Abstract 95: Uncommitted cells and phenotypic plasticity elucidate the complexity of the epithelial-mesenchymal molecular gradient of pleural mesothelioma

Abstract Pleural mesothelioma (PM) comprises three major histologic types: epithelioid, sarcomatoid, and a mixture of the two types termed biphasic. We and others have investigated whole transcriptome profiles of bulk PM samples, identifying a molecular gradient with features of epithelial-mesenchymal transition, which is related to, but not redundant with, histology. To date bulk studies of heterogeneity across PM tumors have not explained how biphasic tumors contain sarcomatoid (mesenchymal) and epithelioid (epithelial) components. We hypothesized that sarcomatoid and epithelioid components arose from distinct tumor clones. We performed integrated bulk and single-cell RNA-sequencing and pathologic analyses of 93 samples from 39 patients’ surgical resections including 3 negative-control pleura cases with non-PM pathology. Single-cell sequencing analysis was performed using the Seq-Well S3 platform. Tumor clones were identified using inferCNV in R. Copy number and somatic variants were identified in adjacent bulk samples of each PM case with optical genome mapping and exome sequencing, respectively. We generated libraries from 19 epithelioid, 3 sarcomatoid, and 13 biphasic cases resulting in an average of 6,657 single cells per case (n=266,262 total cells; UMIs ≥ 300 & features ≥ 80). Among the 32,383 high quality malignant cells, we characterized epithelioid and sarcomatoid programs independent of non-malignant cells in the microenvironment. We observed malignant cells expressing both programs at low levels, termed uncommitted. Uncommitted cells were not outliers in terms of UMIs detected, genes expressed, or mitochondrial transcript content, and were detected in 96.4% of PM cases with at least 50 high quality tumor cells. Most (72.7%) uncommitted cells were isolated from biphasic tumors. Additionally, we observed that individual tumor clones from biphasic tumors exhibited all three cell phenotypes. In one biphasic tumor, five of six clones comprised cells from all three types, where these clones were well-represented across multiple sites with epithelioid content ranging from 10% to 40%. In conclusion, our integrated analysis of multi-site pleural mesothelioma samples suggests that a single tumor clone may be capable of generating all molecular subtypes. Further work aims to identify candidate drivers of this plasticity, to relate single-cell transcriptomic phenotype to histology and to further characterize uncommitted cells. We will also assess the prognostic implications of uncommitted cells content in a wider cohort. Citation Format: David T. Severson, Samuel Freyaldenhoven, Benjamin Wadowski, Travis Hughes, Yin P. Hung, Roderick V. Jensen, William G. Richards, Corinne E. Gustafson, Kimberly Vermilya, Simona Innocenti, Julianne S. Barlow, Matthew B. Cougar, Jamie Anderson, Vivian Wang, Mary N. Dao, Alex K. Shalek, Assunta De Rienzo, Raphael Bueno. Uncommitted cells and phenotypic plasticity elucidate the complexity of the epithelial-mesenchymal molecular gradient of pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 95.