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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2025-02-01
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| author | Magdalena Jurkiewicz Raymond Yeh Catherine A. Shu Susan J. Hsiao Mahesh M. Mansukhani Anjali Saqi Helen Fernandes |
| author_facet | Magdalena Jurkiewicz Raymond Yeh Catherine A. Shu Susan J. Hsiao Mahesh M. Mansukhani Anjali Saqi Helen Fernandes |
| author_sort | Magdalena Jurkiewicz |
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| description | <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. |
| format | Article |
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| issn | 2673-5261 |
| language | English |
| publishDate | 2025-02-01 |
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| spelling | doaj-art-e2259a25b9344ef48c52ab00df1e6d282025-08-20T02:11:17ZengMDPI AGJournal of Molecular Pathology2673-52612025-02-0161510.3390/jmp6010005Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung CancersMagdalena Jurkiewicz0Raymond Yeh1Catherine A. Shu2Susan J. Hsiao3Mahesh M. Mansukhani4Anjali Saqi5Helen Fernandes6Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032, USADepartment of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032, USADepartment of Medicine, Columbia University Irving Medical Center, New York, NY 10032, USADepartment of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032, USADepartment of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032, USADepartment of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032, USADepartment of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032, USA<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.https://www.mdpi.com/2673-5261/6/1/5<i>MET</i> exon 14 skippingDNA NGSRNA sequencinglung adenocarcinoma |
| spellingShingle | Magdalena Jurkiewicz Raymond Yeh Catherine A. Shu Susan J. Hsiao Mahesh M. Mansukhani Anjali Saqi Helen Fernandes Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung Cancers Journal of Molecular Pathology <i>MET</i> exon 14 skipping DNA NGS RNA sequencing lung adenocarcinoma |
| title | Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung Cancers |
| title_full | Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung Cancers |
| title_fullStr | Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung Cancers |
| title_full_unstemmed | Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung Cancers |
| title_short | Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung Cancers |
| title_sort | challenges in amplicon based dna ngs identification of met exon 14 skipping events in non small cell lung cancers |
| topic | <i>MET</i> exon 14 skipping DNA NGS RNA sequencing lung adenocarcinoma |
| url | https://www.mdpi.com/2673-5261/6/1/5 |
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