PS1307 USE OF T CELL‐SPECIFIC RNA IN SITU HYBRIDISATION AS A NOVEL TEST TO DISTINGUISH MALIGNANT (LYMPHOMATOUS) AND BENIGN (INFLAMMATORY) T CELL INFILTRATES

Abstract

Background:The differentiation between benign and malignant (e.g., lymphoma or leukaemia) lymphocytic infiltrates is an important and common clinicopathological question in routine healthcare practice. While this is possible for B cell infiltrates using kappa/lambda immunoglobulin light chain staining, there is not an equivalent for T cell infiltrates, which currently require expensive and time consuming T cell receptor gene rearrangement PCR studies to be undertaken. We have identified two T cell‐specific, mutually exclusively expressed, RNA sequences (TRBC1 and TRBC2), corresponding to the two, alternatively employed, T‐cell receptor beta constant region segments.Aims:We aimed to use our knowledge to develop a new way of distinguishing between benign and malignant (e.g., lymphoma or leukaemia) lymphocytic infiltrates.Methods:We analysed the TRBC1 and TRBC2 gene segments using standard bioinformatics tools, undertook Q‐PCR to investigate relative expression levels and developed TRBC1/TRC2 segment‐specific probes for chromogenic in situ hybridisation (CISH), which we validated on T‐cell lymphoma/ leukaemia lines processed to paraffin and on formalin fixed paraffin embedded (FFPE) sections of T‐cell lymphoma and corresponding benign tissue. We then developed duplex TRBC1/TRC2 segment‐specific CISH probes, which we validated on FFPE sections from a cohort of T‐cell lymphoma (n = 100) and corresponding benign specimens (n = 100).Results:The coding regions of TRBC1 and TRBC2 are very similar at amino acid level, making development of highly specific TRBC1/ TRBC2 diagnostic monoclonal antibodies difficult. However, the 3’ untranslated regions differ substantially. Q‐PCR demonstrated that, in benign populations of peripheral blood mononuclear cells the ratio of TRBC1: TRBC2 was very close to 1:1. Single TRBC1 and TRBC2 CISH staining on serial sections of benign T‐cell infiltrates demonstrated similar numbers of TRBC1+ and TRBC2+ cells. Single staining of both T‐cell lymphoma/ leukaemia lines and FFPE sections of T‐cell lymphoma demonstrated clear TRC1/ TRC2 restriction (i.e., T‐cell monotypia for TCR), with excellent correlation with TRCB1: TRBC2 ratio Q‐PCR results. Duplex (double) CISH staining was successfully developed and optimised on benign lymphocyte populations (figure 1: benign tonsil, polytypic for TCRB: TRBC1‐pink; TRBBC2‐black). We then performed technical validation of our duplex CISH staining and we demonstrate here that it can be used to distinguish between benign (inflammatory) and malignant (lymphomatous) lymphocyte populations in a wide range of tissues.Summary/Conclusion:This is the basis of a novel diagnostic pathology test for T‐cell lymphoma, applicable to FFPE sections, that might replace PCR‐based clonality studies in the majority of cases. It has the potential to transform the routine assessment of T cell infiltrates in a manner analogous to the way kappa/lambda staining has done for B cells. It is therefore likely to impact upon international diagnostic guidelines with global health economic implications.image