Immunophenotyping of intraepithelial lymphocytes in canine chronic enteropathy and intestinal T-cell lymphoma using endoscopic samples.

Human enteropathy-associated T-cell lymphoma (EATL) is considered to be derived from intraepithelial lymphocytes (IELs); however, the origin of canine intestinal T-cell lymphoma (ITCL) remains unclear. Histological, immunohistochemical, and clonality examinations were performed using endoscopically collected canine duodenum samples of mucosal lesions of chronic enteropathy (CE; 73 cases) and ITCL without transmural neoplastic mass lesions (64 cases). Histopathological examinations revealed the intraepithelial accumulation of lymphocytes (called "intraepithelial lymphocytosis") in 54/73 CE cases (74%) and the epitheliotropism of neoplastic lymphocytes in 63/64 ITCL cases (98%). Immunohistochemically, IELs in CE with intraepithelial lymphocytosis (IEL+CE) were diffusely immunopositive for CD3, with scattered immunopositivity for CD5, CD8, CD20, and granzyme B (GRB). The percentage of CD8+ in CD3+ IELs was significantly lower in IEL+CE than in CE without intraepithelial lymphocytosis (IEL-CE). Double-labeling immunohistochemistry revealed a high percentage of GRB expression in CD8- IEL among IEL+CE. Among 64 ITCL cases, CD3 was immunopositive in 64 (100%), CD5 in 22 (34%), CD8 in 8 (13%), CD20 in 12 (19%), CD30 in 13 (20%), and GRB in 49 (77%). In CD3+ cells, Ki67 immunopositivity was highest in ITCL, intermediate in IEL+CE, and lower in IEL-CE. A clonal TCR gene rearrangement was detected in 1/19 IEL-CE cases (5%), 15/54 IEL+CE (28%), and 38/58 ITCL (66%). These results indicate that the immunophenotype of canine ITCL (CD8-GRB+) is similar to that of the increased IELs in CE. The high proliferative activity and clonality of T cells in IEL+CE suggest that canine ITCL originates from these IELs, similar to human EATL.

Atypical multiple myeloma in 3 young dogs.

Three dogs under 12 months old were diagnosed with atypical multiple myeloma (MM), having an aggressive multifocal anaplastic round cell sarcoma in bone marrow, viscera, and/or peripheral blood, which were confirmed by cytology and immunohistochemistry to be of plasma cell origin. The intramedullary sarcomas caused myelophthisis, osteolysis, and hypercalcemia. Complete or free light chain monoclonal gammopathy in the serum and/or urine was demonstrated by protein electrophoresis and immunofixation. The polymerase chain reaction for antigen receptor rearrangement assay performed on 2 cases identified a clonally rearranged immunoglobulin gene. Neoplastic cells lacked expression of CD45, CD3, CD18, CD21, CD34, and MHCII by flow cytometry. Immunohistochemistry revealed MUM1 immunoreactivity of the neoplastic cells. Combining all data, the diagnosis was MM. An aggressive form of MM in young dogs should be a differential diagnosis for patients with an immunoglobulin-productive, B cell-clonal, CD45-negative, MUM1-positive discrete cell neoplasm arising from the bone marrow.

Primary Colorectal Follicular Lymphoma in 3 Dogs.

Primary colorectal follicular lymphomas are rare indolent lymphoid neoplasms in humans that have not been reported in dogs. We describe 3 cases of primary colorectal follicular lymphoma in dogs with histologic and immunohistochemical features similar to their human counterpart. Initial clinical signs in all dogs included tenesmus, hematochezia, and a palpable rectal mass. Two dogs were castrated males and 1 an intact female, between 9 months and 2 years of age, and of varied breeds. All 3 cases of colorectal follicular lymphoma were characterized by proliferation of follicular germinal centers with no polarity or mantle zone and were composed of centrocytes admixed with fewer centroblasts. By immunohistochemistry, lymphoid cells expressed CD20, BCL2, and BCL6 and lacked expression of CD3, CD5, and cyclin D1. Polymerase chain reaction for rearrangements of the immunoglobulin heavy chain confirmed a monoclonal population in all cases. In 2 of the 3 cases, a solitary nodular colorectal mass was excised and appeared curative; however, the third case had multiple colorectal masses and the animal developed multicentric lymphoma. This case series immunohistochemically characterizes and distinguishes colorectal follicular lymphoma from atypical lymphoid hyperplasia.

Coexpression of CD3 and CD20 in Canine Enteropathy-Associated T-cell Lymphoma.

The majority of primary intestinal lymphomas in dogs are T-cell lymphomas, with enteropathy-associated T-cell lymphoma (EATL) large cell type (type 1) being the most common. While most T-cell lymphomas express the T-cell marker CD3, there is increasing evidence that some human and canine T-cell lymphomas coexpress the B-cell marker CD20. We describe 3 cases of CD3+, CD20+, Pax5- EATL type 1 in dogs. All 3 cases had clonal rearrangement of T-cell receptor gamma. Initial clinical signs included weight loss, inappetence, diarrhea, and/or vomiting. The mean age was 9 years (range 3-12). Survival was highly variable ranging from 20 days to longer than 1.6 years. Considering the different chemotherapeutic response of T-cell versus B-cell lymphomas, accurate diagnosis of lymphomas coexpressing CD3 and CD20 as EATL type 1 based on histologic features and clonality results is important. Regardless, the clinical and/or prognostic significance of neoplastic T cells expressing CD20 is unclear.

A Novel and Likely Inherited Lymphoproliferative Disease in British Shorthair Kittens.

An unusual lymphoproliferative disease was identified in multiple closely related British Shorthair (BSH) kittens, suggesting an inherited predisposition to disease. Affected kittens typically developed rapidly progressive and marked generalized lymphadenopathy, moderate splenomegaly, and regenerative and likely hemolytic anemia from 6 weeks of age. Microscopic findings were suggestive of multicentric T-cell lymphoma, but additional testing revealed a polyclonal population of CD3+/CD4-/CD8- "double negative" T cells (DNT cells). This is a novel disease presentation with similarities to the human disorder autoimmune lymphoproliferative syndrome (ALPS), a rare inherited disease causing lymphoproliferation and variable manifestations of autoimmunity. The human disease is most commonly due to the presence of Fas gene mutations causing defective lymphocyte apoptosis, and further investigations of both the mode of inheritance and genetic basis for disease in affected cats are currently in progress.

Distinguishing Intestinal Lymphoma From Inflammatory Bowel Disease in Canine Duodenal Endoscopic Biopsy Samples.

Inflammatory bowel disease (IBD) and intestinal lymphoma are intestinal disorders in dogs, both causing similar chronic digestive signs, although with a different prognosis and different treatment requirements. Differentiation between these 2 conditions is based on histopathologic evaluation of intestinal biopsies. However, an accurate diagnosis is often difficult based on histology alone, especially when only endoscopic biopsies are available to differentiate IBD from enteropathy-associated T-cell lymphoma (EATL) type 2, a small cell lymphoma. The purpose of this study was to evaluate the utility of histopathology; immunohistochemistry (IHC) for CD3, CD20, and Ki-67; and polymerase chain reaction (PCR) for antigen receptor rearrangement (T-cell clonality) in the differential diagnosis of severe IBD vs intestinal lymphoma. Endoscopic biopsies from 32 dogs with severe IBD or intestinal lymphoma were evaluated. The original diagnosis was based on microscopic examination of hematoxylin and eosin (HE)-stained sections alone followed by a second evaluation using morphology in association with IHC for CD3 and CD20 and a third evaluation using PCR for clonality. Our results show that, in contrast to feline intestinal lymphomas, 6 of 8 canine small intestinal lymphomas were EATL type 1 (large cell) lymphomas. EATL type 2 was uncommon. Regardless, in dogs, intraepithelial lymphocytes were not an important diagnostic feature to differentiate IBD from EATL as confirmed by PCR. EATL type 1 had a significantly higher Ki-67 index than did EATL type 2 or IBD cases. Based on the results of this study, a stepwise diagnostic approach using histology as the first step, followed by immunophenotyping and determining the Ki67 index and finally PCR for clonality, improves the accuracy of distinguishing intestinal lymphoma from IBD in dogs.

Mott Cell Differentiation in Canine Multicentric B Cell Lymphoma with Cross-Lineage Rearrangement and Lineage Infidelity in a Dog.

Lymphoma is a severe condition characterized by the proliferation of neoplastic lymphoid cells. A 4-year-old female mongrel dog presented with solitary lymph node enlargement. Significant right prescapular lymphadenopathy and abdominal enlargement were observed during physical examination. A complete blood count revealed lymphocytosis, and a peripheral blood smear revealed lymphoblastosis and Mott cells. Fine needle aspiration cytology (FNAC) of the right prescapular lymph node revealed a predominant population of lymphoblasts and Mott cells. Based on the FNAC and blood smear results, the patient was diagnosed with leukemic state multicentric B-cell lymphoma with Mott cell differentiation. Subsequent PCR for antigen receptor rearrangement and flow cytometry revealed that the patient exhibited cross-lineage rearrangement (CLRA) and lineage infidelity (LI), respectively. CHOP-based chemotherapy was initiated, however, the patient's disease was progressive. The patient died three months after the initial presentation. Mott cell differentiation in canine B-cell lymphoma (MCL) has rarely been reported in the veterinary literature and seems to show an unusual clinical course. To the best of our knowledge, no reports of MCL with CLRA and LI exist. We report the clinical features, diagnosis, and treatment of MCL with CLRA and LI.

Polymerase chain reaction for antigen receptor rearrangement: Benchmarking performance of a lymphoid clonality assay in diverse canine sample types.

BACKGROUND: Polymerase chain reaction for antigen receptor rearrangement (PARR) is a molecular diagnostic tool used for discrimination of lymphoid malignancies in dogs from benign processes. Assay variations have been described and are commercially available, but performance metrics are not uniformly reported. OBJECTIVES: To describe performance (accuracy, sensitivity, specificity) and rigorous benchmarking of a PARR protocol (ePARR) in clinically relevant samples. ANIMALS: One hundred eighty-one client-owned dogs. METHODS: Lymphoma and benign tissues representative of the clinical spectrum with gold standard histopathologic and immunohistochemical diagnoses were collected. Assay development and benchmarking were performed on fresh frozen (FF) tissue, formalin-fixed paraffin-embedded (FFPE) tissue, flow cytometry pellets, and air-dried fine-needle aspirates (FNA). Assay performance was determined for FFPE from 56 dogs (18 B-cell lymphoma, 24 T-cell lymphoma, and 14 non-lymphoma), 80 frozen flow cytometry pellets (66 B-cell lymphoma, 14 T-cell lymphoma, 0 non-lymphoma), and 41 air-dried FNA slides (23 lymphoma, 18 non-lymphoma). RESULTS: For discrimination of lymphoma versus non-lymphoma, ePARR had 92% and 92% sensitivity and specificity on FFPE with 92% accuracy, 85% sensitivity from flow cytometry pellets (non-lymphoma was not evaluated to calculate specificity) with 85% accuracy, and 100% and 100% sensitivity and specificity for FNA with 100% accuracy. Stringent quality control criteria decreased assay success rate without significant performance improvement. Performance metrics were lower in most cases for discrimination of B- or T-cell versus non-B- or non-T-cell samples than for lymphoma versus non-lymphoma. CONCLUSIONS AND CLINICAL IMPORTANCE: These benchmarking data facilitate effective interpretation and application of PARR assays in multiple sample types.