Content area
Full Text
Virchows Arch (2013) 462:409419 DOI 10.1007/s00428-013-1376-6
ORIGINAL ARTICLE
Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep sequencing
Evgeny A. Moskalev & Robert Sthr & Ralf Rieker &
Simone Hebele & Florian Fuchs & Horia Sirbu &
Sergey E. Mastitsky & Carsten Boltze & Helmut Knig &
Abbas Agaimy & Arndt Hartmann & Florian Haller
Received: 22 October 2012 /Revised: 16 December 2012 /Accepted: 23 January 2013 /Published online: 7 March 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Detection of activating EGFR mutations in NSCLC is the prerequisite for individualised therapy with receptor tyrosine kinase inhibitors (TKI). In contrast, mutant downstream effector KRAS is associated with TKI resistance. Accordingly, EGFR mutation status is routinely examined in NSCLC specimens, but the employed methods may have a dramatic impact on the interpretation of results
and, consequently, therapeutic decisions. Specimens with low tumour cell content are at particular risk for false-negative EGFR mutation reporting by sequencing with Sanger chemistry. To improve reliability of detecting clinically relevant mutant variants of EGFR and KRAS, we took full advantage of 454 deep sequencing and developed a two-step amplification protocol for the analysis of EGFR exons 1821 and KRAS exons 2 and 3. We systematically addressed the sensitivity, reproducibility and specificity of the developed assay. Mutations could be reliably identified down to an allele frequency of 0.21.5 %, as opposed to 10 20 % detection limit of Sanger sequencing. High reproducibility (02.1 % variant frequency) and very low background level (0.40.8 % frequency) further complement the reliability of this assay. Notably, re-evaluation of 16 NSCLC samples with low tumour cell content 40 % and
EGFR wild type status according to Sanger sequencing revealed clinically relevant EGFR mutations at allele frequencies of 0.910 % in seven cases. In summary, this novel two-step amplification protocol with 454 deep sequencing is superior to Sanger sequencing with significantly increased sensitivity, enabling reliable analysis of EGFR and KRAS in NSCLC samples independent of the tumour cell content.
Keywords NSCLC . EGFR . KRAS . 454 Deep sequencing . Sensitivity
Introduction
The observation of activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene in...