OBJECTIVE:To evaluate whether concomitant sodium-glucose cotransporter 2 inhibitor (SGLT2i) and progestin therapy is associated with the risk of incident endometrial cancer (EC) and subsequent hysterectomy among patients with endometrial hyperplasia (EH) or benign uterine pathology. METHODS:We conducted a retrospective cohort study using the TriNetX real-world clinical database. Patients with EH, abnormal uterine bleeding, or other benign uterine pathology who received progestin therapy were identified and grouped by concomitant SGLT2i use. Propensity score matching (PSM) was performed to balance baseline characteristics between the SGLT2i + progestin and progestin-only groups. Primary outcomes were incident EC, subsequent hysterectomy, and a composite outcome of EC or hysterectomy. Subgroup analyses were performed by route of progestin administration, uterine pathology, age, and body mass index. Sensitivity analyses were conducted to address potential residual confounding. RESULTS:A total of 486,837 eligible patients were included, including 7605 (1.6%) in the SGLT2i + P group and 479,232 (98.4%) in the P-only group. After 1:1 PSM, 7034 patients remained in each group. During 2-years of follow-up, EC occurred in 29 patients (0.4%) in the SGLT2i + P group and 65 (0.9%) in the P-only group. Compared with progestin alone, SGLT2i + progestin was associated with lower risks of incident EC (HR, 0.43; 95% CI, 0.28-0.67) and subsequent hysterectomy (HR, 0.51; 95% CI, 0.43-0.60). Findings were generally consistent across subgroups and sensitivity analyses. CONCLUSIONS:Concomitant SGLT2i use was associated with lower risks of EC and hysterectomy among patients receiving progestin therapy for non-malignant uterine pathology. Further prospective evaluation is warranted to assess its applicability and underlying mechanisms.

5596 Background: Abemaciclib inhibits cyclin dependent kinases (CDK) 4 and 6 and, in synergy with anti-estrogens, is an FDA approved treatment of ER+ breast cancer. Endometrioid endometrial cancer frequently express the estrogen receptor and harbors PTEN, PI3K, KRAS and CTNNB1 mutations which induce estrogen-mediated upregulation of cyclin D1 and cancer cell proliferation. We hypothesized that the combination of letrozole and abemaciclib will inhibit cellular proliferation, as measured by Ki-67, in patients with endometrial cancer. Methods: This is a single-arm prospective multicenter surgical window of opportunity clinical trial for patients with endometrial cancer planned to undergo definitive surgical management with hysterectomy. Eligibility criteria included endometrioid histology, no prior treatment for endometrial cancer, and a window of greater than 15 days prior to scheduled hysterectomy. Patients received 14 days of abemaciclib 150mg twice daily and letrozole 2.5mg once daily. The primary endpoint was change in Ki-67 (%) from pretreatment biopsy to posttreatment hysterectomy specimen. Secondary endpoints included biological tumor characteristics (MMR, PTEN, PI3K, cyclin D1, pRB) associated with Ki-67 changes. The planned sample size was n=21 evaluable patients which would achieve 80% power with two-sided α = 0.05 to detect a 12.9% decrease in Ki67 expression based on a paired t-test. Results: A total of 27 women were enrolled, 2 patients withdrew consent leaving 25 evaluable patients. The median age was 62 (IQR: 54 - 66) and 19 patients (76%) were menopausal. Most patients were White (68%), 24% identified as Black; 20% were of Hispanic ethnicity. Median BMI was 37 (IQR: 30-40). All cancers were endometrioid with the majority being FIGO grade 1 (76%) and 24% grade 2. Nine (33%) patients had mismatch repair deficient tumors. Twenty-two (88%) patients were stage I, two (8%) stage II, and 1 (4%) stage IV. Mean baseline Ki-67 was 34% (SD 22%). After 2 weeks of treatment with abemaciclib and letrozole mean Ki67 was 14% (SD 16%). The mean Ki-67 reduction was 19% (SD 23%, p<0.001) meeting the prespecified endpoint. We found no significant difference in the magnitude of Ki-67 reduction based upon present or absent staining for pRb, PTEN, cyclin D1, p16, or MMR proteins. Post-menopausal patients had a decrease in Ki-67 of 26% compared to 15% for those who were premenopausal, but this did not reach statistical significance (p=0.5). There were no serious adverse events recorded. The most common study related adverse events were grade 1-2 diarrhea (68%) and grade 1-2 nausea (36%). Two patients had grade 3 hematological events (anemia and neutropenia), which resolved. Conclusions: Abemaciclib and letrozole induced a reduction in cellular proliferation in endometrioid endometrial cancer over a relatively short two week time period prior to surgery, suggesting clinical efficacy. Clinical trial information: NCT04049227 .

Importance:As endometrial cancer (EC) incidence rises, particularly among individuals with obesity and metabolic disorders, effective strategies targeting hormonal and metabolic risks are needed. Objective:To evaluate EC risk in patients with endometrial hyperplasia (EH) or benign uterine pathology treated with progestins vs combined progestins and glucagon-like peptide-1 receptor agonists (GLP-1RAs). Design, Setting, and Participants:This cohort study used TriNetX to analyze EC and hysterectomy among adult women with EH or benign uterine pathology who received progestins between May 1, 2005 (GLP-1RA approval date), and December 31, 2022. Analyses were based on deidentified electronic health records identified via International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes, and data were obtained on February 23, 2025. Four treatment comparisons were analyzed: GLP-1RA plus progestins vs progestins only; GLP-1RA plus progestins vs metformin progestins; triple therapy (GLP-1RA, metformin, and progestins) vs metformin plus progestins; triple therapy vs progestins only. Subgroup analyses GLP-1RA plus progestins vs progestins only stratified patients by progestin route, risk level, body mass index (BMI), and age. Exposures:GLP-1RAs, progestins, and/or metformin. Main outcomes and measures:The primary outcome was the incidence of EC. The secondary outcome was the incidence of subsequent hysterectomy. Results:A total of 18 414 and 426 406 adult female patients received GLP-1RA combined with progestin and progestin alone, respectively (mean [SD] age, 43.1 [10.2] years vs 35.2 [10.9] years). GLP-1RA with progestins was associated with a significantly lower risk of EC compared with progestins only (HR, 0.34 [95% CI, 0.27-0.44]). This protective association remained consistent across subgroups, stratified by progestin route, baseline risk, BMI, and age. GLP-1RA plus progestins also showed a lower EC risk than metformin plus progestins (HR, 0.30 [95% CI, 0.15-0.59]). Triple therapy was more effective in reducing EC risk than dual (metformin plus progestins) (HR, 0.37 [95% CI, 0.25-0.53]) or progestin monotherapy (HR, 0.44 [95% CI, 0.29-0.66]). Hysterectomy rates were lower in the GLP-1RA plus progestins group at 2-year (HR, 0.47 [95% CI, 0.42-0.53]) and 5-year (HR, 0.59 [95% CI, 0.54-0.64]) follow-up. Conclusions and Relevance:In this cohort study of women with benign uterine pathology or endometrial hyperplasia, combined GLP-1RA and progestin was associated with reduced EC risk. Further investigation is warranted to assess its applicability and underlying mechanisms.

Chemotherapy resistance remains a formidable challenge to the treatment of high-grade serous ovarian cancer (HGSOC). The drug-tolerant cells may originate from a small population of inherently resistant cancer stem cells (CSCs) in primary tumors. In contrast, sufficient evidence suggests that drug tolerance can also be transiently acquired by nonstem cancer cells. Regardless of the route, key regulators of this plastic process are poorly understood. Here, we utilized multiomics, tumor microarrays, and epigenetic modulation to demonstrate that SOX9 is a key chemo-induced driver of chemoresistance in HGSOC. Epigenetic upregulation of SOX9 was sufficient to induce chemoresistance in multiple HGSOC lines. Moreover, this upregulation induced the formation of a stem-like subpopulation and significant chemoresistance in vivo. Mechanistically, SOX9 increased transcriptional divergence, reprogramming the transcriptional state of naive cells into a stem-like state. Supporting this, we identified a rare cluster of SOX9-expressing cells in primary tumors that were highly enriched for CSCs and chemoresistance-associated stress gene modules. Notably, single-cell analysis showed that chemo treatment results in rapid population-level induction of SOX9 that enriches for a stem-like transcriptional state. Altogether, these findings implicate SOX9 as a critical regulator of early steps of transcriptional reprogramming that lead to chemoresistance through a CSC-like state in HGSOC.

BACKGROUND:Sleep disturbance is among the most frequent and distressing symptoms reported by gynecologic cancer survivors. Existing evidence-based behavioral sleep interventions are limited by implementation burden, which can decrease adherence. PURPOSE:As part of the preparation phase of the multiphase optimization strategy (MOST), this study solicited stakeholder feedback to maximize adherence in a planned behavioral sleep/circadian intervention optimization trial. METHODS:Thirteen post-treatment survivors of early-stage gynecologic cancer completed the protocol for the planned optimization trial, including simultaneous receipt of all candidate intervention components. This included six weeks of combined sleep restriction, stimulus control, and systematic exposure to morning bright light. Participants then completed a semi-structured interview to provide feedback on their experience. We used a rapid analytic approach to quickly identify actionable feedback from de-identified transcripts. RESULTS:Participants generally reacted positively to the intervention components. Actionable feedback identified recommended protocol modifications and was categorized into four overarching themes: (i) remove barriers to engagement; (ii) revise for clarity; (iii) augment content; and (iv) consider individual circumstances. CONCLUSIONS:Rapid qualitative analysis enabled us to effectively modify our planned study protocol on an expedited timeline. This approach is consistent with the core principles of MOST and can be incorporated into the Preparation phase to enhance optimization efforts.

Supplementary Material and Methods

AbstractPurpose:DNA methylation causes silencing of tumor-suppressor and differentiation-associated genes, being linked to chemoresistance. Previous studies demonstrated that hypomethylating agents (HMA) resensitize ovarian cancer to chemotherapy. NTX-301 is a highly potent and orally bioavailable HMA, in early clinical development.Experimental Design:The antitumor effects of NTX-301 were studied in ovarian cancer models by using cell viability, stemness and ferroptosis assays, RNA sequencing, lipidomic analyses, and stimulated Raman spectroscopy.Results:Ovarian cancer cells (SKOV3, IC50 = 5.08 nmol/L; OVCAR5 IC50 = 3.66 nmol/L) were highly sensitive to NTX-301 compared with fallopian tube epithelial cells. NTX-301 downregulated expression of DNA methyltransferases 1–3 and induced transcriptomic reprogramming with 15,000 differentially expressed genes (DEG, P < 0.05). Among them, Gene Ontology enrichment analysis identified regulation of fatty acid biosynthesis and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase, known features of cancer stem cells. Low-dose NTX-301 reduced the ALDH(+) cell population and expression of stemness-associated transcription factors. Stearoyl-coenzyme A desaturase 1 (SCD), which regulates production of unsaturated fatty acids (UFA), was among the top DEG downregulated by NTX-301. NTX-301 treatment decreased levels of UFA and increased oxidized lipids, and this was blunted by deferoxamine, indicating cell death via ferroptosis. NTX-301–induced ferroptosis was rescued by oleic acid. In vivo, monotherapy with NTX-301 significantly inhibited ovarian cancer and patient-derived xenograft growth (P < 0.05). Decreased SCD levels and increased oxidized lipids were detected in NTX-301–treated xenografts.Conclusions:NTX-301 is active in ovarian cancer models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.

Abstract The dynamic organization of chromatin, governed by epigenetic modifiers, critically regulates cell plasticity and determines cell fate in both normal and malignant stem cells. We hypothesized that chromatin architecture plays a role in regulating transcriptional heterogeneity in ovarian cancer stem cells (OCSCs) to maintain stemness and chemoresistance. PWS microscopy showed that ALDH+ OCSCs derived displayed higher nuclear fractal dimension D signals (P<0.05) than ALDH- non-OCSCs, indicating that chromatin in OCSCs possesses higher packing compared to non-OCSCs. Cut & TAG Sequencing showed that of the 3606-histone repressive H3K27m3 enriched regions (P<0.05, fold change >2), 3208 were detected in OCSCs while 398 peaks were present in non-OCSCs, consistent with findings supporting high D features in OCSCs. The active mark H3K4me3 was enriched at the promoter of stemness-associated genes, including SOX2, ALDH1A3, and FZD7 in OCSCs; and the repressive histone mark H3K27me3 was enriched at the promoter of the epithelial cell marker CDH1 in OCSCs. These results support that the chromatin of OCSCs contains open genomic regions, corresponding to stemness-associated genes and repressed regions associated with genes related to differentiation. RNA sequencing indicated that the coefficient of variance (COV) reflecting transcriptional heterogeneity in OCSCs was higher than in non-OCSCs at baseline. In response to platinum, genes with low baseline expression in OCSCs were most upregulated, supporting increased transcriptional heterogeneity in response to chemotherapy. Inhibitors of epigenetic modifiers (DNMTi, EZH2i, and Dot1Li) induced chromatin re-organization in OCSCs and promoted OCSCs transition toward differentiation, supporting that stemness-associated chromatin structure is targetable. We demonstrated that a Dot1L inhibitor (Dot1Li) targeted OCSCs by decreasing chromatin packing D, resulting in decreased transcriptional heterogeneity. In conclusion, OCSCs harbor highly packed chromatin, contributing to chemoresistance and cell plasticity. Epigenetic modifiers (such as Dot1Li) inhibit stemness by regulating chromatin organization and transcriptional malleability. Citation Format: Yinu Wang, Jane Frederick, Yaqi Zhang, Elizabeth Thomas Bartom, Greta Bauer, Edward Tanner, Vadim Backman, Daniela Matei. Targeting chromatin in ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2580.

OC cells are highly sensitive to NTX301 treatment.

Primer sequences for H3K27Ac ChIP-qPCR

Objective To describe extension of ovarian tissue beyond visible and National Comprehensive Cancer Network recommended margins among patients with BRCA mutations undergoing minimally invasive risk-reducing salpingo-oophorectomy. Methods A prospective study of patients with BRCA mutations who underwent minimally invasive risk-reducing bilateral salpingo-oophorectomy was conducted. Patient enrollment occurred between October 2021 and 2023. Tissue specimens were analyzed according to the Sectioning and Extensively Examining the Fimbriated End protocol. Results Twenty women with BRCA mutations were prospectively enrolled. All patients underwent minimally invasive surgery with 70% undergoing concurrent hysterectomy (n = 14). Approximately half of these procedures were performed with robotic assistance (n = 9, 45%). One patient was admitted overnight (5%); the other nineteen were discharged on the day of surgery (95%). One patient experienced a major complication and required readmission (5%). Extension of ovarian tissue beyond the visible ovary was noted on pathologic examination of six specimens (30%). In one patient this was observed on the left (17%), in three on the right (50%), and in two bilateral extension (33%) was noted. The distance ovarian stroma extended microscopically beyond the visible ovary was between 2 and 14 mm, with a median of 5 mm. Among patients with microscopic extension of ovarian tissue, the majority (n = 5, 83%) had a BRCA2 mutation. Conclusion In women with BRCA mutations undergoing risk-reducing minimally invasive surgery, approximately one third had microscopic extension of ovarian stroma beyond the visible ovary. Current guidelines which recommend resection of at least 20 mm of tissue beyond the visible ovary are likely adequate in this population.

Changes in serum CA-125 levels predict response to chemotherapy and survival in women with advanced epithelial ovarian cancer. Although often used to monitor for recurrence, randomized trial data do not suggest that treatment based on a rising CA-125 alone impacts prognosis if secondary cytoreduction is not under consideration. CA-125 surveillance may still provide benefits to patients who are likely to achieve a complete resection at secondary cytoreduction – assuming that early identification of recurrent disease based on a rising CA-125 level leads to a higher chance of complete gross resection. CA-125 surveillance also provides patients with useful information about prognosis and impending recurrence – regardless of whether this information impacts survival directly. As such, the routine use of CA-125 surveillance should be considered as an individualized option for patients based on disease characteristics and personal preferences.

Supplemental tables include gene lists related to Figure 5, additional experimental data related to Figure S3I, and primers sequence. Table S1. Numbers of spheroids were counted for each cell dilution; Table S2. List of 20 genes that exhibit a significant change between control and FTO-overexpressing OVCAR5 cells in m6A peak levels, and abundance of the corresponding mRNA transcripta; Table S3. List of primers oligonucleotides; Table S4. Summary of the m6A-seq and RNA-seq; Table S5. List of top 50 hypo-methylation & down-regulated genes; Table S6: List of top 50 hypo-methylation &up-regulated genes.

While narrative recommendation letters have long been the norm, the use of standardized letters of evaluation (SLOE) has increased in many specialties. This study investigated SLOE use in gynecologic oncology (GO) fellowship applications nationally to evaluate their discriminatory ability and describe trends based on applicant and letter writer characteristics.

Study Objective: To examine whether there are gender differences in letters of recommendation (LORs) written for residents applying to gynecology surgical fellowships. Design: Retrospective study. Setting: Single, academic institution. Patients: LORs for applicants to gynecology oncology, urogynecology, and minimally invasive gynecology fellowships during the 2019-2020 application cycle. Interventions: Not applicable. Measurements and Main Results: We analyzed the linguistic content of the letters for the presence of 4 summary variables and 21 word categories based on previous studies using validated computerized text analysis software. We used multivari-able analysis using linear mixed models to compare linguistic characteristics of letters by applicant gender. We performed qualitative content analysis on letters and compared the frequency of code themes by gender. The mixed-method design was planned to allow for analysis of domains not captured in text analysis. Among 680 letters written for 186 applicants, 124 (18.2%) were written for men, and 556 (81.8%) were written for women. There were no differences in the least square mean (standard error) word counts for LORs written for men and women applicants, 465 (20.0) vs 458(9.4) words, p = .74. In multivariable analysis, LORs written for men were found to have higher authentic tone and more risk words (p = .005 and p = .03, respectively). LORs written for women contained more communal (relationship-oriented) words (p = .006). The qualitative analysis demonstrated that ability, interpersonal traits, surgical skills, and research were the most often men-tioned themes. Comments about compassion/empathy, leadership potential, teaching, interpersonal skills, and patient rap-port were found more often in letters for men. More doubt raisers (words that raise doubt or concern) were present in letters for men, but letters for both genders had similar levels of negative criticism. In contrast, comments on ability, being "drama-free," and self-awareness were found more often in letters for women. Conclusion: There were gender differences in LORs written for obstetrics and gynecology surgical subspecialty fellowship applicants indicating the presence of gender bias. Journal of Minimally Invasive Gynecology (2023) 30, 406-413. (c) 2023 AAGL. All rights reserved.

Additional experimental results supporting the main figures are included. Figure S1. Expression of key RNA methylation regulators in OC; Figure S2. Flow cytometry analysis for ALDH and CD133 in single cell suspensions derived from human ovarian tumors; Figure S3. FTO overexpression in OC cells; Figure S4. FTO overexpression decreases spheroid formation ability of OC cells dissociated from xenografts; Figure S5. m6A activity and tumor initiation capacity; Figure S6. Gene sets enriched in FTO vs. control OC cells; Figure S7. Expression of PDE4B and PDE1C in FTO overexpressed OC cells; Figure S8. The Genome Browser visualizes the predicted motif binding sites for proteins (IGF2BP2 and IGF2BP3) on the input sequence; Figure S9. Effects of phosphodiesterase inhibitors on stemness features are dependent on FTO; Figure S10. Quality control for RNA-seq; Figure S11. Quality control for MeRIP-seq

TPS5629 Background: Identifying new approaches for the treatment of advanced endometrial cancer (EC) is a priority. A platform study design allows for a faster investigation of novel drug combinations. NRG-GY012 is an ongoing platform study investigating drug combinations with the PARP inhibitor olaparib (O) and the anti-angiogenic agent cediranib (C). Results from the first set of arms established improved PFS for combination cediranib/olaparib over C alone, but did not meet its prespecified statistical significance. In this second set of arms, based on preclinical data, we are investigating the combination of O with an AKT inhibitor (capivasertib), O with durvalumab and C with durvalumab compared to the reference arm of C alone. Methods: This is a multicenter randomized four arm study for patients with recurrent, metastatic or persistent EC. Patients are randomized 1:1:1:1 to cediranib PO 30 mg Daily; olaparib 300 mg PO BID and capivasertib 400mg 4 day on/3 off schedule; olaparib 300 mg PO BID and durvalumab 1500mg q28 days; or the combination of cediranib 20 mg PO Daily 5 days on/2 days and durvalumab 1500mg q28. All treatment cycles are 28 days. Primary endpoint is progression free survival (PFS). The study is powered to detect a doubling in median PFS from 3.6 (based on cediranib alone) to 7.2 months with 90% power, using a one-sided test with α = 0.05 per comparison. Forty patients will be enrolled per arm, with an interim futility analysis planned at 50% information time. Eligibility includes endometroid, serous, and mixed histology EC; at least 1 prior line of chemotherapy (no more than 2 lines for metastatic disease), prior endocrine or immunotherapy alone is allowed; prior treatment with lenvatinib and pembrolizumab is excluded. Archival tumor tissue and blood samples are being collected for translational studies. The study is open across the NRG network; 76 of a planned 168 patients are enrolled to date. Safety analysis at the time of 36 patients enrolled did not demonstrate any new safety signals and the study was approved to continue enrollment. Clinical trial information: NCT03660826 .