Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 May 20;8(57):96684-96696.
doi: 10.18632/oncotarget.18042. eCollection 2017 Nov 14.

Clinical mutational profiling of 1006 lung cancers by next generation sequencing

Peter B Illei  1 , Deborah Belchis  1 , Li-Hui Tseng  1   2 , Doreen Nguyen  1 , Federico De Marchi  1   3 , Lisa Haley  1 , Stacy Riel  1 , Katie Beierl  1 , Gang Zheng  1 , Julie R Brahmer  4 , Frederic B Askin  1 , Christopher D Gocke  1   4 , James R Eshleman  1   4 , Patrick M Forde  4 , Ming-Tseh Lin  1
Affiliations

Clinical mutational profiling of 1006 lung cancers by next generation sequencing

Peter B Illei et al. Oncotarget. .

Abstract

Analysis of lung adenocarcinomas for actionable mutations has become standard of care. Here, we report our experience using next generation sequencing (NGS) to examine AKT1, BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA genes in 1006 non-small cell lung cancers in a clinical diagnostic setting. NGS demonstrated high sensitivity. Among 760 mutations detected, the variant allele frequency (VAF) was 2-5% in 33 (4.3%) mutations and 2-10% in 101 (13%) mutations. A single bioinformatics pipeline using Torrent Variant Caller, however, missed a variety of EGFR mutations. Mutations were detected in KRAS (36% of tumors), EGFR (19%) including 8 (0.8%) within the extracellular domain (4 at codons 108 and 4 at codon 289), BRAF (6.3%), and PIK3CA (3.7%). With a broader reportable range, exon 19 deletion and p.L858R accounted for only 36% and 26% of EGFR mutations and p.V600E accounted for only 24% of BRAF mutations. NGS provided accurate sequencing of complex mutations seen in 19% of EGFR exon 19 deletion mutations. Doublet (compound) EGFR mutations were observed in 29 (16%) of 187 EGFR-mutated tumors, including 69% with two non-p.L858R missense mutations and 24% with p.L858 and non-p.L858R missense mutations. Concordant VAFs suggests doublet EGFR mutations were present in a dominant clone and cooperated in oncogenesis. Mutants with predicted impaired kinase, observed in 25% of BRAF-mutated tumors, were associated with a higher incidence of concomitant activating KRAS mutations. NGS demonstrates high analytic sensitivity, broad reportable range, quantitative VAF measurement, single molecule sequencing to resolve complex deletion mutations, and simultaneous detection of concomitant mutations.

Keywords: cancer; lung; mutation; profiling; sequencing.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. EGFR mutations detected by next generation sequencing
EGFR exon 19 deletion mutation of 3.2% variant allele frequency detected by Integrative Genomics Viewer inspection, but missed by Torrent Variant Caller (A). Complex exon 19 deletion mutation composed of two deletions (c.2235_2236del and c.2241_2250del) and one single nucleotide change (c.2251A>C) (B).
Figure 2
Figure 2
Correlation of variant allele frequencies in 29 tumors with doublet (compound) EGFR mutations (A) and 7 tumors with concomitant EGFR and PIK3CA mutations (B). The r is 0.94 when an outlier (arrowhead in A) was removed. This outlier represents a tumor with a p.T790M germline mutation and a p.L858 somatic mutation. r: Spearman’s rank correlation coefficiency.
Figure 3
Figure 3. Incidence of doublet (compound) EGFR mutations involving different codons
Exon 19 del: exon 19 deletion mutations. Exon 20 ins: exon 20 insertion/duplication mutations.
See this image and copyright information in PMC

References

    1. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350:2129–2139. - PubMed
    1. Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304:1497–1500. - PubMed
    1. Sequist LV, Yang JC, Yamamoto N, O’Byrne K, Hirsh V, Mok T, Geater SL, Orlov S, Tsai CM, Boyer M, Su WC, Bennouna J, Kato T, et al. Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. J Clin Oncol. 2013;31:3327–3334. - PubMed
    1. Keedy VL, Temin S, Somerfield MR, Beasley MB, Johnson DH, McShane LM, Milton DT, Strawn JR, Wakelee HA, Giaccone G. American Society of Clinical Oncology provisional clinical opinion: epidermal growth factor receptor (EGFR) Mutation testing for patients with advanced non-small-cell lung cancer considering first-line EGFR tyrosine kinase inhibitor therapy. J Clin Oncol. 2011;29:2121–2127. - PubMed
    1. Lindeman NI, Cagle PT, Beasley MB, Chitale DA, Dacic S, Giaccone G, Jenkins RB, Kwiatkowski DJ, Saldivar JS, Squire J, Thunnissen E, Ladanyi M. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Arch Pathol Lab Med. 2013;137:828–860. - PMC - PubMed

LinkOut - more resources