Abstract

This report details the diagnosis of lymphoblastic leukemia with suspected B-cell differentiation in a diamondback terrapin (Malaclemys terrapin). There are only nine reports of hemic tumors in turtles.^1-5 Antineoplastic treatment was not attempted in any of the cases. A thorough diagnostic investigation was performed to evaluate the extent of the disease, as this is the first reported case of neoplasia in a diamondback terrapin (J. Harshbarger, personal communication). The diagnostic procedures performed were a complete blood count (CBC), serum chemistry, routine blood films, a bone marrow biopsy, radiographs, immunocytochemistry to type the leukemic cell line, magnetic resonance imaging (MRI), and computed tomography (CT). An antineoplastic treatment protocol of prednisolone, cytosine arabinoside, and chlorambucil was devised (CGC). This abstract describes the investigation of the leukemic disease in the patient, elaborating on the use of immunocytochemical staining as part of the diagnostic procedures that were performed.

The diagnosis of lymphoblastic leukemia was made based on cytologic examination of blood films that revealed a preponderance of large mononuclear cells with intense cytoplasmic basophilia; increased nuclear:cytoplasmic ratio; mild anisokaryosis; round, cleaved, or lobulated nuclei; and rare bi-nucleation. Small well-differentiated lymphocytes, heterophils, basophils, and thrombocytes were scant. Occasional immature erythrocytic cells were also detected. Heterophils occasionally were immature.

Leukemic involvement of the extra-sinusoidal regions of the bone marrow was detected histopathologically. Increased osteoclastic activity was present in some areas of the trabecular bone. Extra sinusoidal myeloid cells and intrasinusoidal erythroid cells were scant. Fragmentation of the samples (attributed to collection and processing artifact) obscured architectural detail in some areas.

Immunocytochemistry is a technique which is able to determine the cell of origin by detecting various membrane and cytoplasmic antigens. The specific set of antigens expressed by a cell population constitutes a unique phenotype. This technique has been applied to a variety of animal species, including reptiles.⁵ The BLA.36 antigen is known to stain a subset of B-cells in humans and has been shown to stain cells in many mammals which correspond to CD79a positive cells, a component of the B-cell receptor. In addition, one of the authors has shown that BLA.36 stains similar B-cell populations in several reptiles (Wojcieszyn, unpublished observations). Results of immunocytochemical staining revealed that the neoplastic cells were positive for the BLA.36 antigen, demonstrating a 3–4+ membrane stain. The neoplastic cells were negative for CD3 antigen, although few circulating small well-differentiated lymphocytes were positive for this marker.

The antineoplastic treatment consisted of prednisone 2.5 mg PO given every 48 hours with cytosine arabinoside 5 mg SC and chlorambucil 1 mg PO given 7 days apart (Table 1; T=0, T=7 days). The patient’s response to the therapy was monitored with a weekly CBC, chemistry, and cytology. The white blood cell (WBC) estimate underwent a steady decline during the first 24 days of treatment. At the start of therapy the WBC estimate was 200 Thd/CMM (reference range 3–8 Thd/CMM), while on day 24 the WBC estimate reached a nadir of 10 Thd/CMM. There were no mitotic figures observed on the cytologic examination performed on day 24. Concurrent increases in blood urea nitrogen (BUN), AST (aspartate transferase), and creatinine phosphokinase (CPK) were observed during the course of treatment. These elevations were likely due to the by-product of the tumor lysis. Tophaceous gout was diagnosed prior to the start of chemotherapy, and was treated with a regime of probenecid, colchicine, and allopurinol.

Table 1. Hematologic parameters of anticancer therapy in a diamondback terrapin.

The anticancer therapy achieved a survival of 46 days. At necropsy a generalized lymphoma and lymphoid leukemia were diagnosed. Acute myocardial necrosis and focal pericardial fibrosis were present along with moderate renal tubular gout. Areas of tumor necrosis were observed, indicating a treatment effect; however, a toxemia with resultant myocardial necrosis and disseminated intravascular coagulation may have occurred from the drug therapy. There was no evidence that the necrotizing event had an effect on the myeloid cell line.

Acknowledgments

The author thanks David Brinker, DVM for financial support and the commitment of the staff of Todds Lane Veterinary Hospital for their assistance. Natalie Antinoff, DVM, Gulf Coast Veterinary Specialists, Houston, TX, for consultation. Michael Katz, MD, Children’s Hospital of the Kings Daughters, Norfolk, VA, for diagnostic imaging services. Greg Lewbart, DVM North Carolina State University College of Veterinary Medicine for resources. Doug Mader, DVM, Marathon Animal Hospital for repeated consultation. Doug Thamm, VMD, University of Wisconsin Madison for consultation. Helmunth Trieshmann, MD, Beth Fairbanks, RT-R, & Bill Hart, RT-R, Orthopaedic Surgery and Sports Medicine Specialists, Newport News, VA, for diagnostic imaging services.

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