Diffuse Small B-Cell Lymphoma: A High-Grade Malignancy.

The most common subtype of lymphoma in the dog is diffuse large B-cell lymphoma (DLBCL). The remaining forms of B-cell lymphoma in dogs are categorized as small-to-intermediate in size and include marginal zone, follicular, mantle cell, and small-cell lymphocytic lymphoma. Marginal zone lymphoma and follicular lymphoma have readily identifiable unique histologic features while other forms of small B-cell lymphoma in the dog are poorly described by histopathology. Forty-seven cases of nodal small B-cell lymphoma identified by flow cytometry (small cell size based on forward scatter) with concurrent histopathology were reviewed. These cases fell into 3 histologic subtypes: marginal zone lymphoma, follicular lymphoma, and a diffuse form of small B-cell lymphoma with consistent features. As a descriptive term, we refer to the latter subtype as diffuse small B-cell lymphoma (DSBCL) until it can be further characterized by gene expression profiling and other molecular tools. Clinical presentation of DSBCL was compared to cases of histologically confirmed DLBCL and clinical follow-up was obtained for 22 of the 27 cases of DSBCL. This subset of diffuse small B-cell lymphoma had an overall median survival of 140 days. The expression of CD21, class II MHC and CD25 by flow cytometry did not differ between DSBCL and the other histologic subtypes of small cell B-cell lymphoma making histopathology the only current method of classification.

Progressive gammopathy and coagulopathy in a young English bulldog.

A restricted polyclonal or biclonal gammopathy resulting in bleeding tendencies was diagnosed in a young, neutered male English bulldog with concurrent splenomegaly, anemia, and severe elevations in IgM and, to a lesser degree, IgA immunoglobulins. There was a positive clinical response to treatment with prednisone and chlorambucil. This case bears similarity to a recently published syndrome of polyclonal gammopathy that is not neoplastic in origin in this breed. Key clinical message: The current case describes the management and clinical course of a recently described syndrome of polyclonal gammopathy in English bulldogs.

Gammapathie et coagulopathie progressives chez un jeune bouledogue Anglais. Une gammapathie polyclonale restreinte ou biclonale résultant en une tendance aux saignements fut diagnostiquée chez un jeune bouledogue Anglais mâle castré, avec une splénomégalie concomitante, de l'anémie et une augmentation sévère des immunoglobulines IgM et, à un degré moindre, des IgA. Une réponse clinique positive au traitement avec de la prednisone et du chlorambucil fut notée. Ce cas comporte des similarités avec un syndrome récemment décrit de gammapathie polyclonale qui ne serait pas d'origine néoplasique chez cette espèce.Message clinique clé :Le présent cas décrit la gestion et l'évolution clinique d'un syndrome récemment décrit de gammapathie polyclonale chez les bouledogues Anglais.(Traduit par Dr Serge Messier).

Reactive mesothelial hyperplasia associated with chronic peritonitis in a 20-year-old Quarter horse.

A 20-year-old gelding was diagnosed with peritonitis and severe reactive mesothelial hyperplasia. Exploratory laparotomy findings were suggestive of a neoplastic etiology; however, additional diagnostics ruled this out and the horse made a full recovery. This report demonstrates the difficulty and value of differentiating between reactive and neoplastic mesothelial processes.

Hyperplasie mésothéliale réactive associée à une péritonite aiguë chez un cheval Quarter horse âgé de 20 ans. Une péritonite et l'hyperplasie mésothéliale réactive grave ont été diagnostiquées chez un hongre âgé de 20 ans. Les résultats d'une laparatomie exploratoire ont suggéré une étiologie néoplasique. Cependant, des diagnostics additionnels ont éliminé cette possibilité et le cheval s'est complètement rétabli. Ce rapport démontre la difficulté et la pertinence de différencier entre les processus mésothéliaux réactif et néoplasique.(Traduit par Isabelle Vallières).

A tissue-specific chromatin loop activates the erythroid ankyrin-1 promoter.

The human ankyrin-1 gene (ANK1) contains 3 tissue-specific alternative promoters. We have shown previously that the erythroid-specific ankyrin 1 (ANK1E) core promoter contains a 5' DNase I hypersensitive site (HS) with barrier insulator function that prevents gene silencing in vitro and in vivo. Mutations in the ANK1E barrier region lead to decreased ANK1 mRNA levels and hereditary spherocytosis. In this report, we demonstrate a second ANK1E regulatory element located in an adjacent pair of DNase I HS located 5.6 kb 3' of the ANK1E promoter at the 3' boundary of an erythroid-specific DNase I-sensitive chromatin domain. The 3' regulatory element exhibits enhancer activity in vitro and in transgenic mice, and it has the histone modifications associated with an enhancer element. One of the ANK1E 3'HS contains an NF-E2 binding site that is required for enhancer function. We show that a chromatin loop brings the 3' enhancer and NF-E2 into proximity with the 5' barrier region including the ANK1E core promoter. These observations demonstrate a model for the tissue-specific activation of alternative promoters that may be applicable to the ∼ 30% of mammalian genes with alternative promoters that exhibit distinct expression patterns.

Suspected primary bone marrow T-cell lymphoid neoplasia causing paraneoplastic hypercalcemia in 11 dogs (2014-2021).

OBJECTIVE: To describe the clinical findings and outcome in hypercalcemic dogs that were diagnosed with T-cell lymphoid neoplasia by bone marrow evaluation. ANIMALS: 11 client-owned dogs, identified retrospectively through 2 diagnostic laboratories between 2014 and 2021. CLINICAL PRESENTATION: Cases presented with hypercalcemia and lacked overt evidence of lymphoid neoplasia in the blood or nonmedullary tissues. T-cell lymphoid neoplasia was diagnosed once the bone marrow was investigated, using a variable combination of cytology, histology, and flow cytometry. RESULTS: The median age at presentation was 5.7 years (range, 4.0 to 8.6 years). All cases were large-breed dogs, and 4 of 11 cases were Golden Retrievers. Dogs presented most commonly for polyuria and polydipsia (72%). Eight cases had neutropenia, and 10 of 11 dogs had reported thrombocytopenia. In all cases, flow cytometry identified an expansion of neoplastic small- to intermediate-sized T cells in the bone marrow that expressed low-class-II major histocompatibility complex. Neoplastic T cells in 10 of 11 cases expressed CD4. Treatments ranged from prednisone alone to multiagent chemotherapy. The median overall survival time was 260 days (range, 25 to 792 days). CLINICAL RELEVANCE: T-cell lymphoid neoplasia diagnosed via bone marrow evaluation that may represent a unique bone marrow T-cell neoplastic entity should be considered in hypercalcemic dogs with isolated cytopenias that lack peripheral lymphocytosis, lymphadenopathy, and organomegaly. Clinical outcome in these cases was variable, which may be related to nonstandardized treatments, but a subset of patients had prolonged survival.

Canine T zone lymphoma is a tumor of mature, previously activated αß T cells.

T cell lymphomas are a diverse group of tumors found in both dogs and humans, originating from various normal T cell types. Identifying the origin of neoplastic lymphocytes can offer valuable insights into the pathogenesis and clinical behavior of these tumors. T zone lymphoma (TZL) in dogs is characterized by the absence of CD45 expression, a strong breed predilection, and its association with adult-onset demodicosis-a condition believed to be linked to immunosuppression. In this study, our aim was to employ transcriptomic and functional data to determine the normal counterpart of TZL. Identifying the normal counterpart may help us understand both how these tumors arise and explain their clinical behavior. Gene expression profiling using NanoString and RNA seq was used to compare the transcriptome between neoplastic T zone cells, normal canine T cells and publicly available gene sets using Gene Set Enrichment Analysis. Mitogen, anti-CD3 stimulation and PMA/ionomycin stimulation were used to assess T cell proliferation in vitro, and intracellular cytokine production was measured by flow cytometry. Gene expression profiling revealed that TZL is most likely derived from an activated or memory alpha-beta T cell but the cells do not fall cleanly into an effector subtype. TZL cells express CD4-specific transcription factors GATA3 and THPOK, even though TZL cells more commonly express CD8, or neither CD4 nor CD8. TZL cells produce high levels of interferon gamma and tumor necrosis factor alpha when stimulated, further supporting the hypothesis that they are derived from an antigen experienced T cell. TZL cells do not proliferate when stimulated through the T cell receptor but will divide when the T cell receptor is bypassed with PMA and ionomycin. The observation that these cells are derived from a mature, previously activated T cell is the first step in understanding the genesis of this unique T cell tumor.

DNA methylation of transposons pattern aging differences across a diverse cohort of dogs from the Dog Aging Project.

Within a species, larger individuals often have shorter lives and higher rates of age-related disease. Despite this well-known link, we still know little about underlying age-related epigenetic differences, which could help us better understand inter-individual variation in aging and the etiology, onset, and progression of age-associated disease. Dogs exhibit this negative correlation between size, health, and longevity and thus represent an excellent system in which to test the underlying mechanisms. Here, we quantified genome-wide DNA methylation in a cohort of 864 dogs in the Dog Aging Project. Age strongly patterned the dog epigenome, with the majority (66% of age-associated loci) of regions associating age-related loss of methylation. These age effects were non-randomly distributed in the genome and differed depending on genomic context. We found the LINE1 (long interspersed elements) class of TEs (transposable elements) were the most frequently hypomethylated with age (FDR < 0.05, 40% of all LINE1 regions). This LINE1 pattern differed in magnitude across breeds of different sizes- the largest dogs lost 0.26% more LINE1 methylation per year than the smallest dogs. This suggests that epigenetic regulation of TEs, particularly LINE1s, may contribute to accelerated age and disease phenotypes within a species. Since our study focused on the methylome of immune cells, we looked at LINE1 methylation changes in golden retrievers, a breed highly susceptible to hematopoietic cancers, and found they have accelerated age-related LINE1 hypomethylation compared to other breeds. We also found many of the LINE1s hypomethylated with age are located on the X chromosome and are, when considering X chromosome inactivation, counter-intuitively more methylated in males. These results have revealed the demethylation of LINE1 transposons as a potential driver of inter-species, demographic-dependent aging variation.

A series of heterogeneous lymphoproliferative diseases with CD3 and MUM1 co-expressed in cats and dogs.

Lymphoma diagnosis in dogs and cats is continually evolving as new subtypes and human correlates are being recognized. In humans, T-cell lymphomas with MUM1 expressed and plasma cell neoplasia or B-cell lymphomas with CD3 expressed aberrantly are reported only rarely. We report here a case series of tumors in dogs and cats with CD3 and MUM1 co-expressed as determined by immunocytochemistry or immunohistochemistry. Lineage was assigned for these tumors by 3 board-certified pathologists and a veterinary immunologist based on review of clinical and cellular features and the results of ancillary testing including PCR for antigen receptor rearrangements, flow cytometry, and serum protein electrophoresis with immunofixation. In cats, 7 of 7 tumors, and in dogs, 3 of 6 tumors with CD3 and MUM1 co-expressed had clonal rearrangement of the immunoglobulin gene or serum monoclonal immunoglobulin, consistent with a diagnosis of a plasma cell neoplasia or myeloma-related disorder with CD3 expressed aberrantly. Disease was often disseminated; notably, 3 of 7 feline cases had cutaneous and/or subcutaneous involvement in the tarsal area. In dogs, 3 of 6 cases had a clonal T-cell receptor gamma result and no clonal immunoglobulin gene rearrangement and were diagnosed as a T-cell tumor with MUM1 expressed. The use of multiple testing modalities in our series of tumors with plasma-cell and T-cell antigens in dogs and cats aided in the comprehensive identification of the lymphoproliferative disease subtype.

Detection and Characterization of Paraproteinemia in Canine Chronic B-cell Lymphocytic Leukemia Using Routine and Free Light Chain Immunofixation.

BACKGROUND: Hyperglobulinemia is reported in 26% of canine chronic B-cell lymphocytic leukemia (B-CLL) cases. However, few cases have been characterized by protein electrophoresis and immunofixation (IF), and the incidence of a monoclonal protein (M-protein) is unknown using these techniques. OBJECTIVE: To characterize and determine the proportion of canine B-CLL cases with an M-protein using plasma protein electrophoresis (PPE), routine and free light chain (fLC) IF, and to assess if productive B-CLL cases express MUM1/IRF4 by cell tube block (CTB). METHODS: PPE, routine (targeting IgG, IgA, IgM, IgG4, and light chain) and fLC IF were performed using 48 dog B-CLL plasma samples from patients diagnosed via peripheral blood flow cytometry. CTB was performed on a separate cohort of 15 patients. RESULTS: Hyperproteinemia (>7.5 g/dL) was present in 17/48 cases (35%). An M-protein was detected in 32/48 cases (67%). Of these, 19/32 cases (59%) had only complete (monoclonal heavy and light chain) M-proteins detected, 10/32 cases (31%) had both complete and fLC M-proteins detected, and 3/32 cases (9%) had only an fLC M-protein detected. IgM was the most common clonal immunoglobulin isotype detected (23 cases). CD21+ cell counts were higher in cases with detectable M-protein. Plasma fLC IF suggested ß-γ region interference, likely caused by clotting proteins. All B-CLL cases consistently expressed PAX5 and did not express MUM1/IRF4. CONCLUSIONS: Most B-CLL cases had an M-protein and were not hyperproteinemic. Most cases with paraproteins had a complete IgM monoclonal gammopathy; a subset had documented fLCs. The prognostic significance of heavy and fLC presence should be evaluated.

Using digital RNA counting to establish flow cytometry diagnostic criteria for subtypes of CD34+ canine acute leukaemia.

Canine acute leukaemia is a heterogeneous neoplasm with multiple phenotypes. Criteria to subtype acute leukaemia by flow cytometry have not been validated. The goal of this study was to develop a panel of antibodies and objective antigen expression criteria for the assignment of lymphoid or myeloid lineage by flow cytometry. We isolated mRNA from the blood of 45 CD34+ acute leukaemia cases and measured expression of 43 genes that represent lymphoid and myeloid lineages using NanoString technology. We determined differentially expressed genes between major groups identified by unsupervised hierarchical clustering. We then evaluated the expression of antigens by flow cytometry to determine if cases could be assigned to a lineage. Two groups were identified by gene expression. Group 1/LYMPH overexpressed lymphoid-associated genes (ex. DNTT) and had a higher percentage of CD5 + CD3- cells by flow cytometry. Group 2/MYELO overexpressed myeloid-associated genes (ex. ANPEP/CD13) and had a higher percentage of class II major histocompatibility complex (MHCII)- CD14+ and/or CD18 + CD4- cells. We proposed that >12.5% CD5 + CD3- cells in the blood was indicative of lymphoid lineage, and > 3.0% CD14 + MHCII- cells or > 18% CD18 + MHCII-CD4- cells was indicative of myeloid lineage. 15/15 cases that met the proposed criteria for acute lymphocytic leukaemia were in LYMPH group and 12/15 cases that met the proposed criteria for acute myeloid leukaemia were in MYELO group. The majority of CD34+ cases that did not meet either immunophenotyping lineage criterion (12/13) clustered within the LYMPH group. In conclusion, currently available antibodies can be useful for determining canine acute leukaemia subtypes.

Clinical outcome and Ki67 evaluation in dogs with nodal small cell B-cell lymphoma diagnosed by flow cytometry.

BACKGROUND: Nodal small cell B-cell lymphoma subtypes in dogs cannot be distinguished by flow cytometry and information regarding treatment, prognosis, and outcome are limited. HYPOTHESIS/OBJECTIVES: Objectives were to describe outcome in dogs with nodal small cell B-cell lymphoma diagnosed by flow cytometry and correlate clinical and laboratory data with survival. We hypothesized that B-cell Ki67 expression measured by flow cytometry is associated with shorter progression free survival (PFS) and overall survival (OS). ANIMALS: Forty-nine dogs with nodal small cell B-cell lymphoma, defined by >80% CD21+ B-cells by flow cytometry and small-sized B-cells by forward scatter. METHODS: Retrospective study reviewing treatment and outcome data extracted from medical records. Percentage of Ki67-expressing B-cells was measured by flow cytometry. Clinical, laboratory, and flow cytometry data were assessed for association with outcome. RESULTS: Median percentage of B-cell Ki67 was 41% (range, 3%-97%). Median PFS was 119 days and median OS was 222 days (n = 49). Among cases treated with CHOP-based chemotherapy (n = 32), median PFS was 70 days, median OS was 267 days, and 50% of cases achieved complete response. Low percentage of B-cell Ki67 (≤11%) was associated with prolonged OS by univariable analysis. Greater age, substage B, high B-cell CD25 expression and low B-cell CD21 and class II major histocompatibility complex expression by flow cytometry were independently associated with shorter OS. CONCLUSIONS AND CLINICAL IMPORTANCE: Most nodal small cell B-cell lymphoma cases had aggressive disease. Low Ki67 expression can help identify cases with better prognosis. Age, substage, and flow cytometry variables are useful prognostic factors.

An aggressive CD4- CD8- T-cell neoplasm in young English bulldogs.

T-cell leukemia/lymphoma accounts for roughly 30% of all types of lymphoproliferative neoplasia in dogs. Two forms of T-cell lymphoma (T-zone and peripheral T-cell lymphoma) exhibit breed-specific predilections. During the course of routine immunophenotyping, we observed a breed-specific presentation of a unique form of T-cell leukaemia in young English bulldogs. To describe the clinical presentation and outcome of a novel T-cell leukaemia in English bulldogs and determine the frequency of this neoplasm in other breeds. The Clinical Hematopathology database, containing immunophenotyping data from peripheral blood of nearly 11 900 dogs, was queried for the phenotype observed in young English bulldogs: CD45+ CD4- CD8- CD5+ CD3+ class II major histocompatibility complex (MHC)-low T-cell leukaemia. Clinical presentation, treatment, and survival data were collected for a subset of cases. Fifty-five English bulldog cases and 64 cases of other breeds were identified. No other breed was represented by >5 cases. Complete medical records were obtained for 50 bulldogs. Median age at diagnosis was 3 years and 76% of cases were male. Median lymphocyte count was 44 286 lymphocytes/µl (range, 1800-317 684/µl) and lymphocytes were described as small to intermediate-sized. Many dogs were thrombocytopenic and had liver and spleen involvement, but not lymphadenopathy. Bulldogs that received multi-agent chemotherapy had longer median survival times (83 days) compared to dogs that received no treatment (6 days) or less aggressive therapy (15 days) (p = .001). Non-bulldogs had similar outcomes. CD4- CD8- class II MHC-low T-cell leukaemia has an aggressive clinical course and predilection for young English bulldogs. Breed-specific presentation suggests an underlying genetic cause.

Clinical outcome and prognostic factors in dogs with B-cell chronic lymphocytic leukemia: A retrospective study.

BACKGROUND: B-cell chronic lymphocytic leukemia (BCLL) in dogs generally is considered an indolent disease, but previous studies indicate a wide range in survival times. OBJECTIVES: We hypothesized that BCLL has a heterogeneous clinical course, similar to chronic lymphocytic leukemia in humans. We aimed to assess presentation and outcome in dogs with BCLL and evaluate the prognostic relevance of clinical and flow cytometric factors. ANIMALS: One hundred and twenty-one dogs with BCLL diagnosed by flow cytometry. Three breed groups were represented: small breed dogs (n = 55) because of increased risk of BCLL; Boxers (n = 33) because of preferential use of unmutated immunoglobulin genes; and other breeds (n = 33). METHODS: Retrospective study reviewing signalment, clinicopathologic data, physical examination findings, treatment, and survival of dogs with BCLL. Cellular proliferation, determined by the percentage of Ki67-expressing CD21+ B-cells by flow cytometry, was measured in 39 of 121 cases. Clinical and laboratory variables were evaluated for association with survival. RESULTS: The median survival time (MST) for all cases was 300 days (range, 1-1644 days). Boxers had significantly shorter survival (MST, 178 days) than non-Boxers (MST, 423 days; P < .0001), and no significant survival difference was found between small breeds and other non-Boxer breeds. Cases with high Ki67 (>40% Ki67-expressing B-cells) had significantly shorter survival (MST, 173 days) than did cases with <40% Ki67 (MST undetermined; P = .03), regardless of breed. Cases with a high lymphocyte count (>60 000 lymphocytes/µL) or clinical signs at presentation had significantly shorter survival. CONCLUSIONS AND CLINICAL IMPORTANCE: B-cell chronic lymphocytic leukemia had a variable clinical course and Boxer dogs and cases with high Ki67 had more aggressive disease.

Immunophenotypic characterization and clinical outcome in cats with lymphocytosis.

BACKGROUND: Lymphocytosis is relatively common in cats, but few studies describe lymphocyte populations or the clinical course associated with different immunophenotypic expansions. HYPOTHESIS/OBJECTIVES: We hypothesized that cats frequently develop non-neoplastic lymphocytosis and that different neoplastic immunophenotypes have variable prognoses. We aimed to characterize the lymphocyte expansions in a large population of cats with lymphocytosis and to assess clinical presentation and outcome in a subset. ANIMALS: Three cohorts of cats older than 1 year with lymphocytosis (>6000/µL) were examined to define immunophenotypic categories (n = 146), evaluate outcome (n = 94), and determine prevalence of immunophenotypes (n = 350). METHODS: Retrospective study of cats with blood submitted for flow cytometry. Medical records (n = 94) were reviewed for clinical data, treatment, and survival information. RESULTS: Five major immunophenotypic categories were identified: B cell, heterogeneous (≥2 lineages expanded), CD4+ T cell, CD4-CD8- (double negative [DN]) T cell, and CD5-low-expressing T cell. B-cell and heterogeneous phenotypes were more consistent with a non-neoplastic process, having polyclonal antigen receptor gene rearrangements, younger age at presentation, lower lymphocyte counts, and prolonged survival. The neoplastic phenotypes, CD4+ T cell, DN T cell, and CD5 low T cell, had different median survival times (752 days [n = 37], 271 days [n = 7], 27.5 days [n = 12], respectively). Among CD4+ T-cell cases, cats with abdominal lymphadenopathy, intestinal involvement, or both and females had shorter survival. Among 350 cats with lymphocytosis, CD4+ T-cell lymphocytosis was most common, followed by heterogeneous and B-cell phenotypes. CONCLUSIONS AND CLINICAL IMPORTANCE: Neoplastic CD4+ T-cell lymphocytosis is common in cats and has a prolonged clinical course compared to aberrant T-cell phenotypes. Cats with heterogeneous and B-cell lymphocyte expansions commonly have non-neoplastic disease.

Clinical features of canine nodal T-cell lymphomas classified as CD8+ or CD4-CD8- by flow cytometry.

Canine T-cell lymphoma (TCL) encompasses a heterogeneous group of diseases with variable clinical presentation, cytomorphology, immunophenotype, and biologic behaviour. The most common types of TCL in dogs involving peripheral lymph nodes include indolent T-zone lymphoma (TZL) and biologically aggressive peripheral T-cell lymphoma (PTCL). TCL phenotypes can be categorized by expression of the surface antigen molecules CD4 and CD8. The majority of TCL cases are CD4+ , with far fewer cases being CD8+ or CD4- CD8- . The clinical features of CD4+ TCLs have been previously described. The less common TCL phenotypes, however, are poorly characterized with little to no information about prognosis. In this retrospective study, we describe and correlate the presenting clinical signs, flow cytometry, and outcomes of 119 dogs diagnosed with nodal, non-TZL, CD8+ or CD4- CD8- TCL by flow cytometry. Skin lesions present at the time of diagnosis were more commonly observed in the CD8+ TCL group. Mediastinal enlargement and/or hypercalcemia were more commonly seen in the CD4- CD8- TCL group. Dogs with either CD8+ or CD4- CD8- TCLs had aggressive clinical disease with median overall survival (OS) times of 198 days and 145 days, respectively. In both groups, neoplastic cell size determined by flow cytometry ranged from small to large, and large cell size was associated with shorter OS times (median OS = 61 days). Cases classified as small cell had a median OS of 257 days. Expression levels of major histocompatibility complex (MHC) class II and CD5 were highly variable among cases but were not prognostically significant in this group of patients.

Polyclonal B-cell lymphocytosis in English bulldogs.

BACKGROUND: English bulldogs disproportionally develop an expansion of small B-cells, which has been interpreted as B-cell chronic lymphocytic leukemia (BCLL). However, clonality testing in these cases has often not been supportive of neoplasia. HYPOTHESIS: English bulldogs have a syndrome of nonneoplastic B-cell expansion. ANIMALS: Eighty-four English bulldogs with small-sized CD21+ B-cell lymphocytosis in the blood as determined by flow cytometry. METHODS: This is a retrospective study. We characterized this syndrome by assessing B-cell clonality, clinical presentation, flow cytometric features, and immunoglobulin gammopathy patterns. We identified 84 cases with CD21+ lymphocytosis among 195 English bulldogs with blood samples submitted to the Colorado State University-Clinical Immunology laboratory for immunophenotyping between 2010 and 2019. Flow cytometry features were compared to normal B-cells and BCLL cases. PCR for antigen receptor rearrangements (PARR) by multiple immunoglobulin primers was performed to assess B-cell clonality. A subset of cases with gammopathy were examined by protein electrophoresis, immunofixation, and immunoglobulin subclass ELISA quantification. RESULTS: Seventy percent (58/83) of cases had polyclonal or restricted polyclonal immunoglobulin gene rearrangements, suggesting nonmalignant B-cell expansion. The median age of all dogs in the study was 6.8 years and 74% were male. The median (range) lymphocyte count was 22 400/µL (2000-384 400/µL) and B-cells had low expression of class II MHC and CD25. Splenomegaly or splenic masses were detected in 57% (26/46) of cases and lymphadenopathy in 11% (7/61). Seventy-one percent (52/73) of cases had hyperglobulinemia and 77% (23/30) with globulin characterization had IgA ± IgM polyclonal or restricted polyclonal gammopathy patterns. CONCLUSIONS AND CLINICAL IMPORTANCE: Polyclonal B-cell lymphocytosis in English bulldogs is characterized by low B-cell class II MHC and CD25 expression, splenomegaly and hyperglobulinemia consisting of increased IgA ± IgM. We hypothesize that this syndrome has a genetic basis.

Validation and method comparison of the use of densitometry to quantify monoclonal proteins in canine sera.

BACKGROUND: Densitometric quantitation using serum protein electrophoresis (SPE) is used to monitor monoclonal proteins (M-proteins) in human patients but has not been validated in the dog. Serum globulin concentrations, species-specific radial immunodiffusion (RID), and ELISAs are currently used in veterinary medicine. OBJECTIVE: We aimed to compare four methods that quantify M-proteins using densitometry and biuret protein (dM-protein) measurements. We also validated the best performing method and compared it with the RID and ELISA methods for measuring canine serum M-protein. METHOD: Serum from six normal dogs and 83 serum samples from 46 dogs with confirmed monoclonal gammopathies were used. A spike and recovery experiment with purified monoclonal IgG and IgM, inter-run and intra-run variability, linearity under dilution, and lower limit of detection were performed. Results of commercial canine RID and ELISA kits for total class-specific immunoglobulin were compared with dM-proteins. RESULTS: The corrected perpendicular drop gating method had <20% error for IgG/γ-globulin and IgM/ß-globulin M-protein quantifications. Linearity (r > .99), intra-run CV (1.1%-2.3%), and inter-run CVs (2.0%-3.5%) were acceptable. Correlation between the RID and densitometry results ranged from r = .25 to r = .88, depending on the class. The RID result was greater than that of the biuret total protein in 26/63 (41%) IgA cases. A panel of IgG, IgA, and IgM RIDs failed to correctly identify an IgM paraproteinemia in 6/6 (100%) cases. Densitometry was not comparable with any other tested method. CONCLUSION: Densitometric quantitation is a valid technique for measuring M-proteins in the ß- and γ-globulin regions. Immunotyping via RID using the tested kit does not appear to detect IgM. Densitometry is recommended for measuring M-proteins in canine patients.

Assessment of immunoglobulin heavy chain, immunoglobulin light chain, and T-cell receptor clonality testing in the diagnosis of feline lymphoid neoplasia.

BACKGROUND: Differentiation between neoplastic and reactive lymphocytic proliferations can be challenging in cats. PCR for antigen receptor rearrangements (PARR) testing is a useful diagnostic tool to assess clonality of a lymphoid population. Previous feline PARR studies evaluated clonality of complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma (TRG) gene rearrangements. OBJECTIVES: We aimed to evaluate the sensitivity and specificity of feline PARR primers targeting complete IGH-VDJ and TRG rearrangements, as well as incomplete IGH-DJ, kappa deleting element (Kde), and immunoglobulin lambda light chain (IGL) gene rearrangements in defined feline neoplasms and nonneoplastic controls. METHODS: Fluorescently labeled PCR primers were designed to amplify complete IGH-VDJ, incomplete IGH-DJ, Kde, IGL, and TRG gene rearrangements in two multiplexed PCR reactions, and PCR products were analyzed by fragment analysis. Fresh tissue samples from 12 flow cytometrically confirmed B-cell lymphomas, 26 cytologically confirmed gastric and renal lymphomas of presumed B-cell origin, 30 flow cytometrically confirmed T-cell leukemias, and 11 negative control cats were tested. RESULTS: Using four immunoglobulin primer sets (IGH-VDJ, IGH-DJ, Kde, and IGL), clonal immunoglobulin rearrangements were detected in 87% (33/38) of the presumed B-cell neoplasms. The IGH-VDJ reaction alone only detected clonality in 50% (19/38) of these cases. TRG rearrangements were clonal in 97% (29/30) of the T-cell leukemia cases. All negative control samples had polyclonal immunoglobulin and TRG rearrangements. CONCLUSIONS: The PARR assay developed in this study is useful for assessing clonality in feline lymphoid neoplasms. Clonality assessment of incomplete IGH-DJ, Kde, and IGL rearrangements helped identify clonal B-cell neoplasms not detected with complete IGH-VDJ PARR alone.

Immunoglobulin G4-related disease in a dog.

Immunoglobulin G4-related disease (IgG4-RD), which affects many organ systems, has been recognized as a distinct clinical entity in human medicine for just over a decade but has not been previously identified in dogs. In humans, IgG4-RD is characterized by diffuse IgG4-positive lymphoplasmacytic infiltrates that commonly lead to increased serum concentrations of IgG4 and IgE, peripheral eosinophilia, tumorous swellings that often include the parotid salivary glands, obliterative phlebitis, and extensive fibrosis. Herein we describe the diagnosis, clinical progression, and successful treatment of IgG4-RD in an 8-year-old female spayed Husky mixed breed dog. Immunoglobulin G4-related disease should be considered as a differential diagnosis for dogs with vague clinical signs, lymphoplasmacytic swellings, restricted polyclonal gammopathy, eosinophilia or some combination of these findings.

Indolent T-cell-rich small B-cell hepatic lymphoma in a Golden Retriever.

An 11-year-old female spayed Golden Retriever presented for an incidentally found liver mass. The hepatic mass and intra-abdominal lymph nodes had a marked heterogeneous T-cell population and far fewer numbers of small clonal B cells. This T-cell-rich small B-cell lymphoma had a unique histological pattern and indolent clinical course.