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e-Poster Display Session

5P - False positives errors in RNA based next generation sequencing of exon 14 skipping mutations in NSCLC


24 Mar 2021


e-Poster Display Session


Moushumi Suryavanshi


Journal of Thoracic Oncology (2021) 16 (suppl_4): S699-S703.


M. Suryavanshi1, S. Mattoo2, U. Batra3, S. Sharma4, D. Kumar5, A. Mehta6

Author affiliations

  • 1 Rajiv Gandhi Cancer Institute and Research Centre, New Delhi/IN
  • 2 Rajiv Gandhi Cancer Institute and Research Center, 110085 - Delhi/IN
  • 3 Rajiv Gandhi Cancer Institute & Research Center, New Delhi/IN
  • 4 Rajiv Gandhi Cancer Institute and Research Centre, 11085 - New Delhi/IN
  • 5 Rajiv Gandhi Cancer Institute and Research Centre, 11085 - Delhi/IN
  • 6 Rajiv Gandhi Cancer Institute and Research Centre, 110085 - New Delhi/IN

Abstract 5P


Exon 14 skipping mutations are found in approximately 3% of patients with NSCLC. Robust approaches for detection of MET exon 14 skipping events are crucial for treatment. About one-third of the mutations occur between exons 13 and 14 at acceptor site of exon 14, and two-thirds occur between exons 14 and 15 at donor site. In addition, MET exon 14 skipping can result from large deletions that can span not only all of exon 14 but large portions of the intronic sequence. This mutation can be detected by sequencing of DNA or RNA or both. DNA based approaches alone are not able to detect more than 60% of these mutations. RNA based sequencing detects the product of exon 14 skipping mutations which is “fusion” of exon 13 to 15 regardless of the underlying genomic event. Most studies have favoured a RNA based approach.


During the period from August 2017 to January 2021, a total of 231 samples of NSCL were assessed by routine clinical application of the Thermofischer Ion Torrent™ Oncomine™ Focus 52 gene Assay. Sequencing data were processed with the Ion Torrent Suite software. This assay detects MET mutations at 3′ end splice donor site by DNA sequencing and RNA sequencing for exon 13 and exon 15 fusion for exon 14 skipping mutation. All positive cases on RNA sequencing were reanalysed by PCR and Sanger sequencing for confirmation.


Exon 13 and exon 15 fusion by RNA was detected in 20 cases. Read counts ranged from 143 to 7980. Two cases had additional MET amplification, one case had EGFR deletion 19,one case had CTNNB1 p.Ser37Phe and KRAS p.Gly12Asp, one case had RET KIF5B Fusion (read count 458), one case had EGFR amplification and in remaining cases exon 13 and exon 15 fusion was the sole abnormality. Only 6 out of 20 cases detected by NGS were confirmed by Sanger sequencing. All cases above the read count of 1607 were detected by sanger sequencing. All true positive cases had exon 3 and exon 15 fusion as the sole abnormality. Cases with MET amplification were also negative on sanger sequencing.


RNA based exon 13 and exon 15 fusion for detection of exon 14 skipping mutations can have false positive calls by Ion torrent-based sequencing and should be confirmed by alternate methods.

Legal entity responsible for the study

The authors.


Rajiv Gandhi Cancer Institute and Research Center, Delhi, India.


All authors have declared no conflicts of interest.

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