Assessment of immunoglobulin heavy chain, immunoglobulin light chain, and T‐cell receptor clonality testing in the diagnosis of feline lymphoid neoplasia

Abstract

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.

Immunophenotypic characterization and clinical outcome in cats with lymphocytosis

“…We previously determined that our IGH‐VDJ PARR reaction only detected 50% of feline B‐cell neoplasms, but targeting additional IG gene rearrangements increased B‐cell PARR sensitivity from 50% to 87% . The sensitivity of our TRG PARR primers to detect clonality in T‐cell neoplasms was 97%.…”

“…Polymerase chain reaction for antigen receptor rearrangements was performed on DNA extracted from blood samples, as previously described, to assess clonality of immunoglobulin (IG) and T‐cell receptor gamma (TRG) gene rearrangements. All 146 cases in the definition cohort and a subset of cases in the outcome cohort had PARR with primers targeting complete IG heavy chain V‐D‐J (IGH‐VDJ) and TRG gene rearrangements.…”

Polyclonal B‐cell lymphocytosis in English bulldogs

“…DNA quantity and quality were determined to be sufficient based on PARR results, as previously described. 17 PARR results were independently interpreted by the authors (E.D. Rout, A.C. Avery), blinded to the signalment and flow cytometry data.…”

“…Detection of incomplete IGH-DJ and IG light-chain rearrangements increases sensitivity in detecting B-cell and plasma cell neoplasms in humans, cats and dogs, as compared to complete IGH-VDJ rearrangements alone. 16,17,22,23 Incomplete IGH-DJ and Kde rearrangements are less prone to somatic hypermutation, and analysis of the IG kappa locus is useful in detecting clonality in canine tumors with somatic hypermutation, such as plasmacytomas. 16,22 The [25][26][27] Familial cases suggest a genetic predisposition and many cases have an isochromosome (3q) abnormality and chromosomal instability.…”

An introduction for the Veterinary Clinical Pathology Special Issue on clinical immunology

“…MacNeill et al tackle the challenge of diagnostic testing for immune‐mediated hemolytic anemia, and LeVine et al review the pathophysiology and diagnostic dilemmas of immune thrombocytopenia . Electrophoresis and immunofixation techniques to characterize serum proteins are reviewed by Moore et al Harris et al validate a densitometric assay to quantify monoclonal proteins in canine sera, and Rout et al assess immunoglobulin heavy and light chain, and T‐cell receptor clonality to diagnose feline lymphoid neoplasia . Bergman et al investigate interferences from canine anti‐mouse antibodies in hormone immunoassays, and Irvine et al assess and compare two immunoassays to measure serum cardiac Troponin I in dogs and cats .…”