M scribed in humans and dogs with neop1a~ia.l-i~

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1 Thrombocytopenia Associated With Neoplasia in Dogs Carol B. Grindem, Edward B. Breitschwerdt, Wayne T. Corbett, Rodney L. Page, and Heather E. Jans Ten percent (21 4/2,59) of all dogs with cancer at North Carolina State University Veterinary Teaching Hospital had thrombocytopenia. The thrombocytopenia was associated with infectious/inflammatory etiologies in 4%. miscellaneous disorders (therapy, bone marrow failure, disseminated intravascular coagulation) in 35%, and neoplasia without identifiable secondary factors in 61 % of cancerbearing dogs. Classifying these dogs by tumor groups revealed the following proportionate ratios: lymphoid, 29%; carcinoma, 28%; sarcoma, 2%; hemic neoplasia, 7%; multiple, 5%; unclassified, 3%; benign, 3%; brain, 3%; and endocrine, 3%. Dogs with hemangiosarcoma, lymphoma, and melanoma were at increased risk of developing thrombocytopenia. Cytotoxic therapy was the major factor increasing the risk of thrombocytopenia in dogs with melanoma. Golden Retrievers were the only breed recognized with a predisposition to develop thrombocytopenia. If thrornbocytopenia is identified in a dog with cancer, we recommend thorough evaluation of the coagulation system before surgery or therapy, and careful consideration of the risks and potential benefits of myelosuppressive or L-asparaginase therapy. J Vet Intern Med 1994;8:4-45. Copyright 1994 by the American College of Veterinary Internal Medicine. ultiple hemostatic abnormalities have been de- M scribed in humans and dogs with neop1a~ia.l-i~ Thromboembolic and hemorrhagic phenomena are important treatment-related complications in hu- mans with cancer.l'9'3,14 Although much has been hypothesized on the pathogenesis of thrombocytopenia in cancer patients, limited information is available on the actual frequency of thrombocytopenia in dogs with neoplasia. The purpose of this study was to document the prevalence rate of thrombocytopenia in dogs with neoplasia at an institutional referral hospital and to evaluate the potential associations between thrombocytopenia and tumor type, signalment, and possible etiology. Materials and Methods Criteria for inclusion in this cross-sectional study were laboratory documentation of thrombocytopenia defined by platelet counts (Coulter Counter, Model S-Plus IV, Coulter Electronics, Inc, Hialeah, FL, or manual counts using hemocytometer and Thrombo Plus, Sarstedt, Princeton, NJ) From the Department of Microbiology, Pathology and Parasitology (Grindem, Corbett), and Companion Animal and Special Species Medicine (Breitschwerdt, Page, Jans). College of Veterinary Medicine, North Carolina State University, Raleigh, NC. Accepted November 22, The authors thank Douglass Corbett and Laura Cullins for technical assistance. Reprint requests: Carol B. Grindem, 47 Hillsborough St, NCSU-CVM, Raleigh, NC Copyright I994 by the American College of Veterinary Internal Medicine /94/86-3$3./ under 2OO,OOO/pL and a histopathologic or cytopathologic diagnosis or neoplasia. I6 Thrombocytopenic dogs were identified from clinical pathology laboratory reports at North Carolina State University, Veterinary Teaching Hospital (NCSU-VTH) between August 1983 and June Medical records were reviewed and data on signalment, physical examination findings, history, laboratory tests, therapy, and biopsy and/or necropsy were abstracted for further analysis. Laboratory data collected at the time ofinitial thrombocytopenia included: platelet count, platelet morphology, packed cell volume (PCV), segmented and band neutrophil counts, concentration of plasma solids (PS), and, if available, the results of coagulation tests (MLA Electra 75, Medical Laboratory Automation, Inc, Mount Vernon, NY, and Thromboplastin-C, Actin and Data-Fi Fibrinogen and Fibrinogen Degradation Products, Dade Diagnostics, Inc, Aguada, Puerto Rico) (prothrombin time [PT], activated partial thromboplastin time [APTT], fibrin degradation products [FDPs], and fibrinogen concentration). Thrombocytopenic and nonthrombocytopenic dogs were grouped by general histological tumor types independently and blindly as to platelet count (sarcoma, carcinoma including melanoma, endocrine, nonlymphoid hematopoietic tumors, benign, unclassified, multiple neoplasms, brain, and lymphoid tumors) and further subdivided by the most common specific tumor types (hemangiosarcoma [HSA], osteosarcoma [OSA], melanoma, nasal carcinoma, squamous cell carcinoma [SCC], pulmonary carcinoma, mast cell tumor, and lymphoma) (Tables 1 and 2). Retrospectively, three of the authors (Breitschwerdt, Jans, and Page) analyzed the history, physical findings, results of laboratory evaluation, and response to treatment or necropsy findings, if available, to determine the putative cause of the thrombocytopenia in each dog. Then dogs were grouped into 1 of the 4 categories as follows: (1) neoplasia without identifiable secondary factors, as defined by no historical, physical, or laboratory cause of thrombocytopenia; (2) infectious/inflammatory disorders, as defined by laboratory documentation of bacteria (microbial culture positive), rickettsia (serologic diagnosis), parasites (microfilaremia or 4 Journal of I'eterinaty Internal Medicine. VoI8. No 6 (November-DecemberJ, 1994: pp 4-45

2 CANINE THROMBOCYTOPENIA AND NEOPLASIA 4 1 Table 1. Data From Thrombocytopenic Dogs With Tumors No. of Platelets/FL % Large % Clumped Age Sex Tumor Type Dogs % (mean? SD) Platelets Platelets (yr; mean? SD) (Female; mean) Sarcoma ,476? 46, ? 3. 57% Carcinoma Endocrine Hemic (Nonlymphoid) Benign Unclassified Multiple Brain Lymphoid ? 54,218 44,333? 43,542 28,733 f 46, ?51, f 56,373 35,8? 51,792 51,333? ,21 f 58, ? f ? k ? f ? % 33% 47% 5% 43% 4% 48% Total ,542?53, ? 3. 51% * Percent of dogs within tumor type that are thrombocytopenic antigenemia), viruses (canine distemper inclusion bodies, fecal electron microscopy, serology), systemic mycoses, or aseptic inflammatory diseases such as pancreatitis; (3) immune-mediated as defined by a positive antinuclear antibody test (ANA) (>8), a positive lupus erythematosus (LE) cell preparation, or thrombocytopenia with megakaryocytic hyperplasia or therapeutic response to immunosuppressive drug therapy or splenectomy; and (4) miscellaneous disorders defined by heterogenous states including trauma, acute hemorrhage, drug therapy, coagulopathies, hyperestrogenism, bone marrow failure, and platelet clumping (Tables 3 and 4). Breed, age, and sex data from all dogs with confirmed neoplasia admitted to the hospital during the same period (1983 to 1989) were compiled for comparison with those of thrombocytopenic dogs with neoplasia. Controls consisted of 16,78 nonthrombocytopenic dogs without neoplasia, 1,845 nonthrombocytopenic dogs with neoplasia, and 773 thrombocytopenic dogs without neoplasia. Controls were hrther subdivided by breed, sex, and type of neoplasia, similar to principal dogs. Baselines for the estimated relative risk (ERR) calculations were the remaining applicable hospital population of dogs after the breed or tumor type of interest was selected. For example, to calculate the ERR of Boxers to develop neoplasia, Boxers with and without neoplasia were compared to the remaining hospital population ofdogs with and without neoplasia. Descriptive statistics were initially used to analyze all data. One-way analysis of variance (ANOVA) was used to detect differences in segmented neutrophil counts, PCV, PS, sex, or age (dependent variables) among general histological tumor groups and specific tumor groups (independent variables). If the AOV was statistically significant at the.5 level, Student s t tests were performed to determine which groups differed., * Yates corrected x2 analysis was used to determine if the principal population (dogs with neoplasia and thrombocytopenia) differed from either control group 1 (nonthrombocytopenic dogs with neoplasia) or control group 2 (nonthrombocytopenic dogs without neoplasia) with regard to breed or A P value of <.5 was used to determine statistical significance in this study. ERR to develop thrombocytopenia with 95% confidence intervals (CI) for dogs with various tumor types (Table 5) was calculated as an odds ratio; the ERR for specific breeds to develop neoplasia, or to develop neoplasia with thrombocytopenia were also calculated as odds ratio^.'^.*^ Specific breeds evaluated included Boxers, Cocker Spaniels, Doberman Pinschers, German Shepherd dogs, Golden Retrievers, Labrador Retrievers, mixed breeds, and Poodles. Other breeds had too few numbers for statistical analysis. Comparisons of platelet counts and coagulation data among tumor types was not performed because dogs were selected based on thrombocytopenia, and platelet counts are biologically related to coagulation test results. Table 2. Data from Thrombocytopenic Dogs With Specific Tumor Types PlateletslpL Age Sex Tumor Type No. of Dogs Percent (mean? SD) (y; mean f SD) (Females) Lymphoma , , f % Hemangiosarcoma ,778? 41, ? % Osteosarcoma ,4? f 2.2 6% Mast cell tumor ,7? 25,32 9.? 1.7 3% Melanoma i- 56, f % Squamous cell carcinoma ,117? 34,28 8.6? % Pulmonary carcinoma f k.9 4% Nasal carcinoma ,6? 59, i 3.5 6% Reference range 2,-5, * Percent of dogs within tumor type that are thrombocytopenic

3 42 GRINDEM ET AL Table 3. Etiologic Comparison of Data From Thrombocytopenic Dogs With Tumors No. of Percent of Etiologic Class Thrombocytopenic Dogs Thrombocytopenic Dogs Platelets (/pl) Age (Y) Sex (% Females) Neoplasia alone ,695 k 52, i Infectious , f 59, k Miscellaneous' , , i All data ,542 k 53, i * Therapy, bone marrow failure, disseminated intravascular coagulation Results Thrombocytopenia was documented in 1% (2 14/ 2,59) of all dogs with cancer at NCSU-VTH between 1983 and Classifying thrombocytopenic dogs by tumor groups revealed the following proportionate ratios: lymphoid 29% (62/2 14), carcinoma 28% (6/ 2 14), sarcoma 2% (42/2 14), nonlymphoid hematopoietic tumors 7% ( 15/2 14), multiple 5% (1/2 14), unclassified 3% (7/2 14), benign 3% (6/214), brain 3% (6/214), and endocrine 3% (6/2 14). Distribution of platelet counts is illustrated in Fig 1. No statistically significant differences in segmented neutrophil count, PCV, PS, or sex were detected as a function of tumor group (Table 1). Thrombocytopenic dogs with lymphoid or unclassified tumors were significantly (P =. 1 ) younger (7.7 t 3. and 7.9 & 3.3 years, respectively) than thrombocytopenic dogs with multiple tumors or carcinomas (1. & 3.2 and years, respectively). Compared with the baseline tumor population, thrombocytopenia occurred more frequently in dogs with carcinomas, hemic tumors, and lymphoid tumors (Table 1). The most common specific tumor types were lymphoma 27% (57/214), melanoma 9% (19/214), HSA 8% (1 /2 14), OSA 5% (1/2 14), mast cell tumor 5% ( 1/2 14), SCC 3% (7/2 14), pulmonary carcinoma 2% (5/2 14), and nasal carcinoma 2% (5/ 2 14) (Table 2). Regarding age, dogs with lymphoma and OSA were significantly younger than dogs with HSA. Dogs with HSA (ERR = 8.3), lymphoma (ERR = 6.1), and melanoma (ERR = 4.9) were at increased risk of developing thrombocytopenia (Table 5). The mean PCV of dogs with HSA (27% k 8.8%) was significantly lower (P <.5) than that of dogs with other tumor types, while dogs with SCC had a significantly higher PCV (mean, 45% k 1.3%). The mean neutrophil count was greater than the reference range for all general tumor types except hemic, benign, and lymphoid, and in most common specific tumor types except OSA, melanoma, and SCC. The neutrophil counts in dogs with melanoma and SCC were significantly lower than in other tumor types, and the neutrophil counts in dogs with OSA were significantly lower than in HSA (Table 2). Although these neutrophil counts were significantly Table 4. Hemostatic Data From Thrombocytopenic Dogs With Tumors PT APTT FDP Fibrinogen Elevation Elevation Elevation Decrease % %> %z %>4 %<ZOO DIC PT APTT PLT (1 O ~/~L; PCV % Tumor type Tested 7.8 s 13.6 s pg/ml mg/dl % (s; Mean f SD) (s; Mean k SD) Mean i SD) (Mean f SD) Sarcoma k26. 95,474k Carcinoma k k ,857 i 49, Endocrine k f ,75Oi5, Hernic (nonlyrnphoid) k i ,6 i 27,493 5 f 13.8 Benign f i , k 44,739 36i 6.8 Unclassified k k 4. 14, i 55,54 26f 9.5 Multiple f k , f 34,29 24 k 3.7 Brain Lymphoid k k5.8 15,458k Hemangiosarcorna k ,9 i 29, k 7.1 Total (%) k i ,862i52'861 34f 1.9 ~ Abbreviations. PT, prothrombin time. APTT, activated partial thromboplastin time, FDP, fibrin degradation products, DIC, disseminated intravascular coagulopathy; PLT, platelets; PCV, packed cell volume

4 CANINE THROMBOCYTOPENIA AND NEOPLASIA 43 Table 5. Estimated Relative Risks by Tumor Type for Developing Thrombocytopenia Estimated 95% Confidence Tumor Type/Subtype Relative Risk Limits Sarcoma 1.85 (1.26,2.71) Hemangiosarcoma 8.38 (4.16, 16.86) Osteosarcoma 1.26 (.6, 2.58) Carcinoma 2.35 ( ) Melanoma 4.9 (2.65, 9.) Nasal carcinoma 1.98 (.58, 5.44) Pulmonary carcinoma 2.92 (.82, 8.55) Squamous cell carcinoma 1.86 (.68,4.34) Lymphoid 6.83 (4.71, 9.89) Nonlymphoid hematopoietic tumors 2.88 ( ) Mast cell tumor 1.66 ( ) Endocrine 1.1 (.37,2.71) Brain 1.87 (.63,4.68) Benign.15 (.5,.33) Unclassified.25 (.1,.54) Multiple.1 (.5,.19) No tumor (baseline).4 (.34,.47) lower, the mean counts were still within reference range and neutropenia rarely occurred in individual dogs. The cause of the variation in the neutrophil counts was unknown except for the low counts related to cytotoxic therapy (melphalen,.5 mg/kg IV q 4 weeks X 6; Alkeran; Burroughs Wellcome Co, Research Triangle Park, NC) in dogs with melanoma. Coagulation profiles were available for 42% of thrombocytopenic dogs with cancer (Table 4). Seventy-two percent (64/89) of dogs tested had one or more abnormal coagulation tests (prolonged APTT or PT, elevated FDPs, or hypofibrinogenemia). Prolongation of APTT and decreased fibrinogen concentration were the most common abnormalities. Dogs with HSA were tested most often and were more likely to have prolonged coagulation times (PT, APTT) and elevated FDPs than those in the other tumor categories. When the thrombocytopenia was grouped into causative subcategories, the results were as follows: neoplasia without identifiable secondary factors in 6 I % ( 1 3/2 14), infectious/inflammatory etiologies in 4% (9/2 14), and miscellaneous disorders (therapy, bone marrow failure, DIC, etc) in 35% (75/2 14) ofthe thrombocytopenic dogs (Table 3). None of the dogs with tumors in this study were diagnosed as having an immune-mediated thrombocytopenia. Dogs in the infectious category had lower platelet counts and were older than dogs in the other etiology groups. The majority of dogs in the miscellaneous categories had lymphoma or melanoma. Cytotoxic treatment was the most common cause of thrombocytopenia in dogs with melanoma. Boxers (P < lo-', x2 = 36.5, ERR = 2.3, CI = 1.75, 3.1) and Golden Retrievers (P < 9.2 X x2 = 15.3, ERR = 1.5, CI = 1.2, 1.76) were at increased risk for development of neoplasia when compared with the general hospital population. Additionally, Golden Retrievers with tumors were more likely to develop thrombocytopenia (P <.2, x2 = 5.2, ERR = 1.8, CI = 1.8,2.9 1). Ofthe thrombocytopenic dogs with neoplasia, 2% were small breed, 25% were medium sized, and 55% were large breed dogs. Discussion Thrombocytopenia was documented in 1% (214/ 2,59) ofall dogs with tumors and 12% (28/1,753) of the dogs with malignant tumors at NCSU-VTH between 1983 and 199. In smaller surveys, thrombocytopenia has previously been reported in 13 to 36% of dogs with ne~plasia.~,~ This is similar to surveys in humans that have reported 1% to 3% tumor-associated thrombocytopenia. ''J~ Potential causes for the differences among surveys include prevalence and stage of specific tumor types and extent of disease, splenic and bone marrow involvement, breeds of dogs, and presence or absence of DIC. In the survey with 36% thrombocytopenia, most of the dogs had large, invasive metastatic tumors at the time of examination, and 58% of the dogs had lymphohematopoietic neoplasms.8 Twenty percent (12/59) of the dogs with solid nonhemic large invasive tumors were thrombocytopenic. Consumptive coagulation was believed to be a contributory factor to the thrombocytopenia in many dogs in that study. In the other study in which 13% (7/53) of dogs with tumors were thrombocytopenic, only dogs with extensive tumor in the spleen or bone marrow were thrombocytopenic; however, 68% of the dogs did have decreased platelet survival.' Ten of 1 1 (91%) dogs with cutaneous vascular tumors were thrombo~ytopenic.~ In this study, dogs with carcinoma (2.3 ERR), hemic neoplasia (2.9 ERR), and lymphoid neoplasia (6.8 ERR) were at greater risk ofdeveloping thrombocytopenia. Part of the risk with carcinoma was due to therapy-associated thrombocytopenia in dogs with melanoma. Also, although in general dogs with sarcoma were not at increased risk of developing thrombocytopenia, dogs with HSA had an ERR of 8.3. Thrombocytopenia was documented in 47% of dogs with HSA, 37% of dogs with lymphoid tumors, 35% of dogs with melanoma, and 24% of dogs with hemic tumors. Our study supports previous reports of an as-

5 44 GRINDEM ET AL sociation between thrombocytopenia and vascular tumors and lymphohematopoietic tumors. Boxers have previously been identified as a breed at increased risk for neoplasia, and our study documents that Golden Retrievers are also at risk. Additionally, Golden Retrievers with neoplasia were predisposed to the development of thrombocytopenia. At least one other hemostatic abnormality was detected in 72% of the tested thrombocytopenic dogs with neoplasia in this study. This is not surprising because 95% or more of human patients with malignancies have hemostatic abnormalities with or without any clinical evidence of thromboembolic or hemor- rhagic disorders. % 4 Decreased fibrinogen concentrations and prolongation of the APTT were the two most frequent abnormalities observed in our study. The APTT is a more sensitive test than the PT, thus explaining why the APTT may be prolonged more often than the PT. Fibrinogen elevation and decreased fibrinogen survival occur in dogs with neoplasia. Fibrinogen is an acute phase reactant that increases with inflammation and is an integral factor in clot formation and hemostasis. Increased consumption (DIC) and decreased production can cause decreased fibrinogen concentrations. Thus, an animal with cancer may have increased or decreased fibrinogen concentrations and thrombosis or hemorrhage, depending on these factors. Therefore, a drug such as L- asparaginase that is associated with inhibition of protein synthesis should be used cautiously in canine cancer patients with coagulopathies.* Microangiopathic hemolytic anemia and DIC have been associated with HSA.23 Probably, because of this, dogs with HSA were more likely to be tested (excluding tumor types with n I 6) for hemostatic defects, and all of -5 57* & PLATELEI mum x ioowu~ Fig 1. Platelet count distribution in thrombocytopenic dogs with neoplasia. these dogs that were tested had at least one abnormal test result. The pathogenesis of the thrombocytopenia associated with neoplasia may include increased platelet consumption, decreased platelet production, immune-mediated destruction, and sequestration of platelets, or a combination of the above. The etiology of the thrombocytopenia in this study was attributed to infectious diseases in 4% of the dogs, miscellaneous causes (bone marrow suppression, therapy, DIC) in 35% of the dogs, and neoplasia without other identifiable causes in 6 1% of the dogs. No immune-mediated thrombocytopenias were documented; however, few dogs were tested for antiplatelet antibodies. Recent work has suggested that antiplatelet antibodies may be present even in nonthrombocytopenic dogs with neoplasia and, therefore, testing for these antibodies may become more routine in the f~ture.~ Additional mechanisms for the abnormal coagulation profiles include DIC, decreased factor or fibrinogen syntheses, renal loss of natural coagulation inhibitors or factors, enhanced fibrinolysis, or presence of acquired or exogenous coagulation inhibitors. The high prevalence of hemostatic defects in thrombocytopenic dogs with neoplasia indicates the need to thoroughly evaluate the coagulation system in such patients before surgery or therapy. Myelosuppressive agents or L-asparaginase should be used in thrombocytopenic dogs that have cancer only after careful consideration of the risks and potential benefits. References 1. Antman KH, Skann AT, Mayer RJ, et al. Microangiopathic hemolytic anemia and cancer: A review. Medicine 1979;58: Brodie GN, Bliss D. Firkin BG. Thrombocytopenia and carcinoma. Br Med J 197; 1 : Feldman BF, Madewell BR, ONeill SO. Disseminated intravascular coagulation: Antithrombin, plasminogen, and coagulation abnormalities in 4 I dogs. J Am Vet Med Assoc I98 I ; 179: 15 I Hargis AM, Feldman BF. Evaluation of hemostatic defects secondary to vascular tumors in dogs: 11 cases ( ). J Am Vet Med Assoc 1991;198: Helfand SC. Platelets and neoplasia. Vet Clin North Am Small Anim Pract 1988; 18: I Helfand SC, Couto CG, Madewell BR. 1mmune.mediated thrombocytopenia associated with solid tumors in dogs. J Am Anim Hosp Assoc 1985;21: Kristensen AT, Weiss DJ, Klausner JS, et al. Platelet dysfunction associated with immune-mediated thrombocytopenia in dogs. J Vet Intern Med 1994;8: Madewell BR, Feldman BF, ONeill S. Coagulation abnormalities in dogs with neoplastic disease. Thrombo Haemostas 198;44:35-38.

6 CANINE THROMBOCYTOPENIA AND NEOPLASIA O Donnell MR, Slichter SJ, Weiden PL, et al. Platelet and fibrinogen kinetics in canine tumors. Cancer Res I98 I ;4 1: O Keefe DA, Couto CG. Coagulation abnormalities associated with neoplasia. Vet Clin North Am Small Anim Pract 1988; 18: I. Rashce H, Dietnch M. Hemostatic abnormalities associated with malignant disease. Eur J Cancer 1977; 13: Schwartz KA, Slichter SJ, Harker LA. Immune-mediated platelet destruction and thrombocytopenia in patients with solid tumours. Br J Haematol 1982; 5 1: Slichter SJ, Harker LA. Hemostasis in malignancy. Ann NY Acad Sci 1974;23: Sun NCJ, McAfee WM, Hum GJ, et al. Hemostatic abnormalities in malignancy, a prospective study of one hundred eight patients. Am J Clin Pathol 1979;71: Zacharski LR, Rickles RF, Henderson WG, et al. Platelets and malignancy. Am J Clin Oncol 1982;5: Grindem CB, Breitschwerdt EB, Corbett WT, et al. Epidemi- ologic survey of thrombocytopenia in dogs: A report on 987 cases. Vet Clin Pathol 1991;2: Armitage P: Statistical Methods in Medical Research. New York, NY: John WileyandSons; 1971: , Statistix 3.1 Manual. St. Paul, MN: Analytical Software, 199, Kahn HA, Sempso CT. Statistical Methods in Epidemiology. New York, NY: Oxford University Press; 1989: Dean AG, Dean JA, Burton AH, et al. 199 Epi Info, Version 5. USD Inc, Stone Mountain, GA. 21. Dorn CR, Pnester WA. Epidemiology. In: Theilen GH, Madewell BR, eds. Veterinary Cancer Medicine, 2nd ed. Philadelphia, PA: Lea and Febiger; 1987: Rogers KS, Barton CL, Benson PA, et al. Effects of L-asparaginase on coagulation values in healthy dogs and dogs with fymphoma. Am J Vet Res 1992;53: Rebar AH, Hahn FF, Halliwell WH, et al. Microangiopathic hemolytic anemia associated with radiation-induced hemangiosarcomas. Vet Pathol 198; 17: