Leiomyoma Research Articles

Preoperative identification from occult leiomyosarcomas in laparoscopic hysterectomy and laparoscopic myomectomy: accuracy of the ultrasound scoring system (PRESS-US).

To assess the diagnostic performance and inter-observer agreement of a PREoperative sarcoma scoring based on ultrasound (PRESS-US) in differentiating uterine leiomyosarcoma (uLMS) from leiomyoma (LM). We conducted a retrospective evaluation of patients who underwent surgery and received standardized ultrasound examinations due to the presence of uterine myoma-like masses. Histological diagnosis was used as the reference standard. The masses were analyzed using morphological uterus sonographic assessment criteria, and the diagnostic accuracy of PRESS-US was evaluated using ROC curve analysis. Kappa (κ) statistics were used to assess the inter-observer agreement between a less experienced and an expert radiologist. Among the 646 patients, 632 (97.8%) were diagnosed with LM, and 14 (2.2%) had uLMS. The malignancy rates for low-risk and high-risk patients were 0.35% and 13.48%, respectively. The optimal PRESS-US cutoff was 17.5, resulting in an AUC of 89.7% (95% CI, 0.79-1.00), with a sensitivity of 85.7% and a specificity of 87.8%. The inter-observer agreement between a less experienced and an expert radiologist was excellent (κ = 0.811, P < 0.001). PRESS-US provides effective risk stratification for uLMS for radiologists with different levels of experience, with high reliability. Subgrouping high-risk patients helps in better risk stratification.

Somatic MED12 Mutations in Myometrial Cells.

Over 70% of leiomyoma (LM) harbor MED12 mutations, primarily in exon 2 at c.130-131 (GG). Myometrial cells are the cell origin of leiomyoma, but the MED12 mutation status in non-neoplastic myometrial cells is unknown. In this study, we investigated the mutation burden of MED12 in myometrium. As traditional Sanger or even NGS sequencing may not be able to detect MED12 mutations that are lower than 0.1% in the testing sample, we used duplex deep sequencing analysis (DDS) to overcome this limitation. Tumor-free myometria (confirmed by pathology evaluation) were dissected, and genomic DNA from MED12 exon 2 (test) and TP53 exon 5 (control) were captured by customer-designed probe sets, followed by DDS. Notably, DDS demonstrated that myometrial cells harbored a high frequency of mutations in MED12 exon 2 and predominantly in code c.130-131. In contrast, the baseline mutations in other coding sequences of MED12 exon 2 as well as in the TP53 mutation hotspot, c.477-488 were comparably low in myometrial cells. This is the first report demonstrating a non-random accumulation of MED12 mutations at c.130-131 sites in non-neoplastic myometrial cells which provide molecular evidence of early somatic mutation events in myometrial cells. This early mutation may contribute to the cell origin for uterine LM development in women of reproductive age.

Age-related features of some plasminogen activation system components content and activity in the blood of benign and malignant uterine body tumors

Purpose of the study. Is to determine the features in the content and activity of some components of the plasminogen activation system in the blood of patients of different ages with leiomyomas (LM) and uterine corpus endometrial cancer (UCEC). Patients and methods. The study was carried out in patients with LM (n = 35) and UCEC T1a-2N0M0 (n = 56) of reproductive, perimenopausal and postmenopausal ages. Using ELISA methods, the content and activity of urokinase (u-PA), tissue plasminogen activator (t-PA), their inhibitor PAI-1, as well as the content of the soluble form of the u-PA receptor (su-PAR) were determined in the blood of patients. The Student´s test was used for statistical processing. Results. Regardless of the nature of the uterus tumor, it has been noted a significant increase in the activity and blood level of PAI-1 (up to 8 times, p &lt; 0.01), especially pronounced in patients with UCEC of reproductive age. It was combined with the absence of changes or with a decreasing in blood level of su-PAR by more than 40 % (p &lt; 0.05). We observed a rise of u-PA blood level without changes in one´s activity in patients with LM of perimenopausal age. And in patients with LM and UCEC of postmenopausal age an increase in u-PA blood level as well as elevation of u-PA activity (up to 3.9 times, p &lt; 0.01–0.05) were noted. There was an increase in the calculated t-PA index (activity per unit mass) by 1.4–2.8 times (p &lt; 0.05) in the most patients. The indicators of LM patients were characterized by the minimum value of the ratio “t-PA activity/u-PA activity in the blood” among all studied subgroups of patients. Age-related features of the researched parameters were observed more often in the cases of LM than in the cases of UCEC. The most pronounced differences between LM and UCEC were observed in patients of reproductive and postmenopausal age, characterized by stable hormonal levels. Conclusion. The participation of the plasminogen activation system in the pathogenesis of tumor lesions of the uterine body has been shown. The system’s “response” to the development of tumors in the uterus has both general characteristics and features that depend on the nature of the tumors and on the age-specific hormonal regulation of the body, which are most pronounced in postmenopausal women. The results obtained can be used in research aimed at clarifying the targets of targeted therapy for LM and UCEC in accordance with the age of patients.

O-264 Profiling Tumor-Initiating Cells in Uterine Leiomyoma through Single-Cell Multi-omics

Abstract Study question What are the molecular and cellular features that contribute to the transformation of myometrial stem cells into tumor-initiating cells within uterine leiomyoma? Summary answer Transformative changes in differentiation and genetic processes, in a distinct subset of M/LM cells drives their progression into a tumorigenic state. What is known already Uterine leiomyomas (LM), which are benign smooth muscle-derived tumours of myometrium, impact approximately 75% of women, causing significant physical and psychological challenges and imposing substantial healthcare costs. The widespread consensus postulates a monoclonal origin for LM, implying their derivation from a dysregulated single multipotent stem cell that could give rise to tumor-initiating cells (TICs). While previous studies have shown cellular heterogeneity in both myometrium (M) and LM, the precise identity of the originating cells is unknown. Study design, size, duration Prospective, observational, and biomedical study conducted at Hospital La Fe (Valencia, Spain) for one year. Eight sample pairs of LM and M underwent single-nuclei RNAseq (snRNA-seq; n = 16) and single-cell proteomic (scP; n = 16) analyses, to generate a detailed transcriptomic and proteomic map decoupled from cell type, state, and spatial location. We further employed single-cell RNA velocity inference for differentiation trajectory [BR1] analysis of mesenchymal cells, followed by the identification of potential driver genes in LM tumorigenesis. Participants/materials, setting, methods Upon obtaining informed consent, we procured LM and M samples from eight patients aged 35-50 undergoing hysterectomies. Part of these samples were preserved in paraffin for spatial transcriptomics utilizing VISIUM (10x Genomics). The remaining tissues were dissociated into single-cell suspensions and underwent scRNA-seq and scP using Chromium Controller and Orbitrap Eclipse Tribid mass spectrometry, respectively. The analysis of the collected data was carried out using publicly accessible R/Python tools. Main results and the role of chance Analysis of snRNAseq (∼52,000 cells) revealed similar cellular compositions in M and LM, including endothelial, perivascular, smooth muscle cells (SMC), fibroblasts, and immune cells. Further trajectory analysis unveiled a subset of myometrial cells with potential as TICs, exhibiting reduced expression of hormone receptors like PGR. Analysis of these cells [BR1] suggested differentiation into two distinct cell types -fibroblasts and SMCs- with elevated hormone expression over pseudotime. Differential analysis between M and LM also showed a dysregulated transcriptomic profile in the TICs within the tumor, as evidenced by upregulation of RAD51B and HMGA2 and downregulation PGR among other genes. These genes are indicative markers, underscoring their significant roles in the tumorigenesis of LM by influencing hormone response and disrupting DNA repair mechanisms. Spatial transcriptomic analysis confirmed these findings and suggested that the distribution of TICs could be ubiquitous, since the expression of potential myometrial-stem markers, was markedly increased only on certain spots within the tissue across both LM and M. Lastly, scProt profiling (∼5,000 cells) also allowed the identification of putative TICs, which showed upregulation in LM of specific proteins involved in replication, transcription, and translation. Limitations, reasons for caution Our study sets the basis for TICs isolation in LM, yet functional validation studies need to be performed to address the clinical potential of our findings. Further studies including more patients, and addressing racial disparities will help to generalize these findings to a broader population. Wider implications of the findings Our findings indicate the presence of a particular subset of M/LM cells whose changes in differentiation path and genetic processes seem to drive the transformation of these cells into a tumorigenic state to develop LM. Targeting these markers might show promise for effectively treating these tumors. Trial registration number NCT04214457

Профілактика ускладнень кесаревого розтину і консервативної міомектомії у вагітних із лейоміомою матки

Aim - to evaluate the effectiveness of the improved technique of cesarean section (CS) with conservative myomectomy (CME) using modern medical and instrumental means іn pregnant women with uterine leiomyoma (LM) type 3-6 by FIGO (International Federation of Gynecology and Obstetrics). Materials and methods. A fragment of a prospective study with a comprehensive examination and selection of optimal delivery tactics for pregnant women with LM at the clinical bases of the Ob/Gyn. Department No. 1 of Shupyk National Healthcare University of Ukraine for the period 2020-2023. The main group (MG, 1) of the study consisted of 37 pregnant women with LM &gt;5 cm type 3-6 according to FIGO, who underwent CS with CME according to an improved technique with the use of modern drugs: tranexamic acid, carbetocin and technical support (argon-plasma coagulation, radio-wave scalpel). The comparison group (CG, 2) included 28 pregnant women with LM &gt;5 cm type 3-6 according to FIGO with CS and CME performed according to the standard method. The control group (KG, 3) was made up of 17 pregnant women without LM, who underwent a planned CS. The reliability of the results was assessed using the Student's t-test. The difference of the obtained indicators was considered to be significant at p&lt;0.05. Results. The improved technique of CS with CME made it possible to reliably reduce the volume of blood loss in MG women compared to CG women (р1,2&lt;0.01) and was statistically insignificant with KG (р1,3&gt;0.05). A decrease in the frequency of complications during surgery and in the postpartum period in the main group was noted: a 2-fold decrease in purulent-inflammatory complications and a faster pace of uterine involution in the first 3 days of the postpartum period (р1,2&lt;0.05). Conclusions. Pregnant women with LM have an increased risk of bleeding, purulent-inflammatory complications, and the frequency of hysterectomies. The improved technique of performing CS with CME makes it possible to reduce the frequency of complications during surgery and postpartum period, preserve the organ in women of reproductive age and helps to avoid repeated surgery in the future for LM. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

Laryngeal leiomyoma: A case report and review of literature

BACKGROUND Leiomyomas (LMs) are mesenchymal tumors that arise from smooth muscle cells. LMs most commonly arise in organs with an abundance of smooth muscle such as the uterus and gastrointestinal tract. Conversely, LMs are rarely detected in the head and neck region. In this study, we report a rare case of laryngeal LM (LLM) and summarized the clinical characteristics of reported LLMs to help clinicians better understand this rare disease and improve its diagnosis, treatment, and postoperative course. CASE SUMMARY A 49-year-old man was admitted to our ENT outpatient clinic with a chief complaint of pharynx discomfort for 2 months. Laryngoscopy performed under topical anesthesia revealed a solitary, pink mass at the tubercle of epiglottis. Surgery via laryngeal endoscopy was performed under general anesthesia, and the lesion was excised easily. Positive immunohistochemical staining for desmin and smooth-muscle actin indicated a smooth muscle origin and the diagnosis was laryngeal leiomyoma. After surgery, the patient’s condition was stable, and he was discharged 2 d after surgery. During the 1-year postoperative period, the patient’s condition remained stable without evidence of recurrence. CONCLUSION Surgical resection is the preferred treatment for LLMs, its early diagnosis and differential diagnosis have important clinical significance.

Abstract 1751: Deep multi-omics analysis of uterine leiomyosarcoma reveals defective HRD signature, a novel gene fusion and the amplification of the poor-prognosis CTHRC1 gene

Abstract Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer representing approximately 2-5% of all uterine malignancies. The molecular heterogeneity and pathogenesis of uLMS are notwell understood. To unravel the intricate molecular landscape of uLMS, we conducted a multi-omics workflow consisting of 4 high-throughput sequencing technologies; short- and long-readwhole genome sequencing (WGS), RNA-Seq, and Tandem mass tag (TMT) labeling enablingmassively parallel proteome proteomic analyses. Our study cohort consisted of 97 fresh frozentissue samples collected from 68 female patients, with 19 uLMS, 37 leiomyoma (LM), and 41normal myometrium (MM). A deep analysis of somatic variants with clinical impact revealed 46variants present in 90% of samples and two actionable therapeutic targets: IDH1_p.Arg132Cysand KRAS_p.Gly12Val. Also, 80% of the samples presented a chromothripsis signature and 60%presented the SBS3 COSMIC signature related to homologous recombination deficiency (HRD).Analysis of the proliferation score in RNA-Seq data revealed a notable difference among thetumor groups, signifying a higher proliferation index in the uLMS group, suggesting a moreaggressive growth pattern with implications for clinical behavior and disease progression. Afterapplying a customized workflow to identify balanced chromosomal abnormalities of genefusions with clinical impact, the proto-oncogene eukaryotic elongation factor 1 alpha (EEF1A1)emerged as a prominent fusion partner. Eight fusions in 7 samples were identified withtranscriptional overexpression in positive cases. At the proteomic level the enrichment analysisunveiled activated pathways associated with the innate immune system, while suppressedpathways pertained to extracellular matrix (ECM) organization and smooth muscle contraction.With an integrated bioinformatics pipeline, we identified copy number amplification of theCTHRC1 gene in 80% of the samples, accompanied by increased gene expression and proteinlevels. This gene is related to collagen remodeling and degradation and has been demonstratedto be upregulated in some solid tumors and is closely associated with malignancy. Survivalcurve analysis highlighted a significantly poorer prognosis in uLMS exhibiting CTHRC1 geneamplification with potential clinical relevance of CTHRC1 amplification as a prognostic indicatorin uLMS. Taken together these results show the overall suppression of ECM and collagenremodeling in uLMS associated with poor prognosis. Additionally, these data provide the firstevidence that the EEF1A1 gene has a strong selective pressure in uLMS underscoring theindispensable role of precision genomics in advancing precision medicine. Citation Format: Raul Maia Falcao, Jorge Estefano de Souza, William Mathieson, Joseph Woodward Carlson, Tirzah Braz Petta. Deep multi-omics analysis of uterine leiomyosarcoma reveals defective HRD signature, a novel gene fusion and the amplification of the poor-prognosis CTHRC1 gene [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 1751.

MRI, clinical, and radiomic models for differentiation of uterine leiomyosarcoma and leiomyoma.

To assess the predictive ability of conventional MRI features and MRI texture features in differentiating uterine leiomyoma (LM) from uterine leiomyosarcoma (LMS). This single-center, IRB-approved, HIPAA-compliant retrospective study included 108 patients (69 LM, 39 LMS) who had pathology, preoperative MRI, and clinical data available at our tertiary academic institution. Two radiologists independently evaluated 14 features on preoperative MRI. Texture features based on 3D segmentation were extracted from T2W-weighted MRI (T2WI) using commercially available texture software (TexRAD™, Feedback Medical Ltd., Great Britain). MRI conventional features, and clinical and MRI texture features were compared between LM and LMS groups. Dataset was randomly divided into training (86 cases) and testing (22 cases) cohorts (8:2 ratio); training cohort was further subdivided into training and validation sets using ten-fold cross-validation. Optimal radiomics model was selected out of 90 different machine learning pipelines and five models containing different combinations of MRI, clinical, and radiomics variables. 12/14 MRI conventional features and 2/2 clinical features were significantly different between LM and LMS groups. MRI conventional features had moderate to excellent inter-reader agreement for all but two features. Models combining MRI conventional and clinical features (AUC 0.956) and MRI conventional, clinical, and radiomics features (AUC 0.989) had better performance compared to models containing MRI conventional features alone (AUC 0.846 and 0.890) or radiomics features alone (0.929). While multiple MRI and clinical features differed between LM and LMS groups, the model combining MRI, clinical, and radiomic features had the best predictive ability but was only marginally better than a model utilizing conventional MRI and clinical data alone.

FOXO3a deregulation in uterine smooth muscle tumors

ObjectiveThe present study aimed to investigate FOXO3a deregulation in Uterine Smooth Muscle Tumors (USMT) and its potential association with cancer development and prognosis. MethodsThe authors analyzed gene and protein expression profiles of FOXO3a in 56 uterine Leiomyosarcomas (LMS), 119 leiomyomas (comprising conventional and unusual leiomyomas), and 20 Myometrium (MM) samples. The authors used techniques such as Immunohistochemistry (IHC), FISH/CISH, and qRT-PCR for the present analyses. Additionally, the authors conducted an in-silico analysis to understand the interaction network involving FOXO3a and its correlated genes. ResultsThis investigation revealed distinct expression patterns of the FOXO3a gene and protein, including both normal and phosphorylated forms. Expression levels were notably elevated in LMS, and Unusual Leiomyomas (ULM) compared to conventional Leiomyomas (LM) and Myometrium (MM) samples. This upregulation was significantly associated with metastasis and Overall Survival (OS) in LMS patients. Intriguingly, FOXO3a deregulation did not seem to be influenced by EGF/HER-2 signaling, as there were minimal levels of EGF and VEGF expression detected, and HER-2 and EGFR were negative in the analyzed samples. In the examination of miRNAs, the authors observed upregulation of miR-96-5p and miR-155-5p, which are known negative regulators of FOXO3a, in LMS samples. Conversely, the tumor suppressor miR-let7c-5p was downregulated. ConclusionsIn summary, the outcomes of the present study suggest that the imbalance in FOXO3a within Uterine Smooth Muscle Tumors might arise from both protein phosphorylation and miRNA activity. FOXO3a could emerge as a promising therapeutic target for individuals with Unusual Leiomyomas and Leiomyosarcomas (ULM and LMS), offering novel directions for treatment strategies.

From Elegance to Complexity: The Intricate Dance of Myometrial Stem Cells Transforming into Leiomyoma Stem Cells: Unraveling the Symphony of Genetic and Epigenetic Refinement

Uterine leiomyomas (LM), commonly referred to as uterine fibroids (UFs), pelvic tumor in women of reproductive age globally and are main indicators of hysterectomies if become symptomatic. The progression of uterine LM involves a complex interplay of hormones, stem cells, growth factors, and genetic/epigenetic irregularities. Both endometrial and myometrial stem/progenitor cells play pivotal roles in the uterus’ response to hormonal stimuli and return to its basal state. LM, arising from a single stem cell of smooth muscle cells with acquired mutations, exhibit diverse cellular transcriptomic patterns influenced by genotype. Affecting over 70% of women at some point in their lives, with significant clinical symptoms, such as pelvic pain, infertility, and heavy uterine bleeding. Recent literature underscores the potential importance of LM stem cells and their paracrine interactions with specialized cells, connecting the gap between medications targeting leiomyoma expansion and potential eradication strategies. Keeping these points in view, this review discusses the current understanding of the involvement of myometrial stem/progenitor cells and the genetic and epigenetic changes occurring in these cells during the pathogenesis of uterine LM.

Survivin-Sodium Iodide Symporter Reporter as a Non-Invasive Diagnostic Marker to Differentiate Uterine Leiomyosarcoma from Leiomyoma.

Leiomyosarcoma (LMS) has been challenging to diagnose because of limitations in clinical and radiographic predictors, as well as the lack of reliable serum or urinary biomarkers. Most uterine masses consist of benign leiomyoma (LM). However, it is currently a significant challenge in gynecology practice to differentiate LMS from LM. This inability poses grave consequences for patients, leading to a high number of unnecessary hysterectomies, infertility, and other major morbidities and possible mortalities. This study aimed to evaluate the use of Survivin-Sodium iodide symporter (Ad-Sur-NIS) as a reporter gene biomarker to differentiate malignant LMS from benign LM by using an F18-NaBF4 PET/CT scan. The PET/CT scan images showed a significantly increased radiotracer uptake and a decreased radiotracer decay attributable to the higher abundance of Ad-Sur-NIS in the LMS tumors compared to LM (p < 0.05). An excellent safety profile was observed, with no pathological or metabolic differences detected in Ad-Sur-NIS-treated animal versus the vehicle control. Ad-Sur-NIS as a PET scan reporter is a promising imaging biomarker that can differentiate uterine LMS from LM using F18-NaBF4 as a radiotracer. As a new diagnostic method, the F18 NaBF4 PET/CT scan can provide a much-needed tool in clinical practices to effectively triage women with suspicious uterine masses and avoid unnecessary invasive interventions.

THU625 Med12 Mutation Activates Tryptophan-Kynurenine-ahr Pathway To Promote Growth Of Uterine Leiomyoma

Abstract Disclosure: A. Zuberi: None. Y. Huang: None. A. Dotts: None. H. Wei: None. J. Coon V: None. T. Lizuka: None. O. Wu: None. O. Sotos: None. D. Chakravarti: None. Y. Dai: None. S.E. Bulun: None. P. Yin: None. A.Z. and Y.H. contributed equally to this work.† Y.D., S.E.B., and P.Y. are co-senior authors. ‡To whom correspondence should be addressed: Serdar E. Bulun, M.D.Division of Reproductive Science in MedicineDepartment of Obstetrics and Gynecology Feinberg School of Medicine at Northwestern University 250 E. Superior Street, Suite 03-2306 Chicago, Illinois 60611-2914 Tel: 312-472-3636 Fax: 312-472-3740 Email: s-bulun@northwestern.edu Ping Yin, M.D., Ph.D. Division of Reproductive Science in Medicine Department of Obstetrics and Gynecology Feinberg School of Medicine at Northwestern University 303 E. Superior Street, Suite 10-111 Chicago, Illinois 60611 Tel: 312-503-1831 Fax: 312-503-0095 Email: p-yin@northwestern.edu. Conflict of interest: The authors have declared that no conflict of interest exists. Uterine leiomyomas (LM) cause heavy menstrual bleeding, anemia and pregnancy loss in approximately 10 million US women. Driver mutations in the mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% LM tumors that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 and a shift of binding sites from proximal promoters to intronic/intergenic regions in mutant (mut-MED12) vs wild-type LM tissues. Integration of cistrome and transcriptome data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene, which was massively upregulated in mut-MED12 LM. TDO2 catalyzes the conversion of tryptophan to kynurenine, which is an aryl hydrocarbon receptor (AHR) ligand and significantly elevated in mut-MED12 LM. Incubation of primary mut-MED12 LM cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis and induced AHR-target gene expression, whereas blocking the TDO2-kynurenine-AHR pathway by siRNA or pharmacologically abolished these effects. Progesterone receptor regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for LM growth. In summary, activation of the TDO2-kynurenine-AHR pathway selectively in mut-MED12 LM induces tumor growth and may inform the development of targeted treatments and precision medicine. Presentation: Thursday, June 15, 2023

THU631 Steroyl-CoA Desaturase Is A Progesterone Receptor Target Gene In Uterine Leiomyoma

Abstract Disclosure: A.S. Komorowski: None. A. Zuberi: None. J.S. Coon V: None. M.L. Anton: None. S.E. Bulun: None. P. Yin: None. Uterine leiomyomas (LM) are common benign neoplasms affecting approximately one in four reproductive-age women. LM can cause menorrhagia, dysmenorrhea, anemia, and infertility. Steroyl-CoA desaturase (SCD) is a delta-9 fatty acid desaturase which converts saturated fatty acids to monounsaturated fatty acids in a reaction that is critical to maintenance of fatty acid homeostasis. Prior work has demonstrated upregulation of SCD in multiple cancer types as part of lipid metabolic reprogramming, and SCD has been identified as a potential drug target for cancer and non-alcoholic steatohepatitis. Growth of LM tumors is progesterone-dependent, and we found that progesterone receptor (PR)-binding sites were enriched in the SCD gene locus. Normalized RNA-seq count demonstrated that knockdown of PR decreased SCD expression in LM cells. Further experiments with knockdown of PR in LM cells demonstrated decreased SCD expression on both quantitative real-time PCR and Western blot. Treatment of LM cells with progesterone for 6 hours lead to mild upregulation of SCD expression. In summary, steroyl-CoA desaturase, an enzyme involved in lipid homeostasis, is upregulated by progesterone and may represent a novel target for treatment of uterine LM. Presentation: Thursday, June 15, 2023

A contrast-enhanced CT-based radiomic nomogram for the differential diagnosis of intravenous leiomyomatosis and uterine leiomyoma.

Uterine intravenous leiomyomatosis (IVL) is a rare and unique leiomyoma that is difficult to surgery due to its ability to extend into intra- and extra-uterine vasculature. And it is difficult to differentiate from uterine leiomyoma (LM) by conventional CT scanning, which results in a large number of missed diagnoses. This study aimed to evaluate the utility of a contrast-enhanced CT-based radiomic nomogram for preoperative differentiation of IVL and LM. 124 patients (37 IVL and 87 LM) were retrospectively enrolled in the study. Radiomic features were extracted from contrast-enhanced CT before surgery. Clinical, radiomic, and combined models were developed using LightGBM (Light Gradient Boosting Machine) algorithm to differentiate IVL and LM. The clinical and radiomic signatures were integrated into a nomogram. The diagnostic performance of the models was evaluated using the area under the curve (AUC) and decision curve analysis (DCA). Clinical factors, such as symptoms, menopausal status, age, and selected imaging features, were found to have significant correlations with the differential diagnosis of IVL and LM. A total of 108 radiomic features were extracted from contrast-enhanced CT images and selected for analysis. 29 radiomics features were selected to establish the Rad-score. A clinical model was developed to discriminate IVL and LM (AUC=0.826). Radiomic models were used to effectively differentiate IVL and LM (AUC=0.980). This radiological nomogram combined the Rad-score with independent clinical factors showed better differentiation efficiency than the clinical model (AUC=0.985, p=0.046). This study provides evidence for the utility of a radiomic nomogram integrating clinical and radiomic signatures for differentiating IVL and LM with improved diagnostic accuracy. The nomogram may be useful in clinical decision-making and provide recommendations for clinical treatment.

O-188 Decoding uterine leiomyoma tumorigenesis using single-cell transcriptomics and single-cell proteomics

Abstract Study question Can single-cell transcriptomics and proteomics contribute to a better understanding uterine of leiomyoma tumorigenesis? Summary answer We demonstrate the significant involvement of the MAPK, PI3K-Akt, and proteoglycan pathways in smooth muscle, endothelial and perivascular cells in leiomyoma tumorigenesis. What is known already Uterine leiomyomas (LM), also known as fibroids, are benign tumors of the uterus that arise from the myometrium. Previous studies have shown cellular heterogeneity in both myometrium and uterine leiomyomas, although cell spatial location within the tissue has not been shown. Further, no data describing single-cell-level proteomic differences and key pathways involved in uterine LM tumorigenesis have yet been reported. Study design, size, duration A prospective, observational, and biomedical study of cohorts was conducted at Hospital La Fe (Valencia, Spain) for one year. Single-cell RNAseq (scRNA-seq; n = 16) and single-cell proteomic (scP; n = 16) analyses were performed on eight sample pairs of LM and matched myometrium (MM), to generate a high-resolution transcriptomic and proteomic map decoupled from cell type, state, and spatial location. Participants/materials, setting, methods After obtaining informed consent, LM and MM samples were collected from eight patients between 35-50 years undergoing hysterectomies. Part of the samples were preserved in paraffin for spatial transcriptomics using VISIUM (10x Genomics). While the remaining tissues were dissociated into single-cell suspensions and subjected to Chromium Controller and Orbitrap Eclipse Tribid mass spectrometry for scRNA-seq and scP, respectively. All data were analyzed using publicly available R/Python tools. Main results and the role of chance After restrictive quality control filtering, we analyzed a total of 52,599 and 5,909 cells by scRNA-seq and scP, respectively. While LM and MM possessed similarities in terms of cellular composition, they displayed differential expression of genes and proteins across all the cell populations studied, particularly in smooth muscle, endothelial and perivascular cells. In LM samples, these cell populations displayed impaired MAPK signaling, which acts as a signal integrator for growth factors, estrogen, and vitamin D. We also observed alterations in the PI3K-Akt and proteoglycan pathways in LM smooth muscle and perivascular clusters, which relate to cell proliferation and tumor growth. Additionally, we encountered a subset of consistently dysregulated genes in all LM populations, which may suggest the existence of a shared tumorigenic pathway independent of cell type. Spatial transcriptomics further demonstrated relationships between cells and their relative locations within the tissue, which we validated by immunofluorescence. Together, our results highlight the relevance of specific cell populations in LM tumorigenesis. Limitations, reasons for caution This study involved a sample cohort limited to Caucasian women; therefore, further studies including more patients, and addressing racial disparities will help to generalize these findings to a broader population. Wider implications of the findings Our work describes an unprecedented transcriptomic and proteomic analysis of LM and MM at single-cell resolution, which supports a novel understanding of myometrial tumorigenesis. Trial registration number NCT04214457

MMP14 expression and collagen remodelling support uterine leiomyosarcoma aggressiveness.

Fibrillar collagen deposition, stiffness and downstream signalling support the development of leiomyomas (LMs), common benign mesenchymal tumours of the uterus, and are associated with aggressiveness in multiple carcinomas. Compared with epithelial carcinomas, however, the impact of fibrillar collagens on malignant mesenchymal tumours, including uterine leiomyosarcoma (uLMS), remains elusive. In this study, we analyse the network morphology and density of fibrillar collagens combined with the gene expression within uLMS, LM and normal myometrium (MM). We find that, in contrast to LM, uLMS tumours present low collagen density and increased expression of collagen-remodelling genes, features associated with tumour aggressiveness. Using collagen-based 3D matrices, we show that matrix metalloproteinase-14 (MMP14), a central protein with collagen-remodelling functions that is particularly overexpressed in uLMS, supports uLMS cell proliferation. In addition, we find that, unlike MM and LM cells, uLMS proliferation and migration are less sensitive to changes in collagen substrate stiffness. We demonstrate that uLMS cell growth in low-stiffness substrates is sustained by an enhanced basal yes-associated protein 1 (YAP) activity. Altogether, our results indicate that uLMS cells acquire increased collagen remodelling capabilities and are adapted to grow and migrate in low collagen and soft microenvironments. These results further suggest that matrix remodelling and YAP are potential therapeutic targets for this deadly disease.

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.

Gene Expression Profile of Uterine Leiomyoma from Women Exposed to Different Air Pollution Levels in Metropolitan Cities of Sao Paulo, Brazil.

Leiomyomas (LMs) are the most frequent uterine benign tumors, representing the leading cause of hysterectomy indications worldwide. They are highly associated with women's reproductive complications, and endocrine disruptors may influence their etiology. In this sense, air pollution represents a relevant hormonal disruptor that acts on key signaling pathways, resulting in tumor development and infertility. Our goal was to evaluate submucosal LM samples from patients living in the metropolitan and Sao Paulo city regions, focusing on genes involved in tumor development and infertility features. Twenty-four patients were selected based on their region of residence and clinical information availability. Several genes were differentially expressed between women living in metropolitan areas and Sao Paulo city. Significant associations were observed between BCL-2, DVL1, FGFR3, and WNT5b downregulation and contraceptive use in the samples from women living in Sao Paulo city. ESR1 and HHAT downregulation was associated with ethnicity. WNT5b and GREM were associated with LM treatment and related pathologies, respectively. In the samples from women living in other cities of the metropolitan region, abortion occurrence was associated with BMP4 upregulation. Although further studies may be necessary, our results showed that air pollution exposure influences the expression of genes related to LM development and female reproductive features.

Accidental Morcellation of Uterine Leiomyosarcoma Influences Relapse Free Survival but Does Not Negatively Influence Overall Survival.

Background: Uterine leiomyosarcoma (LMS) is a rare entity amongst malignant gynaecological tumours and is mostly diagnosed after surgery for benign leiomyoma (LM) of the uterus. As minimal invasive surgery is widely used, the morcellation of LM and the uterus is rather common. As there is little known about the impact of the morcellation of LMS on local and distant metastases, as well as overall survival, we carried out a large-scale retrospective study. Methods: A total of 301 LMS cases from the German Clinical Competence Centre for Genital Sarcomas and Mixed Tumours were analysed. We distinguished morcellated and non-morcellated LMS from pT1 and >pT1 tumours. Fine−Gray competing risks regressions and cumulative incidence rates were computed for the time to local recurrence, distant metastases, and patient death. Results: The recurrence free interval in pT1 LMS was significantly lower in the morcellation group with a 2-year cumulative incidence rate of 49% vs. 26% in non-morcellated LMS (p = 0.001). No differences were seen in >pT1 tumours. Distant metastases were more frequently found in non-morcellated pT1 LMS compared to the morcellated cases (5-year cumulative incidence: 54% vs. 29%, p < 0.001). There was no significant difference in time to death between both groups neither in the pT1 stages nor in >pT1 disease. Subdistribution hazard ratios estimated by multivariable competing risks regressions for the morcellation of pT1 LMS were 2.11 for local recurrence (95% CI 1.41−3.16, p < 0.001) and 0.52 for distant metastases (95% CI 0.32−0.84, p = 0.008). Conclusions: Tumour morcellation is not associated with OS for pT1 tumours. The morcellation of pT1 LMS seems to prolong the time to distant metastases whereas local recurrence is more likely to occur after the morcellation of pT1 LMS.

The Expression of the Immunoproteasome Subunit PSMB9 Is Related to Distinct Molecular Subtypes of Uterine Leiomyosarcoma.

Simple SummaryUterine leiomyosarcoma (uLMS) is a rare, aggressive, and highly heterogeneous tumor. Knockout female mice for the catalytic subunit of the immunoproteasome PSMB9 develops spontaneous uLMS. In this study, we used molecular data from 3 non-related uLMS cohorts that were integrated and analyzed by proteotranscriptomics. We observed overexpression of the immunoproteasome pathway in uLMS, and then further classified the samples as low or high PSMB9 gene expression levels and we provide evidence that; (i) in the group high there is an enrichment of pathways related to the immune system and in the group low, the ECM formation; (ii) samples with high CD8+/PSMB9 ratio shows better OS; and (iii) the main regulator in the high group is IFNγ and in the low, the proto-oncogene SRC. These findings contribute to the understanding of potential therapeutic or prognostic markers in uLMS.Background: Uterine leiomyosarcoma (uLMS) are rare and malignant tumors that arise in the myometrium cells and whose diagnosis is based on histopathological features. Identifying diagnostic biomarkers for uLMS is a challenge due to molecular heterogeneity and the scarcity of samples. In vivo and in vitro models for uLMS are urgently needed. Knockout female mice for the catalytic subunit of the immunoproteasome PSMB9 (MIM:177045) develop spontaneous uLMS. This study aimed to analyze the role of PSMB9 in uLMS tumorigenesis and patient outcome. Methods: Molecular data from 3 non-related uLMS cohorts were integrated and analyzed by proteotranscriptomic using gene expression and protein abundance levels in 68 normal adjacent myometrium (MM), 66 uterine leiomyoma (LM), and 67 uLMS. Results: the immunoproteasome pathway is upregulated and the gene PMSB9 shows heterogeneous expression values in uLMS. Quartile group analysis showed no significant difference between groups high and low PSMB9 expression groups at 3-years overall survival (OS). Using CYBERSORTx analysis we observed 9 out of 17 samples in the high group clustering together due to high M2 macrophages and CD4 memory resting, and high CD8+/PSMB9 ratio was associated with better OS. The main pathway regulated in the high group is IFNγ and in the low is the ECM pathway dependent on the proto-oncogene SRC. Conclusion: these findings suggest 2 subtypes of uLMS (immune-related and ECM-related) with different candidate mechanisms of malignancy.