Mast cell tumors (MCT) are the most common cutaneous

J Vet Intern Med 2003;17:687 692 Biologic Behavior and Prognostic Factors for Mast Cell Tumors of the Canine Muzzle: 24 Cases (1990 2001) Tracy L. Gieger, Alain P. Théon, Jonathan A. Werner, Margaret C. McEntee, Kenneth M. Rassnick, and Hilde E.V. DeCock The medical records of 24 dogs with histologically confirmed mast cell tumors (MCT) of the muzzle were retrospectively evaluated to determine their biologic behavior and prognostic factors. Information on signalment, tumor grade and stage, treatment methods, and pattern of and time to failure and death was obtained from the medical record. Twenty-three dogs were treated with combinations of radiotherapy, surgery, and chemotherapy; 1 dog received no treatment. There were 2 Grade I, 15 Grade II, and 7 Grade III tumors. Tumors were stage 0 (n 8), stage 1 (5), stage 2 (6), stage 3 (4), and stage 4 (1). Mean and median survival times of treated dogs were 36 and 30 months, respectively. Prognostic factors affecting survival time included tumor grade and presence of metastasis at diagnosis. Dogs with Grade I and II tumors survived longer than dogs with Grade III tumors. Variables, including sex, age, gross versus microscopic disease, and treatment type were not found to affect survival. Local control rate was 75% at 1 year and 50% at 3 years. Tumor grade was the only variable found to affect local control. Dogs with Grade I tumors had longer disease-free intervals than those with Grade II tumors, and dogs with Grade II tumors had longer disease-free intervals than dogs with Grade III tumors. Eight of 9 dogs dying of MCT had local or regional disease progression. Muzzle MCT are biologically aggressive tumors with higher regional metastatic rates than previously reported for MCT in other sites. Key words: Canine skin tumors; Mastocytosis; Metastasis. Mast cell tumors (MCT) are the most common cutaneous tumors in dogs, accounting for 7 21% of all skin tumors. 1,2 These tumors are highly variable with regard to location, biologic behavior, and treatment response. Factors including histologic grade, World Health Organization stage, duration of disease, argyrophilic nucleolar organizer region count, completeness of surgical excision, DNA ploidy, tumor location, breed, and abnormal expression of the p53 tumor suppressor gene have been evaluated as prognostic indicators for MCT in dogs. 3 15 Tumor location affected the prognosis of dogs with incompletely resected MCT treated with radiation therapy in 1 study in which tumors of the extremities were associated with a lower metastatic rate, longer disease-free intervals, and longer survival times than tumors located on the trunk. 5 A more recent study determined that location did not affect outcome. 16 No information is available regarding MCT located on the muzzle (Fig 1). The purpose of this study was to analyze retrospectively the biologic behavior and prognosis of dogs with MCT located on the muzzle. From the Veterinary Medical Teaching Hospital (Gieger), Department of Veterinary Pathology, Immunology, and Microbiology (Werner, DeCock), and the Department of Surgical and Radiological Sciences (McEntee, Théon), University of California, School of Veterinary Medicine, Davis, CA; and the Department of Clinical Sciences, Tufts University, N Grafton, MA (Rassnick). Dr Gieger is presently affiliated with the University of Georgia College of Veterinary Medicine, Department of Small Animal Medicine, Athens, GA. Drs McEntee and Rassnick are presently affiliated with the Cornell University College of Veterinary Medicine, Department of Clinical Sciences, Ithaca, NY. Previously presented in part at the 2000 Mid-Year Meeting of the Veterinary Cancer Society in Tucson, AZ. Reprint requests: Dr Gieger, DVM, DACVIM, University of Georgia College of Veterinary Medicine, Department of Small Animal Medicine, Athens, GA 30602; e-mail: tgieger@vet.uga.edu. Submitted November 4, 2002; Revised February 7, 2003; Accepted March 7, 2003. Copyright 2003 by the American College of Veterinary Internal Medicine 0891-6640/03/1705-0011/$3.00/0 Materials and Methods Dogs Twenty-four dogs with histologically confirmed MCT of the muzzle were included in this study. Dogs with complete medical records were selected from the patient populations of the University of California Veterinary Medical Teaching Hospital (n 14), the Tufts University School of Veterinary Medicine Teaching Hospital (n 5), The Berkeley Center for Special Veterinary Services (n 3), and East Bay Veterinary Cancer Specialists (n 2) between January 1990 and January 2001. Medical records were reviewed for signalment, clinicopathologic data, clinical staging, growth characteristics of the tumor, involvement of regional lymph nodes, presence of concurrent disease, treatment methods and associated toxicity, and outcome. Pretreatment evaluation including CBC, serum biochemistry, urinalyses, buffy coat smears and regional lymph node aspiration or biopsy was performed in most cases. Regional lymph node metastasis was defined as a large population of mast cells infiltrating the lymph node or the presence of poorly differentiated mast cells in the lymph node. Thoracic radiographs, abdominal radiographs, ultrasounds, splenic aspirates, and bone marrow aspirates were performed when deemed appropriate. Tumors were staged according to a modified version of the classification system developed by the World Health Organization (Table 1). 5,17 Two pathologists (JAW, HEVD) reviewed all biopsy samples to confirm the diagnosis and grade of the tumors. Tumors were graded according to the system described by Patnaik et al, 4 in which Grade I has the best prognosis and Grade III has the worst. Treatment Methods Twenty-three dogs were treated with surgery, chemotherapy, radiation therapy, or some combination of these modalities; 1 dog did not receive any treatment. Surgical excision was attempted in 16 dogs. Nine dogs were treated with radiation therapy by external beam or interstitial techniques, with a median dose of 48 Gy (mean 45 Gy, range 35 48 Gy). Six dogs were treated with external beam radiation with megavoltage radiation (telecobalt-60 unit with an 80-cm source skin distance, n 5, or a 6-MV linear accelerator with a 100-cm source skin distance, n 1) or orthovoltage radiation (300 kvp orthovoltage unit, n 2). The megavoltage radiation dose was 48 Gy (minimum tumor dose), administered in 12 fractions of 4 Gy on an alternate day schedule, or 16 fractions of 3 Gy given on a daily schedule. The orthovoltage radiation dose (skin dose) was 45 Gy administered in 9 fractions of 5 Gy or 12 fractions of 4 Gy on an alternate

688 Gieger et al Fig 1. Photograph of a dog with a muzzle mast cell tumor. day schedule. One dog was treated with high dose rate brachytherapy ( 192 Ir). Treatment dose was 35 Gy administered in 5 weekly fractions of 7 Gy. Seventeen dogs received chemotherapy. Chemotherapy used a single modality (n 5), in combination with surgery (n 7), or in combination with surgery and radiation therapy (n 5). Protocols included prednisone alone (n 5) a ; prednisone and chlorambucil (n 2) b ; prednisone, vinblastine, and cyclophosphamide (n 3) c,d ; prednisone and CCNU (n 5) e ; prednisone, CCNU, and vinblastine (n 1); and CCNU, hydroxyurea, melphalan, chlorambucil, and cyclophosphamide (n 1). f,g Drug dosage varied among clinicians. Prednisone dosage ranged from 0.5 to 2 mg/kg PO q24h to q48h for 2 weeks to life; vinblastine was 2 mg/m 2 IV once every 21 28 days for 180 days; hydroxyurea was 30 mg/m 2 PO once; melphalan was 2 mg/ m 2 PO once; chlorambucil ranged from 2.5 to 8 mg/m 2 PO q24h to Table 1. dogs. 5 Stage Clinical staging system for mast cell tumors in Description 0 Incompletely excised solitary tumors without regional lymph node involvement 1 Solitary tumors confined to the dermis without regional lymph node involvement 2 Solitary tumors confined to the dermis with regional lymph node involvement 3 Multiple dermal tumors or large infiltrating tumors with or without regional lymph node involvement 4 Tumors with distant metastasis q48h for 1 dose to life; CCNU ranged from 30 to 90 mg/m 2 PO every 28 30 days for 120 180 days; and cyclophosphamide was 50 mg/m 2 PO once or 250 mg/m 2 PO divided over 3 or 4 days every 21 days for 180 days. Data Analysis Follow-up information concerning response to treatment and outcome was obtained by hospital visits or telephone calls to the referring veterinarian or owner. Twenty-three dogs that received treatment were evaluable for survival and local control analysis, and 24 dogs were evaluable for metastasis. Survival time was defined as the time between diagnosis and death attributable to disease progression (ie, local tumor recurrence, metastasis). The local control rate was defined as the percentage of dogs without tumor regrowth at a specific time after treatment. 18 Local tumor progression was confirmed cytologically or histologically. Regional or systemic metastasis was confirmed histologically, cytologically, and radiographically. Differences in distribution of dog and tumor characteristics were analyzed by t-test for continuous variables and Pearson chi-square statistics for categorical variables. Variables examined as indicators of prognosis included sex, age, tumor status (gross disease versus microscopic disease), tumor grade, tumor stage, and treatment type (single versus combined modality). Survival and local control rates were computed by the productlimit method. 19 Actuarial estimates of survival and local control distributions were tested for statistical differences by log-rank statistics. The Cox proportional hazards regression model was used to determine independent prognostic factors of survival and local control. 20 The relative risk of tumor recurrence was estimated by the hazard rate ratio. Differences were considered significant at P.05.

Muzzle Mast Cell Tumors in Dogs 689 Results Patient Characteristics Twenty-three dogs were purebred. Labrador Retrievers (n 8) and Golden Retrievers (n 4) were most often represented. Eleven dogs were neutered males, and 13 dogs were spayed females. Dogs ranged from 2 to 15 years old (mean 7.5 years, median 8 years) and from 4 to 51 kg in body weight (mean 27.5 kg, median 30 kg). No dogs had clinically apparent concurrent disease. Median duration of tumor presence before presentation to the veterinarian was 2.5 months (mean 6.4 months, range 0 48 months). Nine dogs received single-modality treatment, including surgery (n 2), radiation therapy (n 2), and chemotherapy (n 5). Postoperative treatment of local residual disease was accomplished by radiation therapy (n 1), chemotherapy (n 5), or a combination of radiation therapy and chemotherapy (n 4). Dogs with regional lymph node metastasis received chemotherapy in addition to local treatment with surgery (n 5) or surgery combined with radiation therapy (n 2). Laboratory Results CBCs were performed on 21 dogs; results were normal in 17 dogs, 2 dogs had mild nonregenerative anemia, and 2 dogs had eosinophilia. Serum chemistry profiles were performed on 21 dogs; results were normal in 16 dogs, and 5 dogs had minor increases in liver enzyme activities. Urinalyses were performed on 8 dogs; all results were normal. Thoracic radiographs were performed in 10 dogs; results were normal. Abdominal radiographs were performed in 2 dogs; results were normal in both dogs. Abdominal ultrasonography was performed in 13 dogs; results were normal in 11 dogs and abnormal in 2 dogs, which had splenic infiltrates. Splenic aspirates were performed in these 2 dogs and in 8 other dogs, and all results were normal. Buffy coat smears were performed in 16 dogs, with 1 positive result. Bone marrow aspirates were performed in 5 dogs, and results of all aspirates were normal. Tumor Behavior Growth patterns were estimated in 18 dogs with tumors present for at least 1 month before presentation. Three dogs had tumors that displayed intermittent swelling and shrinking, 10 dogs had tumors that gradually enlarged over 1 2 months, and 5 dogs had tumors that grew rapidly over a period of 1 month. No dog had clinical signs of systemic mast cell disease at presentation. Tumor measurements were recorded for 16 dogs, with a median of 1.9 cm at the widest diameter (mean 2.1 cm, range 1 5 cm). Tumor grades included Grade I (n 2), Grade II (15), and Grade III (7). Thirteen of 24 dogs had ipsilateral lymphadenopathy; 11 of these dogs had lymph node evaluations, and metastasis was documented by biopsy or fine-needle aspiration in 7 dogs. Of the 8 enlarged lymph nodes with recorded measurements, the median size was 4 cm (mean 4.3 cm, range 1.5 8 cm). Regional lymph node metastasis was found in 11 of 19 dogs evaluated; 7 of these dogs had clinically enlarged nodes, and 4 dogs had clinically normal nodes. Of the 19 dogs evaluated for metastasis, tumors were categorized as follows: Grade I, 1 dog evaluated, no metastasis; Grade II, 11 dogs evaluated, 7 had metastasis, 4 did not; Grade III, 7 dogs evaluated, 4 had metastasis, 3 did not. Only 1 dog had evidence of systemic disease (positive buffy coat smear). Tumor stages included stage 0 (n 8; Grade I, n 1, Grade II, n 5, Grade III, n 2), stage 1 (n 5; Grade I, n 2, Grade II, n 2, Grade III, n 1), stage 2 (n 6; Grade II, n 3, Grade III, n 3), stage 3 (n 4; Grade II, n 3, Grade III, n 1), and stage 4 (n 1; Grade II). 5 A correlation was found between tumor grade and presence of regional metastasis at diagnosis ( 2 test, P.042). A correlation was not found between presence of metastasis at diagnosis and tumor size, disease duration, or growth rate ( 1 month versus 1 month). Analysis of staging protocols indicated that dogs with Grade III tumors were more likely to have bone marrow aspirate evaluation than dogs with Grade I or II tumors. Prognostic Factors Four dogs died or were euthanized from unrelated causes (pancreatitis, disc disease, malignant histiocytosis, thyroid carcinoma). Eleven dogs were alive with no evidence of disease progression at last follow-up (median 27 months, follow-up range 7 67 months). One dog with a positive buffy coat smear was alive and free of local and systemic disease after 30 months (at the time of this writing). One dog with a Grade II tumor that did not receive any treatment had stable disease for 23 months until it was lost to follow-up. In 23 dogs that received treatment, median survival time ( SE) was 30 ( 9) months. The product-limit estimates of the 1- and 3-year survival rates were 78 ( 9) and 63% ( 10), respectively. Univariate analysis of survival revealed that tumor grade (log-rank, P.002), tumor stage (log-rank, P.031), and presence of metastasis at diagnosis (log-rank, P.019) were statistically significant prognostic indicators of survival times. Dogs with Grade II tumors survived longer than dogs with Grade III tumors (log-rank, P.0007). A difference in survival times was not found between dogs with Grade I and II tumors. Because of the association between presence of metastasis and stage and grade, the Cox proportional hazards regression model was used to eliminate prognostic factors that were no longer significant when adjusted for others. The factors that independently affected survival time were tumor grade (P.0027; Fig 2) and presence of metastasis at diagnosis (P.005; Fig 3), whereas tumor stage no longer had prognostic value. When compared to dogs with no metastases, dogs with metastases had a 7.7-fold increased risk of dying of disease. The product-limit estimates of the 1- and 3-year local control rates were 75 ( 9) and 50% ( 21), respectively (Fig 4). Tumor grade was the only prognostic factor for local control (log-rank, P.0006). A difference in local control was found between Grade II and III tumors (logrank, P.0002), and Grade I and II tumors (log-rank, P.0308).

690 Gieger et al Fig 2. Association between tumor grade and survival rate in 23 dogs with mast cell tumors of the muzzle. Tick marks represent dogs that are alive or whose death was attributable to an unrelated disease., dogs with Grade I and II tumors;, dogs with Grade III tumors. Patterns of Failure Nine dogs died or were euthanized because of recurrent local disease or regional or distant metastasis. In 7 dogs, local or regional tumor progression was the 1st cause of failure. In 1 dog, concurrent regional and distant metastasis (to the spleen, liver, kidney, peritoneum, mesenteric lymph nodes, and skin) was the 1st cause of failure. In 1 dog with no clinical evidence of tumor progression, distant metastasis (liver with concurrent ascites) was the 1st cause of failure. Two dogs with Grade II tumors developed regional metastasis. Five dogs with nodal metastasis at diagnosis had no disease progression in the node. Cox regression analysis revealed that time to metastasis after treatment was inversely associated with tumor grade (P.001) and tumor stage (P.012). High tumor grades and tumor stages were associated with shorter time to metastasis. Discussion MCT of the muzzle in dogs represent a therapeutic challenge because of their location and predilection for regional metastasis. Radical surgery is seldom attempted because of the risk of morbidity (eg, wound dehiscence, poor cosmetic results). Regional lymph node evaluation by fine needle aspiration or biopsy should be attempted in all dogs with head and neck MCT, but results can be confounded by the presence of oral or aural disease and could be technically difficult if lymph nodes are normal in size. The purpose of this study was to document the biologic behavior of muzzle MCT and to identify prognostic factors for tumor control and survival. Patient characteristics of dogs of this study are comparable to those of previous reports. Dogs in this study were mostly purebred (95%) with Golden Retrievers and Labrador Retrievers making up half of the study population. Previous studies have demonstrated that purebred dogs make up the majority of MCT patients, and 1 report documented significantly more Boxers and Labrador Retrievers with MCT when compared to the general hospital population. 4,15,16,21 There were almost equal numbers of Fig 3. Association between presence of metastasis at diagnosis and survival rate in 23 dogs with mast cell tumors of the muzzle. Tick marks represent dogs that are alive or whose death was attributable to an unrelated disease., dogs with no clinical evidence of metastasis at diagnosis;, dogs with clinical evidence of metastasis at diagnosis. males and females, and the median age of dogs in this study was 8 years, which is comparable to other reports. 4,16,21 Tumor grade is the most consistent prognostic indicator in dogs with MCT, and this observation is supported in the current study, in which grade was predictive of local tumor control, survival time, and time to metastasis after treatment. 3 5 In this study, a smaller number of well-differentiated and a larger number of intermediate- and poorly differentiated MCT were represented than in previous reports, in which all tumor locations were included. 4,15,22,23 This difference might represent a shift of grading criteria from earlier studies, or it might represent a true influence of location on tumor grade. An earlier study with similar grading criteria reported a distribution of grades comparable to this study when tumors of the trunk and extremities were combined. 5 However, analysis of tumor grade distribution by location in that study disclosed a higher, but not significant, proportion of Grade II and III tumors located on the trunk versus the extremities. In that study, dogs treated with ir- Fig 4. Tumor control in 23 dogs with mast cell tumors of the muzzle. Tick marks represent dogs that are alive and free of tumor progression or whose death was attributable to an unrelated disease.

Muzzle Mast Cell Tumors in Dogs 691 radiation had a better prognosis when the tumor was located on the extremities than did dogs with tumors on the trunk. To our knowledge, no studies have examined specifically the effects of site on tumor grade. Despite limitations of the grading scheme for MCT and lack of consensus among pathologists regarding grading criteria, this study confirmed the predictive value of tumor grade for tumor behavior and response to treatment in dogs with muzzle MCT. Higher tumor grades were associated with higher risk of metastasis, lower local control rates, and shorter survival times. Tumor grade was also found to affect the time to development of new metastases after treatment, independently of the metastatic status at diagnosis. This finding could indicate that, in dogs with higher grade tumors, metastases were present but not diagnosed at presentation and might not be affected by treatments used in this study. On the other hand, the metastatic process was a later event in dogs with lower grade tumors. Although no statistical conclusions could be drawn, dogs with Grade II tumors developed metastasis only when the primary tumor was not controlled. This observation might provide preliminary evidence that Grade II tumors have a low risk of metastasis, but the risk might increase with persistent or recurring primary tumors. Regional lymph node metastasis was detected in 11 of 19 dogs (58%) evaluated for metastasis at the time of diagnosis. Nodal involvement in 4 dogs with palpably normal lymph nodes emphasizes the importance of regional lymph node evaluation, even in the presence of palpably normal lymph nodes. When compared to tumors on the extremities and trunk, MCT on the muzzle were found to have a higher risk of metastasis. 5,23,24 The difference in tumor behavior of MCT on the muzzle when compared to other cutaneous locations is comparable to the difference in tumor behavior for MCT arising from the oral cavity compared to tumors arising from cutaneous sites. In dogs with Grade II MCT, the incidence of regional metastases was 50% for oral sites and 7% for cutaneous sites. 13 Presence of metastasis at diagnosis affected survival but was not found to affect local control. These findings are comparable to a previous study of dogs with confirmed regional lymph node metastasis treated with radiation therapy for incompletely resected MCT. 25 In that study, median local control duration (41 months) was comparable to that in this study (36 months). In this study, 5 dogs with lymph node metastasis at the time of diagnosis did not have progression of their disease. These data indicate that dogs with regional metastasis should be treated aggressively rather than palliatively to prevent or delay regional disease progression. Despite the high incidence of regional lymph node metastasis in this study, the presence of distant metastasis at the time of diagnosis was uncommon. Evidence of systemic metastasis after therapy also was uncommon, with only 2 dogs developing metastasis after therapy. Analysis of regional and distant metastases in this study should be interpreted with caution because of the lack of a standardized definition of metastasis. In dogs with MCT, uncertainty exists on how to differentiate true nodal metastasis versus migration of nonneoplastic mast cells through the node secondary to chemotactic factors and drainage of the tumor. Lymph node biopsy might be more likely than fine-needle aspiration to confirm true metastasis versus draining mast cells, but this procedure adds morbidity and cost to staging procedures. In this study, 1 dog had a positive buffy coat smear. Mast cells in the buffy coat smear are not specific for mast cell disease and is seen in many other systemic inflammatory conditions. 26 Although the dog in question was classified at stage 4 on the basis of the positive buffy coat smear, it was alive with no evidence of disease at approximately 3 years after diagnosis. In addition, dogs in this study with disseminated MCT had negative buffy coat smears. Buffy coat smears provide evidence to support systemic mastocytosis in dogs with MCT and clinical signs related to mast cell disease (eg, gastrointestinal bleeding, peritumoral edema), but this test should be regarded as nonspecific and insensitive. Tumor stage was not found to affect local control or survival in this study, in contrast to a previous report in which dogs with higher stages of disease had shorter survival times. 5 This observation might provide preliminary evidence that the staging scheme for MCT used in this study is not appropriate for all anatomic sites. Tumor grade should be included in stage grouping of MCT of the face, and probably other locations, to assist in therapeutic decision making. The staging protocol used for detecting MCT metastases, including buffy coat, bone marrow aspirate, abdominal ultrasonography, splenic aspirate, and lymph node aspirate, was of limited usefulness in many dogs but should be considered for dogs with MCT until further studies clarify the usefulness of staging tests for tumors in specific locations. In this study, for example, thorough staging was useful in determining regional lymph node metastasis in dogs with Grade II tumors with normal-sized lymph nodes. Tumor stage was found to affect time to metastasis after treatment independently of tumor grade and presence of metastasis at diagnosis. This finding indirectly could reflect the influence of tumor size or number of tumors in the same patient on the risk of metastasis. However, the data did not allow statistical analysis of the effects of tumor size on metastasis because the exact tumor size was not known in all dogs. This study did not confirm the predictive value of tumor stage for MCT; tumor stage was a prognostic factor in univariate analysis but not multivariate analysis of local control. Tumor stage has been found to have predictive value in dogs with MCT treated with surgery, radiation therapy, and chemotherapy. 5,7,27 This difference in prognostic value might result from the small sample size in this study and the variety of treatment modalities. It also might reflect the high incidence of metastasis for tumors located on the muzzle, which could have obscured the predictive value of tumor size and number of tumors in tumor staging. Other factors, including presence of gross versus microscopic disease, tumor stage, and single- versus multimodality therapy, were not significant factors for local control or survival. This study confirms that tumor location can affect prognosis of dogs with MCT. On the basis of results in this report, MCT of the muzzle should be considered locally aggressive, with an increased potential for local metastasis when compared to MCT at other sites. In this report, local spread or regional metastasis was the cause of euthanasia or death in 8 of 9 dogs who died of MCT. Consequently,

692 Gieger et al these tumors should be treated early in the course of disease with multiple treatment modalities, if possible. This study provides preliminary evidence that tumor staging protocols should include tumor location and tumor grade. Because a small number of dogs were treated with a wide variety of treatment methods in this study, it is difficult to make recommendations regarding specific treatments or to document the relative efficacy of each treatment method. This report provides a basis for improving staging schemes of canine MCT and initiating prospective studies to evaluate treatment modalities best suited to locally aggressive MCT. Footnotes a Deltasone (prednisone), Pharmacia and Upjohn, Kalamazoo, MI b Leukeran (chlorambucil), Glaxo Smith Kline, Research Park Triangle, NC c Cytoxan (cyclophosphamide), Mead Johnson Oncology Products, Princeton, NJ d Vinblastine, Bedford Labs, Bedford, OH e CeeNU (lomustine), Bristol Laboratories, Princeton, NJ f Hydrea capsules (hydroxyurea), Immunex, Seattle, WA g Alkeran (leukeran), Glaxo Smith Kline, Research Park Triangle, NC Acknowledgments The authors thank Steven Atwater, DVM, DACVIM, and Susan Kraegel, DVM, DACVIM, for case contribution and Eric Simonson and Lucy Elphick for technical assistance. References 1. Dorn ER, Taylor D, Schneider R, et al. Survey of animal neoplasms in Alameda and Contra Costa counties, California. J Natl Cancer Inst 1968;40:307 318. 2. Cohen D, Reif SS, Brodey RS, et al. Epidemiological analysis of the most prevalent sites and types of canine neoplasia observed in a veterinary hospital. Cancer Res 1974;34:2859 2868. 3. Bostock DE. The prognosis following surgical removal of mastocytomas in dogs. J Small Anim Pract 1973;14:27 40. 4. Patnaik AK, Ehler WJ, MacEwen EG. Canine cutaneous mast cell tumor: Morphologic grading and survival time in 83 dogs. Vet Pathol 1984;21:469 474. 5. Turrel JM, Kitchell BE, Miller LM, et al. Prognostic factors for radiation treatment of mast cell tumors in 85 dogs. J Am Vet Med Assoc 1988;193:936 940. 6. Kravis LD, Vail DM, Kisseberth WC, et al. Frequency of argyrophilic nucleolar organizer regions in fine-needle aspirates and biopsy specimens from mast cell tumors in dogs. J Am Vet Med Assoc 1996; 209:1418 1420. 7. Bostock DE, Crocker J, Harris K, et al. Nucleolar organiser regions as indicators of post-surgical prognosis in canine spontaneous mast cell tumors. Br J Cancer 1989;59:915 918. 8. Ayl RD, Couto CG, Hammer AS, et al. Correlation of DNA ploidy to tumor histologic grade, clinical variables, and survival in dogs with mast cell tumors. Vet Pathol 1992;29:386 390. 9. Simoes JPC, Schoning P, Butine M. Prognosis of canine mast cell tumors: A comparison of three methods. Vet Pathol 1994;31:637 647. 10. Miller DM. The occurrence of mast cell tumors in young Shar Peis. J Vet Diagn Invest 1995;7:360 363. 11. Ginn PE, Fox LE, Brower JC, et al. Immunohistochemical detection of p53 tumor-suppressor protein is a poor indicator of prognosis for canine cutaneous mast cell tumors. Vet Pathol 2000;37:33 39. 12. Jaffe MH, Hosgood G, Taylor HW, et al. Immunohistochemical and clinical evaluation of p53 in canine cutaneous mast cell tumors. Vet Pathol 2000;37:40 46. 13. Fan TM, Kitchell BE. Retrospective analysis of oral mast cell tumors in dogs. Proceedings of the 19th Annual Veterinary Cancer Society Conference, Woods Hole, MA, 1999:40. 14. Takahashi T, Kadosawa T, Nagase M, et al. Visceral mast cell tumor in dogs: 10 cases (1982 1997). J Am Vet Med Assoc 2000; 216:222 226. 15. Michels GM, Knapp DW, DeNicola DB, et al. 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Recurrence rates and sites for Grade II canine cutaneous tumors following complete surgical excision. J Am Anim Hosp Assoc 2002;38:71 73. 22. Hottendorf GH, Neilson SW. Survey of 300 extirpated canine mastocytomas. Zentralbl Vet 1967;14:272 281. 23. Frimberger AE, Moore AS, LaRue SM, et al. Radiotherapy of incompletely resected, moderately differentiated mast cell tumors in the dog: 37 cases (1989 1993). J Am Anim Hosp Assoc 1997;320 324. 24. LaDue T, Price GS, Dodge R, et al. Radiation therapy for incompletely resected canine mast cell tumors. Vet Radiol Ultrasound 1998;39:57 62. 25. Chaffin K, Thrall DE. Results of radiation therapy in 19 dogs with cutaneous mast cell tumors and regional lymph node metastasis. Vet Rad Ultrasound 2002;43:392 395. 26. McManus PM. Frequency and severity of mastocytemia in dogs with and without mast cell tumors: 120 cases (1995 1997). J Am Vet Med Assoc 1999;215:355 357. 27. Gerritsen RJ, Teske E, Kraus JS, et al. Multi-agent chemotherapy for mast cell tumors in the dog. Vet Quart 1998;20:28 31.