(P 235) INFILTRATION OF MUCOSAL-ASSOCIATED INVARIANT T CELLS (MAITS) INTO CHONDROSARCOMA TUMORS

Objective: Chondrosarcomas are a subtype of sarcoma that arise from cartilage, with few treatment options in the advanced setting. While responses to checkpoint inhibitors have been observed in a minority of patients, improved immunotherapeutic strategies are needed. Here we instituted a retrospective study to comprehensively profile the immune infiltration, spatial transcriptome, and T-cell receptor (TCR) repertoire of chondrosarcoma tumors to better understand the immune landscape.

Methods: Formalin-fixed paraffin-embedded (FFPE) blocks from 24 patients with grade 2 or 3 conventional chondrosarcoma were selected for immunohistochemistry (IHC; CD45, CD3, CD4, CD8, CD20, CD68, CD206, FOX-P3, CD14, MECA-79 and MR-1). In selected cases, 10 um thick scrolls from FFPE blocks were used for in-depth comparison of TCR repertoire. Digital spatial profiling was performed using GeoMx platform on selected cases to assess differential gene-expression profiles. Intra- and peri-tumoral T-cell infiltrate were analyzed using quantitative TCR seq analysis. Mucosal-associated invariant T cells (MAIT cells) were identified based on having specific CDR3 region and a combination of TRAV 1-2 and TRAJ33 or TRAJ20 or TRAJ12; findings were then confirmed using MAIT MATCH search tool with a cut-off value of 0.9 to identify MAIT cells. To further characterize the location of MAITs, TCR sequencing was done on selected punches of FFPE blocks from both tumoral and peritumoral compartments. Finally, because MAIT cells recognize small molecules presented by MHC-I related protein 1 (MR1) as opposed to classical MHC, we tested samples for MR1 expression.

Results: IHC analysis of immune-cell infiltration revealed a significantly (p< 0.001) higher infiltration of multiple immune cells including CD45, CD3, CD4, CD8, CD20, CD68, CD206, FOX-P3, MECA-79 and CD14. These were found at a particularly high level in specific immune clusters found around the peritumoral region. Digital spatial profiling revealed a high inflammatory signal in the peritumoral region. Genes like TNFSF9 and TNFRSF10C, among others, were found to be significantly higher in the peritumoral compared with intra-tumoral regions (p = 7.06 E -04 and 0.009, respectively).

T-cell infiltrate from 11 samples revealed shared CDR3 regions between patients. Interestingly, 76% of the shared CDR3 regions were identified as MAIT cells, and some shared MAIT cells were highly shared (81% of the cases shared the sequences CAVRDSNYQLIW and CAVMDSNYQLIW). TCR sequencing of tumor and peritumoral punches revealed a relative enrichment of MAIT cells in the tumor compared with the peritumor and its clusters despite being less diverse. We found strong consistent expression of MR1 in all but one chondrosarcoma samples (mean MR1 expression, 58%) in both the peritumoral compartment and within the tumor.

Conclusion: Chondrosarcomas are immune-excluded cancers with a significant infiltration of immune cells in the surrounding peritumoral region. Our analysis of TCR repertoire revealed the presence of a subset of conserved immune cells (MAIT) that were found to be enriched in the tumor. Furthermore, a high expression of MR1, the binding ligand of MAIT cells, was found in chondrosarcomas. Additional studies are needed to assess the function and antigen recognition of MAIT cells in the tumor microenvironment of chondrosarcomas.