P3.02b-025 Rapid and Highly Sensitive EGFRdelEx19 and KRAS Exon 2 Mutation Detection in EBUS-TBNA Specimen of Lung Adenocarcinoma: Topic: EGFR Biomarkers

Abstract

First-line treatment with afatinib prolongs overall survival in patients with metastatic non-small-cell lung cancer (NSCLC) harboring EGFR exon 19 deletion mutations. Conversely, somatic KRAS mutations are negative predictors for benefit from EGFR-targeting agents. Rapid availability of these biomarker results is mandatory to prevent delayed or inferior treatments. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is well-established for lung cancer diagnosis and staging. Next generation sequencing (NGS) via targeted resequencing allows simultaneous interrogation for multiple mutations, but has its limitations based on the amount of tumor tissue required and assay times. RT-PCR using Light-Cycler technology (LC-RTPCR) is a rapid and sensitive assay to detect somatic mutations in various tissues from NSCLC patients. The study’s aim was to analyze if LC-RTPCR is feasible for rapid EGFRdelE×19 and KRAS Exon 2 mutation detection in EBUS-TBNA samples and to compare results with results obtained via standard NGS mutation analyses. 48 surplus EBUS-TBNA samples (38 lymph nodes, 10 primary tumor) from 47 patients with pulmonary adenocarcinoma were analyzed. Two samples were collected per lymph node. One was used for routine cytology, the other was freshly frozen (ff). DNA from ff-biopsies was extracted using Genomic DNA buffer set (QIAgen, Germany). Mutation analysis by LC-RTPCR was conducted as previously described. NGS was performed on MiSeq (Illumina) via targeted resequencing using a customized multiplex-PCR panel covering 36 exons from 11 genes. Mutations were annotated with a minimum frequency of 2%. Processing time was approximately 4 days for NGS and 2 hours for LC-RTPCR analyses. NGS of EBUS-TBNA samples was technically feasible for both markers in 22 (46%) samples, for EGFR testing in 32 (67%) samples, and for KRAS in 23 (48%) samples. EGFRdelEx19 mutations were detected in four (8.3% of intention-to-screen), and KRAS Exon 2 mutations in 15 cases (31%) cases. LC-RTPCR was technically feasible in all cases. All mutations detected by NGS were also detected by LC-RTPCR. LC-RTPCR detected three additional KRAS Exon 2 mutations and three additional EGFRdelEx19 mutations. NGS detected additional mutations in 4 cases (2 EGFR Exon 21, 1 KRAS Codon 61, 1 PIK3CA). LC-RTPCR is a rapid, highly sensitive method to detect mutations of immediate therapeutic relevance, such as EGFRdelEx19 and KRAS Exon 2 mutations, in limited EBUS-TBNA specimens from metastatic NSCLC patients. It is of value as rapid and sensitive initial assay in a two-step diagnostic process for first-line treatment decision, incorporating broader biomarker panels as second step.