Tissue and Plasma-Based Highly Sensitive Blocker Displacement Amplicon Nanopore Sequencing for EGFR Mutations in Lung Cancer.

Autor: Akkhasutthikun P; Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.; Center of Excellence in Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand., Kaewsapsak P; Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.; Center of Excellence in Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand., Nimsamer P; Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.; Division of Medical Bioinformatics, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.; Siriraj Long-Read Lab (Si-LoL), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand., Klomkliew P; Center of Excellence in Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand., Visedthorn S; Center of Excellence in Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand., Chanchaem P; Center of Excellence in Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand., Teerapakpinyo C; Chula GenePRO Center, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand., Payungporn S; Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.; Center of Excellence in Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand., Luangdilok S; Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Jazyk: angličtina
Zdroj: Cancer research and treatment [Cancer Res Treat] 2024 Apr; Vol. 56 (2), pp. 455-463. Date of Electronic Publication: 2023 Nov 20.
DOI: 10.4143/crt.2023.1108
Abstrakt: Purpose: The epidermal growth factor receptor (EGFR) mutation is a widely prevalent oncogene driver in non-small cell lung cancer (NSCLC) in East Asia. The detection of EGFR mutations is a standard biomarker test performed routinely in patients with NSCLC for the selection of targeted therapy. Here, our objective was to develop a portable new technique for detecting EGFR (19Del, T790M, and L858R) mutations based on Nanopore sequencing.
Materials and Methods: The assay employed a blocker displacement amplification (BDA)-based polymerase chain reaction (PCR) technique combined with Nanopore sequencing to detect EGFR mutations. Mutant and wild-type EGFR clones were generated from DNA from H1650 (19Del heterozygous) and H1975 (T790M and L858R heterozygous) lung cancer cell lines. Then, they were mixed to assess the performance of this technique for detecting low variant allele frequencies (VAFs). Subsequently, formalin-fixed, paraffin-embedded (FFPE) tissue and cell-free DNA (cfDNA) from patients with NSCLC were used for clinical validation.
Results: The assay can detect low VAF at 0.5% mutant mixed in wild-type EGFR. Using FFPE DNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and Cobas real-time PCR were 98.46%, 100%, and 100%, respectively. For cfDNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and droplet digital PCR were 94.74%, 100%, and 100%, respectively.
Conclusion: The BDA amplicon Nanopore sequencing is a highly accurate and sensitive method for the detection of EGFR mutations in clinical specimens.
Databáze: MEDLINE
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