DNA-Based versus RNA-Based Detection of MET Exon 14 Skipping Events in Lung Cancer

Abstract

Introduction: Genomic variants that lead to MET proto-oncogenem receptor tyrosine kinase (MET) exon 14 skipping represent a potential targetable molecular abnormality in NSCLC. Consequently, reliable molecular diagnostic approaches that detect these variants are vital for patient care.Methods: We screened tumor samples from patients with NSCLC for MET exon 14 skipping by using two distinct approaches: a DNA-based next-generation sequencing assay that uses an amplicon-mediated target enrichment and an RNA-based next-generation sequencing assay that uses anchored multiplex polymerase chain reaction for target enrichment.Results: The DNA-based approach detected MET exon 14 skipping variants in 11 of 856 NSCLC samples (1.3%). The RNA-based approach detected MET exon 14 skipping in 17 of 404 samples (4.2%), which was a statistically significant increase compared with the DNA-based assay. Among 286 samples tested by both assays, RNA-based testing detected 10 positives, six of which were not detected by the DNA-based assay. Examination of primer binding sites in the DNA-based assay in comparison with published MET exon 14 skipping variants revealed genomic deletion involving primer binding sequences as the likely cause of false negatives. Two samples positive via the DNA-based approach were uninformative via the RNA-based approach due to poor-quality RNA.Conclusions: By circumventing an inherent limitation of DNA-based amplicon-mediated testing, RNA-based analysis detected a higher proportion of MET exon 14 skipping cases. However, RNA-based analysis was highly reliant on RNA quality, which can be suboptimal in some clinical samples.

Highly accurate DNA-based detection and treatment results of MET exon 14 skipping mutations in lung cancer

“…Moreover, in many molecular diagnostic laboratories, RNA testing is not part of the routine work flow. Although DNA-based analysis would be easier to implement in most laboratories, accurate METexon14del detection has proven to be challenging on DNA level [22,23]. These mutations are variable in size and position, and can include large intron-exon bordering deletions.…”

“…Commercially available amplicon-based NGS panels for DNA analysis display severe limitations in accuracy, detecting no more than 63 % of literature-reported cases of alterations in MET exon 14 due to their design [24]. Davies et al confirmed that 60 % of the samples positive for METex14del by their RNA-based assay were negative in their DNA-based assay, using one of the commercially available panels described in Poirot et al [23]. The panel that was used (Illumina Trusight tumor 26 assay) detects up to 99 % of donor splice site and Y1003 mutations, but only 8 % of acceptor splice site or branch site mutations, according to in silico analysis performed by Poirot et al Indeed, all 11 MET mutations detected by the DNA assay of Davies et al concerned donor splice site mutations.…”

RNA sequencing: new technologies and applications in cancer research

“…Through the RNA-seq, MET exon 14 mutation and isocitrate dehydrogenase 1 (IDH1) mutation were identified as new potential therapeutic targets in lung adenocarcinoma and chondrosarcoma patients, respectively [80,81]. Several studies have shown that RNA sequencing can effectively improve the detection rate on the basis of DNA sequencing, provide more comprehensive detection results and achieve a better curative effect for targeted therapy [82]. In addition, it has been proved that IDH mutation is a good prognostic marker for glioma by RNA-seq [83].…”

Capmatinib for the treatment of non-small cell lung cancer

“…Testing for these alterations could be included in a broader DNA sequencing panel. However, given the various genomic locations of exon 14 skipping alterations, RNA-based approaches are now being assessed to more comprehensively capture MET exon 14 skipping events in the future [86].…”