BackgroundCanine T‐cell lymphoma (TCL) is clinically and histologically heterogeneous with some forms, such as T‐zone lymphoma (TZL), having an indolent course. Immunophenotyping is an important tool in the classification of TCL in people, and can be equally useful in dogs.Hypothesis/ObjectivesWe hypothesized that loss of expression of the CD45 antigen is a specific diagnostic feature of TZL.AnimalsTwenty dogs with concurrent histology and immunophenotyping by flow cytometry were studied in depth. An additional 494 dogs diagnosed by immunophenotyping were used to characterize the population of dogs with this disease.MethodsLymph node biopsies from 35 dogs with TCL were classified by 2 pathologists using WHO criteria. Twenty lymph nodes were from dogs with CD45− TCL and 15 were from CD45+ TCL. The pathologists were blinded to the flow cytometry findings. Outcome information was sought for the 20 dogs with CD45− lymphoma, and population characteristics of the additional 494 dogs were described.ResultsAll 20 CD45− cases were classified as TZL. The 15 CD45+ cases were classified as aggressive TCL and are described in an accompanying paper. TZL cases had a median survival of 637 days. Examination of 494 additional dogs diagnosed with TZL by immunophenotyping demonstrated that 40% of cases are in Golden Retrievers, are diagnosed at a median age of 10 years, and the majority have lymphadenopathy and lymphocytosis.Conclusions TZL has unique immunophenotypic features that can be used for diagnosis.

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

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.

Background: Differentiation between neoplastic and reactive lymphocytic proliferations can be challenging in cats. PCR for antigen receptor rearrangements (PARR) testing is a useful diagnostic tool to assess clonality of a lymphoid population.Previous feline PARR studies evaluated clonality of complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma (TRG) gene rearrangements. Objectives:We aimed to evaluate the sensitivity and specificity of feline PARR primers targeting complete IGH-VDJ and TRG rearrangements, as well as incomplete IGH-DJ, kappa deleting element (Kde), and immunoglobulin lambda light chain (IGL) gene rearrangements in defined feline neoplasms and nonneoplastic controls. Methods: Fluorescently labeled PCR primers were designed to amplify complete IGH-VDJ, incomplete IGH-DJ, Kde, IGL, and TRG gene rearrangements in two multiplexed PCR reactions, and PCR products were analyzed by fragment analysis. Fresh tissue samples from 12 flow cytometrically confirmed B-cell lymphomas, 26 cytologically confirmed gastric and renal lymphomas of presumed B-cell origin, 30 flow cytometrically confirmed T-cell leukemias, and 11 negative control cats were tested. Results: Using four immunoglobulin primer sets (IGH-VDJ, IGH-DJ, Kde, and IGL), clonal immunoglobulin rearrangements were detected in 87% (33/38) of the presumed B-cell neoplasms. The IGH-VDJ reaction alone only detected clonality in 50% (19/38) of these cases. TRG rearrangements were clonal in 97% (29/30) of the T-cell leukemia cases. All negative control samples had polyclonal immunoglobulin and TRG rearrangements. Conclusions: The PARR assay developed in this study is useful for assessing clonality in feline lymphoid neoplasms. Clonality assessment of incomplete IGH-DJ, Kde, and IGL rearrangements helped identify clonal B-cell neoplasms not detected with complete IGH-VDJ PARR alone. K E Y W O R D S antigen receptor rearrangement, lymphoma, T-cell receptor gamma S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Rout ED, Burnett RC, Yoshimoto JA, Avery PR, Avery AC. Assessment of immunoglobulin heavy chain, immunoglobulin light chain, and T-cell receptor clonality testing in the diagnosis of feline lymphoid neoplasia. Vet Clin Pathol. 2019;48(Suppl. 1):45-58. https ://doi.

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.