Canine lymphoma (cL) is a common type of neoplasia in dogs with a wide range of clinical presentation, anatomical location, cytology and histopathology features. Though the precise aetiology remains unclear, the environment, habitat and genetic predisposition are stated to be involved. Most of the affected dogs present with multicentric, intermediate to high-grade B-cell metastatic lymphadenopathy, hallmark of the multi-faceted patho-clinical syndrome. Hyper-calcinaemia is characteristic of occasional T cell immunophenotype. Chemotherapy, in combination with doxorubicin based multi-drug regimen, remains the treatment of choice. With regression of malignancy, increased life span of 10-14 months duration is reasonably expected¹.

A male neutered 63.6 kg Swiss Mountain dog,7 years and 9 months old, was presented to the Milford Veterinary Clinic (MVC) on 16.07.2021 for not doing well with the history of lethargy, respiratory distress, and occasional vomiting. The owner noticed a big swelling under the pet’s mandible just 5 days earlier. The patient with pre-existing arthritis and painful hind quarters developed locomotion issues. Physical examination revealed swollen mandibular, prescapular, prefemoral and popliteal lymph nodes. Fine Needle Aspirate (FNA) samples from the mandibular and popliteal lymph nodes for cytology were referred to the diagnostic laboratory. Two view: right lateral (RL) and ventrodorsal (VD) thoracic and abdominal survey radiographs (Fig. 2, 3) were taken in the clinic. The haemogram (Table 1) revealed pronounced lymphocytosis and monocytosis, and the blood chemistry profile (Table 2) markedly increased serum SDMA and ALP titres, with concurrent hypochloraemia, indicating impaired structural and functional patency of the internal organs. Blood-stained residual faecal matter, emitting extremely foul smell (16.07.2021), and marked reduction in the body weight (9.2 kg) within a few weeks time prompted in-depth analysis of the patient’s previous case records. During the process of routine dental cleaning in the clinic (23.11.2020) six small growths in the oral mucosa were detected and surgically excised. The patient was presented with highly painful hind quarters, and compromised locomotion on 01.02.2021, and in the next visit to the clinic on 17.04.2021 hip dysplasia had accentuated. Suitable diet change was advised with oral home medicines comprising joint supplements.

The cytopathology profile of the FNA slides (Fig. 1): lymphocytes with clumped chromatin and scant fine granular basophilic cytoplasm, many of these cells exhibiting indistinct nucleolus. The mitotic figures are very few. Occasional macrophages with phagocytized cell debris, free nuclei, some intact blood cells, lipid droplets, and lympho glandular entities are visible. The case is diagnosed as canine lymphoma with corroborative evidence^2,3.

Polymerase chain reaction for Antigen Receptor Rearrangements Assay (PARR assay) is a highly dependable molecular diagnostic tool in which a specific area of DNA is amplified and separated by size. The targeted area comprises the unique genes, coding for the antigen-binding region in B cells (Ig genes) and T cells (T cell receptor genes). The monoclonal neoplastic lymphocytes have receptor genes with the identical sequence and size. Thus, lymphoma PCR products consistently form a single band. Since the normal/reactive hyperplasia lymphoid tissues contain many lymphocytes with receptors of markedly varying size, PCR products exhibit polyclonal bands on gel electrophoresis. Therefore, PARR assay is being increasingly used to differentiate, with high fidelity, neoplastic transformation from reactive hyperplasia, determination of lineage (B or T cell), staging of lymphoma, and detection of residual disease post-chemotherapy⁴. It also provides useful cues on the concurrent involvement of the haemopoietic system⁵.

The PARR protocol involves sample collection (e.g. air-dried fine needle aspirate cytopathology, FNAC slides from the regional lymph nodes), and the sequential laboratory procedures: extraction and genomic purification of DNA from the cells in the FNAC slide, gently recovered with a scalpel, amplification with appropriate primers and optimized cycles frequency, timing, and temperature to maximize the yield. The PCR products (in suitable buffer, e.g. 1x 100 Tris EDTA) are subjected to polyacrylamide gel electrophoresis (100V, 90 minutes), stained with ethidium bromide dye, andvisualized under UV light. The positive reaction (clonal) is evidenced by a prominent, discrete band. The reaction is negative (polyclonal), if no band or a series of diffuse bands is seen⁶.

In the instant case, PARR assay report on the FNA sample from the lymph nodes: Immunoglobulin gene Clonal; T-cell receptor gene Polyclonal; the results indicate clonally rearranged immunoglobulin gene, pointing to B-cell lymphoma (Referral: Clinical ImmunoPathology Laboratory, Colorado State University, Fort Collins).

Generalized canine lymphoma (cL), involving a wide range of peripheral lymph nodes, accounts for the majority (∼70%) of all lymphoma malignancies in dogs^7,8. Lymphomas may also be present in the extra-nodal formats: with mediastinal, abdominal (gastrointestinal, hepatic splenic, renal), cutaneous, ocular, pulmonary, and CNS involvement¹. Multicentric cL has been classified into five stages, defined by the World Health Organization⁹: limited to a solitary lymph node (stage 1), involving several lymph nodes in the same region(stage II), widespread but not painful lymphadenopathy (stage III), with secondary involvement of the liver and/or spleen (stage IV), or of the haemopoietic system (stage V). Lymphoma phenotype (B-cell, vs. T-cell origin) is a highly dependable prognostic indicator. The diagnostic protocol includes immunochemical staining of the FNA slides/biopsy samples from the affected lymph node(s), PARR test on the collateral slides, and flow cytometry^2,3.

In the instant case, the haemogram (16.07.2021) revealed marked leukocytosis, resulting from the significantly increased % of circulatory lymphocytes and monocytes (Table 1). Monocytosis was apparent in the earlier CBC report (17.04.2021). This observation strongly suggests lymphoma: WHO classification⁹, stage V. The blood chemistry profile (Table 2) showed an exceptionally high SDMA value (32 μg/dl; reference interval 0-14μg/dl), indicating early renal dysfunction. The markedly increased ALP titre indicated impaired hepatic function. The survey radiographs (Fig. 2,3) pointed to metastases in the lungs, liver and other internal organs. With deranged cell homeostasis, the patient’s clinical condition deteriorated exponentially. The patient was presented again on 22.07.2021 with the complaint of remaining off feed. Tarry residual stools (floor drops) were presumably the consequence of bleeding in the upper gastro-intestinal tract. In view of the poor prognosis, the well-informed owner finally opted for the pet’s euthanasia.

Auto cell counter H=High L=Low

Catalyst H= High L=Low

References