B‐cell lymphoma with Mott cell differentiation in a cat

A 12‐year‐old, male castrated Domestic Shorthair cat was presented to Animal Medical Center of Gifu Univeristy with anorexia and vomiting. Physical examination revealed an enlarged left tonsil and right mandibular lymph node (approximately 2–3× the normal size), and a submucosal mass on the right side of the epiglottis (1.5 × 2.0 cm). On computed tomography images, an enlarged left tonsil, and enlarged right mandibular, right pharyngeal, and left and right cervical lymph nodes were observed. Cytologic examination of smears of tonsil and lymph nodes revealed numerous medium‐ to large‐sized neoplastic lymphoid cells, approximately half of which contained one or several light‐blue homogenous globoid cytoplasmic inclusions (5–10 μm), which stained magenta with periodic acid–Schiff (PAS) stain. Histopathologic examination of the left tonsil revealed diffuse proliferation of medium‐ to large‐sized neoplastic lymphoid cells effacing the original lymphoid architecture. Half of the cells contained one or several eosinophilic globoid cytoplasmic inclusions, which stained magenta with PAS and showed positive immunohistochemical reactions for immunoglobulin M (IgM) and λ light chain. Neoplastic lymphoid cells were also CD20⁺, Pax5⁺, and MUM1⁺, and CD3^?. Thus, the neoplastic lymphoid cells expressed a B‐cell immunophenotype, and the globoid cytoplasmic inclusions represented an aberrant IgM λ light chain accumulation, similar to Russell bodies. B‐cell lymphoma with Mott cell differentiation was diagnosed based on cytologic, histopathologic, and immunohistochemical features. This is the first report of B‐cell lymphoma with Mott cell differentiation in a cat.     

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.     

Biphenotypic B‐cell lymphoma in 2 cats

The clinical and pathologic features of biphenotypic B‐cell lymphoma in 2 cats are reported. Clinical presentation varied from multiple cutaneous masses identified on the thigh in one cat to signs of lethargy from acute hemorrhage due to neoplastic infiltration of one kidney in the other. Cytology and histopathology confirmed round cell neoplasia in both cats and immunochemical staining demonstrated expression of both B‐ and T‐lymphocyte markers by the neoplastic cells in both animals. In PCR analysis of antigen receptor gene rearrangement, clonal rearrangement of B‐cell receptor genes and polyclonal T‐cell receptor gene rearrangement were demonstrated in both lymphomas. These findings were consistent with a diagnosis of B‐cell lymphoma with aberrant CD3 expression in both cases. Clinical progression of disease post diagnosis was rapid in both cats, suggesting a poor prognosis for this lymphoma type. Although bigenotypic receptor rearrangement of lymphoma cells appears relatively common, this is the first known report of actual biphenotypic lymphoma in cats.     

A tumor-related lymphoid progenitor population supports hierarchical tumor organization in canine B-cell lymphoma

Background: Tumors have heterogeneous properties, which could be explained by the existence of hierarchically and biologically distinct tumor cells such as tumor‐initiating cells (TICs). This model is clinically important, as TICs are promising targets for cancer therapies. However, TICs in spontaneous B‐cell lymphoma have not been conclusively identified. Hypothesis/Objectives: Tumor cells with a progenitor phenotype exist in B‐cell lymphoma, reflecting a hierarchical organization. Animals: Twenty‐eight client‐owned dogs with previously untreated B‐cell lymphoma and 6 healthy dogs. Methods: This was a prospective study. Flow cytometry was used to identify lymphoid progenitor cells (LPCs) that coexpressed hematopoietic progenitor antigens CD34, CD117, and CD133, with lymphoid differentiation markers CD21 and/or CD22 in B‐cell lymphoma. The polymerase chain reaction for antigen receptor rearrangements was used to analyze clonality and relatedness of tumor populations. A xenograft model with NOD/SCID/IL‐2Rγ^?/? mice was adapted to expand and serially transplant primary canine B‐cell lymphoma. Results: LPCs were expanded in lymph nodes from 28 dogs with B‐cell lymphoma compared with 6 healthy dogs (P= .0022). LPCs contained a clonal antigen receptor gene rearrangement identical to that of the bulk of tumor cells. Canine B‐cell lymphoma xenografts in recipient mice that maintained LPCs in the tumors were recurrently observed. Conclusions and Clinical Importance: These results suggest the presence of a hierarchy of tumor cells in B‐cell lymphoma as has been demonstrated in other cancers. These findings have the potential to impact not only the understanding of lymphoma pathogenesis but also the development of lymphoma therapies by providing novel targets for therapy.     

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.     

Eosinophilic meningomyelitis associated with T‐cell lymphoma in a cat

A 12‐year‐old cat was presented for evaluation of progressive tetraparesis. Magnetic resonance imaging of the cervical spine demonstrated T2‐hyperintensity, and contrast enhancement within the C4–C7 spinal cord, with marked meningeal contrast enhancement and segmental nerve root thickening. Lumbar cerebrospinal fluid contained 407 total nucleated cells/μL, with 99% eosinophils. The cat transiently improved with prednisolone, clindamycin, and ivermectin therapy, but subsequently worsened and was euthanized. Necropsy revealed an asymmetric infiltration predominantly of the white matter, meninges, and nerve roots of the C4–C6 spinal cord segments by an unencapsulated, poorly demarcated neoplasm composed of atypical lymphocytes admixed with eosinophils, causing perivascular hemorrhage and lytic necrosis. The neoplastic cells were immunoreactive for CD3, ultimately confirming T‐cell lymphoma.     

RNA‐transfected CD40‐activated B cells as a vaccine strategy in canine malignancies

Introduction: Cell‐based vaccine strategies using dendritic cells as cellular adjuvant have entered phase III trials in humans and have been found to be safe, feasible, and potentially efficacious. Canine patients are generally smaller than adult human patients, which makes production of canine dendritic cell (DC) vaccines problematic, given patient size and the small number of available DC precursors. Here we describe feasibility studies of a novel cell‐based vaccine strategy which uses CD40‐activated B‐cells (CD40‐B) loaded with RNA. This strategy is based on our observations that RNA‐transfected human CD40‐B can drive anti‐tumor T cell responses. One advantage of using CD40‐B cells is the ability to expand this cell population ex vivo, allowing for the numbers of cells required for therapeutic vaccines. Methods: Twenty milliliters of blood were drawn from 6 normal dogs and 5 canine lymphoma patients. Peripheral blood mononuclear cells were separated by Ficoll centrifugation. Culture conditions for B cell activation were optimized using CD40‐ligand, canine IL‐4, and Toll‐like receptor stimulus with CpGoligodinucleotides (ODN). Cyclosporine was added to eliminate peripheral T lymphocytes. Proliferation and activation of CD40‐B cells were demonstrated by CFSE dilution of B cells quantified by flow cytometry. Gene transfer was achieved by mRNA electroporation. Results: Marked in vitro stimulation and proliferation of canine peripheral B cells were achieved with soluble trimeric CD40L, canine IL‐4, and ODN. CD40‐B cells showed dramatic upregulation of MHC class II molecules and CD21 (B‐cell activation marker). After two weeks in culture, cells were negative for CD3 and CD4. Canine CD40‐B cells were efficiently transfected with mRNA, with >60% of CD40‐B expressing green fluorescent protein after GFP mRNA electroporation. Conclusion: RNA‐transfected CD40‐B cells can be efficiently generated from normal and tumor‐bearing dogs. These results provide rationale to test tumor RNA‐transfected CD40‐B as a novel therapeutic approach to treating canine malignancies. Clinical trials in canine lymphoma have been proposed.     

Class II major histocompatibility complex expression and cell size independently predict survival in canine B-cell lymphoma

Background: Class II major histocompatibility complex (MHC) is an independent predictor of outcome in human B‐cell lymphoma. We assessed class II expression together with other markers for their impact on prognosis in canine B‐cell lymphoma. Hypothesis: Low class II MHC expression, large cell size, and expression of CD34 will predict a poorer outcome in canine B‐cell lymphoma. Expression of CD5 and CD21 on tumor cells also may be associated with outcome. Animals: One hundred and sixty dogs with cytologically confirmed lymphoma. Methods: Patient signalment, treatment type, and flow cytometry characteristics were analyzed for their influence on outcome. A multivariable predictive model of survival was generated using 2/3 of the patients and validated on the remaining 1/3 of the dataset. Results: Class II MHC expression had a negative association with mortality and relapse. Treatment type also influenced relapse and mortality, whereas cell size and patient age was only associated with mortality. CD34, CD21, and CD5 expression was not associated with disease outcome. The constructed model performed variably in predicting the validation group's outcome at the 6‐month time point. Conclusions and Clinical Importance: Low levels of class II MHC expression on B‐cell lymphoma predict a poor outcome, as in human B‐cell lymphoma. This finding has implications for the use of dogs to model human lymphomas. Class II expression, cell size, treatment, and age can be combined to predict mortality with a high level of specificity.     

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.     

Spontaneous Malignant T-cell Lymphoma in a Young Adult Crl:CD (SD) Rat

We investigated a case of spontaneous malignant T-cell lymphoma observed in a 19-week-old male Crl:CD (SD) rat. The rat showed paralysis beginning 1 week before euthanasia. Hematological examination revealed marked lymphocytosis without distinct atypia. Macroscopically, hepatosplenomegaly and partial atrophy of the thoracic spinal cord were observed. Microscopically, neoplastic cells infiltrated into the liver, splenic red pulp, bone marrow and epidural space of the thoracic spinal cord, while no neoplastic cells were observed in the thymus and lymph nodes. Moreover, the spinal cord showed focal degeneration due to compression by marked infiltration of neoplastic cells in the subdural space. The neoplastic cells were generally small-sized round cells that had a round nucleus with/without a single nucleolus and scanty cytoplasm. Immunohistochemically, the neoplastic cells were positive for CD3 and CD8 and negative for CD79α. Judging from these results, the present tumor in this young adult rat was diagnosed as malignant T-cell lymphoma.     

The Utility of Flow Cytometry for the Diagnosis of Cranial Mediastinal Malignancy in Canine Patients

Introduction:  The most common neoplasms located in the anterior mediastinum in the canine are thymoma and lymphoma. Distinguishing between the two is a diagnostic challenge. Treatment and prognosis for these diseases differs significantly. Thymomas contain a population of normally developing T cells. The majority of these T cells exhibit an immature phenotype, characterized by co‐expression of CD4 and CD8. This phenotype is rarely seen on neoplastic lymphocytes. The purpose of this study was to determine if analysis by flow cytometry could discriminate thymoma from lymphoma based on these cell surface markers.
Methods:  Fine needle aspirates were obtained from ten canine patients with mediastinal masses. Cells were analyzed by flow cytometry using a panel of T and B cell markers.
Results:  Six cases with 10% or greater CD4 + CD8+ cells were diagnosed with thymoma and were confirmed by histopathology. Four cases had fewer than 5% CD4 + CD8+ cells, having lymphocytes expressing CD4 only (3 cases) or CD21, a B cell marker(1 case). These were confirmed as lymphoma by cytology and/or a clonality assay. The sensitivity and specificity of this assay when used in the diagnostic work‐up for suspected thymoma was 100%.
Conclusion:  Flow cytometry may provide important, complementary information in the diagnostic work‐up of the canine patient with a mediastinal mass.     

Complex mammary carcinoma with metastases to lymph nodes,subcutaneous tissue,and multiple joints in a dog

An 8‐year‐old, intact female, mixed‐breed dog presented to the Oklahoma State University Boren Veterinary Medical Teaching Hospital for evaluation of progressive lameness and joint effusion of multiple joints. Physical examination revealed joint effusion of the elbow, hock, and stifle joints bilaterally, enlarged left axillary and right popliteal lymph nodes, a subcutaneous mass over the left elbow, and a subcutaneous mass involving the left second and third mammary glands. Cytologic examination of the mammary mass, enlarged lymph nodes, and joint fluid from most affected joints revealed a monomorphic population of loosely cohesive neoplastic epithelial cells. The patient was humanely euthanized, and subsequent necropsy with histopathologic examination revealed a complex mammary carcinoma with metastases to enlarged lymph nodes, subcutaneous tissue over the left elbow, and the synovium of multiple joints. Immunohistochemical stains were performed and showed diffusely positive pan cytokeratin, CK8/18, and CK19 staining in the neoplastic luminal epithelial cells of the mammary carcinoma, synovium, and lymph nodes, and showed diffusely positive vimentin staining of the myoepithelial cells. Myoepithelial calponin positivity was diffuse in the mammary mass and lymph nodes but minimal in the synovium. Only the mammary mass showed p63 positivity. Metastatic mammary neoplasia is relatively common in dogs; however, metastasis to the synovium has only been reported once previously in the literature. This is the first case utilizing immunohistochemistry for confirmation and characterization of metastases.     

Diagnosis of mediastinal masses in dogs by flow cytometry

BACKGROUND: Biopsy of mediastinal masses can be invasive, but the procedure may be necessary if cytology of a mass aspirate is inconclusive. The 2 most common mediastinal masses, lymphoma and thymoma, may both be comprised of small lymphocytes. We investigated the ability of flow cytometry to distinguish between these 2 neoplasms. HYPOTHESIS: Flow cytometry of mediastinal mass aspirates may provide a definitive diagnosis of thymoma or lymphoma, reducing the need for biopsy. ANIMALS: Dogs with mediastinal masses presenting to the Veterinary Teaching Hospital/Animal Cancer Center were included in the study. METHODS: Aspirates obtained over 2 years that met the inclusion criteria (i.e. sufficient viable cells and a definitive diagnosis by means other than flow cytometry) were analyzed by flow cytometry to determine the percentage of cells expressing B- and T-cell markers, and co-expressing CD4 and CD8. RESULTS: All cases of thymoma (n = 6) consisted of > or = 10% lymphocytes coexpressing CD4 and CD8, a phenotype that is characteristic of thymocytes, whereas 6 of 7 lymphomas contained <2% CD4+CD8+ lymphocytes. The CD4+CD8+ lymphoma could be readily distinguished flow cytometrically from thymoma by light scatter properties. The phenotypes of the remaining lymphomas were CD4+ T cell (4), CD34+ (1) and B cell (1). CONCLUSIONS: Our studies demonstrate that flow cytometry is a useful tool for discriminating mediastinal masses. Lymphocyte-rich mediastinal masses could be unambiguously identified by flow cytometry in 13/13 cases.     

Myoclonus and hypercalcemia in a dog with poorly differentiated lymphoproliferative neoplasia

A 1‐year, 8‐month‐old Rhodesian Ridgeback was presented with obtundation, ambulatory tetraparesis, and myoclonus. Initial clinical findings included ionized hypercalcemia with an apparent marked increase in parathyroid hormone, thrombocytopenia, and nonregenerative anemia. Low numbers of circulating atypical cells were noted on blood film evaluation. Brain magnetic resonance imaging identified an extra‐axial contrast enhancing subtentorial lesion, and cerebrospinal fluid (CSF) analysis documented a marked atypical lymphocytic pleocytosis. Flow cytometry performed on the CSF demonstrated expression of only CD45, CD90, and MHC class II, with Pax5 positivity on subsequent immunohistochemistry. The final diagnosis was of B‐cell lymphoblastic lymphoma or acute leukemia, given the distribution of disease and the presence of significant bone marrow infiltration alongside an aggressive clinical course. The unusual immunophenotype of the neoplastic cells and hypercalcemia presented antemortem diagnostic challenges, highlighting the need for a multidisciplinary approach and caution in the interpretation of clinical abnormalities in cases with multiple comorbidities.     

Immunohistochemical analysis of expression patterns of tumor necrosis factor receptors on lymphoma cells in enzootic bovine leukosis

Tumor necrosis factor-alpha (TNF-alpha) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5+/CD11b+), B-1b (CD5-/CD11b+) and B-2 (conventional B) (CD5-/CD11b-). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII, which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.     

Development of the polymerase chain reaction assay based on the canine genome database for detection of monoclonality in B cell lymphoma

From the canine genome database and its bioinformatic analysis, we identified conserved sequences within the vast majority of 61 variable segments and 1 joining segment of the immunoglobulin heavy chain (IgH) gene, and designed optimal primers for polymerase chain reaction (PCR) amplification directed at these conserved sequences to evaluate the monoclonality of IgH in canine B cell lymphoma. Using the primers, a PCR-based assay was performed on fine-needle aspiration samples of normal, hyperplasia, and malignant lymph nodes and lymphoma cell lines. All fine-needle aspiration samples of five B cell lymphoma cases and the B cell lymphoma line GL-1 exhibited clonal amplification, whereas no amplification was observed in the samples from normal and hyperplasia lymph nodes, cases of T cell lymphoma, and the T cell lymphoma line CL-1. The primers we designed clearly distinguished malignant B lymphocytes from normal, reactive, and malignant T lymphocytes, indicating a potential utility of the primers for PCR-based routine clinical examination for canine B cell lymphoma.