Technical Evaluation of a Novel Digital PCR Platform for Detecting EGFR/KRAS Mutations in NSCLC Archived Plasma Specimens

Introduction: KRAS p.G12C hot spot mutations has rapidly modified diagnostic algorithm for lung cancer patients electing Non-Small Cell Lung Cancer (NSCLC) patients to target treatment. As regards, patients that harbor this hallmark showed a clinical benefit in terms of progression-free survival (PFS) and overall survival (OS) in comparison with control group under target treatment. In this scenario, KRAS p.G12C mutation requires optimized testing strategy in diagnostic routine practice. Although the widespread diffusion of NGS platforms, a not negligible percentage of Italian diagnostic institutions adopt singleplex technology (RT-PCR, dPCR) for molecular testing. Here, we aim to technically validate a novel dPCR system (QIAcuity™ Digital PCR System, Qiagen; Hilden, Germany) on a retrospective series of cfDNA samples from previously tested with a custom NGS system. (1,2)Methods: n = 50 liquid biopsy specimens (n = 25 KRAS/EGFR mutated and n = 25 wild type for actionable KRAS/EGFR mutations) from diagnostic routine NSCLC patients previously tested with a custom NGS panel were retrieved from our internal archival. Each sample was tested by adopting n = 5 KRAS and n = 3 EGFR commercially available dPCR assays on QIAcuity™ Digital PCR System (Qiagen; Hilden, Germany); an ultra-deep dPCR walk-away platform that automatizes molecular analysis. Technical sensitivity, technical specificity, and concordance rate between “gold standard” NGS system and QIAcuity™ Digital PCR System (Qiagen; Hilden, Germany) were assessed. Results: Overall, all specimens were successfully analyzed with dPCR system. In details, 24 out of 25 mutated and 21 out of 24 wild type cases were detected. A technical sensitivity, specificity, and a concordance rate of 96.0 % (24/25), 88.0 % (22/25) and 92.0 % (46/50) were evaluated taking into account a MAF cut-off ≥ 0.2 % and a partition number of 100 positive partitions in wild-type channel. Conclusion: Qiacuity (Qiagen; Hilden, Germany) platform enables accurate molecular analysis of diagnostic routine specimens. Optimized technical workflow is required to technically implement this platform in diagnostic routine setting.