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Abstract 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 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 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 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

Abstract 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.

Abstract 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 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 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.

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.

The non-Hodgkin lymphomas (NHLs) are a heterogeneous family of lymphoid malignancies that are among the most common neoplasms of both dogs and humans. Owing to shared molecular, signaling, incidence, and pathologic features, there is a strong framework supporting the utilization of canine lymphoma as a comparative, large animal model of human NHL. In alignment with the biologic similarities, the current approach towards the diagnosis and classification of canine lymphoma is based upon the human World Health Organization guidelines. While this approach has contributed to an increasing appreciation of the potential biological scope of canine lymphoma, it has also become apparent that the most appropriate diagnostic philosophy must be multimodal, namely by requiring knowledge of microscopic, immunophenotypic, and clinical features before establishing a final disease diagnosis. This review seeks to illustrate the comparative similarities and differences in the diagnosis of canine lymphoma through the presentation of the microscopic and immunophenotypic features of its most common forms.

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.

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.

Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (p(raw) = 2.3 × 10⁻⁶, p(genome) = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p < 0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation.