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Background  Oncogenic mutations are powerful predictive biomarkers for molecularly targeted cancer therapies. For mutation detection patients have to undergo invasive tumor biopsies. Alternatively, archival samples are used which may no longer reflect the actual tumor status. Circulating tumor cells (CTC) could serve as an alternative platform to detect somatic mutations in cancer patients. We sought to develop a sensitive and specific assay to detect mutations in the  EGFR  gene in CTC from lung cancer patients.    Methods  We developed a novel assay based on real-time polymerase chain reaction (PCR) and melting curve analysis to detect activating  EGFR  mutations in blood cell fractions enriched in CTC. Non-small-cell lung cancer (NSCLC) was chosen as disease model with reportedly very low CTC counts. The assay was prospectively validated in samples from patients with  EGFR -mutant and  EGFR -wild type NSCLC treated within a randomized clinical trial. Sequential analyses were conducted to monitor CTC signals during therapy and correlate mutation detection in CTC with treatment outcome.    Results  Assay sensitivity was optimized to enable detection of a single  EGFR -mutant CTC/mL peripheral blood. CTC were detected in pretreatment blood samples from all 8  EGFR -mutant lung cancer patients studied. Loss of  EGFR -mutant CTC signals correlated with treatment response, and its reoccurrence preceded relapse.    Conclusions  Despite low abundance of CTC in NSCLC oncogenic mutations can be reproducibly detected by applying an unbiased CTC enrichment strategy and highly sensitive PCR and melting curve analysis. This strategy may enable non-invasive, specific biomarker diagnostics and monitoring in patients undergoing targeted cancer therapies.

plos one

Development of a Highly Sensitive and Specific Method for Detection of Circulating Tumor Cells Harboring Somatic Mutations in Non-Small-Cell Lung Cancer Patients