LAUSR

Mechanisms behind the evolution of normal pancreas tissue into pancreatic ductal adenocarcinoma (PDAC) are still poorly understood. More thorough characterization of mutational processes and development of clones is possible with single-cell DNA sequencing (scDNA-seq) of normal pancreas tissue. We developed a custom panel with 596 amplicons targeting over 200 genes known to be important in PDAC for use with Mission Bio’s Tapestri platform. We have recently optimized this method with nuclei which have been extracted from thirty frozen normal pancreas tissues, each from a different patient. Of these tissues, twenty-five were derived from rapid autopsies of patients with brain tumors to minimize the possibility of tumor cells being present in the pancreas. Normal pancreas tissue was verified by hematoxylin and eosin staining. Nuclei were extracted using the S2 Genomics Singulator 100 and counted with DAPI using the Countess 3 FL to ensure the concentration was adequate and that the sample was clean without excessive debris before library preparation and sequencing. Bulk whole exome sequencing (WES) and digital droplet polymerase chain reaction (ddPCR) were performed on several of these sample to validate certain mutations seen in scDNA-seq. Patients of a variety of ages ranging from five to 74 and with various risk factors including smoking and diabetes were chosen to better understand the impact on clonal expansion and genetic patterns present in normal pancreas tissue. For each of the thirty samples, an average of 2464 good quality nuclei were sequenced with over 70 reads per cell per amplicon on average. Mutations that were present in high quantities of cells were confirmed with WES and ddPCR. We found that older patients and patients that either smoke or those with diabetes had not only an increased number of mutations, but each individual cell, on average, had a significantly higher mutation count. The associated clones were also much greater in size in the higher-risk patients’ tissues. Furthermore, there was an increase in heterogeneity in the form of mutually exclusive clusters of cells. These results highlight the effects of various risk factors on normal pancreas tissue and potentially describe novel mechanisms of tumorigenesis. Citation Format: Elias-Ramzey R. Karnoub, Haochen Zhang, Shigeaki Umeda, Ignas Masilionis, Ronan Chaligné, Christine Iacobuzio-Donahue. Genetic characterization of normal pancreas tissue using single-cell technology [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 1517.