Journal Article

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

Schmid, Kurt Werner, Herold, Thomas, Schuler, Martin, Breitenbuecher, Frank, Freitag, Lutz, Gauler, Thomas C., Hoffarth, Sandra, Köhler, Jens, Worm, Karl, Cortes-Incio, Diana, Kasper, Stefan

2014

Overview

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. 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. 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. 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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Adult / Aged / Analysis / Antineoplastic Agents - therapeutic use / Assaying / Bioindicators / Biology / Biomarkers / Biomarkers, Tumor - genetics / Biopsy / Blood / Blood cells / Cancer / Cancer metastasis / Cancer treatment / Carcinoma, Non-Small-Cell Lung - diagnosis / Carcinoma, Non-Small-Cell Lung - drug therapy / Carcinoma, Non-Small-Cell Lung - genetics / Carcinoma, Non-Small-Cell Lung - pathology / Care and treatment / Consortia / Deoxyribonucleic acid / Development and progression / DNA / Epidermal growth factor receptors / Female / Genes / Genetic aspects / Genetic testing / Genomics / Growth factors / Health aspects / Humans / Hybridization / Kinases / Lung cancer / Lung diseases / Lung Neoplasms - diagnosis / Lung Neoplasms - drug therapy / Lung Neoplasms - genetics / Lung Neoplasms - pathology / Lungs / Male / Medical research / Medicine / Melting / Melting curve / Metastasis / Methods / Middle Aged / Mutagenesis / Mutants / Mutation / Neoplasm Recurrence, Local - diagnosis / Neoplasm Recurrence, Local - genetics / Neoplasm Recurrence, Local - pathology / Neoplasm Recurrence, Local - prevention & control / Neoplastic Cells, Circulating - metabolism / Neoplastic Cells, Circulating - pathology / Non-small cell lung cancer / Non-small cell lung carcinoma / Nucleic Acid Denaturation / Oncology / Pathology / Patients / Peripheral blood / Polymerase chain reaction / Real-Time Polymerase Chain Reaction - methods / Receptor, Epidermal Growth Factor - genetics / Sensitivity and Specificity / Single-Cell Analysis / Tumor cells / Tumors