Non-invasive and Repeatable EGFR Analysis Using Novel Enrichment Method for Circulating Tumor Cells.

e22033 Non-invasive and repeatable EGFR analysis using novel enrichment method for circulating tumor cells Background: The first-line treatment in confirmed NSCLC (non-small cell lung cancer) patients harboring EGFR mutation is the epidermal growth factor receptor inhibitors (EGFR-TKIs) therapy. A bronchoscopy with transbronchial biopsy or CT guided needle biopsy for EGFR mutation screening may accompany complication, such as infection, bleeding or pneumothorax. However, use of enriched circulating tumor cells (CTCs) from patients’ blood might overcome these risks by means of non-invasive and repeated EGFR mutation analysis. Methods: The blood samples (10 ml) were obtained from NSCLC patients harboring EGFR mutation, and processed through Cytogen Protocolto enrich CTCs. In brief, peripheral blood mononuclear cell fraction was passed through micro fabricated porous filter (CytoGen), and EpCAM antibody was used for positive selection. The identification of enriched CTC was performed by immunofluorescent staining for EpCAM, CD45 and DAPI. The DNAs were extracted from enriched CTCs and used as templates for multi-step real-time PCR. The LOD (limit of detection) values were optimized in spike-in experiments. And, the mutation was identified by both EGFR dependent Real-time PCR and sequencing. Results: The recovery rate by micro fabricated porous filters was about 80% in spike-in experiments for establishing platform. The EGFR mutations were detected by multi-step Real-time PCR method in as low as 10~20 cells per ml of blood. The DNAs ( > 350 ng) were obtained from enriched CTCs through micro fabricated porous filter, pre-amplified, and analyzed for at least 7 mutations using real-time PCR. Among other EGFR mutations, deletion of exon 19 and L858R point mutation are commonly detected. However, the mutation relevant to exon 18 was not detected. Conclusions: In this study, we showed that CTCs enriched using micro fabricated porous filter can be used for EGFR mutation analysis. This new approach may provide insight establishing the personalized cancer therapeutic strategies based on them to molecular profiles in NSCLC. Background: Methods: Results: Conclusions: