Comparison of Targeted Next‐Generation Sequencing (NGS) and Real‐Time PCR in the Detection of EGFR, KRAS, and BRAF Mutations on Formalin‐Fixed, Paraffin‐Embedded Tumor Material of Non‐Small Cell Lung Carcinoma—Superiority of NGS

Abstract

The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

Analytical Validation of BRAF Mutation Testing from Circulating Free DNA Using the Amplification Refractory Mutation Testing System

“…The statement is evidence based and supported by 5 studies, 169e173 comprising 1 PCS, 172 2 PRCSs, 171,173 and 2 RCSs. 169,170 All included studies were assessed for quality and none were found to have methodologic flaws that would raise concerns about the studies' findings (SDC Table 22).…”

Next-generation sequencing analysis of lung and colon carcinomas reveals a variety of genetic alterations

“…The additional deletion in the EGFR gene was concomitant with the G12D KRAS mutation in one patient, and with a V600E BRAF mutation in another patient. KRAS, BRAF and EGFR mutations are normally exclusive (9) but concomitant KRAS and EGFR mutations have already been described (10,11). The identification of concomitant mutations should increase with the higher sensitivity of NGS technologies.…”

Tissue Requirements and DNA Quality Control for Clinical Targeted Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Samples: A Mini-Review of Practical Issues

“…Most studies showed successful sequencing results in samples with >20% tumor content [3,5,8]. An EGFR mutation was called for in all samples with tumor-cell content >22.8% [3].…”

“…However, demand for improved sensitivity of direct sequencing and resolving the inconvenience of multiple sequential testing practices for real-time PCR has increased [2,3]. Targeted NGS can overcome these limitations of DS or real-time PCR, and showed that NGS tests are accurate and feasible to detect clinically relevant cancer mutations as daily routine diagnostics [4][5][6]. Thus, targeted NGS has been suggested for routine diagnostics and is now widely used in the molecular pathology laboratories [4][5][6].…”