Using primary antibodies against epithelial markers, disseminated tumor cells and CTC have been detected in bone marrow and blood from patients with various malignancies. Among a large variety of alternative strategies applied for CTCenrichment and analysis, an automated system for immunocytometric detection and quantitation of CTC has been developed, which was validated in breast, prostate, colorectal and lung 14alpha-hydroxy-Sprengerinin-C cancer patients. Interestingly, the absolute amount of CTC detected in advanced stage cancer patients varied considerably, depending on CTC-isolation techniques. While high numbers of CTC were detected in patients with small-cell lung cancer, absolute CTC numbers and dynamic range in NSCLC seemed too low for broad clinical applicability. Nevertheless, it has become increasingly clear that CTC populations are phenotypically heterogenous and epithelial-marker independent enrichment approaches are warranted to display the full picture of CTC in a cancer Forsythin patient at a given time point. Several recent reports supported this notion by consistently showing higher CTC numbers when comparing epithelial-marker-independent CTC enrichment techniques with the widely applied CTC isolation by selection of EpCAM and CKpositive cells using the CellSearch system. Against this background we reasoned whether probing for cancer-specific somatic mutations instead of microscopy-based parameters enhances the sensitivity of CTC detection in NSCLC. We have chosen EGFR DelEx19 mutations as a model to develop a highly sensitive, specific and robust method for mutation detection in mixtures of genomic DNA that were extracted from CTC-enriched peripheral blood cell populations. The novel method applied in this study has been designed to detect the vast majority of EGFR DelEx19 mutations, notably all possible mutant variants disrupting codons 746 and 747 of the EGFR gene. In contrast to a prior study analyzing only EpCAM-positive blood cells we have chosen an unbiased, epithelial markerindependent CTC enrichment strategy where samples were split and CTC enrichment was achieved by a dual approach, depleting leucocytes in one half of a sample and selection of EpCAMpositive cells in the other half. This takes into account the phenotypic variability of CTC, which may lose one or several epithelial markers while being shed from a primary tumor or metastases. In pilot experiments, Immunofluorescence staining of selected CTC samples from NSCLC patients indeed showed heterogeneous populations of CTCs displaying epithelial, mesenchymal and mixed phenotypes. Applying this unbiased CTC-enrichment approach, we achieved a 100% detection rate in a pilot cohort of 8 patients with EGFR-mutant NSCLC. Most interestingly, we observed an association of EGFRmutant CTC with treatment response and outcome. These findings are most likely to be attributed to the novel and highly sensitive mutation detection method applied in this study. It has been recognized for some time that detection of mutations at levels below 1% is not reliably possible by next generation sequencing applications due to intrinsic error rates of this technology. Only very recently improved technologies were reported, rendering detection of mutations at levels of 0,1% and below possible. In keeping with these results, we were not able to detect EGFR Del19 mutations by next generation sequencing in genomic DNA of samples displaying unambiguous mutation signals in our newly developed mutation detection assay. These findings indicate that our newly developed mutation detection assay apparently displays higher sensitivity than standard next generation sequencing techniques. Another remarkable aspect of our mutation detection assay was the observation that different EGFR DelEx19 mutations gave rise to different melting curves.