Abstract 7147: Ultra-high throughput single-nuclei RNAs sequencing of malignant pleural mesothelioma samples in the BEAT-MESO clinical trial

Abstract Background: Malignant Pleural Mesothelioma (MPM) is a rare cancer with limited treatment options. Large-scale deep biological description of intratumor heterogeneity at single-cell resolution is still missing. Recent studies of fresh tumor biopsies analyzed only a few patients, limiting resolution of population heterogeneity. Objective: To describe at single-cell resolution in FFPE tissues the cancer cell and tumor microenvironment heterogeneity of untreated MPM patients. Method: Of the 400 enrolled patients in the BEAT-MESO clinical trial (NCT03762018), 220 patients had available tumor blocks for single-nuclei preparation. The study compared the chemotherapy and bevacizumab combination with and without atezolizumab (anti-PD-L) treatment. All patients signed the ICFs and approved the translational study. Single nuclei were prepared using the snPATHO protocol followed by 10x Genomics FLEX pipeline. Samples were sequenced on a Novaseq X platform. Results: 179 of the available 220 samples had sufficient tumor content to perform the assay. In total, we detected 792’000 cells. We defined cell type identities using singleR with a combined reference map from non-small cell lung cancer (NSCLC) and a small mesothelioma cohort. We could readily identify cell types of the tumor microenvironment. The most abundant cell types beside tumor cells were cancer associated fibroblast/CAFS (median of 22% of all cells) and macrophages (14% of all cells). We could also define T cell heterogeneity, whereas CD4 TCM cells were the most abundant and detected in all patients, in contrast with proliferating CD4 or CD8 T cells which were absent in 68% of patients, suggestive of rich abundance of tumor reactive T cells. Among immune checkpoints, the expression of TIGIT was the highest, followed by LAG3 and PD1 expression. Tregs were rare and most frequently expressed TIGIT similar to CD8EM and CD4CM cells. We could also define an important heterogeneity of cancer-associated fibroblasts as well as of endothelial cells. Endothelial cell heterogeneity reflected on the proximity to B cells and tertiary lymphoid structures. In contrast, to previous single-cell RNA seq analysis of a small cohort of MPM, we could readily detect neutrophil granulocytes in the tumors. We will also provide data on matching H&E evaluation of TLSs. Depending on the maturation of clinical data, we will also present comparison with clinical parameters. Conclusions: Our results show the outstanding power to perform snRNAseq of fixed tissues from clinical trials. The method is readily compatible with large phase III clinical trials, and provide a much deeper insight into tumor heterogeneity than bulk RNAseq with deconvolution would do. Citation Format: Daria Buszta, Maxim Norkin, Jonathan Bac, Sanjay Popat, Anthony Pope, Riyaz Shah, Toby Talbot, Julia Giner, Wold-Dieter Janthur, Ernest Nadal, Annamaria Catino, David Gilligan, Amy Roy, Zoi Tsourti, Patrick Vagenknecht, Georgia Dimopoulou, Roswitha Kammler, Stephen P. Finn, Enriqueta Felip, Solange Peters, Rolf Stahel, Raphael Gottardo, Krisztian Homicsko. Ultra-high throughput single-nuclei RNAs sequencing of malignant pleural mesothelioma samples in the BEAT-MESO clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7147.