Plasminogen activator inhibitor 1 is associated with high-grade serous ovarian cancer metastasis and is reduced in patients who have received neoadjuvant chemotherapy.

Introduction: High-grade serous ovarian cancer (HGSOC) is the most prevalent and deadliest subtype of epithelial ovarian cancer (EOC), killing over 140,000 people annually. Morbidity and mortality are compounded by a lack of screening methods, and recurrence is common. Plasminogen-activator-inhibitor 1 (PAI-1, the protein product of SERPIN E1) is involved in hemostasis, extracellular matrix (ECM) remodeling, and tumor cell migration and invasion. Overexpression is associated with poor prognosis in EOC. Platelets significantly increase PAI-1 in cancer cells in vitro, and may contribute to the hematogenous metastasis of circulating tumor cells (CTCs). CTCs are viable tumor cells that intravasate and travel through the circulation-often aided by platelets - with the potential to form secondary metastases. Here, we provide evidence that PAI-1 is central to the platelet-cancer cell interactome, and plays a role in the metastatic cascade. Methods: SK-OV-3 cells where PAI-1 had been silenced, treated with healthy donor platelets, and treated with platelet-conditioned medium were used as an in vitro model of metastatic EOC. Gene expression analysis was performed using RNA-Seq data from untreated cells and cells treated with PAI-1 siRNA or negative control, each with and without platelets. Four cohorts of banked patient plasma samples (n = 239) were assayed for PAI-1 by ELISA. Treatment-naïve (TN) whole blood (WB) samples were evaluated for CTCs in conjunction with PAI-1 evaluation in matched plasma. Results and discussion: Significant phenotypic changes occurring when PAI-1 was silenced and when platelets were added to cells were reflected by RNA-seq data, with PAI-1 observed to be central to molecular mechanisms of EOC metastasis. Increased proliferation was observed in cells treated with platelets. Plasma PAI-1 significantly correlated with advanced disease in a TN cohort, and was significantly reduced in a neoadjuvant chemotherapy (NACT) cohort. PAI-1 demonstrated a trend towards significance in overall survival (OS) in the late-stage TN cohort, and correlation between PAI-1 and neutrophils in this cohort was significant. 72.7% (16/22) of TN patients with plasma PAI-1 levels higher than OS cutoff were CTC-positive. These data support a central role for PAI-1 in EOC metastasis, and highlight PAI-1's potential as a biomarker, prognostic indicator, or gauge of treatment response in HGSOC.

Ex vivo expansion of circulating tumour cells (CTCs).

Circulating tumour cells (CTCs) are a critical intermediate step in the process of cancer metastasis. The reliability of CTC isolation/purification has limited both the potential to report on metastatic progression and the development of CTCs as targets for therapeutic intervention. Here we report a new methodology, which optimises the culture conditions for CTCs using primary cancer cells as a model system. We exploited the known biology that CTCs thrive in hypoxic conditions, with their survival and proliferation being reliant on the activation of hypoxia-inducible factor 1 alpha (HIF-1α). We isolated epithelial-like and quasi-mesenchymal CTC phenotypes from the blood of a cancer patient and successfully cultured these cells for more than 8 weeks. The presence of CTC clusters was required to establish and maintain long-term cultures. This novel methodology for the long-term culture of CTCs will aid in the development of downstream applications, including CTC theranostics.

Procoagulant activity in high grade serous ovarian cancer patients following neoadjuvant chemotherapy-The role of the activated protein C pathway.

INTRODUCTION: Ovarian cancer patients are at high risk of thrombosis particularly during chemotherapy treatment however the mechanism is not understood. The aim of this study is to investigate the role of the activated protein C (aPC) pathway in the procoagulant activity observed in ovarian cancer patients undergoing neoadjuvant chemotherapy. PATIENTS AND METHODS: Thrombin generation was determined before and after addition of thrombomodulin (TM) in high grade serous ovarian cancer (HGSOC) patients treated with neoadjuvant chemotherapy (n = 29) compared with HGSOC patients who were chemo naïve (n = 23) and patients with benign tumours (n = 29). Plasma expression of proteins from the aPC pathway was analysed. mRNA expression was determined in endothelial (EA.hy926) and ovarian (OAW42) cell lines following addition of carboplatin and paclitaxel. RESULTS: Lower levels of ETP (p < 0.007; p < 0.003) and peak thrombin (p < 0.0008; p < 0.0018) were found in the neoadjuvant group compared with both chemo naïve and benign groups. Following addition of TM, ETP (p < 0.0005) and peak thrombin (p < 0.0049) were higher in the neoadjuvant group compared with the benign controls indicating an increase in aPC resistance. Increased TM and lower levels of protein S were found in the neoadjuvant group compared with benign controls (p < 0.05; p < 0.003). Factor V levels were increased in the neoadjuvant group compared with the chemo naïve group (p < 0.05). Carboplatin and paclitaxel altered the expression of EPCR and thrombomodulin in OAW42 cells with a modest effect on EA.hy926 cells. CONCLUSION: Chemotherapy induced procoagulant activity in HGSOC is associated with an alteration in expression of key members of the aPC pathway. This acquired aPC resistance may explain the procoagulant phenotype associated with ovarian cancer patients undergoing chemotherapy.

Patients' experience and compliance with extended low molecular weight heparin prophylaxis post-surgery for gynecological cancer: a prospective observational study.

OBJECTIVE: Gynaecological cancer patients have a high risk of developing venous thromboembolism (VTE). There is limited information on patient experience and compliance with an extended low molecular weight heparin prophylaxis in this setting. The aim of this study was to assess patient compliance, satisfaction and experience with the extended low molecular weight heparin prophylaxis after major surgery for gynaecological cancer. METHODS: This was a prospective observational study conducted in a large tertiary center for gynaecological cancer between July 2017-March 2018. Consecutive patients undergoing surgery for gynaecological cancer who received low molecular weight heparin prophylaxis for four weeks following surgery were recruited. All participants received a log book to record all injections, side effects, and questionnaire to be completed at the end of the study. RESULTS: A total of 106 patients completed and returned the VTE prophylaxis logbook and questionnaire. Sixty-six (62%) patients received low molecular weight heparin for 28 days, twenty-five (24%) for 26-27 days, and 15 (14%) for less than 26 days. The median number of days of therapy was 28 days (range; 12-28 days). Reasons for missed or stopped injections included: forgetfulness(n=12), medical procedures (n=6), pain (n=5), incorrect prescription (n=4), patient choice (n=3), cost (n=2), physician request (n=2), non-availability of person administering the injections (n=1) or unknown (n=5). Sixty-one (58%) patients self-administered the injections. Patients who had the injection performed by a third person were twice as likely to experience pain compared to patients who self-administered (OR 2.81, p=0.003). Eighty-nine (84%) patients self-reported side effects during low molecular weight heparin prophylaxis including: bruising (75%), pain after injections (49%), itchiness (9%), swelling (9%) or other (8%). Although 83 (78%) patients were satisfied with injections, 91 (86%) admitted they would much prefer a tablet form. CONCLUSIONS: Compliance with standard recommended regimen of 28-days prophylaxis was completed by 62% of patients. Majority of patients (86%) reported a preference for a tablet form, if one was available.

A novel serum microRNA panel to discriminate benign from malignant ovarian disease.

Ovarian cancer is the seventh most common cancer in women and the most frequent cause of gynaecological malignancy-related mortality in women. Currently, no standardized reliable screening test exists. MicroRNA profiling has allowed the identification of signatures associated with diagnosis, prognosis and response to treatment of human tumours. The aim of this study was to determine if a microRNA signature could distinguish between malignant and benign ovarian disease. A training set of 5 serous ovarian carcinomas and 5 benign serous cystadenomas were selected for the initial experiments. The validation set included 20 serous ovarian carcinomas and 20 benign serous cystadenomas. The serum/plasma focus microRNA Exiqon panel was used for the training set. For the validation set a pick and mix Exiqon panel, which focuses on microRNAs of interest was used. A panel of 4 microRNAs (let-7i-5p, miR-122, miR-152-5p and miR-25-3p) was significantly down regulated in cancer patients. These microRNAs target WNT signalling, AKT/mTOR and TLR-4/MyD88, which have previously been found to play a role in ovarian carcinogenesis and chemoresistance. let-7i-5p, miR-122, miR-152-5p and miR-25-3p could act as diagnostic biomarkers in ovarian cancer.

Co-operative radical pelvic surgery: a role for the gynecologist in vaginal reconstruction using a uterine myoserosal flap in urological and anorectal cancer surgery.

This study describes a new technique for reconstructing the vagina and vestibule after radical extirpative surgery for urological and anorectal malignancy. The uterus is always excised when exenterative surgery is performed for gynecological cancer. The use of the uterus as a graft gives the gynecologic oncologist/reconstructive surgeon a role in the multidisciplinary team with urologists when the anterior vaginal wall and vestibule are excised and with the anorectal surgeons when the posterior vaginal wall and perineum are excised for nongynecological cancers. In some such cases, only the anterior or posterior wall of the vagina may be excised, leaving a healthy full-length, one-third, or half-circumference vaginal sleeve. A myoserosal flap is raised from the in situ uterus. The ectocervix is excised, and the adnexa are detached or excised. The uterus is opened to generate a hexagonal flap. The endometrium and endocervix are excised/ablated with electrocautery. The flap is advanced to the edge of the remaining anterior vestibule or reconstituted perineum. The serosal surface of the uterus forms the new wall of the vagina and undergoes metaplastic transformation to squamous epithelium within 3 months. The very satisfactory anatomical and functional outcome means that this technique merits further evaluation.

Pre-Treatment of platinum resistant ovarian cancer cells with an MMP-9/MMP-2 inhibitor prior to cisplatin enhances cytotoxicity as determined by high content screening.

Platinum resistance is a major cause of treatment failure in ovarian cancer. We previously identified matrix metalloproteinase 9 (MMP-9) as a potential therapeutic target of chemoresistant disease. A2780cis (cisplatin-resistant) and A2780 (cisplatin-sensitive) ovarian carcinoma cell lines were used. The cytotoxic effect of MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) alone or in combination with cisplatin was determined using high content screening. Protein expression was examined using immunohistochemistry and ELISA. Co-incubation of cisplatin and an MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) resulted in significantly greater cytotoxicity as compared to either treatment alone in a cisplatin resistant MMP-9 overexpressing cell line; A2780cis. In addition, pre-incubating with MMP-9i prior to cisplatin further enhances the cytotoxic effect. No significant difference was observed in MMP-9 protein in tissue but a trend towards increased MMP-9 was observed in recurrent serum. We propose that MMP-9/MMP-2i may be utilized in the treatment of recurrent/chemoresistant ovarian cancers that overexpress MMP-9 mRNA but its role in vivo remains to be evaluated.