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A Comparison of Methods for EGFR Mutation Testing in Non–Small Cell Lung Cancer

Young, Elizabeth C. PhD*; Owens, Martina M. MSc*; Adebiyi, Idowu MSc; Bedenham, Tina BSc; Butler, Rachel PhD; Callaway, Jonathan BSc§; Cranston, Treena BSc; Crosby, Charlene MBiolSci; Cree, Ian A. MBChB, PhD; Dutton, Laura BSc#; Faulkes, Catherine MSc**; Faulkner, Claire PhD; Howard, Emma PhD#; Knight, Julia MSc††; Huang, Yuanxue PhD††; Lavender, Louise PhD**; Lazarou, Lazarus P. BSc; Liu, Hongxiang PhD††; Mair, Debbie MSc‡‡; Milano, Antonio PhD§§; Sandell, Stacey BSc§; Skinner, Alison BSc§; Wallace, Andrew PhD#; Williams, Maggie BSc; Spivey, Vicky PhD∥∥; Goodall, John PhD∥∥; Frampton, Jonathan PhD∥∥; Ellard, Sian PhD*; on behalf of the Clinical Molecular Genetics Society (CMGS) Scientific Subcommittee

doi: 10.1097/PDM.0b013e318294936c
Original Articles

EGFR mutation testing of tumor samples is routinely performed to predict sensitivity to treatment with tyrosine kinase inhibitors for patients with non–small cell lung cancer. At least 9 different methodologies are employed in UK laboratories, and the aim of this study was to compare the sensitivity of different methods for the detection of EGFR mutations. Participating laboratories were sent coded samples with varying mutation loads (from 0% to 15%) to be tested for the p.Leu858Arg (p.L858R) missense mutation and c.2235_2249del exon 19 deletion. The p.L858R mutation and deletions within exon 19 of the EGFR gene account for ∼90% of mutation-positive cases. The 11 laboratories used their standard testing method(s) and submitted 15 sets of results for the p.L858R samples and 10 for the exon 19 deletion. The p.Leu858Arg (p.L858R) mutation was detected at levels between 1% and 7.5% by Sanger sequencing, pyrosequencing, real-time polymerase chain reaction (PCR), amplification refractory mutation system, and capillary electrophoresis single-strand conformation analysis. The c.2235_2249del mutation was detected at 1% to 5% by fragment size analysis, Sanger sequencing or real-time PCR. A mutation was detected in 24/25 (96%) of the samples tested which contained 5% mutated DNA. The 1% sensitivity claimed for commercial real-time PCR-targeted EGFR tests was achieved and our results show greater sensitivity for the Sanger sequencing and pyrosequencing screening methods compared to the 10% to 20% detection levels cited on clinical diagnostic reports. We conclude that multiple methodologies are suitable for the detection of acquired EGFR mutations.

*Molecular Genetics Department, Royal Devon and Exeter NHS Trust, Exeter

Molecular Genetics, Institute of Medical Genetics, Cardiff and Vale NHS Trust, Cardiff, Wales

Oxford Medical Genetics Laboratories, Oxford University Hospitals NHS Trust, Oxford

§Wessex Regional Genetics Laboratory, Salisbury Health Care NHS Trust, Wiltshire

Bristol Genetics Laboratory, Southmead Hospital, Bristol

Department of Pathology, Warwick Medical School, University Hospitals Coventry and Warwickshire, Coventry

#Regional Molecular Genetics Service, Genetic Medicine (6th Floor), St Mary’s Hospital, Manchester

**Molecular Diagnostics, Royal Surrey County Hospital

‡‡Molecular Diagnostics, The Institute of Cancer Research and Royal Marsden NHS Foundation Trust, Surrey

††Molecular Malignancy Laboratory, Department of Histopathology, Addenbrooke’s Hospital, Cambridge University Hospitals NHS Foundation Trust

∥∥Horizon Discovery Ltd., Waterbeach, Cambridge

§§Sheffield Diagnostic Genetics Service, Sheffield, UK

R.B. has had past consultancy posts with Roche, Astra Zeneca, Boehringer Ingelheim, and Pfizer; and her institution is in receipt of a grant from Cancer Research, UK. A.W. has current consultancy posts with Roche and Astra Zeneca. The remaining authors declare no conflict of interest.

Reprints: Sian Ellard, PhD, Molecular Genetics Department, Royal Devon and Exeter NHS Foundation Trust, Barrack Road, Exeter, EX2 5AD, UK (e-mail:

© 2013 by Lippincott Williams & Wilkins.