Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Assessment of the clinical application of detecting EGFR, KRAS, PIK3CA and BRAF mutations in patients with non-small cell lung cancer using next-generation sequencing.

Author(s): Xu X, Yang Y, Li H, Chen Z, Jiang G, Fei K

Publication: Scand J Clin Lab Invest, 2016, Vol. , Page 1-7

PubMed ID: 27215271 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the concordance of genotyping calls obtained by NGS using the NextDaySeq lung panel on an Ion Torrent and by real-time qPCR using the Human KRAS, PIK3CA and BRAF Gene Mutations Detection Kits in NSCLC specimens. Researchers were blinded when comparing techniologies. Genotyping discrepancies were further investigated by additional real-time qPCR assays (Roche Cobas KRAS and EGFR Mutation Test panels), Sanger sequencing, and/or additional NGS using a custom AmpliSeq panel. Areas with >10% tumor content were selected from 188 resected NSCLC specimens. Although specimen processing details were not provided, the authors state that microdissected fresh specimens were snap-frozen and used when an FFPE specimen was not available. DNA was extracted from five or more 5µm FFPE sections using QIAamp DNA FFPE Tissue Kit and from frozen specimens using QIAamp DNA Tissue Kit. The resultant DNA was stored at -20˚C until use. NGS using the NextDaySeq lung panel on an Ion Torrent achieved >500x coverage in 96.2% of specimens with 96.8% uniformity. 

   Summary of Findings:

   Of the 109 mutations identified using the NextDaySeq lung panel, 89 were also identified with real-time qPCR and 18 were not included in the real-time qPCR panel.  Additionally, 10 mutations originally identified by qPCR were not found using the NextDaySeq lung panel. Thus, if real-time qPCR was considered the reference method after the initial assay, NGS had a sensitivity of 89.9%, specificity of 97.5%, PPV of 97.8%, and NPV of 88.6%. However, further analysis showed that the 10 mutations originally missed by NGS were in fact detected but at a level below the default VAF cut-off of 1%. Similarly, the two mutations identified by NGS but originally missed by real-time qPCR were present after repeating the initial assay and when investigated using the COBAS real-time qPCR assays. Of the 18 variants identified by NGS but not included in the real-time qPCR panels, the 13 that occurred at a VAF of more than 10% were confirmed by Sanger sequencing but the remaining five which occurred at a VAF of 2.2-7.3% were not detected by Sanger sequencing but were confirmed with the use of AmpliSeq deep sequencing. 

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing
   DNA	SNP assay
   DNA	Real-time qPCR
   DNA	DNA sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Next generation sequencing Specific	Technology platform	Real-time PCR Sanger sequencing
   Real-time qPCR Specific	Targeted nucleic acid	Human KRAS Gene Mutations Detection Kit Human BRAF Gene Mutations Detection Kit Human PIK3CA Gene Mutations Detection Kit Roche Cobas EGFR Mutation Test panels Roche Cobas KRAS Mutation Test panels
   DNA sequencing Specific	Targeted nucleic acid	EGFR exons 18, 19, 20, and 21 KRAS exons 2 and 3 PIK3CA exons 9 and 20 BRAF exons 11 and 15
   Next generation sequencing Specific	Targeted nucleic acid	NextDaySeq Lung panel AmpliSeq Panel

   View more