Abstract
BackgroundOncogenic 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.MethodsWe 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.ResultsAssay 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.ConclusionsDespite 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.
Highlights
Activating EGFR mutations define a group of genetically dependent pulmonary adenocarcinomas, which are exquisitely sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKI)
Therapeutic decisions in patients with metastatic Non-small-cell lung cancer (NSCLC) are guided by the EGFR mutation status, which is determined in tumor biopsies [1]
Optimal mutation detection sensitivity was achieved by a combination of designed hybridization probes imperfectly binding to EGFR Exon 19 sequences harboring a deletion at amino acid position 746 or 747, locked-nucleic acids (LNA) suppressing amplification of wildtypic EGFR sequences and preventing hybridization of probes to wildtypic EGFR Exon 19 sequences and applying asymmetric polymerase chain reaction (PCR) conditions preferentially amplifying the DNA strand hybridization probes bind to
Summary
Activating EGFR mutations define a group of genetically dependent pulmonary adenocarcinomas, which are exquisitely sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKI). Therapeutic decisions in patients with metastatic NSCLC are guided by the EGFR mutation status, which is determined in tumor biopsies [1]. Acquisition of such biopsies may be hazardous to the patient. Enumeration of CTC has been correlated with clinical outcomes and treatment response [2,3,4,5,6] These studies have applied immunocytochemical detection of protein markers, mostly neglecting genomic biomarkers such as EGFR mutation status. For mutation detection patients have to undergo invasive tumor biopsies. 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
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