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Abstract Background Polymerase chain reaction for antigen receptor rearrangement (PARR) is a molecular diagnostic tool used for discrimination of lymphoid malignancies in dogs from benign processes. Assay variations have been described and are commercially available, but performance metrics are not uniformly reported. Objectives To describe performance (accuracy, sensitivity, specificity) and rigorous benchmarking of a PARR protocol (ePARR) in clinically relevant samples. Animals One hundred eighty-one client-owned dogs. Methods Lymphoma and benign tissues representative of the clinical spectrum with gold standard histopathologic and immunohistochemical diagnoses were collected. Assay development and benchmarking were performed on fresh frozen (FF) tissue, formalin-fixed paraffin-embedded (FFPE) tissue, flow cytometry pellets, and air-dried fine-needle aspirates (FNA). Assay performance was determined for FFPE from 56 dogs (18 B-cell lymphoma, 24 T-cell lymphoma, and 14 non-lymphoma), 80 frozen flow cytometry pellets (66 B-cell lymphoma, 14 T-cell lymphoma, 0 non-lymphoma), and 41 air-dried FNA slides (23 lymphoma, 18 non-lymphoma). Results For discrimination of lymphoma versus non-lymphoma, ePARR had 92% and 92% sensitivity and specificity on FFPE with 92% accuracy, 85% sensitivity from flow cytometry pellets (non-lymphoma was not evaluated to calculate specificity) with 85% accuracy, and 100% and 100% sensitivity and specificity for FNA with 100% accuracy. Stringent quality control criteria decreased assay success rate without significant performance improvement. Performance metrics were lower in most cases for discrimination of B- or T-cell versus non-B- or non-T-cell samples than for lymphoma versus non-lymphoma. Conclusions and Clinical Importance These benchmarking data facilitate effective interpretation and application of PARR assays in multiple sample types.

Cats have the highest incidence of lymphoma among all animal species. Lymphoma accounts for 41% of all malignant tumors in cats and is responsible for 90% of hematopoietic tumors in felines. Biopsies are considered the gold standard for diagnosis. Polymerase chain reaction (PCR)-based clonality assessment of antigen receptor gene rearrangements can be a valuable complementary tool for identifying infiltrating B-and T-lymphocyte clones. Many studies have focused on intestinal cases but few have addressed mediastinal lymphoma. This study aims to: (1) investigate the clonality patterns of lymphoma samples from various anatomical sites, with a particular focus on mediastinal lymphoma, and (2) evaluate the sensitivity and specificity of the clonality analysis of pleural effusion samples in comparison with cytology, histology, immunohistochemistry, and immunocytochemistry for diagnosing mediastinal lymphoma. There were 82 cases, divided into 49 formalin-fixed and paraffin-embedded biopsy specimens (FFPE), 22 cell pellets, and 11 fresh tissue. This study examined the sensitivity and specificity of PCR for antigen receptor rearrangement (PARR) compared to immunohistochemistry (IHC) and immunocytochemistry. For T-cell receptor gamma chain genes, PARR demonstrated a sensitivity of 58.33% for both fresh tissue and FFPE samples, with a specificity of 100%. Cell pellet analysis exhibited a sensitivity of 64.71% and maintained 100% specificity. A combined analysis of fresh tissue and FFPE with cell pellets showed a sensitivity of 62.07%. For IGH, the sensitivity for fresh tissue and FFPE samples was 56.25%, while cell pellet analysis showed a sensitivity of 62.50%. When considering fresh tissue and FFPE samples, the sensitivity was 57.14%. In conclusion, molecular techniques have emerged as valuable tools for detecting lymphoma, especially in cases where traditional diagnostic methods yield inconclusive results, such as mediastinal lymphoma. While biopsy may not always be feasible, cytology and cell pellets obtained from pleural effusion offer alternative immunocytochemistry and molecular analysis samples, provided they are of sufficient quality and quantity. All sample types considered in this study were suitable for PARR to aid in cases with inconclusive results. Therefore, the sample selection should be tailored to the clinical situation.

Abstract Background Histopathology, immunohistochemistry, and molecular clonality testing are metrics frequently used to diagnose chronic enteropathy (CE) in cats. However, normal values for these metrics have been based mainly on samples from cats that were relatively young, specific pathogen-free, or both. Objectives To describe results of histopathology, immunohistochemistry, and clonality testing of endoscopically-derived biopsy specimens of the upper small intestinal tract from a cohort of clinically healthy client-owned cats. Animals Twenty clinically healthy client-owned cats ≥3 years of age. Methods Tissue specimens were collected from the stomach and duodenum and evaluated single blinded by a board-certified pathologist. In addition, samples were evaluated by routine immunohistochemistry and clonality testing. Cats were followed after the procedure for signs of CE. Results Integrated results from histopathology, immunohistochemistry, and clonality testing were interpreted as consistent with small cell lymphoma (SCL; n = 12), emerging SCL (n = 1), lymphocytic enteritis (n = 6), and pseudoclonality (n = 1). On follow-up, 3 cats eventually developed clinical signs of CE, of which 2 were euthanized 295 and 654 days post-endoscopy. The remaining 17 cats did not show clinical signs of CE after a median of 709 days (range, 219-869 days). Conclusions and Clinical Importance Intestinal biopsy specimens from clinically healthy client-owned cats commonly had abnormal findings on histopathology, immunohistochemistry, clonality testing, or some combination of these without apparent clinical relevance. Current diagnostic metrics for diagnosing CE in cats may need modification to be applicable to the general population of cats.

Lymphomas are among the most important and common malignant tumors in cats. Differentiating lymphomas from reactive lymphoid proliferations can be challenging, so additional tools such as clonality assessment by PCR are important in diagnosis finding. Several PCR assays have been developed to assess clonality in feline lymphomas. For T-cell lymphomas TRG (T-cell receptor gamma) genes are the preferred target whereas for B-cell lymphomas most primer sets target immunoglobulin heavy chain (IGH) genes. Here we compare commonly used diagnostic primer sets for the assessment of clonality in feline lymphomas under controlled conditions (i.e., identical sample set, PCR setup, amplicon detection system). Formalin-fixed and paraffin-embedded samples from 31 feline T-cell lymphomas, 29 B-cell lymphomas, and 11 non-neoplastic controls were analyzed by PCR combined with capillary electrophoresis. We show that the combination of the primer sets published by Weiss et al. and Mochizuki et al. provided the best results for T-cell clonality, i.e., correctly assigns most populations as clonal or polyclonal. For B-cell clonality, the combination of the primer sets by Mochizuki et al. and Rout et al. gave the best results when omitting the Kde gene rearrangement due to its low specificity. This study rigorously evaluated various primer sets under uniform experimental conditions to improve accuracy of lymphoma diagnostic and provides a recommendation for achieving the highest diagnostic precision in lymphoma clonality analysis.

Abstract Background Differentiation of lymphocytic-plasmacytic enteropathy (LPE) from small cell lymphoma (SCL) in cats can be challenging. Hypothesis/Objective Histology-guided mass spectrometry (HGMS) is a suitable method for the differentiation of LPE from SCL in cats. Animals Forty-one cats with LPE and 52 cats with SCL. Methods This is a retrospective clinicopathologic study. Duodenal tissue samples of 17 cats with LPE and 22 cats with SCL were subjected to HGMS, and the acquired data were used to develop a linear discriminate analysis (LDA) machine learning algorithm. The algorithm was subsequently validated using a separate set of 24 cats with LPE and 30 cats with SCL. Cases were classified as LPE or SCL based on a consensus by an expert panel consisting of 5-7 board-certified veterinary specialists. Histopathology, immunohistochemistry, and clonality testing were available for all cats. The panel consensus classification served as a reference for the calculation of test performance parameters. Results Relative sensitivity, specificity, and accuracy of HGMS were 86.7% (95% confidence interval [CI]: 74.5%-98.8%), 91.7% (95% CI: 80.6%-100%), and 88.9% (95% CI: 80.5%-97.3%), respectively. Comparatively, the clonality testing had a sensitivity, specificity, and accuracy of 85.7% (95% CI: 72.8%-98.7%), 33.3% (95% CI: 14.5%-52.2%), and 61.5% (95% CI: 48.3%-74.8%) relative to the panel decision. Conclusions and Clinical Importance Histology-guided mass spectrometry was a reliable technique for the differentiation of LPE from SCL in duodenal formalin-fixed paraffin-embedded samples of cats and might have advantages over tests currently considered state of the art.