PROJECT SUMMARY While cancer is known for its heterogeneity, with distinct subtypes observed within each organ system, the source of this heterogeneity, and the relationship between subtype definition and clinical outcomes, remain contested. Studies in animal models of pancreatic ductal adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC) suggest that the cell-of-origin of individual tumors has a strong influence on subtype, defined transcriptionally, and tumor aggressiveness, defined by survival and metastatic potential. In particular, mouse PDAC derived from exocrine acinar cells predominantly expresses a “classical” subtype signature, and is slower to develop than duct-derived tumors, while mouse HCC derived from hepatocytes is more aggressive than that derived from hepatocyte precursor cells. The tools for lineage-tracing and genetic manipulation used in mouse studies cannot be applied to human patients, however, leaving an important knowledge gap regarding the cell of origin of human PDAC and HCC and its influence on tumor subtype and clinical outcomes. To address these gaps, we propose a study focusing on the cell-of-origin concept in PDAC and HCC. We have observed that non-random somatic mutations in highly-expressed genes of normal cells create a "lineage mark" in tumor cells, serving as permanent traces of the cell of origin. We hypothesize that cell-of- origin can serve as a novel biomarker in PDAC and HCC, with potential implications for patient stratification and treatment decisions. In Aim 1, we will establish a sequencing-based paradigm for relating the cell-of-origin in human PDAC to tumor subtype and clinical behavior. By leveraging existing whole genome sequencing (WGS) and RNA-seq data, as well as applying an innovative targeted deep-sequencing approach to locally-collected samples, we will validate and expand our findings of indels in acinar-specific genes associated with PDAC. The wet-lab/dry-lab pipeline developed in this aim will be directly applicable to prospectively-collected human samples. Aim 2 aims to determine the timing and spatial specificity of cell-of-origin mutations in HCC. By analyzing existing single-cell RNA-seq (scRNA-seq) data, we will identify de novo indels in liver-expressed genes. This analysis will shed light on the timing of lineage-specific indel mutations during the transition from normal liver tissue to HCC, and can be applied to PDAC and other cancers, as scRNA-seq analysis increasingly enters the clinic. Overall, this research has significant implications for improving patient outcomes and lays the foundation for future investigations into the mechanisms underlying PDAC and HCC development in humans.