Search

Background: Epigenetic alterations in ctDNA are highly promising as a source of novel potential liquid biopsy biomarkers and comprise a very promising liquid biopsy approach in ovarian cancer, for early diagnosis, prognosis and response to treatment. Methods: In the present study, we examined the methylation status of six gene promoters (BRCA1, CST6, MGMT, RASSF10, SLFN11 and USP44) in high-grade serous ovarian cancer (HGSOC). We evaluated the prognostic significance of DNA methylation of these six gene promoters in primary tumors (FFPEs) and plasma cfDNA samples from patients with early, advanced and metastatic HGSOC. Results: We report for the first time that the DNA methylation of SLFN11 in plasma cfDNA was significantly correlated with worse PFS (p = 0.045) in advanced stage HGSOC. Conclusions: Our results strongly indicate that SLFN11 epigenetic inactivation could be a predictor of resistance to platinum drugs in ovarian cancer. Our results should be further validated in studies based on a larger cohort of patients, in order to further explore whether the DNA methylation of SLFN11 promoter could serve as a potential prognostic DNA methylation biomarker and a predictor of resistance to platinum-based chemotherapy in ovarian cancer.

Simple Summary In the present study we evaluated the frequency and the clinical relevance of ESR1 mutations in high-grade serous ovarian cancer (HGSOC). Drop-off droplet digital PCR (ddPCR) was first used to screen for ESR1 mutations in primary tumors (formalin-fixed paraffin-embedded, FFPEs) from HGSOC patients and plasma cell-free DNA (cfDNA) samples from advanced and metastatic ovarian cancer patients. We further used the recently developed ESR1-NAPA assay to detect individual ESR1 mutations in drop-off ddPCR-positive samples. We report for the first time the presence of ESR1 mutations in 15% of FFPEs and in 13.8% of plasma cfDNA samples from advanced and metastatic ovarian cancer patients. ESR1 mutations have been recently associated with resistance to endocrine therapy in metastatic breast cancer and their detection has led to the development and current evaluation of novel, highly promising therapeutic strategies. In ovarian cancer there have been just a few reports on the presence of ESR1 mutations. The aim of our study was to evaluate the frequency and the clinical relevance of ESR1 mutations in high-grade serous ovarian cancer (HGSOC). Drop-off droplet digital PCR (ddPCR) was first used to screen for ESR1 mutations in 60 primary tumors (FFPEs) from HGSOC patients and in 80 plasma cell-free DNA (cfDNA) samples from advanced and metastatic ovarian cancer patients. We further used our recently developed ESR1-NAPA assay to identify individual ESR1 mutations in drop-off ddPCR-positive samples. We report for the first time the presence of ESR1 mutations in 15% of FFPEs and in 13.8% of plasma cfDNA samples from advanced and metastatic ovarian cancer patients. To define the clinical significance of this finding, our results should be further validated in a large and well-defined cohort of ovarian cancer patients. CA extern

Simple Summary In the present study, we examined the methylation status of six gene promoters (BRCA1, CST6, MGMT, RASSF10, SLFN11 and USP44) in high-grade serous ovarian cancer (HGSOC). We evaluated the prognostic significance of DNA methylation of these six gene promoters in primary tumors (FFPEs) and plasma cfDNA samples from patients with early, advanced and metastatic HGSOC. We report for the first time that the DNA methylation of SLFN11 in plasma cfDNA was significantly correlated with worse PFS (p = 0.045) in advanced stage HGSOC. Our results strongly indicate that SLFN11 epigenetic inactivation could serve as a potential prognostic DNA methylation biomarker and a predictor of resistance to platinum-based chemotherapy in ovarian cancer. Abstract Background: Epigenetic alterations in ctDNA are highly promising as a source of novel potential liquid biopsy biomarkers and comprise a very promising liquid biopsy approach in ovarian cancer, for early diagnosis, prognosis and response to treatment. Methods: In the present study, we examined the methylation status of six gene promoters (BRCA1, CST6, MGMT, RASSF10, SLFN11 and USP44) in high-grade serous ovarian cancer (HGSOC). We evaluated the prognostic significance of DNA methylation of these six gene promoters in primary tumors (FFPEs) and plasma cfDNA samples from patients with early, advanced and metastatic HGSOC. Results: We report for the first time that the DNA methylation of SLFN11 in plasma cfDNA was significantly correlated with worse PFS (p = 0.045) in advanced stage HGSOC. Conclusions: Our results strongly indicate that SLFN11 epigenetic inactivation could be a predictor of resistance to platinum drugs in ovarian cancer. Our results should be further validated in studies based on a larger cohort of patients, in order to further explore whether the DNA methylation of SLFN11 promoter could serve as a potential prognostic DNA methylation biomarker and a predictor of resistance to platinum-based chemotherapy in ovarian cancer.

Ovarian cancer still remains the most lethal female cancer, since in most cases it is diagnosed at an advanced stage. Usually after completion of primary treatment chemoresistance occurs, and recurrent disease is finally observed. Liquid biopsy, based on minimally invasive and serial blood tests, has the advantage of following tumor evolution in real time, offering novel insights on precision medicine. Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating cell-free microRNAs (cfmiRNAs) and circulating exosomes represent the major components of liquid biopsy analysis. Liquid biopsy has been already implemented in ovarian cancer, and most studies so far are mainly focused on CTCs and ctDNA. This review is mainly focused on the clinical potential of circulating miRNAs and exosomes as a source of liquid biopsy biomarkers in ovarian cancer diagnosis, prognosis, and response to treatment.

Ovarian cancer has the worst survival rate because it is typically diagnosed at advanced stage. Despite treatment, the disease commonly recurs due to chemo-resistance. Liquid biopsy, based on minimally invasive blood tests, has the advantage of following tumor evolution in real time, offering novel insights on cancer prevention and treatment. Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating cell-free microRNAs (cfmiRNAs) and circulating exosomes represent the major components of liquid biopsy. In this chapter, we provide an overview of recent research on CTCs, ctDNA, cfmiRNAs and exosomes in ovarian cancer. We also focus on the clinical value of liquid biopsy in early diagnosis, prognosis, treatment response, as well as screening in the general population.

// Lydia Giannopoulou 1 , Issam Chebouti 2 , Kitty Pavlakis 3 , Sabine Kasimir-Bauer 2 , Evi S. Lianidou 1 1 Analysis of Circulating Tumor Cells Laboratory, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, University Campus, Athens, 15771, Greece 2 Department of Gynecology and Obstetrics, University Hospital of Essen, University of Duisburg-Essen, Essen, D-45122, Germany 3 Pathology Department, IASO Women’s Hospital, Marousi, Athens, 15123, Greece Correspondence to: Evi S. Lianidou, email: lianidou@chem.uoa.gr Keywords: circulating tumor DNA, RASSF1A, high-grade serous ovarian cancer, methylation specific PCR, high resolution melting analysis Received: April 10, 2016 Accepted: November 11, 2016 Published: February 10, 2017 ABSTRACT 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.

Objective Estrogen receptor, coded by the ESR1 gene, is highly expressed in epithelial ovarian cancer. ESR1 gene is frequently methylated in many types of gynecological malignancies. However, only a few studies attempted to investigate the role of ESR1 methylation and its clinical significance in ovarian cancer so far. The aim of our study was to examine ESR1 methylation status in primary tumors and corresponding circulating tumor DNA of patients with high-grade serous ovarian cancer (HGSC). Methods ESR1 methylation was detected by a highly specific and sensitive real-time methylation-specific PCR assay. Two groups of HGSC samples were analyzed: group A (n = 66 primary tumors) and group B (n = 53 primary tumors and 50 corresponding plasma samples). Results ESR1 was found methylated in both groups of primary tumors: in 32/66 (48.5%) of group A and in 15/53 (28.3%) of group B. 19/50 (38.0%) corresponding plasma samples of group B were also methylated for ESR1. A significant agreement for ESR1 methylation was observed between primary tumors and paired plasma ctDNA samples (P = 0.004). Interestingly, the presence of ESR1 methylation in primary tumor samples of group B was significantly correlated with a better overall survival (P = 0.027) and progression-free survival (P = 0.041). Conclusions We report for the first time the presence of ESR1 methylation in plasma ctDNA of patients with HGSC. The agreement between ESR1 methylation in primary tumors and paired ctDNA is statistically significant. Our results indicate a correlation between the presence of ESR1 methylation and a better clinical outcome in HGSC patients.

Ovarian cancer remains the most lethal disease among gynecological malignancies despite the plethora of research studies during the last decades. The majority of patients are diagnosed in an advanced stage and exhibit resistance to standard chemotherapy. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) represent the main liquid biopsy approaches that offer a minimally invasive sample collection. Both have shown a diagnostic, prognostic and predictive value in many types of solid malignancies and recent studies attempted to shed light on their role in ovarian cancer. This review is mainly focused on the clinical value of both CTCs and ctDNA in ovarian cancer and, more specifically, on their potential as diagnostic, prognostic and predictive tumor biomarkers.

Background: The estrogen receptor is highly expressed in epithelial ovarian cancer and represents the main target for endocrine therapy. The ESR1 gene is frequently methylated in many types of gynecological malignancies and previous studies have shown an inverse correlation between ESR1 methylation and gene expression. Few studies attempted to investigate the role of ESR1 methylation in ovarian cancer so far, and the clinical significance of ESR1 methylation status is not as yet clear. Methods: The ESR1 methylation status was examined in primary tumors and corresponding circulating tumor DNA (ctDNA) samples of patients with high-grade serous ovarian cancer. For the detection of methylation we applied a novel highly specific and sensitive real-time methylation specific PCR (real-time MSP) assay. Two groups of primary tumor samples were recruited (training group, n=66 and validation group, n=61), along with the corresponding plasma samples (n=58) for the validation group. ESR1 methylation was also analyzed in a small group of 16 normal fallopian tube samples. Results: ESR1 was found methylated in both groups of primary tumor samples and in the corresponding plasma samples of the validation group. More specifically, ESR1 methylation was detected in 32/66 (48.5%) and 17/61 (27.9%) primary tumor samples of the training and the validation group, respectively, and in 23/58 (39.7%) corresponding plasma samples. A significant agreement between ESR1 methylation in primary tumors and paired ctDNA was observed in 40/56 (71.4%) samples of the validation group (P=0.004, k=0.360). Almost all normal fallopian tube samples (15/16) were found methylated. Interestingly, the presence of ESR1 methylation in the primary tumor samples of the validation group was nearly significantly correlated (P=0.057) with a better overall survival. Conclusions: We detected for the first time ESR1 methylation in ctDNA of patients with high-grade serous ovarian cancer. The agreement between ESR1 methylation in the primary tumors and paired ctDNA is statistically significant. Our results indicate a potential correlation between ESR1 methylation and better overall survival in high-grade serous ovarian cancer patients. Citation Format: Lydia Giannopoulou, Sofia Mastoraki, Areti Strati, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi Lianidou. ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4587.

Introduction: ESR1 methylation is frequently observed in many types of solid malignancies, but few studies attempted to investigate ESR1 methylation status in ovarian cancer so far. We examined for the first time ESR1 methylation in plasma samples of high-grade serous (HGSC) ovarian cancer patients, using a novel real-time MSP assay. Materials and methods: We first developed and validated a highly specific and sensitive real-time MSP assay for ESR1 methylation. A commercially available 100% methylated standard was used as positive control in all experiments. We then evaluated ESR1 methylation in plasma (2mL) of 59 pre-surgery, mainly advanced stage HGSC patients. A control group of 51 plasma samples from healthy women was recruited for the evaluation of specificity of our assay. All cfDNA samples isolated from plasma were subjected to sodium bisulfite (SB) treatment and were then analyzed by real-time MSP for ESR1 methylation. For the normalization of the results, we used ACTB as a reference gene. The real-time PCR assay for ACTB was designed to amplify specifically SB treated DNA. Results: The real-time MSP assay is highly sensitive, as it detects down to 0.1% of ESR1 methylation in the presence of 99.9% non-methylated sequences. ESR1 methylation was detected in 18/59 (30.5%) high-grade serous ovarian patients, but only in 1/51 (2.0%) of healthy women. All results were obtained after normalization with ACTB as a reference gene. Conclusion: We report for the first time that ESR1 methylation can be detected in cfDNA of high-grade serous ovarian cancer patients, but not in healthy women. Further investigation is required to determine the clinical significance of ESR1 methylation status in cfDNA of HGSC patients. We also aim to evaluate our findings in a larger group of patients. Citation Format: Lydia Giannopoulou, Sophia Mastoraki, Areti Strati, Isaam Chebouti, Sabine Kasimir-Bauer, Evi S. Lianidou. ESR1 methylation in plasma cfDNA of patients with high-grade serous ovarian cancer [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 5691. doi:10.1158/1538-7445.AM2017-5691

Introduction: RASSF1A promoter methylation is frequent in high grade serous ovarian cancer (HGSC), the most common histological subtype. We examined RASSF1A promoter methylation in primary tumors, adjacent normal tissues and corresponding plasma samples of patients with HGSC, using real-time methylation specific PCR (real-time MSP) and methylation-sensitive high-resolution melting analysis (MS-HRMA). Materials and methods: A training group of 78 primary HGSC FFPEs was first analyzed using both real-time MSP and MS-HRMA. Our validation group consisted of 61 primary HGSC FFPEs, 58 adjacent tissues (analyzed using both real-time MSP and MS-HRMA), and 59 corresponding plasma samples (analyzed using real-time MSP). The specificity of both assays was evaluated by analyzing a group of 16 fallopian tube samples from healthy individuals. OVCAR29 and IGROV1 cell lines were used as positive controls. Results: In the training group, RASSF1A promoter methylation was detected in 33/78 (42.3%) by real-time MSP and in 33/78 (42.3%) by MS-HRMA (Agreement = 94.9%, P = 0.001, k = 0.895). The validation group results are shown in Table 1. According to the semi-quantitative MS-HRMA, RASSF1A promoter methylation was detected at significantly lower percentages in the adjacent morphologically normal tissues, compared to the paired primary tumors. In corresponding plasma samples, methylation was detected in 15/59 (25.4%) (Agreement with paired tumors = 60.7%, P = 0.259, k = 0.143). Both real-time MSP and MS-HRMA revealed no RASSF1A promoter methylation in the small group of normal fallopian tubes (n = 16). Conclusion: RASSF1A promoter is highly methylated in primary tumors and at lower percentages in the adjacent normal tissues. In all cases, MS-HRMA gave comparable results with real-time MSP. Evaluation of the clinical significance of RASSF1A promoter methylation in corresponding plasma requires further investigation. Table 1RASSF1A promoter methylationMethodPrimary tumor samples (validation group) N = 61Adjacent tissues N = 58Agreement (paired samples, N = 57)Real time MSP25/61 (41%)17/58 (29.3%)48/57(84.2%), P = 0.001, k = 0.652MS-HRMA28/61 (45.9%)21/58 (36.2%)51/57(89.5%), P = 0.001, k = 0.782Agreement (methods)58/61(95.1%), P = 0.001, Cohen's kappa = 0.90050/58(86.2%),P = 0.001, Cohen's kappa = 0.689 Citation Format: Lydia Giannopoulou, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi S. Lianidou. RASSF1A gene promoter methylation in primary tumors, adjacent morphologically normal tissues and plasma samples of patients with high grade serous ovarian cancer. [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 442.

Introduction: Impaired DNA methylation patterns hold promise as cancer biomarkers. SOX17, a member of the SOX family of transcription factors, is conserved in many species and plays critical roles in the regulation of development and stem/precursor cell function, at least partly through repression of the canonical Wnt/beta-catenin signaling pathway. Global analysis of CpG island hypermethylation and gene expression in colorectal cancer cell lines revealed that SOX17 gene silencing is associated with DNA hypermethylation. We have already shown that this gene is hypermethylated in circulating tumor cells and circulating tumor DNA of patients with breast cancer (Chimonidou et al, Clin Chem 2011, Chimonidou et al, Clin Chem, 2013). High grade serous ovarian cancer (HGSC) is the most common histological subtype of ovarian cancer, with a worse disease prognosis and overall survival. In this study, we investigated for the first time the status of SOX17 promoter methylation in patients with high grade serous ovarian cancer. Materials and methods: HGSC tumors (FFPEs) from ovarian cancer patients belonging to an independent group (n = 78) and a training group (n = 47) were analyzed. For the evaluation of the specificity of our results, a small group of 10 corresponding normal tissues from the first ten individuals of the training group was also analyzed. Genomic DNA was isolated from all samples, and then was modified by Sodium Bisulfite (SB). All SB converted DNA samples were analyzed by a real time Methylation Specific PCR (MSP) assay, for detecting SOX17 promoter methylation. The IGROV1 ovarian cancer cell line was used as a positive control in all procedures. Results: In the training group, SOX17 promoter methylation was detected in 25/47 ovarian tumor samples (53.2%) and in 5/10 corresponding normal tissues. The same 5/10 matched pathological samples of the training group, were also found methylated for the SOX17 gene. In the independent group, SOX17 promoter methylation was observed in 61/78 tumor samples (78.2%). Conclusion: Our results indicate for the first time that SOX17 promoter methylation is a frequent event in high grade serous ovarian cancer. Further investigation is required to determine the prognostic significance of SOX17 promoter methylation status in ovarian cancer and correlate these findings with patients’ clinicopathological characteristics. Citation Format: Lydia Giannopoulou, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi S. Lianidou. SOX17 gene promoter methylation in high-grade serous epithelial ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3822. doi:10.1158/1538-7445.AM2015-3822

Breast cancer is a genetically complex disease and is frequently associated with nonrandom chromosomal alterations. The occurrence of aberrations involving chromosomes 1 and 17 in malignant tissues of breast cancer patients has not been studied systematically. The numeric aberrations of chromosomes 1 and 17 were detected by nonisotopic in situ hybridization on paraffin-embedded tissue sections from 44 invasive breast carcinomas (42 cases available for chromosome 17) and were correlated with clinicopathologic parameters, patients' survival, p53, and c-erbB-2 proteins. Chromosome 17 and 1 aneuploidy were observed in the majority of breast carcinomas with equal percentages of polysomy and monosomy for chromosome 17 and predominance of polysomy for chromosome 1. Monosomy of chromosome 17 was significantly associated with positive lymph nodes and negative estrogen receptor (ER) immunohistochemical expression. Patients with chromosome 17 monosomy were at greater risk of death. Ductal carcinoma displayed a greater percentage of chromosome 1 polysomy than lobular ones. A statistically significant association was demonstrated between chromosome 1 polysomy and higher nuclear grade. Patients with chromosome 1 aneuploidy were at greater risk of death, and especially those with ER negativity. Aneuploid patients with c-erbB-2(-)/PR(-) phenotype demonstrated lower survival rates. These data suggest a possible susceptibility of chromosome 17 to losses and gains and chromosome 1 to gains. Chromosome 17 monosomy and chromosome 1 aneuploidy may be useful prognostic markers in breast cancer patients.