Abstract B021: Development of an ultrasensitive sequencing platform for blood-based mutation profiling of pancreas cancer patients

Abstract Introduction: One barrier to improving Pancreatic Ductal Adenocarcinoma (PDAC) patient outcomes is the absence of effective, non-invasive biomarkers for disease screening, treatment monitoring, and identification of therapeutic targets. While sequencing-based approaches to detect mutant circulating tumor DNA (ctDNA) in blood have transformed clinical management in several cancer types, use of these ‘liquid biopsies’ has lagged behind in PDAC due to low shedding of PDAC tumor cells. Nevertheless, intact circulating tumor cells have been documented in even the earliest stages of PDAC and precede the limits of disease detection on imaging or gross histology in mouse models. This suggests that circulating tumor cells could server as a PDAC biomarker if the sensitivity of sequencing could be increased. Methods: Point mutations in the gene KRAS are found in up to 95% of PDAC patients and hence represent a potential biomarker target for this cancer. To detect these pathogenic mutations in KRAS, we will adapt Maximum Depth Sequencing (MDS) for exon 2 of this gene by testing various primers and reaction conditions. The sensitivity of the K-MDS assay was optimized for blood by spiking PDAC cells with known KRAS mutations into healthy control blood at various dilutions. Finally, to evaluate the clinical sensitivity, we have collected paired blood draws from 200 advanced PDAC patients in prospective fashion, one to be tested with a commercial ctDNA test and one by K-MDS. Results: We optimized MDS to sequence exon 2 of KRAS and successfully detected a single mutant template in >107recovered independently barcoded templates (e.g. 107 individual KRAS alleles). We then optimized the assay for human blood and demonstrated that K-MDS could reproducibly detect a KRAS mutation corresponding to PDAC cells spiked into healthy control whole blood at the lowest dilution tested, namely one cell per ml of blood. This corresponds to a sensitivity of one mutated fragment per one million KRAS fragments, or 1000x more sensitive than currently available commercial assays. We are currently performing K-MDS on prospective PDAC cohort, with completion of this study expected this fall. Conclusions: We developed a highly sensitive sequencing assay cable of detecting PDAC ctDNA in whole blood. With successful validation in PDAC blood samples, this assay could provide a powerful tool for non-invasive PDAC mutation profiling and disease monitoring. Citation Format: Ryne Ramaker, James Abbruzzese, Chris Counter. Development of an ultrasensitive sequencing platform for blood-based mutation profiling of pancreas cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B021.