BackgroundAssuming that tumor cell dissemination requires a shift to a mesenchymal phenotype, we analyzed the incidence of epithelial-to-mesenchymal-transition (EMT)-like circulating tumor cells (CTCs) in ovarian cancer patients and inquired, how their molecular phenotypes respond to platinum-based chemotherapy and influence outcome.ResultsBefore surgery, overall detection rate for epithelial CTCs was 18%. EMT-like CTCs were more frequently observed (30%) and were mutually exclusive to epithelial CTCs in the majority of patients (82%). After chemotherapy, EMT-like CTCs increased up to 52%, accompanied by the “de novo” emergence of PI3Kα+/Twist+ EMT-like CTCs. Before surgery, PI3K+ EMT-like CTCs in combination with epithelial CTCs indicated decreased OS (p = 0.02) and FIGO I-III patients with residual tumor burden after surgery were more likely to be positive for EMT-like CTCs after chemotherapy (p = 0.02). In the latter group, epithelial CTCs alone significantly correlated with decreased PFS and OS (p = 0.02, p = 0.002), supported by an additional inclusion of PI3K+ CTCs (OS, p = 0.001).Materials and MethodsBlood samples of 91 ovarian cancer patients before surgery and 31 matched samples after adjuvant chemotherapy were evaluated for CTCs with the AdnaTest ovarian cancer and EMT-1, analyzing the epithelial-associated transcripts EpCAM, Muc-1 and CA125 and the EMT-associated transcripts PI3Kα, Akt-2 and Twist.ConclusionsPlatinum-based chemotherapy seems to select for EMT-like CTCs in ovarian cancer patients and provokes a shift towards PI3Kα and Twist expressing CTCs, which may reflect clonal tumor evolution towards therapy resistance. It has to be determined, whether this CTC subgroup may serve as a biomarker to identify patients at high risk.

The RASSF1A promoter is frequently methylated in high-grade serous ovarian cancer (HGSC). We examined RASSF1A promoter methylation in primary tumors, adjacent morphologically tumor cell-free tissues and corresponding circulating tumor DNA (ctDNA) samples of patients with HGSC, using a real-time methylation specific PCR (real-time MSP) and a methylation-sensitive high-resolution melting analysis (MS-HRMA) assay for the detection and semi-quantitative estimation of methylation, respectively. Two groups of primary HGSC tumor FFPE samples were recruited (Group A n=67 and Group B n=61), along with matched adjacent morphologically tumor cell-free tissues (n=58) and corresponding plasma samples (n=59) for group B. Using both assays, RASSF1A promoter was found highly methylated in primary tumors of both groups, and at lower percentages in the adjacent morphologically tumor cell-free tissues. Interestingly, RASSF1A promoter methylation was also observed in ctDNA by real-time MSP. Overall survival (OS) was significantly associated with RASSF1A promoter methylation in primary tumor samples using MS-HRMA (P=0.023). Our results clearly indicate that RASSF1A promoter is methylated in adjacent tissue surrounding the tumor in HGSC patients. We report for the first time that RASSF1A promoter methylation provides significant prognostic information in HGSC patients.

BackgroundWe recently showed that the presence of ERCC1+CTCs is an independent predictive biomarker for platinum-resistance and poor prognosis of ovarian cancer. The goal of our current research was to determine how the auxiliary assessment of ERCC1-transcripts influences overall CTC-detection rate. We extended this investigation from an initially predictive setting to paired pre- and post-therapeutic blood analysis in order to see, whether ERCC1+CTCs dynamics mirror response to chemotherapy.Methods65 Paired blood samples (10ml) of primary ovarian cancer patients at primary diagnosis and after chemotherapy were studied for CTCs with the AdnaTest Ovarian Cancer (QIAGEN Hannover GmbH). We analyzed the tumor-associated transcripts EpCAM, MUC-1 and CA-125. ERCC1-transcripts were investigated in a separate approach by singleplex RT-PCR.RESULTSAuxiliary assessment of ERCC1-transcripts enhanced the overall CTC-detection rate up to 17%. ERCC1+CTCs (defined as positive for one of the AdnaTest markers plus ERCC1-positivity) were detected in 15% of patients at primary diagnosis and in 12% after chemotherapy. The presence of ERCC1+CTCs after chemotherapy correlated with platinum-resistance (P=0.01), reduced PFS (P=0.0293) and OS (P=0.0008) and their persistence indicated poor post-therapeutic outcome (PFS: P=0.005; OS: P=0.0058). Interestingly, the assessment of ERCC1-transcripts alone was sufficient for the detection of prognostic relevant ERCC1-expressing CTCs.ConclusionAuxiliary assessment of ERCC1-transcripts expands the phenotypic spectrum of CTC detection and defines an additional overlapping fraction of ERCC1-expressing CTCs, which are potentially selected by platinum-based chemotherapy. Specifically, we suggest that ERCC1+CTCs could additionally be useful as a surrogate for monitoring platinum-based chemotherapy and to assess the post-therapeutic outcome of ovarian cancer.