BACKGROUND: Platinum resistance constitutes one of the most recognized clinical challenges for ovarian cancer. Notably, the detection of the primary tumor-based excision repair cross-complementation group 1 (ERCC1) protein by immunohistochemistry was recently shown to be inaccurate for the prediction of platinum resistance. On the basis of the previous finding that circulating tumor cells (CTC) in the blood of ovarian cancer patients are prognostically significant, and given our hypothesis that the negative prognostic impact of CTC may arise from a cellular phenotype associated with platinum resistance, we asked whether expression of the excision repair cross-complementation group 1 (ERCC1) gene in the form of the ERCC1 transcript in CTC may be a suitable blood-based biomarker for platinum resistance. METHODS: The presence of CTC was analyzed by immunomagnetic CTC enrichment (n = 143 patients) targeting the epithelial epitopes epithelial cell adhesion molecule (EPCAM) (also known as GA733-2) and mucin 1, cell surface associated (MUC1), followed by multiplex reverse-transcription PCR to detect the transcripts EPCAM, MUC1, and mucin 16, cell surface associated (MUC16) (also known as CA125), including ERCC1 transcripts in a separate approach. ERCC1 expression in primary tumors was comparatively assessed by immunohistochemistry, using the antibody 8F1. RESULTS: At primary diagnosis, the presence of CTC was observed in 14% of patients and constituted an independent predictor of overall survival (OS) (P = 0.041). ERCC1-positive CTC (ERCC1+CTC) were observed in 8% of patients and constituted an independent predictor, not only for OS but also for progression-free survival (PFS) (P = 0.026 and P = 0.009, respectively). More interestingly, we discovered the presence of ERCC1+CTC at primary diagnosis to be likewise an independent predictor of platinum resistance (P = 0.010), whereas ERCC1 expression in corresponding primary tumor tissue predicted neither platinum resistance nor prognosis. CONCLUSIONS: The presence of ERCC1+CTC can serve as a blood-based diagnostic biomarker for predicting platinum resistance at primary diagnosis of ovarian cancer.

The presence of circulating tumor cells (CTCs) in the blood of ovarian cancer patients was shown to correlate with decreased overall survival, whereby CTCs with epithelial–mesenchymal‐transition (EMT) or stem‐like traits are supposed to be involved in metastatic progression and recurrence. Thus, investigating the transcriptional profiles of CTCs might help to identify therapy resistant tumor cells and to overcome treatment failure. For this purpose, we established a multi‐marker panel for the molecular characterization of single CTCs, detecting epithelial (EpCAM, Muc‐1, CK5/7), EMT (N‐cadherin, Vimentin, Snai1/2, CD117, CD146, CD49f) and stem cell (CD44, ALDH1A1, Nanog, SOX2, Notch1/4, Oct4, Lin28) associated transcripts.First primer specificity and PCR‐performance of the multiplex‐RT‐PCRs were successfully validated on genomic DNA and cDNA isolated from OvCar3 cells. The assay sensitivity of the epithelial panel was evaluated by adding defined numbers of tumor cells into the blood of healthy donors and performing a subsequent immunomagnetic tumor cell enrichment (AdnaTest OvarianCancerSelect), resulting in a 100% concordance for the epithelial markers EpCAM and Muc‐1 to the AdnaTest OvarianCancerDetect. Additionally, by processing blood from ovarian cancer patients, high assay sensitivity could be verified. In blood of healthy donors no signals for epithelial markers were detected, for EMT and stem cell markers, however, signals were obtained mainly originating from leukocytes which calls for single cell analysis.To that aim by using the ovarian cancer cell line OvCar3, we successfully established a workflow enabling the characterization of single CTCs. It consists of a density gradient‐dependent enrichment for nucleated cells, a depletion of CD45‐positive cells of hematopoietic origin followed by immunofluorescent labeling of CTCs by EpCAM and Muc‐1. Single CTCs are then isolated by micromanipulation and processed for panel gene expression profiling. Finally, fifteen single CTCs from three ovarian cancer patients were analyzed and found to be positive for stem cell (CD44, ALDH1A1, Nanog, Oct4) and EMT markers (N‐cadherin, Vimentin, Snai2, CD117, CD146). Albeit, inter‐cellular and intra/inter‐patient heterogeneity and co‐expression of epithelial, mesenchymal and stem cell transcripts on the same CTC was observed.We have established a robust workflow to perform sensitive single cell panel gene expression analysis without the need of pre‐amplification steps. Our data point towards a heterogeneous expression of stem cell and EMT associated transcripts in ovarian cancer CTCs.

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

BACKGROUND: Ovarian cancer is the leading cause of death among malignancies in women. Despite advances in treatment, >50% of patients relapse. For disease monitoring, the identification of a blood-based biomarker would be of prime interest. In this regard, noncoding RNAs, such as microRNA (miRNA) or small nuclear RNA (snRNA), have been suggested as biomarkers for noninvasive cancer diagnosis. In the present study, we sought to identify differentially expressed miRNA/snRNA in sera of ovarian cancer patients and investigate their potential to aid in therapy monitoring. METHODS: miRNA/snRNA abundance was investigated in serum (n = 10) by microarray analysis and validated in an extended serum set (n = 119) by reverse-transcription quantitative PCR. RESULTS: Abundance of U2-1 snRNA fragment (RNU2-1f) was significantly increased in sera of ovarian cancer patients (P < 0.0001) and paralleled International Federation of Gynecology and Obstetrics stage as well as residual tumor burden after surgery (P < 0.0001 and P = 0.011, respectively). Moreover, for patients with suboptimal debulking, preoperative RNU2-1f concentration was associated with radiographic response after chemotherapy and with platinum resistance (P = 0.0088 and P = 0.0015, respectively). Interestingly, according to the RNU2-1f abundance dynamics, persistent RNU2-1f positivity before surgery and after chemotherapy identified a subgroup of patients with high risk of recurrence and poor prognosis. CONCLUSIONS: This is the first report to suggest that a circulating snRNA can serve as an auxiliary diagnostic tool for monitoring tumor dynamics in ovarian cancer. Our results provide a rationale to further investigate whether this high-risk patient group may benefit from additional therapies that are directly applied after chemotherapy.

Background: Extracellular vesicle (EV)-associated microRNAs (miRNAs) have been suggested as promising biomarkers for blood-based cancer diagnosis. However, one of the major limitations for the use of EVs with diagnostic purpose is the lack of standardized EV-profiling techniques. In this regard, the objective of our study was to design an integrated next-generation sequencing (NGS)-based workflow for analyzing the signature of EV-associated miRNA in the plasma of platinum-resistant ovarian cancer patients. Methods: For EV-extraction, different enrichment methods were compared (ExoQuick vs. exoRNeasy). NGS was performed with the Illumina platform. Results: We established an integrated NGS-based workflow, including EV-enrichment with the ExoQuick system, which resulted in an optimal RNA-yield and consistent small RNA libraries. We applied this workflow in a pilot cohort of clinically documented platinum-sensitive (n=15) vs. platinum-resistant (n=15) ovarian cancer patients, resulting in a panel of mature EV-associated miRNAs (including ovarian cancer associated miR-181a, miR-1908, miR-21, miR-486 and miR-223), which were differentially abundant in the plasma of platinum-resistant patients. Conclusions: This is the first study, analyzing the profile of EV-associated miRNAs in platinum-resistant ovarian cancer patients. We provide rationale to further validate these miRNA candidates in an independent set of patients, in order to characterize their biomarker potential as predictors for platinum-resistance.

Rationale: Fibrosis promotes the maintenance of atrial fibrillation (AF), making it resistant to therapy. Improved understanding of the molecular mechanisms leading to atrial fibrosis will open new pathways toward effective antifibrotic therapies. Objective: This study aims to decipher the mechanistic interplay between PLK2 (polo-like kinase 2) and the profibrotic cytokine OPN (osteopontin) in the pathogenesis of atrial fibrosis and AF. Methods and Results: Atrial PLK2 mRNA expression was 10-fold higher in human fibroblasts than in cardiomyocytes. Compared with sinus rhythm, right atrial appendages and isolated right atrial fibroblasts from patients with AF showed downregulation of PLK2 mRNA and protein, along with increased PLK2 promotor methylation. Genetic deletion as well as pharmacological inhibition of PLK2 induced profibrotic phenotype conversion in cardiac fibroblasts and led to a striking de novo secretion of OPN. Accordingly, PLK2-deficient (PLK2 knockout) mice showed cardiac fibrosis and were prone to experimentally induced AF. In line with these findings, OPN plasma levels were significantly higher only in patients with AF with atrial low-voltage zones (surrogates of fibrosis) compared with sinus rhythm controls. Mechanistically, we identified ERK1/2 as the relevant downstream mediator of PLK2 leading to increased OPN expression. Finally, oral treatment with the clinically available drug mesalazine, known to inhibit ERK1/2, prevented cardiac OPN overexpression and reversed the pathological PLK2 knockout phenotype in PLK2 knockout mice. Conclusions: Abnormal PLK2/ERK1/2/OPN axis function critically contributes to AF-related atrial fibrosis, suggesting reinforcing PLK2 activity and/or OPN inhibition as innovative targets to prevent fibrosis progression in AF. Mesalazine derivatives may be used as lead compounds for the development of novel anti-AF agents targeting fibrosis.