Uterine Sarcoma Research Articles

Combined inhibition of EZH2 and histone deacetylases as a potential epigenetic therapy for human uterine sarcoma cells.

e17620 Background: Uterine sarcoma is strongly associated with poor prognosis, high rates of recurrence, and metastasis. However, the molecular mechanisms underlying the origin and development of uterine sarcoma and treatment options are limited. Enhancer of zeste homolog 2 (EZH2) and histone deacetylase (HDACs) play an important role in cancer progression. This study aimed to assess the effect of targeting both EZH2 and HDACs on the phenotype of uterine sarcoma (US) cells. Methods: Tazemetostat (EPZ-6438, E7438), a potent, and selective EZH2 inhibitor, was first approved by the US Food and Drug Administration (FDA) in 2020. Entinostat (MS-275, SNDX-275) is one of the strong inhibitors for HDAC1 and HDAC3, Vorinostat is used to treat cutaneous T-cell lymphoma, Tucidinostat (Chidamide, HBI-8000, CS-055) is a low nanomolar inhibitor of HDAC1, 2, 3, and 10. Uterine sarcoma cell line (MES-SA) was treated with different concentrations (1.56-200 mM) of Tazemetostat and/or Entinostat, Vorinostat, Tucidinostat (Chidamide). for 24, 48, 72 h, respectively. Further mechanistic confirmation was carried out on best combination in both 2D and 3D culturing using IHC and real time PCR. Results: Our data showed that Entinostat synergistically (with a combination index 0.539) augments the cytotoxic activity of Tazemetostat in both 2D and spheroids in vitro model systems. The IC50 values for Tazemetostat were significantly reduced to 4.5 mM after combination treatment with 6.25 mM Entinostat for 48 h. Entinostat enhanced the antiproliferative effect of Tazemetostat with a decrease in the expression of proliferative markers Ki67(P < 0.0001) and PCNA (P < 0.01). Furthermore, combination treatment significantly increased US cell apoptosis, concomitantly with increased both BAX and Caspase-3 levels and decreased Bcl-2 (P < 0.0001) using 2D and spheroid in vitro model (P < 0.001). The finding was confirmed by IHC analysis in the US cell-derived spheroids. Conclusions: Our studies demonstrate that Entinostat, in combination with Tazemetostat proves significantly superior to either single treatment. Dual targeting EZH2 and HDACs may provide a promising treatment option for this aggressive cancer disease.

Uterine sarcoma patients who underwent morcellation: Who are they? Results of the prospective intergroup real-world registry for gynecological sarcoma in Germany (REGSA- NOGGO RU1).

e17632 Background: Uterine sarcomas represent a group of rare malignant mesenchymal neoplasms with most types of them being associated with poor prognosis. In clinical practice, it is challenging to distinguish them from the most common benign tumors in women, so-called leiomyomas. Due to erroneous diagnosis, there is a risk of morcellating an occult sarcoma. This often leads to the distribution of malignant tissue in the abdominal cavity and a worse prognosis in most of the patients. In order to better understand this high-risk group of patients we analyzed their characteristics and management based on data of the real-world German registry for gynecological sarcoma (REGSA). Methods: REGSA is a real-world multicenter prospective registry including patients with gynecologic sarcomas in the primary and the recurrent situation of disease. For this work, we performed a descriptive analysis with data from all available patients with documented morcellation of uterine sarcoma. Results: 8.7% (n=76) of a total of 874 patients (registration period 09/2015-05/2022) were morcellated. Those 76 patients had a median age of 50.5 years. Thirty-seven patients (48.7%) were ≥50 years of age at the time of surgery. Leiomyosarcoma was the most common histopathological subtype (n=42; 50.3%). Fifty-eight patients (76.3%) had laparoscopic operations. A subtotal hysterectomy was the most common procedure used in 40.8% (n=31). Diagnostic curettage was performed in 5.3% of all cases (n=4). The predominant preoperative symptom was "bleeding disorders" in 19 patients (25.0%). Follow-up treatment after morcellation was heterogeneous. 17 patients received chemotherapy, with docetaxel and gemcitabine being the most frequently used regimen (n=8; 47.1%), followed by combinations with anthracyclines in 5 patients (29.4%). Conclusions: Many patients were over 50 years of age at the time of morcellation, although it is undisputed that the risk of morcellating occult uterine sarcoma increases with age. Whether curettage has diagnostic value needs to be further investigated. There is a high clinical need to define a consensus on further management of this high-risk group, regarding adjuvant therapy and follow-up care for there seems to be a great deal of uncertainty regarding adjuvant management.

Molecular tumor profiling and therapy selection in advanced gynecological cancers: A retrospective cohort analysis from the Australian Molecular Screening and Therapeutics (MoST) Program.

5526 Background: Gynecological (gyne) cancers are highly diverse with distinct sites of origin and histological subtypes that can limit development of evidence-based therapies. Comprehensive genomic profiling (CGP) has an increasing role in characterizing clinically relevant molecular subsets and in identifying actionable biomarkers with potential to guide therapy selection. Methods: MoST (ACTRN12616000908437) is an Australia-wide precision oncology program for adult patients (pts) with advanced cancers and limited treatment options. Tumor specimens are analysed by CGP and genomic results tiered by the level of evidence supporting matched therapies (https://topograph.info). Pts are followed for subsequent treatment and survival. Here, we present data for the MoST gyne cancer cohort. Results: Between Sept 2016 and Oct 2022, 533 gyne tumors were sequenced, with included pts having a minimum 4 months (mo) follow-up. Key histotypes included: ovarian serous (n=162) and non-serous (n=46) epithelial tumors; uterine carcinomas (n=99) and sarcomas (n=108); cervical carcinomas (n=51); germ cell and sex-cord stromal tumors (n=18); carcinosarcomas (n=33); and vulva/vaginal tumors (n=16). The median number of reported variants was 3 (IQR 2-5) and tumor mutational burden 3.1 mut/Mb (IQR 1.3—5.5, Range 0—284); 9 (2%) tumors were microsatellite unstable; and 22 (4%) had high level loss-of-heterozygosity (confirmed on validated genome-wide assays). The most common pathways involved in this pan-gyne cohort were: p53 (n=287, 54%), PI3K/AKT (n=227, 43%), cell cycle (n=182, 34%), and mitogen-activated protein kinase (n=91, 17%). Pathogenic mutations in BRCA1/2 were identified in 29 tumors (5%; 18 in ovarian serous cancers), whereas 81 tumors (15%) had alterations in other homologous recombination genes, most commonly uterine sarcomas (n=32, 30%). Actionable genomic biomarkers matched to a clinically active treatment (TOPOGRAPH tier 1-3) were identified in 207 (39%) tumors. In 19 pts (3.5%) who received Tier 1-3 matched therapy after CGP, a trend towards longer survival was observed (median OS 22.3 mo, 95% CI 17.3 to not reached) compared to those receiving unmatched therapy (n=65, median OS 14.9 mo, 10.2 to 22.1, p=0.052) following CGP. No OS difference was seen in pts who received matched investigational therapy (Tier 3B/4, n=31, median OS 17.0 mo, 8.1 to NR) versus only unmatched therapy (n=118, median OS 14.0 mo, 11.3 to 22.5; HR 0.97, 0.57 to 1.64; p=0.91). Conclusions: CGP identified actionable genomic results in nearly half of advanced gyne cancers, with enrichment in particular histotypes that supports testing above current standard of care. Trends in prolonged OS with subsequent matched therapies may be better realized with earlier testing and improved drug/clinical trial access.

E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways.

E7050 is an inhibitor of VEGFR2 with anti-tumor activity; however, its therapeutic mechanism remains incompletely understood. In the present study, we aim to evaluate the anti-angiogenic activity of E7050 in vitro and in vivo and define the underlying molecular mechanism. It was observed that treatment with E7050 markedly inhibited proliferation, migration, and capillary-like tube formation in cultured human umbilical vein endothelial cells (HUVECs). E7050 exposure in the chick embryo chorioallantoic membrane (CAM) also reduced the amount of neovessel formation in chick embryos. To understand the molecular basis, E7050 was found to suppress the phosphorylation of VEGFR2 and its downstream signaling pathway components, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. Moreover, E7050 suppressed the phosphorylation of VEGFR2, FAK, Src, Akt, JNK, and p38 MAPK in HUVECs exposed to MES-SA/Dx5 cells-derived conditioned medium (CM). The multidrug-resistant human uterine sarcoma xenograft study revealed that E7050 significantly attenuated the growth of MES-SA/Dx5 tumor xenografts, which was associated with inhibition of tumor angiogenesis. E7050 treatment also decreased the expression of CD31 and p-VEGFR2 in MES-SA/Dx5 tumor tissue sections in comparison with the vehicle control. Collectively, E7050 may serve as a potential agent for the treatment of cancer and angiogenesis-related disorders.

High negative prediction for the Basel sarcoma score: Sonographic assessment of features suspicious of uterine sarcoma

IntroductionIn the management of uterine myomas, laparoscopic surgery with morcellation enables a minimal invasive procedure. Cases of unsuspected uterine sarcoma dissemination have been reported and led to regulative restrictions. To help to distinguish preoperatively myomas from sarcomas, we assessed the value of six sonographic criteria (Basel Sarcoma Score, BSS) in a prospective outpatient cohort of consecutive patients with uterine masses. Material and methodsWe prospectively evaluated all patients presenting with myoma-like masses planned for surgery with standardized ultrasound examination. BSS including the following criteria was investigated: rapid growth in past three months, high blood flow, atypical growth, irregular lining, central necrosis and oval solitary lesion. For each criterion, a score 0/1 was given. BSS (0–6) equals the sum of all given scores. Histological diagnosis was used as reference. ResultsAmong 545 patients, 522 had the final diagnosis of myoma, 16 had peritoneal masses with sarcomatous components (PMSC), and seven had other malignancies. Median BSS for PMSC was 2.5 (range: 0–4) vs 0 for myomas (range: 0–3). The most common sonographic criteria leading to a false positive score in myomas were rapid growth in past three months and high blood flow. For the detection of sarcomatous masses with BSS threshold of >1, sensitivity was 93.8%, specificity 97.9%, and positive predictive value (PPV) and negative predictive value (NPV) were 57.7% and 99.8%, respectively (AUC 0.95). ConclusionBSS can help distinguishing between myomas and sarcomatous masses, with high NPV. Caution is required when >1 criterion is present. As a simple tool, it could easily be integrated into routine myoma sonographic examination and help develop standardized assessment of uterine masses for better preoperative triage.

Prognostic factors in patients with uterine sarcoma: the SARCUT study

ObjectiveUterine sarcomas are a rare and heterogeneous group of malignancies that include different histological sub-types. The aim of this study was to identify and evaluate the impact of the different...

Prognostic factors in undifferentiated uterine sarcoma: a subanalysis of the SARCUT study.

The aim of this study was to analyze the prognostic factors related to the recurrence rate and overall survival of patients with undifferentiated uterine sarcoma. An international multicenter study involving 43 international centers, the SARCUT study, collected 966 uterine sarcoma cases; among them 39 cases corresponded to undifferentiated uterine sarcoma and where included in the present subanalysis. The risk factors related to the oncological outcomes where analyzed. The median age of the patients was 63 (range 14-85) years. Seventeen (43.5%) patients presented FIGO stage I. The 5-year overall survival (OS) was 15.3% and 12-months disease-free survival (DFS) 41%. FIGO stage I was significantly associated with a better prognosis. In addition, patients who received adjuvant radiotherapy showed significant longer disease-free survival compared to those without adjuvant radiotherapy (20.5 vs. 4.0months, respectively; p = 0.04) and longer overall survival (34.7 vs. 18.2months, respectively; p = 0.05). Chemotherapy administration was associated with shorter DFS (HR 4.41, 95% CI 1.35-14.43, p = 0.014). Persistent disease after primary treatment (HR = 6.86, 95% CI 1.51-31.09, p = 0.012) and FIGO stage IV (HR 4.12, 95%CI 1.37-12.44, p = 0.011) showed significant worse prognosis for OS. FIGO stage seems to be the most important prognostic factor in patients with undifferentiated uterine sarcoma. Adjuvant radiotherapy seems to be significantly associated also to a better disease-free and overall survival. On the contrary, the role of chemotherapy administration remains unclear since was associated to a shorted DFS.

Pathological characteristics and immune microenvironment of SMARCA4-deficient undifferentiated uterine sarcoma

SMARCA4-deficient undifferentiated uterine sarcoma (SDUS) is a highly invasive single-gene malignant tumor caused by mutations in the SMARCA4 gene. SDUS has a poor prognosis, with no established treatment strategy at present. Further, there is a lack of relevant research on the role of the immune microenvironment in SDUS worldwide. Here, we report a case of SDUS that was diagnosed and analysed using morphological, immunohistochemical, and molecular detection techniques, along with the analysis of the immune microenvironment. By immunohistochemistry, the tumor cells showed retained INI-1 expression, focal CD10 expression, and loss of BRG1, CK-pan, synaptophysin, desmin, and ER expression. Further, some of the immune cells expressing CD3 and CD8 had infiltrated into the SDUS, but no PD-L1 expression was detected. The multiple immunofluorescent staining results showed that a proportion of the immune cells and SDUS cells expressed CD8/CD68/PD-1/PD-L1. Therefore, our report will help in the diagnostic awareness of SDUS.

Pancancer Analysis Revealed the Value of RAC2 in Immunotherapy and Cancer Stem Cell.

To investigate the oncogenic effect and clinical significance of RAC2 in pancarcinoma from the perspective of tumor immunity and cancer stem cell. After in-depth mining of TCGA, GEO, UCSC, and other databases, basic information of the RAC2 gene and its expression in tumor tissues as well as the relationship between RAC2 and tumor were analyzed based on survival, mutation, immune microenvironment, tumor stemness, and enrichment analysis on related pathways. RAC2 mRNA expression was increased in most tumor tissues and was associated with their prognosis. Compared to normal tissues, the RAC2 mutation rate was higher in patients with skin melanoma, uterine sarcoma, and endometrial cancer. RAC2 had a strong relation with immune cell infiltration, immunomodulators, immunotherapy markers, cancer stem cell of THYM, and immune-related pathways. This study explored the potential importance of RAC2 in the prognosis, immunotherapy, and cancer stem cell of 33 cancers, laying the foundation for mechanistic experiments and its future application in clinical practice. However, the results using bioinformatics methods could be affected by the differences in patients across databases. Thus, the present results were preliminary and required further experimental validation.

Lymph node dissection in uterine leiomyosarcomas: A matched-pair study

ObjectiveTo assess the impact of the lymph node dissection (LND) in the disease-free (DFS) and overall survival (OS) of the women treated surgically of uterine leiomyosarcoma (ULMS). Material and methodsA multicentric retrospective study was conducted among European countries collecting patients diagnosed of uterine sarcoma (SARcoma of the UTerus - SARCUT study). A total of 390 ULMS were selected for the present study to compare patients who underwent LND and those who did not. A further matched-pair subanalysis identified 116 women, 58 pairs (58 with LND and 58 without it) comparable in age, tumor size, surgical procedures, extrauterine disease and adjuvant treatment. Demographic data, pathology results and follow-up were abstracted from medical records and analyzed. Disease-free (DFS) and overall survival (OS) were studied using Kaplan-Meier curves and Cox regression analysis. ResultsAmong the 390 patients, the 5-year DFS was significantly higher in no-LDN group comparing to the LDN group (57.7% vs. 33.0%; HR 1.75, 95% CI 1.19–2.56; p = 0.007), but not the 5-year OS (64.6% vs. 64.3%; HR 1,10 95% CI 0,77–1,79; p = 0.704). In the matched-pair subanalysis, there were no statistical differences between the study groups. The 5- year DFS was 50.5% in the no-LND and 33.0% in the LND group (HR 1.38; 95% CI 0,83–2.31; p = 0,218) and the 5-year OS was 59.7% and 64.3% respectively (HR 0.81; 95% CI 0,45–1,49; p = 0,509). ConclusionsLND performed in women diagnosed of ULMS have no impact neither in the disease-free nor in the overall survival compared to patients without LDN in a complete homogeneous group.

PO-1563 The role of postoperative radiotherapy in uterine sarcomas: a single institution analysis.

PO-1563 The role of postoperative radiotherapy in uterine sarcomas: a single institution analysis.

PO-1428 Treatment outcomes in Uterine Sarcomas: A Real-world experience

PO-1428 Treatment outcomes in Uterine Sarcomas: A Real-world experience

The Functional Role and Regulatory Mechanism of FTO m6A RNA Demethylase in Human Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis and high rates of recurrence and metastasis. The origin and molecular mechanism underlying and driving its clinical and biological behavior remain largely unknown. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications in contributing to the pathogenesis of uLMS. However, the connection between reversible m6A RNA methylation and uLMS pathogenesis remains unclear. In this study, we assessed the role and mechanism of FTO m6A RNA demethylase in the pathogenesis of uLMS. Immunohistochemistry analysis revealed that the levels of RNA demethylases FTO and ALKBH5 were aberrantly upregulated in uLMS tissues compared to adjacent myometrium with a significant change by histochemical scoring assessment (p < 0.01). Furthermore, the inhibition of FTO demethylase with its small, potent inhibitor (Dac51) significantly decreased the uLMS proliferation dose-dependently via cell cycle arrest. Notably, RNA-seq analysis revealed that the inhibition of FTO with Dac51 exhibited a significant decrease in cell-cycle-related genes, including several CDK members, and a significant increase in the expression of CDKN1A, which correlated with a Dac51-exerted inhibitory effect on cell proliferation. Moreover, Dac51 treatment allowed the rewiring of several critical pathways, including TNFα signaling, KRAS signaling, inflammation response, G2M checkpoint, and C-Myc signaling, among others, leading to the suppression of the uLMS phenotype. Moreover, transcription factor (TF) analyses suggested that epitranscriptional alterations by Dac51 may alter the cell cycle-related gene expression via TF-driven pathways and epigenetic networks in uLMS cells. This intersection of RNA methylation and other epigenetic controls and pathways provides a framework to better understand uterine diseases, particularly uLMS pathogenesis with a dysregulation of RNA methylation machinery. Therefore, targeting the vulnerable epitranscriptome may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer.

The Role of MicroRNAs in Uterine Leiomyosarcoma Diagnosis and Treatment.

Uterine sarcomas are rare gynecological tumors arising from the myometrium or the connective tissue of the endometrium with a relatively poor prognosis. MicroRNAs (miRNAs) represent small, single-stranded, non-coding RNA molecules that can function as oncogenes or tumor suppressors under certain conditions. The current review aims at studying the role of miRNAs in uterine sarcoma diagnosis and treatment. In order to identify relevant studies, a literature review was conducted using the MEDLINE and LIVIVO databases. The search terms "microRNA" and "uterine sarcoma" were employed, and we were able to identify 24 studies published between 2008 and 2022. The current manuscript represents the first comprehensive review of the literature focusing on the particular role of miRNAs as biomarkers for uterine sarcomas. miRNAs were found to exhibit differential expression in uterine sarcoma cell lines and interact with certain genes correlating with tumorigenesis and cancer progression, whereas selected miRNA isoforms seem to be either over- or under-expressed in uterine sarcoma samples compared to normal uteri or benign tumors. Furthermore, miRNA levels correlate with various clinical prognostic parameters in uterine sarcoma patients, whereas each uterine sarcoma subtype is characterized by a unique miRNA profile. In summary, miRNAs seemingly represent novel trustworthy biomarkers for the diagnosis and treatment of uterine sarcoma.

Development of an Algorithm to Differentiate Uterine Sarcoma from Fibroids Using MRI and LDH Levels

This study aimed to establish an evaluation method for detecting uterine sarcoma with 100% sensitivity using MRI and serum LDH levels. One evaluator reviewed the MRI images and LDH values of a total of 1801 cases, including 36 cases of uterine sarcoma and 1765 cases of uterine fibroids. The reproducibility of the algorithm was also examined by four evaluators with different imaging experience and abilities, using a test set of 61 cases, including 14 cases of uterine sarcoma. From the MRI images and LDH values of 1801 cases of uterine sarcoma and uterine fibroids, we found that all sarcomas were included in the group with a high T2WI and either a high T1WI, an unclear margin, or high LDH values. In addition, when cases with DWI were examined, all sarcomas had high DWI. Among the 36 sarcoma cases, the group with positive findings for T2WI, T1WI, margins, and serum LDH levels all had a poor prognosis (p = 0.015). The reproducibility of the algorithm was examined by four evaluators and the sensitivity of sarcoma detection ranged from 71% to 93%. We established an algorithm to distinguish uterine sarcoma if tumors in the myometrium with low T2WI and DWI are present.

Uterine Endometrial Stromal Tumors With Pure Low-Grade Morphology Harboring YWHAE::NUTM2 Fusions: Report of a Case Series Emphasizing Potential for High-Grade Transformation and Aggressive Behavior.

Uterine endometrial stromal sarcomas (ESS) with YWHAE::NUTM2 gene fusions are typically morphologically high-grade tumors composed of atypical round cells, sometimes associated with a low-grade fibromyxoid component; they are currently included in the category of high-grade ESS (HGESS). We report 5 morphologically pure low-grade endometrial stromal tumors harboring YWHAE::NUTM2 fusions, including 1 endometrial stromal nodule (ESN) and 4 low-grade endometrial stromal sarcomas (LGESS), an association only occasionally reported previously. Patients ranged from 30 to 51 (mean=43) years and tumors from 4.5 to 7.5cm (mean=5.7). All were stage I at diagnosis (confined to the uterus). Microscopically, the 4 LGESS showed extensive "tongue-like" invasion of the myometrium, and the ESN was entirely confined to the endometrium with no myometrial invasion. All tumors were composed entirely of morphologically uniform bland ovoid cells resembling proliferative endometrial stroma. A fibromyxoid component was seen in 1 LGESS and the ESN; in the LGESS, this was the sole component. Atypical round cells characteristic of YWHAE::NUTM2 HGESS were not identified. Mitotic count ranged from <1 to 13 per 10 high-power fields (mean: 3). CD10 was positive in 2/4 (focal), estrogen receptor in 5/5 (focal=1; diffuse=4), progesterone receptor in 5/5 (focal=1; diffuse=4) and cyclin D1 was diffusely positive in 3/4. Follow-up was available in all 5 patients and ranged from 6 to 159 months (mean=72). Two patients with LGESS had recurrent disease at 15 and 155 months; 1 showed predominantly LGESS with rare round cells in the initial recurrence and pure HGESS in a subsequent recurrence, while the other patient's recurrent tumor was predominantly HGESS (90%) in a background of focal fibromyxoid LGESS (10%). Both patients rapidly progressed and died of disease within 5 months of high-grade recurrence. We show that rare cases of morphologically pure low-grade endometrial stromal tumors, some but not all with a fibromyxoid component, harbor YWHAE::NUTM2 fusions and may recur rapidly, with transformation to HGESS and aggressive behavior. Our findings suggest that at least a subset of YWHAE::NUTM2 HGESS evolves from LGESS. We suggest that cyclin D1 and CD10 staining should be performed in all LGESS. Diffuse staining for cyclin D1 and/or negative or focal staining for CD10 should suggest the possibility of a YWHAE::NUTM2 fusion, and appropriate molecular testing should be undertaken. Since no single morphological or immunohistochemical parameter is entirely sensitive for fusion status, we also suggest that testing for a YWHAE::NUTM2 gene fusion should be considered in all cases of LGESS and, if a fusion is present, this should raise the possibility of subsequent high-grade transformation and aggressive behavior, even though such cases should still be categorized as LGESS. Although seemingly rare, ESN and LGESS with YWHAE::NUTM2 fusions may be under-recognized due to a lack of routine fusion testing.

Abstract 6232: Ulipristal acetate, a selective progesterone receptor modulator, induces cell death via inhibition of STAT3/CCL2 signaling pathway in uterine sarcoma

Abstract Purpose: Ulipristal acetate (UPA) is used for the treatment of uterine leiomyoma as a selective progesterone receptor modulator. Until now, there has been no experimental research on the effect of UPA on uterine sarcoma. In this study, we examined the efficacy of UPA in uterine sarcoma with in vitro and in vivo animal models. Experimental Design: The cytotoxicity of UPA was determined in uterine sarcoma cell lines (MES-SA, SK-UT-1, and SK-LMS-1) by MTT and caspase-3 activity assays. A migration assay was done using a Transwell chamber, and MMP-2 was measured with its specific ELISA kit. Apoptotic genes and signaling pathways affected by UPA were analyzed by complementary DNA (cDNA) microarray of uterine sarcoma cell lines and western blot, respectively. The in vivo efficacy of UPA was examined in uterine sarcoma cell line- and patient-derived xenograft mice models. Results: UPA inhibited cell growth and increased apoptosis in cell lines including uterine sarcoma (MES-SA) and leiomyosarcoma (SK-UT-1 and SK-LMS-1) in a dose-dependent manner. UPA also inhibited cell migration and the expression of MMP-2. cDNA microarray analysis revealed that CCL2 was commonly down-regulated by UPA in investigated cell lines. The UPA-mediated inhibition of CCL2 expression was confirmed by ELISA. The phosphorylation and the total expression of STAT3, downstream from the CCL2 signaling pathway, was also inhibited by UPA. We also found that UPA reduced cell growth and increased caspase-3 activity in primary culture cells from leiomyosarcoma patient-derived xenografts (PDX-C). Moreover, we found that STAT3/CCL2 inhibition by UPA was independent of the expression of progesterone receptor. In vivo studies with MES-SA and SK-UT-1 cell line-derived xenografts and a PDX model with leiomyosarcoma demonstrated that UPA significantly decreased tumor growth. Conclusions: UPA had significant anti-tumor effects in uterine sarcoma through the STAT3/CCL2 signaling pathway and might be a potential therapeutic agent to treat this disease. Citation Format: Jae Ryoung Hwang, Young-Jae Cho, Ji-Yoon Ryu, Ju-Yeon Choi, Jung-Joo Choi, Jeong-Won Lee. Ulipristal acetate, a selective progesterone receptor modulator, induces cell death via inhibition of STAT3/CCL2 signaling pathway in uterine sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6232.

Abstract 6021: Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas

Abstract Background: Uterine leiomyosarcoma (LMS) comprises 60% to 70% of the uterine sarcoma (US). These tumors present high rates of recurrence and metastasis, and the patients’ survival rates are very low (20% in five years). It is known that somatic mutations and modifications in the DNA methylation levels are associated with several types of neoplasms. Previously, our group identified missense and loss of function mutations in KMT2D, CREBBP, ATM, TSC2, and GNAS, using a genetic screening method in LMS samples. Based on these results, we decided to investigate whether a genetic and epigenetic crosstalk could help to understand the biological processes involved in the LMS origin, development, and pathogenesis. The present study aimed to evaluate the KMT2D, CREBBP, ATM, TSC2 and GNAS gene expression profile in LMS cell line, and their methylation levels in formalin-fixed and paraffin-embedded (FFPE) patients´ samples. Methods: We selected 15 LMS - FFPE samples obtained via surgical procedures performed between 2012 and 2019 at the Instituto do Cancer do Estado de São Paulo (ICESP). Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit and we evaluated methylation levels using the Illumina Infinium Methylation EPIC BeadChip system 850k, including samples treated with bisulfite-treated DNA. Leiomyoma (LM) (THESCs CRL-4003) and LMS (SK-UT-1) cell lines were cultivated for gene expression analysis. Cells were growth for 24, 48, 72, and 96 hours, and the total RNA was extracted by TRIzol. High-Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis and for gene expression evaluation, real-time PCR reactions were performed using TaqMan Universal PCR Master Mix and inventoried TaqMan probes. Results: KMT2D and TSC2 showed increased expression in LMS compared to LM (cut-off ≤ -2 and ≥ 2 for down and upregulation, respectively), with higher expression in 24 hours of KMT2D [Fold Regulation (FR): 2.77] and TSC2 (FR: 2.58). In contrast, CREBBP was downregulated, with lowest expression in 72 hours (FR: -6.57). ATM was upregulated at 24, 48 with higher expression in 72 hours (FR: 2.32), and GNAS was downregulated, with lowest expression in 96 hours (FR: -4.31). Moreover, the methylation analyzes showed an expressive hypomethylation in LMS samples, compared to myometrium, for KMT2D (β value: 0.45; p= 0.004); CREBBP (β value: 0.51 p &amp;lt; 0.0001); ATM (β value: 0.47 p &amp;lt; 0.0001); TSC2 (β value: 0.53 p &amp;lt; 0.0001) and GNAS (β value: 0.41 p &amp;lt; 0.0001). In conclusion, our study showed that potentially pathogenic mutations may be associated with an aberrant hypomethylation profile, as well as increased expression of KMT2D, TSC2, ATM and loss expression of CREBBP and GNAS in LMS. The interconnection of these genetic and epigenetic events is essential for understanding the complex biology of these tumors, in addition to enabling identification of biomarkers. Citation Format: Laura G. dos Anjos, Daniela Bizinelli, Katia C. Carvalho. Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6021.

Abstract 996: Identification of molecular biomarkers differentiating malignant uterine leiomyosarcoma from benign leiomyoma

Abstract Uterine leiomyosarcomas are rare, aggressive cancers that represent the most common type of uterine sarcomas. They frequently harbor genetic alterations in TP53, RB1, PTEN, and ATRX. Their benign counterpart, uterine leiomyomas (or fibroids) are very common tumors that may significantly affect women’s quality of life. The majority of leiomyomas harbor genetic alterations affecting either MED12, HMGA2, or FH. A third of leiomyosarcomas have been reported to harbor leiomyoma-associated driver mutations, suggesting that some leiomyomas may have undergone malignant transformation. The leiomyoma/leiomyosarcoma diagnosis is largely based on histopathology. This can be specifically challenging in differentiating leiomyosarcomas from a subset of leiomyomas that display histopathological features resembling malignancy. The aim of this study was to identify molecular biomarkers that can accurately differentiate benign and malignant uterine smooth muscle tumors. 3’RNA sequencing of 48 leiomyosarcomas and 44 leiomyomas revealed that leiomyosarcomas are uniquely characterized by dysregulation of the retinoblastoma pathway. We confirmed upregulation of several members of this pathway, including TOP2A and CDK1, in a publicly available transcriptomic dataset of 49 uterine leiomyosarcomas. The second objective of this study was to explore the gene expression profile of leiomyosarcomas carrying leiomyoma-associated driver mutations. This revealed that unlike leiomyomas, leiomyosarcomas do not form distinct gene expression profiles based on the presence of leiomyoma-associated driver mutations such as MED12. However, leiomyosarcomas with a MED12 mutation showed upregulation of RAD51B and ADAM12, which we have previously highlighted as biomarkers of leiomyomas harboring a MED12 mutation. We identified upregulation of IRS4 and characteristic deletions affecting COL4A5 and COL4A6 in four in-house and three additional uterine leiomyosarcomas from The Cancer Genome Atlas (TCGA) collection. Similar driver alterations have previously been reported in a small subset of leiomyomas but not in leiomyosarcomas. In conclusion, leiomyosarcomas display a gene expression profile characterized by dysregulation of genes related to the retinoblastoma pathway. These include potential biomarkers that could be used in a clinical setting to differentiate leiomyosarcomas from leiomyomas. Citation Format: Sara Khamaiseh, Riitta Koivisto-Korander, Terhi Ahvenainen, Ralf Bützow, Miika Mehine, Pia Vahteristo. Identification of molecular biomarkers differentiating malignant uterine leiomyosarcoma from benign leiomyoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 996.

Abstract 6585: OncoKB, MSK’s precision oncology knowledge base

Abstract OncoKB, Memorial Sloan Kettering Cancer Center’s (MSK) precision oncology knowledge base (www.oncokb.org), is an FDA-recognized* somatic variant database that contains information about the oncogenic effect and clinical implications of genomic alterations in cancer. Since its 2016 public release, OncoKB has grown to include annotation for &amp;gt;5,770 alterations in ~700 cancer-associated genes. OncoKB data is integrated into the cBioPortal for Cancer Genomics and used to annotate &amp;gt;12,000 MSK patient sequencing reports annually, encompassing both solid tumor and hematological malignancies. Users in academic, commercial and hospital settings outside MSK can programmatically access OncoKB data via its web API with an OncoKB license, which is free for academic research. To date, users from ~ 1400 institutions across &amp;gt;70 countries have licensed access to OncoKB annotations. The OncoKB Therapeutic (Tx) Levels of Evidence assign tumor-type specific clinical actionability to individual mutational events based on data supporting whether an alteration is predictive of response to matched targeted therapies. To date, OncoKB includes 44 Level 1 genes (included in the FDA drug label), 23 Level 2 genes (included in professional guidelines), 33 Level 3A genes (predictive of drug response in well-powered clinical studies), 27 Level 4 genes (predictive of drug response based on compelling biological evidence), and 11 R1/R2 resistance genes. In 2022, several major content additions were made to OncoKB based on key shifts in the precision oncology landscape. For example, OncoKB included 2 new tumor-agnostic FDA drug approvals, dabrafenib + trametinib and selpercatinib for BRAF V600E and RET fusion-positive solid tumors respectively (Level 1), capturing 5 tumor-agnostic FDA drug approvals to date. OncoKB promoted ERBB2 oncogenic mutations and FGFR1 fusions to Level 1 following their inclusion as patient eligibility criteria in FDA drug labels for trastuzumab deruxtecan (NSCLC) and pemigatinib (myeloid/lymphoid neoplasms) respectively. NCCN guidelines for uterine sarcoma and pancreatic cancer listed PARP-inhibition for BRCA-mutant disease, making them Level 2 in these indications. Lastly, previously considered undruggable targets, TP53 Y220C and KRAS G12D, were included in OncoKB based on compelling evidence demonstrating response to allele-targeting drugs, PC14586 and RMC-6263, respectively. In sum, 7 novel clinically actionable biomarkers (Levels 1-4) and 11 follow-on precision oncology therapies for existing leveled biomarkers were added to OncoKB in 2022. Current OncoKB efforts are focused on prioritized high-volume cancer gene curation for annotation of whole exome/genome data, annotation of germline alterations and development of a clinical trials matching system. *FDA recognition of OncoKB is partial and limited to the information clearly marked on www.oncokb.org. Citation Format: Sarah P. Suehnholz, Moriah Nissan, Hongxin Zhang, Ritika Kundra, Calvin Lu, Amanda Dhaneshwar, Nicole Fernandez, Stephanie Carrero, Maria E. Arcila, Marc Ladanyi, Michael F. Berger, Aijazuddin Syed, Rose Brannon, Ross Levine, Ahmet Dogan, Ezra Rosen, Alexander Drilon, David B. Solit, Nikolaus Schultz, Debyani Chakravarty. OncoKB, MSK’s precision oncology knowledge base. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6585.