SPLENIC MARGINAL ZONE LYMPHOMA IN AN ASIAN SMALL-CLAWED OTTER (AONYX CINEREA) Author(s): Nancy L. Stedman, D.V.M., Ph.D., Dipl. A.C.V.P., and Zachary V. Mills, B.S., M.S. Source: Journal of Zoo and Wildlife Medicine, 45(3):719-722. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2014-0014R.1 URL: http://www.bioone.org/doi/full/10.1638/2014-0014R.1

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Journal of Zoo and Wildlife Medicine 45(3): 719–722, 2014 Copyright 2014 by American Association of Zoo Veterinarians

SPLENIC MARGINAL ZONE LYMPHOMA IN AN ASIAN SMALLCLAWED OTTER (AONYX CINEREA) Nancy L. Stedman, D.V.M., Ph.D., Dipl. A.C.V.P., and Zachary V. Mills, B.S., M.S.

Abstract: Severe splenomegaly was found during routine examination of a clinically normal 7-yr-old male Asian small clawed otter. The spleen and three enlarged splenic lymph nodes were immediately removed. The spleen weighed 310 g (approximately 8% of body weight). The spleen and resected lymph nodes were diffusely infiltrated by coalescing sheets of neoplastic lymphocytes that occasionally surrounded remnants of preexisting lymphoid follicles. Immunohistochemical confirmation of B lymphocyte origin and microscopic pattern were consistent with primary splenic marginal zone lymphoma (MZL) with metastasis to the splenic lymph nodes. The otter received no additional treatment and survived for 16 mo following splenectomy. Necropsy confirmed metastasis to multiple abdominal and extra-abdominal lymph nodes, liver, and kidney, and renal failure related to glomerulosclerosis. The prolonged survival in this otter is typical for MZL, an indolent form of B-cell lymphosarcoma that spreads slowly to the abdominal and extra-abdominal lymph nodes. Key words: Aonyx cinerea, Asian small-clawed otter, lymphoma, lymphosarcoma, mustelid, spleen.

BRIEF COMMUNICATION A 7-yr-old sexually intact male Asian smallclawed otter (Aonyx cinerea) underwent routine examination as part of a preventative medicine program. The animal was behaviorally normal but was found to have a severely enlarged and firm spleen that was not present at last examination performed 16 mo previously. Because of the severity of splenic enlargement, the spleen and three adjacent splenic lymph nodes were surgically removed. An enlarged pancreaticoduodenal lymph node was also identified at surgery, but because of its proximity to the pancreas, removal was not attempted. Bloodwork performed at the time of surgery revealed the following when compared with institutional reference intervals for this species: mild anemia characterized by decreased erythrocytes (4.3 million/dl; decreased from previous value of 5.3 million/dl and reference interval of 5.0–8.6 million/dl), slightly decreased hematocrit (31%; decreased from previous value of 37% but not reference interval of 31–53%), and decreased hemoglobin (10.0 mg/ dl; decreased from previous value of 12.3 mg/dl and reference interval of 10.7–18.4 mg/dl); hyperproteinemia (10.2 g/dl; reference interval 5.6–7.9 g/dl) related to hyperglobulinemia (7.8 g/dl; reference interval 2.9–5.1 g/dl), and hyperphosphatemia (12.9 mg/dl; reference interval 3.2–7.9 mg/dl). Blood urea nitrogen (BUN) and creatiFrom Busch Gardens Tampa, 3605 East Bougainvillea Avenue, Tampa, Florida 33612, USA (Stedman, Mills). Correspondence should be directed to Dr. Stedman ([email protected]).

nine were at the high end of internal reference intervals (BUN 42 mg/dl; reference interval 16– 43 mg/dl and creatinine 0.8 mg/dl; reference interval 0.3–0.8 mg/dl). Findings were consistent with early renal disease and hyperglobulinemia of undetermined cause. Unfortunately, the hyperglobulinemia was not characterized further. Reference intervals used for comparison were calculated as the nonparametric central 95% of at least 233 data points for each parameter obtained from healthy collection adult Asian small-clawed otters. The resected spleen weighed 310 g (8% of presurgical body weight of 3.855 kg), and had a nodular appearance related to multifocal to coalescing infiltrates of grey–white, homogeneous soft tissue throughout the organ (Fig. 1). Similar tissue infiltrated the three enlarged splenic lymph nodes. Microscopically, imprints of the spleen revealed a monomorphic population of lymphocytes approximately 6–8 microns in diameter with a single nucleolus in most nuclei. Histopathology confirmed severe effacement and replacement of the splenic architecture by sheets of neoplastic lymphocytes forming expansive nodules reminiscent of follicles but without any distinct follicular architecture. Neoplastic lymphocytes had moderate, lightly eosinophilic cytoplasm. Nuclei were central and round to oval nuclei with vesicular chromatin and a single nucleolus in most cells. The mitotic index was 4 per 10 4003 fields. Occasionally, a central core of nonneoplastic lymphocytes consistent with remnants of a follicle (‘‘fading’’ germinal center) was present within the nodules of neoplastic lymphocytes. In the splenic lymph nodes, this nodular architecture with

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Figure 1. Spleen; Asian small-clawed otter. The spleen is enlarged with a nodular texture. Enlarged splenic lymph nodes are also present along the hilum.

central follicular remnants was more prominent than in the spleen, and paracortical tissue was also entrapped between the nodules of neoplastic lymphocytes in the splenic lymph nodes (Fig. 2). Immunohistochemistry was performed with the use of antibodies against CD3 (product code A 0452, Dako North America Inc., Carpenteria, California 93013, USA; working dilution 1 : 400), CD79a (clone JCB117, Dako; working dilution 1 : 200), BLA36 (clone BLA.36, Dako; working dilution 1 : 400), and canine CD18 (kindly provided by Dr. Peter Moore; working dilution 1 : 10). All immunohistochemical staining was performed on a DakoCytomation autostainer in accordance with the manufacturer’s recommendations. Neoplastic lymphocytes exhibited weak to moderate cytoplasmic staining for CD79a and BLA36, and no staining for CD3, confirming a Bcell origin neoplasm. Nonneoplastic lymph node tissue stained appropriately with these antibodies, but no cells exhibited any staining for CD18. The morphologic diagnosis based upon the pattern of the infiltrates, microscopic appearance of the neoplastic lymphocytes, and B-cell origin was splenic marginal zone lymphoma (MZL) with metastasis to the splenic lymph nodes. No additional treatment was administered other than splenectomy. Sixteen months after splenectomy, the otter began to exhibit lethargy and inappetance. Physical exam revealed marked ulceration of the rostral mandibular and maxillary gingiva and mucocutaneous junctions of the rostral labia. Radiographs confirmed soft tissue masses in the cranial thorax and abdomen. Bloodwork revealed worsening anemia (erythrocytes 2.24 million/dl; hematocrit 24%; hemoglobin 5.4 mg/dl); persistent hyperproteinemia (7.9 g/dl) related to hyper-

globulinemia (6.2 g/dl), and worsening hyperphosphatemia (17.1 mg/dl). BUN and creatinine were markedly elevated (BUN 232 mg/dl and creatinine 3.5 mg/dl). Because of radiographic evidence of probable widespread neoplasia and clinicopathologic evidence of renal failure, the otter was euthanized. Gross necropsy confirmed moderate to severe enlargement of the mediastinal/tracheobronchial, hepatic, gastric, pancreaticoduodenal, renal, sublumbar, inguinal, axillary, prescapular, and cervical lymph nodes. A single 6mm-diameter, off-white, soft tissue mass was identified in the cranial pole of the right kidney, and several 6–10-mm- diameter soft tissue masses were present in several liver lobes. Microscopic evaluation of the enlarged lymph nodes and the

Figure 2. Splenic lymph node; Asian small-clawed otter. (a) Neoplastic lymphocytes retain a nodular architecture surrounding nonneoplastic lymphocytes consistent with ‘‘fading’’ germinal centers. Pre-existing paracortical tissue is present between the nodules. Hematoxylin and eosin. Bar ¼ 200 lm. (b) Neoplastic lymphocytes have moderate eosinophilic cytoplasm, vesicular nuclei, and a single nucleolus in most cells. Hematoxylin and eosin. Bar ¼ 20 lm.

STEDMAN AND MILLS—OTTER SPLENIC MARGINAL ZONE LYMPHOMA

liver and kidney masses confirmed metastatic lymphoma. Kidneys also had moderate to marked basement membrane thickening in most glomeruli, occasional senescent glomeruli, hyaline material within tubules, and mild, diffuse interstitial fibrosis. Ulcers noted grossly in the mouth were related to vascular necrosis. The final diagnosis was metastatic marginal zone lymphoma in multiple lymph nodes, liver, and right kidney with glomerulosclerosis, terminal renal failure, and uremic stomatitis. Because metastatic disease in the kidney was unilateral and focal, renal failure was related to glomerulosclerosis and not metastasis. Differential diagnoses for this B-cell–origin neoplasm were splenic MZL, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). In dogs, these forms of B-cell lymphosarcoma can be distinguished by the following criteria: MZL is characterized by identification of an inner remnant collapsed mantle cell cuff or heterogeneous follicular center, single large nucleolus, and relatively low mitotic rate; FL is characterized by a uniform population of centroblasts and centrocytes across the follicle; and DLBCL is characterized by a diffuse arrangement of neoplastic lymphocytes in sheets with a relatively high mitotic rate.11 In this otter, the arrangement of neoplastic lymphocytes as discrete nodules around heterogeneous follicular centers, relatively low mitotic rate, and vesicular chromatin pattern with single nucleoli in the neoplastic lymphocytes were consistent with MZL originating in the spleen and metastasizing to multiple abdominal and extra-abdominal lymph nodes. MZL is a form of B-cell origin, indolent lymphosarcoma that has been described in humans, dogs, and cats. It may arise as a consequence of chronic antigenic stimulation and follicular lymphoid hyperplasia.6 Similar to humans, MZL in dogs is associated with clonal rearrangement of immunoglobulin heavy chain genes, an indolent disease course, and prolonged survival.5,12 In dogs with primary splenic MZL, neoplasia begins as a focal splenic tumor with gradual metastasis to the abdominal lymph nodes, and eventually to the extra-abdominal lymph nodes.11 In dogs, splenectomy can be beneficial with or without adjunct chemotherapy, although in humans, administration of anti-CD20 antibodies with or without chemotherapy is efficacious without splenectomy and may be used in patients that are poor surgical candidates.5,6,8 This otter was treated with splenectomy alone without adjunct chemotherapy because frequent handling

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and anesthesia of this animal to administer medications would have been difficult. The prolonged survival in this otter suggests splenic MZL in this species may also have an indolent disease course, and may be amenable to splenectomy as the sole treatment. Hyperglobulinemia was not characterized further in this case, and the cause for this finding is uncertain. In humans, MZL is only reported to be associated with hyperglobulinemia when it occurs in mucosa-associated lymphoid tissue in the thymus of patients with concurrent autoimmune disease.7 Therefore, it is doubtful hyperglobulinemia in this otter was directly related to the presence of MZL. It is also possible hyperglobulinemia in this otter was a response to the same stimulus that predisposed to MZL. Except for routine vaccination, the otter had no known history of chronic exposure to any antigen that could have predisposed to either condition. In humans, glomerulopathies are also rarely reported in association with lymphocytic leukemia and lymphoma, but manifest most commonly as minimal change disease.4 Therefore, the relationship between lymphoma and glomerulosclerosis in this otter is also uncertain. With the exception of domestic ferrets, lymphoma is uncommonly reported in mustelids, and limited to published and anecdotal reports of intestinal large granular lymphoma in a fisher and Eurasian otter, and multicentric or cerebral lymphoma and lymphocytic leukemia in sea otters.1,3,10 Asian small-clawed otters are relatively common in zoos, but there are only two published reports of neoplasia in this species, although in the authors’ experience neoplasia occurs with some frequency in geriatric animals.2,9 This case illustrates the importance of routine examination of animals in zoological institutions to detect and treat clinically inapparent disease in a timely manner, and the importance of classifying lymphosarcoma for prognostic purposes and to determine appropriate treatment options. Acknowledgments: Special thanks to Abbie Butler and the staff of the Histology Lab at the University of Georgia for performing immunohistochemical staining.

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cytes in a fisher (Martes pennanti) and a Eurasian otter (Lutra lutra). J Zoo Wildl Med. 2010;41:309–315. 2. Dalton LM, Robeck TR, Young WG. Squamous cell carcinoma on the tongue of an Asian small-clawed otter (Amblonyx cinereus) In: Proc Int Assoc Aquat An Med; 1997. p. 136. 3. Kim JH, Kim BH, Kim JH, Yoo MJ, Kim DY. Lymphosarcoma in a sea otter (Enhydra lutris). J Wildl Dis 2002;38:616–617. 4. Mallouk A, Pham PT, Pham PC. Concurrent FSGS and Hodgkin’s lymphoma: case report and literature review on the link between nephrotic glomerulopathies and hematological malignancies. Clin Exp Nephrol. 2006;10:284–289. 5. O’Brien D, Moore PF, Vernau W, Peauroi JR, Rebhun RB, Rodriguez Jr CO, Skorupski KA. Clinical characteristics and outcome in dogs with splenic marginal zone lymphoma. J Vet Intern Med. 2013;27: 949–954. 6. Sagaert X, Tousseyn T. Marginal zone B-cell lymphomas. Discov Med. 2010;10:79–86. 7. Shimizu K, Ishii G, Nagai K, Yokose T, Ishizawa K, Tamaru J, Yoshida J, Nishimura M, Ochiai A. Extranodal marginal zone B-cell lymphoma of mucosaassociated lymphoid tissue (MALT lymphoma) in the thymus: report of four cases. Jpn J Clin Oncol. 2005;35: 412–416.

8. Stefanello D, Valenti P, Zini E, Comazzi S, Gelain ME, Roccabianca P, Avallone G, Caniatti M, Marconato L. Splenic marginal zone lymphoma in 5 dogs (2001–2008). J Vet Intern Med. 2011;25:90–93. 9. Swenson J, Carpenter JW, Janardhan KS, KetzRiley C, Brinkman E. Paresis in an Asian small clawed otter (Aonyx cinereus) associated with vertebral and ischial osteolysis caused by a malignant lymphangiosarcoma. J Zoo Wildl Med. 2008;39:236–243. 10. Tanaka N, Izawa T, Kashiwagi-Yamamoto E, Kuwamura M, Ozaki M, Nakao T, Yamate J. Primary cerebral T-cell lymphoma in a sea otter (Enhydra lutris). J Vet Med Sci. 2013;75:1667–1669. 11. Valli VE, San Myint M, Barthel A, Bienzle D, Caswell J, Colbatzky F, Durham A, Ehrhart EJ, Johnson Y, Jones C, Kiupel M, Labelle P, Lester S, Miller M, Moore P, Moroff S, Roccabianca P, RamosVara J, Ross A, Scase T, Tvedten H, Vernau W. Classification of canine malignant lymphomas according to the World Health Organization Criteria. Vet Pathol. 2011;48:198–211. 12. Valli VE, Vernau W, de Lorimier LP, Graham PS, Moore PF. Canine indolent nodular lymphoma. Vet Pathol. 2006;43:241–256. Received for publication 27 January 2014