title: Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples.
abstract: Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA.
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York. Electronic address: borsul@mskcc.org.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Borsu
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: Julie
authors.initials: J
authors.lastname: Intrieri
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: Linta
authors.initials: L
authors.lastname: Thampi
authors.affiliation: Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: Helena
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: Gregory
authors.initials: G
authors.lastname: Riely
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: Khedoudja
authors.initials: K
authors.lastname: Nafa
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: Raghu
authors.initials: R
authors.lastname: Chandramohan
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York; Human Oncology & Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: Marc
authors.initials: M
authors.lastname: Ladanyi
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Arcila
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics; D000077192:Adenocarcinoma of Lung; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D014408:Biomarkers, Tumor; D003062:Codon; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; D003062:Codon; D066246:ErbB Receptors