The highly heterogeneous properties and dynamic immunogenic features of osteosarcoma (OS) lead to limited therapeutic efficacy. The oncogenetic mechanisms of OS await further explanation, and new insights into therapeutic targets for OS remain urgent. We used single-cell RNA-sequencing data to identify cell type-specific differentially expressed genes between normal human osteocytes and OS cells. Subsequently, we analyzed the differential expression of leptin receptor (LEPR) in OS cell lines through in vitro experiments. We assessed the prognostic potential of LEPR and immune cell infiltration status associated with differential LEPR expression in OS patients. By integrating protein quantitative trait loci and methylation quantitative trait loci datasets with OS genome-wide association studies summary statistics, we performed Mendelian randomization analysis to elucidate the causal effect of LEPR on OS risk and identify cytosine-phosphate-guanine sites that may influence LEPR expression and OS risk. We observed lower LEPR expression in OS cell clusters compared to normal osteocyte clusters. RT-qPCR results indicated that the LEPR gene showed consistent differential expression in OS cells. Survival analysis revealed that lower LEPR expression was associated with worse survival outcomes in OS patients. OS patients with high LEPR expression tended to exhibit more immune cell infiltration. Mendelian randomization analysis indicated that genetically-determined LEPR was inversely associated with OS risk. We identified 8 methylation cytosine-phosphate-guanine sites within the LEPR promoter region causally associated with both lower LEPR protein expression and higher OS risk. Our integrated multi-omics analyses supported a causal association between LEPR expression and OS risk, suggesting that LEPR may offer protective benefits for OS patients. The relationship between LEPR and OS risk highlights the potential of LEPR as an early prognostic biomarker and may provide new insights into the etiology of OS.

Trabectedin, prototype of the ecteinascidin class of drugs, is a known second or later line therapeutic option for advanced soft tissue sarcoma (STS). We evaluated the antitumor activity of two novel synthetic trabectedin derivatives, ecubectedin and PM54, in selected patient-derived xenograft (PDX) STS models. In total 364 NMRInu/nu mice were transplanted bilaterally with two leiomyosarcoma (LMS), two dedifferentiated liposarcoma (DDLPS), one synovial sarcoma (SynSa) and one CIC-rearranged sarcoma (CRS) PDX models. Mice were randomized to six groups and treated via tail vein injection with 1) vehicle, 2) doxorubicin, 3) trabectedin, 4) lurbinectedin, 5) ecubectedin, or 6) PM54. Treatment was given on days 1, 8 and 15, and mice were sacrificed on day 16. The SynSa experiment included extra mice to investigate post-treatment xenograft evolution. Antitumor activity was assessed by tumor volume measurement, histopathologic and immunohistochemical analysis. In all LMS and DDLPS models, ecubectedin and PM54 led to tumor growth delay compared to trabectedin. Histopathological evaluation of treated tumors showed moderately increased antitumor activity of novel ecteinascidins compared to trabectedin. The CRS showed tumor shrinkage in response to ecubectedin (63% regression from baseline) and PM54 (24% regression from baseline) while the SynSa showed tumor volume stabilization. Both translocation-related models showed significant antitumor effect on histopathological evaluation in response to the novel drugs, compared to trabectedin. Ecubectedin and PM54 have modest antitumor activity in STS PDX models, with the strongest effects seen in the translocation-related sarcoma models, showing the potential of this class of drugs in treatment of STS.

Ewing sarcoma (EwS) is characterized by chimeric fusions such as EWSR1::FLI1. Chromosome gain or loss is prevalent in EwS, and this chromosome instability (CIN) phenotype correlates with poor treatment outcomes. We previously showed that CENP-A, a centromere-specific histone H3 variant, interacts with EWSR1 via its SYGQ2 motif, an interaction extending to the EWSR1::FLI1 fusion protein. CENP-A is highly expressed in EwS cells, driven by EWSR1::FLI1. EwS cells exhibit significant CIN, mitigated by depleting EWSR1::FLI1 or CENP-A, and frequently assemble neocentromeres. Using CENP-A ChIP-seq, we identify neocentromere-associated DNA sequences, and ChIP assays confirm EWSR1::FLI1 binds these sequences, suggesting EWSR1::FLI1 recruits CENP-A to drive neocentromere assembly. Notably, overexpressing EWSR1::FLI1, but not CENP-A, induces neocentromeres in non-EwS cells. These neocentromeres likely produce dicentric chromosomes, explaining the high incidence of chromosome bridges and chromoplexy underlying the CIN phenotype in EwS.

Myoepithelial carcinoma is an uncommon malignant tumor of neoplastic myoepithelial cells. Myoepithelial carcinoma may arise in salivary glands or within soft tissues, and myoepithelial carcinomas of salivary and soft tissue types are generally regarded as distinct. Fusions involving EWSR1 are a molecular hallmark of soft tissue myoepithelial carcinomas; however, salivary myoepithelial carcinomas typically lack these fusions. We report an unusual high-grade myoepithelial carcinoma arising in the submandibular gland of a man in his 20s, initially diagnosed as extraskeletal Ewing sarcoma due to an EWSR1 rearrangement identified by fluorescence in situ hybridization (FISH). The resection specimen demonstrated a round cell tumor with multiple growth patterns and high-grade features. No benign pleomorphic adenoma component was identified. Tumor cells were positive for pankeratin, S100, and SOX10, and were only weakly positive for CD99. Next-generation sequencing unearthed an EWSR1::CREB1 fusion, which has been identified in a handful of diverse soft tissue neoplasms. While EWSR1 signal rearrangements by FISH have been described, a bona fide fusion involving EWSR1 is novel, if this tumor does indeed represent a myoepithelial carcinoma of salivary origin. No primary salivary tumors with a CREB1 fusion partner have been described. We highlight the possibility that myoepithelial carcinoma may arise within the major salivary glands, and that definitive distinction between salivary myoepithelial carcinoma and soft tissue myoepithelial carcinoma is quite difficult, even with molecular data.

Primary malignant bone tumors (PMBTs) are rare neoplasms arising from bone tissue. Limited evidence suggests an association between reduced serum 25(OH) vitamin D3 levels and PMBT incidence. The active metabolite 1,25(OH)2 vitamin D3 (1,25D3) modulates tumor cell proliferation, apoptosis, and migration through interactions with the vitamin D receptor (VDR). This study investigated the effects of 1,25D3 on osteosarcoma (MG-63, SaOS-2), chondrosarcoma (SW-1353), and Ewing's sarcoma (RD-ES) cell lines. Expression of key components of vitamin D metabolism, including 1α-hydroxylase (CYP27B1), 24-hydroxylase (CYP24A1), VDR, protein disulfide-isomerase A3 (PDIA3), and the 1,25D3-responsive bone gamma-carboxyglutamate protein (BGLAP), was analyzed at mRNA and protein levels. VDR nuclear translocation was assessed by Western blotting. Cell viability, cell cycle progression, apoptosis, and migration were evaluated using metabolic assays, flow cytometry, immunocytochemistry, wound healing, and live-cell imaging. Treatment with 1,25D3 upregulated CYP24A1 in osteosarcoma cells, while BGLAP expression was detected in both osteosarcoma and SW-1353 cells. All examined cell lines expressed VDR and PDIA3, and 1,25D3 induced VDR nuclear translocation. MG-63 cell viability decreased by 23%, whereas SW-1353 cell viability decreased by 10%, with no significant changes observed in SaOS-2 and RD-ES cells. In MG-63 cells, 1,25D3 induced a significant accumulation of cells in the G1 phase, consistent with cell-cycle arrest, whereas only minor cell-cycle alterations were observed in the other cell lines. Interestingly, 1,25D3 reduced the proportion of apoptotic osteosarcoma cells by 18-30%. The migration of MG-63 cells was significantly reduced by 1,25D3, and an inhibitory trend, albeit not statistically significant, was also observed in RD-ES cells. In conclusion, 1,25D3 modulated cell viability, apoptosis, and migration in PMBT cell models, with distinct, cell-type-specific responses. These findings suggest a potential role for 1,25D3 in regulating PMBT growth and invasiveness.

Primary pulmonary artery sarcoma (PPAS) is a rare malignancy often misdiagnosed as pulmonary thromboembolism. Antemortem diagnosis is often difficult because of the tumor's deep intravascular location and challenges in obtaining tissue samples. We report a 72-year-old man with pulmonary artery intimal sarcoma diagnosed antemortem using endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). Contrast-enhanced computed tomography demonstrated a heterogeneously enhancing intraluminal pulmonary artery lesion, and positron emission tomography/computed tomography revealed intense fluorodeoxyglucose uptake (SUVmax 18.4). Histopathological examination showed atypical spindle-shaped cells positive for vimentin and focally positive for α-smooth muscle actin, consistent with intimal sarcoma. The patient received radiation therapy and doxorubicin-based chemotherapy but died 25 weeks after admission. Consistent with previous reports, this case illustrates the aggressive clinical course and diagnostic challenges of PPAS. It also highlights the importance of considering PPAS in patients with atypical pulmonary embolism and demonstrates the utility of EUS-FNA as a minimally invasive approach for obtaining a definitive diagnosis of centrally located pulmonary artery tumors.