Abstract 3076: A fully automated q-PCR-based circulating tumor cell analysis using the Alere TM q-Analyzer test platform

Background: Circulating Tumor Cell (CTC) analysis has emerged as a promising new diagnostic field for cancer patients towards the estimation of risk for metastatic relapse and metastatic progression providing unique information for therapeutic strategies. The reliable clinical utility of CTCs, however, relies on the standardization of assays used for the detection of CTCs. Here, we report a fully integrated and robust system for the analysis of distinct molecular marker expression profiles of CTCs in blood samples of metastatic breast cancer (MBC) patients. Material and Methods: The CTC test platform consists of the portable q-Analyzer instrument and a disposable test-specific cartridge accommodating all required reagents for the processing of lysates of enriched CTCs from whole blood samples, thus removing subjectivity from the test process by eliminating any dependence on the test environment. Due to the full automation of the actual process, handling is limited to the loading of 100µl of enriched cell lysate obtained from CTC Alere™ AdnaTest BreastCancerSelect onto the test cartridge and insertion into the instrument. Released mRNA is selectively captured to a solid phase and subjected to reverse transcription providing a template for target-specific amplification and real-time quantification of the tumor-associated transcripts EPCAM, MUC1, HER2, ESR1 and PGR. Real-time monitoring is achieved by utilizing the Competitor Monitored Amplification (CMA) combining the benefits of quantitative real-time PCR and microarray analysis for the analysis of expression profiles of multiple genes in a single reaction sample. The implementation of an additional control, assessing the level of contaminating leucocytes present in the enriched sample, provides true quality monitor for the purity of the sample and means to substantially improve the test specificity. Results: Spiking two and five T47D cells into 5 ml blood of 44 healthy donors and evaluating 83 non-spiked healthy donor blood samples, analytical sensitivity revealed 100% recovery and 95% specificity, respectively. The duplicated inter-assay coefficient of variation was