Pervasive early dissemination in pancreatic cancer uncovered by tissue-paired plasma whole-genomes

In pancreatic cancer, 50% of the patients are diagnosed at the metastatic stage due to a lack of symptoms. The overall survival rate dramatically reduces from 44% in early-stage resectable patients to 3% in metastatic cases. However, even in early-stage patients, >75% of them still recur after resection and adjuvant therapies. Therefore, there is an urgent need to develop a sensitive strategy to detect this disease earlier before it spreads. Characterizing circulating tumor DNA (ctDNA) in plasma is an effective approach to detect and monitor cancer. Whole-genome sequencing (WGS) tracks all the tumor mutations simultaneously, and has a higher sensitivity than targeted approaches, especially in samples with extremely low tumor burden. In this study, to investigate the ctDNA dynamics and early dissemination in pancreatic cancer, we established a cohort of 1,013 samples from 277 donors, including plasma WGS at 20-60x, alongside germline DNA WGS, tissue WGS and transcriptomic sequencing. Using a tumor-guided approach, we found that the early-stage resectable cases shed very little ctDNA (<1% tumor fraction, TFx). Plasma TFx levels were elevated at the metastatic stage, but were also dependent on metastatic tissue site, where patients with liver metastases had higher TFx than the ones with only non-hepatic lesions. We further discovered the tumor-intrinsic features that were related to increasing ctDNA shedding, such as whole-genome duplication (WGD), high cell cycle activity and non-glandular morphology, as well as extrinsic features related to reduced shedding, including a reactive microenvironment and B cell immunity. Our analysis on tumor clonal architecture revealed that subclonal mutations were more frequently detected than the clonal ones in plasma samples with low ctDNA levels or from early-stage patients, which strongly suggests that dissemination from early-stage primary tumors mostly derived from subclones. In the longitudinal plasma, we observed that subclones seeded metastasis years before imaging diagnosis. This study with a unique large cohort of paired tumor and plasma sequencing data provided a comprehensive insight on ctDNA dynamics, disease monitoring and early detection in pancreatic cancer.