A Clinicopathologic and Ultrastructural Study of Undifferentiated Malignant Tumors of the Oral Cavity in Dogs

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1 Vet. Pathol. 23: (1986) A Clinicopathologic and Ultrastructural Study of Undifferentiated Malignant Tumors of the Oral Cavity in Dogs A. K. PATNAIK, P. H. LIEBERMAN, R. A. ERLANDSON, E. G. MACEWEN, AND A. I. HURVITZ Department of Pathology and Donaldson-Atwood Cancer Clinic; The Animal Medical Center, New York, Ny, and Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY Abstract. Undifferentiated malignant tumors of the oral cavity were diagnosed in six dogs under 2 years of age. The dogs were examined because of pain and swelling of the upper molar or premolar areas. In all six dogs, the tumors were initially misdiagnosed as infections or carnasal abscesses. The differential diagnosis included malignant lymphoma, osteosarcoma, mesenchymal chondrosarcoma, embryonal rhabdomyosarcoma, and malignant melanoma. Electron microscopy of three neoplasms showed that there were no specific features characteristic of carcinoma or sarcoma. Immunoperoxidase studies for cytokeratins, epithelial membrane antigen, actin, myosin, desmin, and vimentin were also negative. We conclude that these tumors be designated undifferentiated malignant tumors of the oral cavity until histogenesis is established. Benign and malignant neoplasms in dogs under 2 years of age are ra~e.~,~,~ In one study of neoplasms in the dog, malignant tumors accounted for only 3.2% of the tumors in dogs under two years old.s Lymphoma, leukemia, and osteosarcoma were among the neoplasms reported in the young dogs.5 The purpose of this report is to describe six cases of undifferentiated neoplasm of the oral cavity in dogs under 2 years of age. Materials and Methods The case records of six dogs under two years old with undifferentiated tumors of the oral cavity diagnosed over a 15-year period were reviewed. Only those cases with complete case records and follow-up information were chosen for the study. Diagnosis was based on histologic examination of tissue specimens. Specimens were processed routinely, fixed in 10% buffered formalin, and stained with hematoxylin and eosin. Selected slides were stained with Mayer s mucicarmine, Schmorl s femc femcyanide reduction stain for melanin, and Gridley s reticulum stains. Tissues for electron microscopy were fixed in Karnovsky s fixative (paraformaldehyde and glutaraldehyde), buffered with s-collidine, post fixed in osmium tetroxide, and embedded in Maraglas epoxy resin. Immunoperoxidase procedures were used according to the methods of SternbergerI4 and further modification by Erlandson et al. Antibodies against three human epidermal keratins designated AE1, AE2 and AE3 were generously supplied by Dr. Tung-Tien Sien, Department of Dermatology, New York University School of Medicine. Antibodies against actin and myosin were from Miles-Yeda, Ltd. Antibodies against epithelial membrane antigen (EMA) and desmin were from DAKO, Inc. Antibodies against vimentin were supplied privately by DAKO, Inc. Three of the cases were examined immunohistologically. Results Undifferentiated, malignant neoplasm of the oral cavity was diagnosed in six young dogs (Table 1). The average age of the dogs was 12.5 months (range, 6 to 22 months). Three dogs were 12 months old or younger. Four dogs were males and two were females. Three dogs were German shepherds, and five were large-breed dogs. Most dogs were examined because of pain and swelling ofthe upper molar or premolar areas. As the tumors grew, suborbital swelling, facial edema, and difficulty eating were observed. Other clinical findings included exophthalmos, loose molar and premolar teeth, nasal and ocular discharge, and ulceration and bleeding from the mouth. Radiographs showed soft tissue swelling and bony erosion. The only abnormal finding on hematologic and biochemical testing was high WBC counts (range, 13,000 to 33,000 WBC/mm3). In five dogs, the primary neoplasm, located in the right side of the oral cavity, was an ill-defined, soft, gray-tan, hemorrhagic tumor involving the upper premolar and molar areas. The tumor extended into maxillary, nasal, and orbital tissues, and sometimes into brain tissue. In the sixth dog (dog 2), the neoplasm involved the soft palate and surrounding tissue. The salivary glands were not involved in any dog. Five dogs were necropsied. Diffuse metastases were 170

2 Undifferentiated Tumors of the Oral Cavity in Six Dogs 171 Table 1. Clinical and pathologic findings in six dogs with undifferentiated malignant neoplasm of the oral cavity. No. Breed Sex Age (mo) Clinical Signs on Physical Examination Gross Pathology Sites of Metastasis (microscopic) Survival (days) After Diagnosis 1 German shepherd 2 Giant schnauzer 3 6 German shepherd German shepherd Mixed breed Doberman pinscher M 9 Firm, fluctuating swelling R* side of face with exophthalmos, R nasal disease, & enlarged R mandibular & pharyngeal lymph nodes; lung metastasis on X-ray F 14 Mass in back of mouth & soft palate; anorexia & lethargy; submandibular swelling 1 month s duration; lung metastasis on X-ray M 6 Swelling under R eye; loose molars (1 st & 2nd) extracted 1 week before examination M 12 Swelling of R nasal & buccal areas; no bone involvement F 22 Mass behind eye of 2 /2 month s duration; no bone involvement initially (surgery two times before examination) M 13 Bleeding from nose & mouth; swelling on R side of face & mandible Mass with deformity involving R maxilla & nasal passage; ulceration of gum & palate Grayish, friable mass involving soft palate & nasal cavity bilaterally Mass involving R maxilla extending to turbinates, palate & gum Mass involving buccal & nasal cavities & intraorbital tissue Mass involving R eye, nose, frontal sinuses & cranial cavity; no oral lesion Ulcerated mass involving R molar arcade & surrounding tissue Brain, lungs, lymph nodes, liver, heart, kidneys, adrenal glands, mesentery, intestines, gastric mucosa, testes Brain, lungs, lymph nodes, liver, heart, kidneys, adrenal glands, mesentery, intestines, pancreas, ovaries, mammary glands Brain, lungs, lymph nodes, liver, heart, kidneys, adrenal glands, mesentery, pleura, gastric mucosa, skeletal muscle Lungs, pleura Liver, lymph nodes, adrenal glands Lymph nodes present in all but dog 5 in which only liver metastasis was seen. The primary and metastatic tumors were characterized by groups of small to medium-sized cells with scant stroma (Figs. 1-3). The cells were round or ovoid to spindle-shaped with round to elongated nuclei containing small amounts of chromatin and granular cytoplasm. There were large areas of necrosis often coagulative. The number of mitotic cells per high power field ranged from 23 to 30. There was little morphologic variation among the neoplasms and the metastatic sites. In all cases there was histologic evidence of tumor extension into the maxillary and frontal sinuses, tur- binates, and adjoining bones, and, in three cases, the cranial cavity. The most common sites of metastasis were lymph nodes (five), lungs (four), liver (four), heart (three), kidneys (three), and brain (three). Because of rapid progression of disease and poor prognosis, all dogs were killed. The average time from diagnosis to death was 22 days (range, 2 to 60 days). The two dogs that lived for 42 to 60 days had multiple surgical resections. In three cases (dogs 1, 2, 5), tissue specimens were examined by transmission electron microscopy; they had remarkably similar ultrastructural features. The tumors consisted of patternless sheets of loosely-organized, polygonal and spindle-shaped cells in stroma

3 172 Patnaik et al. Fig. 1. Highly cellular neoplasm; small to medium-sized cells, little stroma, and many mitotic cells. HE. Fig. 2. Small to medium-sized, spindle and round pleomorphic cells. HE. containing scattered collagen fibrils (Figs. 4-6, 8,000 x and Figs. 7,8, 12,000 x). The irreguiarly-shaped nuclei displayed clumped chromatin and one to three nucleoli (Fig. 4). The relatively scanty cytoplasm contained scattered cisternae of rough endoplasmic reticulum, inconspicuous mitochondria, and a small Golgi apparatus (Figs. 7,8). Polysomes were prominent in many of the neoplastic cells (Fig. 8). Except for scattered, rudimentary cell junctions (Figs. 7, 8), no structures indicating tumor cell differentiation were seen. Immunoperoxidase studies for AE1, AE2, AE3, EMA, actin, myosin, desmin, and vimentin were negative. Fig. 3. Higher magnification of Fig. 2. Pleomorphic cells with many mitotic figures invading the mucosa. Discussion Because of the age and clinical signs of the dogs of this report, their lesions were diagnosed as infections or carnasal abscesss, and thus accurate diagnoses were delayed. Neoplasms were suspected when there was no response to antibiotics and when evidence of metastasis was observed. The differential diagnosis in young dogs with clinical Fig. 4. Low magnification electron micrograph of tumor in dog 1. Haphazardly arranged, undifferentiated cells with scanty cytoplasm. Irregularly-shaped nuclei, coarsely clumped chromatin, and multiple nucleoli. Fig. 5. Low magnification electron micrograph of tumor in dog 2. Loosely organized neoplastic cells surrounding lymphocytes. Fig. 6. Low magnification electron micrograph of tumor in dog 5. Undifferentiated neoplastic cells. Nuclei are less atypical than in dog 1. --t

4 Undifferentiated Tumors of the Oral Cavity in Six Dogs 173

5 174 Patnaik et al. Fig. 7. Electron micrograph of tumor in dog 1. Tumor cells joined by two rudimentary cell junctions. There are no diagnostic structures. Fig. 8. Electron micrograph of tumor in dog 1. Two polygonal tumor cells joined by rudimentary cell junction adjacent to a spindle-shaped cell. Polysomes are prominent. signs similar to those of our dogs includes malignant lymphoma, osteosarcoma, mesenchymal chondrosarcoma, embryonal rhabdomyosarcoma, and malignant melanoma. The light microscopic features of the tumors in our dogs excluded all of the~e.~s~-l~ Also, melanomas usually develop in older dog^.^,^^ Ultrastructural examination of tissue specimens from three tumors confirmed results of light microscopy that these tumors were undifferentiated neoplasms. Numerous polysomes often were found in rapidly-growing anaplastic tumor cells.6 Features of epithelial differentiation, i.e., tonofilaments, lumens, microvilli, and basement membrane were not evident making a diagnosis of carcinoma untenable. The presence of cell junctions ruled out lymphoma, and the absence of premelanosomal organelles is not compatible with malignant melanoma. Rudimetary cell junctions have been seen in poorly-differentiated carcinomas and, occasionally, sarcomas.6 The cells of embryonal rhabdomyosarcoma usually are partially surrounded by basement membrane and contain arrays of actin and myosin filaments in the cytoplasm. The fine structural findings indicate an undifferentiated tumor and do not permit further subclassification. Immunocytochemical studies further support the truly undifferentiated nature of this neoplasm. In humans also, there are neoplasms which are not possible to subclassify with presently available techniques. Acknowledgements Authors thank the Bodman Foundation for support, A. Christine MacMurray for editorial assistance, Dr. T.-T. Sien and DAKO, Inc. for supplying antibodies for immunocytochemistry, and Maryann D. Gangi and Daisy Jimenez- Joseph for the immunocytochemical procedures. References Azar HA, Espinoza CG, Richman AV, Saba SR, Wang T: Undifferentiated large cell malignancies: an ultrastructural and immunocytochemical study. Hum Pathol 13:323, 1982 Brodey RS: Canine and feline neoplasia. Adv Vet Sci Comp Med 14:309, 1970 Cohen D, Brodey RS, Chen SM: Epidemiologic aspects

6 Undifferentiated Tumors of the Oral Cavity in Six Dogs 175 of oral and pharyngeal neoplasms of the dog. Am J Vet Res , Cohen D, Reif JS, Brodey RS, Keiser H: Epidemiological analysis of the most prevalent sites and types of canine neoplasia observed in a veterinary hospital. Cancer Res 34:2859, Dorn CR, Taylor DO, Schneider R, Hibbard HH, Klauber MR: Survey of animal neoplasms in Alameda and Contra Costa counties, California. 11. Cancer morbidity in dogs and cats from Alameda County. J Natl Cancer Inst 40:307, Erlandson RA: Diagnostic Transmission Electron Microscopy of Human Tumors: The Interpretation of Submicroscopic Structures in Human Neoplastic Cells, pp. 33, 107. Masson Publishing USA, Inc., New York, Erlandson RA, Cardon-Cardo C, Higgins PJ: Histogenesis of benign pleomorphic adenoma (mixed tumor) of the major salivary glands. An ultrastructural and immunohistochemical study. Am J Surg Pathol8:803, Hulland TJ: Tumors of muscle. In: Tumors in Domestic Animals, 2nd ed., ed. Moulton JE, p. 75. University of California Press, Berkeley, Moulton JE, Dungworth DL Tumors of the lymphoid and hemopoietic tissues. In: Tumors in Domestic Animals, 2nd ed., ed. Moulton JE, p University of California Press, Berkeley, Patnaik AK, Lieberman PH, Erlandson RA, Liu S-K: Canine sinonasal skeletal neoplasms: chondrosarcomas and osteosarcomas. Vet Pathol 21:475, Pool RR: Tumors of bone and cartilage. In: Tumors in Domestic Animals, 2nd ed., ed. Moulton JE, p University of California Press, Berkeley, Seibold HR: Juvenile alveolar rhabdomyosarcoma in a dog. Vet Pathol 11:558, Stannard AA, Pulley LT: Tumors of the skin and soft tissue. In: Tumors in Domestic Animals, 2nd ed., ed. Moulton JE, p. 62. University of California Press, Berkeley, Sternberger LA: Immunocytochemistry, 2nd ed., p. 82. John Wiley and Sons, New York, 1979 Request reprints from Dr. A. K. Patnaik, Department of Pathology, Donaldson-Atwood Cancer Clinic, Animal Medical Center, New York, NY (USA).