Use of an ultra-sensitive sequencing platform to detect mutant KRAS in the whole blood of pancreatic cancer patients.

Pancreatic Ductal Adenocarcinoma (PDAC) is a leading cause of cancer death and mortality is increasing. A contributor to poor outcomes is the absence of non-invasivebiomarkers for disease screening, treatment monitoring, and identification of therapeutic targets. Blood-based profiling of circulating tumor DNA (ctDNA) using Next Generation Sequencing (NGS) has addressed these needs in several cancer types, but available commercial ctDNA assays are not as effective in PDAC. To address this unmet clinical need, we developed an ultra-sensitive sequencing assay to detect mutant in the whole blood of PDAC patients. We adapted the bacterial Maximum Depth Sequencing (MDS) assay for Human whole blood MDS (hMDS) to improve upon the sensitivity of NGS by barcoding DNA fragments with unique molecular identifiers prior to performing multiple rounds of first-strand synthesis to resolve sequencing errors. Analytic sensitivity was evaluated by spiking PDAC cells at various dilutions into control blood isolated from 10 individuals and assaying the mixture by hMDS in triplicate. Clinical sensitivity was then evaluated by collecting paired blood draws from 200 advanced PDAC patients in prospective fashion, one to be tested with a commercial ctDNA test and one by hMDS. ThehMDS assay reproducibly detected PDAC cells at dilutions as low as one cell per mL or one mutated fragment per million fragments. Thus, hMDS reached an analytic sensitivity 1000x higher than NGS. Clonal mutations were detected in 179 of the first 194 patient samples (92.2%), surpassing historical commercial detection rates of 50% by commercial ctDNA assays. Mutations were reproducibly detected in replicate analysis of forward and reverse DNA strands. Weak clonal activating mutations were also detected in several non-cancer, control patients. We developed an assay capable of sensitively detecting PDAC ctDNA in whole blood. Formal comparison to commercial testing is ongoing, but preliminary results suggest this assay could be a sensitive tool for non-invasive PDAC mutation profiling and disease monitoring. The presence of weak clonal mutations in control patients has motivated development of a multiplex platform capable of screening for mutations in multiple driver genes.

Duke Scholars

Author John Strickler Medicine, Medical Oncology