Plasma EGFR T790M mutation detection in NSCLC patients using a combined exosomal RNA and circulating tumor DNA qPCR assay

• Published in: European journal of cancer (1990) 2016-12, Vol.69, p.S3-S3
• Main Authors: Castellanos-Rizaldos, E, Grimm, D.G, Tadigotla, V, Bentink, S, Hurley, J, Healy, J, Neal, P.L, Bentis, C, McMullen, E, Tun, R, Karlovich, C, Brock, G, Krug, A, Noerholm, M, Skog, J.K
• Format: Article
• Language: English
• Subjects: Apoptosis; Assaying; Bioassays; Biomarkers; Biopsy; Deoxyribonucleic acid; DNA; Epidermal growth factor receptors; Hematology, Oncology and Palliative Medicine; Historical account; Inhibitors; Lung cancer; Molecular chains; Mutation; Non-small cell lung carcinoma; Operators; Patients; Plasma; Polymerase chain reaction; Regression analysis; Regression models; Ribonucleic acid; RNA; Sensitivity; Sensitivity analysis; Therapy; Tissue analysis; Tumors
• ISSN: 0959-8049; 1879-0852
• DOI: 10.1016/S0959-8049(16)32611-9

Background Non-small cell lung cancer (NSCLC) comprises ~85% of all diagnosed lung cancers and targeted EGFR inhibitor therapy is available for patients with known EGFR activating mutations in their tumor. The T790M mutation in exon 20 of EGFR is a primary mechanism of acquired resistance to first generation EGFR inhibitors. Approximately 60% of patients that are refractory to EGFR inhibitor therapy harbor this mutation. This genetic alteration has also been found in tumors from treatment naive patients. Therefore, in addition to being used as a biomarker for patient stratification before treatment, T790M can be used to monitor for the emergence of resistance EGFR inhibitors. Obtaining tissue biopsies from NSCLC is challenging, and as many as 30% of patients have no tissue for molecular analysis of EGFR, therefore monitoring the mutations in blood as a liquid biopsy have proven useful. In this study, we combined the information derived from the dying cellular processes (e.g., apoptosis and necrosis) on cell free DNA (cfDNA) and living processes (exosomal RNA release). Materials and Methods We have developed ExoDX Lung (T790M), a Laboratory Developed Test (LDT) under Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations. The assay uses the Amplification Refractory Mutation detection System (ARMS) for the qualitative and quantitative detection of T790M in exon 20 of EGFR in cfDNA and exosomal RNA, obtained using our extraction procedure (EXO52). The assay was tested on plasma from 160 patient samples. Of these, 53 of the NSCLC samples were classified as EGFR T790M positive at baseline by tissue analysis (i.e. prior to treatment with mutant-selective inhibitor of EGFR) and 107 were either NSCLC samples negative by FFPE, or were obtained from individual or pooled healthy donors. Within the 53 samples classified as T790M positive by FFPE, ~40% (21/53) were patients with intrathoracic (M0-M1a) disease or unknown M stage (MX) that have historically been very difficult to detect by cfDNA analysis. Additional analytical validation for this assay was performed using 98 individual spike-ins of varying concentrations of T790M (0.75-2660 copies/mL) into healthy pooled plasma by different operators and on different days. Results: Average analytical sensitivity and specificity across 100 bootstraps using a l1-regularized logistic regression model on clinical samples in this cohort of 160 samples was determined to be 87% (±10%) and 98% (±4%), respectively