A COM MEEME W 3139‘?” at? $8LEUED CAP-{ENE m H\ E E6?" ‘Siixég Timers {3cm “to Dawn (If M. S. I’ECHEGM’Q STATE UNWERSETY Dibakar P‘migmhi i967 m ., LIBRARY % Michigan State Univeréty THESIS ABSTRACT A COMPARATIVE STUDY OF SELECTED CANINE SKIN NEOPLASMS by Dibakar Panigrahi A study was made of the canine skin neoplasms diagnosed as trans- missible venereal tumor, adnexal carcinoma or basal cell carcinoma in the Department of Pathology over a 10-year period. Of the 459 neoplasms in these groups, 43 were diagnosed as transmissible venereal tumor, 302 as adnexal carcinoma, and 114 as basal cell carcinoma. These neo- plasms were compared with regard to breed, sex and age incidences and primary location in the body. Microscopic study was made of 25 trans- missible venereal tumors, 25 adnexal carcinomas and 25 basal cell car— cinomas. Histochemical studies were done on 22, 17 and 19 neOplasms, respectively, from the 3 groups. Of the 25 transmissible venereal tumors studied microscopically, only 3 had the typical histologic characteristics described for this ne0plasm. Most of the remaining neOplasms in this group histologically resembled the adnexal carcinoma. This discrepancy probably represents a change in the diagnostic criteria used in recent years in evaluating skin neoplasms of dogs. The 3 neoplasms typical of transmissible venereal tumors occurred on the genital organs and in the neighboring area. The adnexal carcinoma appears to represent a distinct group of neoplasms seen in dogs and is probably sometimes diagnosed as histio- cytoma or extragenital venereal tumor. It occurs frequently in the Dibakar Panigrahi skin of young dogs. Boxer dogs are commonly affected. The term atypical epithelioma is suggested by the author for this group of neoplasms, for the following reasons: 1. The neoplastic cells appear to originate from the stratum germi— nativum of the stratified squamous epithelium of the epidermis and other areas, such as lips and oral cavity, and the epithelium of hair follicles. 2. The cells are atypical due to the fact that they are pleo- morphic and do not resemble the parent cells from which they originate. This neoplasm may easily be confused with the transmissible venereal tumor when the latter is present in extragenital sites. No specific criteria could be established in this study to differentiate atypical epithelioma from transmissible venereal tumor. The use of special stains within the limitations of fixed tissues is not very helpful in their differentiation. Microscopic characteristics, history and the area of location of the neoplasm are of some value to the pathologist. More work with histochemistry on fresh tissues, chromosome counts, tissue culture, and electron microscopy is needed to define the nature of the ne0plastic cells. The basal cell carcinoma is microscopically distinct from the trans- missible venereal tumor and adnexal carcinoma. The tumor occurred frequently in Cocker Spaniel dogs. Older dogs were affected. These neoplasms were commonly located in the head and neck areas. The occur- rence of glycogen in the cytoplasm of the neoplastic cells in some of them is of interest. The average greatest nuclear diameter is signifi- cantly greater in basal cell carcinoma than in transmissible venereal tumor or adnexal carcinoma. A COMPARATIVE STUDY OF SELECTED CANINE SKIN NEOPLASMS by Dibakar Panigrahi A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1967 ACKNOWLEDGEMENTS The author is deeply indebted and wishes to express his gratitude and appreciation to the following for their generous help in making this study possible: To Dr. G. L. Waxler, major professor, for his personal interest in this study and for his valuable suggestions, help, and guidance, which were so useful in the completion of this thesis. To Dr. C. C. Morrill, Chairman of the Department of Pathology, for providing opportunity to do graduate studies in the department and for his help and guidance during the entire period of study. To Dr. R. F. Langham, for his enthusiasm in this study, the large amount of time he devoted to the project, and his many suggestions. To Dr. V. L. Sanger, for his time spent in the correction of this manuscript and for his many useful suggestions. To the American Cancer Society Institutional Research Grant, for providing financial assistance for this project. To Dr. J. L. Gill,‘Department of Dairy, for assisting in evalua— tion of the statistical data. To the histotechnicians of the Department of Pathology, especially to Mrs. Mae Sunderlin, for her assistance in preparation of materials used in this study. Finally, the author is grateful to his colleagues for their help and encouragement during his training in this department. ii To my uncle Sri. Baikuntha nath Panigrahi iii INTRODUCTION. REVIEW OF LITERATURE. MATERIALS AND METHODS RESULTS Transmissible Venereal Histiocytoma . TABLE OF CONTENTS Basal Cell Carcinoma . Transmissible Venereal Adnexal Carcinoma. Basal Cell Carcinoma . DISCUSSION. SUMMARY Transmissible Venereal Adnexal Carcinoma. Basal Cell Carcinoma LIST OF REFERENCES. VITA. Tumor Tumor iv Page l3 14 18 20 20 47 58 7O 7O 73 78 86 88 93 Table LIST OF TABLES Page Breed and sex incidences of 3 skin tumor types. . . . . 21 Microscopic characteristics of transmissible venereal tumor, adnexal carcinoma, and basal cell carcinoma. . . 24 Histochemical characteristics of transmissible, venereal tumor, adnexal carcinoma, and basal cell carCinoma O O O . O O O O O O O O O D O O O O O I O O O O 40 Figure 10 ll 12 13 LIST OF FIGURES Page Transmissible venereal tumor. Uniform appearing neoplastic cells arranged.ixlsheets. The supporting stroma is scanty. . . . . . . . . . . . . . 34 Transmissible venereal tumor. Note uniformity in the shape of the neOplastic cells and hyperchromatism Of Cell nUClei O O O O O O O O O O O O O O O O I O O O 34 Transmissible venereal tumor. Higher magnification of Figure 2. Note hyperchromatism . . . . . . . . . . 35 Transmissible venereal tumor, with very small amount of reticulum, mostly around capillaries. . . . . . . . 35 Transmissible venereal tumor, with moderate amount of reticulum distributed irregularly in another area of the same section as Figure 4. . . . . . . . . . . . 36 Unclassified neoplasm. Note variation in shape of nuclei and hyperchromatism. One giant cell seen. Nucleoli are not prominent . . . . . . . . . . . . . . 37 Unclassified neoplasm, same as Figure 6. Note presence of mitotic figures in large numbers . . . . . 37 Adnexal carcinoma. Neoplastic cells have invaded the subcutaneous fat and clustered around fat vaCUOleS O O O O I O O O O O O O O O O O I .0 O I O O O 49 Adnexal carcinoma. Note necrosis in the cellular area 0 O C O O O O O O O O O O O O O O O O I O O O O O 49 Adnexal carcinoma. Neoplastic cells originating from epidermis O O O O O O O O O O O O O O O V. O .0 O 0 50 Adnexal carcinoma. Same as Figure 10. Note origin of the neoplastic cells from epidermis . . . . . . . . 50 Adnexal carcinoma. Higher magnification of Figure 11. Note origin of neoplastic cells from epidermis and the variation in their shape . . . . . .._ . . . . 51 Adnexal carcinoma. The neOplastic cells appear to originate from the epithelium of the hair follicle. Also note the marked variation in the shape of the nuclei . . . .‘. . . . . . . . . . . . . . . . . . . . 51 vi Figure 14 15 l6 l7 18 19 20 21 22 23 24 25 26 27 28 Adnexal carcinoma. The neOplastic cells appear to originate from the epithelial cells of two hair fOlliCleS O O O I O O O O O O O 0 O O O O O I O O O O O Adnexal carcinoma. Individualization of neOplastic cells due to subepidermal edema. Also some cells appear to originate from the epidermis . . . . . . . . Adnexal carcinoma. Highly cellular neoplasm with very little visible supporting stroma° . . . . . . . . Adnexal carcinoma. Note indistinct cytoplasmic out- lines with some variation in the shape of the nuclei. A few mitoses are also seen. . . . . . . . . . . Adnexal carcinoma. Higher magnification of Figure 17. Note variation in the shape of the nuclei . . . Adhexal carcinoma. Higher magnification of Figure 8. Note variation in the shape of the nuclei. . . . . Adnexal carcinoma. Large amounts of reticulum seen around small groups of cells . . . . . . . . . . . . Adnexal carcinoma. Moderate amount of reticulum distributed irregularly around groups of cells . . . . Adnexal carcinoma. Moderate amount of reticulum irregularly distributed around and between neo- plastic cells. . . . . . . . . . . . . . . . . . . . Adnexal carcinoma. Very small amount of reticulum present 0 O O O O O O O O O O O O O O I O 6 O 0 0 O O O Basal cell carcinoma. Note apparent origin of the neoplastic cells from the epidermal basal cells. . Basal cell carcinoma. Solid nests of neOplastic cells with necrosis of the cells in the center . . . . Basal cell carcinoma. Neoplastic cells arranged in solid nests surrounded by connective tissue stroma . . Basal cell carcinoma. NeOplastic cells arranged in small solid nests surrounded by large amounts of Cellular stroma. I O O O O O O O I O I O O O O Basal cell carcinoma. Neoplastic cells arranged in serpentine cords and surrounded by large amounts of connective tissue stroma . . . . . . . . . . vii Page 52 52 54 54 55 55 56 56 57 57 6O 6O 61 61 62 Figure 29 3O 31 32 33 34 35 36 37 38 39 Page Basal cell carcinoma. Neoplastic cells arranged in cords with their long axes perpendicular to the basement membrane. Also note large amount of nuclear chromatin. . . . . . . . . . . . .'. . . .'. . . . . . 62 Basal cell carcinoma. Small nests of cells forming tubule-like structures . . . . . . . . . . . . . . . . 63 Basal cell carcinoma. Small nests of neOplastic cells with the peripheral cell nuclei arranged with long axes perpendicular to the basement membrane . . . 63 Basal cell carcinoma. Small nests of cells separated by small amount of stroma. Note formation of gland- like Structures I I I I I I I I I I I . I I I I I I I I I 64 Basal cell carcinoma. Note formation of a cyst-like structure with desquamated neoplastic cells in its lumen I I I I I I I I I I I I I I I I I I I I I I I I I 64 Basal cell carcinoma. Moderate amount of reticulum arranged irregularly around groups of neOplastic cellSI I I I I I I I I I I I I I I I I I I I I I I I I 66 Basal cell carcinoma. Note presence of large amount of reticulum distributed around small groups of neoplastic cells . . . . . . . . . . . . . . . . . . . 66 Basal cell carcinoma. Note presence of moderate amount of reticulum mostly distributed around nests of cells and occasionally coursing from the periphery toward the center. . . . . . . . . . . . . . . . . . . 67 Basal cell carcinoma. Note presence of deeply stain- ing, periodic acid—Schiff-positive material in the cytoplasm of the neoplastic cells. . . . . . . . . . . 68 Basal cell carcinoma. Note presence of only very small amounts of PAS-positive material after dia- stase digestion. . . . . . . . . . . . . . .‘. . . . . 68 Basal cell carcinoma. Note presence of moderate numbers of mast cells in the stroma surrounding the neoplastic cells . . . . . . . . . . . . . . . . . . . 69 viii INTRODUCTION Although transmissible venereal tumors of dogs were reported nearly a century ago, the actual cell type involved in the formation of this tumor is not unanimously agreed upon. Probably no neOplasm of dogs has aroused so much interest and confusion among research workers as has this one. Some investigators have suggested the name "histio- cytoma" (Mulligan, 1948) based on the histologic characteristics of the tumor, while others do not accept this nomenclature. A group of tumors occurring primarily in the skin of dogs, with histological characteristics somewhat similar to the transmissible venereal tumor, has been encountered in the Department of Pathology, Michigan State University. These tumors have been considered as origi- nating from the basal cell layer of the epidermis and adnexal structures of the skin and accordingly designated as "adnexal carcinomas". These would have probably been referred to as "extragenital venereal" tumors by some investigators (Smith and Jones, 1966). Although basal cell carcinomas are quite distinct in their mor- phologic characteristics, they were included in this study because they can be confused with the transmissible venereal tumors in extragenital locations and with adnexal carcinomas. The objectives of this study were: 1. To study the morphologic characteristics of the neoplastic cells from the 3 tumor types. 2 2. To demonstrate any possible differences between these neoplasms by the use of special stains. 3. To summarize the incidence by breed, age, sex and anatomical location. REVIEW OF LITERATURE Transmissible Venereal Tumor The transmissible venereal tumor of dogs is a unique biological entity and has created considerable interest among workers in both veterinary and human medical research. The origin of the neoplastic cells is yet to be ascertained. Several names, such as "infective sarcomata" (Smith and Washbourn, 1899), "endothelioma" (Beebe and Ewing, 1906), "transmissible lympho— sarcoma" (Feldman, 1932), "contagious lymphosarcoma" (DeMonbreun and Goodpasture, 1934), "venereal sarcoma" (Stubbs and Furth, 1934), "neuro- blastoma" (Jackson, 1936, 1944), "histiocytoma" (Mulligan, 1948), and "transmissible venereal tumor" (Bloom, Paff, and Noback, 1951) have been applied to this tumor since the first report that appeared nearly a century ago (Novinsky, 1877). Almost all the classifications, however, have been based on the histologic characteristics and general behavior of the tumors. The transmissible nature of this tumor, both by coitus and experimental tranSplantation, has been well established in numerous experiments. The most important of thesexexperiments have been conducted by Smith and Washbourn (1898), White (1902), Sticker (1904, 1906a), Beebe and Ewing (1906), Beebe (1907), Wade (1908), Stubbs and Furth (1934), DeMonbreun and Goodpasture (1934), Bloom _£y§1, (1951), and Karlson and Mann (1952). The tumors were successfully transplanted into fox cubs (Sticker, 1906b; Wade, 1908), but almost all the laboratory animals, including cats and chickens, were refractory to experimental 3 4 transfers (Smith and Washbourn, 1898; Sticker, 1904, 1906M: DeMonbreun and Goodpasture, 1934). Development of immunity to further transplants following spontaneous regression of the transmissible venereal tumors has been reported by most workers in this field. But to the knowledge of the author, no one has eXplained the mechanism reSponsible for such reaction. The rela— tively benign nature of the neoplasm has also been recognized, although a few reports have dealt with its metastasis to lymph nodes, spleen, liver, and eye (Feldman, 1929; Rust, 1949; Karlson and Mann, 1952; Barron, Saunders, Seibold and Heath, 1963). Microorganisms were sus- pected by some early workers of being responsible for the etiology and transmission of the tumor, but subsequent works proved that it could be transplanted only by viable tumor cells and no organisms were involved. Shimkin (1955) cited Novinsky (1877) as being the first worker who experimentally transmitted this tumor to puppies in his laboratory. He has been referred to as the "forefather of experimental oncology" (Stewart, Snell, Dunham, and Schlyen, 1959). Smith and Washbourn (1898, 1899) reported the incidence of this neoplasm in "highly-bred" dogs. In,1 dog they noted metastatic tumor cells in other organs. The authors stated that the malignancy seen in ”highly—bred" and ill-nourished dogs might not manifest such characteris- tics in healthy animals. The tumor they studied had the histologic structure of "round—cell sarcoma" with very little stroma. "Contagious tumors” were observed by White (1902) in 2 purebred female bulldogs. One of these animals, which had a vaginal growth, also developed a mammary tumor. Histologic examination of tumors from both these sites revealed small, closely packed round cells with delicate 5 stroma. The author concluded that transmission of these tumors might be an example of transplantation rather than infection. Beebe and Ewing (1906) observed a number of these neoplasms in New York City. Microsc0pic examination revealed the arrangement of tumor cells in cords supported by thin strands of connective tissue. They believed that it was a true malignant neoplasm and thought that the transplanted cells actually gave rise to the tumor in the hosts. These writers diagnosed the tumor as an "alveolar sarcoma" or "endothelioma". Beebe (1907) implanted the tumors into dogs by several methods with some interesting results. He stated, "An immune animal may again become susceptible by any process which would be likely to diminish his general resistance". He did not believe that any microorganism was responsible for either origin or transmission of the transmissible venereal tumors. The neoplasm under his observation had a benign nature in the beginning, but subsequent metastasis caused cachexia and even- tually death of the animals. Several bacterial endotoxins injected into the tumor tissues and at a considerable distance from them in experimental animals caused necrosis and subsequent regression of the tumors until they disappeared (Beebe and Tracy, 1907). However, 1 dog treated with suspensions of "Staphylococcus pyogenes aureus" had no tumor regression. Whole blood transfusion from spontaneously recovered and naturally immune animals to the tumor-bearing dogs caused complete regression in 7 out of 10 dogs. Only 1 dog died without showing any evidence of regression (Crile and Beebe, 1908). The authors believed the regres- sion was probably due to transfer of some immune material through the blood. 6 Wade (1908) successfully transplanted the tumors through 8 tumor generations. In 1 case (2nd generation), secondary foci were noted in several visceral organs. He emphasized that the implanted cells stimu- lated the connective tissue cells of the host through intracellular viruses to give rise to new tumors, although he could not demonstrate the presence of any such microorganisms. This theory, however, has not been accepted by other workers. Wade noted acute interstitial nephritis, which he thought was due to a soluble toxin from the tumor cells, in many of his experimental dogs. This has not been reported by others, possibly because kidneys have not been routinely included in their studies. Huebner (1922) reported the involvement of both eyes of a 2-year— old male Fox Terrier with metastatic tumors. The dog also had a penile tumor that had been removed previously. Several lymph nodes had meta— static foci. Huebner called this tumor a "small-celled, medullary round cell sarcoma". His attempts to transmit the tumor to other dogs met with no success. It is therefore doubtful that the tumor he was dealing with was a true venereal tumor. However, his diagnosis cannot be ruled out completely, as other workers have not been successful in trans- planting the tumors into all dogs they studied. Glass (1923) reported on the incidence of the tumor in the United States and thought that the disease was carried through English Bulldogs to this country and subsequently spread to other dogs. Attempts to transplant the tumor by vaginal scarification and plant— ing under the rectus fascia of the abdominal wall in 2 dogs were disap— pointing (Novak and Craig, 1927). Although the tumors were removed from the vagina, their failure to grow in transmission experiments remained unexplained. 7 Feldman (1929, 1932), in his comprehensive review, called the tumor a "transmissible lymphosarcoma”. His diagnosis was based on the cellular morphology, and he believed that the tumor was arising from undifferen- tiated lymphocytes, although definite proof was lacking. Feldman listed 81 tumors of dogs, of which 7 (8.6%) fell into this classification. All the tumors listed by him were from mongrels. Two tumors from the same dog, 1 from the orbit and another from below the skin of the frontal region, were described as resembling the genital tumors. However, no transmission experiments were done to define the nature of the orbital neOplasm. Microsc0pic examination revealed the presence of large, irregu- lar cells with fairly granular cytoplasm. Cell nuclei were large, with much chromatin and slightly eccentric nucleoli. Many mitotic figures were seen. Histochemical methods were negative for intercellular reticulum fibers. Stubbs and Furth (1934) expressed doubts regarding the lymphocytic origin of the cells constituting the venereal sarcoma of dogs. They stated, "Evidence for the view that the tumor cells are lympho- cytes is wanting. ...The cells in this growth were never seen maturing into typical lymphocytes. For this reason its designation as lymphosarcoma has no basis." They also thought that the cells were not histiocytes; no phagocytosis was observed in fixed tissues. The term "endothelioma" was not acceptable to them, as the origin of the cells from the endothelium was inapparent. DeMonbreun and Goodpasture (1934) made significant research contri- butions. Histochemical investigation revealed the absence of mitochondria, which they thought might have been masked by the large numbers of fat globules that were present in the tumor cells. Their attempts to stain 8 the cells, by both supravital and intravital methods, were regarded as failures. They postulated that the tumors probably were derived from the cells of the lymphocytic series. In one of their experiments, they noted a destructive action of serum from rabbits immunized with tumor tissue emulsion on the tumor cells iguzitgg. No tumor could be produced in dogs following inoculation of cells previously exposed to the serum. An interesting and useful report was presented by Jackson (1936) regarding the various differences between the basal cell tumor and the contagious venereal tumor of dogs, which he thought might be confused with each other when occurring in unusual locations. He observed a striking similarity in the microsc0pic characteristics of venereal tumors and heart-base tumors of dogs. This led him to suspect a com- mon origin for both the tumors. However, transmission eXperiments, which might have thrown some light on the apparent differences between them, were not conducted. Jackson (1944) demonstrated the presence of numerous lipid globules in the tumor cells by a specially devised acetic—carbol—Sudan method. He thought that the globules had been overlooked by previous workers. This supported the observation of DeMonbreun and Goodpasture (1934). From Ireland, an increased incidence of "infectious venereal granu- loma" in Greyhounds was described by Gleason (1947). He noted that the histologic characteristics resembled those of roundecell sarcoma, al- though the stroma was better developed in venereal granuloma. Lacroix and Riser (1947) reported 52 "transmissible lymphosarcomas" in dogs, of which 2 had penile locations. The neoplasm was common in Boston and Scottish Terriers, and a common location was the ear. Dogs below 1 year of age had the highest incidence. 9 Some interesting observations were published by Mulligan (1945, 1948, 1949). In the beginning he suSpected some hormonal relationships with the incidence of venereal tumors. Mulligan studied 5 skin tumors of dogs and concluded that they were similar, microsc0pically, to lymphosarcoma (lymphoblastic type). He did not consider them as extra— genital venereal sarcomas because he had neverf6und the latter in hundreds of dogs observed in his laboratory over a period of 8 years. Even though he considered this tumor as lymphosarcoma, subsequently the name "histiocytoma" was suggested by him. He concluded that the pre— viously reported venereal sarcoma may have been a histiocytoma. The lymphoid nature of the "transmissible lymphosarcoma" was accepted by Rust (1949). Of the 12 tumors he observed, only 1 had meta- static lesions in visceral organs. Bloom 33 31. (1951) clarified much of the long—existing confusion regarding the histogenesis of transmissible venereal tumors of dogs. After extensive studies on different aspects of the neoplasm they indi— cated that the cells were mature end—cells of reticulo-endothelial origin, as they did not grow further in in_yi££2_experiments. Histo- chemical studies revealed the presence of fine granules of mitochondria, Golgi elements, and moderate amounts of acid phosphatase in the nuclei and traces in the cytoplasm of tumor cells. They also observed a considerable number of lipid droplets in degenerate cells. In intact cells they were present as "bound lipids" (Jackson, 1944). Cytochrome oxidase, peroxidase, phospholipids, glycogen, and PAS—positive materials were absent. The authors disagreed with the use of the term histio- cytoma and suggested retaining the name "transmissible venereal tumor". 10 Reviewing the "transmissible venereal tumor" of dogs, Karlson and Mann (1952) referred to it as a true neoplasm. They did some significant studies of the transplantation of this tumor to other dogs. Using 601 dogs in 62 groups, they transmitted for 40 tumor generations. The highest incidence (95%) was in the 15th generation. However, only 5 dogs from the entire group had metastatic growths. Surprisingly, there was no significant change in the microsc0pic character of the tumors, even after several passages. Cotchin (1954) stated that the incidence of "transmissible venereal tumor" in dogs was lower than in previous years. In another report Cotchin (1956) listed 8 instances of the transmissible venereal tumor among 104 genital tumors of dogs, 3 from the vagina and 5 from the male genital organs. In the beginning he expressed doubt regarding its neoplastic nature, but subsequently he believed it to be a true ne0plasm. A high incidence of "venereal sarcoma" in dogs in Indonesia was recorded by Ressang, Titus, Sikar, and Nabib (1958). Their studies of these neoplasms revealed a wide clinical and histologic variation from the European data. Moulton (1961) gave a comprehensive review regarding the incidence and gross and microsc0pic characteristics of this tumor. He expressed doubt that the venereal tumors reported in extragenital sites were true venereal tumors, as transmission experiments to prove their identity were lacking. He indicated the possibility of confusing this tumor with the mastocytoma and histiocytoma occurring in the skin. The first and, to date, the only experiment, to the knowledge of the writer, regarding the nutritional requirements of the "transmissible 11 venereal sarcoma" cells was published by Prier (1963). He attempted to cultivate the cells in the laboratory on different commercial media. Luxuriant growth with maximum cell yield was achieved on Puck's medium with addition of 20% calf serum. Addition of 50 mg. of cysteine per liter of the medium caused 85% reduction in the cell yield, whereas no detectable growth was noted when this amino acid was added in levels of more than 250 mg. per liter. However, low levels of cystine or cysteine in the base medium were essential for production of viable cells in large numbers. Addition of methionine in levels above 25 mg. per liter of the medium caused degeneration of the cells. Barron, Saunders, Seibold, and Heath (1963) studied 2 additional transmissible venereal tumors involving eyes of dogs and thought that the tumor might have some affinity for intraocular metastasis. Several other reports also confirm their observation (Huebner, 1922; Feldman, 1929; Rust, 1949; Karlson and Mann, 1952). A valuable contribution was made by Makino (1963) on chromosome numbers, their distribution, and DNA content in the tumor cells. This was a new approach toward classifying the neoplasm. His observation included 17 tumors obtained from different areas of Japan. He noted morphologic and numerical alterations and structural changes in chromo- somes of tumor cells. The chromosome number was 59 instead of the nor— mal 78 (76 autosomes and a pair of sex elements); 17 of them were meta— centric and 42 acrocentric. An_increased DNA content in the cells was suggested to be due to either increased mitosis or change in total volume of chromosomes. Weber, Nowell, and Hare (1965) confirmed the observations of Makino (1963) by chromosome counts. Their experiments with an artificially 12 transplanted "venereal sarcoma" and a primary "venereal sarcoma" with multiple metastases revealed the presence of identical karyotypes in both. However, among the total of 59 chromosomes, 15 metacentric and 44 acrocentric forms were noted as compared to 17 and 42, respectively, in the report by Makino. Both the tumors under their investigation were removed from the genital organs of dogs in the State of Pennsylvania. The authors commented on the histologic characteristics of the tumors and thought they were quite useful in differentiating this tumor from histiocytoma, mastocytoma, and lymphosarcoma. Referring to this tumor as "histiosarcoma", Runnells, Monlux, and Monlux (1965) stated that the tumor is composed of histiocytes. Con- finement of this tumor to the genitalia and occasionally to the skin of young dogs (2 to 6 years of age) was mentioned. Referring to the "transmissible venereal tumor" of the dog, Smith and Jones (1966) mentioned its frequent location near or on the geni— talia. Another tumor with similar histologic characteristics that occurred in the skin other than genitalia of young dogs was described as "extragenital venereal tumor" or "round-cell tumor". According to their observation, this tumor was a different entity from the transmis— sible venereal tumor. They demonstrated the presence of normal chromo— somes, both in number and morphology, in the extragenital venereal tumor cells, in contrast to the reduced number and abnormal morphology of chromosomes in cells of transmissible venereal tumor. This observa- tion is definitely significant but disagrees with the conclusions drawn by Mulligan (1948). 13 Histiocytoma Mulligan (1948) suggested the name "histiocytomd' for a group 6f neoplasms located in the skin and on the genital organs of predominantly young dogs. His diagnosis was based chiefly on the microscopic charac- teristics of the tumors. Two of 21 tumors were located on the genital organs. Histochemical studies revealed the absence of lipids. Reticu- lum fibers, which he thought were part of the stroma, were present. To defend his diagnosis, Mulligan (1949) stated, "The cutaneous connective tissue is the natural habitat of the histiocyte,not the case with any cell of the lymphoid series. When a neOplasm, not of fibroblastic or of mast cell type, is noted in this tissue, the histiocyte is the logical cell to suspect, whether cutaneous or genital locations are involved. So called venereal lymphosarcoma is apparently histiocytoma." The human counterpart of histiocytoma was mentioned as "xanthoma" (Mulligan, 1961). The author also suggested deleting the term "venereal sarcoma". Ottosen (1949) studied several skin neoplasms in dogs. His study included "histiocytic sarcomata" in 10 dogs. Almost all the tumors were recorded in older dogs (about 10 years of age). This statement does not agree with that of Mulligan (1948), in which the latter noted an in- creased tumor incidence in younger dogs (up to 5 years of age). Ottosen noted giant cells in the tumors, an observation which has not been men- tioned in other reports. Also, there were numerous fat globules of uni- form size in the tumor cells. This again contradicts the original report of Mulligan in which he mentioned the absence of fat globules in the tumor cells. It seems that Ottosen probably dealt with a group of tumors quite different from the histiocytomas originally reported. 14 Bloom gtnal. (1951) conducted studies of these tumors and suggested eliminating the term histiocytoma, as the tumor cells were not histiocytes. Head (1953) reported the incidence of "hiStiocytic cell sarcoma" in young dogs. There were more male than female dogs affected. Although the author did not observe any metastatic lesions, 7 tumors recurred following attempted excision. However, no gross or microscopic descrip- tions of the tumors were listed. Howard and Nielsen (1965) listed some interesting findings in their survey of tumors from Boxers and other dogs. In their study the inci- dence of histiocytoma in the skin of the Boxer was nearly 4 times as high as encountered in other breeds. Basal Cell Carcinoma The basal cell carcinoma was first reported by Jacob (1827). This neOplasm has also been referred to as "hair-matrix tumor" (Haythorn, 1931), "adnexal carcinoma" (Foot, 1947), "basal cell epithelioma", "basalioma" (Nielsen and Cole, 1960), "basal cell tumor" (Moulton, 1961), and "rodent ulcer" (Runnells £3 21-: 1965). The origin of the tumor cells has created controversies among dif- ferent workers. Owen (1930) stated, "The term basal cell carcinoma is used to designate carcinoma arising from the basal cell layer of the epidermis and retaining to a large extent the characteristics of basal cells." She also suggested the use of thin tissue sections (2 to 3 microns) for demonstration of intercellular bridges in the tumors. Multicentric origin of the tumors from the basal cells of epidermis and skin adnexa has been reported by several authors (Haythorn, 1931; Foot, 1947; Mulligan, 1949; Moulton, 1961; Smith and Jones, 1966). The 15 benign nature of this neoplasm has also been recognized, although Head (1953) included 2 tumors that had metastatic foci. Using several special staining techniques, Haythorn (1931) noted the presence of rudimentary hair shafts in the tumors. He postulated the origin of these tumors from the hair-matrix. Feldman (1932) reported 2 cases of "so-called basal-cell carcinoma" of dogs. The tumors grew slowly and intercellular bridges were noted by him in one of the sections. Jackson (1936) emphasized the resemblance of the tumor cells to the basal cells of stratified squamous epithelium and its derivatives and anticipated the possibility of confusion of this tumor with "con- tagious venereal tumor" of the dog when occurring in unusual locations. Microscopic examinations of 8 tumors from dogs revealed the uniform appearance of tumor cells. Two chief patterns of cellular arrangements were described: (1) groups of cells with their long axes perpendicular to the cell columns and (2) groups of cells forming alveolar structures. Extensive studies of the basal cell carcinomas of man revealed their origin from the specialized basal cells of the dermal adnexa rather than from basal cells of the stratified squamous epithelium (Foot, 1947). Foot suggested the term "adnexal carcinoma" for these tumors. Mulligan (1948) listed "basal cell carcinomas" which originated in the hair follicles. The cells resembled the epidermal basal cells, although their continuity with the latter was not apparent. Mulligan (1949) described "basal cell (hair follicle) carcinomas" and believed that their origin was from hair follicles, epidermis, and lining epithelium of epidermal inclusion cysts. Formation of structures 16 resembling hair follicles by the tumor cells was also noted. A statistical review of 47 "basal cell and sweat gland" tumors from a total of 510 tumors of skin, subcutaneous tissue, and associated structures was presented by Cotchin (1951). In another report Cotchin (1954) included 51 "basal cell and glandular" tumors of dogs, although there was no mention of the actual basal cell types he studied. No gross or microsc0pic descriptions of these tumors were given. Head (1953) examined tumors from dogs and divided them into 2 groups: (1) superficial origin and (2) subepidermal origin. Two of the 28 tumors in the first group metastasized to the regional lymph nodes and l to the lungs. The second group of 21 tumors had the char- acteristic Medusa head appearance histologically and did not manifest any metastases, although 3 recurred after attempted removal. Frequent occurrence of the tumors of both groups in male dogs was indicated. Histologic studies of "basal cell epitheliomas" by Goltz, Fusaro and Jarvis (1959) with special staining techniques revealed the absence of glycogen in most of the tumors. However, a few tumors that dif— ferentiated to form "adenoid or trichoepitheliomatous" structures had glycogen in them. Nielsen and Cole (1960) did a comprehensive review of the tumors in dogs. In their study the term "basal cell tumor" was used to desig- nate the epithelial tumors that did not have any differentiation. They studied 33 tumors, of which 26 were located in the head and neck area. Three tumors recurred due to incomplete excision. However, no metastasis was observed by the authors. There were 2 histologic patterns of cells: (1) ribbon type and (2) solid type, the former being more common. The solid type was more malignant than the ribbon type. l7 Thrasher (1961) reported 2 primary "basal cell carcinomas," 1 in the skin and another from the mammary gland. However, their microscopic characteristics were not described. Sams, Smith, and Finlayson (1963) demonstrated delicate unbranched argerphilic fibers in the human "basal cell epitheliomas". Most of the fibers disappeared on treatment with 0.5 M acetic acid solution, revealing their collagenous rather than true reticular nature. The presence of large amounts of acid mucopolysaccharides was noted by them especially in the stroma having numerous fibroblasts, although their presence in the intercellular spaces was variable. Small amounts of neutral polysaccharides were also seen within the cell nests. An in- crease in the number of mast cells was noted in the stroma surrounding the tumors. MATERIALS AND METHODS The tissues used for this study were assembled from the collection of the Department of Pathology, Michigan State University. They had been sent to the department by several private practitioners and the Small Animal Clinic, Michigan State University, for histopathologic diagnosis. The instances in which these neoplastic conditions were diag- nosed (from June 1956 to December 1965) were recorded, along with the available data, in the form of tables under the following headings: breed, age, sex, location in the body, and degree of malignancy and metastasis, if any. All the tissues had been fixed in 10% formalin and embedded in paraffin. Hematoxylin and eosin (H & E) stained sections were examined micro- scopically. Those sections having heavy inflammatory processes and post-mortem changes due to imprOper fixation were considered to be unfit for study and hence were discarded. Available paraffin blocks for the selected slides were collected, and sections were cut at a thickness of 6 microns. Sections were stained with Gomori's one-step trichrome stain and periodic acid-Schiff (PAS) with and without diastase digestion (Armed Forces Institute of Pathology, 1960). Another stain, outlined by Gurr (1962), was used to demonstrate the presence of reticulum fibers in the tumors. May-Grunwald-Giemsa stain (Armed Forces Institute of Pathology, 1960) was applied to the tissues to stain mast cells and to eliminate mast cell tumors if any had been inadvertently included. 18 19 All the sections were examined for ulceration, necrosis, amount and type of stroma, inflammatory cells, hyperchromatism, size and shape of nuclei, and morphology of neoplastic cells. Measurement of the size of the nuclei in their longest diameter was done with an ocular micrometer fitted to the microscope and calibrated by stage micrometer. Ten representative neoplasms from each group were included and 100 nuclei from each section were measured. The results were statistically evaluated for any difference in size of the nuclei between these 3 groups. An average of 5 high-power fields (hpf) was examined to enumerate the average number of mitoses present. Photomicrographs were taken from representative sections. The description of these neoplasms, by Moulton (1961), was accepted as a guide during the entire study. RESULTS Transmissible Venereal Tumor Breed incidence. Of 43 transmissible venereal tumors diagnosed, infor- mation regarding breed affected was available in 40 cases. Of these, 10 (25%) were Boxers, 6 (15%) were Boston Terriers, and 4 (10%) were Cocker Spaniels. This is recorded in TABLE 1. Sex distribution. Of 36 dogs for which information was available, 18 tumors were recorded in male dogs and 18 in females. Age distribution.‘ The range in age of affected animals was 4 months to 13 years; the mean age was 5 years. Primary location. Among 43 recorded instances, only 2 tumors were re- moved from the male genitalia. One was from the glans penis and the other from the inside of the prepuce. Two tumors, 1 each from the tongue and lip areas, were also reported. Others were from the skin on various parts of the body. Metastases. Although data from referring veterinary practitioners may have been deficient, metastatic foci were reported in the mandibular and sublingual lymph nodes in l instance. 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