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 (r2 = 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.
Highlights
Limited to rare tumors such as chronic myeloid leukemia (CML) or gastrointestinal stromal tumors (GIST), the success of treating mutationally activated kinases with kinase inhibitors has reached the group of the frequent ‘‘solid’’ tumors and has thereby profoundly changed clinical oncology
We show how the limited sensitivity of methods that are widely applied to clinical mutation diagnostics could lead to a critical inaccuracy in genetic patient stratification
Despite the limited size of our sample set, these results underscore the inadequacy of dideoxy sequencing for clinical cancer gene mutation diagnostics
Summary
Limited to rare tumors such as chronic myeloid leukemia (CML) or gastrointestinal stromal tumors (GIST), the success of treating mutationally activated kinases with kinase inhibitors has reached the group of the frequent ‘‘solid’’ tumors and has thereby profoundly changed clinical oncology. In light of the current efforts to systematically sequence the genomes of all major tumor types (http://www.icgc.org/ or www.cancergenome.nih.gov), the list of genetically defined tumors that are susceptible to a specific therapeutic intervention is likely to expand dramatically in the few years. Together, these observations suggest a rapidly growing need for sensitive and accurate mutation detection in clinical specimens as part of routine clinical care. The tumor composition as well as multiple types of non-neoplastic cells affect the detection of tumor-specific somatic mutations in a background of non-mutant, ‘‘wild-type’’ alleles. Conventional sequencing approaches lack sensitivity for the detection of such rare alleles
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