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Abstract Background Xenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers. Method Microarray technology (array comparative genomic hybridization (aCGH) and micro RNA arrays) was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0) were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106) were selected for further validation by real time polymerase chain reaction (RT-PCR). Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods. Results The most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0). MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, EWSR1, FLI1 and their fusion gene (EWS-FLI1). Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers. Conclusion In the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, FLI1, EWSR1, and the EWS-FLI1 fusion genes.

Abstract Background Ewing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to EWS/FLI1 gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas. Methods Array comparative genomic hybridization (CGH) and expression arrays were used to screen for copy number alterations and expression changes in ESFT patient samples. A total of 31 ESFT samples were analyzed by aCGH and in 16 patients DNA and RNA level data, created by expression arrays, was integrated. Time of the follow-up of these patients was 5–192 months. Clinical outcome was statistically evaluated by Kaplan-Meier/Logrank methods and RT-PCR was applied on 42 patient samples to study the gene of the highest interest. Results Copy number changes were detected in 87% of the cases. The most recurrent copy number changes were gains at 1q, 2, 8, and 12, and losses at 9p and 16q. Cumulative event free survival (ESFT) and overall survival (OS) were significantly better (P < 0.05) for primary tumors with three or less copy number changes than for tumors with higher number of copy number aberrations. In three samples copy number imbalances were detected in chromosomes 11 and 22 affecting the FLI1 and EWSR1 loci, suggesting that an unbalanced t(11;22) and subsequent duplication of the derivative chromosome harboring fusion gene is a common event in ESFT. Further, amplifications on chromosomes 20 and 22 seen in one patient sample suggest a novel translocation type between EWSR1 and an unidentified fusion partner at 20q. In total 20 novel ESFT associated putative oncogenes and tumor suppressor genes were found in the integration analysis of array CGH and expression data. Quantitative RT-PCR to study the expression levels of the most interesting gene, HDGF, confirmed that its expression was higher than in control samples. However, no association between HDGF expression and patient survival was observed. Conclusion We conclude that array CGH and integration analysis proved to be effective methods to identify chromosome regions and novel target genes involved in the tumorigenesis of ESFT.

Erythropoietin-producing hepatocellular A2 (EphA2) is a member of the Eph tyrosine kinase receptor family that has been linked to various biological processes. In tumors, EphA2 overexpression is associated with noncanonical pathway activation, tumor progression, and a poor prognosis, which has emphasized its importance as a marker of malignancy. Studies on numerous cancer models have highlighted EphA2's dual and often contradictory action, which can be attributed to EphA2′s interactions involving multiple pathways and different ligands, as well as the heterogeneity of the tumor microenvironment. In this review, we summarize the main mechanisms underlying EphA2 dysregulation in cancer, highlighting its molecular complexity. Then, we analyze therapies that have been developed over time to counteract its action. We discuss the limitations of the described approaches, emphasizing the fact that the goal of new options is high specificity without losing therapeutic efficacy. For this reason, immunotherapy or the emerging field of targeted protein degradation with proteolysis-targeting chimeras (PROTACs) may represent a promising solution that can be developed based on a deeper understanding of the molecular mechanisms sustaining EphA2 oncogenic activity. [ABSTRACT FROM AUTHOR]

Introduction: Extraskeletal Ewing sarcoma (EEwS) is a rare malignant tumor, and current international recommendations indicate systemic and local treatment like bone Ewing sarcoma (BEwS); to the best of our knowledge, very few studies tried to explore the clinical and genetic characteristics of this tumor, and the most appropriate treatment strategy remains uncertain. Methods: We reviewed 35 EEwS cases enrolled at Rizzoli Orthopedic Institute in Bologna, Italy, between 1988–2022. We performed RNA sequencing in 18 Ewing sarcoma cases, including 12 BEwSs and 6 EEwSs. We analyzed overall survival (OS), local relapse-free survival (LRFS), and metastasis-free survival (MFS) and the risk factors associated to survival. Results: Unsupervised hierarchical clustering showed no differences in the transcriptional profile between EEwS and BEwS. Five-year OS was 67% (95% confidence interval [CI]: 47–80), 5-year LRFS was 61% (95% CI: 43–75), and 5-year MFS was 55% (95% CI: 38–70). Recurrent tumors, larger than 8 cm, and elevated lactate dehydrogenase (LDH) serum value resulted to be negative prognostic factors. Conclusions: The finding/detection of a genetic profile that is indistinguishable between EEwS and BEwS confirms the view that the two subgroups belong to the same tumor entity and supports the use of a single therapeutic approach for Ewing sarcoma, regardless of the site of origin. Statistical evaluation showed that size bigger than 8 cm, elevated LDH, and recurrent tumors had a worse prognosis, suggesting a risk-stratification method for identifying patients for specific therapy treatment. However, larger, multicenter, prospective trials are called for to validate our findings. [ABSTRACT FROM AUTHOR]

Ewing sarcoma (EwS), a highly aggressive malignancy affecting children and young adults, is primarily driven by a distinctive oncogenic fusion, the EWSR1‐ETS, whose activity is a key source of epigenetic and clinical heterogeneity. CD99 is constantly present in EwS cells, known to modulate the EwS genetic profile and tumor malignancy. However, the relevance of CD99 alone, or in association with EWSR1‐ETS chimeras, is poorly understood. We explored the dynamic relationship between CD99 and EWS::FLI1, the main fusion observed in EwS, by means of model systems with inducible expression of either molecule. The transcriptomic dynamics of cells with or without expression of EWS::FLI1 or CD99 were analyzed and correlated with tumor cell growth. The CD99‐associated EwS gene profile was found to have commonalities with the profile induced by EWS::FLI1, but also peculiar differences. Both EWS::FLI1 and CD99 are regulated targets of the DREAM complex, but the CD99 expression specifically impacted genes that are the targets of FOXM1 and are involved in the setting of the G2/M phase of the cell cycle. Most CD99‐regulated FOXM1‐targeted genes were found to correlate with bad prognosis in two public clinical datasets (R2 platform), further supporting the clinical relevance of CD99‐mediated regulation of EwS gene expression. [ABSTRACT FROM AUTHOR]

Rhabdomyosarcoma (RMS), the most common form of sarcoma typical of pediatric age, arises from the malignant transformation of the mesenchymal precursors that fail to differentiate into skeletal muscle cells. Here, we investigated whether the protein phospholipase C δ4 (PLCδ4), a member of the PLC family involved in proliferation and senescence mechanisms of mesenchymal stromal stem cells, may play a role in RMS. Our molecular and morpho-functional data reveal that PLCδ4 is highly expressed in the fusion-negative, p53-positive, SMARCB1 heterozygous mutated embryonal RMS (ERMS) cell line A204, while it is poorly expressed in the ERMS cell lines RD (fusion-negative, MYC amplification, N-RAS (Q61H), homozygous mutated p53) and Hs729 (homozygous mutated p53) and the alveolar rhabdosarcoma (ARMS) cell line SJCRH30 (RH30; fusion positive, heterozygous mutated RARA, polyheterozygous mutated p53). To characterize the role of PLCδ4, the RD cell line was stably transfected with wild-type PLCδ4 (RD/PLCδ4). Overexpressed PLCδ4 mainly localized to the nucleus in RD cells and contributed to the phosphorylation of PRAS40 (T246), Chk2(T68), WNK1(T60), and Akt 1/273 (S473), as revealed by proteome profiler array analysis. Overexpression of PLCδ4 in RD cells enhanced cyclin B1 expression and resulted in G2/M-phase cell cycle arrest. In contrast, siRNA-mediated knockdown of PLCδ4 in A204 cells resulted in reduced cyclin B1 expression. Our study identifies a novel role for nuclear PLCδ4 as a regulator of cyclin B1 via Akt-dependent phosphorylation. The modulation of PLCδ4 expression and its downstream targets could represent a crucial signaling pathway to block embryonal RMS cell proliferation. [ABSTRACT FROM AUTHOR]

DNA methylation is a key epigenetic regulatory mechanism that plays a critical role in a variety of cellular processes, including the regulation of cell fate during development, maintenance of cell identity, and genome stability. DNA methylation is tightly regulated by enzymatic reactions and its deregulation plays an important role in the development of cancer. Specific DNA methylation alterations have been found in pediatric solid tumors, providing new insights into the development of these tumors. In addition, DNA methylation profiles have greatly contributed to tune the diagnosis of pediatric solid tumors and to define subgroups of patients with different risks of progression, leading to the reduction in unwanted toxicity and the improvement of treatment efficacy. This review highlights the dysregulated DNA methylome in pediatric solid tumors and how this information provides promising targets for epigenetic therapies, particularly inhibitors of DNMT enzymes (DNMTis). Opportunities and limitations are considered, including the ability of DNMTis to induce viral mimicry and immune signaling by tumors. Besides intrinsic action against cancer cells, DNMTis have the potential to sensitize immune-cold tumors to immunotherapies and may represent a remarkable option to improve the treatment of challenging pediatric solid tumors. [ABSTRACT FROM AUTHOR]