CLASSICAL HODGKIN DISEASE: PRESENCE OF TIGIT MRNA IN PERITUMORAL LYMPHOCYTES

Abstract

Background: T cell Ig and ITIM domains (TIGIT) has been recognized as an immune checkpoint receptor able to negatively regulate T cell functions. Our group (Annibali O et al. Sci Rep. 2021) demonstrated TIGIT expression in lymphocytes around the RS cells using immunohistochemistry in 56% of evaluated patients. (19/34) Aim of this study was to assess the in situ TIGIT mRNA expression in CHL, in order to verify the effective intracellular transcription of TIGIT receptor in peritumoral lymphocytes that resulted positive at immunohistochemical analysis. Material and methods: 34 formalin fixed-paraffin embedded samples, in which was tested TIGIT protein expression, were selected. The TIGIT protein expression status was determined by immunohistochemical investigations with TiGIT antibody, as previously described. Seriate sections from FFPE lymph nodes were submitted for RNAscope Assay. Results: Among 34 enrolled cases, 15 resulted negative for TIGIT immunohistochemistry on lymphocytes within the tumor environment and were classified score 0. 10 cases showed a sparse, faintly stained non-tumoral lymphocytes within the tumor environment, near the HRS cells and were classified as score 1. 5 cases showed the presence of a discrete quote of non-tumoral lymphocytes with moderate membrane staining around the HRS cells and were reported as score 2. 4 cases demonstrated evidence of a circle of non-tumoral lymphocytes with intense membrane staining, surrounding the HRS cells corresponding to the score 3.RNAscope was successfully applied to the whole FFPE histological sections of the 34 HL cases. TIGIT probe is represented as red dots/spots within the lymphocytes surrounding the RS cell. After slides were visualized, each case was assigned a semi-quantitative score based on the mRNA expression level of the TIGIT gene according to the scoring system provided by the RNAscope manufacturer. The remaining cases scored a 0 with no mRNA probe signals observed in the lymphocytes. Low mRNA expression was observed in ten cases marked as 1 which is equivalent to 1–3 probe signals per positive lymphocyte. Five cases showed moderate mRNA expression with a score of 2 representing 4–10 probe signals per positive lymphocyte. 4 cases showed high mRNA expression with a score of 3, representing > 11 probe signals per positive lymphocyte. With QuPath software, we observed an increase in the number of positive cells in relation to protein expression status. In fact, at score 0 corresponds 0% positive cells, at score 1+ corresponds 8% positive cells, at score 2+ corresponds 33% positive cells and at score 3+ corresponds 54.9% positive cells. Conclusion: Our results confirm the presence of immunoescape through the expression of TIGIT in patients with HL, based on the expression of the mRNA coding for TIGIT and validate the score system proposed for the immunohistochemical reaction. Encore Abstract—previously submitted to EHA 2023 Keyword: Hodgkin lymphoma No conflicts of interests pertinent to the abstract.