Characterization of a PCR‐based lymphocyte clonality assay as a complementary tool for the diagnosis of feline lymphoma

Abstract

Differentiation between resident mature lymphocyte populations and small cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. In this prospective study, diagnostic sensitivity and specificity of different primer sets for routine diagnosis of feline TCR gamma (TCRG) and complete IG heavy chain (IGH) gene rearrangements were assessed. Fine needle aspirates from 20 feline lymphoma cases and lymph node material from 10 cats without hematopoietic neoplasia were subjected to clonality testing. Feline lymphoma cell lines and previously confirmed patient material served as positive control. Detection limits for clonal populations within a polyclonal background was 90% for B-cells and 50% for T-cells. Diagnostic sensitivity and specificity of the clonality assay were 70% and 90%. Overall diagnostic accuracy was 77%, positive predictive value 93% and negative predictive value 60%.

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

“…Additionally, we are designing IGL PARR primers to target other IGLV subgroups, which could increase the sensitivity of IGL PARR. The traditionally employed IGH‐VDJ assay only detected 50% of the B‐cell neoplasms in this study, which is within the range detected by some of the other publications assaying this region . This could be partly due to the types of samples selected; for example, if the B‐cell neoplasms sampled have a high degree of somatic hypermutation, primer annealing could be compromised.…”

“…The reported sensitivity for detecting a clonal TRG by PCR in T‐cell neoplasms has been relatively high, with 5/7 studies reporting sensitivities ≥79% . The sensitivity for detecting a clonal IGH receptor among feline B‐cell neoplasms has been historically lower, with sensitivity values ranging from 34% to 89% . One difficulty in detecting B‐cell neoplasms is that the IGH receptor can undergo somatic hypermutation, altering nucleotides at primer sites, which compromises primer binding.…”

“…Another challenge in assessing the diagnostic accuracy of the feline PARR assay in finding a gold standard to classify patients accurately. Most studies use histology and immunohistochemistry as a gold standard, but histology has limitations in both correctly detecting feline lymphoma and identifying lineage, especially in challenging cases with mixed B cells and T cells …”

Relationship of the mucosal microbiota to gastrointestinal inflammation and small cell intestinal lymphoma in cats

“…Small cell GI LSA closely resembles feline inflammatory bowel disease (IBD) in its clinical presentation,2 with both diseases having poorly defined etiologies. Various risk factors including genetic and molecular alterations,3, 4, 5 diet,3, 6, 7 and chronic inflammation8, 9, 10 have been linked to the development of both disorders. Still other studies have suggested an association between chronic mucosal inflammation and the progression of IBD to pronounced dysplasia in the GI tract 11, 12, 13, 14…”

Differentiation of lymphocytic‐plasmacytic enteropathy and small cell lymphoma in cats using histology‐guided mass spectrometry

“…8 The sensitivity and specificity of clonality assays in human and veterinary medicine vary widely with sensitivities between 70.0 and 97.6% and specificities between 54.3-98.7%. 7,[9][10][11][12][13][14][15] These wide ranges are the result of a combination of technical and biological variability. 16 Because of the inherently high error rate for clonality assays, there are rigorous preanalytical, analytical, and postanalytical standards published in human medicine under the EuroClonality/BIOMED-2 clonality standardization group guidelines.…”