Biomarker For Epithelial Ovarian Cancer Research Articles

Upregulation of miR-874-3p and miR-874-5p inhibits epithelial ovarian cancer malignancy via SIK2.

Based on miR-874 expression levels in the GSE47841 microarray, we hypothesized that the mature products of miR-874, miR-874-3p, or miR-874-5p, would inhibit epithelial ovarian cancer (EOC) cell proliferation, metastasis, and chemoresistance. We first examined miR-874-3p and miR-874-5p expression levels in primary EOC tumor tissue samples and found that they were significantly decreased. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation and transwell assays revealed that miR-874-3p and miR-874-5p significantly inhibit EOC cell proliferation, migration, and invasion. Then, using MTT and soft agar assays of paclitaxel-treated Caov3 and SKOV3 cells transfected with miR-874-3p and miR-874-5p, we found that miR-874-3p and miR-874-5p enhance EOC cell chemosensitivity. We then confirmed that serine/threonine-protein kinase 2 (SIK2) was a target gene of miR-874-3p and miR-874-5p. Overall, the results of this study indicate that SIK2 expression can serve as a prognostic biomarker for EOC and that miR-874-3p and miR-874-5p have the potential to enhance clinical treatment of EOC.

PO-110 AKT expression is associated with poor clinical outcome in ovarian cancer

IntroductionOvarian cancer (OC) is the third most common gynaecological cancer among women worldwide and it is associated with the highest mortality rate among gynaecologic malignancies. There is an urgent need to refine classification of ovarian cancer and identify novel targets. The PI3K/AKT/mTOR pathway is an intracellular signalling pathway important in regulating the cell cycle. Therefore, it is directly related to cellular quiescence, proliferation and cancer. We aimed to investigate the prognostic role of AKT in ovarian cancer.Material and methodsInvestigation of the expression of AKT in ovarian epithelial cancer was carried out on tissue microarrays of 525 consecutive ovarian epithelial cancer cases treated at Nottingham University Hospitals (NUH) between 1997 and 2010 and their expression was correlated to clinicopathological outcomes as well as to recurrence free survival (RFS).Results and discussionsHigh cytoplasmic AKT expression was significantly associated with serous type of tumour (p=0.042). Furthermore, High AKT protein expression was correlated with poor outcome in terms of Recurrence Free Survival (RFS) (p=0.034). Moreover, by selecting only stage one disease positive AKT expression showed significantly higher chance of worse RFS.ConclusionAKT protein is a prospectively valuable recurrence predictive biomarker of epithelial ovarian cancer.

PO-113 Investigation of the role of IRF4 in melanoma cells

IntroductionOvarian cancer (OC) is the third most common gynaecological cancer among women worldwide and it is associated with the highest mortality rate among gynaecologic malignancies. There is an urgent need to refine classification of ovarian cancer and identify novel targets. The PI3K/AKT/mTOR pathway is an intracellular signalling pathway important in regulating the cell cycle. Therefore, it is directly related to cellular quiescence, proliferation and cancer. We aimed to investigate the prognostic role of AKT in ovarian cancer.Material and methodsInvestigation of the expression of AKT in ovarian epithelial cancer was carried out on tissue microarrays of 525 consecutive ovarian epithelial cancer cases treated at Nottingham University Hospitals (NUH) between 1997 and 2010 and their expression was correlated to clinicopathological outcomes as well as to recurrence free survival (RFS).Results and discussionsHigh cytoplasmic AKT expression was significantly associated with serous type of tumour (p=0.042). Furthermore, High AKT protein expression was correlated with poor outcome in terms of Recurrence Free Survival (RFS) (p=0.034). Moreover, by selecting only stage one disease positive AKT expression showed significantly higher chance of worse RFS.ConclusionAKT protein is a prospectively valuable recurrence predictive biomarker of epithelial ovarian cancer.

Abstract 3309: Circulating epigenetic biomarkers for the early detection of ovarian cancer

Abstract Background Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Greater than 75% of cases are diagnosed with advanced stage, which is associated with 5-year survival rates of less than 20% compared with 92% if caught early. Previous attempts at discovering early detection biomarkers for EOC have largely failed because of reliance on sub-optimal discovery approaches and the lack of comprehensive patient cohorts. DNA methylation changes are among the earliest alterations in cancer and methylation represents a stable and robust modification on DNA. In addition, several groups including ours have reported the detection of tumor-associated methylation changes in cfDNA extracted from plasma. Accordingly, in this study, we performed comprehensive DNA methylation analysis by reduced representation bisulfite sequencing (RRBS) to identify DNA methylation biomarkers that can be detected in circulation and used for the early detection of ovarian cancer. Methods For the biomarker discovery phase, a cohort of 23 stage 1 serous EOC tissues, and 10 unmatched, adjacent normal tissues from fallopian tubes were analyzed by RRBS. We used Metilene, a binary segmentation algorithm combined with a two-dimensional statistical test, to detect differentially methylated regions (DMRs). For validation, we performed targeted bisulfite amplicon sequencing. Briefly, bisulfite primers were designed to regions of interest, optimized, multiplexed and sequenced to depths ranging from 500-25,000X on the Illumina MiSeq platform. The validation cohort consisted of cfDNA isolated from the plasma of 51 independent EOCs and 10 normal individuals as well as the genomic DNA from tissue used in the discovery cohort. Results We identified 10972 statistically significant (q-value <0.05) DMRs between stage 1 EOC and normal samples. Of these, 227 were hypermethylated and had an average differential methylation value of greater than 30% in tumors compared to normal. Unsupervised clustering analysis of these DMRs demonstrated clear separation of tumor from normal, with the possibility of three distinct molecular subtypes of serous EOC. Six DMRs were prioritized and further validated in both tissue and plasma. Targeted bisulfite amplicon sequencing performed revealed hypermethylation in greater than 50% of tumor samples across the DMRs. Additionally, the area under the curve (AUC) for the DMRs interrogated ranged from 0.91 and 0.94. Conclusion In conclusion, our RRBS based comprehensive biomarker discovery identified a six-DMR panel that demonstrated preliminary robust diagnostic accuracy in EOC samples. Further evaluation of these DMRs in multicenter retrospective and prospective cohorts might lead to the development of a non-invasive diagnostic, as well as a population-screening assay for OC patients. Citation Format: Gerald C. Gooden, Christophe Legendre, Monique Spillman, Ajay Goel, Bodour Salhia. Circulating epigenetic biomarkers for the early detection of 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 3309.

The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

Glycosyltransferase enzyme GCNT3, has been proposed as a biomarker for prognosis in colorectal cancer (CRC). Our study goes in depth into the molecular basis of GCNT3 role in tumorigenesis and drug resistance, and it explores its potential role as biomarker in epithelial ovarian cancer (EOC). High levels of GCNT3 are associated with increased sensibility to 5-fluoracil in metastatic cells. Accordingly, GCNT3 re-expression leads to the gain of anti-carcinogenic cellular properties by reducing cell growth, invasion and by changing metabolic capacities. Integrated transcriptomic and proteomic analyses reveal that GCNT3 is linked to cellular cycle, mitosis and proliferation, response to drugs and metabolism pathways. The vascular epithelial growth factor A (VEGFA) arises as an attractive partner of GCNT3 functions in cell invasion and resistance. Finally, GCNT3 expression was analyzed in a cohort of 56 EOC patients followed by a meta-analysis of more than one thousand patients. This study reveals that GCNT3 might constitute a prognostic factor also in EOC, since its overexpression is associated with better clinical outcome and response to initial therapy. GCNT3 emerges as an essential glycosylation-related molecule in CRC and EOC progression, with potential interest as a predictive biomarker of response to chemotherapy.

Correlación de la captación de 18F-FDG de la PET/TC con el Ki67 de la inmunohistoquímica en el cáncer epitelial de ovario pretratamiento

ObjectiveStandardised uptake value (SUV) and volumetric parameters such as metabolic tumour volume (MTV) and total lesion glycolysis (TLG) from 18F-FDG PET/CT are useful criteria for disease prognosis in pre-operative and post-treatment epithelial ovarian cancer (EOC). Ki67 is another prognostic biomarker in EOC, associated with tumour aggressiveness. The aim of this study is to evaluate the association between 18F-FDG PET/CT measurements and Ki67 in pre-treatment EOC to determine if PET/CT parameters could non-invasively predict tumour aggressiveness. Material and methodsA pre-treatment PET/CT was performed on 18 patients with suspected or newly diagnosed EOC. Maximum SUV (SUVmax), mean SUV (SUVmean), whole-body MTV (wbMTV), and whole-body TLG (wbTLG) with a threshold of 30% and 40% of the SUVmax were obtained. Furthermore, Ki67 index (mean and hotspot) was estimated in tumour tissue specimens. Immunohistochemical findings were correlated with PET parameters. ResultsThe mean age was 57.0 years old (standard deviation 13.6 years). A moderate correlation was observed between mean Ki67 index and SUVmax (r=0.392), SUVmean 30% (r=0.437), and SUVmean 40% (r=0.443), and also between hotspot Ki67 index and SUVmax (r=0.360), SUVmean 30% (r=0.362) and SUVmean 40% (r=0.319). There was a weaker correlation, which was inversely negative, between mean and hotspot Ki67 and volumetric PET parameters. However, no statistical significant differences were found for any correlations. ConclusionsSUVmax and SUVmean were moderately correlated with Ki67 index, whereas volumetric PET parameters overall, showed a weaker correlation. Thus, SUVmax and SUVmean could be used to assess tumour aggressiveness in pre-treatment EOC.

S100A7 Regulates Ovarian Cancer Cell Metastasis and Chemoresistance Through MAPK Signaling and Is Targeted by miR-330-5p.

Epithelial ovarian cancer (EOC) is the most common cause of gynecological cancer-associated death. The high mortality rate is largely due to early stage metastasis and posttreatment recurrence. Identifying crucial regulators of EOC cells as well as ways to target them promises to improve the disease's prognosis. The S100 calcium-binding protein A7 (S100A7) has been shown to promote cell proliferation, migration, invasion, and tumor metastasis. In this study, we have investigated the role that S100A7 plays in regulating EOC cells. We have also identified the microRNA protein miR-330-5p, a known suppressor of oncogenesis and chemoresistance, as an inhibitor of S100A7 activity. We employed both fresh EOC tissue specimens as well as EOC cell lines Caov3 and SKOV3. S100A7 levels were analyzed using quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry. siRNA was used to knockdown S100A7. Cell proliferation, colony formation, and Transwell migration and invasion assays were performed on EOC cell lines with and without cisplatin treatment. TargetScan was used to identify miR-330-5p as a regulator of S100A7. Luciferase reporter plasmids were constructed with either the wild-type or mutant S100A7 3'UTR to determine the effect of miR-330-5p on S100A7. S100A7 was significantly overexpressed in human EOC tissue specimens and EOC cell lines Caov3 and SKOV3. Knocking down S100A7 reduced EOC cell proliferation, migration, and invasion. Furthermore, S100A7 knockdown cells demonstrated increased sensitivity to the chemotherapeutic cisplatin. Finally, miR-330-5p was shown to target the S100A7 3'UTR and to reduce EOC cell growth by inhibiting S100A7 expression. As a regulator of EOC cell proliferation, metastasis, and chemoresistance, S100A7 represents a potential prognostic biomarker for EOC as well as a treatment target. Because miR-330-5p functions as an inhibitor of EOC cell growth and S100A7 expression, it promises to improve EOC outcomes. Further research into the clinical significance of both S100A7 and miR-300-5p is warranted.

Metabolomic profiling suggests long chain ceramides and sphingomyelins as a possible diagnostic biomarker of epithelial ovarian cancer

IntroductionEpithelial ovarian cancer (EOC) is a disease with a poor survival rate mostly due to its discovery in late stages. The aim of this study was to investigate the metabolomic profile of ovarian cancer with the intention of identifying and describing novel biomarkers with diagnostic potential. Material and methodsTargeted serum metabolomic profiling was performed on 15 patients with ovarian cancer, 21 healthy controls and 21 patients with benign ovarian conditions, using HPLC–TQ/MS. ResultsPanel of 49 top performing biomarkers shows separation between EOC and controls with 87% specificity and 87% sensitivity with AUC of 93% (CI 90%–95%). Using only 5 top biomarkers, specificity of 80% and sensitivity of 83% was achieved on extended sample set. Most significant biomarkers belong to sphingolipid classes, especially long chain ceramides and sphingomyelins. Different concentrations of various fatty acid chain lengths ceramides and sphingomyelins are also implying their respective roles in cell proliferation and growth inhibition. ConclusionLong chain ceramides and sphingomyelins may serve as a novel biomarker for epithelial ovarian cancer detection and may also offer insight into the role of sphingolipid metabolism in cell proliferation.

Serum CA125 in combination with ferritin improves diagnostic accuracy for epithelial ovarian cancer

Introduction: CA125 has poor sensitivity and low specificity for detecting early ovarian cancer. Serum ferritin is elevated in many malignancies. We evaluated the performance of ferritin alone and in combination with CA125 as a diagnostic tool to detect epithelial ovarian cancer (EOC).Methods: CA125 and ferritin were detected in the serum of 50 healthy control (HC), 50 women with benign gynaecological conditions and 124 women with EOC. The relationship between serum ferritin and CA125 and each of the clinicopathological parameters was assessed, and their diagnostic accuracy for discriminating ovarian cancer determined.Results: Serum ferritin and CA125 were higher in patients with EOC compared to HCs and patients with benign conditions (both p < 0.001). There was no relationship between levels of ferritin and CA125. Both ferritin and CA125 discriminated HC from EOC (p < 0.05), but ferritin showed better diagnostic accuracy than CA125 (p = 0.048). Ferritin was superior to CA125 in discrimination early EOC (p = 0.002), but in advanced stages, CA125 was superior (p = 0.026). A combination of ferritin and CA125 marginally increases the diagnostic accuracy to discriminate EOC from HCs.Conclusion: Ferritin discriminates between HCs and EOC patients, especially in early stage disease. The combination of serum ferritin and CA125 provides the higher diagnostic accuracy to screen for EOC. Serum ferritin could serve as an EOC biomarker to complement the standard CA125 test.

Serum S100 calcium binding protein A4 (S100A4, metatasin) as a diagnostic and prognostic biomarker in epithelial ovarian cancer

Ovarian cancer is the leading cause of gynaecologic cancer death in developed countries and often presents at an advanced stage. The absence of specific symptoms and lack of reliable early diagnost...

UPLC-MS/MS based diagnostics for epithelial ovarian cancer using fully sialylated C4-binding protein.

Serum levels of fully sialylated C4-binding protein (FS-C4BP) are remarkably elevated in patients with epithelial ovarian cancer (EOC) and can be used as a marker to distinguish ovarian clear cell carcinoma from endometrioma. This study aimed to develop a stable, robust and reliable liquid chromatography–hybrid mass spectrometry (UPLC-MS/MS) based diagnostic method that would generalize FS-C4BP as a clinical EOC biomarker. Glycopeptides derived from 20 μL of trypsin-digested serum glycoprotein were analyzed via UPLC equipped with an electrospray ionization time-of-flight mass spectrometer. This UPLC-MS/MS-based diagnostic method was optimized for FS-C4BP and validated using sera from 119 patients with EOC and 127 women without cancer. A1958 (C4BP peptide with two fully sialylated biantennary glycans) was selected as a marker of FS-C4BP because its level in serum was highest among FS-C4BP family members. Preparation and UPLC-MS/MS were optimized for A1958, and performance and robustness were significantly improved relative to our previous method. An area under the curve analysis of the FS-C4BP index receiver operating characteristic curve revealed that the ratio between A1958 and A1813 (C4BP peptide with two partially sialylated biantennary glycans) reached 85%. A combination of the FS-C4BP index and carbohydrate antigen-125 levels further enhanced the sensitivity and specificity.

Recomendaciones para la determinación de biomarcadores en cáncer de ovario epitelial. Consenso nacional de la Sociedad Española de Anatomía Patológica y de la Sociedad Española de Oncología Médica

Advances in the understanding of the histological and molecular characteristics of ovarian cancer now allow 5subtypes to be identified, leading to a more refined therapeutic approach and improved clinical trials. Each of the subtypes has specific histological features and a particular biomarker expression, as well as mutations in different genes, some of which have prognostic and predictive value. CA125 and HE4 are examples of ovarian cancer biomarkers used in diagnosis and follow-up. Currently, somatic or germinal mutations on BRCA1 and BRCA2 genes are the most important biomarkers in epithelial ovarian cancer, having prognostic and predictive value. In this article, a group of experts from the Spanish Society of Medical Oncology and the Spanish Society of Pathology review the histological and molecular characteristics of the 5subtypes of ovarian cancer and describe the most useful biomarkers and mutations for diagnosis, screening and tailored treatment strategy.

Fully sialylated alpha-chain of complement 4-binding protein (A2160): a novel prognostic marker for epithelial ovarian carcinoma.

Fully sialylated alpha-chain of complement 4-binding protein (A2160) is a member of the glycoprotein family and has recently been identified as a diagnostic biomarker for epithelial ovarian cancer. This study examined the utility of A2160 as a prognostic biomarker for this disease. This is a retrospective analysis of prospectively collected plasma samples from 93 women with stage I-IV epithelial ovarian cancer who underwent primary cytoreductive surgery between 2009 and 2014. Pretreatment A2160 levels were correlated to clinico-pathological factors and survival outcome. Women with advanced-stage disease had significantly higher 2160 levels compared to those with early stage disease (stage I-II versus III-IV, median 2.17-2.70 versus 5.31-8.70U/mL, P<0.01). Women with high-grade serous ovarian carcinoma had higher A2160 levels compared to other histologies (6.60 versus 3.01U/mL, P=0.05). Women who had suboptimal cytoreduction had significantly higher A2160 levels than those who achieved optimal/complete cytoreduction (7.02 versus 2.30-3.17U/mL, P<0.01). On univariable analysis, higher A2160 levels were significantly associated with decreased progression-free survival (64-100 versus 1-33%ile, 5-year rates 53.4 versus 78.9%, P=0.029). After controlling for age, CA-125 level, cytoreductive status, histology, and stage, higher A2160 levels remained an independent prognostic factor for decreased progression-free survival (adjusted-hazard ratio (HR) 2.48, 95% confidence interval (CI) 1.01-6.11, P=0.049). Similarly, higher A2160 levels were independently associated with decreased cause-specific survival on multivariable analysis (adjusted-HR 3.07, 95% CI 1.19-7.93, P=0.021). Our study suggests that A2160 may be a useful prognostic biomarker for epithelial ovarian cancer, and higher pretreatment levels of A2160 predicts poor survival outcome.

Increased HE4 mRNA Expression Correlates with High Level Of eIF3a mRNA And Better Survival in Women with Epithelial Ovarian Cancer.

Human epididymis protein 4 (HE4) is one of the most promising biomarkers for epithelial ovarian cancer (EOC). The majority of previous studies utilized the serum level or tissue protein expression of HE4 based upon immunohistochemistry (IHC) to evaluate the role of HE4 in the diagnosis, prognosis, and surveillance of EOC, but very little is known about HE4 mRNA expression. Eukaryotic translation initiation factor 3a (eIF3a) is implicated in oncogenesis and has been investigated extensively as a potential biomarker for malignancy. We previously reported a positive correlation between IHC expressions of eIF3a and HE4 in EOC. In the present study, we performed RT-PCR to determine mRNA expressions of HE4 and eIF3a in 30 normal ovarian tissues, 45 benign, 20 borderline and 94 malignant ovarian tumors. The association of HE4 and eIF3a mRNA expressions with clinicopathological characteristics and patient survivals was investigated. IHC was also performed in the same participants to investigate the correlation between mRNA and protein levels of HE4. HE4 mRNA level was found to be 48.42 ± 74.55 (mean ± SD, range: 0.01-343.99), significantly higher in primary EOC than in the borderline tumor, benign tumor, and normal ovarian tissue (P<0.001). The cutoff value was 13.99 for HE4 to discriminate malignant from benign tumors at 68.1% sensitivity and 93.0% specificity. By Spearman's correlation test, HE4 mRNA expression was indicated to positively correlate with serum CA125 level (r=0.530, P<0.001). Higher HE4 mRNA expression was associated with decreased frequency of lymph node metastasis (P=0.038) and better overall survival (OS) (P=0.007) in primary EOC. Multivariable analysis showed an independent prognostic value of the relative mRNA level of HE4 greater than one for OS (Hazard Ratio, 0.069, 95%CI, 0.009-0.530, P=0.010). eIF3a mRNA expression in women with primary EOC was 0.95 ± 1.19 (mean ± SD, range: 0.06-7.46), which was in a positive linear correlation with HE4 mRNA expression (r=0.310, P=0.002). In the present study, the HE4 mRNA level was unparalleled with IHC expression of HE4 (P>0.05). Collectively, our study revealed that increased HE4 mRNA expression correlates with high level of eIF3a mRNA and better survival in women with EOC, which calls for further investigations.

Evaluation of serum CCL18 as a potential biomarker for ovarian cancer.

Early detection and differentiation diagnosis of a pelvic mass (PM) is crucial in improving the prognosis of patients with epithelial ovarian cancer (EOC). C-C motif chemokine ligand 18 (CCL18) was reported as a chemokine-mediated tumor-related inflammation that can be detected in serum and may correlate with cancer patients' prognosis. We performed this study to investigate the relationship between CCL18 levels and clinical characteristics of EOC patients, and to explore their diagnostic and prognostic values. CCL18 serum concentrations were detected by ELISA in 187 patients with EOC, 126 patients with benign PMs, and 118 healthy controls. CCL18 serum levels were analyzed in the context of patients' clinicopathological information, and ROC analyses were performed to determine the effect of CCL18 on distinguishing benign and malignant PMs. The ability of CCL18 to serve as an EOC biomarker was compared with CA125. Further survival analyses were carried out to assess the prognostic value of CCL18 in EOC patients. Mean serum CCL18 levels were elevated in benign PM patients and were even higher in EOC patients than in healthy controls; furthermore, high CCL18 expression was associated with worse International Federation of Gynecology and Obstetrics (FIGO) staging and predicted a poorer survival of the patient. When compared with CA125, although the sensitivity and negative predictive values (NPV) of serum CCL18 were lower, its specificity and positive predictive values (PPV) were higher. Serum CCL18 was elevated in patients with EOC and could serve as a new tumor biomarker, which also predicted a poor survival of the patient.

Pyridoxine 5\u2032-phosphate oxidase is a novel therapeutic target and regulated by the TGF-\u03b2 signalling pathway in epithelial ovarian cancer

Pyridoxine 5′-phosphate oxidase (PNPO) is an enzyme that converts pyridoxine 5′-phosphate into pyridoxal 5′-phosphate (PLP), an active form of vitamin B6 implicated in several types of cancer. However, the role of PNPO and its regulatory mechanism in epithelial ovarian cancer (EOC) are unknown. In the present study, PNPO expression in human ovarian tumour tissue and its association with the clinicopathological features of patients with EOC were examined. Further, the biological function of PNPO in EOC cells and in xenograft was evaluated. We demonstrated for the first time that PNPO was overexpressed in human EOC. Knockdown of PNPO induced EOC cell apoptosis, arrested cell cycle at G2/M phase, decreased cell proliferation, migration and invasion. Xenografts of PNPO-shRNA-expressing cells into the nude mouse attenuated tumour growth. PNPO at mRNA and protein levels in EOC cells was decreased after transforming growth factor-β1 (TGF-β1) treatment. The inhibitory effect of TGF-β1 on PNPO expression was abolished in the presence of SB-431542, a TGF-β type I receptor kinase inhibitor. Moreover, we found that TGF-β1-mediated PNPO expression was at least in part through the upregulation of miR-143-3p. These data indicate a mechanism underlying PNPO regulation by the TGF-β signalling pathway. Furthermore, PLP administration reduced PNPO expression and decreased EOC cell proliferation, suggesting a feedback loop between PLP and PNPO. Thus, our findings reveal that PNPO can serve as a novel tissue biomarker of EOC and may be a potential target for therapeutic intervention.

Interleukin 6 Present in Inflammatory Ascites from Advanced Epithelial Ovarian Cancer Patients Promotes Tumor Necrosis Factor Receptor 2-Expressing Regulatory T Cells.

Epithelial ovarian cancer (EOC) remains a highly lethal gynecological malignancy. Ascites, an accumulation of peritoneal fluid present in one-third of patients at presentation, is linked to poor prognosis. High levels of regulatory T cells (Tregs) in ascites are correlated with tumor progression and reduced survival. Malignant ascites harbors high levels of Tregs expressing the tumor necrosis factor receptor 2 (TNFR2), as well as pro-inflammatory factors such as interleukin 6 (IL-6) and tumor necrosis factor (TNF). IL-6 is also associated with poor prognosis. Herein, we study the effect of IL-6 and TNF present in ascites on the modulation of TNFR2 expression on T cells, and specifically Tregs. Ascites and respective peripheral blood sera were collected from 18 patients with advanced EOC and soluble biomarkers, including IL-6, sTNFR2, IL-10, TGF-β, and TNF, were quantified using multiplexed bead-based immunoassay. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with cell-free ascites for 48 h (or media as a negative control). In some experiments, IL-6 or TNF within the ascites were neutralized by using monoclonal antibodies. The phenotype of TNFR2+ Tregs and TNFR2- Tregs were characterized post incubation in ascites. In some experiments, cell sorted Tregs were utilized instead of PBMC. High levels of immunosuppressive (sTNFR2, IL-10, and TGF-β) and pro-inflammatory cytokines (IL-6 and TNF) were present in malignant ascites. TNFR2 expression on all T cell subsets was higher in post culture in ascites and highest on CD4+CD25hiFoxP3+ Tregs, resulting in an increased TNFR2+ Treg/effector T cell ratio. Furthermore, TNFR2+ Tregs conditioned in ascites expressed higher levels of the functional immunosuppressive molecules programmed cell death ligand-1, CTLA-4, and GARP. Functionally, TNFR2+ Treg frequency was inversely correlated with interferon-gamma (IFN-γ) production by effector T cells, and was uniquely able to suppress TNFR2+ T effectors. Blockade of IL-6, but not TNF, within ascites decreased TNFR2+ Treg frequency. Results indicating malignant ascites promotes TNFR2 expression, and increased suppressive Treg activity using PBMC were confirmed using purified Treg subsets. IL-6 present in malignant ovarian cancer ascites promotes increased TNFR2 expression and frequency of highly suppressive Tregs.

Altered expression of different GalNAc‑transferases is associated with disease progression and poor prognosis in women with high-grade serous ovarian cancer.

Protein glycosylation perturbations are implicated in a variety of diseases, including cancer. Aberrant glycosylation in cancer is frequently attributed to altered expression of polypeptide GalNAc transferases (GalNAc‑Ts) - enzymes initiating mucin-type O-glycosylation. A previous study from our group demonstrated that one member of this family (GALNT3) is overexpressed in epithelial ovarian cancer (EOC), and GALNT3 expression correlated with shorter progression-free survival (PFS) in EOC patients with advanced disease. As considerable degree of redundancy between members of the GalNAc‑Ts gene family has been frequently observed, we decided to investigate whether other members of this family are essential in EOC progression. Insilico analysis based on publically available data was indicative for altered expression of five GalNAc‑Ts (GALNT2, T4, T6, T9 and T14) in ovarian high-grade serous carcinoma (HGSC) samples compared to non-tumoral (control) ovarian tissue. We analyzed protein expression of these GalNAc‑Ts in EOC cells and tumors by western blotting, followed by immunohistochemical (IHC) evaluation of their expression in EOC tumor and control samples using tissue microarrays (TMAs). Western blot analyses were indicative for low expression of GALNT2 and strong expression of GALNT6, T9 and T14 in both EOC cells and tumors. These observations were confirmed by IHC. GALNT2 displayed significantly lower expression, while GALNT6, GALNT9 and GALNT14 showed significantly higher expression in HGSC tumors compared to control tissue. Importantly, GALNT6 and GALNT14 expression correlated with poor prognosis of serous EOC patients. Moreover, our results suggest for overlapping functions of some GalNAc‑Ts, more specifically GALNT3 and GALNT6, in directing EOC progression. Our results are indicative for a possible implication of different members of the GalNAc‑T gene family in modulating EOC progression, and the potential use of GALNT6 and GALNT14 as novel prognostic EOC biomarkers. These data warrant future studies on the role of members of the GalNAc‑Ts gene family in ovarian tumorigenesis.

HOXA9 and SOX1 - a promising DNA methylation based diagnostic biomarker for epithelial ovarian cancer

HOXA9 and SOX1 - a promising DNA methylation based diagnostic biomarker for epithelial ovarian cancer

Erratum to: Recommendations for biomarker testing in epithelial ovarian cancer: a National Consensus Statement by the Spanish Society of Pathology and the Spanish Society of Medical Oncology.

Because of advances in the understanding of histological and molecular characteristics in ovarian cancer, it is now possible to recognize the existence of five subtypes, which in turn has allowed a more refined therapeutic approach and better design of clinical trials. Each of these five subtypes has specific histological features and a particular biomarker expression, as well as mutations in different genes, some of which have prognostic and predictive value. CA125 and HE4 are examples of ovarian cancer biomarkers used in the diagnosis and follow-up of these malignancies. Currently, somatic or germinal mutations on BRCA1 and BRCA2 genes are the most important biomarkers in epithelial ovarian cancer having prognostic and predictive value. This article will review the histological and molecular characteristics of the five subtypes of ovarian cancer, describing the most important biomarkers and mutations that can guide in diagnosis, screening and tailored treatment strategy.