Abstract 5038: Study on acquisition timing of <i>KRAS</i> mutations in the pancreatic ductal epithelium

Abstract Introduction: Pancreatic cancer is a highly aggressive malignancy with a poor prognosis, emphasizing the urgent need for early detection to improve outcomes. In the multistep carcinogenesis of pancreatic cancer, KRAS mutations are acquired at the earliest stage. However, the exact timing of acquiring KRAS mutations in the pancreatic ductal epithelium remains insufficiently understood. Methods: Non-cancerous regions of the pancreas, distant from macroscopic tumors, were obtained from surgical specimens. The main pancreatic ducts were isolated, spread open manually, and excised into pieces ∼1 mm in size to create pancreatic duct organoids. DNA was extracted and subjected to whole-exome sequencing (WES) with matched control DNA. Phylogenetic trees were reconstructed based on shared mutations. Single-cell-derived organoids were established from some bulk organoids and analyzed via WES to determine the mutation accumulation rate of pancreatic ductal epithelium. The timing of KRAS mutation acquisition was estimated based on the mutation rates and the height of the branches to which KRAS mutations were allocated. Results: A total of 430 organoid samples were analyzed from 25 pancreatic cancer patients and 23 subjects without pancreatic cancer (1-21 samples per case; median: 8). A median of 33 mutations per sample (range: 8-141) was identified. 18 KRAS mutations were detected in 16 cancer patients, while 4 mutations in 4 non-cancer subjects, suggesting a higher prevalence of KRAS-mutated clones in the pancreatic ductal epithelium of cancer patients. In the phylogenetic analysis, 13 out of 22 KRAS mutations were allocated to the trunk, and KRAS mutant clones expanded to 6 samples on average (2-20). In contrast, KRAS-negative clones rarely expanded across multiple samples. We analyzed 56 single-cell-derived organoids from 19 cases, including 7 KRAS mutant cells. KRAS wild-type cells showed a positive correlation between mutation number and age, with a rate of 0.29 mutations per exome per year. KRAS mutant cells displayed a significantly higher mutation number compared to KRAS wild-type cells, suggesting an increased mutation rate of KRAS mutant cells. KRAS mutations were acquired at various ages (21-44), with some occurring as early as the early 20s. Conclusions: In this study, we elucidated the evolutionary history of clones in non-cancerous pancreatic ductal epithelium. Our findings will contribute to a better understanding of early events of pancreatic carcinogenesis. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Sachiko Minamiguchi, Takayuki Anazawa, Kazuyuki Nagai, Hiroshi Seno, Seishi Ogawa. Study on acquisition timing of KRAS mutations in the pancreatic ductal epithelium [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 5038.