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Background: To explore the safety and utility of combining low dose single‐agent doxorubicin with a canine specific anti‐CD20 monoclonal antibody (1E4‐cIgGB) in client owned dogs with untreated B‐cell lymphoma. Animals: Forty‐two client‐owned dogs with untreated B‐cell lymphoma. Methods: A prospective, single arm, open label clinical trial of dogs with B‐cell lymphoma were enrolled to receive 1E4‐cIgGB and doxorubicin in addition to 1 of 3 immunomodulatory regimens. B‐cell depletion was monitored by flow cytometry performed on peripheral blood samples at each visit. Results: Dogs demonstrated a statistically significant depletion in CD21+ B‐cells 7 days following the first antibody infusion (median fraction of baseline at 7 days = 0.04, P <.01) that persisted throughout treatment (median fraction of baseline at 21 days = 0.01, P <.01) whereas CD5+ T‐cells remained unchanged (median fraction of baseline at 7 days = 1.05, P =.88; median fraction of baselie at 7 days = 0.79, P =.42; Figure 1; Supplemental Table 3). Recovery of B‐cells was delayed, with at Day 196, only 6/17 dogs (35%) remaining on the study had CD21+ counts >0.5 of baseline, indicating sustained B cell depletion at 4+ months after the final treatment. 1E4‐cIgGB was well tolerated with only 1 dog exhibiting a hypersensitivity event within minutes of the last antibody infusion. Conclusions: The canine 1E4‐cIgGB anti‐CD20 monoclonal antibody is apparently safe when administered with doxorubicin and effectively depletes B‐cells in dogs with DLBCL. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The authors present insights on prions, which are hypothesized as infectious agents devoid of instructional nucleic acid and propagate themselves without a genetic code, instead encipher their infectious nature structurally, within the protein conformation itself. Topics discussed include prion interaction during infectious prion disease, the significant role of innate immunity in combating and abetting peripheral prion pathogenesis and the ability of B cells to trap prions.

Examines the resistance of herpes stromal keratitis (HSK) to an IgG2a-derived peptide. HSK as a leading cause of human blindness; Association with allotypic variation in immunoglobulin genes; Shared expression of peptide subsequences.

Large granular lymphocytes (LGLs) have only been anecdotally reported in HIV infection. We previously reported an LGL lymphocytosis in FIV-infected cats associated with a rise in FIV proviral loads and a marked neutropenia that persisted during chronic infection. Extensive immunophenotyping of peripheral blood mononuclear cells in cats chronically infected with FIV were identified LGLs as CD8lo + FAS + ; this cell population expanded commensurate with viral load. CD8lo + FAS + cells expressed similar levels of interferon-γ compared to CD8lo + FAS + cells from FIV-naive control animals, yet CD3ɛ expression, which was increased on total CD8 + T cells in FIV-infected cats, was decreased on CD8lo + FAS + cells. Down-modulation of CD3 expression was reversed after culturing PBMC for 3 days in culture with ConA/IL-2. We identified CD8lo + FAS + LGLs to be polyclonal T cells lacking CD56 expression. Blood smears from HIV-infected individuals and SIVmac239-infected rhesus macaques revealed increased LGLs compared to HIV/SIV negative counterparts. In humans, there was no correlation with viral load or treatment and in macaques the LGLs arose in acute SIV infection with increases in viremia. This is the first report describing and partially characterizing LGL lymphocytosis in association with lentiviral infections in three different species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 <.0001, HR 6.265 [95% CI 3.947‐9.945]). The BORR for RAB‐treated dogs was 73.2% (50.9% complete response [CR], 22.3% partial response [PR]) and 5.6% (0% CR, 5.6% PR) for placebo‐treated dogs (P <.0001). One month after the last treatment, 37 RAB‐treated dogs (33%) were progression free compared with no placebo‐treated dogs (P <.0001). The most common adverse events observed in the RAB group were diarrhea (87.5%), decreased appetite (68.3%), and vomiting (68.3%) and were generally low grade and reversible. Serious adverse events were reported in 24 RAB‐treated (20%) and 5 placebo‐treated dogs (13%). Conclusions and Clinical Importance: Rabacfosadine demonstrated statistically significant antitumor efficacy in dogs with lymphoma when administered every 21 days for up to 5 treatments as compared to placebo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: T‐zone lymphoma (TZL), an indolent disease in older dogs, comprises approximately 12% of lymphomas in dogs. TZL cells exhibit an activated phenotype, indicating the disease may be antigen‐driven. Prior research found that asymptomatic aged Golden Retrievers (GLDRs) commonly have populations of T‐zone‐like cells (phenotypically identical to TZL) of undetermined significance (TZUS). Objective: To evaluate associations of inflammatory conditions, TZL and TZUS, using a case‐control study of GLDRs. Animals: TZL cases (n = 140), flow cytometrically diagnosed, were identified through Colorado State University's Clinical Immunology Laboratory. Non‐TZL dogs, recruited through either a database of owners interested in research participation or the submitting clinics of TZL cases, were subsequently flow cytometrically classified as TZUS (n = 221) or control (n = 147). Methods: Health history, signalment, environmental, and lifestyle factors were obtained from owner‐completed questionnaires. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated using multivariable logistic regression, obtaining separate estimates for TZL and TZUS (versus controls). Results: Hypothyroidism (OR, 0.3; 95% CI, 0.1‐0.7), omega‐3 supplementation (OR, 0.3; 95% CI, 0.1‐0.6), and mange (OR, 5.5; 95% CI, 1.4‐21.1) were significantly associated with TZL. Gastrointestinal disease (OR, 2.4; 95% CI, 0.98‐5.8) had nonsignificantly increased TZL odds. Two shared associations for TZL and TZUS were identified: bladder infection or calculi (TZL OR, 3.5; 95% CI, 0.96‐12.7; TZUS OR, 5.1; 95% CI, 1.9‐13.7) and eye disease (TZL OR, 2.3; 95% CI, 0.97‐5.2; TZUS OR, 1.9; 95% CI, 0.99‐3.8). Conclusions and Clinical Importance: These findings may elucidate pathways involved in TZUS risk and progression from TZUS to TZL. Further investigation into the protective association of omega‐3 supplements is warranted. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, and immunoglobulin heavy variable region (IGHV) gene mutational status is an important prognostic marker. IGHV mutational status has not been previously examined in canine CLL. We sequenced the IGHV-D-J rearrangements from 55 canine patients with CLL, including 36 non-Boxer and 19 Boxer dogs. The majority of non-Boxers (75%) had mutated IGHV genes, whereas the majority of Boxers (79%) had unmutated IGHV genes. IGHV3-41 and IGHV3-67 gene usage was significantly higher in Boxers with CLL compared to non-Boxers. Additionally, 11 Boxers with large B-cell lymphoma and the normal IGHV repertoire of six control dogs (three Boxers and three non-Boxers) were sequenced. IGHV3-41 was preferentially used in Boxers with other forms of lymphoma and without lymphoproliferative disease. However, preferential use of unmutated IGHV genes was unique to Boxers with CLL, suggesting Boxers may be a valuable model to investigate unmutated CLL. [ABSTRACT FROM AUTHOR]

Dogs, with their breed-determined limited genetic background, are great models of human disease including cancer. Canine B-cell lymphoma and hemangiosarcoma are both malignancies of the hematologic system that are clinically and histologically similar to human B-cell non-Hodgkin lymphoma and angiosarcoma, respectively. Golden retrievers in the US show significantly elevated lifetime risk for both B-cell lymphoma (6%) and hemangiosarcoma (20%). We conducted genome-wide association studies for hemangiosarcoma and B-cell lymphoma, identifying two shared predisposing loci. The two associated loci are located on chromosome 5, and together contribute ~20% of the risk of developing these cancers. Genome-wide p-values for the top SNP of each locus are 4.6×10-7 and 2.7×10-6, respectively. Whole genome resequencing of nine cases and controls followed by genotyping and detailed analysis identified three shared and one B-cell lymphoma specific risk haplotypes within the two loci, but no coding changes were associated with the risk haplotypes. Gene expression analysis of B-cell lymphoma tumors revealed that carrying the risk haplotypes at the first locus is associated with down-regulation of several nearby genes including the proximal gene TRPC6, a transient receptor Ca2+-channel involved in T-cell activation, among other functions. The shared risk haplotype in the second locus overlaps the vesicle transport and release gene STX8. Carrying the shared risk haplotype is associated with gene expression changes of 100 genes enriched for pathways involved in immune cell activation. Thus, the predisposing germ-line mutations in B-cell lymphoma and hemangiosarcoma appear to be regulatory, and affect pathways involved in T-cell mediated immune response in the tumor. This suggests that the interaction between the immune system and malignant cells plays a common role in the tumorigenesis of these relatively different cancers. [ABSTRACT FROM AUTHOR]

Background Mast cell tumors (MCTs) are the most common skin tumors in dogs and exhibit variable biologic behavior. Mutations in the c-kit proto-oncogene are associated with the tumorigenesis of MCTs, resulting in growth factor-independent and constitutive phosphorylation of the KIT receptor tyrosine kinase (RTK). Toceranib (TOC) phosphate (Palladia®) is a KIT RTK inhibitor that has biological activity against MCTs. Despite these benefits, patients ultimately develop resistance to TOC. Therefore, there is a need to identify distinguishing clinical and molecular features of resistance in this population. Results The canine C2 mastocytoma cell line contains an activating mutation in c-kit. Three TOCresistant C2 sublines (TR1, TR2, TR3) were established over seven months by growing cells in increasing concentrations of TOC. TOC inhibited KIT phosphorylation and cell proliferation in a dose-dependent manner in the treatment-naïve, parental C2 line (IC50 < 10 nM). In contrast, the three sublines were resistant to growth inhibition by TOC (IC50 > 1,000 nM) and phosphorylation of the KIT receptor was less inhibited compared to the TOCsensitive C2 cells. Interestingly, sensitivity to three structurally distinct KIT RTK inhibitors was variable among the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents vinblastine and lomustine. Sequencing of c-kit revealed secondary mutations in the juxtamembrane and tyrosine kinase domains of the resistant sublines. These included point mutations in TR1 (Q574R, M835T), TR2 (K724R), and TR3 (K580R, R584G, A620S). Additionally, chronic TOC exposure resulted in c-kit mRNA and KIT protein overexpression in the TOC-resistant sublines compared to the parental line. C2, TR1, TR2, and TR3 cells demonstrated minimal P-glycoprotein (P-gp) activity and no functional P-gp. Conclusions This study demonstrates the development of an in vitro model of acquired resistance to targeted therapy in canine MCTs harboring a c-kit-activating mutation. This model may be used to investigate the molecular basis of and strategies to overcome TOC resistance. [ABSTRACT FROM AUTHOR]

Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development. However, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude that this system has broad applications for in vitro preclinical development for B-cell malignancies. [ABSTRACT FROM AUTHOR]

Abstract: A novel canine lymphoma cell line, OSW, was established from the malignant pleural effusion of a dog with peripheral T-cell lymphoma. The immunoprofile as determined by flow cytometry was as follows: positive for CD45, CD49d, CD18, CD11a; weakly positive for CD11b, CD11c, CD11d; and negative for CD45RA, CD1a, CD1c, CD3, TCRαβ, TCRγδ, CD4, CD5, CD8a, CD8b, CD90(Thy1), CD21, MHCII, CD14(TUK4), CD34, and MPO. Immunocytochemistry of cytospin preparations was negative for cytoplasmic CD3, CD79a, and MPO, but was positive for CD20. The cell line had an oligoclonal T-cell receptor gamma (TCRγ) gene rearrangement. Array comparative genomic hybridization (aCGH) and single locus probe (SLP) analysis showed that there were copy number increases of loci on dog chromosome 13 (CFA 13), and copy number decreases were evident for regions of CFA 11, 22, 26, 30 and 32, which include several of the more common chromosomal aberrations reported previously in canine lymphoma. The OSW cell line grows rapidly in vitro and is tumorigenic as a xenograft in SCID/NOD mice. OSW represents one of only a few reported canine lymphoma cell lines and is the most thoroughly characterized. This cell line and xenograft represent significant in vitro and in vivo models, respectively, for comparative and translational lymphoma research. [Copyright &y& Elsevier]

We do not know the relationship of IgG4-RD in dogs and cases previously diagnosed as hypereosinophilia syndrome but suspect that at least some cases of canine IgG4-RD have been diagnosed as idiopathic hypereosinophilia syndrome in the past. Except for the two cases of IgG4-producing multiple myeloma published as part of our evaluation of the immunofixation protocol,[8] serum IgG4 increases in dogs have been much lower in the non-IgG4-RD cases than was observed in our IgG4-RD case, as judged by the electrophoretogram changes. Documentation of increased serum IgG4 concentration is a diagnostic criterion of IgG4-RD in humans and may help solidify the association of IgG4-RD and dogs with chronic pancreatitis. [Extracted from the article]