High-grade Endometrial Cancer Research Articles

High-grade Endometrial Carcinomas With Solid Basaloid Morphology and Geographic Necrosis Lacking Definitive Pilomatrix-like Features: Clinicopathologic Characteristics Including Aggressive Behavior and Novel Molecular Events.

High-grade endometrioid carcinomas occasionally demonstrate solid basaloid morphology with geographic necrosis (SB-GN). This pattern is among the defining features of pilomatrix-like high-grade endometrioid carcinoma (PiMHEC), a recently proposed tumor type which is additionally characterized by the presence of shadow cells, abnormal beta-catenin/CTNNB1 mutations, strong CDX2 expression, and poor outcomes. Clinicopathologic overlap between PiMHEC and other high-grade endometrial cancers with SB-GN has not been established. We screened 300 endometrial carcinomas on tissue microarray for SB-GN histology and performed a detailed whole-section morphologic review, immunohistochemical analysis, and next-generation sequencing on all cases bearing this pattern. Four (1.3%) demonstrated SB-GN. All 3 with clinical follow-up had extremely aggressive behavior despite being MMR-deficient; in contrast, only 27% of other MMR-deficient high-grade carcinomas recurred. One SB-GN case met most of the previously outlined diagnostic criteria for PiMHEC including abnormal beta-catenin/CTNNB1 (p.S37P variant) and strong CDX2 expression; notably, however, shadow cells were absent. This case also demonstrated a KRAS p.A59T pathogenic variant. The other 3 cases also lacked shadow cells; the 2 with sequencing data bore no CTNNB1 abnormalities but showed likely oncogenic variants involving the pilomatrixoma-associated gene FGFR2. All 3 cases with molecular results also bore somatic Notch pathway (NOTCH1/NOTCH2/NOTCH3) variants. The single case treated with immunotherapy showed complete and sustained response with regression of bone metastases despite abnormal beta-catenin/CTNNB1, which has been associated with immunotherapeutic resistance. These data suggest that the SB-GN pattern may connote a poor prognosis even in the absence of overt pilomatrix-like differentiation, and that novel molecular events may have implications for the treatment of these tumors.

Assessing para-aortic nodal status in high-grade endometrial cancer patients with negative pelvic sentinel lymph node biopsy.

To determine the accuracy of pelvic sentinel lymph node biopsy (SLN) in detecting positive para-aortic (PA) lymph nodes in high-grade uterine cancer, and to determine the recurrence rate in patients with high-grade uterine cancers who did not receive adjuvant chemotherapy based on negative pelvic SLNs. This was a retrospective cohort study of patients with newly diagnosed, high-grade endometrial cancer who underwent surgery, including pelvic SLNs with or without PA node dissection, at a tertiary care institution between 2015 and 2020. Baseline demographics, surgical management, pathology data, and outcomes were analyzed using descriptive statistics, and survival analysis. Postoperative histology of the 110 patients meeting inclusion criteria was 45.5% grade 3 endometrioid, 36.4% serous, 10.9% clear cell, and 7.3% carcinosarcoma. On final pathology, 63.7% were stage 1, and 23.6% were stage 3C with positive nodes. A total of 63 patients (57.3%) had a PA lymph node dissection (56 bilateral, 7 unilateral) in addition to the pelvic SLN. Among this group, 5.8% (95% confidence interval 1.2%-16.0%) had a positive PA node despite a negative pelvic SLN. Among those with a negative pelvic SLN and no adjuvant chemotherapy (n = 75), the rate of distant recurrence was 14.7%, and 3-year recurrence-free survival was 71.9%. The rate of isolated PA node metastasis in high-grade endometrial cancers despite a negative pelvic SLN may be significantly higher than the accepted rate of isolated PA node metastasis in low-grade endometrial cancer. This supports adjuvant treatment decisions continuing to incorporate primary tumor pathology and molecular classification.

Abstract C165: Interrogating the Tumor and Immune Microenvironment of Endometrial Cancers Among a Diverse Cohort of Cases

Abstract Endometrial cancer (EC) is the sixth most common gynecological malignancy, with an increasing incidence worldwide over the last two decades. African American women are more likely to be diagnosed with endometrial cancer than Caucasian women. Unfortunately, African American women are more frequently diagnosed with high-grade EC, which occurs 2-3 times more often compared to their white and Hispanic counterparts. Additionally, African American women have higher mortality rates from EC than Caucasian women, highlighting a significant cancer disparity among these patients. Uterine EC disparities are induced by complex ecosystems composed of cellular niches with distinct genotypes, phenotypes, and spatial transcriptomic landscapes. In estrogen-dependent EC tumorigenesis, the endometrium undergoes uncontrollable proliferation, progressing from normal to atypical hyperplasia and ultimately to carcinoma. The heterogeneity of EC tumors complicates therapeutic targeting, rendering EC a challenging disease to cure. This study aims to investigate the genetic and transcriptomic features found in the tumor microenvironment of African American patients with EC. Previous research has shown that the molecular classification of EC tumors can be categorized as estrogen-driven, hypermutant, microsatellite instable, or lacking a specific molecular profile. Current models do not adequately reflect the oncogenic origin and pathological progression in patients. The cohort of EC cases includes primary, metastatic, and normal tumor tissues classified as Uterine Endometrial Adenocarcinoma, along with two Serous Papillary cases with varying tumor grades (low to high grade). Spatial transcriptomics and whole exome sequencing were performed on these samples. Spatial transcriptomics will identify the alterations in gene expression and the spatial landscape of the cell neighborhoods driving tumor heterogeneity. Whole exome sequencing will provide insight into the germline and somatic variants present in EC, offering a deeper understanding of the potential genetic drivers in African American patients. The genes expressed across the EC tumor landscape are implicated in cell proliferation, epithelial- mesenchymal transition, DNA damage response, and immune response. The analysis will also show the transition of normal to malignant endometrium harboring high tumor mutational burden and microsatellite instability when comparing paired samples. This comprehensive approach aims to elucidate the genetic and transcriptomic underpinnings contributing to the disparities observed in African American patients with EC, ultimately informing better-targeted therapies and improving outcomes. Citation Format: Danyelle Paine, John Carpten. Interrogating the Tumor and Immune Microenvironment of Endometrial Cancers Among a Diverse Cohort of Cases [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C165.

The relationship between imaging-based body composition parameters and disease prognosis in patients with endometrial cancer.

Obesity is known as a risk factor for endometrial cancer (EC). Only a few studies investigate the relationship between sarcopenia and sarcopenic obesity and EC. In this study, our aim was to investigate the relationship between the cross-sectional imaging-based body composition parameters and the disease prognosis in low-grade (LG) and high-grade (HG) EC. We conducted a retrospective study in women diagnosed with low and high-grade EC between January 2014 and May 2022 who had abdominal MRI and thorax CT as a part of routine staging workup. We used the skeletal muscle index (SMI) at the level of the third lumbar vertebra to assess sarcopenia on CT. The T2-weighted sequence at the level of the L2-L3 intervertebral disc is used for visceral fat area (VFA), subcutaneous fat area (SFA), and total fat area (TFA). Two radiologists in consensus, calculated the parameters. A total of 250 EC patients (144 low-grade EC, 106 high-grade EC).Sarcopenia was observed in 122 (48.8%) patients, and sarcopenic obesity was found in 82 (32.8%) patients. Although there was an increase in VFA in cases with high-grade EC, there was no significant difference in terms of SFA. Additionally, the frequency of sarcopenia and sarcopenic obesity was higher in cases with high-grade EC. There was no association between sarcopenia and age, histological type, FIGO staging, or comorbidity in the univariate analysis. However, BMI was found to be associated with sarcopenia. Quantitative radiological measurement of sarcopenia, sarcopenic obesity, and body fat composition can be used as novel parameters in the prediction of disease prognosis in endometrial cancer.

HER2-negative or low expression as an unfavorable prognostic factor in patients with stage I/II uterine carcinosarcoma.

Uterine carcinosarcoma (UCS) is uncommon high-grade endometrial cancer with limited treatment options. We evaluated the prognostic significance of human epidermal growth factor receptor 2 (HER2) expression and HER2 gene amplification within large cohorts of UCS, and clarify clinicopathologic characteristics of HER2-low UCS. We examined HER2 protein expression in 148 patients of UCS using in vivo diagnostic HER2 immunohistochemistry (IHC) kits and HER2 gene amplification using fluorescence in situ hybridization (FISH) in 72 patients. HER2 IHC score was evaluated according to the latest American Society of Clinical Oncology/College of American Pathologists criteria for gastric cancer, which was negative in 41 patients, low expression of 1+ was observed in 57 patients, and HER2 high expression was observed in 50 patients (2+ in 38 and 3+ in 12 patients). There was no significant statistical difference in clinicopathological characteristics based on HER2 protein expression status. HER2 negative and low expression compared to high expression revealed poor overall survival in stage I/ II. The concordance between IHC and FISH results were relatively low compared to other cancer types (HER2 IHC score 1+, 2+, and 3+ were 5%, 15%, and 50%), and combining these results was not efficient as a prognostic factor in UCS. In contrast, the HER2 IHC score alone was a prognostic factor in stage I/II UCS. HER2 low group did not show specific clinicopathologic features. Since the HER2 IHC score low in advanced UCS is a predictive factor, stratification of UCS using HER2 IHC score for HER2 IHC score low group and developing adjuvant therapy may be proposed in the near future.

Comparative Assessment of miR-185-5p and miR-191-5p Expression: From Normal Endometrium to High-Grade Endometrial Cancer.

Endometrial cancer (EC) is a significant cause of cancer-related deaths in women. MicroRNAs (miRs) play a role in cancer development, acting as oncogenes or tumor suppressors. This study evaluated the diagnostic potential of hsa-miR-185-5p and hsa-miR-191-5p in EC and their correlation with clinical and histopathological features. A cross-sectional study analyzed formalin-fixed, paraffin-embedded tissue samples from 59 patients: 18 with EC, 21 with endometrial hyperplasia (EH), 17 with normal endometrium (NE), and 3 with endometrial polyps (EPs). Quantitative reverse transcription-polymerase chain reaction and TaqMan probes were used for miR expression analysis. The Shapiro-Wilk test was used to analyze the normal distribution of the data. Subsequently, parametric or non-parametric tests were used to evaluate the associations between the expression levels of each miR and clinical parameters. Both miRs were underexpressed in some precursor and malignant lesions compared to certain NE subtypes and benign lesions. Specifically, hsa-miR-185-5p showed underexpression in grade 3 EC compared to some NE and EH subtypes (FC: -57.9 to -8.5, p < 0.05), and hsa-miR-191-5p was underexpressed in EH and EC compared to secretory endometrium and EPs (FC: -4.2 to -32.8, p < 0.05). SETD1B, TJP1, and MSI1 were common predicted target genes. In conclusion, hsa-miR-185-5p and hsa-miR-191-5p are underexpressed in EC tissues, correlating with histopathological grades, highlighting their potential as diagnostic biomarkers and their role as tumor suppressors in EC.

A first-in-human, phase 1 study evaluating oral TACC3 inhibitor, AO-252, in advanced solid tumors.

TPS3176 Background: TACC3 is the most oncogenic member of the transforming acidic coiled-coil domain-containing protein (TACC) family, which have important roles in microtubule and centrosome-related processes. TACC3 is overexpressed in multiple cancers with centrosome amplification caused by genomic aberrations such as TP53 mutations, leading to chromosome instability and worse prognosis. AO-252 is a computationally designed, small molecule inhibitor of TACC3 protein-protein interactions. Binding assays and kinome screening suggested the specificity of AO-252 to TACC3. AO-252 targets the interactions of TACC3 with Clathrin/KIFC1, BRDA1 and MBD2/HAT complexes regulating mitosis, DNA damage response and epigenetic functions and was confirmed using co-immunoprecipitation (co-IP) assays. Gene expression analysis in multiple cell lines further confirmed the various TACC3 pathways affected by AO-252. In vitro, AO-252 showed low nanomolar potency in &gt;200 cell line panel covering multiple different cancer indications and spared the normal cells. In vivo, AO-252 demonstrated strong tumor growth inhibition or regression in multiple xenograft models including triple-negative breast (TNBC), high-grade serous ovarian (HGSOC) and endometrial cancer with TP53 mutation/loss with a good therapeutic index. Methods: This first-in-human, multicenter, open-label phase I dose escalation and expansion study evaluates the safety, tolerability, maximum tolerated dose, and recommended phase 2 dose (RP2D) of AO-252 in solid tumors (NCT06136884). Patients with unresectable or metastatic TNBC, platinum-resistant HGSOC, primary peritoneal, fallopian-tube and serous endometrial cancers with documented TP53 mutation/loss, who have failed on at least 1 prior line of systemic therapy in advanced/metastatic setting (maximum 4 for TNBC and endometrial carcinoma) are eligible for enrollment. Part 1 follows an accelerated titration dose-escalation for cohort 1 and 2 and a traditional 3+3 study design from cohort 3. A maximum of 54 patients will be treated in the dose escalation, patients receiving AO-252 orally once or twice daily in a 28-day cycle. Part 2 consists of dose-expansion of a maximum of 30 patients into 2 RP2D levels of AO-252. Primary objectives are to evaluate the safety and tolerability as noted by dose-limiting toxicities, adverse events, serious adverse events, laboratory test results, electrocardiograms, vital signs, physical exams, and ECOG performance status. Secondary objectives are to determine the antitumor activity assessed by objective response rate per RECIST1.1, duration of response and disease control rate and to determine the pharmacokinetics of single and repeated doses of AO-252. Potential biomarkers relating to treatment outcome will be evaluated as exploratory endpoints. Enrollment in Cohort 1 is completed and enrollment to Cohort 2 will begin in February 2024. Clinical trial information: NCT06136884 .

High-Grade Endometrial Cancer: Molecular Subtypes, Current Challenges, and Treatment Options.

Although many recent advancements have been made in women's health, perhaps one of the most neglected areas of research is the diagnosis and treatment of high-grade endometrial cancer (EnCa). The molecular classification of EnCa in concert with histology was a major step forward. The integration of profiling for mismatch repair deficiency and Human Epidermal Growth Factor 2 (HER2) overexpression, can further inform treatment options, especially for drug resistant recurrent disease. Recent early phase trials suggest that regardless of subtype, combination therapy with agents that have distinct mechanisms of action is a fruitful approach to the treatment of high-grade EnCa. Unfortunately, although the importance of diagnosis and treatment of high-grade EnCa is well recognized, it is understudied compared to other gynecologic and breast cancers. There remains a tremendous need to couple molecular profiling and biomarker development with promising treatment options to inform new treatment strategies with higher efficacy and safety for all who suffer from high-grade recurrent EnCa.

Copy number signatures and CCNE1 amplification reveal the involvement of replication stress in high-grade endometrial tumors oncogenesis

PurposeManaging high-grade endometrial cancer in Martinique poses significant challenges. The diversity of copy number alterations in high-grade endometrial tumors, often associated with a TP53 mutation, is a key factor complicating treatment. Due to the high incidence of high-grade tumors with poor prognosis, our study aimed to characterize the molecular signature of these tumors within a cohort of 25 high-grade endometrial cases.MethodsWe conducted a comprehensive pangenomic analysis to categorize the copy number alterations involved in these tumors. Whole-Exome Sequencing (WES) and Homologous Recombination (HR) analysis were performed. The alterations obtained from the WES were classified into various signatures using the Copy Number Signatures tool available in COSMIC.ResultsWe identified several signatures that correlated with tumor stage and disctinct prognoses. These signatures all seem to be linked to replication stress, with CCNE1 amplification identified as the primary driver of oncogenesis in over 70% of tumors analyzed.ConclusionThe identification of CCNE1 amplification, which is currently being explored as a therapeutic target in clinical trials, suggests new treatment strategies for high-grade endometrial cancer. This finding holds particular significance for Martinique, where access to care is challenging.

Abstract 935: Co-clinical endometrial cancer (EC) patient-derived organoids (PDO) to assess functional effects of replication stress (RS)-targeting agents

Abstract Background: EC is the most commonly diagnosed gynecologic cancer in the US and has both rising incidence and mortality. Uterine serous cancer (USC) and uterine carcinosarcoma (UCS) are high grade ECs characterized by TP53 mutation and molecular alterations suggesting high replication stress (RS) and cell cycle dysregulation. These molecular alterations could engender sensitivity to RS-targeting strategies such as WEE1 inhibition. In a single arm phase 2 of 35 patients, the WEE1 inhibitor adavosertib demonstrated clinical activity in recurrent USC, with an overall response rate of 29.4%. To support the development of predictive biomarkers of sensitivity to RS targeting agents, we report on the establishment of co-clinical EC PDOs from pre- and on-treatment biopsies from patients with USC or UCS enrolled to a translational trial of adavosertib. Methods: Patients with USC or UCS consented for trial participation underwent pre- and on-treatment biopsies while receiving adavosertib monotherapy. Biopsies were dissociated and passaged as 3D organoid cultures in Matrigel in an optimized media. Established PDOs were seeded in 3D microfluidic devices, treated with adavosertib in a dose-dependent manner and assessed for viability after 6 days of treatment by dual labeling live/dead imaging. IC50 values were calculated using log(inhibitor) vs response - variable slope (four parameters) model in GraphPad Prism. To assess the effect of adavosertib on replication fork speed, DNA fiber assays were conducted with PDOs treated with adavosertib for 72 hours. Finally, FFPE blocks were created from PDOs for assessment of DNA damage repair biomarkers by IHC. Results: 19 biopsies were obtained from 11 participating patients. 6 biopsies did not yield sufficient tumor cells for PDO generation. From the remaining 13 samples, 5 PDOs were successfully established (3 USC and 2 UCS), with a higher success rate when 3 biopsy cores were received (3 of 4; 75%) compared to when 2 biopsy cores were received (2 of 9; 22 %). Dual labeling live/dead imaging of PDOs treated with adavosertib resulted in IC50 values ranging from 113nM to 856nM. DNA fiber assays revealed consistent decrease in fork speed with adavosertib exposure, consistent with the hypothesized effect of adavosertib in increasing RS in these cells. A dose dependent effect on replication fork speed was observed, with greater decreases in fork speed with increasing concentrations of adavosertib. Due to the limited number of samples and the number of PDOs generated from on-treatment as opposed to pre-treatment biopsies, clinical correlation between PDO adavosertib sensitivity and clinical activity was not formally assessed. IHC analysis of potential DNA damage biomarkers is ongoing. Conclusions: We describe the successful establishment of co-clinical EC PDOs for functional evaluation of sensitivity to RS targeting agents. Citation Format: Elena Ivanova, Aisha L. Saldanha, Shrabasti Roychoudhury, Bose Kochupurakkal, Ari P. Zlota, Clare E. Padrick, Ha V. Vo, Eli K. Greenberg, Hannah Sawyer, Carina Feeney, Courtney H. Qi, Swati Narayan, Jennifer D. Curtis, Nabihah Tayob, Marisa R. Nucci, Panagiotis Konstantinopoulos, Dipanjan Chowdhury, Geoffrey Shapiro, Cloud P. Paweletz, Ursula A. Matulonis, Joyce F. Liu. Co-clinical endometrial cancer (EC) patient-derived organoids (PDO) to assess functional effects of replication stress (RS)-targeting agents [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 935.

Abstract A019: Autologous patient-derived organoid-immune cell co-culture platform for therapeutics discovery in high-grade endometrial cancer

Abstract Endometrial cancer is the most common gynecologic malignancy with increasing incidence and mortality rates. Recurrent endometrial cancers, which are mostly from high-grade endometrial cancers (HGEC), have limited treatment options and display extremely poor response rate upon chemotherapies, thus showing very poor prognosis. Immunotherapy revolutionized the field of cancer medicine, but lack of scalable and sustainable pre-clinical models for HGEC poses a significant challenge in studying critical immunological aspects of HGEC. We therefore established a comprehensive endometrial cancer biobank comprising primary tissues, patient-derived organoids (PDO), and their matching immune cells from all existing HGEC types, enabling us to establish an autologous interaction between cancer and immune cells in vitro. Inactivation of antigen processing and presentation (APP) pathways is one of the major mechanisms for immune evasion in many cancer types including HGEC. Using a subset of our biobank, we performed bulk RNA-seq on 11 normal-cancer PDO pairs and found that genes involved in APP pathways, MHC-II in particular, are downregulated in HGEC organoids. We found that dampened MHC expression can be reverted by external stimuli such as IFNγ and Tazemetostat. We then assessed the effects of enhanced MHC-I and -II expression mediated by the two molecules on cancer-immune cell interactions by setting up organoid-immune cell co-cultures using autologous PBMC, CD4, and CD8 T cells. Our results suggest that cell signaling and epigenetic manipulations on HGEC can be exploited to enhance immune cell-mediated apoptosis. Using our co-culture platform, we next evaluated the efficacy of different immunotherapeutic modalities such as NK cell transfer, bi-specific T-cell engagers, and CAR-T cells on HGEC. Our proof-of-principle experiments demonstrate the utility of our co-culture system in studying the autologous cancer-immune cell interactions from the same patient ex vivo. Our sustainable and scalable testing platform could be used to assess the safety and efficacy of current therapeutics or to identify new therapeutics that enhance immune responses against advanced cancers lacking effective treatment options such as HGEC. Citation Format: Charlie Chung, Aaron Nizam, Brian Yueh, Santhilal Subhash, Marina Frimer, Gary L. Goldberg, Semir Beyaz. Autologous patient-derived organoid-immune cell co-culture platform for therapeutics discovery in high-grade endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A019.

Abstract IA009: Racial disparities in high grade endometrial cancers

Abstract Endometrial cancer (EC) is the most common gynecological cancer in the United States, with approximately 66,200 cases diagnosed annually. Incidence rates have been increasing over the past two decades, especially in non-white and younger populations. Additionally, population-based studies have shown that the increases are greatest among the more aggressive histologic subtypes. In unadjusted analyses, incidence rates are highest among non-Hispanic white women (NHW); however, when the greater prevalence of hysterectomies in non-Hispanic black women (NHB) is accounted for, the incidence among NHB women exceeds that of other racial and ethnic groups. In addition, survival after an EC diagnosis is significantly lower for NHB women at every stage of diagnosis, and for all histologic subtypes. EC is currently the 6th leading cause of cancer-related deaths among women but will likely overtake ovarian cancer (the 5th leading cause) before the end of the decade—it has already done so for NHB women. Factors influencing survival exist across the cancer care spectrum, starting at diagnosis and continuing through treatment and beyond. A portion of the differences in EC survival can be explained by the histologic subtype of the tumor. Low grade endometrioid ECs are the most common histologic type and tend to have a good prognosis. High grade tumors, such as serous cancers, are more common among NHB women and have poorer prognosis. A better understanding of the molecular characteristics of these more aggressive tumors is warranted, as they are increasing in incidence and cause a disproportionate amount of morbidity and mortality. To date, most studies that have included molecular profiling do not have large enough numbers or participants from diverse populations to examine whether mutations that may be clinically significant differ by race and ethnicity in high grade endometrial cancers. In a population of women with high grade EC (endometrioid, serous, clear cell, mixed) identified from two academic hospitals in Detroit, Michigan, we performed whole exome sequencing on tumor and normal tissue from 285 women. We hypothesized that somatic mutations in specific genes may vary by race and ultimately contribute to the survival disparity consistently identified in population-based studies. We report findings from the two most common high grade histologic subtypes, endometrioid (n=51 NHB, n=60 NHW) and serous (n=85 NHB, n=37 NHW). Established and emerging therapeutic targets were selected as a priori for the initial analysis. Tumor mutational burden (TMB), microsatellite instability status (MSI), mutations in genes in the PIK3 pathway, POLE, ARID1A and p53 were all examined. Findings highlight the need to consider histologic subtype in conjunction with race and ethnicity, as failure to do so may erroneously confound the estimates. Larger studies in diverse populations will be critical to disentangle the contribution of ancestry, histology and somatic mutations to the survival disparities seen after an EC diagnosis. Citation Format: Michele L. Cote. Racial disparities in high grade endometrial cancers [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA009.

Abstract B008: Modeling radiation sensitivity in patient-derived organoid models of endometrial cancer

Abstract Endometrial cancer is the 4th most common cancer in women, with increasing incidence and deaths each year from 2008 to 2017. The typical treatment for high grade and advanced endometrial cancer includes cytoreductive surgery followed by radiation and/or chemotherapy, depending on stage, grade, and other risk factors. Despite advances in molecular classification of endometrial cancer, there is a paucity of data on the mechanisms of sensitivity and resistance to radiation and chemotherapy. One reason for this lack of understanding is because, clinically, surgery is not typically offered in the setting of recurrence; thus, there are no available specimens to assess how the tumor evolves in response to therapy. Preclinical models can in part overcome this roadblock, yet there are very few preclinical models of endometrial cancer that accurately recapitulate the heterogeneity of human disease. Our objective was to define how endometrial tumors respond to radiation and chemotherapy using patient-derived organoid models. First, we have established a living biorepository that includes over 30 patient-derived organoid models of high risk or advanced endometrial cancer, with available clinical response data. Specifically, tumor specimens were obtained at the time of primary surgery and digested to generate single cell suspensions, which were then resuspended in basement membrane extract (BME), plated as three-dimensional domes, and cultured in specialized media. Organoids with a diameter of &amp;gt;50 µm typically formed in 48-96 hours after initial plating. For therapeutic studies, organoids were plated as BME domes in 96-well plates, followed by exposure to increasing levels of radiation (2-16 Gy) and/or chemotherapy (carboplatin+paclitaxel, 10 nM – 10 µM). Drug effects were monitored using an automated kinetic live-cell imaging platform (Cytation5) in which media was supplemented with fluorescent dyes to multiplex interrogation of cell health and induction of apoptosis. To date, we have observed differential sensitivity to radiation and chemotherapy in patient-derived organoid models of high risk and advanced endometrial cancer as determined by changes in organoid number and size and induction of apoptosis. These studies are the first to examine the therapeutic effects of radiation in endometrial cancer patient-derived organoid models. Ongoing studies are correlating response to therapy in patient-derived organoid models with clinical patient outcomes. We are also performing pre- and post-treatment transcriptomic analyses to define gene signatures of response to radiation and cytotoxic chemotherapy in endometrial cancer organoid models. The ultimate goal is to use these heterogeneous preclinical models to identify pretreatment biomarkers of response that can be translated to a clinical diagnostic test. Citation Format: Sofia Gabrilovich, Kaitriana Powell-Smith, Hiruni Sumanasiri, Emily Symons, Emily Hill, Miles Pufall, Donna Santillan, Kristina W. Thiel. Modeling radiation sensitivity in patient-derived organoid models of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B008.

Abstract IA012: The role of the serine/threonine phosphatase PP2A in endometrial cancer development and progression: lessons learned from mutations and small molecules

Abstract Uterine cancer is the most common gynecologic malignancy in the United States with approximately 60,000 new diagnoses each year. While many cases of uterine endometrial carcinoma (EMCA) have favorable outcomes with recurrence-free long-term survival, outcomes for the high-grade, treatment-refractory histological subtypes, including uterine serous carcinoma (USC) and uterine carcinosarcoma (UCS), remain an important clinical problem. Uterine serous carcinomas and uterine carcinosarcomas tumors are the most aggressive subtypes of uterine cancer, displaying a high propensity to invade the peritoneal cavity and to metastasize to distant sites. Accordingly, 50-70% of high-grade tumors exhibit extrauterine spread at time of initial surgery and have limited treatment options. The inherent aggressiveness of these tumors suggests that they are intrinsically equipped to disseminate and propagate tumors at secondary sites. The molecular drivers of this phenotype, however, have yet to be completely characterized. A lack of established and validated molecular drivers of the high-grade subtypes, especially those that are responsible for the high rate of metastasis, has limited the use of targeted treatment strategies. However, we and other groups have shown that PPP2R1A, the scaffolding subunit of the tumor suppressor protein phosphatase 2A, is mutated in up to 40% of USC patients, and these recurrent mutations are found at both the primary and metastatic sites. These mutations are heterozygous, and two specific mutations: P179R and S256F, occur exclusively within these high-grade subtypes of uterine cancer (i.e., are not found in any other cancer type). PP2A, is a family of serine/threonine phosphatases, where the active heterotrimeric form of the enzyme is comprised of a catalytic (C) subunit, a scaffolding (A) subunit, and a substrate directing regulatory (B) subunit. In aggregate, PP2A is considered a tumor suppressor protein, but certain heterotrimers have tumor promoting roles. Here we present data related to the molecular and biological basis through which these recurrent pathogenic mutations drive endometrial cancer development and progression using disease relevant cellular and in vivo model systems. Additionally, building from previously published work from our group, we have developed a novel series of small molecule molecular glues (PMGs) that specifically correct the mutant heterotrimer driving tumor regressions in both cellular and patient derived xenograft models of high-grade endometrial cancer. Collectively, this data suggests that recurrent pathogenic mutations in the PPP2R1A gene drive endometrial cancer pathogenesis and these mutations can be therapeutically targeted using a series of pharmaceutically tractable small molecules. Citation Format: Goutham Narla. The role of the serine/threonine phosphatase PP2A in endometrial cancer development and progression: lessons learned from mutations and small molecules [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA012.

Estrogen Receptor (ER) and Progesterone Receptor (PgR) Expression in Endometrial Cancer—An Immunohistochemical Assessment

Endometrial cancer (EC) is one of the most common types of cancer in Poland and worldwide. Many risk factors lead to the pathogenesis of this disease, such as lifestyle choices, BMI, the medicines used in breast cancer therapy, and Lynch syndrome. EC cells show the expression of estrogen receptors (ERs) and progesterone receptors (PgR). These receptors occur in multiple isoforms and have a significant influence on the operation of cells. The loss of ER and PgR expression is associated with a poor prognosis. We assessed tissue slides that were obtained from 103 women with EC diagnoses of various grades, stages, and histological types. In this study, we used computer image analyses to increase the objectivity of the assessment. We proved that, in the tissue of patients with high-grade (G3) EC, the expression of PgR is significantly lower than that in the tissues of patients with low-grade EC. We also observed that PgR is significantly expressed in EC with a low FIGO stage and in the endometroid type of EC (which rarely becomes malignant compared to serous type). The expression of ERb1 was lower in patients with EC at the IV FIGO stage than in patients with stage III EC. These findings confirm that the loss of ER and PgR expression is connected with a poor prognosis.

Assessment of oncological safety and utility of hysteroscopy in high grade endometrial cancers: Results from an Israel gynecologic oncology group study

ObjectiveTo compare survival measures of women with Stage I high-grade endometrial cancer who underwent either hysteroscopy or a non-hysteroscopic procedure as a diagnostic procedure. Study design298 patients with stage I high grade endometrial cancer who underwent surgery between 2002 and 2014. Patients were divided into two groups: hysteroscopy and non-hysteroscopy (curettage or office endometrial biopsy). Clinical, pathological, and survival measures were compared between the groups. High grade histology included endometroid grade −3, uterine serous papillary carcinoma, clear cell carcinoma, and carcinosarcoma. ResultsThere were 71 patients in the hysteroscopy group and 227 patients in the non-hysteroscopy group. The median follow-up was 52 months (range 12–120 months). There were no differences between the groups in the 5-year recurrence-free survival (73.9 % vs. 79.7 %; p = 0.65), disease-specific survival (79.3 % vs. 83.6 %; p = 0.87), and overall survival (65.7 % vs. 80.3 %; p = 0.35). ConclusionHysteroscopic diagnosis in women with early-stage and high-grade endometrial cancer does not adversely affect the survival outcomes.

Abstract A023: Preclinical development of PKMYT1 and WEE1 inhibitor combinations

Abstract Background: CCNE1-amplification or overexpression are prevalent alterations in multiple tumor types, including high-grade serous ovarian, endometrial, and gastro-esophageal cancers. CCNE1-amplification is associated with high levels of replication stress, genomic instability, and resistance to chemotherapy. Previously, CRISPR-enabled synthetic lethal screening identified that loss of the CDK1 regulator Membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) is synthetic lethal with CCNE1-overexpression, leading to the development of lunresertib - a first-in-class, potent, selective, orally bioavailable PKMYT1 inhibitor currently being investigated in phase I clinical trials (NCT04855656, NCT05147272, NCT05147350). In CCNE1 overexpressing cells, lunresertib treatment leads to reduction of inhibitory CDK1-T14 phosphorylation and activated CDK1 in S-phase, triggering premature mitosis, chromosome pulverization, and ultimately cell death. In CCNE1-amplified tumor models, lunresertib exhibits antitumor activity as monotherapy. We hypothesized that further activation of CDK1 by reducing the inhibitory phosphorylation site at CDK1-pY15 by WEE1 inhibition would increase the antitumor activity of lunresertib. Methods: In vitro cell viability studies were used to determine the level of synergy between lunresertib and the WEE1 inhibitor Debio-0123 in engineered as well as tumor-derived CCNE1-high models. The lunresertib/Debio-0123 combination mechanism of action was investigated using high content imaging and immunoblotting for DNA damage and cell cycle markers in cultured cells. Finally, the pre-clinical anti-tumor activity and tolerability of combination lunresertib/Debio-0123 was evaluated in vivo using CCNE1-amplified tumor xenografts in mice. Results: Combination of low doses of lunresertib with Debio-0123 selectively induced synergistic cytotoxicity and catastrophic DNA damage in CCNE1-overexpressing cells, without affecting wild type counterparts. Increased DNA damage resulted from robust activation of CDK1 in S-phase, causing rapid induction of premature mitosis. Combination of lunresertib and Debio-0123 abrogated phosphorylation of both Thr14 and Tyr15 inhibitory CDK1 sites at doses that led to only partial dephosphorylation in both monotherapy settings. Mechanistically, synergistic CDK1 dephosphorylation was partially mediated by activation of CDC25B phosphatase. In a mouse model, the combination of RP-6306 and Debio-0123 was well-tolerated and demonstrated tumor regressions in a CCNE1-overexpressing tumor xenograft model at sub-therapeutic monotherapy doses. Conclusions: Lunresertib and the WEE1 inhibitor Debio-0123 elicit strong synergistic activity in preclinical models of CCNE1-overexpressoin due to rapid and complete CDK1 activation. In a xenograft tumor model, intermittent dosing of lunresertib/WEE1i lead to tumor regressions at sub-therapeutic monotherapy doses. Together these findings provide mechanistic rationale for clinical development of PKMYT1 and WEE1 inhibitor combinations. Citation Format: David Gallo, Fenil Shah, Rino Stocco, Prasamit Baruah, Jimmy Fourtounis, Rosie Kryczka, Marc L Hyer, Anne Roulston, C Gary Marshall, Michal Zimmermann, Stephen Morris, Michael Zinda. Preclinical development of PKMYT1 and WEE1 inhibitor combinations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A023.

Abstract A061: Understanding health-seeking delays for post-menopausal bleeding among Haitian women

Abstract Introduction: Black women have the highest risk of Endometrial cancer (EC) among any racial group in the US. When disaggregated, Haitian women specifically have been shown to have a higher incidence of high-grade EC, present at more advanced stages, and have lower overall survival compared to US-born Blacks. There is no screening test for EC, and women are diagnosed when they develop symptoms. The most common symptom of EC is postmenopausal bleeding (PMB). Little data exist about understanding/perception of PMB and subsequent care-seeking delays in this population of at-risk women. Utilizing the Safer and Anderson model of total patient delay, our objective was to qualitatively evaluate perceptions of PMB and how these may guide care-seeking behaviors among a healthy population of at-risk postmenopausal Haitian women. Methods: Nineteen women were included. Participants were 1) Haitian Black women; 2) ≥ 50 years of age; 3) living in South Florida; and 4) without a personal or family history of breast or gynecologic cancer. Participants were recruited through a mix of purposive and snowball sampling strategies via multiple channels (community outreach, radio advertisements, social media). Women participated in focus group discussions, conducted in Creole, and guided by a semi-structured interview guide designed to capture perceptions about PMB. Sessions were audio recorded, transcribed, and coded by a team of 5 raters. Dedoose software was used for content analyses and identification of emerging themes. Results: Three key themes that aligned primarily within the Safer and Andersen constructs of appraisal delay and illness delays were identified: 1) There was a significant lack of knowledge about the meaning of PMB. Resumption of fertility, PMB as a normal part of aging, and interpreting the symptom as reflective of a benign disease (e.g., food poisoning) were common. Cancer was not a primary concern in the setting of PMB, and there was consistent low perceived severity of the symptom. In fact, there was a conveyed sense of invulnerability to cancer purely due to the nature of being Haitian. 2) Women would preferentially use cultural-specific coping strategies in lieu of seeking immediate medical care. Such strategies included use of home remedies such as teas, and prayer/reliance on God. 3) Appraisal of PMB and discussion of severity involves consultation with spiritual leaders and others in the community for guidance. There was a perception that community members may dissuade the pursuit of traditional medical care. The use of the Internet as a facilitator for care was not considered primary. Conclusion: This study underscores the complexity of factors impacting potential help-seeking delays among Haitian women experiencing PMB. Our findings highlight the need for culturally-tailored, comprehensive educational interventions to increase awareness of the meaning of PMB and its health relevance to improve earlier care-seeking and timely diagnosis of EC. Citation Format: Maurice J. Chery, Vanessa Morales, Lashae Rolle, Alejandra Casas, Diana Zapata, Sophia H.L. George, Matthew P. Schlumbrecht. Understanding health-seeking delays for post-menopausal bleeding among Haitian women [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr A061.

Data-independent acquisition for proteomic applications in early-stage endometrial cancer progression.

Most endometrial cancer (EC) patients are diagnosed at an early-stage (FIGO stage I or II), with a favorable prognosis. However, some high-grade, early-stage EC patients have unexpected recurrences and undesirable results, the molecular alterations that underlie these tumors are far from being fully understood. Our goal was to use proteome analysis to examine dysregulated pathways in this specific subgroup of EC. We used data-independent acquisition (DIA) quantitative proteomics to analyze cancer and matched paracancerous tissues from 20 EC patients (10 high-grade and 10 low-grade). Immunohistochemistry (IHC) analysis was used to validate protein expression of six hub genes. In total, 7107 proteins were quantified, while 225 downregulated and 366 upregulated proteins in high-grade cancer tissues, 130 downregulated and 413 upregulated proteins in high-grade paracancerous tissues. The pathway associated with oxidative phosphorylation (OXPHOS) was upregulated and have similar expression patterns in high-grade EC tissues and matched paracancerous tissues. OXPHOS-related protein, ATP5F1D showed the best classification and diagnostic ability in distinguishing high-grade from low-grade EC. In both cancer and paracancerous tissues, double-label immunofluorescence demonstrated ITGA4 and COL4A1 co-localized at the basal membrane. Our present works elucidate that metabolism reprogramming is associated with high-grade, early-stage EC, particularly OXPHOS is upregulated. Noticeably, the paracancerous tissues have undergone molecular changes similar to cancer tissues, maybe they have signal exchange via secreted proteins (ITGA4 and COL4A1). The upregulation of OXPHOS-related proteins may be the potential biomarker for EC diagnosis, and targeting OXPHOS metabolism might be an effective treatment for high-grade, early-stage EC.

Urine metabolomics for assessing fertility-sparing treatment efficacy in endometrial cancer: a non-invasive approach using ultra-performance liquid chromatography mass spectrometry

ObjectiveThis study aimed to reveal the urine metabolic change of endometrial cancer (EC) patients during fertility-sparing treatment and establish non-invasive predictive models to identify patients with complete remission (CR).MethodThis study enrolled 20 EC patients prior to treatment (PT) and 22 patients with CR, aged 25–40 years. Eligibility criteria consisted of stage IA high-grade EC, lesions confined to endometrium, normal hepatic and renal function, normal urine test, no contraindication for fertility-sparing treatment and no prior therapy. Urine samples were analyzed using ultraperformance liquid chromatography mass spectrometry (UPLC-MS), a technique chosen for its high sensitivity and resolution, allows for rapid, accurate identification and quantification of metabolites, providing a comprehensive metabolic profile and facilitating the discovery of potential biomarkers. Analytical techniques were employed to determine distinct metabolites and altered metabolic pathways. The statistical analyses were performed using univariate and multivariate analyses, logistic regression and receiver operating characteristic (ROC) curves to discover and validate the potential biomarker models.ResultsA total of 108 different urine metabolomes were identified between CR and PT groups. These metabolites were enriched in ascorbate and aldarate metabolism, one carbon pool by folate, and some amino acid metabolisms pathways. A panel consisting of Baicalin, 5beta-1,3,7 (11)-Eudesmatrien-8-one, Indolylacryloylglycine, Edulitine, and Physapubenolide were selected as biomarkers, which demonstrated the best predictive ability with the AUC values of 0.982/0.851 in training/10-fold-cross-validation group, achieving a sensitivity of 0.975 and specificity of 0.967, respectively.ConclusionThe urine metabolic analysis revealed the metabolic changes in EC patients during the fertility-sparing treatment. The predictive biomarkers present great potential diagnostic value in fertility-sparing treatments for EC patients, offering a less invasive means of monitoring treatment efficacy. Further research should explore the mechanistic underpinnings of these metabolic changes and validate the biomarker panel in larger, diverse populations due to the small sample size and single-institution nature of our study.