The gene expression profile and cell of origin of canine peripheral T-cell lymphoma.

BACKGROUND: Peripheral T-cell lymphoma (PTCL) refers to a heterogenous group of T-cell neoplasms with poor treatment responses and survival times. Canine PTCL clinically and immunophenotypically resembles the most common human subtype, PTCL-not otherwise specified (PTCL-NOS), leading to interest in this canine disease as a naturally occurring model for human PTCL. Gene expression profiling in human PTCL-NOS has helped characterize this ambiguous diagnosis into distinct subtypes, but similar gene expression profiling in canine PTCL is lacking. METHODS: Bulk RNA-sequencing was performed on tumor samples from 33 dogs with either CD4+ (26/33), CD8+ (4/33), or CD4-CD8- (3/33) PTCL as diagnosed by flow cytometry, and sorted CD4+ and CD8+ lymphocytes from healthy control dogs. Following normalization of RNA-seq data, we performed differential gene expression and unsupervised clustering methods. Gene set enrichment analysis was performed to determine the enrichment of canine CD4+ PTCL for human PTCL-NOS, oncogenic pathways, and various stages of T-cell development gene signatures. We utilized gene set variation analysis to evaluate individual canine CD4+ PTCLs for various human and murine T-cell and thymocyte gene signatures. Cultured canine PTCL cells were treated with a pan-PI3K inhibitor, and cell survival and proliferation were compared to DMSO-treated controls. Expression of GATA3 and phosphorylated AKT was validated by immunohistochemistry. RESULTS: While the canine CD4+ PTCL phenotype exhibited a consistent gene expression profile, the expression profiles of CD8+ and CD4-CD8- canine PTCLs were more heterogeneous. Canine CD4+ PTCL had increased expression of GATA3, upregulation of its target genes, enrichment for PI3K/AKT/mTOR signaling, and downregulation of PTEN, features consistent with the more aggressive GATA3-PTCL subtype of human PTCL-NOS. In vitro assays validated the reliance of canine CD4+ PTCL cells on PI3K/AKT/mTOR signaling for survival and proliferation. Canine CD4+ PTCL was enriched for thymic precursor gene signatures, exhibited increased expression of markers of immaturity (CD34, KIT, DNTT, and CCR9), and downregulated genes associated with the T-cell receptor, MHC class II associated genes (DLA-DQA1, DLA-DRA, HLA-DQB1, and HLA-DQB2), and CD25. CONCLUSIONS: Canine CD4+ PTCL most closely resembled the GATA3-PTCL subtype of PTCL-NOS and may originate from an earlier stage of T-cell development than the more conventionally posited mature T-helper cell origin.

Atypical multiple myeloma in 3 young dogs.

Three dogs under 12 months old were diagnosed with atypical multiple myeloma (MM), having an aggressive multifocal anaplastic round cell sarcoma in bone marrow, viscera, and/or peripheral blood, which were confirmed by cytology and immunohistochemistry to be of plasma cell origin. The intramedullary sarcomas caused myelophthisis, osteolysis, and hypercalcemia. Complete or free light chain monoclonal gammopathy in the serum and/or urine was demonstrated by protein electrophoresis and immunofixation. The polymerase chain reaction for antigen receptor rearrangement assay performed on 2 cases identified a clonally rearranged immunoglobulin gene. Neoplastic cells lacked expression of CD45, CD3, CD18, CD21, CD34, and MHCII by flow cytometry. Immunohistochemistry revealed MUM1 immunoreactivity of the neoplastic cells. Combining all data, the diagnosis was MM. An aggressive form of MM in young dogs should be a differential diagnosis for patients with an immunoglobulin-productive, B cell-clonal, CD45-negative, MUM1-positive discrete cell neoplasm arising from the bone marrow.

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

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

Progressive gammopathy and coagulopathy in a young English bulldog.

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

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

Genome-wide association analysis of canine T zone lymphoma identifies link to hypothyroidism and a shared association with mast-cell tumors.

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.

Genome-wide association study identifies shared risk loci common to two malignancies in golden retrievers.

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.

Genome-wide assessment of recurrent genomic imbalances in canine leukemia identifies evolutionarily conserved regions for subtype differentiation.

Leukemia in dogs is a heterogeneous disease with survival ranging from days to years, depending on the subtype. Strides have been made in both human and canine leukemia to improve classification and understanding of pathogenesis through immunophenotyping, yet classification and choosing appropriate therapy remains challenging. In this study, we assessed 123 cases of canine leukemia (28 ALLs, 24 AMLs, 25 B-CLLs, and 46 T-CLLs) using high-resolution oligonucleotide array comparative genomic hybridization (oaCGH) to detect DNA copy number alterations (CNAs). For the first time, such data were used to identify recurrent CNAs and inclusive genes that may be potential drivers of subtype-specific pathogenesis. We performed predictive modeling to identify CNAs that could reliably differentiate acute subtypes (ALL vs. AML) and chronic subtypes (B-CLL vs. T-CLL) and used this model to differentiate cases with up to 83.3 and 95.8 % precision, respectively, based on CNAs at only one to three genomic regions. In addition, CGH datasets for canine and human leukemia were compared to reveal evolutionarily conserved copy number changes between species, including the shared gain of HSA 21q in ALL and ∼25 Mb of shared gain of HSA 12 and loss of HSA 13q14 in CLL. These findings support the use of canine leukemia as a relevant in vivo model for human leukemia and justify the need to further explore the conserved genomic regions of interest for their clinical impact.

Development of an in vitro model of acquired resistance to toceranib phosphate (Palladia®) in canine mast cell tumor.

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 TOC-resistant 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 TOC-sensitive 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.

A novel unstable duplication upstream of HAS2 predisposes to a breed-defining skin phenotype and a periodic fever syndrome in Chinese Shar-Pei dogs.

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.

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

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

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

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

Safety and biologic activity of a canine anti-CD20 monoclonal antibody in dogs with diffuse large B-cell lymphoma.

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.

Inactivation of Plasmodium spp. in plasma and platelet concentrates using riboflavin and ultraviolet light.

BACKGROUND: Photochemical treatment of blood products could help prevent transfusion-transmitted malaria and reduce the need for donor deferrals. In this study we evaluated the effectiveness of riboflavin and ultraviolet (UV) light against both Plasmodium falciparum, which causes the most severe form of human malaria, and Plasmodium yoelii, an in vivo murine model for malaria. STUDY DESIGN AND METHODS: Plasma and platelet (PLT) concentrates were inoculated with either P. falciparum- or P. yoelii-infected red blood cells (RBCs). Aliquots from each unit were collected after inoculation, after addition of riboflavin, and after treatment. In vitro P. falciparum growth was assessed using thin blood films of duplicate samples at 24, 48, 72, and 96 hours. P. yoelii parasitemia was followed in mice for 14 days postinoculation. RESULTS: In the in vitro studies, the mean P. falciparum parasitemia increased 12- to 19-fold in pretreatment samples, both before and after addition of riboflavin, after 96-hour culture. Few parasites were observed in Mirasol-treated units at 24 hours; those that were observed were degenerating. Through the remainder of the 96-hour culture period, cultures of treated samples were negative. In the in vivo study, mouse plasma containing P. yoelii-infected RBCs had a mean starting titer of 4.6 log mouse infectious dose 50%/mL. No infectious parasite was detected in treated samples. CONCLUSION: Treatment with riboflavin and UV light was effective at reducing viable P. falciparum in both PLT and plasma products by at least 3.2 logs. Additionally, an at least 4.4-log reduction was observed with P. yoelii.

Primary cutaneous gamma delta T-cell lymphoma with brain involvement and hemophagocytic syndrome.

Primary cutaneous gamma delta T-cell lymphoma is a rare diagnosis with only 40 reported cases. We describe a case of cutaneous gamma delta T-cell lymphoma with hemophagocytic syndrome and brain involvement that was not apparent morphologically on skin biopsy and was diagnosed as perifolliculitis and lobular panniculitis. The biopsy was sent later for molecular studies to the University of Washington, which demonstrated a T-cell clone. This case demonstrates that a T-cell clone may be present in a skin biopsy without morphologic or immunophenotypic evidence of lymphoma.

T-cell-rich, large B-cell lymphoma in the brain of a horse.

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.

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

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

Genome wide exploration of the methylome in aggressive B-cell lymphoma in Golden Retrievers reveals a conserved hypermethylome.

Few recurrent DNA mutations are seen in aggressive canine B cell lymphomas (cBCL), suggesting other frequent drivers. The methylated island recovery assay (MIRA-seq) or methylated CpG-binding domain sequencing (MBD-seq) was used to define the genome-wide methylation profiles in aggressive cBCL in Golden Retrievers to determine if cBCL can be better defined by epigenetic changes than by DNA mutations. DNA hypermethylation patterns were relatively homogenous within cBCL samples in Golden Retrievers, in different breeds and in geographical regions. Aberrant hypermethylation is thus suspected to be a central and early event in cBCL lymphomagenesis. Distinct subgroups within cBCL in Golden Retrievers were not identified with DNA methylation profiles. In comparison, the methylome profile of human DLBCL (hDLBCL) is relatively heterogeneous. Only moderate similarity between hDLBCL and cBCL was seen and cBCL likely cannot be accurately classified into the subtypes seen in hDLBCL. Genes with hypermethylated regions in the promoter-TSS-first exon of cBCL compared to normal B cells often also had additional hyper- and hypomethylated regions distributed throughout the gene suggesting non-randomized repeat targeting of key genes by epigenetic mechanisms. The prevalence of hypermethylation in transcription factor families in aggressive cBCL may represent a fundamental step in lymphomagenesis.

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

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

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

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