Abstract 2230: Combination of immunomagnetic tumor cell enrichment and molecular profiling for the detection of circulating tumor cells in blood of ovarian cancer patients and comparison to disseminated tumor cells in the bone marrow
Authors: Wimberger Pauline , Heubner Martin , Aktas Bahriye , Hauch Siegfried , Kimmig Rainer , Kasimir-Bauer Sabine 
Published: February 2011

Background: Disseminated tumor cells (DTCs) in the bone marrow (BM) were shown to be of prognostic significance in gynaecological cancers. Since BM aspiration is less accepted by patients compared to blood drawing, it would be highly desirable to replace BM aspiration by blood analysis, especially for therapy monitoring. For ovarian cancer, no blood test for detection and characterization of circulating tumor cells (CTCs) has been established so far. In this pilot trial, a new blood test based on immunomagnetic enrichment and subsequent molecular profiling in peripheral blood of patients with ovarian cancer was evaluated. Results were compared with the detection of DTCs in the BM. Methods: Two x 5 ml blood were analyzed from ovarian cancer patients before (n=40) or after (n=28) chemotherapy. CTCs were immunomagnetically enriched by targeting two different epitopes of EpCAM, MUC-1 and Mesothelin. The mRNA of these cells was isolated with oligo-dT magnetic beads and analyzed for EpCAM, MUC-1, CA 12-5 and beta-Actin as an internal PCR control by multiplex RT-PCR with the AdnaTest OvarianCancer (AdnaGen AG, Langenhagen, Germany). The PCR products were quantified on the Agilent Bioanalyzer 2100. Specificity was determined using blood of healthy donors. BM aspirates of 51 patients were analyzed in duplicate by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 and evaluated for DTCs with the ARIOL system (Applied Imaging) according to the ISHAGE evaluation criteria. Results: Control experiments using healthy donor blood samples yielded an overall specificity of &gt; 90%. Recovery was determined by the detection of a low number of target cells (5 IGROV cells spiked into 5 ml blood of healthy donors) revealing a recovery rate of 95%. Before chemotherapy, CTCs were detected in 11/40 patients (28%) expressing EpCAM (73%), MUC-1 (73%) and CA 12-5 (27%), respectively. After chemotherapy, the overall detection rate for CTCs was 43% (12/28 patients), thereof EpCAM (50%), MUC-1 (67%) and CA 12-5 (33%), respectively. The overall detection rate for DTCs in the BM was 21% (12/51 patients). A comparison between DTCs in the BM and CTCs in blood resulted in 24 DTCs-/CTCs- patients, 5 DTCs+/CTCs+ patients, 7 DTCs+/CTCs- patients and 15 DTCs-/CTCs+ patients, respectively. Conclusion: A combination of immunomagnetic tumor cell enrichment and a multi-gene RT-PCR is able to detect CTCs in ovarian cancer patients. This approach might help to identify molecular targets for alternative therapeutic interventions. Furthermore, due to the weak correlation between CTCs and DTCs we conclude that (1) the clinical relevance of these cells may be different and (2) that blood analysis cannot replace BM analysis at the moment but could give information complimentary to that obtained by DTCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2230.