A case of T-cell chronic lymphocytic leukemia progressing to Richter syndrome with central nervous system involvement in a dog

An 8-year-old neutered Beagle dog was presented with polyuria and polydipsia. Routine clinicopathologic testing showed a significant lymphocytosis and proteinuria. Lymphocytes were of small to intermediate in size with a mature morphology. Infectious disease screening was negative. PCR for antigen receptor gene rearrangements showed a clonal T-cell receptor (TCR) rearrangement consistent with T-cell chronic lymphocytic leukemia (CLL). Bone marrow cytology showed <30% lymphocytes, while the proportion in splenic fine-needle aspirate cytology was considered increased. The dog was initially monitored but started on prednisolone and chlorambucil therapy 2 months later due to worsening clinical signs and progressive lymphocytosis. After an additional 2 weeks, the dog developed multifocal spinal pain and single-node lymphadenomegaly. Cytology of the lymph node showed a monomorphic population of large lymphoblasts consistent with lymphoma. Cytology of a cerebrospinal fluid sample also showed large lymphoblasts. PCR for antigen receptor gene rearrangement at both sites showed a clonal TCR rearrangement of the same molecular size as in the initial leukemic cells. The dog was diagnosed with a transformation of the CLL to Richter syndrome (RS) with involvement of the central nervous system (CNS). Therapy was started with L-asparaginase and an increased dose of prednisolone; however, the dog was euthanized due to progressive clinical signs. To our knowledge, this is the first report of canine RS with direct involvement of the CNS.     

B‐cell intestinal lymphoma with Mott cell differentiation in a 1‐year‐old miniature Dachshund

Abstract: A 1‐year‐old intact female miniature Dachshund was presented with hematochezia, vomiting, and diarrhea of more than 1‐week duration. An abdominal mass was palpated, which at exploratory surgery was found to be a 7‐cm‐long thickened section of ileum. The thickened ileum was resected. Impression smears revealed numerous small‐ to medium‐sized lymphocytes, with a smaller number of cells resembling Mott cells. The Mott‐like cells contained multiple pale vacuoles that were positive for periodic acid‐Schiff (PAS) in wet‐fixed smears, consistent with Russell bodies. Histologic evaluation of the surgically excised ileum revealed 2 populations of neoplastic lymphoid cells. The majority were uniform medium‐sized lymphocytes with hyperchromatic oval or round nuclei and inconspicuous nucleoli. The remaining cells resembled Mott cells, which contained several PAS‐positive eosinophilic globules in the cytoplasm, occasionally compressing the nucleus. The majority of neoplastic cells stained positively for vimentin, CD20, CD79a, and Pax‐5, but were negative for CD3 and lysozyme; 43.5% of cells stained positively for Ki‐67. The Mott cells were strongly positive for immunoglobulin but were negative for Pax‐5. Using electron microscopy, a homogenous substance of intermediate electron density was observed frequently in the cisternae of rough endoplasmic reticulum in the cytoplasm of the Mott cells, and rarely in the perinuclear cisternae of the lymphoid cells, corresponding to the site of immunoglobulin staining. Monoclonal rearrangement of immunoglobulin heavy‐chain (IgH) gene was observed by PCR testing for lymphocyte–antigen receptor rearrangement. The morphologic features, immunophenotype, and IgH gene rearrangement verified the lymphoid cells were neoplastic (mature cell type) and had a B‐cell phenotype, with evidence of immunoglobulin production and differentiation into Mott cells. This case was unusual because of the age of the dog and because most intestinal lymphomas are T‐cell phenotype. The Mott cell morphology also differed from typical mature B‐cell lymphoma types and may be a unique B‐cell lymphoma variant.     

Tumor staging in a Beagle dog with concomitant large B-cell lymphoma and T-cell acute lymphoblastic leukemia

An 8-y-old spayed female Beagle dog was presented with peripheral lymphadenomegaly. Lymph node cytology and flow cytometry led to the diagnosis of large B-cell lymphoma (LBCL). We detected minimal percentages of LBCL cells in peripheral blood and bone marrow samples. However, a monomorphic population of neoplastic cells different from those found in the lymph node was found in the bone marrow. T-cell acute lymphoblastic leukemia was suspected based on flow cytometric immunophenotyping. PCR for antigen receptor rearrangement (PARR) revealed clonal rearrangement of both B-cell and T-cell receptors, and the presence of both neoplastic clones in the lymph node, peripheral blood, and bone marrow. The dog was treated with multi-agent chemotherapy but died 46 d following diagnosis. Tumor staging and patient classification are needed to accurately establish a prognosis and select the most appropriate therapeutic protocol.     

Feline large granular lymphocyte lymphoma: An Italian Society of Veterinary Oncology (SIONCOV) retrospective study

Feline large granular lymphocyte (LGL) lymphoma is an uncommon subtype of lymphoma characterized by a grave prognosis and scarce response to chemotherapy. There are limited reports on clinico‐pathological and prognostic factors. One‐hundred and 9 cats with newly diagnosed LGL lymphoma that underwent initial staging (including hematology, serum biochemistry, thoracic radiographs and abdominal ultrasound), and followed‐up were retrospectively evaluated. LGL lymphoma was localized within the gastrointestinal tract with or without extra‐intestinal involvement in 91.7% of the cases, and at extra‐gastrointestinal sites in 8.3%. Symptoms were frequent. Anemia (31.2%) and neutrophilia (26.6%) were commonly observed, and 14 (12.8%) cats had neoplastic circulating cells. Frequent biochemistry abnormalities included elevated ALT (39.4%) and hypoalbuminemia (28.4%). Twenty (54.1%) of 37 cats had elevated serum LDH. Treatment varied among cats, and included surgery (11%), chemotherapy (23%), corticosteroids (38.5%) and no treatment (27.5%). Median time to progression (MTTP) was 5 days, and median survival time (MST) 21 days. MST was significantly shorter in the case of substage b, circulating neoplastic cells, lack of chemotherapy administration, and lack of treatment response. A small subset of cats (7.3%) survived more than 6 months, suggesting that a more favorable clinical course can be found among LGL lymphoma patients.     

Detection of monoclonality in intestinal lymphoma with polymerase chain reaction for antigen receptor gene rearrangement analysis to differentiate from enteritis in dogs

The diagnosis of canine intestinal lymphoma by morphological examination is challenging, especially when endoscopic tissue specimens are used. The utility of detection of antigen receptor gene rearrangement by polymerase chain reaction (PARR) in canine lymphoma has been well established, but its usefulness to distinguish enteritis and intestinal lymphoma remains unclear. In this retrospective study we assessed clonality of 29 primary canine intestinal lymphoma, 14 enteritis and 15 healthy control cases by PARR analysis, using formalin‐fixed, paraffin‐embedded full‐thickness tissue specimens. We could detect monoclonal rearrangements in 22 of 29 canine intestinal lymphomas [76%; 95% confidence interval (CI) 56–90%] and polyclonal rearrangements in all of the enteritis and healthy control cases (100%; CI 88–100%). We revealed a predominance of T‐cell phenotype compared to B‐cell phenotype (85%; CI 65–96% and 15%; CI 4–35%, respectively). We showed that PARR analysis contributes to differentiation of canine intestinal lymphoma from enteritis and to phenotyping of lymphomas.     

Erythrophagocytic low‐grade extranodal T‐cell lymphoma in a cat

Abstract: A 13‐year‐old male castrated domestic shorthair cat was presented to the referring veterinarian with a 2‐month history of weight loss and lethargy. Splenomegaly, hepatomegaly, nonregenerative anemia, neutropenia, and hyperbilirubinemia were noted. Results of testing for feline immunodeficiency virus, feline leukemia virus, Toxoplasma gondii, and Mycoplasma sp. were negative. On cytologic examination of aspirates from the enlarged spleen and liver, a population of erythrophagocytic round cells was observed. Splenectomy and a liver biopsy were done which revealed a population of CD3+/CD79a– erythrophagocytic mononuclear round cells localized in the hepatic and splenic sinusoids. T‐cell PARR (PCR for antigen receptor gene rearrangements) analysis of bone marrow and spleen demonstrated a single band indicative of a clonal proliferation of T cells. Based on the marked splenomegaly, sinusoidal infiltration, lack of lymphadenopathy, and results of cytology, PARR, and immunophenotyping, a diagnosis of low‐grade extranodal T‐cell lymphoma was made. The cat was treated with chlorambucil and prednisolone; clinical and laboratory abnormalities resolved and the cat has remained clinically normal for 2.5 years. To our knowledge, this report documents the first case of an erythrophagocytic T‐cell lymphoma in a cat. The clinicopathologic findings were suggestive of hepatosplenic T‐cell lymphoma, a neoplasm described previously only in humans and dogs.     

Malignant transformation of T-cell large granular lymphocyte leukemia in a dog

An 8-year-old spayed female Golden Retriever was referred to us for evaluation of mild lymphocytosis. The peripheral lymphocytes were comprised of mostly large granular lymphocytes (LGLs), and flow cytometry showed that they were mostly CD3+8+ T lymphocytes. Clonal rearrangement of the T-cell receptor gene was identified in the peripheral blood, and the dog was therefore diagnosed with LGL chronic leukemia. The dog was subclinical without treatment until hospitalization on day 154, at which point the lymphocytes looked like lymphoblasts and the surface markers changed to CD3-8-. This was regarded as malignant transformation from LGL chronic leukemia to the acute type. Sequential chemotherapy was started, but the dog died on day 190. Necropsy revealed tumor cell infiltration into the heart, skin, and brain.     

Histopathologic features, immunophenotyping, clonality, and eubacterial fluorescence in situ hybridization in cats with lymphocytic cholangitis/cholangiohepatitis

Feline lymphocytic cholangitis is a poorly characterized disease complex with respect to histologic lesions, immunophenotype, and etiopathogenesis. Seventy-eight cases of feline lymphocytic cholangitis (n = 51) and feline hepatic lymphoma (n = 27) were reviewed using standardized histopathology, immunophenotyping (B cell and T cell), polymerase chain reaction for T-cell receptor (TCR) gene rearrangement, and fluorescence in situ hybridization (FISH) for eubacteria. Five histopathologic features in cases of lymphocytic cholangitis assisted in its differentiation from hepatic lymphoma: bile duct targeting (n = 32, 62.7%), ductopenia (n = 9, 17.6%), peribiliary fibrosis (n = 37, 72.5%), portal B-cell aggregates (n = 36, 70.6%), and portal lipogranulomas (n = 38, 74.5%). The majority of lymphocytic cholangitis cases (n = 35, 68.6%) were T cell predominant; 15 (29.4%) had an equal mix of B cells and T cells, and 1 (1.9%) had a B cell-predominant infiltrate; 66.6% of hepatic lymphoma cases were T-cell lymphomas. TCR clonality results were unexpected, with 17.1% of cases of lymphocytic cholangitis having clonal or oligoclonal populations and with T-cell lymphomas having variable TCR clonality (63.6% clonal or oligoclonal, 36.3% polyclonal). The majority of lymphocytic cholangitis (n = 32 of 36, 88.8%) and all hepatic lymphoma cases had no detectable eubacteria using FISH. As demonstrated here, bile duct targeting, ductopenia, peribiliary fibrosis, portal B-cell aggregates, and portal lipogranulomas are lymphocytic cholangitis features that, along with polyclonal TCR (83%), help differentiate it from hepatic lymphoma. No strong evidence was found implicating in situ bacterial colonization as an etiopathogenesis of lymphocytic cholangitis.     

Characterization of a PCR‐based lymphocyte clonality assay as a complementary tool for the diagnosis of feline lymphoma

Differentiation between resident mature lymphocyte populations and small cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR‐based immunoglobulin (IG) and T‐cell receptor (TCR) clonality testing for confirmation. In this prospective study, diagnostic sensitivity and specificity of different primer sets for routine diagnosis of feline TCR gamma (TCRG) and complete IG heavy chain (IGH) gene rearrangements were assessed. Fine needle aspirates from 20 feline lymphoma cases and lymph node material from 10 cats without hematopoietic neoplasia were subjected to clonality testing. Feline lymphoma cell lines and previously confirmed patient material served as positive control. Detection limits for clonal populations within a polyclonal background was 90% for B‐cells and 50% for T‐cells. Diagnostic sensitivity and specificity of the clonality assay were 70% and 90%. Overall diagnostic accuracy was 77%, positive predictive value 93% and negative predictive value 60%.     

Melting curve analysis in canine lymphoma by calculating maximum fluorescence decrease

PARR is widely used in the diagnostics of canine lymphoma. In human and veterinary medicine, melting curve analysis (MCA) has successfully been introduced to facilitate the process. Since visual interpretation of melting curves can be rather subjective, the purpose of this study was to develop an objective interpretation of melting curves by calculating the maximum fluorescence decrease (dF_max) within a defined rise of temperature. Lymph node aspirates and blood of 34 dogs with lymphoma and 28 control dogs were tested. 27/34 lymphoma cases were correctly detected to be monoclonal (sensitivity 79%). 2/28 control dogs showed a monoclonal rearrangement (specificity 93%). B‐ and T‐cell neoplasia were still detectable using DNA amount as low as 10 ng. In serial dilutions of tumor DNA with DNA of normal tonsils, the detection limit was 25% for B‐cell lymphomas and 100% for T‐cell lymphoma, suggesting that PCR conditions could still be optimized.     

The Utility of PCR for Antigen Receptor Rearrangement (PARR) in Staging and Predicting Prognosis in Canine Lymphoma

Introduction:  In lymphoid neoplasia, molecular assays to confirm clonality rely on the fact that lymphoid cells normally contain DNA regions that are unique in sequence, resulting from recombination of the V, D, and J genes. Based on the belief that malignancies are clonal, the presence of a single, clonal rearrangement can indicate neoplasia. In humans with lymphoid malignancies, PCR for antigen receptor rearrangement has been studied as a molecular staging test and a therapeutic monitoring tool. The purpose of this study was to determine the utility of PARR for molecular staging and predicting prognosis in canine lymphoma.
Methods:  Patients with lymphoma who had complete clinical staging were included in the study. All had PARR of pre treatment lymph node, blood and/or bone marrow. All were assigned a clinical stage (WHO 1–5) and a PCR stage (PS) where 0 indicated no clonal expansion in the blood, and 5 indicated the presence of a clonal expansion. In addition to stage, other factors that could affect prognosis were evaluated.
Results:  Eighty six patients were included. Clinical stage was distributed as follows: 3 = 44, 4 = 21, and 5 = 21. Sixty two patients had clonal rearrangements detected in the lymph node and were able to be PCR staged. Seventeen were PS 0 and 45 were PS 5. PCR stage was not prognostic for DFI or survival.
Conclusion:  PARR is sensitive at detecting blood/bone marrow infiltration in canine lymphoma. It is not a useful prognostic tool in the diverse population in this study.     

Increased frequency of CD45 negative T cells (T zone cells) in older Golden retriever dogs

T zone lymphoma (TZL) is characterized by the clonal expansion of T cells lacking expression of the pan‐leukocyte antigen CD45 (TZ cells). A strong breed predisposition is observed in Golden retrievers. This study aimed to confirm aberrant CD45 mRNA expression and determine if Golden retrievers without clinical lymphoma have an increased frequency of circulating TZ cells. Gene expression analysis on confirmed TZL cases showed a significant decrease in CD45 expression compared to normal dogs. Peripheral blood samples from senior dogs, 242 Golden retrievers and 42 non‐Golden retrievers, without evidence of lymphoproliferative disease were assessed for the presence of TZ cells by flow cytometry. Thirty‐one percent of Golden retrievers had TZ cells compared to 14% of non‐Golden retrievers. Thirty‐four percent of Golden retrievers with TZ cells had a clonal T cell receptor gamma (TRG) gene rearrangement. Interestingly, 20% of Golden retrievers without TZ cells also had a clonal TRG rearrangement. Golden retrievers may have an increased risk of TZL due to an increased frequency of TZ cells.     

Cutaneous T‐cell lymphoma – Sézary syndrome in a Boxer

A 7‐year‐old male Boxer with a 3.5‐year history of atopy and food hypersensitivity was presented with multiple poorly circumscribed nodules and maculae of the skin and tongue, and jaundiced mucosal membranes. Cytologic and histopathologic examination of the skin lesions revealed cutaneous epitheliotropic lymphoma. Cells were CD3⁺ and CD8⁺ in flow cytometry. The CBC showed a moderate leukocytosis with 16% atypical lymphocytes with irregularly cleaved nuclei. Flow cytometric phenotyping of peripheral blood showed an elevated proportion of the CD8⁺ T‐lymphocyte subpopulation, indicating a malignant population of T‐cell origin, and the electropherogram of the PCR antigen receptor rearrangement produced a monoclonal peak for TCRγ. Liver enzyme activities were markedly increased and abdominal ultrasound examination showed increased echogenicity of the liver and enlarged abdominal lymph nodes. Fine‐needle aspirates of the liver confirmed infiltration with lymphocytes exhibiting the same morphology as the cells detected in skin and peripheral blood. Treatment was induced with L‐asparaginase, lomustine, and prednisone. Partial clinical remission of the skin and tongue lesions was achieved within 10 days, and hematologic abnormalities resolved. Despite further treatment with L‐asparaginase and lomustine, the dog relapsed within one month and was euthanized. Presence of malignant lymphocytes in skin, peripheral blood, and liver indicate a rare variant of leukemic cutaneous T‐cell lymphoma, equivalent of Sézary syndrome in a dog. This case report describes the use of flow cytometry as a complementary tool for lymphocyte characterization of skin lesions for the first time.     

Disseminated lymphoma with large granular lymphocyte morphology diagnosed in a horse via abdominal fluid and transtracheal wash cytology

A 22‐year‐old Tennessee Walking Horse mare was presented to the Auburn University Large Animal Teaching Hospital with a 3‐day history of lethargy, anorexia, and mild signs of colic. The mare had a several‐month history of weight loss and refractory cough. Physical examination revealed an increased respiratory rate, and crackles and wheezes were heard on thoracic auscultation. Thoracic ultrasonographic examination showed disseminated, minor, bilateral comet tail‐like lesions on the parietal pleural surfaces. Abdominal ultrasonographic examination was unremarkable. Trans‐rectal palpation revealed a firm small colon impaction with concomitant diarrhea. Laboratory data were characterized by a very pronounced acute inflammatory leukogram with severe neutropenia and significant left shift, evidence of hepatocellular damage/necrosis, cholestasis, and possibly mixed metabolic alkalosis and acidosis. On cytologic evaluation of a peritoneal fluid sample, there were many large granular lymphocytes (LGL). Large numbers of LGL were also observed on cytologic examination of a subsequent transtracheal wash. The final cytologic interpretation was disseminated lymphoma with LGL morphology. Due to worsening of the clinical signs and poor prognosis, the mare was euthanized. On necropsy and in histopathologic examination, disseminated lymphoma with LGL morphology was noted in a mesenteric lymph node, lungs, liver, spleen, kidneys, and right dorsal colon. Lymphoma with LGL morphology is rarely diagnosed in the horse. This report provides unique cytologic findings of a case of disseminated lymphoma with LGL morphology in a horse, confirmed with histopathologic evaluation.     

B‐cell lymphoma with Mott cell differentiation in two young adult dogs

Abstract: Two young adult dogs with gastrointestinal signs were each found to have an intra‐abdominal mass based on physical examination and diagnostic imaging. On exploratory laparotomy, small intestinal masses and mesenteric lymphadenopathy were found in both dogs; a liver mass was also found in dog 1. Cytologic and histologic examination of intestinal and liver masses and mesenteric lymph nodes revealed 2 distinct lymphoid cell populations: lymphoblasts and atypical Mott cells. With Romanowsky stains, the atypical Mott cells contained many discrete, clear to pale blue cytoplasmic inclusions consistent with Russell bodies that were positive by immunohistochemistry for IgM and CD79a in both dogs and for IgG in dog 2. The Mott cells and occasional lymphoblasts stained strongly positive with periodic acid‐Schiff. Using flow cytometric immunophenotyping in dog 1, 60% of peripheral blood mononuclear cells and 85% of cells in an affected lymph node were positive for CD21, CD79a, IgM, and MCH II, indicative of B‐cells. With electron microscopy, disorganized and dilated endoplasmic reticulum was seen in Mott cells in tumors from both dogs. Antigen receptor gene rearrangement analysis of lymph node and intestinal masses indicated a clonal B‐cell population. Based on cell morphology, tissue involvement, and evidence for clonal B‐cell proliferation, we diagnosed neoplasms involving Mott cells. To the authors' knowledge, this is the second report of Mott cell tumors or, more appropriately, B‐cell lymphoma with Mott cell differentiation, in dogs. More complete characterization of this neoplasm requires further investigation of additional cases. This lymphoproliferative disease should be considered as a differential diagnosis for canine gastrointestinal tumors.     

Large granular lymphocyte lymphoma in the skin and urinary bladder of a dog

A 10-year-old female Cavalier King Charles Spaniel presented with hematuria, pollakiuria and skin rash. Based on the histopathological and cytological examination of the skin and bladder mucosa, the dog was diagnosed with large granular lymphocytic (LGL) lymphoma of the bladder and skin. The dog responded well to the initial chemotherapy with nimustine for 3 months. Since recurrence of skin erosion and bladder wall thickening were observed, the dog was subsequently administered chemotherapy with other anticancer drugs, including chlorambucil, vincristine, doxorubicin, L-asparaginase, cytosine arabinoside, and cyclophosphamide. The dog survived for 11 months and died due to tumor-related disseminated intravascular coagulation. This is the first report of a canine case of LGL lymphoma in the skin and bladder.     

Gamma delta T‐cell large granular lymphocyte lymphoma in a dog

A 2‐year and 6‐month‐old female neutered Labrador Retriever with Horner syndrome, megaesophagus, and a mediastinal mass was referred to the Queen Mother Hospital for Animals of the Royal Veterinary College. A large granular lymphocyte (LGL) lymphoma was diagnosed on cytology; flow cytometric analysis revealed a γδ T‐cell phenotype (CD3+, CD5+, CD45+, TCRγδ+, CD4?, CD8?, CD34?, CD21?). Chemotherapy was started with a combination of lomustine, vincristine, procarbazine, and prednisolone, followed by bleyomicin. Euthanasia was elected by the owners, due to progressive deterioration and lack of quality of life, 28 days after diagnosis. This is the first cytologic and immunophenotypic characterization of a canine γδ T‐cell lymphoma with LGL morphology and probably of mediastinal origin. The role of chemotherapy in delaying the disease progression remains unknown.     

Cervical thymoma originating in ectopic thymic tissue in a cat

Abstract: An 11‐year‐old female spayed domestic shorthair cat was referred to The Ohio State University Veterinary Teaching Hospital (OSU‐VTH) for evaluation of a 6 × 4 × 3.5 cm mass in the left midcervical region causing increased respiratory sounds and lateral deviation of the trachea. A fine needle aspirate of the mass was obtained before referral and the cytology results were compatible with a reactive lymph node. Immunocytochemistry showed increased numbers of CD3+ T lymphocytes and small numbers of CD20+ and CD79a+ medium to large lymphocytes. Differential diagnoses from the referral pathologist were T‐cell‐rich B‐cell lymphoma and feline Hodgkin's‐like lymphoma. A subsequent fine needle aspirate performed at the OSU‐VTH showed similar results. On flow cytometry the majority of cells were CD3+ T lymphocytes that were double positive for CD4 and CD8 (73%), compatible with either a double‐positive (CD4+CD8+) T‐cell lymphoma or lymphocytes from ectopic thymic tissue. The mass was surgically removed. Histopathology and immunohistochemistry of the mass revealed a predominant population of CD3+ small lymphocytes and small numbers of medium to large lymphocytes with moderate anisocytosis and anysokaryosis. A population of cytokeratin‐positive epithelial cells surrounded small microcystic structures filled with eosinophilic material and structures interpreted as Hassall's corpuscles. These findings were consistent with thymic tissue and a diagnosis of ectopic thymoma was made. PCR results for lymphocyte antigen receptor rearrangement (PARR) were negative. The cat had no evidence of disease 16 months after removal of the mass. To our knowledge this is the first report of an ectopic cervical thymoma in a cat. The clinical and diagnostic features of this unusual case will be useful in helping veterinarians and pathologists obtain a presurgical diagnosis and establish a prognosis for similar lesions.