Primary EOC Research Articles

Lipolysis-stimulated lipoprotein receptor-targeted antibody-drug conjugate demonstrates potent antitumor activity against epithelial ovarian cancer

BackgroundEpithelial ovarian cancer (EOC) is a lethal malignant tumor, for which new treatment options are urgently required. Lipolysis-stimulated lipoprotein receptor (LSR) is widely expressed in EOC, and it is associated with poor prognosis. In this study, we developed an antibody-drug conjugate (ADC) targeting LSR as a new therapeutic approach to EOC. MethodsWe, herein, developed novel anti-LSR monoclonal antibodies (mAbs) and an LSR-ADC by conjugating monomethyl auristatin E as a payload. We subsequently evaluated the in vitro and in vivo (on xenograft models) antitumor effect of the LSR-ADC. ResultsAn overexpression of LSR was observed not only in the primary EOC tumor but also in its lymph node and omental metastases. The EOC cell lines NOVC7-C and OVCAR3 strongly expressed LSR (as compared to ES2 cells). Both the anti-LSR mAb and the LSR-ADC were able to specifically bind to LSR-positive cells and were rapidly internalized and trafficked to the lysosomes. The LSR-ADC demonstrated a potent antitumor effect against NOVC-7C and OVCAR3, but little activity against ES2 cells. In vitro, the LSR-ADC exhibited a potent antitumor effect against NOVC-7C and OVCAR3. Moreover, in the OVCAR3 xenograft models as well as in the patient-derived xenograft models of LSR-positive EOC, the LSR-ADC significantly inhibited tumor growth. The LSR-ADC also suppressed the omental/bowel metastases in OVCAR3-Luc xenografts and improved the median survival. ConclusionThe developed LSR-ADC demonstrated a significant antitumor activity against LSR-positive EOC cell lines and tumors. Our preclinical data support the use of the LSR-ADC as a novel therapy for patients with LSR-positive ovarian cancer.

TWIST1 induces proteasomal degradation of β-catenin during the differentiation of ovarian cancer stem-like cells

Ovarian cancer (OC) is one of the leading gynecologic cancers worldwide. Cancer stem-like cells are correlated with relapse and resistance to chemotherapy. Twist1, which is involved in ovarian cancer stem-like cell differentiation, is positively correlated with CTNNB1 in different differentiation stages of ovarian cancer cells: primary epithelial ovarian cancer cells (primary EOC cells), mesenchymal spheroid-forming cells (MSFCs) and secondary epithelial ovarian cancer cells (sEOC cells). However, the expression of β-catenin is inversed compared to CTNNB1 in these 3 cell states. We further demonstrated that β-catenin is regulated by the protein degradation system in MSFCs and secondary EOC but not in primary EOC cells. The differentiation process from primary EOC cells to MSFCs and sEOC cells might be due to the downregulation of β-catenin protein levels. Finally, we found that TWIST1 can enhance β-catenin degradation by upregulating Axin2.

Safety and tolerability of Hyperthermic Intraperitoneal Chemotherapy (HIPEC) in ovarian cancer: clinical results from a phase I trial

Objectives: To demonstrate safety and tolerability of HIPEC in ovarian cancer. Methods: A phase I, single-institution feasibility study was designed to evaluate the safety and feasibility of HIPEC at time of optimal cytoreductive surgery (CRS) in ovarian cancer patients, followed by optional normothermic intraperitoneal chemotherapy, at City of Hope National Comprehensive Cancer Center (Duarte, CA). Primary endpoint of the trial was to evaluate the safety and feasibility of HIPEC at time of optimal cytoreductive surgery (CRS). Progression-free survival was a secondary end-point. Exploratory endpoints included the assessment of molecular markers in tumors. Eligibility criteria included epithelial ovarian cancer (stage III or IV), primary or recurrent, ECOG 0-1, and appropriate surgical candidates. 35 patients were enrolled between 2015 and 2019, who underwent optimal CRS (gross residual disease less than 1 cm), and received HIPEC intra-operatively with cisplatin at 75 mg/m2 for 60 minutes immediately following optimal CRS. Patients were stratified as either 1) primary disease (n=19), defined as having received HIPEC at time of interval CRS following neoadjuvant chemotherapy for primary EOC; 2) recurrent disease (n=16), defined as having received HIPEC at time of interval or upfront CRS for recurrent (salvage) EOC. Adjuvant chemotherapy was given at the discretion of the treating oncologist following HIPEC. Results: Median age was 58 years (36-74 years), with a mean BMI of 26. Primary (interval) patients represented 54% of patients, with pretreatment clinical stages III (53%) and IV (47%). Recurrent (salvage) patients represented 45.7%, with platinum-sensitive (75%) and platinum-resistant (25%). The most common histological subtype was high grade serous (69%). All primary (interval) patients underwent neoadjuvant chemotherapy (3-4 cycles) prior to interval CRS and HIPEC. The majority of recurrent patients (81%) did not receive neo-adjuvant chemotherapy. Thirty-three patients underwent closed technique and two underwent laparoscopic technique. Total mean operating time was 8.4 hours (4.6-15.3). Mean length of stay was 10 days (3-30). All patients underwent optimal cytoreduction (69% with complete R0 resection). Peritoneal carcinomatosis index (PCI) ranged 2 to 30 (median of 8). Patients with PCI /= 8 whose PFS was 12.6 (p=0.04). Time to initiation of adjuvant chemotherapy in recurrent patients was 8.4 weeks. 22.9% of the ovarian cancer cohort was gBRCA mutated. Median PFS for primary patients was 12.6 months (95% CI 6.4; NR); with median follow up of 22.1 months (95% CI 12.6, 31.5). Median PFS for recurrent patients was 18.8 months (95% CI 7.2; 28.7), with median follow up of 36.3 months (95% CI 21.1, 51.5). There were no grade 4 or 5 AEs. Anemia was the most common Grade 3 AE (43%), followed by liver dysfunction and electrolyte abnormalities. The most common AE of any grades were abdominal pain (86%), anemia (77%), and nausea (66%). Two patients (6%) had grade 3 acute kidney injury and one patient had grade 3 chronic kidney disease. Sodium thiosulfate was introduced to the trial in January 2018, following which there were no grade 1-4 acute or chronic kidney injuries. Download : Download high-res image (61KB) Download : Download full-size image Conclusions: HIPEC in ovarian cancer was well tolerated, without any grade 4 or 5 AEs. Renal toxicities were mitigated with sodium thiosulfate. Patients with a low PCI ( /= 8.

Abstract 3186: Differential expression of PD-L1 between primary and metastatic Middle-Eastern epithelial ovarian carcinoma and its clinico-pathological correlation

Abstract Ovarian cancer (OC) is one of the most common gynecologic cancer, which has the worst prognosis and highest mortality rate. The lack of curative treatment and the high relapse rate, especially in advanced OC, continues to present a clinical challenge, highlighting the need for new therapeutic strategies. This study was conducted to compare the expression of PD-L1 in Middle-Eastern primary epithelial ovarian carcinoma to that in peritoneal metastases and its correlation with clinico-pathological parameters. In total, 194 treatment naïve paired EOC and peritoneal metastases were analyzed by immunohistochemistry for PD-L1 expression. Clinico-pathological information was available for all patients. Significant differences in PD-L1 expression were found between primary EOC and peritoneal metastasis (p < 0.0001). We found discordant tumor cell PD-L1 expression between primary tumors and corresponding peritoneal metastasis in 34% (66/194) of cases. Furthermore, PD-L1 expression in peritoneal metastasis samples was significantly associated with adverse prognostic factors, such as high proliferative index (Ki67) (p = 0.0039) and high histologic grade (p = 0.0330). In conclusion, the heterogeneity of PD-L1 expression in EOC suggests its assessment as a potential biomarker for PD-L1 blockade may require analysis of metastatic lesions. Citation Format: Sandeep K. Parvathareddy, Abdul K. Siraj, Padmanaban Annaiyappanaidu, Felisa De Vera, Yan Kong, Zeeshan Qadri, Saeeda O. Ahmed, Hassan AlDossari, Khawla S. Al-Kuraya. Differential expression of PD-L1 between primary and metastatic Middle-Eastern epithelial ovarian carcinoma and its clinico-pathological correlation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3186.

Optimal treatment duration of bevacizumab (BEV) combined with carboplatin and paclitaxel in patients (pts) with primary epithelial ovarian (EOC), fallopian tube (FTC) or peritoneal cancer (PPC): A multicenter open-label randomized 2-arm phase 3 ENGOT/GCIG trial of the AGO Study Group, GINECO, and NSGO (AGO-OVAR 17/BOOST, GINECO OV118, ENGOT Ov-15, NCT01462890).

5501 Background: GOG-0218 established the addition of BEV 15 mg/kg every 3 weeks (q3w) for 15 months to standard front-line chemotherapy for advanced ovarian cancer, but the optimal BEV duration remained unknown. We report primary results from a randomized phase 3 trial designed to address this question. Methods: Eligible pts with FIGO stage IIB–IV EOC, FTC, or PPC and ECOG PS ≤2 underwent primary cytoreductive surgery followed by 6 cycles of chemotherapy (paclitaxel 175 mg/m2 + carboplatin AUC 5 q3w) and BEV (15 mg/kg q3w). Pts were randomized to receive BEV for either 15 months (standard arm BEV15) or 30 months (experimental arm BEV30), stratified by FIGO stage/residual tumor (stage IIB–IIIC/no residual tumor vs stage IIB–IIIC/residual tumor or stage IV). The primary endpoint was investigator-assessed progression-free survival (PFS) according to RECIST v1.1. Secondary endpoints were overall survival (OS), objective response rate, quality of life, safety, and tolerability. The trial was designed with 80.2% power to detect a hazard ratio (HR) of 0.66 favoring BEV30 (2-sided log-rank test, 5% significance level, 10% dropout rate) after 697 PFS events. The trial was funded by F. Hoffmann-La Roche Ltd., performed according to ENGOT model A. Results: From Nov 2011 to Aug 2013, 927 women (83% with EOC) from 161 centers were randomized. Baseline characteristics were balanced between arms; median age was 61 years, 96% had ECOG PS 0/1, 58% had no residual tumor, and 77% had high-grade serous histology. Serious adverse events of special interest for BEV occurred in 51/448 pts (11%) vs 61/442 pts (14%) receiving BEV15 vs BEV30, respectively: hypertension (2.7%/4.5%), thromboembolic event (2.2%/3.2%), fistula (3.1%/1.1%), gastrointestinal perforation (0.2%/0.9%), proteinuria (0.7%/1.4%), hemorrhage (0.2%/0.9%), and myocardial infarction (0%/1.1%). Efficacy is shown in the table. Conclusions: Longer treatment with BEV for up to 30 months improves neither PFS nor OS in pts with primary EOC, FTC, or PPC. Therefore BEV treatment duration of 15 months remains standard of care. Clinical trial information: NCT01462890. [Table: see text]

Assessment of Lymph Node Involvement with PET-CT in Advanced Epithelial Ovarian Cancer. A FRANCOGYN Group Study.

The objective of our study is to evaluate the diagnostic performance of positron emission tomography/computed tomography (PET-CT) for the assessment of lymph node involvement in advanced epithelial ovarian, fallopian tubal or peritoneal cancer (EOC). This was a retrospective, bicentric study. We included all patients over 18 years of age with a histological diagnosis of advanced EOC who had undergone PET-CT at the time of diagnosis or prior to cytoreduction surgery with pelvic or para-aortic lymphadenectomy. We included 145 patients with primary advanced EOC. The performance of PET-CT was calculated from the data of 63 patients. The sensitivity of PET-CT for preoperative lymph node evaluation was 26.7%, specificity was 90.9%, PPV was 72.7%, and NPV was 57.7%. The accuracy rate was 60.3%, and the false-negative rate was 34.9%. In the case of primary cytoreduction (n = 16), the sensitivity of PET-CT was 50%, specificity was 87.5%, PPV was 80%, and NPV was 63.6%. The accuracy rate was 68.8%, and the false negative rate was 25%. After neoadjuvant chemotherapy (n = 47), the sensitivity of PET-CT was 18.2%, specificity was 92%, PPV was 66.7%, and NPV was 56.1%. The accuracy rate was 57.5%, and the false negative rate was 38.3%. Due to its high specificity, the performance of a preoperative PET-CT scan could contribute to the de-escalation and reduction of lymphadenectomy in the surgical management of advanced EOC in a significant number of patients free of lymph node metastases.

Evaluating class III antiarrhythmic agents as novel MYC targeting drugs in ovarian cancer

ObjectiveTo evaluate the utility of amiodarone and its derivative dronedarone as novel drug repositioning candidates in EOC and to determine the potential pathways targeted by these drugs. MethodsDrug-predict bioinformatics platform was used to assess the utility of amiodarone as a novel drug-repurposing candidate in EOC. EOC cells were treated with amiodarone and dronedarone. Cell death was assessed by Annexin V staining. Cell viability and cell survival were assessed by MTT and clonogenics assays respectively. c-MYC and mTOR/Akt axis were evaluated as potential targets. Effect on autophagy was determined by autophagy flux flow cytometry. Results“DrugPredict” bioinformatics platform ranked Class III antiarrhythmic drug amiodarone within the top 3.9% of potential EOC drug repositioning candidates which was comparable to carboplatin ranking in the top 3.7%. Amiodarone and dronedarone were the only Class III antiarrhythmic drugs that decreased the cellular survival of both cisplatin-sensitive and cisplatin-resistant primary EOC cells. Interestingly, both drugs induced degradation of c-MYC protein and decreased the expression of known transcriptional targets of c-MYC. Furthermore, stable overexpression of non-degradable c-MYC partially rescued the effects of amiodarone and dronedarone induced cell death. Dronedarone induced higher autophagy flux in EOC cells as compared to amiodarone with decreased phospho-AKT and phospho-4EBP1 protein expression, suggesting autophagy induction due to inhibition of AKT/mTOR axis with these drugs. Lastly, both drugs also inhibited the survival of EOC tumor-initiating cells (TICs). ConclusionsWe provide the first evidence of class III antiarrhythmic agents as novel c-MYC targeting drugs and autophagy inducers in EOC. Since c-MYC is amplified in >40% ovarian tumors, our results provide the basis for repositioning amiodarone and dronedarone as novel c-MYC targeting drugs in EOC with potential extension to other cancers.

Ad5NULL-A20: A Tropism-Modified, αvβ6 Integrin-Selective Oncolytic Adenovirus for Epithelial Ovarian Cancer Therapies.

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvβ6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues.Experimental Design: Ad5NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvβ6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed.Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins, and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvβ6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non-tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20-treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5.Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvβ6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215-24. ©2018 AACR.

Abstract B66: miRNA 3’UTR activity driven enrichment of ovarian tumor-initiating cells (TICs) to overcome the barriers of heterogeneity and TIC plasticity

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Tumor recurrence is a major barrier towards improving patient outcome in EOC. Tumor-initiating cells (TICs) survive platinum-based therapies and contribute to tumor recurrence, at which stage the tumor is often difficult to eradicate, and thus TICs form the root cause of poor patient survival in EOC. Unfortunately, no TIC-targeting drugs to date are being evaluated in EOC patient trials. Isolating majority of TIC clones present in EOC tumors for functional analysis remains elusive to date and hence, understanding the complexity of TIC functioning remains a hurdle to be overcome in TIC therapeutics. TIC regulators can differ between tumors across patients and also in tumors within a patient. Tumor heterogeneity and TIC plasticity are the major challenges towards deciphering TIC functioning in EOC. Studying EOC cell populations with TIC properties is often limited to approaches driven by enrichment based on expression of stem-cell markers, which do not faithfully reflect TIC functioning in the context of heterogeneity and plasticity. Hence, pathway-based enrichment of TICs provide a reliable approach to functionally enrich for TIC subpopulations in EOC. miRNAs are small RNA molecules that regulate post-transcriptional gene silencing by binding mostly to the 3’UTR of their potential targets and targeting them for degradation through miRNA-induced silencing complexes (miRISCs). Isolation and characterization of TIC subpopulations in EOC based on miRNA activity can offer a reliable approach to functionally enrich for TICs in EOC. We have developed a novel miRNA-sensor based functional platform driven by miR-181a 3’UTR activity with mCHERRY fluorescence as the readout. miR-181a expression was upregulated in primary recurrent EOC tumors that were clinically resistant to platinum chemotherapy, suggesting a potential role for this miRNA in regulating TIC properties in EOC. Accordingly, miR-181a high subpopulation isolated from both cisplatin-sensitive and cisplatin-resistant/recurrent primary high-grade serous (HGSOC) ovarian tumor cells using miR-181a sensor were enriched in TIC properties in vitro and in vivo. Cisplatin treatment of primary EOC cells enriched miR-181a high subpopulation and reprogrammed miR-181a low non-TICs into miR-181a high TICs. Mechanistically, Wnt signaling was upregulated in miR-181a high ovarian tumor cells, showing that miRNA sensor platform can enrich TICs based on stem-cell pathway activity. Taken together, we provide the first evidence of TIC enrichment and analysis in EOC based on miRNA 3’UTR activity that provides with a valuable tool to broaden the understanding of TIC regulatory mechanisms in EOC in “real time,” with potential extension to other cancers by overcoming the barriers of tumor heterogeneity and TIC plasticity. miRNA sensor platform will provide a TIC pharmacologic screening tool to enable the discovery of novel miRNA-inhibiting TIC-targeting drugs in EOC. Citation Format: Anil Belur Nagaraj, Peronne Joseph, Matthew Knarr, Olga Kovalenko, Arshia Surti, Analisa DiFeo. miRNA 3’UTR activity driven enrichment of ovarian tumor-initiating cells (TICs) to overcome the barriers of heterogeneity and TIC plasticity. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B66.

Superior in vitro and in vivo activity of trastuzumab-emtansine (T-DM1) in comparison to trastuzumab, pertuzumab and their combination in epithelial ovarian carcinoma with high HER2/neu expression

BackgroundEpithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. The objective of this study was to compare the anti-tumor activity of HER2/neu-targeting monoclonal antibodies, trastuzumab (T), pertuzumab (P), combination of trastuzumab and pertuzumab (T+P) and trastuzumab-emtansine (T-DM1) in EOC with high HER2/neu expression. MethodsPrimary EOC cell lines were established and cell blocks were analyzed for HER2/neu expression. Cytostatic, apoptotic and antibody-dependent cell-mediated cytotoxicity (ADCC) activities of T, P, T+P and T-DM1 were evaluated in vitro. The in vivo antitumor activity was tested in xenograft models with 3+ HER2/neu expression. ResultsHigh (3+) HER2/neu expression was detected in 40% of the primary EOC cell lines. T, P, T+P, and T-DM1 were similarly effective in inducing strong ADCC against primary EOC cell lines expressing 3+ HER2/neu. The combination of T and P was more cytostatic when compared with that of T or P used alone (p<0.0001 and p<0.0001, respectively). T-DM1 induced significantly more apoptosis when compared with T+P (p<0.0001). Finally, T-DM1 was significantly more effective in tumor growth inhibition in vivo in EOC xenografts overexpressing HER2/neu when compared to T alone, P alone and T+P (p=0.04). ConclusionIn vitro and in vivo experiments with 3+ HER2/neu expressing EOC revealed limited anti-tumor activity of T or P. T-DM1 showed superior anti-tumor activity to T and P as single agents and as a combination. Our preclinical data support the design of clinical studies with T-DM1 for the treatment of chemotherapy-resistant EOC overexpressing HER2/neu.

The impact of type and number of bowel resections on anastomotic leakage risk in advanced ovarian cancer surgery

ObjectiveTo identify risk factors for anastomotic leakage (AL) in patients undergoing primary advanced ovarian cancer surgery and to evaluate the prognostic implication of AL on overall survival in these patients. MethodsWe analyzed our institutional database for primary EOC and included all consecutive patients treated by debulking surgery including any type of full circumferential bowel resection beyond appendectomy between 1999 and 2015. We performed logistic regression models to identify risk factors for AL and log-rank tests and Cox proportional hazards models to evaluate the association between AL and survival. ResultsAL occurred in 36/800 (4.5%; 95% confidence interval [3%–6%]) of all patients with advanced ovarian cancer and 36/518 (6.9% [5%–9%]) patients undergoing bowel resection during debulking surgery. One hundred fifty-six (30.1%) patients had multiple bowel resections. In these patients, AL rate per patient was only slightly higher (9.0% [5%–13%]) than in patients with rectosigmoid resection only (6.9% [4%–10%]), despite the higher number of anastomosis. No independent predictive factors for AL were identified. AL was independently associated with shortened overall survival (HR 1.9 [1.2–3.4], p=0.01). ConclusionIn the present study, no predictive pre- and/or intraoperative risk factors for AL were identified. AL rate was mainly influenced by rectosigmoid resection and only marginally increased by additional bowel resections.

SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows promising antitumor activity in epithelial ovarian carcinoma with HER2/Neu expression

BackgroundEpithelial ovarian cancer (EOC) is an aggressive and heterogeneous disease. <10% of EOC demonstrate HER2/neu 3+ receptor over-expression. However, moderate to low (i.e., 2+ and 1+) HER2/neu expression is reported in up to 50% of EOC. The objective of this study was to compare the anti-tumor activity of SYD985, a novel HER2-targeting antibody-drug conjugate (ADC), to trastuzumab emtansine (T-DM1) in EOC models with differential HER2/neu expression. MethodsThe cytotoxicity of SYD985 and T-DM1 was evaluated using ten primary EOC cell lines with 0/1+, 2+, and 3+ HER2/neu expression in antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability and bystander killing experiments. Finally, the in vivo activity of SYD985 and T-DM1 was also studied in ovarian cancer xenografts. ResultsSYD985 and T-DM1 induced similar ADCC in the presence of peripheral blood lymphocytes (PBL) against EOC cell lines with differential HER2/neu expression. In contrast, SYD985 was 3 to 42 fold more cytotoxic in the absence of PBL when compared to T-DM1 (p<0.0001). Unlike T-DM1, SYD985 induced efficient bystander killing of HER2/neu 0/1+ tumor cells when admixed with HER2/neu 3+ EOC cells. In vivo studies confirmed that SYD985 is significantly more active than T-DM1 against HER2/neu 3+ EOC xenografts. ConclusionsSYD985 is a novel ADC with remarkable activity against EOC with strong (3+) as well as moderate to low (i.e., 2+ and 1+) HER2/neu expression. SYD985 is more potent than T-DM1 in comparative experiments and unlike T-DM1, it is active against EOC demonstrating moderate/low or heterogeneous HER2/neu expression.

Central nervous system metastatic epithelial ovarian cancer. Clinical parameters and prognostic factors: a multicenter study of Anatolian Society of Medical Oncology.

The authors assessed the clinical parameters and potential prognostic features in patients with CNS metastatic disease. Clinical data of the 33 patients from the participating centers were retrospectively collected and analyzed. Median age at the time of CNS metastasis was 57 years. Median time from the diagnosis of primary EOC until CNS metastatic disease was 22 months. Nearly half (45.5%) of the patients had single CNS metastatic lesions and all patients in the study group except two received radiotherapy as palliative treatment. Median overall survival (OS) from the time of CNS metastasis was 15 months (0-66). At univariate analysis only number of brain metastatic lesions (p = 0.001) and presence of extracranial disease (p = 0.004) were strongly associated with OS whereas multimodal treatment, size of metastatic lesions, platinum sensitivity, age, grade, and disease stage at presentation were not. Development of CNS metastasis carries a poor prognosis, however patients with single metastatic lesions and only intracranial metastatic disease can have prolonged survival after appropriate palliative management of their disease.

Optimizing the treatment of ovarian cancer: Neoadjuvant chemotherapy and interval debulking versus primary debulking surgery for epithelial ovarian cancers likely to have suboptimal resection

ObjectiveTo: a) identify prognostic factors in patients with epithelial ovarian cancer treated with neoadjuvant chemotherapy (NACT) and interval debulking surgery (IDS), and b) compare post-surgical survival between patients treated with NACT/IDS for presumed unresectable disease and stage IIIC/IV patients who underwent suboptimal primary debulking surgery (PDS). MethodsThis was a retrospective study of consecutive stage IIIC or IV patients undergoing IDS after NACT at Mayo Clinic from January 2007 to December 2013. A subset of patients receiving NACT/IDS for the indication of unresectable disease were matched 1:1 on age and stage to a cohort of patients who underwent suboptimal PDS between 2003 and 2011. Hazard ratios and corresponding 95% confidence intervals were estimated from Cox proportional hazards models. ResultsWe identified 87 patients treated with NACT/IDS: the median OS and PFS following surgery was 2.4 and 1.0years, respectively. Factors associated with significantly worse OS were older age (adjusted HR 1.60 per 10-year increase in age, 95% CI 1.18, 2.16) and elevated CA-125 before IDS (adjusted HR 2.30 for CA-125 >35U/mL, 95% CI 1.25, 4.23). Number of adjuvant chemotherapy cycles administered did not have a significant effect on survival. In the matched cohort analysis of presumed unresectable cases undergoing NACT/IDS vs suboptimal PDS cases (n=45 each), the NACT/IDS group had a significant OS advantage (HR 0.53; 95% CI 0.32, 0.88), and fewer patients experienced a 30-day postoperative Accordion grade 3/4 complication (11% vs 36%, P=0.01). ConclusionsYounger age and normalization of CA-125 prior to IDS are associated with improved survival with NACT/IDS. For primary EOC where resection to residual disease of 1cm or less is unlikely, NACT/IDS is associated with improved survival and reduced perioperative morbidity compared to PDS. As these patients are likely best served by NACT/IDS, more reliable predictors of resectability would be valuable.

Metabolic phenotyping for monitoring ovarian cancer patients.

Epithelial ovarian cancer (EOC) is the most deadly of the gynecological cancers. New approaches and better tools for monitoring treatment efficacy and disease progression of EOC are required. In this study, metabolomics using rapid resolution liquid chromatography mass spectrometry was applied to a systematic investigation of metabolic changes in response to advanced EOC, surgery and recurrence. The results revealed considerable metabolic differences between groups. Moreover, 37, 30, and 26 metabolites were identified as potential biomarkers for primary, surgical and recurrent EOC, respectively. Primary EOC was characterized by abnormal lipid metabolism and energy disorders. Oxidative stress and surgical efficacy were clear in the post-operative EOC patients. Recurrent EOC patients showed increased amino acid and lipid metabolism compared with primary EOC patients. After cytoreductive surgery, eight metabolites (e.g. l-kynurenine, retinol, hydroxyphenyllactic acid, 2-octenoic acid) corrected towards levels of the control group, and four (e.g. hydroxyphenyllactic acid, 2-octenoic acid) went back again to primary EOC levels after disease relapse. In conclusion, this study delineated metabolic changes in response to advanced EOC, surgery and recurrence, and identified biomarkers that could facilitate both understanding and monitoring of EOC development and progression.

Abstract A02: CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: A new actionable pathway for ovarian cancer patients.

Abstract Epithelial Ovarian Cancers (EOCs) consist in a heterogeneous group of tumors. Late diagnosis and platinum-resistant recurrences make EOCs the most lethal gynecological malignancy. The central role played by CDKs (Cyclin-Dependent Kinase) in several cellular mechanisms, such as control of cell cycle progression, DNA repair and transcription and the association of their activity in different apoptotic pathways, make them attractive targets to overcome platinum-resistance in EOCs. We explored the possibility that the activity of one or more CDK was necessary to overcome the platinum-induced cell death in EOC cells. To this aim we used an unbiased functional genomic screening targeting, with specific shRNAs, all the 23 members of the CDKs family alone or in association with platinum. This screening identified CDK6 as the CDK most significantly involved in the regulation of platinum sensitivity in EOC cells since CDK6 silencing did not affect cell survival per se but strongly increased platinum-induced cell death. The effect of CDK6 silencing on the sensitivity to both carboplatinum and cisplatin was confirmed in a larger panel of EOC cell lines using multiple shRNAs. Next, the use of CDK6 dominant negative (CDK6D163N) and constitutively active (CDK6R31C) mutants demonstrated that CDK6 kinase activity was necessary to protect from platinum-induced death. Accordingly, an orally active CDK4/CDK6 inhibitor, PD0332991, was able to sensitize EOC cells to CBDCA or CDDP treatment both in vitro and in vivo, in a CDK6-dependent, CDK4-independent manner. To understand the molecular mechanism whereby CDK6 regulates platinum-induced cell death in EOC we focused on specific CDK6 phosphorylation targets and demonstrated that the transcription factor FOXO3a was a relevant downstream target of CDK6. Functional and biochemical analyses showed that upon platinum exposure, CDK6 binds and phosphorylates FOXO3a on a specific Serine residue. We identified the Cyclin D3/CDK6 complex as the one principally involved in FOXO3a phosphorylation and proved that FOXO3a phosphorylation by the Cyclin D3/CDK6 complex leaded to increased FOXO3a protein stability. Accordingly, FOXO3a downregulation sensitizes EOC cells to platinum while FOXO3a overexpression in CDK6 silenced cells was able to rescue the effect of CDK6 silencing on platinum-induced cell death. Mechanistically, we showed that the CDK6/FOXO3a axis was necessary to regulate the DNA damage response in EOC cells through the regulation of ATR/CHK1 pathway since, after platinum treatment, the knock-down of CDK6 or FOXO3a induced the downregulation of ATR and the decrease of CHK1 phosphorylation. Accordingly, we identified in the ATR promoter three putative FOXO3a binding sites and, using chromatin immunoprecipitation, we confirmed that ATR was a FOXO3a transcriptional target, in platinum treated EOC cells. Inhibition of CDK6, FOXO3a or ATR increased platinum dependent-cell death via the induction of the so-called Premature Chromosome Condensation (PCC), a lethal event that selectively kills cancer cells when the ATR/CHK1 pathway is inhibited in the presence of damaged DNA. Overall we demonstrated that in EOC platinum sensitivity could be increased by specific CDK6 inhibition also using a clinically available small molecule, the PD0332991. We are currently analyzing if in primary EOC the expression of CDK6, FOXO3a and/or Cyclin D3 could be used as biomarkers to select those patients that may benefit from a combination therapy based on platinum and CDK6 inhibition, further increasing the translational relevance of our work. Citation Format: Alessandra Dall'Acqua, Schiappacassi Monica, Gustavo Baldassarre. CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: A new actionable pathway for ovarian cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A02.

Abstract B63: CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: a new actionable pathway for ovarian cancer patients

Abstract Epithelial Ovarian Cancers (EOC) consist in a heterogeneous group of tumors. Late diagnosis and platinum-resistant recurrences make EOC the most lethal gynecological malignancies. The central role played by CDKs (Cyclin-Dependent Kinase) in some cellular mechanisms, like control of cell cycle progression, DNA repair and transcription and their activity in apoptotic pathways, make them attractive targets to overcome platinum-resistance in EOC. We explored if the activity of any CDK was necessary to overcome the platinum-induced cell death in EOC cells. We used an unbiased functional genomic screening targeting, with specific shRNAs, all the 23 members of the CDKs family alone or in association with platinum. This screening identified CDK6 as the most significantly involved in the regulation of platinum sensitivity since CDK6 silencing did not affect cell survival per se but strongly increased platinum-induced cell death. The effect of CDK6 silencing on the sensitivity to platinum drugs was confirmed in a larger panel of EOC cell lines. The use of CDK6 dominant negative (CDK6D163N) and constitutively active (CDK6R31C) mutants showed that CDK6 kinase activity was necessary to protect from platinum-induced death. Thus, an orally active CDK4/CDK6 inhibitor, PD0332991, was able to sensitize EOC cells to platinum treatment both in vitro and in vivo, in a CDK6-dependent CDK4-independent manner. To unveil the mechanism whereby CDK6 regulates platinum-induced cell death in EOC we focused on specific CDK6 phosphorylation targets and demonstrated that the transcription factor FOXO3a was a relevant downstream target of CDK6. Functional and biochemical analyses showed that upon platinum exposure, CDK6 binds and phosphorylates FOXO3a on a specific Serine residue. We identified the Cyclin D3/CDK6 complex as the one mainly involved in FOXO3a phosphorylation and proved that FOXO3a phosphorylation by the Cyclin D3/CDK6 complex leaded to increased FOXO3a protein stability. Moreover, FOXO3a downregulation sensitizes EOC cells to platinum while FOXO3a overexpression in CDK6 silenced cells was able to rescue the effect of CDK6 silencing on platinum-induced cell death. We showed that the CDK6/FOXO3a axis was necessary to regulate the DNA damage response in EOC cells through the regulation of ATR/CHK1 pathway, since after platinum treatment the silencing of both CDK6 or FOXO3a induced the downregulation of ATR and the decrease in CHK1 phosphorylation. We identified in the ATR promoter three putative FOXO3a binding sites and, using chromatin immunoprecipitation, we confirmed that ATR is a FOXO3a transcriptional target. Hence, inhibition of CDK6, FOXO3a or ATR increased platinum dependent-cell death via the induction of the so-called Premature Chromosome Condensation (PCC), a lethal event that selectively kills cancer cells when the ATR/CHK1 pathway is inhibited in the presence of damaged DNA. We demonstrated that in EOC, platinum sensitivity could be increased by specific CDK6 inhibition also using a clinically available small molecule, the PD0332991. We are currently analyzing if in primary EOC the expression of CDK6, FOXO3a and/or Cyclin D3 could be used as biomarkers to select patients that may benefit from a combination therapy based on platinum and CDK6 inhibition further increasing the translational relevance of our work. Citation Format: MONICA B. SCHIAPPACASSI, Alessandra Dall'acqua, Gustavo Baldassarre. CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: a new actionable pathway for ovarian cancer patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B63.

Time to diagnosis of Type I or II invasive epithelial ovarian cancers: a multicentre observational study using patient questionnaire and primary care records.

ObjectiveTo compare time to diagnosis of the typically slow‐growing Type I (low‐grade serous, low‐grade endometrioid, mucinous, clear cell) and the more aggressive Type II (high‐grade serous, high‐grade endometrioid, undifferentiated, carcinosarcoma) invasive epithelial ovarian cancer (iEOC).DesignMulticentre observational study.SettingTen UK gynaecological oncology centres.PopulationWomen diagnosed with primary EOC between 2006 and 2008.MethodsSymptom data were collected before diagnosis using patient questionnaire and primary‐care records. We estimated patient interval (first symptom to presentation) using questionnaire data and diagnostic interval (presentation to diagnosis) using primary‐care records. We considered the impact of first symptom, referral and stage on intervals for Type I and Type II iEOC.Main outcome measuresPatient and diagnostic intervals.ResultsIn all, 78% of 60 Type I and 21% of 134 Type II iEOC were early‐stage. Intervals were comparable and independent of stage [e.g. median patient interval for Type I: early‐stage 0.3 months (interquartile range 0.3–3.0) versus late‐stage 0.3 months (interquartile range 0.3–4.5), P = 0.8]. Twenty‐seven percent of women with Type I and Type II had diagnostic intervals of at least 9 months. First symptom (questionnaire) was also similar, except for the infrequent abnormal bleeding (Type I 15% versus Type II 4%, P = 0.01). More women with Type I disease (57% versus 41%, P = 0.04) had been referred for suspected gynaecological cancer. Median time from referral to diagnosis was 1.4 months for women with iEOC referred via a 2‐week cancer referral to any specialty compared with 2.6 months (interquartile range 2.0–3.7) for women who were referred routinely to gynaecology.ConclusionOverall, shorter diagnostic delays were seen when a cancer was suspected, even if the primary tumour site was not recognised to be ovarian. Despite differences in carcinogenesis and stage for Type I and Type II iEOC, time to diagnosis and symptoms were similar. Referral patterns were different, implying subtle symptom differences. If symptom‐based interventions are to impact on ovarian cancer survival, it is likely to be through reduced volume rather than stage‐shift. Further research on histological subtypes is needed.Tweetable abstractNo difference in time to diagnosis for Type I versus Type II invasive epithelial ovarian cancers.

MiR-211 suppresses epithelial ovarian cancer proliferation and cell-cycle progression by targeting Cyclin D1 and CDK6.

BackgroundEpithelial ovarian cancer (EOC) is a significant cause of morbidity and mortality. MicroRNAs play important roles in cancer development and progression. The microRNA miR-211 is localized on intron 6 of the Trpm1 gene at 15q13-q14, a locus that is frequently lost in neoplasms. Its function and loss-of-function have been described in normal and cancer cells and tissues. miR-211 is known to be dysregulated in ovarian cancer: however, its function and the downstream effect of its loss-of-function in ovarian cancer have not been described before.MethodsWe analyzed miR-211 expression in clinical samples of primary EOC tissues compared to normal epithelial ovarian tissues and in the EOC cell lines: OVCAR3, Caov3, OVCA429, SKOV3 and A2780 compared to human ovarian surface epithelial cells. We then investigated the effect of miR-211 on EOC cell proliferation and apoptosis by counting cell numbers, MTT, colony formation, cell cycle, and PI/Annexin V staining assays. A luciferase reporter system was developed to assess miR-211 regulation of the predicted targets. Expression level of discovered targets and correlation with miR-211 expression were analyzed in EOC tissues. Finally, OVCAR3 stably expressing miR-211 or control cells were injected subcutaneously into mice to determine in vivo effect of miR-211 on tumorigenesis.ResultsWe found that the expression of miR-211 is downregulated in EOC tissues and cell lines compared to normal epithelial ovarian tissue and human ovarian surface epithelial cells, respectively. miR-211 was found to arrest cells in the G0/G1-phase, inhibit proliferation and induce apoptosis. Cyclin D1 and CDK6 were found to be direct targets of miR-211, and when overexpressed in miR-211-expressing EOC cells, could restore proliferative ability. Finally, in vitro investigation confirmed that miR-211 is a tumor suppressor that controls Cyclin D1 and CDK6 expression.ConclusionsOur results demonstrate that miR-211 is a tumor suppressor that controls expression of Cyclin D1 and CDK6, and that its downregulation results in overexpression of Cyclin D1 and CDK6 which increases proliferation ability of EOC cells to proliferate compared to normal cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0322-4) contains supplementary material, which is available to authorized users.

Stathmin regulates mutant p53 stability and transcriptional activity in ovarian cancer

Stathmin is a p53-target gene, frequently overexpressed in late stages of human cancer progression. Type II High Grade Epithelial Ovarian Carcinomas (HG-EOC) represents the only clear exception to this observation. Here, we show that stathmin expression is necessary for the survival of HG-EOC cells carrying a p53 mutant (p53MUT) gene. At molecular level, stathmin favours the binding and the phosphorylation of p53MUT by DNA-PKCS, eventually modulating p53MUT stability and transcriptional activity. Inhibition of stathmin or DNA-PKCS impaired p53MUT–dependent transcription of several M phase regulators, resulting in M phase failure and EOC cell death, both in vitro and in vivo. In primary human EOC a strong correlation exists between stathmin, DNA-PKCS, p53MUT overexpression and its transcriptional targets, further strengthening the relevance of the new pathway here described. Overall our data support the hypothesis that the expression of stathmin and p53 could be useful for the identification of high risk patients that will benefit from a therapy specifically acting on mitotic cancer cells.