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Abstract Bone sarcomas are malignant tumors of mesenchymal origin. Complete surgical resection is the cornerstone of multidisciplinary treatment. However, advanced, unresectable forms remain incurable. A crucial step towards addressing this challenge involves comprehending the molecular mechanisms underpinning tumor progression and metastasis, laying the groundwork for innovative precision medicine-based interventions. We previously showed that tyrosine kinase receptor Ephrin Type-A Receptor 2 (EphA2) is overexpressed in bone sarcomas. EphA2 is a key oncofetal protein implicated in metastasis, self-renewal, and chemoresistance. Molecular, genetic, biochemical, and pharmacological approaches have been developed to target EphA2 and its signaling pathway aiming to interfere with its tumor-promoting effects or as a carrier for drug delivery. This review synthesizes the main functions of EphA2 and their relevance in bone sarcomas, providing strategies devised to leverage this receptor for diagnostic and therapeutic purposes, with a focus on its applicability in the three most common bone sarcoma histotypes: osteosarcoma, chondrosarcoma, and Ewing sarcoma.

Anticancer therapy urgently needs the development of novel strategies. An innovative molecular target is represented by circular RNAs (circRNAs), single-strand RNA molecules with the 5′ and 3′ ends joined, characterized by a high stability. Although circRNA properties and biological functions have only been partially elucidated, their relationship and involvement in the onset and progression of cancer have emerged. Specific targeting of circRNAs may be obtained with antisense oligonucleotides and silencing RNAs. Nanotechnology is at the forefront of research for perfecting their delivery. Continuous efforts have been made to develop novel nanoparticles (NPs) and improve their performance, materials, and properties regarding biocompatibility and targeting capabilities. Applications in various fields, from imaging to gene therapy, have been explored. This review sums up the smart strategies developed to directly target circRNAs with the fruitful application of NPs in this context.

Acute myeloid leukemia (AML) is a complex hematologic malignancy with high morbidity and mortality. Nucleophosmin 1 (NPM1) mutations occur in approximately 30% of AML cases, and NPM1-mutated AML is classified as a distinct entity. NPM1-mutated AML patients without additional genetic abnormalities have a favorable prognosis. Despite this, 30–50% of them experience relapse. This study aimed to investigate the potential of total RNAseq in improving the characterization of NPM1-mutated AML patients. We explored genetic variations independently of myeloid stratification, revealing a complex molecular scenario. We showed that total RNAseq enables the uncovering of different genetic alterations and clonal subtypes, allowing for a comprehensive evaluation of the real expression of exome transcripts in leukemic clones and the identification of aberrant fusion transcripts. This characterization may enhance understanding and guide improved treatment strategies for NPM1mut AML patients, contributing to better outcomes. Our findings underscore the complexity of NPM1-mutated AML, supporting the incorporation of advanced technologies for precise risk stratification and personalized therapeutic strategies. The study provides a foundation for future investigations into the clinical implications of identified genetic variations and highlights the importance of evolving diagnostic approaches in leukemia management.

Ewing sarcoma (EWS) is an aggressive pediatric bone tumor characterized by unmet clinical needs and an incompletely understood epigenetic heterogeneity. Here, we considered CD99, a major surface molecule hallmark of EWS malignancy. Fluctuations in CD99 expression strongly impair cell dissemination, differentiation, and death. CD99 is also loaded within extracellular vesicles (EVs), and the delivery of CD99-positive or CD99-negative EVs dynamically exerts oncogenic or oncosuppressive functions to recipient cells, respectively. We undertook mass spectrometry and functional annotation analysis to investigate the consequences of CD99 silencing on the proteomic landscape of EWS cells and related EVs. Our data demonstrate that (i) the decrease in CD99 leads to major changes in the proteomic profile of EWS cells and EVs; (ii) intracellular and extracellular compartments display two distinct signatures of differentially expressed proteins; (iii) proteomic changes converge to the modulation of cell migration and immune-modulation biological processes; and (iv) CD99-silenced cells and related EVs are characterized by a migration-suppressive, pro-immunostimulatory proteomic profile. Overall, our data provide a novel source of CD99-associated protein biomarkers to be considered for further validation as mediators of EWS malignancy and as EWS disease liquid biopsy markers.

BackgroundAdvanced and unresectable bone and soft tissue sarcomas (BSTS) still represent an unmet medical need. We demonstrated that the alkylating agent trabectedin and the PARP1-inhibitor olaparib display antitumor activity in BSTS preclinical models. Moreover, in a phase Ib clinical trial (NCT02398058), feasibility, tolerability and encouraging results have been observed and the treatment combination is currently under study in a phase II trial (NCT03838744).MethodsDifferential expression of genes involved in DNA Damage Response and Repair was evaluated by Nanostring® technology, extracting RNA from pre-treatment tumor samples of 16 responder (≥6-month progression free survival) and 16 non-responder patients. Data validation was performed by quantitative real-time PCR, RNA in situ hybridization, and immunohistochemistry. The correlation between the identified candidate genes and both progression-free survival and overall survival was investigated in the publicly available dataset “Sarcoma (TCGA, The Cancer Genome Atlas)”.ResultsDifferential RNA expression analysis revealed an 8-gene signature (CDKN2A, PIK3R1, SLFN11, ATM, APEX2, BLM, XRCC2, MAD2L2) defining patients with better outcome upon trabectedin+olaparib treatment. In responder vs. non-responder patients, a significant differential expression of these genes was further confirmed by RNA in situ hybridization and by qRT-PCR and immunohistochemistry in selected experiments. Correlation between survival outcomes and genetic alterations in the identified genes was shown in the TCGA sarcoma dataset.ConclusionsThis work identified an 8-gene expression signature to improve prediction of response to trabectedin+olaparib combination in BSTS. The predictive role of these potential biomarkers warrants further investigation.

The knowledge of exosome impact on sarcoma development and progression has been implemented in preclinical studies thanks to technological advances in exosome isolation. Moreover, the clinical relevance of liquid biopsy is well established in early diagnosis, prognosis prediction, tumor burden assessment, therapeutic responsiveness, and recurrence monitoring of tumors. In this review, we aimed to comprehensively summarize the existing literature pointing out the clinical relevance of detecting exosomes in liquid biopsy from sarcoma patients. Presently, the clinical utility of liquid biopsy based on exosomes in patients affected by sarcoma is under debate. The present manuscript collects evidence on the clinical impact of exosome detection in circulation of sarcoma patients. The majority of these data are not conclusive and the relevance of liquid biopsy-based approaches in some types of sarcoma is still insufficient. Nevertheless, the utility of circulating exosomes in precision medicine clearly emerged and further validation in larger and homogeneous cohorts of sarcoma patients is clearly needed, requiring collaborative projects between clinicians and translational researchers for these rare cancers.

Gastrointestinal stromal tumors (GISTs) are rare, mesenchymal tumors of the gastrointestinal tract, characterized by either KIT or PDGFRA mutation in about 85% of cases. KIT/PDGFRA wild type gastrointestinal stromal tumors (wtGIST) account for the remaining 15% of GIST and represent an unmet medical need: their prevalence and potential medical vulnerabilities are not completely defined, and effective therapeutic strategies are still lacking. In this study we set a patient-derived preclinical model of wtGIST to investigate their phenotypic features, along with their susceptibility to cellular immunotherapy with cytokine-induced killer lymphocytes (CIK) and interferons (IFN). We generated 11 wtGIST primary cell lines (wtGISTc). The main CIK ligands (MIC A/B; ULBPs), along with PD-L1/2, were expressed by wtGISTc and the expression of HLA-I molecules was preserved. Patient-derived CIK were capable of intense killing in vitro against wtGISTc resistant to both imatinib and sunitinib. We found that CIK produce a high level of granzyme B, IFNα and IFNγ. CIK-conditioned supernatant was responsible for part of the observed tumoricidal effect, along with positive bystander modulatory activities enhancing the expression of PD-L1/2 and HLA-I molecules. IFNα, but not In, had direct antitumor effects on 50% (4/8) of TKI-resistant wtGISTc, positively correlated with the tumor expression of IFN receptors. wtGIST cells that survived IFNα were still sensitive to CIK immunotherapy. Our data support the exploration of CIK immunotherapy in clinical studies for TKI-resistant wtGIST, proposing reevaluation for IFNα within this challenging setting.

This work describes the set-up of a shared platform among the laboratories of the Alleanza Contro il Cancro (ACC) Italian Research Network for the identification of fusion transcripts in sarcomas by using Next Generation Sequencing (NGS). Different NGS approaches, including anchored multiplex PCR and hybrid capture-based panels, were employed to profile a large set of sarcomas of different histotypes. The analysis confirmed the reliability of NGS RNA-based approaches in detecting sarcoma-specific rearrangements. Overall, the anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories.

Bone sarcomas are a group of heterogeneous malignant mesenchymal tumors. Complete surgical resection is still the cornerstone of treatment, but, in the advanced/unresectable setting, their management remains challenging and not significantly improved by target- and immuno-therapies. We focused on the tyrosine kinase Eph type-A receptor-2 (EphA2), a key oncoprotein implicated in self-renewal, angiogenesis, and metastasis, in several solid tumors and thus representing a novel potential therapeutic target. Aiming at better characterizing its expression throughout the main bone sarcoma histotypes, we investigated EPHA2 expression in the Cancer Cell Lines Encyclopedia and in public datasets with clinical annotations. looking for correlations with molecular, histopathological and patients’ features and clinical outcomes in a total of 232 osteosarcomas, 197 Ewing’s sarcomas, and 102 chondrosarcomas. We observed EPHA2 expression in bone sarcoma cell lines. We demonstrated higher EPHA2 expression in tumor tissues when compared to normal counterparts. A significant correlation was found between EPHA2 expression and Huvos grade (osteosarcoma) and with worse overall survival (dedifferentiated chondrosarcoma). Next, we characterized EPHA2 expression and activation in bone sarcoma primary tissues and in patient-derived xenografts generated in our laboratory to verify their reliability as in vivo models of osteosarcoma, Ewing’s sarcoma and chondrosarcoma. Furthermore, for the first time, we demonstrated EPHA2 expression in chondrosarcoma, suggesting its potential key role in this histotype. Indeed, we observed a significant dose-dependent antitumor effect of the EphA2-inhibitor ALW-II-41-27 in patient-derived in vitro models. In conclusion, EphA2 targeting represents a promising novel therapeutic strategy against bone sarcomas.

Abstract Background Enhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death. Methods We investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role. Results Trabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing. Conclusions PARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.

Background: The evidence on high-dose ifosfamide (HD-IFO) use in patients with relapsed osteosarcoma is limited. We performed a retrospective study to analyze HD-IFO activity. Methods: Patients with osteosarcoma relapsed after standard treatment [methotrexate, doxorubicin, cisplatin +/− ifosfamide (MAP+/−I)] with measurable disease according to RECIST1.1 were eligible to ifosfamide (3 g/m2/day) continuous infusion (c.i.) days 1–5 q21d. RECIST1.1 overall response rate (ORR) (complete response (CR) + partial response (PR)), progression-free survival at 6-month (6m-PFS), duration of response (DOR), and 2-year overall survival (2y-OS) were assessed. PARP1 expression and gene mutations were tested by immunohistochemistry and next-generation sequencing. Results: 51 patients were included. ORR was 20% (1 CR + 9 PR). Median DOR was 5 months (95%CI 2–7). Median PFS, 6m-PFS, OS, and 2y-OS were 6 months (95%CI 4–9), 51%, 15 months (10–19), and 30%, respectively. A second surgical complete remission (CR2) was achieved in 26 (51%) patients. After multivariate analysis, previous use of ifosfamide (HR 2.007, p = 0.034) and CR2 (HR 0.126, p < 0.001) showed a significant correlation with PFS and OS, respectively. No significant correlation was found between outcomes and PARP1 or gene mutations. Conclusions: HD-IFO should be considered as the standard first-line treatment option in relapsed osteosarcoma and control arm of future trial in this setting.

Phage display is a nanotechnology with limitless potential, first developed in 1985 and still awaiting to reach its peak. Awarded in 2018 with the Nobel Prize for Chemistry, the method allows the isolation of high-affinity ligands for diverse substrates, ranging from recombinant proteins to cells, organs, even whole organisms. Personalized therapeutic approaches, particularly in oncology, depend on the identification of new, unique, and functional targets that phage display, through its various declinations, can certainly provide. A fast-evolving branch in cancer research, immunotherapy is now experiencing a second youth after being overlooked for years; indeed, many reports support the concept of immunotherapy as the only non-surgical cure for cancer, at least in some settings. In this review, we describe literature reports on the application of peptide phage display to cancer immunotherapy. In particular, we discuss three main outcomes of this procedure: (i) phage display-derived peptides that mimic cancer antigens (mimotopes) and (ii) antigen-carrying phage particles, both as prophylactic and/or therapeutic vaccines, and (iii) phage display-derived peptides as small-molecule effectors of immune cell functions. Preclinical studies demonstrate the efficacy and vast potential of these nanosized tools, and their clinical application is on the way.

Metastatic bone and soft tissue sarcomas often relapse after chemotherapy (CHT) and molecular targeted therapy (mTT), maintaining a severe prognosis. A subset of sarcoma cancer stem cells (sCSC) is hypothesized to resist conventional drugs and sustain disease relapses. We investigated the immunotherapy activity of cytokine induced killer cells (CIK) against autologous sCSC that survived CHT and mTT. The experimental platform included two aggressive bone and soft tissue sarcoma models: osteosarcoma (OS) and undifferentiated-pleomorphic sarcoma (UPS). To visualize putative sCSC we engineered patient-derived sarcoma cultures (2 OS and 3 UPS) with a lentiviral sCSC-detector wherein the promoter of stem-gene Oct4 controls the expression of eGFP. We visualized a fraction of sCSC (mean 24.2 ± 5.2%) and confirmed their tumorigenicity in vivo. sCSC resulted relatively resistant to both CHT and mTT in vitro. Therapeutic doses of doxorubicin significantly enriched viable eGFP+sCSC in both OS (2.6 fold, n = 16) and UPS (2.3 fold, n = 29) compared to untreated controls. Treatment with sorafenib (for OS) and pazopanib (for UPS) also determined enrichment (1.3 fold) of viable eGFP+sCSC, even if less intense than what observed after CHT. Sarcoma cells surviving CHT and mTT were efficiently killed in vitro by autologous CIK even at minimal effector/target ratios (40:1 = 82%, 1:4 = 29%, n = 13). CIK immunotherapy did not spare sCSC that were killed as efficiently as whole sarcoma cell population. The relative chemo-resistance of sCSC and sensitivity to CIK immunotherapy was confirmed in vivo. Our findings support CIK as an innovative, clinically explorable, approach to eradicate chemo-resistant sCSC implicated in tumor relapse.

Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause. The most recent hypotheses on IPF pathogenesis suggest a central role of epithelial cell damage, followed by a dysregulated molecular cross talk between epithelial cells and fibroblasts. Thus, IPF progression has often been assimilated to that of cancer, and several signaling patterns appear to be disrupted in both diseases. Here, we analyze the expression in an IPF series of a panel of molecules, which are known to play a role in tumorigenic process. Our findings, although preliminary, reveal that IPF landscape is enriched in neoplastic potential expressed in a context of complex genomic polyclonality and cellular heterogeneity. These results provide a rationale for further investigations aimed to exploit—in a similar fashion to cancer—targeted therapies for a “precision medicine” approach to IPF.

The good outcome of hematopoietic stem cell (HSC) transplantation is hampered by low doses of CD34+ cell infusion. Transplanted HSCs undergo a replicative stress that causes accelerated senescence due to rapid telomere shortening. The expansion of human cord blood HSCs is instrumental in obtaining a large number of "good quality" cells, in terms of telomere length and telomerase activity compared to adult HSCs.