Cancer Res Treat.  2024 Apr;56(2):455-463. 10.4143/crt.2023.1108.

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

• Akkhasutthikun P
• Kaewsapsak P
• Nimsamer P
• Klomkliew P
• Visedthorn S
• Chanchaem P
• Teerapakpinyo C
• Payungporn S
• Luangdilok S

• KMID: 2554336
• DOI: http://doi.org/10.4143/crt.2023.1108

Abstract

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.

Keyword

Non?small cell lung carcinoma; mutation; Blocker displacement amplification; Nanopore sequencing

Figure

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  Fig. 1. (A) The principle of blocker displacement amplification (BDA)–based polymerase chain reaction amplicon and Nanopore sequencing for epidermal growth factor receptor (EGFR) mutations. Formalin-fixed, paraffin-embedded (FFPE) and cell-free DNA (cfDNA) samples were performed based on BDA using blocker probe (light green line) bind with the wild-type allele (dark green circle) to prevent amplification. Conversely, it could not bind to the mutant allele (red circle), leading to amplification. Nanopore sequencing was used for following mutant allele enrichment. (B) Bioinformatic pipeline for variant detection (created with Biorender.com).

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  Fig. 2. The blocker displacement amplification (BDA) amplicon Nanopore sequencing was evaluated for variant allele frequency (VAF) enrichment (red bar), compared to without the blocker probes (blue bar). (A) The VAF enrichment of each mutant epidermal growth factor receptor (EGFR) from lung cancer cell lines. The detection of low VAF from mutant EGFR was mixed in wild-type EGFR with different ratios input from 5% to 0.5%. The VAF after BDA enrichment is shown in exon 19 (B), exon 20 (C), and exon 21 (D).

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  Fig. 3. The calculated cutoff for detecting epidermal growth factor receptor (EGFR) mutations in exon 19 (A), exon 20 (C), and exon 21 (E) from formalin-fixed, paraffin-embedded DNA. For cell-free DNA, the calculated cutoff for detecting EGFR mutations in exon 19 (B), exon 20 (D), and exon 21 (F). The red dots represent mutant mixed in wild-type alleles with ranging ratios of 5% to 0.1%. The blue dots represent the wild-type patient samples. LOD, limits of detection.

Reference

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