J Pathol Transl Med.  2021 Sep;55(5):324-329. 10.4132/jptm.2021.07.15.

Robust home brew fragment sizing assay for detection of MET exon 14 skipping mutation in non–small cell lung cancer patients in resource constrained community hospitals

• Mehta A
• Nathany S
• Chopra A
• Mattoo S
• Kumar D
• Panigrahi MK

• KMID: 2520171
• DOI: http://doi.org/10.4132/jptm.2021.07.15

Abstract

Background
A mutation/deletion involving donor or acceptor sites for exon 14 results in splicing out of exon 14 of the mesenchymal epithelial transition (MET) gene and is known as “MET exon 14 skipping” (ΔMET14). The two recent approvals with substantial objective responses and improved progression-free survival to MET inhibitors namely capmatinib and tepotinib necessitate the identification of this alteration upfront. We herein describe our experience of ΔMET14 detection by an mRNA-based assay using polymerase chain reaction followed by fragment sizing.
Methods
This is a home brew assay which was developed with the concept that the transcripts from true ΔMET14 will be shorter by ~140 bases than their wild type counterparts. The cases which were called MET exon 14 skipping positive on next-generation sequencing (NGS) were subjected to this assay, along with 13 healthy controls in order to establish the validity for true negatives.
Results
Thirteen cases of ΔMET14 mutation were detected on NGS using RNA-based sequencing. Considering NGS as a gold standard, the sizing assay using both gel and capillary electrophoresis that showed 100% specificity for both with concordance rates of 84.6% and 88.2% with NGS, respectively, were obtained.
Conclusions
Owing to the cost-effective nature and easy to use procedures, this assay will prove beneficial for small- and medium-sized laboratories where skilled technical personnel and NGS platforms are unavailable.

Keyword

MET exon 14 skipping; Lung neoplasms; Fragment sizing assay; Home brew; Electrophoresis

Figure

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  Fig. 1. Image illustrates the difference in size of the fragments between wild type and mutant ∆MET14mRNA.

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  Fig. 2. Primer design for fragment sizing assay for ∆MET14. The bases highlighted in yellow comprise the forward primer while those in green make the reverse primer. Nucleotides in blue belong to exon 14 (National Center for Biotechnology Information).

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  Fig. 3. (A) Analysis of two representative cases on polymerase chain reaction (PCR)–based gel electrophoresis and automated electrophoresis using Tape Station. a, DNA ladder; b, positive control for ∆MET14; c, sample positive for ∆MET14; d, sample negative for ∆MET14. (B) Mapped reads for ∆MET14 using next-generation sequencing–based assay. Good mapped reads for ∆MET14 (upper left panel). False call due to reads showing big deletions and polymorphism resulting in poor mapping quality (upper right panel). Integrative Genomics Viewer showing fewer mapped reads (low mutant allele frequency which may lead to a negative result of PCR-based assays (lower panel).

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  Fig. 4. Consort diagram showing an overview of cases included in the study along with the test results.

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  Fig. 5. Integrative Genomics Viewer image showing the coverage of MET exon 14 in the Oncomine Focus Assay.

Reference

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