment containing cartilage The bearer was an 849 day old A • C female from had been implanted died after 41 days with 2 tumors, the 20th brother-by-sister generation of Line 9935. She each containing bone and cartilage. The growths were died 65 days after excision of the primary growth with then excised from the left sides of the 8 remaining rats no recurrence, but with an early squamous cell can- and spaced serial sections were examined. Five ap- cer of the lower jaw. peared to be pure adenocarcinomas, and 3 were mixed Fragments of the primary mammary tumor were im- tumors corresponding in morphology with the other planted in the subcutaneous tissues of both sides of half of the graft, previously examined. each of 6 males of the same inbred line as the bearer Results.--On the 89th day after implantation 1 of the of the primary growth. The grafts all grew progres- 5 rats in which half of the graft and the tumor on the sively, and microscopic examination of each of the left side had shown only adenocarcinoma was selected resultant neoplasms revealed areas of adenocarcinoma and 10 rats were inoculated with fragments from the (Fig. 2) not unlike the primary tumor. In addition, growth on the right side. A section of the tumor used 2 tumors showed areas of fibrosarcoma (Fig. 3) with for inoculation of generation 6A is shown in Fig. 8. only a few scattered epithelial elements, and 3 con- For the other series a tumor obviously containing tained large areas of squamous cell carcinoma (Fig. bone was selected from the 3 that had grafts containing 4) and adenoacanthoma (Fig. 5). fibrosarcoma, and mixed tumors on the left side. Ten The second generation consisted of 8 females simi- females were inoculated with grafts from this tumor larly implanted with grafts from one of the tumors (Fig. 9). All the grafts in generation 6B grew pro- from the first transplanted generation. All the grafts gressively and all the resultant tumors were distinctly grew progressively and microscopic study revealed bony. Seven proved to be mixed tumors and 3 were tumors showing all the morphological variations ob- osteochondrosarcomas. In succeeding generations all served in the previous generation and, in addition, the growths have been bony and a decreasing percent- 3 growths that contained areas of bone and cartilage age have contained epithelial remnants. Fig. 10 is a (Fig. 6). The presence of bone was detected in the representative section of one of these tumors, showing gross at the time of the postmorten examination as well malignant stroma and adult bone. Lung metastases as in the histologic sections. are frequent from growths of this series, and all have Mixed tumors continued to appear in the third and been bony in structure. Fig. 11 shows the lungs and fourth transplanted generations, but it was obvious pericardium studded with small bony metastases from

Fu;. l.--Primar\ sp~,ntanc~,u~ tmn~,r. R2572. Ma2. X~)5. Fro. 2.--Adcm~carcinoma ill fir.st ~cncrati~m transplant. R2572 IA. Mag. X }~5. [q(;. 3.--l'ibr~sarcon/a in fir,~t generation transplant. R2572 1A. Mag. y,, 95. Fl(;. 4.--First gcncration transplant.~howing a squamous ceil carcimmm and adcnocarcmoma. R2572 IA. Mag. X 95.

Fro. 5.--Aden.acanthoma in first generation transplant. R2572 1A. Mag. Xt~5. Fw,. 6.--Second generation transplant showing cartilage and adcm~carciuoma. R2572 2A. Mag. )K 95. FIc. 7.--Fourth generation transplant. Mixed tumor showing cartilage, bone, and adcnocarcmoma. R2572/4A. Mag. X 95. Fro. 8.--Adcnocarcinoma in fifth generation transplant. R2572/5A to 6A. Mag. X 93.

a tumor of generation 8B, and Fig. 12 is a section thelium, but this tumor now appears to be a pure through one of these metastatic loci. adenocarcinoma. Metastases from this component had The 5 succeeding generations from generation 6A, not been observed until the tenth generation. Fig. which was composed solely of adenocarcinoma, have 13 shows extensive involvement of the lungs with contained no bone or cartilage. In the tenth generation metastases from one of the tumors in the tenth genera-

TABLE II: TRANSPLANTATION HISTORY BY GENERATION OF R2572 Classification A , ,-.~ No. Age Days Tpl, rAdeno- Sq. cell Osteo- Lung of inoc., to tumor car- car- Fibro- chondro- meta- rats Sex Gen. days death history cinoma cinoma sarcoma sarcoma stases 6 c~ 1A 71 227 q-+ 6 3 2 0 0 8 ~ 2A 83 148 ++ 8 4 6 3 0 9 ~ 3A 100 147 ++ 9 0 4 2 0 10 9 4A 122 132 ++ 10 0 4 3 0 9 ~ 5A 248 106 ++ 9 0 5 4 1 10 ~ 6A 156 69 -+-+ 10 0 0 0 0 10 ~ 6B 113 96 ++ 7 0 10 10 1 i I ~ 7A 149 67 qt-+ 11 1 0 0 0 8 ~ 7B 335 125 Fq- 5 0 8 8 2 10 9 8A 170 75 ++ 10 0 0 0 0 10 ~ 8B 403 97 ++ 3 0 10 10 4 2 ~ 9A 182 56 -k+ 2 0 0 0 0 6 c~ 9B 576 72 ++ 1 0 2 6 2 4 ~ 9C 135 52 ++ I) 0 1 3 0 38 ~ 10A 119 107 -~+ 38 0 4? 0 7 8 c~ llA 94 66 ++ 8 0 0 0 0 5 ~ IIA 91 77 ++ 5 0 0 0 0

Fro. 9.---Mixcd ttun(," in fourth generation transphmt sh(Jxving b~mc, cartilage, and adcnocarcin(,l~a. R2572,,'5A to 6B. Mag. X !)5. FI(;. l{).--Scvcnth generation transplant showing adult bone and malignant stroma. R2572,,'7B. Mag. X 95.

4 of the 38 growths examined showed a somewhat tion, and a section (Fig. 14) reveals the epithelial more cellular stroma than is characteristic, and are nature of one of these secondary growths. therefore shown in Table II in the fibrosarcoma column Remarks.--Spontaneous mixed tumors of rats oc- with a question mark. One growth in the seventh curred frequently in the mammary gland, uterus, generation contained a few islands of squamous epi- and face. Metastases from some of these were corn-

Fro. l l.--Eighth generation transplant showing extensive bony metastases in the lung and pericardium. R2572/8B. Fro. 12.--Eighth generation transplant showing extensive bony metastasis in the lung. R2572/SB. Mag. X 95. FIa. 13.--Tenth generation transplant showing extensive lung metastases from the epithelial component. Fro. 14.--Lung metastasis of the tenth generation transplant. R2572/10A. Mag. X 95.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1945 American Association for Cancer Research. Spontaneous Malignant Mixed Tumors of the Rat, and the Successful Transplantation and Separation of both Components from a Mammary Tumor

Wilhelmina F. Dunning, Maynie R. Curtis and Mark E. Maun

Cancer Res 1945;5:644-651.

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