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.
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.
Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland.
Katja
K
Tuononen
Satu
S
Mäki-Nevala
Virinder Kaur
VK
Sarhadi
Aino
A
Wirtanen
Mikko
M
Rönty
Kaisa
K
Salmenkivi
Jenny M
JM
Andrews
Aino I
AI
Telaranta-Keerie
Sari
S
Hannula
Sonja
S
Lagström
Pekka
P
Ellonen
Aija
A
Knuuttila
Sakari
S
Knuutila
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D056726:Genetic Association Studies; D005820:Genetic Testing / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D054643:INDEL Mutation; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D060888:Real-Time Polymerase Chain Reaction; D018631:ras Proteins / Q000235:genetics*
D005404:Fixatives; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; C512478:EGFR protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins