Abstract 580: The broad surveillance and unbiased detection of circulating tumor DNA using next-generation sequencing without a priori knowledge of somatic mutations is a prognostically significant biomarker in pancreatic ductal adenocarcinoma
Authors: Affolter Kajsa E. , Hellwig Sabine , Nix David A. , Bronner Mary P. , Thomas Alun , Fuertes Carrie L. , Hamil Cindy L. , Garrido-Laguna Ignacio , Scaife Courtney L. , Mulvihill Sean J. , Underhill Hunter R. 
Published: July 2021

Abstract
Background. Detection of low frequency tumor-derived variants in circulating cell-free DNA (ccfDNA; i.e., circulating tumor DNA, ctDNA) without a priori knowledge of mutations in the matched solid tumor is confounded by next-generation sequencing (NGS) noise and constrained by theoretical and practical limitations associated with low DNA yield from plasma. Here, we leverage technical replicates (two libraries prepared and sequenced independently from each extracted ccfDNA sample) to simultaneously suppress NGS-associated errors and improve detection of ctDNA derived from pancreatic ductal adenocarcinomas (PDACs).
Methods. Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III) undergoing surgery aimed at complete total resection. For 11 patients, ccfDNA was also collected post-operatively within 100 days of surgery. Two technical replicates of ccfDNA libraries using molecular barcodes were independently generated using 2 mL plasma equivalents of ccfDNA for each library. Subsequently, libraries were capture-enriched using a 118 gene panel (~124 kb) and underwent NGS (paired-end, 125x2; HiSeq 2500). Consensus sequence determination and position-specific error modeling were coupled with restricting variant identification to nonreference alleles present in both patient replicates that corresponded to COSMIC-reported pathogenic mutations in PDAC.
Results. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients (35.7%) with a variant allele frequency ranging from 0.08% to 1.59% (median: 0.25%). Of the nine ctDNA variants identified, five (55.6%) had a corresponding mutation in tumor DNA. The estimated median overall survival in patients with and without detection of pre-operative ctDNA was significantly different (312 vs. 826 days, respectively; χ2=5.3, P=0.0207). Using mutations identified in tumor tissue DNA to guide detection of pre-operative ctDNA yielded a similar survival analysis (369 vs 1,011 days, respectively; χ2=5.6, P=0.018). Detection of ctDNA in post-operative ccfDNA with or without guidance from tumor tissue mutations was not associated with outcomes. Applying the noise reduction strategy used to detect ctDNA in PDAC patients eliminated false positives from healthy controls.
Conclusions. Acquisition of NGS data in replicate coupled with in silico error suppression strategies enabled detection of ctDNA without a priori knowledge of tumor variants, which overcomes a major obstacle limiting ccfDNA applications. Pre-operative ctDNA detected during a broad and untargeted survey of ccfDNA with NGS is a non-invasive, prognostic biomarker to help guide PDAC patient management.
Citation Format: Kajsa E. Affolter, Sabine Hellwig, David A. Nix, Mary P. Bronner, Alun Thomas, Carrie L. Fuertes, Cindy L. Hamil, Ignacio Garrido-Laguna, Courtney L. Scaife, Sean J. Mulvihill, Hunter R. Underhill. The broad surveillance and unbiased detection of circulating tumor DNA using next-generation sequencing without a priori knowledge of somatic mutations is a prognostically significant biomarker in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 580.