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T-cell–rich, large B-cell lymphoma (TCRLBCL) is the most commonly diagnosed type of lymphoma in horses. Here we describe the clinical signs, neuropathology, immunohistochemistry (IHC), and PCR for antigen receptor rearrangement (PARR) analysis results of a TCRLBCL in the brain of an 8-y-old male Quarter Horse that was euthanized after acute anorexia, tremors, head pressing, falling, blindness, incoordination, and seizures. Autopsy revealed a firm, smooth, pale-yellow mass that expanded both lateral ventricles and the adjacent subcortical white matter. Histologically, the mass consisted of a densely cellular neoplasm composed of large, CD79+ neoplastic B-lymphocytes admixed with sheets of small, CD3+ reactive T-lymphocytes, Iba1+ histiocytes, MUM1+ plasma cells, and rare eosinophils supported by a fine fibrovascular stroma. Formalin-fixed, paraffin-embedded tissue scrolls were retrieved and subjected to PARR analysis, which revealed a clonal reaction in the immunoglobulin gene and a polyclonal reaction for the T-lymphocyte receptor gene, consistent with a neoplastic B-lymphocyte and reactive T-lymphocyte proliferation. The diagnosis of TCRLBCL was suspected histologically and confirmed based on IHC and PARR analysis.

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.

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 that12.5% 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.

Supplementary Tables 1-2 from Distinct B-Cell and T-Cell Lymphoproliferative Disease Prevalence among Dog Breeds Indicates Heritable Risk

Immunophenotypes in lymphoproliferative diseases (LPD) are prognostically significant, yet causative factors for these conditions, and specifically those associated with heritable risk, remain elusive. The full spectrum of LPD seen in humans occurs in dogs, but the incidence and lifetime risk of naturally occurring LPD differs among dog breeds. Taking advantage of the limited genetic heterogeneity that exists within dog breeds, we tested the hypothesis that the prevalence of LPD immunophenotypes would differ among different breeds. The sample population included 1,263 dogs representing 87 breeds. Immunophenotype was determined by the presence of clonal rearrangements of immunoglobulin heavy chain or T-cell receptor γ chain. The probability of observing the number of B-cell or T-cell tumors in a particular breed or breed group was compared with three reference populations. Significance was computed using χ2 test, and logistic regression was used to confirm binomial predictions. The data show that, among 87 breeds tested, 15 showed significant differences from the prevalence of LPD immunophenotypes seen across the dog population as a whole. More significantly, elevated risk for T-cell LPD seems to have arisen ancestrally and is retained in related breed groups, whereas increased risk for B-cell disease may stem from different risk factors, or combinations of risk factors, arising during the process of breed derivation and selection. The data show that domestic dogs provide a unique and valuable resource to define factors that mediate risk as well as genes involved in the initiation of B-cell and T-cell LPD.

BACKGROUND 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 leukemia in young English bulldogs. OBJECTIVE To describe the clinical presentation and outcome of a novel T-cell leukemia in English bulldogs and determine the frequency of this neoplasm in other breeds. METHODS 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)ᶫ° T-cell leukemia. Clinical presentation, treatment, and survival data were collected for a subset of cases. RESULTS 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/ul (range, 1,800-317,684/ul) 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=0.001). Non-bulldogs had similar outcomes. CONCLUSION CD4- CD8- class II MHCᶫ° T-cell leukemia has an aggressive clinical course and predilection for young English bulldogs. Breed-specific presentation suggests an underlying genetic cause. This article is protected by copyright. All rights reserved.

Tumor draining lymph nodes (TDLNs) function as an anatomic niche which primes immune cells to generate anti-cancer responses. While tumors are frequently staged based on the presence or absence of TDLN metastasis, mechanisms for how tumors impact the TDLN microenvironment and immunologic responses are incompletely understood. Dogs with spontaneously arising tumors provide an opportune model to study immuno-oncology, sharing many similarities to human patients with their environment, clinical care, and pathobiology. To evaluate how tumors impact TDLN responses, we conducted single cell RNA sequencing (scSeq) on cryopreserved TDLNs of dogs with osteosarcoma (n=4) and healthy controls (n=2). We obtained an average of 3,984 cells per sample (range 1,603-5,259) with a total of 4,780 cells from healthy samples and 19,121 cells from TDLN samples. All samples were integrated into one dataset, then unsupervised clustering was completed. Each unique cell cluster was assigned an identity using canonical markers and reference mapping to human datasets. Following cell classification, we observed notable changes in gene expression across all cells isolated from the LNs, with most differences arising from changes to the transcriptome of myeloid cells. Although still preliminary, the data also demonstrate differences in the abundance of T, B, and myeloid cells between healthy and tumor draining LNs. Taken together, this dataset provides indications of the changes TDLNs undergo in dogs with osteosarcoma. Citation Format: Samuel A. Brill, Dylan T. Ammons, Anne C. Avery, Douglas H. Thamm. Evaluation of tumor draining lymph nodes in dogs with spontaneously arising osteosarcoma using single-cell sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4465.

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 40% Ki67‐expressing B‐cells) had significantly shorter survival (MST, 173 days) than did cases with 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.

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.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).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.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). CD21Most 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.

Despite high initial response rates, a subset of dogs with B-cell lymphoma responds less robustly to CHOP-based chemotherapy and experiences shorter survival. One hundred and four dogs with nodal B-cell lymphoma were treated with a response-based CHOP (RBCHOP) protocol modified based on response to individual drugs during the first chemotherapy cycle. Dogs achieving complete (CR) or partial response (PR) at week 3, following treatment with vincristine and cyclophosphamide, received RBCHOP 1 (n = 72), a protocol sequentially rotating vincristine, cyclophosphamide, and doxorubicin. Dogs without a detectable response at week 3 that subsequently achieved CR or PR following treatment with doxorubicin received RBCHOP 2 (n = 14), in which four doses of doxorubicin were given consecutively followed by vincristine and cyclophosphamide. Dogs that failed to respond at week 3 and then to doxorubicin at week 5 assessment were offered rescue chemotherapy (RBCHOP 3, n = 18). Median progression free survival (PFS) and overall survival time (OST) were similar between RBCHOP 1 (PFS 210 days, OST 354 days) and RBCHOP 2 (PFS 220 days, OST 456 days), but significantly shorter for RBCHOP 3 (PFS 34 days, OST 80.5 days, P < 0.001). No presenting signalment nor hematologic variable differentiated patient cohort, however, dogs in RBCHOP 2 and RBCHOP 3 were more likely to have a lymphocytosis at diagnosis (P = 0.02 and 0.04, respectively). Protocol modification based on response during the first cycle resulted in similar toxicity profiles and outcomes to previously published variants of CHOP, and prognosis remained poor for dogs failing to respond during the first treatment cycle.

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.

A 10-year-old intact male Golden Retriever was presented to The Ohio State University Veterinary Medical Center for acute, non-painful facial swelling of the right mandibular region. On physical examination, the right mandibular swelling was found to represent marked lymphadenopathy of the submandibular lymph node. At this time, marked lymphadenopathy of the prescapular and popliteal lymph nodes was also appreciated. The CBC showed a moderate leukocytosis (38.4 × 109 cells/L, reference interval [RI] 4.8-13.9 × 109 cells/L) characterized by a moderate lymphocytosis (28.4 × 109 cells/L, RI 1.0-4.6 × 109 cells/L). Evaluation of peripheral blood and enlarged prescapular and popliteal lymph nodes revealed two morphologically different populations of homogeneous lymphocytes, with the lymphocyte population in the lymph nodes being distinct from that in the blood smear. Flow cytometry of peripheral blood revealed CD45-, CD5+, CD4-, CD8-, variably CD21+ neoplastic lymphocytes compatible with T-zone lymphocytes due to the absence of CD45 expression. Flow cytometry of the lymph node aspirate indicated a distinct population of CD21+ lymphocytes consistent with a B-cell phenotype along with a smaller proportion of the T-zone lymphocytes observed in the blood confirming the presence of two distinct populations of neoplastic lymphocytes, one involving T cells, and the other involving B cells.

Canine B-cell lymphoma is a clinically heterogenous disease; however, it is generally treated as a single disease entity. The purpose of this clinical trial was to prospectively evaluate naïve canine B-cell lymphoma patients using histopathology, flow cytometry (FC) and a standardized chemotherapy protocol to better define subsets of this disease that may respond differently to treatment. Sixty-four dogs with naïve multicentric B-cell lymphoma were treated with a standardized 19-week CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy protocol. Most of the dogs (84.3%) were diagnosed with diffuse large B-cell lymphoma (DLBCL), followed by nodal marginal zone (7.8%), small B-cell (4.7%), Burkitt-like (1.6%) and follicular lymphoma (1.6%). FC confirmed the diagnosis of B-cell lymphoma in all cases. There were no clear phenotyping differences between the subtypes of B-cell lymphoma detectable by our FC panel. The histologic subtypes in this study exhibited a range of forward scatter values on flow cytometry, but all of the DLBCL cases were higher than a value of 469, while the only cases with a lower forward scatter value were follicular lymphoma and diffuse small B-cell lymphoma. Dogs with DLBCL had a significantly better objective response rate to the CHOP protocol (96.3%) than the non-DLBCL subtypes (70%, P = .024). The median progression-free survival time for patients with DLBCL (233 days) was significantly longer than that of all other histopathologic subgroups combined (163 days, P = .0005).

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.

Background Rabacfosadine (RAB, Tanovea‐CA1) is a novel chemotherapy agent conditionally approved for the treatment of lymphoma in dogs. Hypothesis/Objectives To determine the efficacy and safety of RAB in dogs with lymphoma. Animals One hundred and fifty‐eight client‐owned dogs with naïve or relapsed multicentric lymphoma were prospectively enrolled from January to October 2019. Methods Dogs were randomized to receive RAB or placebo at a 3 : 1 ratio. Treatment was given every 21 days for up to 5 treatments. Study endpoints included progression‐free survival (PFS), overall response rate (ORR) at a given visit, best overall response rate (BORR), and percent progression free 1 month after treatment completion. Safety data were also collected. Results The median PFS was significantly longer in the RAB group compared to placebo (82 vs 21 days; P

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

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.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.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.The corrected perpendicular drop gating method had20% error for IgG/γ-globulin and IgM/β-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.Densitometric quantitation is a valid technique for measuring M-proteins in the β- 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.

T-cell lymphomas (TCL) are a diverse group of neoplasms with variable diagnostic features, pathophysiologies, therapeutic responses and clinical outcomes. In dogs, TCL includes indolent and aggressive tumours such as T-zone lymphoma (TZL) and peripheral T-cell lymphoma (PTCL), respectively. Delineation of molecular subtypes and investigation into underlying pathophysiologies of aggressive TCLs remains inadequate. We investigate the correlations between flow cytometry and histopathology of 73 cases of nodal TCL. The majority of cases (82.2%) were characterized as CD4+ TCL by flow cytometry. Fewer cases were classified as CD8+ TCL (6.8%) or CD4- CD8- TCL (11.0%). All cases, regardless of immunophenotype, exhibited conserved histologic features consistent with the WHO classification of PTCL. Histologic subsets of PTCL corresponding to immunophenotypic features were not identified. Neoplastic cell size determined by flow cytometry correlated significantly with mitotic rate. RNA-seq was performed on a subset of CD4+ PTCL cases (n = 6) and compared with sorted control CD4+ T-cells. The gene expression pattern of CD4+ PTCL was similar between all cases regardless of breed. PTCL was enriched in pathways representing G-coupled protein receptor signalling, extracellular matrix remodelling and vascular development, immune signalling and mitotic activity. Furthermore, global gene expression changes were consistent with downregulation of PTEN signalling and upregulation of the MTOR-PI3K-ATK axis. In this study, we evaluated the correlations between flow cytometry, histopathology and gene expression within a large cohort of nodal TCLs. We further demonstrate the ability of flow cytometry to identify a subtype of T-cell lymphoma, CD4+ PTCL, with a uniform histomorphology and gene expression profile.

Background T zone lymphoma (TZL), a histologic variant of peripheral T cell lymphoma, represents about 12% of all canine lymphomas. Golden Retrievers appear predisposed, representing over 40% of TZL cases. Prior research found that asymptomatic aged Golden Retrievers frequently have populations of T zone-like cells (phenotypically identical to TZL) of undetermined significance (TZUS), potentially representing a pre-clinical state. These findings suggest a genetic risk factor for this disease and caused us to investigate potential genes of interest using a genome-wide association study of privately-owned U.S. Golden Retrievers. Results Dogs were categorized as TZL (n = 95), TZUS (n = 142), or control (n = 101) using flow cytometry and genotyped using the Illumina CanineHD BeadChip. Using a mixed linear model adjusting for population stratification, we found association with genome-wide significance in regions on chromosomes 8 and 14. The chromosome 14 peak included four SNPs (Odds Ratio = 1.18–1.19, p = .3 × 10− 5–5.1 × 10− 5) near three hyaluronidase genes (SPAM1, HYAL4, and HYALP1). Targeted resequencing of this region using a custom sequence capture array identified missense mutations in all three genes; the variant in SPAM1 was predicted to be damaging. These mutations were also associated with risk for mast cell tumors among Golden Retrievers in an unrelated study. The chromosome 8 peak contained 7 SNPs (Odds Ratio = 1.24–1.42, p = 2.7 × 10− 7–7.5 × 10− 5) near genes involved in thyroid hormone regulation (DIO2 and TSHR). A prior study from our laboratory found hypothyroidism is inversely associated with TZL risk. No coding mutations were found with targeted resequencing but identified variants may play a regulatory role for all or some of the genes. Conclusions The pathogenesis of canine TZL may be related to hyaluronan breakdown and subsequent production of pro-inflammatory and pro-oncogenic byproducts. The association on chromosome 8 may indicate thyroid hormone is involved in TZL development, consistent with findings from a previous study evaluating epidemiologic risk factors for TZL. Future work is needed to elucidate these mechanisms.

Emerging details of the gene expression and mutational features of canine lymphoma and leukemia demonstrate areas of similarities and differences between disease subsets in the humans and dogs. Many features of canine diffuse large B-cell lymphoma resemble the ABC form of human DLBCL, including constitutive activation of the NF-kB pathway, and almost universal presence of double expressing MYC/BCL2 lymphomas. Frequent TRAF3 mutations and absence of BCL6 expression are differences with the human disease that need further exploration. Canine peripheral T-cell lymphoma is more common in dogs than in people and behaves in a similarly aggressive manner. Common features of canine and human PTCL include activation of the PI3 kinase pathways, loss of PTEN, and the tumor suppressor CDKN2. There is insufficient data available yet to determine if canine PTCL exhibits the GATA3-TBX21 dichotomy seen in people. Common to all forms of canine lymphoproliferative disease are breed-specific predilections for subsets of disease. This is particularly striking in PTCL, with the Boxer breed being dramatically overrepresented. Breed-specific diseases provide an opportunity for uncovering genetic and environmental risk factors that can aid early diagnosis and prevention.

Key Clinical Message 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.

Background KIT inhibitors, such as toceranib (TOC), and vinblastine (VBL) have not been prospectively compared in the treatment of macroscopic mast cell tumors (MCTs). Also, it is unknown whether VBL or TOC is superior for treating MCT without c‐kit mutations. Hypothesis/Objectives To determine the value of KIT genotyping and localization in treatment decisions for dogs with macroscopic MCT. We hypothesized that c‐kit mutated MCT would have a better response to TOC than VBL. Animals Eighty‐eight client‐owned dogs with macroscopic MCT. Methods Prospective, randomized trial. Dogs were randomized to TOC (2.75 mg/kg EOD) or VBL (2.5 mg/m2 weekly × 4 then EOW) by KIT localization and c‐kit mutation status using an adaptive randomization scheme. Results Sixty dogs were allocated to TOC and 28 to VBL. Of the dogs receiving TOC, 20% had c‐kit mutations, compared to 30% receiving VBL (P = 0.74). Overall response rates were 46% (TOC) and 30% (VBL) (odds ratio = 1.56 [0.62–3.92]; P = 0.28). Median progression‐free survival (PFS) for dogs receiving VBL was 78 days (7–1,521) and for TOC 95.5 (14–990); hazard ratio (HR) = 1.34 [0.72–2.50]; P = 0.36. Median overall survival (OS) was 241.5 days (10–1,521) for the VBL group and 159 (20–990) for the TOC group; HR = 0.80 ([0.45–1.41]; P = 0.44). Conclusions and Clinical Importance Neither PFS nor OS was significantly different between treatment groups. As the proportion of dogs with c‐kit mutations was not different between treatment groups in this population of dogs, c‐kit mutation status did not predict treatment response.

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.

Canine T-zone lymphoma (TZL) is a subtype of T-cell lymphoma characterized by unique histologic pattern and cytomorphology, immunophenotypic loss of CD45 expression, and an indolent clinical behaviour. Dogs with TZL typically present with 1 or more enlarged lymph nodes and/or lymphocytosis. We describe a novel extranodal presentation of TZL involving the tongue. Twelve dogs with tongue masses were diagnosed with lingual TZL based on a variable combination of immunophenotyping via flow cytometry, cytology, histopathology, immunohistochemistry and/or PCR for antigen receptor rearrangement (PARR) assay. Eleven dogs exhibited concurrent lymphocytosis and/or lymph node enlargement. Three cases were initially diagnosed as plasma cell tumours based on histology alone, thereby revealing a potential diagnostic challenge. Seven dogs achieved clinical remission and 4 achieved stable disease following variable treatment, consistent with the indolent nature of typical TZL involving the lymph nodes and peripheral blood. In 1 case the TZL resulted in progressive disease and failure to respond to treatment. In this case, the TZL exhibited histologic features of a higher grade neoplasm. This case series highlights a unique presentation of TZL and identifies a new differential diagnosis for lingual neoplasia. In this study, we characterize the clinical presentation, diagnostic features and patient outcomes of 12 dogs with lingual TZL.

A 5-year-old male neutered Bernese Mountain Dog was presented for cutaneous plasmacytoma, which was treated by surgical excision. Four months later, the dog developed multiple skin masses, hyphema, pericardial and mild bicavitary effusions, myocardial masses, and marked plasmacytosis in the peripheral blood. Circulating plasma cells expressed CD34 and MHC class II by flow cytometry. Immunocytochemistry demonstrated that these cells were strongly positive for multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM-1) and weakly to moderately positive for Pax5. The dog was hypoglobulinemic but had a monoclonal IgA gammopathy detected by serum immunofixation electrophoresis. The PCR analysis of antigen receptor gene rearrangements (PARR) by fragment analysis using GeneScan methodology revealed that plasmacytoid cells in the original cutaneous plasmacytoma and peripheral blood had an identical immunoglobulin heavy chain gene (IgH) rearrangement, indicating that both populations were derived from the same neoplastic clone. Canine cutaneous plasmacytoma rarely progresses to a malignant form and plasma cell leukemia is rarely diagnosed in the dog. This report describes a case of cutaneous plasmacytoma progressing to plasma cell leukemia with a rapid and aggressive clinical course. This report also highlights the utility of flow cytometry, immunocytochemistry, immunofixation electrophoresis, and PARR by fragment analysis using GeneScan methodology in the diagnosis of this hematopoietic neoplasm.

Canine cutaneous mast cell tumor (MCT) is the most common canine skin tumor and exhibits variable biologic behavior. Signaling through the KIT receptor tyrosine kinase promotes cellular proliferation and survival and has been shown to play a role in MCT progression. Despite investigations into numerous biomarkers and the proposal of several grading schemas, no single marker or grading system can accurately predict outcome in canine MCT. The first aim of this study was to develop an immunohistochemical assay to measure phosphorylated KIT (pKIT) to investigate its association with 2 commonly used grading systems and other established prognostic markers for canine MCT. Thirty-four archived MCTs were evaluated for expression of pKIT and Ki-67, KIT localization, mitotic count, mutations in exons 8 and 11 in c-kit, and grading by the Patnaik and 2-tier systems. Expression of pKIT was significantly ( P < .05) correlated with the 2-tier grading scheme and c-kit mutation. Correlation approached significance ( P = .06) with Mitotic Index (MI) and Ki-67. An additional aim was to determine whether pKIT labeling provides a pharmacodynamic marker for predicting response to the receptor tyrosine kinase inhibitor toceranib (TOC). MCTs from 4 of 7 patients demonstrated a partial response to TOC. pKIT expression was assessed by immunohistochemistry in biopsies obtained before and 6 hours after the patients were treated with TOC. Reduced pKIT expression after TOC treatment was demonstrated in 3 of the 4 patients with a partial response compared to 1 of the 3 nonresponders. Collectively, these results demonstrate that immunohistochemical detection of pKIT may be a clinically relevant assay to evaluate the activation status of the major oncogenic pathway in canine MCT.

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.

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.

BACKGROUND Flow cytometry (FC) is used increasingly in veterinary medicine for further characterization of hematolymphoid cells. Guidelines for optimizing assay performance and interpretation of results are limited, and concordance of results across laboratories is unknown. OBJECTIVES This study aimed to determine inter-investigator agreement on the interpretation of FC results from split samples analyzed in different laboratories using various protocols, cytometers, and software; and on the interpretation of archived FC standard (FCS) data files contributed by the different investigators. METHODS This was a multicenter observational cross-sectional study. Anticoagulated blood or lymph node aspirate samples from nine client-owned dogs were aliquoted and shipped to participating laboratories. Samples were analyzed with individual laboratory-developed protocols. In addition, FCS files from a set of separate samples from 11 client-owned dogs were analyzed by participating investigators. A person not associated with the study tabulated the results and interpretations. Agreement of interpretations was assessed with Fleiss' kappa statistic. RESULTS Prolonged transit times affected sample quality for some laboratories. Overall agreement among investigators regarding the FC sample interpretation was strong (κ = 0.86 ± 0.19, P

One of the primary objectives of the Oncology-Pathology Working Group (OPWG), a joint initiative of the Veterinary Cancer Society and the American College of Veterinary Pathologists, is for oncologists and pathologists to collaboratively generate consensus documents to standardize aspects of and provide guidelines for oncologic pathology. Consensus is established through critical review of peer-reviewed literature relevant to a subgroup's particular focus. Subsequent acceptance and approval of the document by the OPWG membership at large establishes consensus. The intent of this publication is to help educate practitioners and pathologists on the value of diagnostics related to the KIT receptor tyrosine kinase for canine cutaneous mast cell tumours and to provide a guide for the use of these tests in veterinary medicine. This document represents the opinions of the OPWG and the authors and does not constitute a formal endorsement by the American College of Veterinary Pathologists or the Veterinary Cancer Society.

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.

Minimally invasive techniques used to evaluate canine peripheral lymphadenopathy (PLN), including fine needle aspiration biopsy with cytological evaluation (FNAB-C) and flow cytometry (FC), have benefits and limitations. The cell block (CB) method is an alternate processing technique in which fine needle aspirate biopsy samples are concentrated, fixed, and embedded in paraffin for routine histological processing/staining. Utilizing three observers, we determined the diagnostic value of the CB in evaluating canine PLN across six categories (non-diagnostic, reactive, inflammatory/infectious, probable lymphoma and lymphoma, metastatic neoplasia) and correlated findings to immunophenotypic and clonal antigen receptor rearrangement results in canine nodal lymphoma. Eighty-five paired FNAB-C and CB samples were evaluated from canine patients presenting to the University of Minnesota Veterinary Oncology or Internal Medicine services. Diagnostic quality samples were obtained in 55/85 (65%) CB and 81/85 (95%) FNAB-C samples, respectively, and nodal pathology impacted CB diagnostic yield. Overall percent agreement between diagnostic-quality FNAB-C and CB samples was 86%, but increased to 95% if the categories of lymphoma and probable lymphoma were combined. There was 100% agreement for both the diagnoses of metastatic neoplasia and reactive lymph nodes and 92% agreement for the diagnosis of lymphoma/probable lymphoma. Using immunohistochemistry (IHC), CB samples correctly immunophenotyped 22/23 (96%) cases of B-cell lymphoma, but only 1/6 (17%) cases of T-cell lymphoma. IHC was not completed on nine cases of lymphoproliferative disease because of insufficient cellularity. When the CB method (CBM) yielded diagnostic quality samples there was good to excellent agreement with FNAB-C samples and CB samples were suitable for some IHC tests.

The purpose of this cross-sectional study was to examine differences in the geographic distribution of two distinct subtypes of canine lymphoma (CL), B-cell lymphoma (BCL) and T-zone lymphoma (TZL), in the USA while accounting for heritable risks associated with the outcome of disease through inclusion of only one breed of dog. This study included 454 Golden retrievers and associations between geographic areas of the USA and the phenotypic variant of lymphoma were examined using multivariable logistic regression. There was a detectable difference in the geographic distribution of BCL and TZL with dogs in the Northeast [odds ratio (OR) = 3.4, 95% confidence interval (CI) = 1.6–7.0] and East North Central regions (OR = 12.1, 95%CI = 3.6–40.5) being more likely to be diagnosed with TZL as compared to dogs in the Mountain region of the USA. The finding of non-random geographic distribution of lymphoma subtypes suggest that environmental risk factors may contribute to the development of different types of CL.

Background B-cell chronic lymphocytic leukemia (B-CLL) is the most common hematopoietic malignancy in humans in the developed world and the primary risk factor is genetic. Dogs also develop B-CLL, but there is no systematic description of the disease in dogs. Understanding the epidemiology of B-CLL in dogs may help practitioners recognize the disease and position the dog as a model for future genetic studies. Objectives To describe B-CLL presentation in dogs, its clinicopathologic findings, and breed predisposition. Animals Four hundred and ninety-one dogs with B-CLL and 5,673 control dogs with suspicion of a lymphoproliferative disorder (LPD). Methods Retrospective cross-sectional study of dogs for which samples were submitted to the Colorado State University Clinical Immunology Laboratory for immunophenotyping between 2010 and 2014. To assess breed predilection, dogs with B-CLL were compared to those with suspicion of other LPDs using logistic regression. Results The median age was 11 years with no sex predilection. Half of the dogs presented with peripheral lymphadenopathy or splenomegaly and 26% had anemia. Eleven small-breed dogs had significantly increased odds of B-CLL. In addition, English Bulldogs had an increased risk and a unique presentation: these dogs were diagnosed at a median of 6 years and expressed lower class II MHC and CD25. Conclusions B-cell chronic lymphocytic leukemia is overrepresented in small-breed dogs. Future genetic studies of these breeds may identify genetic risk factors. The unique presentation of English Bulldogs provides evidence of multiple forms of this disease. Additional studies are necessary to determine whether presenting signs are associated with survival.

OBJECTIVE To describe cytologic characteristics of renal fine-needle aspirate (FNA) samples from dogs, evaluate proportions of cytologic specimens deemed adequate for interpretation (diagnostic yield), assess diagnostic utility of cytologic examination for neoplastic and nonneoplastic diseases, and characterize ultrasonographic features of evaluated kidneys to determine whether the imaging characteristics could be used to inform cytologic interpretations. DESIGN Retrospective, observational study. SAMPLE 102 cytologic specimens and 97 ultrasonographic studies from 100 dogs. PROCEDURES Medical records were reviewed to identify dogs that underwent ultrasound-guided renal FNA. Slides were categorized as adequate or inadequate for interpretation; adequate slides were used for retrospective cytologic diagnosis. Sensitivity, specificity, and predictive values of cytologic examination for detection of neoplastic and nonneoplastic conditions were calculated by comparison with histologic or lymphoid cell clonality assay results. Ultrasonographic characteristics of neoplastic and nonneoplastic renal lesions were described. RESULTS 74 of 102 (72%) specimens had slides adequate for interpretation; 26 were included in the diagnostic accuracy analysis. Sensitivity of cytologic examination was 78% and 50% for detection of neoplastic and nonneoplastic conditions, respectively, with specificities of 50% and 77%, respectively; sensitivity for detection of lymphoma was 100%. Ultrasonographic appearance of kidneys with confirmed neoplasia varied; masses were most commonly found in kidneys with carcinoma (5/5), lymphoma (5/7), or other neoplasia (3/4) and absent in kidneys with nonneoplastic conditions (n = 5). CONCLUSIONS AND CLINICAL RELEVANCE Renal FNA specimens were adequate for interpretation at rates comparable with those reported for other organs and were considered clinically useful for diagnosis of neoplasia. Imaging characteristics may potentially aid differentiation between neoplastic and nonneoplastic lesions; however, further investigation is needed.