Preclinical 3D model screening reveals digoxin as an effective therapy for a rare and aggressive type of endometrial cancer.

OBJECTIVE: Dedifferentiated endometrial carcinoma (DDEC) characterized by SWItch/Sucrose Non-Fermentable (SWI/SNF) complex inactivation is a highly aggressive type of endometrial cancer without effective systemic therapy options. Its uncommon nature and aggressive disease trajectory pose significant challenges for therapeutic progress. To address this obstacle, we focused on developing preclinical models tailored to this tumor type and established patient tumor-derived three-dimensional (3D) spheroid models of DDEC. METHODS: High-throughput drug repurposing screens were performed on in vitro 3D spheroid models of DDEC cell lines (SMARCA4-inactivated DDEC-1 and ARID1A/ARID1B co-inactivated DDEC-2). The dose-response relationships of the identified candidate drugs were evaluated in vitro, followed by in vivo evaluation using xenograft models of DDEC-1 and DDEC-2. RESULTS: Drug screen in 3D models identified multiple cardiac glycosides including digoxin and digitoxin as candidate drugs in both DDEC-1 and DDEC-2. Subsequent in vitro dose-response analyses confirmed the inhibitory activity of digoxin and digitoxin with both drugs showing lower IC50 in DDEC cells compared to non-DDEC endometrial cancer cells. In in vivo xenograft models, digoxin significantly suppressed the growth of DDEC tumors at clinically relevant serum concentrations. CONCLUSION: Using biologically precise preclinical models of DDEC derived from patient tumor samples, our study identified digoxin as an effective drug in suppressing DDEC tumor growth. These findings provide compelling preclinical evidence for the use of digoxin as systemic therapy for SWI/SNF-inactivated DDEC, which may also be applicable to other SWI/SNF-inactivated tumor types.

High throughput screening identifies dasatinib as synergistic with trametinib in low grade serous ovarian carcinoma.

BACKGROUND: Low grade serous ovarian carcinoma (LGSOC) is a distinct histotype of ovarian cancer characterised high levels of intrinsic chemoresistance, highlighting the urgent need for new treatments. High throughput screening in clinically-informative cell-based models represents an attractive strategy for identifying candidate treatment options for prioritisation in clinical studies. METHODS: We performed a high throughput drug screen of 1610 agents across a panel of 6 LGSOC cell lines (3 RAS/RAF-mutant, 3 RAS/RAF-wildtype) to identify novel candidate therapeutic approaches. Validation comprised dose-response analysis across 9 LGSOC models and 5 high grade serous comparator lines. RESULTS: 16 hits of 1610 screened compounds were prioritised for validation based on >50% reduction in nuclei counts in over half of screened cell lines at 1000 nM concentration. 11 compounds passed validation, and the four agents of greatest interest (dasatinib, tyrosine kinase inhibitor; disulfiram, aldehyde dehydrogenase inhibitor; carfilzomib, proteasome inhibitor; romidepsin, histone deacetylase inhibitor) underwent synergy profiling with the recently approved MEK inhibitor trametinib. Disulfiram demonstrated excellent selectivity for LGSOC versus high grade serous ovarian carcinoma comparator lines (P = 0.003 for IC50 comparison), while the tyrosine kinase inhibitor dasatinib demonstrated favourable synergy with trametinib across multiple LGSOC models (maximum zero interaction potency synergy score 46.9). The novel, highly selective Src family kinase (SFK) inhibitor NXP900 demonstrated a similar trametinib synergy profile to dasatinib, suggesting that SFK inhibition is the likely driver of synergy. CONCLUSION: Dasatinib and other SFK inhibitors represent novel candidate treatments for LGSOC and demonstrate synergy with trametinib. Disulfiram represents an additional treatment strategy worthy of investigation.

Molecular Surrogate Subtypes of Ovarian and Peritoneal Low-grade Serous Carcinoma.

Low-grade serous carcinoma (LGSC) is an uncommon histotype of ovarian carcinoma, accounting for ~3% of cases. There is evidence that survival of peritoneal LGSC (pLGSC) is longer than that of ovarian LGSC (oLGSC). Key molecular alterations of LGSC have been established, including loss of CDKN2A and PR expression, MAPK pathway alterations, and loss of USP9X expression. We hypothesized that LGSC could be subclassified into clinically applicable molecular subtypes by a few surrogate tests similar to endometrioid carcinomas using a hierarchical decision tree based on the strength of the prognostic associations of the individual alterations. Our study included 71 LGSCs. Immunohistochemistry for CDKN2A, ER, PR, NF1, and USP9X and sequencing for KRAS, NRAS, and BRAF were performed. Our data showed the co-occurrence of key molecular alterations, and despite suggestive trends, hierarchical molecular subtyping did not provide significantly different stratification of patients according to survival in this cohort. We confirmed that patients diagnosed with pLGSC have a longer survival than high-stage oLGSC, with the intriguing observation that normal CDKN2A and PR status were associated with excellent survival in pLGSC. Therefore, CDKN2A and PR status might aid in the classification of indeterminate implants, where abnormal findings favor pLGSC over noninvasive implants. Molecular subtypes should be further evaluated in larger cohorts for their prognostic and potentially predictive value.

The spectrum of MAPK-ERK pathway genomic alterations in gynecologic malignancies: Opportunities for novel therapeutic approaches.

OBJECTIVE: To investigate the incidence of MAPK/ERK pathway genomic alterations among patients with gynecologic malignancies. METHODS: We accessed the American Association of Cancer Research Genomics Evidence of Neoplasia Information Exchange publicly available dataset (v13.0). Patients with malignant tumors of the ovary, uterus, and cervix were identified. Following stratification by tumor site and histology, we examined the prevalence of MAPK/ERK pathway gene alterations (somatic mutation, and/or structural chromosome alterations). We included the following RAS-MAPK pathway genes known to be implicated in the dysregulation of the pathway; KRAS, NRAS, BRAF, HRAS, MAP2K1, RAF1, PTPN11, NF1, and ARAF. Data from the OncoKB database, as provided by cBioPortal, were utilized to determine pathogenic gene alterations. RESULTS: We identified a total of 10,233 patients with gynecologic malignancies; 48.2% (n = 4937) with ovarian, 45.2% (n = 4621) with uterine and 6.6% (n = 675) with cervical cancer respectively. The overall incidence of MAPK pathway gene alterations was 21%; the most commonly altered gene was KRAS (13%), followed by NF1 (7%), NRAS (1.3%), and BRAF (1.2%). The highest incidence was observed among patients with mucinous ovarian (71%), low-grade serous ovarian (48%), endometrioid ovarian (37%), and endometrioid endometrial carcinoma (34%). CONCLUSIONS: Approximately 1 in 5 patients with a gynecologic tumor harbor a MAPK/ERK pathway genomic alteration. Novel treatment strategies capitalizing on these alterations are warranted.

Survey of NF1 inactivation by surrogate immunohistochemistry in ovarian carcinomas.

OBJECTIVE: Inhibition of the MAPK pathway by MEK inhibitors (MEKi) is currently a therapeutic standard in several cancer types, including ovarian low-grade serous carcinoma (LGSC). A common MAPK pathway alteration in tubo-ovarian high-grade serous carcinoma (HGSC) is the genomic inactivation of neurofibromin 1 (NF1). The primary objectives of our study were to survey the prevalence of NF1 inactivation in the principal ovarian carcinoma histotype as well as to evaluate its associations with clinico-pathological parameters and key biomarkers including BRCA1/2 status in HGSC. METHODS: A recently commercialized NF1 antibody (clone NFC) was orthogonally validated on an automated immunohistochemistry (IHC) platform and IHC was performed on tissue microarrays containing 2140 ovarian carcinoma cases. Expression was interpreted as loss/inactivated (complete or subclonal) versus normal/retained. RESULTS: Loss of NF1 expression was detected in 250/1429 (17.4%) HGSC including 11% with subclonal loss. Survival of NF1-inactivated HGSC patients was intermediate between favorable BRCA1/2 mutated HGSC and unfavorable CCNE1 high-level amplified HGSC. NF1 inactivation was mutually exclusive with CCNE1 high-level amplifications, co-occurred with RB1 loss and occurred at similar frequencies in BRCA1/2 mutated versus wild-type HGSC. NF1 loss was found in 21/286 (7.3%) endometrioid carcinomas with a favorable prognostic association (p = 0.048), and in 4/64 (5.9%) LGSC, mutually exclusive with other driver events. CONCLUSIONS: NF1 inactivation occurs in a significant subset of BRCA1/2 wild-type HGSC and a subset of LGSC. While the functional effects of NF1 inactivation need to be further characterized, this signifies a potential therapeutic opportunity to explore targeting NF1 inactivation in these tumors.

Establishment and validation of preclinical models of SMARCA4-inactivated and ARID1A/ARID1B co-inactivated dedifferentiated endometrial carcinoma.

OBJECTIVE: Dedifferentiated endometrial cancer (DDEC) is an uncommon and clinically highly aggressive subtype of endometrial cancer characterized by genomic inactivation of SWItch/Sucrose Non-Fermentable (SWI/SNF) complex protein. It responds poorly to conventional systemic treatment and its rapidly progressive clinical course limits the therapeutic windows to trial additional lines of therapies. This underscores a pressing need for biologically accurate preclinical tumor models to accelerate therapeutic development. METHODS: DDEC tumor from surgical samples were implanted into immunocompromised mice for patient-derived xenograft (PDX) and cell line development. The histologic, immunophenotypic, genetic and epigenetic features of the patient tumors and the established PDX models were characterized. The SMARCA4-deficienct DDEC model was evaluated for its sensitivity toward a KDM6A/B inhibitor (GSK-J4) that was previously reported to be effective therapy for other SMARCA4-deficient cancer types. RESULTS: All three DDEC models exhibited rapid growth in vitro and in vivo, with two PDX models showing spontaneous development of metastases in vivo. The PDX tumors maintained the same undifferentiated histology and immunophenotype, and exhibited identical genomic and methylation profiles as seen in the respective parental tumors, including a mismatch repair (MMR)-deficient DDEC with genomic inactivation of SMARCA4, and two MMR-deficient DDECs with genomic inactivation of both ARID1A and ARID1B. Although the SMARCA4-deficient cell line showed low micromolecular sensitivity to GSK-J4, no significant tumor growth inhibition was observed in the corresponding PDX model. CONCLUSIONS: These established patient tumor-derived models accurately depict DDEC and represent valuable preclinical tools to gain therapeutic insights into this aggressive tumor type.

Low-grade serous ovarian cancer: expert consensus report on the state of the science.

Compared with high-grade serous carcinoma, low-grade serous carcinoma of the ovary or peritoneum is a less frequent epithelial ovarian cancer type that is poorly sensitive to chemotherapy and affects younger women, many of whom endure years of ineffective treatments and poor quality of life. The pathogenesis of this disease and its management remain incompletely understood. However, recent advances in the molecular characterization of the disease and identification of novel targeted therapies with activity in low-grade serous carcinoma offer the promise of improved outcomes. To update clinicians regarding recent scientific and clinical trial advancements and discuss unanswered questions related to low-grade serous carcinoma diagnosis and treatment, a panel of experts convened for a workshop in October 2022 to develop a consensus document addressing pathology, translational research, epidemiology and risk, clinical management, and ongoing research. In addition, the patient perspective was discussed. The recommendations developed by this expert panel-presented in this consensus document-will guide practitioners in all settings regarding the clinical management of women with low-grade serous carcinoma and discuss future opportunities to improve research and patient care.

BRCA mutations lead to XIAP overexpression and sensitise ovarian cancer to inhibitor of apoptosis (IAP) family inhibitors.

BACKGROUND: We tested the hypothesis that inhibitor of apoptosis family (IAP) proteins may be altered in BRCA1-mutated ovarian cancers and that could affect the sensitivity to IAP inhibitors. METHODS: The levels of IAP proteins were evaluated in human cancers and cell lines. Cell lines were used to determine the effects of IAP inhibitors. The in vivo effects of treatments were evaluated in PDX mouse models. RESULTS: Expression of X-linked inhibitor of apoptosis (XIAP) is increased in BRCA1-mutated cancers and high levels are associated with improved patient outcomes after platinum chemotherapy. XIAP overexpression is mediated by NF-kB activation and is associated with an optimisation of PARP. BRCA1-mutated cell lines are particularly sensitive to IAP inhibitors due to an inhibitory effect on PARP. Both a BRCA1-mutated cell line with acquired resistance to PARP inhibitors and one with restored BRCA1 remain sensitive to IAP inhibitors. Treatment with IAP inhibitors restores the efficacy of PARP inhibition in these cell lines. The IAP inhibitor LCL161 alone and in combination with a PARP inhibitor, exhibited antitumour effects in PDX mouse models of resistant BRCA2 and 1-mutated ovarian cancer, respectively. CONCLUSION: A clinical trial may be justified to further investigate the utility of IAP inhibitors.

Genomic analysis of low-grade serous ovarian carcinoma to identify key drivers and therapeutic vulnerabilities.

Low-grade serous ovarian carcinoma (LGSOC) is associated with a poor response to existing chemotherapy, highlighting the need to perform comprehensive genomic analysis and identify new therapeutic vulnerabilities. The data presented here represent the largest genetic study of LGSOCs to date (n = 71), analysing 127 candidate genes derived from whole exome sequencing cohorts to generate mutation and copy-number variation data. Additionally, immunohistochemistry was performed on our LGSOC cohort assessing oestrogen receptor, progesterone receptor, TP53, and CDKN2A status. Targeted sequencing identified 47% of cases with mutations in key RAS/RAF pathway genes (KRAS, BRAF, and NRAS), as well as mutations in putative novel driver genes including USP9X (27%), MACF1 (11%), ARID1A (9%), NF2 (4%), DOT1L (6%), and ASH1L (4%). Immunohistochemistry evaluation revealed frequent oestrogen/progesterone receptor positivity (85%), along with CDKN2A protein loss (10%) and CDKN2A protein overexpression (6%), which were linked to shorter disease outcomes. Indeed, 90% of LGSOC samples harboured at least one potentially actionable alteration, which in 19/71 (27%) cases were predictive of clinical benefit from a standard treatment, either in another cancer's indication or in LGSOC specifically. In addition, we validated ubiquitin-specific protease 9X (USP9X), which is a chromosome X-linked substrate-specific deubiquitinase and tumour suppressor, as a relevant therapeutic target for LGSOC. Our comprehensive genomic study highlighted that there is an addiction to a limited number of unique 'driver' aberrations that could be translated into improved therapeutic paths. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hormone receptor expression and outcomes in low-grade serous ovarian carcinoma.

OBJECTIVE: Low-grade serous ovarian carcinomas (LGSC) are frequently ER/PR positive, though the mechanisms by which ER/PR regulate prognosis or anti-estrogen treatment efficacy are poorly understood. We studied ER/PR expression in LGSC tumors and cell lines to evaluate patient outcomes and cellular treatment responses. METHODS: LGSC tumors and patient-derived cell lines were studied from patients with advanced-stage (III/IV) disease. Tumor samples and clinical data were obtained from the Canadian Ovarian Experimental Unified Resource (COEUR-tissue microarray) and the Ovarian Cancer Research (OvCaRe) tissue bank. ER/PR expression was assessed by both Western blot and immunohistochemistry (IHC). Two different IHC scoring systems (simple and Allred) were used. Cox regression was used to identify factors (age, disease residuum, ER/PR status, etc.) associated with progression-free (PFS) and overall survival (OS). Estradiol and tamoxifen proliferation and viability experiments were performed in LGSC cell lines. RESULTS: In 55 LGSC cases studied, median follow-up was 56 months (range 1-227). Fifty-three (96%) cases strongly expressed ER whereas 37 (67%) expressed PR. Cox-regression analysis showed that residuum (p < 0.001) was significantly associated with PFS, whereas both ER Allred score (p = 0.005) and residuum (p = 0.004) were significant for OS. None of the LGSC cell lines expressed PR. Loss of PR and ER expression over time was detected in LGSC tumors and cell lines respectively. Estrogen and tamoxifen treatment did not alter LGSC cell proliferation or viability in-vitro. CONCLUSIONS: In patients with advanced LGSC, higher ER Allred scores were significantly associated with better overall survival. ER/PR expression changed over time in both LGSC tumors and cell lines. Better translational research models are needed to elucidate the molecular mechanisms of ER/PR signalling in LGSC.

Low-grade serous carcinoma (LGSC): A Canadian multicenter review of practice patterns and patient outcomes.

OBJECTIVE: Patients with advanced low-grade serous carcinoma (LGSC) have poor long-term survival rates. As a rare histotype, there are uncertainties regarding the use of current therapies. Thus, we studied practice patterns and treatment outcomes as part of a national initiative to better understand and improve the care of women with advanced LGSC. METHODS: This retrospective cohort study was conducted in 5 Canadian referral institutions from 2000 to 2016. Data collection and pathology reporting were standardized. Outcome measures included overall survival (OS), progression-free survival (PFS), progression-free intervals (PFI), and time to next treatment (TTNT). Cox regression analysis was used to evaluate the effects of clinical and pathologic factors on outcomes and prognosis. RESULTS: There were 134 patients (stage II-IV) with a median follow-up of 32.4 months (range 1.6-228). Four primary treatments were compared across institutions: 1) surgery followed by chemotherapy (56%), 2) neoadjuvant chemotherapy (NACT) followed by surgery (27%), 3) surgery alone (9%), and 4) surgery followed by anti-hormone therapy (4%). Primary platinum/paclitaxel chemotherapy was used in 81%. Patients treated with NACT had worse PFS. Multivariable Cox regression analysis identified lesser residual disease, younger age, and primary peritoneal origin as variables significantly associated with better OS/PFS (p < 0.03). One institution had significantly better PFS than the others (p = 0.025), but this finding could be related to a higher frequency of primary peritoneal LGSC. PFI and TTNT intervals in patients with relapsed disease were not significantly different after the first relapse irrespective of treatment type. CONCLUSIONS: There are notable differences in practice patterns across Canada. This underscores the need for ongoing strategies to measure, evaluate and achieve optimal patient outcomes for women with advanced LGSC.

Low-grade serous ovarian cancer: State of the science.

In January 2019, a group of basic, translational, and clinical investigators and patient advocates assembled in Miami, Florida, to discuss the current state of the science of low-grade serous carcinoma of the ovary or peritoneum-a rare ovarian cancer subtype that may arise de novo or following a diagnosis of serous borderline tumor. The purpose of the conference was to review current knowledge, discuss ongoing research by established researchers, and frame critical questions or issues for future directions. Following presentations and discussions, the primary objective was to initiate future collaborations, uniform database platforms, laboratory studies, and clinical trials to better understand this disease and to advance clinical care outside the boundaries of single academic institutions. This review summarizes the state of the science in five principal categories: epidemiology and patient outcomes, pathology, translational research, patient care and clinical trials, and patients' perspective.

Mismatch repair deficiency and prognostic significance in patients with low-risk endometrioid endometrial cancers.

OBJECTIVES: Mismatch repair deficiency is observed in 25%-30% of all endometrial cancers. This can be detected by the absence of mismatch repair protein staining on immunohistochemistry, and is used as a screen for Lynch syndrome. Only 10% of women with mismatch repair deficiency have Lynch syndrome, but mismatch repair deficiency may still have prognostic significance. The objective of this study was to compare clinical outcomes between mismatch repair-deficient and mismatch repair-proficient low-risk endometrioid endometrial cancers (stage IA, grade 1 or 2). METHODS: This was a retrospective population-based cohort study of all low-risk endometrioid endometrial cancers (stage IA, grade 1 or 2) from the Vancouver Coastal Health Authority region from February 2011 to January 2016 that were assessed for mismatch repair deficiency. Any other histology, stage, or grade was excluded from the study. Primary outcome measures were progression-free survival and overall survival calculated using Kaplan-Meier method and log-rank tests. Cox proportional hazards model estimated the association between mismatch repair deficiency and recurrence and death after adjustment for covariates, expressed as hazard ratios (HRs). Secondary outcome measures were recurrence rates expressed per 100 person-years (p100py). RESULTS: There were 475 patients diagnosed with low-risk endometrioid endometrial cancer, including 131 with mismatch repair-deficient (27.6%) and 344 with mismatch repair-proficient (72.4%) tumors. Women with mismatch repair-deficient tumors had worse progression-free survival (24 months; p=0.0082) and higher recurrence rates (3.56 p100py) compared with those with mismatch repair-proficient tumors (27 months; 1.21 p100py, p=0.04). The absolute number of recurrences was overall low. There were 11 recurrences out of 131 mismatch repair-deficient cases (8.4%) and 14 out of 344 mismatch repair proficient cases (4.1%). After adjustment for age, lymphovascular space invasion status, adjuvant therapy, and post-operative grade, mismatch repair-deficient status remained associated with a higher risk of recurrence (HR 3.56, 95% CI 2.01 to 5.95). There was no significant difference in overall survival between mismatch repair groups (mismatch repair-proficient group 27.5 months vs 25.0 months in the deficient group) (HR 1.23, 95% CI 0.49 to 3.10). CONCLUSION: In low-risk stage IA grade 1 or 2 endometrioid endometrial cancers, mismatch repair deficiency is associated with a higher recurrence rate than mismatch repair proficiency after adjustment for covariates, implying that mismatch repair deficiency reflects a different biology in endometrial cancer.

Markers of MEK inhibitor resistance in low-grade serous ovarian cancer: EGFR is a potential therapeutic target.

BACKGROUND: Although low-grade serous ovarian cancer (LGSC) is rare, case-fatality rates are high as most patients present with advanced disease and current cytotoxic therapies are not overly effective. Recognizing that these cancers may be driven by MAPK pathway activation, MEK inhibitors (MEKi) are being tested in clinical trials. LGSC respond to MEKi only in a subgroup of patients, so predictive biomarkers and better therapies will be needed. METHODS: We evaluated a number of patient-derived LGSC cell lines, previously classified according to their MEKi sensitivity. Two cell lines were genomically compared against their matching tumors samples. MEKi-sensitive and MEKi-resistant lines were compared using whole exome sequencing and reverse phase protein array. Two treatment combinations targeting MEKi resistance markers were also evaluated using cell proliferation, cell viability, cell signaling, and drug synergism assays. RESULTS: Low-grade serous ovarian cancer cell lines recapitulated the genomic aberrations from their matching tumor samples. We identified three potential predictive biomarkers that distinguish MEKi sensitive and resistant lines: KRAS mutation status, and EGFR and PKC-alpha protein expression. The biomarkers were validated in three newly developed LGSC cell lines. Sub-lethal combination of MEK and EGFR inhibition showed drug synergy and caused complete cell death in two of four MEKi-resistant cell lines tested. CONCLUSIONS: KRAS mutations and the protein expression of EGFR and PKC-alpha should be evaluated as predictive biomarkers in patients with LGSC treated with MEKi. Combination therapy using a MEKi with EGFR inhibition may represent a promising new therapy for patients with MEKi-resistant LGSC.

BRCA mutation testing for first-degree relatives of women with high-grade serous ovarian cancer.

BACKGROUND: Women with high-grade serous ovarian cancer (HGSC) have a 20% chance of carrying a BRCA1 or 2 mutation. Not all undergo genetic testing, and there is a large legacy group of untested patients. Their female first-degree relatives (FDR) may not qualify for testing unless they have specific ethnicity, or personal/family cancer history. We conducted a cost-effectiveness analysis to evaluate risk-reducing strategies for these FDR who are ineligible for testing. METHODS: A Markov Monte Carlo simulation model estimated the costs and benefits of 3 strategies for female FDR of HGSC patients whose BRCA status is unknown: (1) no BRCA testing; (2) universal BRCA testing, followed by risk-reducing bilateral salpingo-oophorectomy (RRBSO) for mutation carriers; (3) universal RRBSO, without BRCA testing. Effectiveness was estimated in quality-adjusted life year (QALY) gains over a 50-year time horizon. Sensitivity analyses accounted for uncertainty around various parameters. RESULTS: Universal BRCA testing for female FDR of women with HGSC yielded a higher average QALY gain at acceptable cost compared to no BRCA testing, with an incremental cost-effectiveness ratio of $7888 per QALY. Universal BRCA testing was more effective and less costly than universal RRBSO (19.20 QALYs vs. 18.52 QALYs, and $10,135 vs. $14,231, respectively). Results were stable over wide ranges of plausible costs and estimates. Compliance with hormone replacement therapy had to exceed 79.3% for universal RRBSO to be the most effective strategy. CONCLUSION: BRCA mutation testing should be offered to all female first-degree relatives of women with high-grade serous ovarian cancer when BRCA mutation status is unknown.

Synergistic effect of MEK inhibitor and metformin combination in low grade serous ovarian cancer.

OBJECTIVE: Low-grade serous ovarian cancer (LGSOC) constitutes 5-8% of epithelial ovarian cancers and is refractory to chemotherapy. We and others have shown metformin to cause significant growth inhibition in high-grade ovarian cancer both in vitro and in vivo. Here, we aimed to analyze if metformin was effective in inhibiting proliferation of LGSOC alone and in combination with MEK inhibitor. METHODS: Three LGSOC lines (VOA1056, VOA1312 and VOA5646) were treated with metformin, trametinib or 2-deoxyglucose (2DG) alone or in combination with metformin. Proliferation was measured by MTT assay over a period of four days. Protein expression was measured by western blotting. Seahorse Analyzer was used to measure effect of metformin on glycolysis and mitochondrial respiration. RESULTS: All LGSOC cell lines showed significant inhibition with metformin in a dose- and time-dependent manner. Trametinib significantly inhibited the growth of Ras mutated LGSOC lines (VOA1312 and VOA1056), while VOA5646 cells without RAS mutation did not show any response. Metformin and trametinib combination showed synergistic inhibition of RAS mutated VOA1312 and VOA1056 cells, but not for non-Ras mutated VOA5646 cells. Metformin and trametinib increased phosphorylated AMPK expression in LGSOC lines with combination showing stronger expression. Trametinib decreased 42/44 mitogen activated kinase phosphorylation in all cell lines, while metformin and combination had no significant effect. 2-DG significantly inhibited glycolysis in all LGSOC lines and combination with metformin showed synergistic inhibitory effect. CONCLUSIONS: Metformin alone or in combination with MEK and glycolytic inhibitors may be a potential therapy for LGSOC, a cancer that is indolent but chemo-resistant.

Costs and Benefits of Extended Endocrine Strategies for Premenopausal Breast Cancer.

Background: After completing 5 years of adjuvant tamoxifen, women with estrogen receptor (ER)-positive breast cancer benefit from 5 more years of endocrine therapy, either with tamoxifen or an aromatase inhibitor (AI). For premenopausal women, ovarian ablation (OA) would be required before starting an AI (OA/AI). According to the SOFT/TEXT studies, OA/AI improves 5-year disease-free survival compared with tamoxifen alone, suggesting that OA/AI could be superior to tamoxifen as extended endocrine therapy. The long-term costs and consequences of premature menopause from OA are unknown, but could be estimated through a cost-effectiveness analysis. Methods: A Markov chain Monte Carlo simulation model estimated the costs and benefits of 3 extended endocrine strategies in a hypothetical cohort of premenopausal women with ER-positive early breast cancer: (1) no further treatment; (2) tamoxifen for 5 years (extended tamoxifen); or (3) OA/AI for 5 years. Effectiveness was measured in years of life expectancy gain. Sensitivity analyses accounted for uncertainty surrounding various parameters. Monte Carlo simulation estimated the number of adverse events and deaths from each strategy in the US population over a 40-year period. Results: Extended tamoxifen yielded a higher average life expectancy gain than OA/AI (17.31 vs 17.06 years) at lower average cost ($3,550 vs $14,312). For 18,000 premenopausal ER-positive women, the simulation estimated 13,236, 12,557, and 11,338 deaths with no further treatment, extended tamoxifen, and OA/AI, respectively, but an additional 1,897 deaths from OA, for a total of 13,235 deaths associated with OA/AI. After 24.6 years of follow-up, more women are expected to die from OA/AI than extended tamoxifen. Conclusions: For premenopausal women with ER-positive cancer who have completed adjuvant tamoxifen, another 5 years of tamoxifen is the preferable extended endocrine strategy. The potential long-term health consequences of OA could affect overall survival when it precedes the use of an AI.

Long-term consequences of ovarian ablation for premenopausal breast cancer.

The TEXT and SOFT trials concluded that an aromatase inhibitor (AI) with ovarian ablation (OA) yields a higher 5-year disease-free survival than tamoxifen alone in premenopausal ER+ high-risk early breast cancer. However, the long-term health consequences and costs of OA, either by GnRH agonist or oophorectomy, have not been evaluated. The objective was to conduct a cost-effectiveness analysis comparing tamoxifen to OA with AI. Markov Monte Carlo simulation model estimated the costs and benefits of 3 endocrine strategies: (1) tamoxifen; (2) GnRH agonist with AI (GnRHa-AI); (3) bilateral salpingo-oophorectomy with AI (BSO-AI). Effectiveness was measured in life expectancy gain (years), and costs were averaged over a lifetime (USD 2015). Adverse events and deaths from each strategy were modeled in the United States population over a time horizon of 40 years. For women without prior chemotherapy (low-risk), tamoxifen alone was more effective (18.03 years) and less costly ($1566) than GnRHa-AI (17.66 years, $93,692) or BSO-AI (17.63 years, $25,892). For those with prior chemotherapy (high-risk), BSO-AI was more costly but more effective (16.78 years, $25,368) than tamoxifen alone (16.55 years, $1523) with an ICER of $102,290, while GnRHa-AI yielded an ICER of $443,376. The simulation estimated 787 and 577 deaths attributable to OA among 9320 high-risk women after BSO-AI and GnRHa-AI, respectively. There may be a role for ovarian ablation in premenopausal women with ER+ high-risk early breast cancer; however, this analysis raises concerns about the long-term health consequences of ovarian ablation and the potential effects on overall survival.

BAF250a Expression in Atypical Endometriosis and Endometriosis-Associated Ovarian Cancer.

BACKGROUND AND OBJECTIVE: Atypical endometriosis (AE) is thought to be a precursor lesion to the ovarian cancer subtypes associated with endometriosis, namely, endometrioid and clear cell carcinomas. ARID1A encodes a nuclear protein (BAF250a) governing chromatin remodeling, and mutations in ARID1A have been found in 30% to 50% of clear cell and endometrioid ovarian cancers. As BAF250a expression loss by immunohistochemistry (IHC) has been documented in the endometriosis precursor lesions closely associated with these ovarian cancers subtypes, our goal was to further study the association between BAF250a expression in cases of AE with and without an associated cancer. METHODS: Three separate databases were screened for suspected cases of AE. Based on a detailed review of the pathology reports, we selected cases likely to contain AE for slide review. After slide review, tissue blocks were recalled to perform IHC for BAF250a in the associated cancer, AE, or typical endometriosis when present. RESULTS: There were 35 cases of endometriosis-associated cancer and 8 cases of AE not associated with cancer. Atypical endometriosis was found on pathology review in 23 endometriosis-associated cancer cases (66%). In the 35 cancer cases, BAF250a IHC showed loss of expression in 14 cases. Atypical endometriosis was present in 10 of these cases, 6 of which showed BAF250a loss (60%). BAF250a loss was not observed in the 8 cases of AE not associated with cancer or in the contiguous AE of 13 cases, whereby BAF250a expression was retained in the associated cancer. CONCLUSIONS: BAF250a loss in AE is consistently associated with the development of BAF250a-negative endometriosis-associated cancers and appears to be an early event in most of these cases. This research provides additional evidence that in the absence of cancer, BAF250a expression should be evaluated as a biomarker of cancer risk in patients diagnosed with AE.