Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung Cancers

Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung Cancers

<b>Introduction:</b> <i>MET</i> Exon 14 skipping alterations are drivers of non-small cell lung carcinoma (NSCLC) with responses to tyrosine kinase inhibitors. Amplicon-based DNA NGS assays (DNA NGSs) for the detection of <i>MET</i>ex14 skipping can yield false-ne...

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Main Authors: Magdalena Jurkiewicz, Raymond Yeh, Catherine A. Shu, Susan J. Hsiao, Mahesh M. Mansukhani, Anjali Saqi, Helen Fernandes
Format: Article
Language:English
Published: MDPI AG 2025-02-01
Series:Journal of Molecular Pathology
Subjects:
<i>MET</i> exon 14 skipping
DNA NGS
RNA sequencing
lung adenocarcinoma
Online Access:https://www.mdpi.com/2673-5261/6/1/5
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Summary:<b>Introduction:</b> <i>MET</i> Exon 14 skipping alterations are drivers of non-small cell lung carcinoma (NSCLC) with responses to tyrosine kinase inhibitors. Amplicon-based DNA NGS assays (DNA NGSs) for the detection of <i>MET</i>ex14 skipping can yield false-negative results. We examined the efficacy of <i>MET</i>ex14 skipping with a DNA NGS and reflex RNA-based NGS (RNA NGS) strategy. <b>Materials and Methods</b>: Clinical cases with definitive or suspected lung adenocarcinoma (LungCa), lacking driver mutations with targeted DNA NGS, underwent the RNA NGS to identify oncogenic drivers. Samples with <i>MET</i>ex14 skipping identified on reflex RNA NGSs were confirmed with Sanger sequencing. <b>Results:</b><i>MET</i>ex14 skipping events were detected in 22/762 (2.9%) samples by DNA NGS. RNA NGS identified 10 additional samples, for an overall frequency of 32/762 (4.1%). All 22 <i>MET</i>ex14 DNA variants affected the donor splice site. Sanger sequencing revealed that missed <i>MET</i>ex14 variants were largely deletions spanning the ~30 bp intronic region upstream of the splice acceptor site. The failure of DNA NGS to detect all <i>MET</i>ex14 mutants was due to a lack of coverage of the 3′ splice acceptor site of intron 13, branch point, and polypyrimidine tract. <b>Conclusions:</b> Modification of amplicon-based DNA hotspot assays, with primers that cover both donor and acceptor splice sites, can identify a larger number of <i>MET</i>ex14 skipping events. Clinical data show that patients with advanced NSCLC and <i>MET</i>ex14 variants responded to targeted therapy.
ISSN:2673-5261

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