TRBC1/TRBC2 RNA In Situ Hybridization as a Diagnostic Approach for Canine and Feline T-Cell Lymphoma: A Proof-of-Concept Study

Abstract

Background/Objectives: T-cell lymphomas are relatively common in veterinary species, yet current diagnostic tools such as PCR-based clonality assays often lack sensitivity and specificity. In humans, we recently developed two related tissue-based diagnostic approaches based on the differential detection of the mutually exclusively expressed TCRbeta1 and 2 (TCRβ1 and 2) constant region proteins, or the corresponding <i>TRBC1</i> and <i>TRBC2</i> transcripts. Analogous to the detection of kappa/lambda light chains for the diagnosis of B-cell/plasma cell neoplasms in human clinical practice, our TCRβ1/2 diagnostic assay has the potential to transform veterinary diagnostic workflows. Methods: We identified and confirmed the sequences of the relevant <i>TRBC1</i> and <i>TRBC2</i> sequences in both cats and dogs, focusing on the 3′ untranslated region (UTR), where there is the least sequence homology between <i>TRBC1</i> and <i>TRBC2</i>. To allow us to design appropriate probe sequences, we confirmed a lack of 3′UTR in either species, and we observed limited 3′ untranslated region UTR sequence polymorphism in the cat but not in the dog 3′UTR. We designed BaseScope™ RNA in situ hybridization probes targeting the 3′ UTR to distinguish between <i>TRBC1</i> and <i>TRBC2</i> transcripts in formalin-fixed paraffin-embedded tissues. Results: In normal tissues, we found the <i>TRBC2</i>:<i>TRBC1</i> expression ratio to be similar to the 1.2:1 ratio in humans, between 1:1 and 3:1, skewing towards <i>TRBC2</i>, in both dogs and cats. These findings were corroborated using quantitative reverse transcription PCR. Applying our in situ hybridization probes to cases of T-cell lymphoma in dogs and cats, we demonstrated that an assay for differential expression of <i>TRBC1</i> and <i>TRBC2</i> in T-cell populations could identify clonal T-cell populations, as in human diagnostics. If further studies corroborate this proof-of-concept study, TRBC1/2 detection could obviate the need for slow, complex and expensive multiplexed PCR-based (PCR for antigen receptor rearrangements (PARR)) clonality assays. Conclusions: This study provides proof-of-concept data for a novel diagnostic approach that could simplify and substantially improve the accuracy of lymphoma diagnostics in veterinary medicine, by detecting <i>TRBC1</i>/<i>2</i> transcripts.