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PLoS One. 2011 May 5;6(5):e19601. doi: 10.1371/journal.pone.0019601.

Benchmarking of mutation diagnostics in clinical lung cancer specimens.

Author information

1
Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne, Cologne, Germany.

Abstract

Treatment of EGFR-mutant non-small cell lung cancer patients with the tyrosine kinase inhibitors erlotinib or gefitinib results in high response rates and prolonged progression-free survival. Despite the development of sensitive mutation detection approaches, a thorough validation of these in a clinical setting has so far been lacking. We performed, in a clinical setting, a systematic validation of dideoxy 'Sanger' sequencing and pyrosequencing against massively parallel sequencing as one of the most sensitive mutation detection technologies available. Mutational annotation of clinical lung tumor samples revealed that of all patients with a confirmed response to EGFR inhibition, only massively parallel sequencing detected all relevant mutations. By contrast, dideoxy sequencing missed four responders and pyrosequencing missed two responders, indicating a dramatic lack of sensitivity of dideoxy sequencing, which is widely applied for this purpose. Furthermore, precise quantification of mutant alleles revealed a low correlation (r(2) = 0.27) of histopathological estimates of tumor content and frequency of mutant alleles, thereby questioning the use of histopathology for stratification of specimens for individual analytical procedures. Our results suggest that enhanced analytical sensitivity is critically required to correctly identify patients responding to EGFR inhibition. More broadly, our results emphasize the need for thorough evaluation of all mutation detection approaches against massively parallel sequencing as a prerequisite for any clinical implementation.

PMID:
21573178
PMCID:
PMC3088700
DOI:
10.1371/journal.pone.0019601
[Indexed for MEDLINE]
Free PMC Article

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