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Pyrosequencing for EGFR Mutation Detection: Diagnostic Accuracy and Clinical Implications

Sahnane, Nora PhD*; Gueli, Rossana MD; Tibiletti, Maria G. BSc*; Bernasconi, Barbara PhD*; Stefanoli, Michele BSc*; Franzi, Francesca MD*; Pinotti, Graziella MD; Capella, Carlo MD*; Furlan, Daniela BSc*

doi: 10.1097/PDM.0b013e3182893f55
Original Articles

EGFR-activating mutations predict responsiveness to EGFR tyrosine kinase inhibitors (TKIs) in non–small cell lung cancer (NSCLC) patients. Mutation screening is crucial to support therapeutic decisions and is commonly conducted using dideoxy sequencing, although its sensitivity is suboptimal in clinical settings. To evaluate the diagnostic performance of pyrosequencing and dideoxy sequencing, we examined EGFR mutation status in a retrospective cohort of 53 patients with NSCLCs clinically selected for TKI therapy and whose clinical outcome was available. Moreover, pyrosequencing quantitative results were compared with EGFR amplification data. EGFR mutations were investigated by pyrosequencing and by dideoxy sequencing. Detection rates of both methods were determined by titration assays using NCI-H1975 and HCC-827 cell lines. Increased EGFR copy number was assessed by fluorescence in situ hybridization (FISH). Pyrosequencing showed a higher detection rate than dideoxy sequencing. Tumor control rate of cases with mutant and wild-type EGFR was 86% and 29%, respectively. EGFR amplification was significantly associated with EGFR mutation and a positive correlation between high percentages of mutant alleles and clinical response to TKI was observed. We concluded that pyrosequencing is more sensitive than dideoxy sequencing in mutation screening for EGFR mutations. Detection rate of dideoxy sequencing was suboptimal when low frequencies of mutant alleles or low tumor cell contents were observed. Pyrosequencing enables quantification of mutant alleles that correlates well with increased EGFR copy number assessed by FISH. Pyrosequencing should be used in molecular diagnostic of NSCLC to appropriately select patients who are likely to benefit from TKI therapy.

*Department of Surgical and Morphological Sciences, Anatomic Pathology Unit, University of Insubria, Varese, Italy

Oncology Unit, Ospedale di Circolo, Varese, Italy

N.S. and R.G. contributed equally.

Supported in part by the University of Insubria.

No fundings were received from National Institutes of Health (NIH), Wellcome Trust, Howard Hughes Medical Institute (HHMI), or other organizations.

The authors declare no conflict of interest.

Reprints: Nora Sahnane, PhD, Department of Surgical and Morphological Sciences, Anatomic Pathology Unit, Ospedale di Circolo, University of Insubria, Via O. Rossi, 9, 21100 Varese, Italy (e-mail:

© 2013 by Lippincott Williams & Wilkins.