The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.

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Introduction: Glutathione S-transferase 1 (GSTP1) has been reported to function as tumor suppressor gene in various types of human cancers. GSTP1 inactivation is associated with CpG island promoter hypermethylation in the majority of prostate cancers (PCs). The initiation of minimal residual disease (MRD) and especially the detection of circulating tumor cells (CTCs) in patients’ peripheral blood represents a negative prognostic parameter for recurrence-free survival. The aim of the present study was to assess the methylation status of the GSTP1 gene in CTCs that were isolated, using the CellCollector® (GILUPI, GmbH), a novel clinical device designed for the in vivo isolation of EpCAM-positive CTCs. Patients and methods: In-vivo isolation of CTCs was performed by using CellCollector® from high-risk prostate cancer patients (n=97) and 20 healthy volunteers. For all these patients, the Ab coated region of the CellCollector® was washed in PBS , cut, and stored in Trizol reagent till analysis and DNA was further prior to the analysis isolated DNA was modified by sodium bisulfite (SB) and subjected to a real time MSP assay specific for GSTP1 methylation. In all cases, peripheral blood was also collected and used for CTC analysis by Immunostaining and the CellSearch® system. Results: All DNA samples were first checked for their quality. Based on the quality evaluation of all available DNA samples, only 63 DNAs were further qualified for analysis. GSTP1 promoter was found methylated in 12/63 (19%) the EpCAM positive fraction of in-vivo isolated CTCs. Moreover, in 5/12 (41.7%) patients for which GSTP1 promoter was found methylated, CTCs were also detected by the CellSearch® and 7 /12 (58.3%) for which GSTP1 promoter was also found positive, CTCs were also detected by the Immunostaining. Conclusion: GSTP1 promoter is methylated in in-vivo isolated CTCs from high-risk prostate cancer patients. GSTP1 promoter methylation in in-vivo isolated CTCs should be prospectively validated as a novel tumor biomarker for prostate cancer patients in a large cohort of patients. Acknowledgements: This research has been co-financed by the European Union (European Regional Development Fund - ERDF) and Greek national funds through the Operational Program ‘‘Competitiveness and Entrepreneurship’’ of the National Strategic Reference Framework (NSRF) - Research Funding Program: “ERA-NET on Translational Cancer Research (TRANSCAN) Joint Transnational Call for Proposals 2011 (JTC 2011) on: “Validation of biomarkers for personalised cancer medicine Citation Format: Athina N. Markou, Panagiotis Paraskevopoulos, Marifili Lazaridou, Shukun Chen, Thomas Kroneis, Monika Świerczewska, Joanna Budna, Andra Kuske, Tobias M. Gorges, Maciej Zabel, Peter Sedlmayr, Catherine Alix-Panabieres, Klaus Pantel, Evi Lianidou. GSTP1 promoter methylation in in-vivo isolated CTCs from high-risk prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1716. doi:10.1158/1538-7445.AM2017-1716

Introduction: CTC have been verified as prognostic markers for disease progression in various cancer types. The main aim of the EU project “CTC-SCAN” is to validate the number of CTC isolated from patient's blood as a prognostic marker for relapse in high-risk prostate cancer patients treated with primary radical prostatectomy or radiotherapy. In this study, we present our results on gene expression profiling of CTCs that were isolated, using the CellCollector, a novel clinical device designed for the in vivo isolation of EpCAM-positive CTCs. Patients and methods: We first developed and validated 3 multiplex and 3 single-plex highly sensitive RT-qPCR assays amplifying:a)Epithelial markers:CK-19,EpCAM,E-CAD & PBGD, b)Stem cell markers:PSCA,ALDH1,CD133& HPRT, c) EMT markers: TWIST, VIM, N-CAD and B2M and d)PSA, e)TMPRSS2-ERG fusion, f)Plastin-3. 62 patients and 36 healthy volunteers participated in the study. After in vivo isolation, total RNA was extracted from captured cells,followed by cDNA synthesis. RT-qPCR was performed for the molecular characterization of captured cells. In all cases, peripheral blood was also collected for CTC analysis by CellSearch and the EPISPOT. Results: The findings of our study are summarized in Table 1. Briefly, in 13/15(87%) samples, in which at least one cell was detected by CellSearch, we detected the expression of at least one gene. In 28/47 samples, negative by CellSearch, we detected the expression of several genes by the developed RT-qPCR assays. In 9/14 samples that were exclusively found to be positive by EPISPOT for PSA immunospots, at least one of the analyzed genes was also expressed. Conclusions: In vivo isolation of CTC in combination with a downstream molecular analysis is minimally invasive, and in combination with high specific and sensitive RT-qPCR assays for CTC detection and molecular characterization seems very promising. Comparison studies with the CellSearch and the EPISPOT have shown a higher sensitivity, but a poor agreement. in vivo(n = 18)in vitro(n = 18)GENEPOSITIVE(%)POSITIVE(%)POSITIVE(%)CK-1920(32%)1(0.5%)0(0%)E-CAD0(0%)0(0%)0(0%)EpCAM16(26%)1(0.5%)0(0%)CD1330(0%)0(0%)0(0%)ALDH111(18%)0(0%)0(0%)PSCA0(0%)0(0%)0(0%)VIMENTIN10(16%)0(0%)0(0%)TWIST15(24%)0(0%)0(0%)N-CAD11(18%)0(0%)0(0%)PLASTIN-36(10%)0(0%)0(0%)PSA6(10%)0(0%)0(0%)TMPRSS:ERG0(0%)0(0%)0(0%)CELLSEARCH(cut-off>1cells/ml)15(24%)0(0%)EPISPOT15(24%)0(0%) Citation Format: Athina N. Markou, Marifili Lazaridou, Panagiotis Paraskevopoulos, Shukun Chen, Thomas Kroneis, Monika Swierczewska, Joanna Budna, Andra Kuske, Tobias M. Gorges, Maciej Zabel, Peter Sedlmayr, Catherine Alix-Panabieres, Klaus Pantel, Evi S. Lianidou. Molecular characterization of in-vivo isolated EpCAM-positive circulating tumor cells in high-risk prostate cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4960.