EMT-like circulating tumor cells in ovarian cancer patients are enriched by platinum-based chemotherapy

Abstract

Background: Assuming 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.

Results: Before 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 methods: Blood 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.

Conclusions: Platinum-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.

Keywords: Akt-2; PI3Kα; circulating tumor cells; epithelial-to-mesenchymal-transition; ovarian cancer.

Figure 1. Overall detection frequency of epithelial and EMT-like CTCs in ovarian cancer

The bar chart illustrates overall detection rates of epithelial and EMT-like CTCs in ovarian cancer patients before surgery and after chemotherapy. Percentages for the two classes of CTCs were calculated independently from each other and, in both cases, refer to the whole study population (before surgery: n = 91, after chemotherapy n = 31). A patient was considered “epithelial CTC-positive” or “EMT-like CTC-positive”, if at least one of the epithelial markers or one of the EMT-associated markers was detectable, respectively.

Figure 2. Phenotypic overlap of epithelial and EMT-like CTCs

The pie chart depicts the overlap of epithelial and EMT-like CTCs in ovarian cancer patients before surgery and after chemotherapy. Percentages were calculated in reference to all patients with overall CTC-positivity. Besides patients with exclusively epithelial (blue) and exclusively EMT-like CTCs (red), there were also patients, harbouring both CTC populations in their blood (green).

Figure 3. Heterogeneity of EMT-associated CTC-phenotypes and their response to platinum-based chemotherapy

The figure summarizes molecular phenotypes of EMT-like CTCs and their response to platinum-based chemotherapy. (A) The bar chart shows the marker distribution in epithelial CTC-positive patients. Percentages for EpCAM and Muc-1 were calculated independently from each other and in reference to only those patients with positivity for epithelial CTCs. (B) The stacked bar chart illustrates the marker distribution of epithelial CTC-positive patients, also considering dual-positivity for EpCAM and Muc-1. (C) The bar chart shows the marker distribution in EMT-like CTC-positive patients. Percentages for PI3Kα, Akt-2 and Twist were calculated independently from each other and in reference to only those patients with positivity for EMT-like CTCs. (D) The stacked bar chart illustrates the marker distribution in EMT-like CTC-positive patients, now also considering dual- or triple positivity for EMT-associated transcripts.

Figure 4. Prognostic relevance of epithelial and EMT-like CTCs

The Kaplan-Meier plots show prognostic relevance of different CTC-subtypes at primary diagnosis. (A, B): epithelial CTCs in FIGO I-III patients (thus without distant metastasis) (C): epithelial or PI3Kα-positive CTCs in the unselected patient cohort (D, E): epithelial or PI3Kα-positive EMT-like CTCs in FIGO I-III patients. Red curves represent patients, positive for the respective CTC-subtype(s); green curves represent patients, negative for the indicated CTC-subtype(s).