1413 An integrated tumor, immune and genetic landscape of pleural mesothelioma to unleash effective immunotherapy avenues

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Background Pleural mesothelioma (PM) is an aggressive cancer linked to asbestos exposure. Our understanding of links between the tumor mutational signature, myeloid and tumor-infiltrating lymphocytes (TIL) phenotype and functional states are lacking. To address this paucity of knowledge, we performed multi-omic profiling of the tumor immune microenvironment (TIME) of PM cases to provide insights that could drive novel therapeutic approaches for patients with surgically resectable tumors. Methods PM tumor tissue from patients who underwent surgical resection was collected and confirmed by pathologist as epithelioid and biphasic histological subtypes. In some cases, tumor adjacent (TA) pleura was harvested. Twenty-six samples (22 epithelioid, 4 biphasic) were processed for bulk RNA-seq with seventeen (15 epithelioid, 2 biphasic) for whole exome sequencing. Single cell (sc) RNA-seq with matched scTCR-seq and scBCR-seq (10x Genomics) was performed on 6 TAs and 16 tumors. Twenty-four patients had recorded exposure to asbestos, 18 were former smokers, 25 received chemotherapy with treatment ending an average of 67 days prior to surgery,13 patients received a preoperative (chemo)-immunotherapy, and 5 patients did not have preoperative treatment. All analyzes were performed using in-house pipelines with downstream analysis in R. Wilcoxon Sum Ranking test was used for differentially expressed genes (adjusted p-value significance: 0≤***<0.001≤**<0.01). Results The dominant mutation identified in analyzed cohort was BAP1 (6/17 (35.3%), CNV: 2/17 (11.8%)). HLA loss of heterozygosity was not identified. Median tumor mutation burden was low (0.8). Cibersort-abs immune infiltration prediction identified the presence of M2 macrophages, B cells and memory CD4 T cell subsets. scRNA-seq profiling confirmed presence of identified immune subtypes, and clusters of Tregs and effector memory CD8+ T cells. Among analyzed tumors, most showed small to large expansion of CD8+ clones, with two tumors dominated by hyper and largly expanded clones. Overall, TIL showed upregulation of checkpoint receptors (PD-1***, LAG3***, TIM3***, TIGIT***), and the transcription factor, TOX***. However, specific hyperexpanded clones showed heterogeneous cytotoxic features (granzymes, IFNg and TNF). Interestingly, MSLN+ tumor cells showed diverse transcriptomic features across patients. Furthermore, we identified several clusters of cancer associated fibroblasts and normal cells within the TIME. Conclusions Bulk RNA-seq and scRNA-seq showed presence of a suppressive TIME. Hyperexpanded TCRs were identified suggesting potential anti-tumor response despite its dysfunctional phenotype. Our ongoing efforts are focused on receptor-ligand interaction predictions as well as identifying predicted neo-antigens and drivers of tumor progression to refine future therapeutic strategies in this rare disease. Acknowledgements This study was supported in part by the Dillon & L Bourg Mesothelioma Fund , the Re & RM Kennedy Lung Cancer Fund, the Fleming Endowed Fund, the MDA Rare Tumor Initiative, MDA Patient Mosaic, the NIH CCSG Award (CA016672 (Institutional Tissue Bank (ITB), Research Histology Core Lab (RHCL), ORION and ATGC sequencing core), and the TMP-IL MDA Moonshots Platform. We would like to thank the MDA Thoracic and Cardiovascular Surgery nurses and staff for their support of this study and the patients who consented to be included in the research protocol. Ethics Approval The study was performed under the MDA IRB approved LAB08-0380 protocol following patient informed consent.

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