Dual-function platform for chemotherapy prediction and mutation detection in pancreatic cancer by t CAM-seq

Predicting chemotherapy response by chromatin accessibility and detecting actionable mutations are two independent paradigms, but here we demonstrate their integration in an all-in-one platform. We addressed two fundamental questions, 1) whether clinically actionable mutations can be detected from a Tn5-accesible genome instead of a traditional whole genome or exome, and 2) whether the higher depth of sequencing would compromise the quantitative ability of chromatin accessibility. We designed the panel using Illumina's standard deep-sequencing platform, encompassing the canonical driver mutations of pancreatic ductal adenocarcinoma (PDAC), e.g., KRAS, TP53, BRCA1/2 etc. On the same panel, we included our previously discovered 1092-chromatin signature regions that distinguishes gemcitabine resistant and sensitive tumors. We named this method t argeted c hromatin a ccessibility and m utation sequencing ( tCAM-seq ). First, we applied this tCAM-seq approach on four PDAC cell lines (AsPC1, BxPC3, MiaPaCa2, and PANC1), which accurately identified canonical driver mutations. The 1092-chromatin signature exhibited distinct accessibility patterns between gemcitabine resistant (AsPC1, BxPC3) and gemcitabine sensitive (MiaPaCa2, PANC1) cell lines. Next, we performed a head-to-head comparison of tCAM-seq with ATAC-seq on an archival cohort of Tn5-accessible DNA libraries prepared from surgically resected PDAC patients (n=24) between 2015 - 2017 at MSKCC. We observed high concordance (median R²=0.77, range 0.61-0.85) among the two methods in terms of chromatin accessibility estimation. Our tCAM-seq maintained the predictive accuracy, even with >100× sequencing read depth. Kaplan-Meier analysis (n=24) of overall survival demonstrated a significant segregation between chemotherapy responder and non-responder patients (p=0.0184, HR=0.2958, 95% CI=0.066-1.323; median follow-up 8.98 years) using the tCAM-seq -derived 1092-chromatin accessibility profiles. Owing to the higher sequencing depth, tCAM-seq identified canonical driver mutations with high confidence which were otherwise not possible with traditional bulk ATAC-seq . Our results suggest clinically actionable mutations in PDAC reside within “accessible” chromatin regions and therefore can be detected from the Tn5-accessible genome, while accurately quantifying the chromatin accessibility profiles on the same sample. This clinically translatable “dual-purpose” tCAM-seq panel surpasses mutation-only panels by enabling both mutation-matched targeted therapies and prediction of chemotherapy response from the same assay. This will enable oncologists to provide a more comprehensive and better-informed treatment decision.