Genomic insights into uterine leiomyosarcoma: Unraveling KMT2D, CREBBP, NOTCH2, TSC2, ATM, and GNAS signatures.

Uterine Leiomyosarcoma (ULMS), characterized by diagnostic and therapeutic challenges, remains poorly understood despite advancements. Previous research identified genetic mutations in ULMS samples, suggesting their role in pathogenesis. This study deepened the exploration, focusing on specific genes (KMT2D, CREBBP, NOTCH2, ATM, TSC2, GNAS), validating mutations, examining gene methylation, and correlating with clinical features. Thirty samples, including 15 Formalin-Fixed Paraffin-Embedded (FFPE) and 15 fresh-frozen ULMS, were collected, comprising 3 ULMS, 3 Uterine Leiomyomas (ULM), 3 Myometrial samples (MM), and 6 other Uterine Sarcomas (US) for each sample set. Two cell lines, ULM (THESCs - CRL ‒ 4003) and ULMS (SK-UT-1 ‒ HTB-114), were used for comprehensive analysis. FFPE samples underwent genomic DNA extraction using the QIAamp DNA FFPE Tissue Kit, and methylation levels were assessed using the Illumina Infinium Methylation EPIC BeadChip 850k system. Genomic DNA and RNA were extracted using the Trizol method. qRT-PCR with TaqMan assays analyzed gene expression. Mutations in CREBBP, NOTCH2, and GNAS were sequenced using the Sanger method. Results indicated downregulation in CREBBP, GNAS, and NOTCH2 genes in ULMS. Sequencing revealed specific mutations (e.g., c.4063G>A in CREBBP, T>A at position 78 in NOTCH2). A pronounced reduction in CpG region methylation suggested a unique epigenetic alteration in ULMS. Methylation with clinical outcomes identified ATM as a potential independent prognostic biomarker. In conclusion, this study provides a comprehensive view of ULMS molecular complexity, potentially influencing clinical practices.