Abstract 3076: A fully automated q-PCR-based circulating tumor cell analysis using the Alere TM q-Analyzer test platform
Authors: Hubold Stephan , Loncarevic Ivan , Thiele Jana , Bredemeier Maren , Klemm Heidi , Klabunde Heike , Michel Danny , Kimmig Rainer , Hauch Siegfried , Aktas Bahriye , Ermantraut Eugen , Kasimir-Bauer Sabine 
Published: May 2015

Abstract
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 &lt;2.5% for each individual transcript. The overall agreement for detecting CTCs between the manual Alere™ AdnaTest BreastCancerDetect and this procedure proved to be 93% with high corresponding individual concordance for MUC1 (κ=1), EPCAM (κ=1), PGR (κ=0.97) and a lower agreement for HER2 (κ=0.46) and ESR1 (κ=0.74). Data were confirmed in 10 MBC patients and more samples are currently evaluated.
Conclusion: The Alere™ q CTC Breast Test presents a robust and sensitive test platform, addressing the growing need for standardization which is a prerequisite for multicentric evaluations. We believe that our approach ultimately will pave the way of CTC markers into clinical practice. Moreover, the test facilitates comparisons and improvements of different CTC enrichment methods.
Citation Format: Stephan Hubold, Ivan Loncarevic, Jana Thiele, Maren Bredemeier, Heidi Klemm, Heike Klabunde, Danny Michel, Rainer Kimmig, Siegfried Hauch, Bahriye Aktas, Eugen Ermantraut, Sabine Kasimir-Bauer. A fully automated q-PCR-based circulating tumor cell analysis using the Alere TM q-Analyzer test platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3076. doi:10.1158/1538-7445.AM2014-3076