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Background: Malignant peripheral nerve sheath tumour (MPNST) is an aggressive orphan disease commonly affecting adolescents or young adults. Current knowledge of molecular tumour biology has been insufficient for development of rational treatment strategies. We aimed to discover molecular subtypes of potential clinical relevance., Methods: Fresh frozen samples of MPNSTs (n = 94) and benign neurofibromas (n = 28) from 115 patients in a European multicentre study were analysed by DNA copy number and/or transcriptomic profiling. Unsupervised transcriptomic subtyping was performed and the subtypes characterized for genomic aberrations, clinicopathological associations and patient survival., Findings: MPNSTs were classified into two transcriptomic subtypes defined primarily by immune signatures and proliferative processes. "Immune active" MPNSTs (44%) had sustained immune signals relative to neurofibromas, were more frequently low-grade (P = 0.01) and had favourable prognostic associations in a multivariable model of disease-specific survival with clinicopathological factors (hazard ratio 0.25, P = 0.003). "Immune deficient" MPNSTs were more aggressive and characterized by proliferative signatures, high genomic complexity, aberrant TP53 and PRC2 loss, as well as high relative expression of several potential actionable targets (EGFR, ERBB2, EZH2, KIF11, PLK1, RRM2). Integrated gene-wise analyses suggested a DNA copy number-basis for proliferative transcriptomic signatures in particular, and the tumour copy number burden further stratified the transcriptomic subtypes according to patient prognosis (P < 0.01)., Interpretation: Approximately half of MPNSTs belong to an "immune deficient" transcriptomic subtype associated with an aggressive disease course, PRC2 loss and expression of several potential therapeutic targets, providing a rationale for molecularly-guided intervention trials., Funding: Research grants from non-profit organizations, as stated in the Acknowledgements., Competing Interests: Declaration of interests Authors declare that they have no competing interests related to this study. Payments/honoraria from companies for written or oral presentations or lectures are reported by K.B. (Novartis) and O.C.L (Nykode) and for consulting by O.C.L. (Novartis Norway). K.B. also reports participation on a Data Safety Monitoring Board or Advisory Board for GSK, Bayer, NEC OncoImmunity, and Incyte., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Introduction: Surgical resection of pulmonary metastases has been associated with increased survival at 5 years for osteosarcoma, but limited information is available on long-term outcome, role of repeated metastasectomies, and surgical sequelae in a pediatric population. We analyzed a consecutive series of children, adolescents, and young adults (AYA) treated by repeated lung metastasectomies during a period >40 years to estimate the clinical benefit and potential cure rate of salvage surgery., Methods: All patients who underwent lung metastasectomy for osteosarcoma at the IRCCS Istituto Ortopedico Rizzoli of Bologna, University of Modena, and IRCCS Istituto Nazionale Tumori of Milan from May 1973 to January 2014 were included. Overall survival (OS) at 20 years from the first metastasectomy was calculated., Results: A total of 463 consecutive children and AYA were analyzed. Median age was 15.9 years (range 0.2-23.2 years) and median follow-up 18.6 years. The 5- and 20-year OS were 34.0% and 29.7% (95% CI 25.5-34.0%). Among the 138 (29.8%) alive patients, 42 (30.4%) had disease recurrence cured by repeated metastasectomies. Disease-free interval from primary tumor, number of metastases, and complete resection were the most relevant survival predictors at multivariable model analysis., Discussion: The extended follow-up of this consecutive series shows that repeated lung metastasectomy can achieve a permanent cure when offered to properly selected patients with metastases from osteosarcoma.

Background: Among sarcomas, which are rare cancers with an incidence of <6 per 100.000/year cases, ultra-rare sarcomas have an incidence of approximately ≤1/1,000,000/year cases and altogether account for ~20% of all soft tissue sarcomas (STS) and bone sarcomas. The Italian Sarcoma Group has recently performed a non-interventional, retrospective TrObs study with data from 512 anthracycline-pretreated patients with advanced multiple STS histologies and treated with trabectedin (Palmerini, Cancers 2021; ClinicalTrials.gov Identifier: NCT02793050)., Methods: A post-hoc analysis of case series to evaluate the efficacy and safety of trabectedin on patients with ultra-rare and other rare translocation-related sarcomas included in TrObs study was performed. Main outcomes comprised investigator-assessed overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and safety., Results: Thirty-six patients (18 women) with ultra-rare and other rare sarcoma and a median age of 53.0 years (range: 22-81) were included. Most patients had solitary fibrous tumor (SFT; n=11) followed by epithelioid sarcoma (n=5), malignant peripheral nerve sheath tumor (MPNST; n=4), extraskeletal myxoid chondrosarcoma (EMC; n=3), desmoplastic small round cell tumor (DSRCT; n=3), and alveolar soft part sarcoma (ASPS), rhabdomyosarcoma and clear cell sarcoma (n=2 each). Thirty-five patients had metastatic disease and 23 patients received trabectedin as a second-line treatment. Among 35 patients evaluable for response, two patients with SFT and ASPS had a partial response and one patient with DSRCT obtained a complete response, reaching an ORR of 8.6% (95% CI: 2.8-23.4%). Among patients with an ORR, 6-months PFS was 100% in patients with ASPS, 45.7% in patients with SFT and 33.3% in those with DSRCT. Two patients with epithelioid sarcoma and myoepithelioma had disease stabilization lasting >24 months. Nine patients had at least one grade 3/4 adverse event, mostly being bone marrow toxicity (n=6)., Conclusions: Trabectedin has some anti-tumor activity in some ultra-rare and other rare sarcomas, particularly translocation-related sarcomas, with the well-known manageable safety profile., Competing Interests: EP has served on an advisory board for Takeda, Amgen, Daiichi Sankyo, Lilly, Eusa Pharma, Deciphera, and SynOx Therapeutics and has received research support from Bristol Myers Squibb, Pfizer, PharmaMar, and Daiichi Sankyo. ABr is a consultant for Eli Lilly, Eisai, Glaxo-Smith Kline, Pharmamar and has received travel grants from PharmaMar, Takeda, and Ipsen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Palmerini, Sanfilippo, Grignani, Buonadonna, Romanini, Badalamenti, Ferraresi, Vincenzi, Comandone, Pizzolorusso, Brunello, Gelsomino, De Pas, Ibrahim, Gurrieri, Grosso, Zanelli, Pantaleo, Milesi, Ciuffreda, Ferrari, Marchesi, Quattrini, Righi, Setola, Carretta, Casali, Picci and Ferrari.)

The phase III clinical study of adjuvant liposomal muramyl tripeptide (MTP-PE) in resected high-grade osteosarcoma (OS) documented positive results that have been translated into regulatory approval, supporting initial promise for innate immune therapies in OS. There remains, however, no new approved treatment such as MTP-PE for either metastatic or recurrent OS. Whilst the addition of different agents, including liposomal MTP-PE, to surgery for metastatic or recurrent high-grade osteosarcoma has tried to improve response rates, a mechanistic hiatus exists in terms of a detailed understanding the therapeutic strategies required in advanced disease. Here we report a Bayesian designed multi-arm, multi-centre, open-label phase II study with randomisation in patients with metastatic and/or recurrent OS, designed to investigate how patients with OS might respond to liposomal MTP-PE, either given alone or in combination with ifosfamide. Despite the trial closing because of poor recruitment within the allocated funding period, with no objective responses in eight patients, we report the design and feasibility outcomes for patients registered into the trial. We demonstrate the feasibility of the Bayesian design, European collaboration, tissue collection with genomic analysis and serum cytokine characterisation. Further mechanistic investigation of liposomal MTP-PE alone and in combination with other agents remains warranted in metastatic OS., (© 2022. The Author(s).)

Background: According to retrospective osteosarcoma series, ABCB1/P-glycoprotein (Pgp) overexpression predicts for poor outcomes. A prospective trial to assess a risk-adapted treatment strategy using mifamurtide in Pgp+ patients was performed., Methods: This was a phase 2, multicenter, uncontrolled trial including patients 40 years old or younger with nonmetastatic extremity high-grade osteosarcoma stratified according to Pgp expression. All patients received high-dose methotrexate, doxorubicin, and cisplatin (MAP) preoperatively. In Pgp+ patients, mifamurtide was added postoperatively and combined with MAP for a good histologic response (necrosis ≥ 90%; good responders [GRs]) or with high-dose ifosfamide (HDIFO) at 3 g/m 2 /d on days 1 to 5 for a histologic response < 90% (poor responders [PRs]). Pgp- patients received MAP postoperatively. After an amendment, the cumulative dose of methotrexate was increased from 60 to 120 g/m 2 (from 5 to 10 courses). The primary end point was event-free survival (EFS). A postamendment analysis was performed., Results: In all, 279 patients were recruited, and 194 were included in the postamendment analysis: 70 (36%) were Pgp-, and 124 (64%) were Pgp+. The median follow-up was 51 months. For Pgp+ patients, 5-year EFS after definitive surgery (null hypothesis, 40%) was 69.8% (90% confidence interval [CI], 62.2%-76.2%): 59.8% in PRs and 83.7% in GRs. For Pgp- patients, the 5-year EFS rate was 66.4% (90% CI, 55.6%-75.1%)., Conclusions: This study showed that adjuvant mifamurtide, combined with HDIFO for a poor response to induction chemotherapy, could improve EFS in Pgp+ patients. Overall, the outcomes compared favorably with previous series. Mifamurtide and HDIFO as salvage chemotherapy are worth further study., (© 2022 The Authors. Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Primary spindle cell and pleomorphic sarcomas of bone represent an exceedingly rare group of mesenchymal malignancies that include soft tissue histotypes, as malignant peripheral nerve sheath tumor. Outside the head and neck region, only 36 cases of primary malignant peripheral nerve sheath tumor of bone have been described. We retrieved from our archives eight cases of primary malignant peripheral nerve sheath tumor of bone arising outside the head and neck region, describing their clinical, radiological, and morphologic features. Our series, in which all but one patient died of diseases after a median of seven months, confirms that primary malignant peripheral nerve sheath tumors of bone are aggressive tumors. Pathologists should be aware of this rare histotype. More aggressive and active adjuvant treatments should be investigated., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The value of neoadjuvant chemotherapy in soft tissue sarcoma (STS) is not completely understood. This study investigated the benefit of neoadjuvant chemotherapy according to prognostic stratification based on the Sarculator nomogram for STS., Methods: This study analyzed data from ISG-STS 1001, a randomized study that tested 3 cycles of neoadjuvant anthracycline plus ifosfamide (AI) or histology-tailored (HT) chemotherapy in adult patients with STS. The 10-year predicted overall survival (pr-OS) was estimated with the Sarculator and was stratified into higher (10-year pr-OS < 60%) and lower risk subgroups (10-year pr-OS ≥ 60%)., Results: The median pr-OS was 0.63 (interquartile range [IQR], 0.51-0.72) for the entire study population, 0.62 (IQR, 0.51-0.70) for the AI arm, and 0.64 (IQR, 0.51-0.73) for the HT arm. Three- and 5-year overall survival (OS) were 0.86 (95% confidence interval [CI], 0.82-0.93) and 0.81 (95% CI, 0.71-0.86) in lower risk patients and 0.69 (95% CI, 0.70-0.85) and 0.59 (95% CI, 0.51-0.72) in the higher risk patients (log-rank test, P = .004). In higher risk patients, the 3- and 5-year Sarculator-predicted and study-observed OS rates were 0.68 and 0.58, respectively, and 0.85 and 0.66, respectively, in the AI arm (P = .04); the corresponding figures in the HT arm were 0.69 and 0.60, respectively, and 0.69 and 0.55, respectively (P > .99). In lower risk patients, the 3- and 5-year Sarculator-predicted and study-observed OS rates were 0.85 and 0.80, respectively, and 0.89 and 0.82, respectively, in the AI arm (P = .507); the corresponding figures in the HT arm were 0.87 and 0.81, respectively, and 0.86 and 0.74, respectively (P = .105)., Conclusions: High-risk patients treated with AI performed better than predicted, and this adds to the evidence for the efficacy of neoadjuvant AI in STS., Lay Summary: People affected by soft tissue sarcomas of the extremities and trunk wall are at some risk of developing metastasis after surgery. Preoperative or postoperative chemotherapy has been tested in clinical trials to reduce the chances of distant metastasis. However, study findings have not been conclusive. This study stratified the risk of metastasis for people affected by sarcomas who were included in a clinical trial testing neoadjuvant chemotherapy. Exploiting the prognostic nomogram Sarculator, it found a benefit for chemotherapy when the predicted risk, based on patient and tumor characteristics, was high., (© 2021 American Cancer Society.)

MRP-1 is implicated in multidrug resistance and was described as prognostic in high-risk patients with soft-tissue sarcoma (STS) in a previous study. The current research aimed to validate MRP-1 prognostic/predictive value in localized sarcomas treated with anthracyclines plus ifosfamide within the ISG-1001 phase III study. In addition, the inhibitory activity on MRP-1 was investigated in preclinical studies to identify new combinations able to increase the efficacy of standard chemotherapy in STS. MRP-1 expression was assessed by IHC in tissue microarrays from patients with STS and tested for correlation with disease-free survival (DFS) and overall survival (OS). In vitro studies tested the efficacy of MRP-1 inhibitors (nilotinib, ripretinib, selumetinib, and avapritinib) in sarcoma cell lines. The effect of combinations of the most active MRP-1 inhibitors and chemotherapy was measured on the basis of apoptosis. MRP-1 was evaluable in 231 of 264 cases who entered the study. MRP-1 expression (strong intensity) was independently associated with worse DFS [HR, 1.78; 95% confidence interval (CI), 1.11-2.83; P = 0.016], in the multivariate analysis, with a trend for a worse OS (HR, 1.78; 95% CI, 0.97-3.25; P = 0.062). In vitro studies showed that the addition of MRP-1 inhibitors (nilotinib or avapritinib) to doxorubicin plus palifosfamide, significantly increased cell death in SK-UT-1 and CP0024 cell lines. MRP-1 is an adverse predictive factor in localized high-risk patients with STS treated with neoadjuvant anthracyclines plus ifosfamide followed by surgery. In vitro findings support the clinical assessment of the combination of chemotherapy and MRP-1 inhibitors as a promising strategy to overcome the drug ceiling effect for chemotherapy., (©2021 American Association for Cancer Research.)

Introduction: The role of chemotherapy for patients with dedifferentiated chondrosarcoma (DDCS) is still under discussion. Here, we present the outcome in patients with DDCS treated with intensive chemotherapy from the EUROpean Bone Over 40 Sarcoma Study., Materials and Methods: The chemotherapy regimen included doxorubicin, ifosfamide and cisplatin. Postoperative methotrexate was added in case of poor histological response. Toxicity was graded based on the National Cancer Institute expanded common toxicity criteria, version 2.0, and survival was analysed using Kaplan-Meier curves, log-rank tests and univariate Cox regression models., Results: Fifty-seven patients with DDCS (localised, 34 [60%]; metastatic, 23 [40%]) aged 42-65 years were included. Surgical complete remission (SCR) was achieved in 36 (63%) patients. The median overall survival (OS) was 24 months (95% confidence interval, 22-25), and the 5-year OS was 39%. Patients with extremity localisation had a 5-year OS of 49% compared with 29% in patients with a central tumour (P = 0.08). Patients with localised disease had a 5-year OS of 46%, whereas patients with metastatic disease had a 5-year OS of 29% (P = 0.12). Patients in SCR had a 5-year OS of 49%, whereas patients not in SCR had a 5-year OS of 23% (P = 0.004). Chemotherapy toxicity was considerable but manageable. There was no treatment-related death, and 39 (70%) patients received ≥6 cycles of the planned nine chemotherapy cycles., Conclusions: Adding intensive chemotherapy to surgery for treatment of DDCS is feasible and shows favourable survival data compared with previous reports. With the limitations of data from a non-controlled trial, we conclude that chemotherapy could be considered in the management of patients aged >40 years., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S.B. has reported grants from Deutsche Krebshilfe, Deutsche Forschungsgemeinschaft and the European Science Foundation and personal fees from Lilly, Bayer, Pfizer, Novartis, Isofol and Clinigen; E.P. has reported advisory roles for Amgen, Daiichi Sankyo, Eli Lilly, EUSA Pharma and Deciphera, received research support from Bristol Myers Squibb, Pfizer and PharmaMar and received travel support from Lilly, PharmaMar and Takeda; L.K. has reported honoraria from Bayer; P.R. has reported advisory roles for Exactech and Stryker; A.F. has reported honoraria from Ipsen Pharma, PharmaMar and Lilly; P.C. has reported honoraria for the speaker, consultancy or advisory role from Bayer, Deciphera, Eisai, Eli Lilly, Nektar Therapeutics and Pfizer and received research funds from Advenchen Laboratories, Amgen Dompé, AROG Pharmaceuticals, Bayer, Blueprint Medicines, Daiichi Sankyo, Deciphera, Eisai, Eli Lilly, Epizyme Inc., Glaxo, Karyopharm Pharmaceuticals, Novartis, Pfizer and PharmaMar; P.R. has reported honoraria from Bayer, Clinigen, BMS, Roche, MSD, Deciphera, Novartis, Pfizer, PharmaMar, Lilly and Amgen; I.H., S.F., K.S.H., P.P., S.H.-N., D.M.D., C.B., B.B., E.St., M.G., T.K., M.E., V.F., M.K., R.B., D.B., O.B., A.C., R.S., R.B., T.K., L.H., G.A., B.-N.H., E.Se., A.F., F.K., G.H.-W. and S.S. have declared no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Aim: The aim is to evaluate which of the existing scoring systems of histological response to neoadjuvant chemotherapy best stratifies the clinical outcome of patients with localized Ewing sarcoma of bone., Methods: 474 patients with diagnosis of localized Ewing sarcoma of bone were included. The median follow-up was 13.5 years., Results: The overall survival and the disease-free survival (DFS) were 70.8% and 63.9% at 5 years. The percentage of histological response to neoadjuvant chemotherapy ranged between 5% and 100% (mean 83%). The agreement between Bologna System and the different percentual cut-offs of histological response to neoadjuvant chemotherapy was high, with kappa statistics of 0.83 for a cut-off of ≥90%; 0.86 for a cut-off of ≥95%; 0.79 for a cut-off of ≥96% and 0.61 for a cut-off of 100%. Statistically higher DFS rates for good responders compared to poor responders were found when using each given system. Model performance indicators showed that Bologna system had a lower AIC score and a higher c-statistics to predict DFS. When the patients classified as good responders using the different percentual cut-offs of histological response to neoadjuvant chemotherapy, were instead re-classified using the Bologna system, statistical differences were noted in DFS within each specific group., Conclusions: All scoring tools to evaluate histological response to neoadjuvant chemotherapy offer good predictive value for DFS in localized Ewing's sarcoma of bone. The Bologna system better stratifies those patients with histological response to neoadjuvant chemotherapy between 90 and 99%, representing a more reliable scoring tool in this subset., Competing Interests: Declaration of competing interest All authors affirm that we have no actual or potential conflicts of interest, including any financial, personal, or other relationships with other people or organizations., (Copyright © 2021 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Purpose: The main objective was to evaluate the activity and tolerability of TEMIRI as a front-line treatment in primary disseminated Ewing sarcoma (PDMES) using the RECIST 1.1 criteria. The secondary objectives included the assessment of toxicity and the performance status/symptom changes., Methods: Between 2012 and 2018, patients with PDMES received two courses of temozolomide 100 mg/sqm/day + irinotecan 50 mg/sqm/day for 5 days every 3 weeks as an amendment to the Italian Sarcoma Group/Associazione Italiana EmatoIogia ed Oncologia Pediatrica (ISG/AIEOP) EW-2 protocol (EUDRACT#2009-012353-37, Vers. 1.02)., Results: Thirty-four patients were enrolled. The median age at diagnosis was 19 years (range 3-55). After TEMIRI, the RECIST response was as follows: a partial response in 20 (59%) patients, stable disease in 11 (32%), and disease progression in 3 (9%). The ECOG/Lansky score was improved in 25/34 (73.5%) cases, and a reduction or disappearance of pain was observed in 31/34 patients (91%). The incidence of grade 3-4 toxicity was 3%. The 3-year event-free survival (EFS) and overall survival (OS) were 21% (95% CI 6-35%) and 36% (95% CI: 18-54%), respectively., Conclusion: the smooth handling and encouraging activity demonstrated by up-front TEMIRI did not change the EFS in PDMES, so this result suggests the need for the further evaluation of the efficacy of TEMIRI in combination with conventional treatments in non-metastatic patients., Competing Interests: The authors declare no conflict of interest.

Purpose: To analyze toxicity and outcome predictors in Ewing sarcoma patients with lung metastases treated with busulfan and melphalan (BU-MEL) followed by whole-lung irradiation (WLI)., Methods: This retrospective study included 68 lung metastatic Ewing Sarcoma patients who underwent WLI after BU-MEL with autologous stem cell transplantation, as part of two prospective and consecutive treatment protocols. WLI 12 Gy for <14 years old and 15 Gy for ≥14 years old patients were applied at least eight weeks after BU-MEL. Toxicity, overall survival (OS), event-free survival (EFS) and pulmonary relapse-free survival (PRFS) were estimated and analyzed., Results: After WLI, grade 1-2 and grade 3 clinical toxicity was reported in 16.2% and 5.9% patients, respectively. The five-year OS, EFS and PRFS with 95% confidence interval (CI) were 69.8% (57.1-79.3), 61.2% (48.4-71.7) and 70.5% (56.3-80.8), respectively. Patients with good histological necrosis of the primary tumor after neoadjuvant chemotherapy showed a significant decreased risk of pulmonary relapse or death compared to patients with poor histological necrosis., Conclusions: WLI at recommended doses and time interval after BU-MEL is feasible and might contribute to the disease control in Ewing sarcoma with lung metastases and responsive disease. Further studies are needed to explore the treatment stratification based on the histological response of the primary tumor.

Cells of the monocyte macrophage lineage form multinucleated giant cells (GCs) by fusion, which may express some cell cycle markers. By using a comprehensive marker set, here we looked for potential replication activities in GCs, and investigated whether these have diagnostic or clinical relevance in giant cell tumor of bone (GCTB). GC rich regions of 10 primary and 10 first recurrence GCTB cases were tested using immunohistochemistry in tissue microarrays. The nuclear positivity rate of the general proliferation marker, replication licensing, G1/S-phase, S/G2/M-phase, mitosis promoter, and cyclin dependent kinase (CDK) inhibitor reactions was analyzed in GCs. Concerning Ki67, moderate SP6 reaction was seen in many GC nuclei, while B56 and Mib1 positivity was rare, but the latter could be linked to more aggressive ( p = 0.012) phenotype. Regular MCM6 reaction, as opposed to uncommon MCM2, suggested an initial DNA unwinding. Early replication course in GCs was also supported by widely detecting CDK4 and cyclin E, for the first time, and confirming cyclin D1 upregulation. However, post-G1-phase markers CDK2, cyclin A, geminin, topoisomerase-2a, aurora kinase A, and phospho-histone H3 were rare or missing. These were likely silenced by upregulated CDK inhibitors p15 INK4b , p16 INK4a , p27 KIP1 , p53 through its effector p21 WAF1 and possibly cyclin G1, consistent with the prevention of DNA replication. In conclusion, the upregulation of known and several novel cell cycle progression markers detected here clearly verify early replication activities in GCs, which are controlled by cell cycle arresting CDK inhibitors at G1 phase, and support the functional maturation of GCs in GCTB., Competing Interests: MM reports non-related funding from the German Ministry for Education and Research (MI-I 01ZZ1801E) and non-related consultancy for Siemens Healthineers and SmartReporting GmbH. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Maros, Balla, Micsik, Sapi, Szendroi, Wenz, Groden, Forsyth, Picci and Krenacs.)

The Italian Sarcoma Group performed this retrospective analysis of patients with advanced soft tissue sarcoma, pretreated with ≥1 anthracycline-based treatment, and treated with trabectedin every three weeks. Primary endpoint was to describe real-life use of trabectedin across Italy. Secondary endpoints included objective response rate (ORR) and safety. Overall, 512 patients from 20 Italian centers were evaluated. Leiomyosarcoma (37.7%)/liposarcoma (30.3%) were the most prevalent histological types (abbreviated as L-sarcoma). Patients received a median of four trabectedin cycles (range: 1-40), mostly as a second-line treatment (~60% of patients). The ORR was 13.7% superior ( p < 0.0001) in patients with L-sarcoma compared with patients with non-L-sarcoma (16.6% vs. 9.0%). Median progression-free survival (PFS) was 5.1 months, whereas median overall survival (OS) was 21.6 months. Significantly better PFS and OS were observed in patients with L-sarcoma, those with objective responses and/or disease stabilization, treated in an early line and treated with reduced dose. Bone marrow toxicity (61.4%) and transaminase increases (21.9%) were the most common grade 3/4 adverse events. The results of this real-life study suggest that trabectedin is an active treatment, which is mostly given as a second-line treatment to patients with a good performance status and high-grade, metastatic L-sarcoma (clinical trial information: NCT02793050).

Given the potential for neuroendocrine differentiation in Ewing's sarcoma family of tumors (ESFT), we aimed to determine neuroendocrine expression in a large series of genetically-confirmed ESFT and its prognostic significance in clinically-localised neoplasms (n = 176). Slides prepared from tissue microarrays were stained for Insulinoma-associated protein 1 (INSM1), CD56, chromogranin-A and synaptophysin. INSM1 expression was present in 59% of ESFT, while synaptophysin, chromogranin-A and CD56 were expressed in only 13%, 8% and 5% of ESFT, respectively. Histological subtypes were only significantly correlated with INSM1 (p = 0.032) or CD56 (p = 0.016) immunoexpression. Regarding prognosis, no significant association was found between INSM1, synaptophysin or chromogranin-A immunoexpression and progression-free survival (PFS) or overall survival (OS). Despite the low proportion of tumors with CD56 immunoreactivity, CD56 expression was shown to correlate with both poor PFS (p < 0.001) and poor OS (p < 0.001) in the present series. In conclusion, neuroendocrine differentiation is often present in ESFT, and in the present study INSM1 expression in particular was found to be higher than previously described in Ewing's tumors. Nevertheless, this finding does not distinguish these tumors from other round cell tumors that may show focal or diffuse neuroendocrine differentiation. CD56 expression could be used as a prognostic factor in ESFT, although given the results herein obtained, we recommend a prospective validation in independent series including localized and disseminated tumors in ESFT., (Published by Elsevier GmbH.)

Background: Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma of uncertain origin, marked by specific chromosomal translocations involving the NR4A3 gene, and usually characterized by an indolent course. Surgery (with or without radiotherapy) is the treatment of choice in localized disease. The treatment for advanced disease remains uncertain. In order to better evaluate prognostic factors and outcome, a retrospective pooled analysis of patients with EMC treated at three Italian Sarcoma Group (ISG) referral centers was carried out., Methods: All patients with localized EMC surgically treated from 1989 to 2016 were identified. Diagnosis was centrally reviewed according to WHO 2013. Only patients with NR4A3 rearrangement were included., Results: Sixty-seven patients were identified: 13 (20%) female, 54 (80%) male. Median age was 56 years (range 18-84). Numbers and type of translocation were: 50 (80%) NR4A3-EWS, 10 (16%) NR4A3-TAF15, 1 (2%) NR4A3-TCF12, and 1 (2%) NR4A3-TFG. Median follow-up was 55 months (range 2-312). Five- and ten-year overall survival rates were 94% (86-100 95%CI) and 84% (69-98 95%CI). Thirty-five (52%) patients relapsed: 9 had local recurrence (LR) and 26 had distant metastasis (5 with concomitant LR). The 5- and 10-year disease-free survival rates (DFS) were 51% (38-65 95%CI) and 20% (7-33 95%CI). Size of the primary tumor was significantly related to distant metastasis-free survival (DMFS) (p = 0.004). Patients carrying the NR4A3-EWS translocation had a trend in favor of better DFS (p = 0.08) and DMFS (p = 0.09) compared with the patients with NR4A3-TAF15., Conclusions: Prolonged survival can be expected in patients with EMC, in spite of a high rate of recurrence. Size is significantly associated with distant relapse. The type of NR4A3 translocation could influence outcome.

Background: Giant cell tumor of bone (GCTB) is a rare osteoclastogenic stromal tumor. GCTB can rarely undergo malignant transformation. This post hoc analysis evaluated and classified malignancies in patients with GCTB who received denosumab., Methods: This analysis was conducted on patients with pathologically confirmed GCTB and measurable active disease treated with denosumab 120 mg subcutaneously once every 4 weeks, with loading doses on study days 8 and 15, as part of a phase 2, open-label, multicenter study. We identified potential cases of malignancy related to GCTB through an independent multidisciplinary review or medical history, associated imaging or histopathologic reports, and disease course. The findings were summarized and no statistical analysis was performed., Results: Twenty of five hundred twenty-six patients (3.8%) who received at least one dose of denosumab were misdiagnosed with GCTB that was later discovered to be malignancies: five primary malignant GCTB, five secondary malignant GCTB, four sarcomatous transformations, and six patients with other malignancies (giant cell-rich osteosarcoma, undifferentiated pleomorphic sarcoma, spindle cell sarcoma, osteogenic sarcoma, phosphaturic mesenchymal tumor of mixed connective tissue type, and fibrosarcoma/malignant fibrous histiocytoma). Many malignancies were present before denosumab was initiated (8 definitive cases, 7 likely cases), excluding potential involvement of denosumab in these cases. Signs associated with potential misdiagnoses of GCTB included poor mineralization with denosumab treatment, rapid relapse in pain, or a failure of the typical dramatic improvement in pain normally observed with denosumab., Conclusions: Although rare, GCTB can undergo malignant transformation, and rates in this study were consistent with previous reports. Signs of poor mineralization or lack of response to denosumab treatment may warrant close monitoring., Trial Registration: clinicaltrials.gov , ( NCT00680992 ). Registered May 20, 2008.

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/m 2 /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.

Background: Osteofibrous dysplasia-like adamantinoma (OFD-AD) and classic adamantinoma (AD) are rare, neoplastic diseases with only limited data supporting current treatment protocols. We believe that our retrospective multicenter cohort study is the largest analysis of patients with adamantinoma to date. The primary purpose of this study was to describe the disease characteristics and evaluate the oncological outcomes. The secondary purpose was to identify risk factors for local recurrence after surgical treatment and propose treatment guidelines., Methods: Three hundred and eighteen confirmed cases of OFD-AD and AD for which primary treatment was carried out between 1985 and 2015 were submitted by 22 tertiary bone tumor centers. Proposed clinical risk factors for local recurrence such as size, type, and margins were analyzed using univariable and multivariate Cox regression analysis., Results: Of the 318 cases, 128 were OFD-AD and 190 were AD. The mean age at diagnosis was 17 years (median, 14.5 years) for OFD-AD and 32 years (median, 28 years) for AD; 53% of the patients were female. The mean tumor size in the OFD-AD and AD groups combined was 7.8 cm, measured histologically. Sixteen percent of the patients sustained a pathological fracture prior to treatment. Local recurrence was recorded in 22% of the OFD-AD cases and 24% of the AD cases. None of the recurrences in the OFD-AD group progressed to AD. Metastatic disease was found in 18% of the AD cases and fatal disease, in 11% of the AD cases. No metastatic or fatal disease was reported in the OFD-AD group. Multivariate Cox regression analysis demonstrated that uncontaminated resection margins (hazard ratio [HR] = 0.164, 95% confidence interval [CI] = 0.092 to 0.290, p < 0.001), pathological fracture (HR = 1.968, 95% CI = 1.076 to 3.600, p = 0.028), and sex (female versus male: HR = 0.535, 95% CI = 0.300 to 0.952, p = 0.033) impacted the risk of local recurrence., Conclusions: OFD-AD and AD are parts of a disease spectrum but should be regarded as different entities. Our results support reclassification of OFD-AD into the intermediate locally aggressive category, based on the local recurrence rate of 22% and absence of metastases. In our study, metastatic disease was restricted to the AD group (an 18% rate). We advocate wide resection with uncontaminated margins including bone and involved periosteum for both OFD-AD and AD., Level of Evidence: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Background: The Skeletal Oncology Research Group (SORG) machine learning algorithm for predicting survival in patients with chondrosarcoma was developed using data from the Surveillance, Epidemiology, and End Results (SEER) registry. This algorithm was externally validated on a dataset of patients from the United States in an earlier study, where it demonstrated generally good performance but overestimated 5-year survival. In addition, this algorithm has not yet been validated in patients outside the United States; doing so would be important because external validation is necessary as algorithm performance may be misleading when applied in different populations., Questions/purposes: Does the SORG algorithm retain validity in patients who underwent surgery for primary chondrosarcoma outside the United States, specifically in Italy?, Methods: A total of 737 patients were treated for chondrosarcoma between January 2000 and October 2014 at the Italian tertiary care center which was used for international validation. We excluded patients whose first surgical procedure was performed elsewhere (n = 25), patients who underwent nonsurgical treatment (n = 27), patients with a chondrosarcoma of the soft tissue or skull (n = 60), and patients with peripheral, periosteal, or mesenchymal chondrosarcoma (n = 161). Thus, 464 patients were ultimately included in this external validation study, as the earlier performed SEER study was used as the training set. Therefore, this study-unlike most of this type-does not have a training and validation set. Although the earlier study overestimated 5-year survival, we did not modify the algorithm in this report, as this is the first international validation and the prior performance in the single-institution validation study from the United States may have been driven by a small sample or non-generalizable patterns related to its single-center setting. Variables needed for the SORG algorithm were manually collected from electronic medical records. These included sex, age, histologic subtype, tumor grade, tumor size, tumor extension, and tumor location. By inputting these variables into the algorithm, we calculated the predicted probabilities of survival for each patient. The performance of the SORG algorithm was assessed in this study through discrimination (the ability of a model to distinguish between a binary outcome), calibration (the agreement of observed and predicted outcomes), overall performance (the accuracy of predictions), and decision curve analysis (establishment on the ability of a model to make a decision better than without using the model). For discrimination, the c-statistic (commonly known as the area under the receiver operating characteristic curve for binary classification) was calculated; this ranged from 0.5 (no better than chance) to 1.0 (excellent discrimination). The agreement between predicted and observed outcomes was visualized with a calibration plot, and the calibration slope and intercept were calculated. Perfect calibration results in a slope of 1 and an intercept of 0. For overall performance, the Brier score and the null-model Brier score were calculated. The Brier score ranges from 0 (perfect prediction) to 1 (poorest prediction). Appropriate interpretation of the Brier score requires comparison with the null-model Brier score. The null-model Brier score is the score for an algorithm that predicts a probability equal to the population prevalence of the outcome for every patient. A decision curve analysis was performed to compare the potential net benefit of the algorithm versus other means of decision support, such as treating all or none of the patients. There were several differences between this study and the earlier SEER study, and such differences are important because they help us to determine the performance of the algorithm in a group different from the initial study population. In this study from Italy, 5-year survival was different from the earlier SEER study (71% [319 of 450 patients] versus 76% [1131 of 1487 patients]; p = 0.03). There were more patients with dedifferentiated chondrosarcoma than in the earlier SEER study (25% [118 of 464 patients] versus 8.5% [131 of 1544 patients]; p < 0.001). In addition, in this study patients were older, tumor size was larger, and there were higher proportions of high-grade tumors than the earlier SEER study (age: 56 years [interquartile range {IQR} 42 to 67] versus 52 years [IQR 40 to 64]; p = 0.007; tumor size: 80 mm [IQR 50 to 120] versus 70 mm [IQR 42 to 105]; p < 0.001; tumor grade: 22% [104 of 464 had Grade 1], 42% [196 of 464 had Grade 2], and 35% [164 of 464 had Grade 3] versus 41% [592 of 1456 had Grade 1], 40% [588 of 1456 had Grade 2], and 19% [276 of 1456 had Grade 3]; p ≤ 0.001)., Results: Validation of the SORG algorithm in a primarily Italian population achieved a c-statistic of 0.86 (95% confidence interval 0.82 to 0.89), suggesting good-to-excellent discrimination. The calibration plot showed good agreement between the predicted probability and observed survival in the probability thresholds of 0.8 to 1.0. With predicted survival probabilities lower than 0.8, however, the SORG algorithm underestimated the observed proportion of patients with 5-year survival, reflected in the overall calibration intercept of 0.82 (95% CI 0.67 to 0.98) and calibration slope of 0.68 (95% CI 0.42 to 0.95). The Brier score for 5-year survival was 0.15, compared with a null-model Brier of 0.21. The algorithm showed a favorable decision curve analysis in the validation cohort., Conclusions: The SORG algorithm to predict 5-year survival for patients with chondrosarcoma held good discriminative ability and overall performance on international external validation; however, it underestimated 5-year survival for patients with predicted probabilities from 0 to 0.8 because the calibration plot was not perfectly aligned for the observed outcomes, which resulted in a maximum underestimation of 20%. The differences may reflect the baseline differences noted between the two study populations. The overall performance of the algorithm supports the utility of the algorithm and validation presented here. The freely available digital application for the algorithm is available here: https://sorg-apps.shinyapps.io/extremitymetssurvival/., Level of Evidence: Level III, prognostic study.

Objective: Aim of the manuscript is to discuss how to improve margins in sacral chordoma., Background: Chordoma is a rare neoplasm, arising in half cases from the sacrum, with reported local failure in >50% after surgery., Methods: A multidisciplinary meeting of the "Chordoma Global Consensus Group" was held in Milan in 2017, focusing on challenges in defining and achieving optimal margins in chordoma with respect to surgery, definitive particle radiation therapy (RT) and medical therapies. This review aims to report on the outcome of the consensus meeting and to provide a summary of the most recent evidence in this field. Possible new ways forward, including on-going international clinical studies, are discussed., Results: En-bloc tumor-sacrum resection is the cornerstone of treatment of primary sacral chordoma, aiming to achieve negative microscopic margins. Radical definitive particle therapy seems to offer a similar outcome compared to surgery, although confirmation in comparative trials is lacking; besides there is still a certain degree of technical variability across institutions, corresponding to different fields of treatment and different tumor coverage. To address some of these questions, a prospective, randomized international study comparing surgery versus definitive high-dose RT is ongoing. Available data do not support the routine use of any medical therapy as (neo)adjuvant/cytoreductive treatment., Conclusion: Given the significant influence of margins status on local control in patients with primary localized sacral chordoma, the clear definition of adequate margins and a standard local approach across institutions for both surgery and particle RT is vital for improving the management of these patients., Competing Interests: Declaration of competing interest The authors have declared no conflicts of interest., (Copyright © 2020 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Angiosarcoma of bone (B-AS) is a rare malignant tumor of vascular origin. The aim of this retrospective study is to report on treatments and prognosis. Data were collected from the EMSOS website. 80 patients in 9 centers included: 51 male/29 female; median age 54 years (range 17 to 92); 56% with localized disease, 44% metastatic. Primary tumor surgery: 76% (30% amputation, 26% intralesional margins); radiotherapy (RT): 41%; chemotherapy (CT): 47% (56% in metastatic, 41% in localized cases). With a median follow-up of 31 months (range 40 to 309), 5-year overall survival (OS) was 27% (95%CI 16-30): 41% (95%CI 25-56) for localized patients, and 8% (95%CI 0-20) for metastatic (p = 0.002). In metastatic patients, 1 year OS was significantly influenced by chemotherapy response: 67% (95CI% 29-100) for those who responded or had stable disease (n = 7), and 18% (95CI% 0-41) for patients with progressive disease (n = 11), p 0.002. The surgical complete remission (SCR) status was pivotal in localized patients (5-year OS 45% for SCR, 17% no SCR, p = 0.03); also 5-year OS was significantly influenced by age and site of the tumor. After multivariate analysis, the addition of radiotherapy to surgery significantly influenced the disease-free survival (DFS) rate, whereas the use of chemotherapy lost the significance showed at the univariate analysis. Overall, patients with metastatic B-AS have a dismal prognosis, with a prolonged survival in case with a response to chemotherapy. Experimental trials with more active systemic treatment regimens are needed. In patients with localized disease, the patient's age and site of the tumor are prognostic factors and any effort must be made to achieve an SCR status. No definitive conclusions can be drawn from our data on the use of adjuvant chemotherapy, while the use of adjuvant radiotherapy might improve DSF in patients surgically free of disease.

Purpose: To determine whether the administration of histology-tailored neoadjuvant chemotherapy (HT) was superior to the administration of standard anthracycline plus ifosfamide neoadjuvant chemotherapy (A+I) in high-risk soft tissue sarcoma (STS) of an extremity or the trunk wall., Patients and Methods: This was a randomized, open-label, phase III trial. Patients had localized high-risk STS (grade 3; size, ≥ 5 cm) of an extremity or trunk wall, belonging to one of the following five histologic subtypes: high-grade myxoid liposarcoma (HG-MLPS); leiomyosarcoma (LMS), synovial sarcoma (SS), malignant peripheral nerve sheath tumor (MPNST), and undifferentiated pleomorphic sarcoma (UPS). Patients were randomly assigned in a 1:1 ratio to receive three cycles of A+I or HT. The HT regimens were as follows: trabectedin in HG-MLPS; gemcitabine plus dacarbazine in LMS; high-dose prolonged-infusion ifosfamide in SS; etoposide plus ifosfamide in MPNST; and gemcitabine plus docetaxel in UPS. Primary and secondary end points were disease-free survival (DFS) and overall survival (OS), estimated using the Kaplan-Meier method and compared using Cox models adjusted for treatment and stratification factors. The study is registered at ClinicalTrials.gov (identifier NCT01710176)., Results: Between May 2011 and May 2016, 287 patients (UPS: n = 97 [33.8%]; HG-MLPS: n = 65 [22.6%]; SS: n = 70 [24.4%]; MPNST: n = 27 [9.4%]; and LMS: n = 28 [9.8%]) were randomly assigned to either A+I or HT. At the final analysis, with a median follow-up of 52 months, the projected DFS and OS probabilities were 0.55 and 0.47 (log-rank P = .323) and 0.76 and 0.66 (log-rank P = .018) at 60 months in the A+I arm and HT arm, respectively. No treatment-related deaths were observed., Conclusion: In a population of patients with localized high-risk STS, HT was not associated with a better DFS or OS, suggesting that A+I should remain the regimen to choose whenever neoadjuvant chemotherapy is used in patients with high-risk STS.

Importance: Osteosarcoma, the most common malignant bone tumor in children and adolescents, occurs in a high number of cancer predisposition syndromes that are defined by highly penetrant germline mutations. The germline genetic susceptibility to osteosarcoma outside of familial cancer syndromes remains unclear., Objective: To investigate the germline genetic architecture of 1244 patients with osteosarcoma., Design, Setting, and Participants: Whole-exome sequencing (n = 1104) or targeted sequencing (n = 140) of the DNA of 1244 patients with osteosarcoma from 10 participating international centers or studies was conducted from April 21, 2014, to September 1, 2017. The results were compared with the DNA of 1062 individuals without cancer assembled internally from 4 participating studies who underwent comparable whole-exome sequencing and 27 173 individuals of non-Finnish European ancestry who were identified through the Exome Aggregation Consortium (ExAC) database. In the analysis, 238 high-interest cancer-susceptibility genes were assessed followed by testing of the mutational burden across 736 additional candidate genes. Principal component analyses were used to identify 732 European patients with osteosarcoma and 994 European individuals without cancer, with outliers removed for patient-control group comparisons. Patients were subsequently compared with individuals in the ExAC group. All data were analyzed from June 1, 2017, to July 1, 2019., Main Outcomes and Measures: The frequency of rare pathogenic or likely pathogenic genetic variants., Results: Among 1244 patients with osteosarcoma (mean [SD] age at diagnosis, 16 [8.9] years [range, 2-80 years]; 684 patients [55.0%] were male), an analysis restricted to individuals with European ancestry indicated a significantly higher pathogenic or likely pathogenic variant burden in 238 high-interest cancer-susceptibility genes among patients with osteosarcoma compared with the control group (732 vs 994, respectively; P = 1.3 × 10-18). A pathogenic or likely pathogenic cancer-susceptibility gene variant was identified in 281 of 1004 patients with osteosarcoma (28.0%), of which nearly three-quarters had a variant that mapped to an autosomal-dominant gene or a known osteosarcoma-associated cancer predisposition syndrome gene. The frequency of a pathogenic or likely pathogenic cancer-susceptibility gene variant was 128 of 1062 individuals (12.1%) in the control group and 2527 of 27 173 individuals (9.3%) in the ExAC group. A higher than expected frequency of pathogenic or likely pathogenic variants was observed in genes not previously linked to osteosarcoma (eg, CDKN2A, MEN1, VHL, POT1, APC, MSH2, and ATRX) and in the Li-Fraumeni syndrome-associated gene, TP53., Conclusions and Relevance: In this study, approximately one-fourth of patients with osteosarcoma unselected for family history had a highly penetrant germline mutation requiring additional follow-up analysis and possible genetic counseling with cascade testing.

Cancer causes inflammation as it progresses through healthy tissue. The differentiation of tumoral growth from the surrounding inflammatory change is paramount in planning surgeries seeking to preserve function. This retrospective study aims at illustrating how a careful use of imaging (computed tomography (CT)/magnetic resonance imaging (MRI)) can help to draw the line between infiltration and inflammation. Out of 72 cases of parosteal osteosarcoma in our institution we selected 22 which had pretreatment imaging, and out of those, 14 that had both MRI and CT. Using Fisher's exact test, we evaluated the performance of each technique on accurately diagnosing medullary tumor infiltration, using histological analysis as a gold standard. All cases (14/14) demonstrated medullary abnormality on MRI, but only 6/14 (42.9%) demonstrated abnormality on CT. The 8/14 cases with MRI abnormality but no CT abnormality (57.1%) showed inflammation with no tumoral cells present on histological analysis. In the cases where the two examinations showed medullary abnormality (6/14) histology demonstrated tumoral infiltration. MRI demonstrated high sensitivity and negative predictive value, but low specificity and low positive predictive value and accuracy (P=1). CT demonstrated high sensitivity, specificity, high positive and negative predictive values and accuracy (P = 0.000333). MRI is highly sensitive for the detection of medullary abnormality but lacks specificity for tumor invasion. Correlation with CT is recommended in all cases of positive MR to add specificity for tumors. The adequate use of the two imaging methods allows to differentiate between inflammatory change and tumoral infiltration in POS, relevant for surgical planning.

Objective: Giant cell tumor of bone (GCTB) is a frequently recurring locally aggressive osteolytic lesion, where pathological osteoclastogenesis and bone destruction are driven by neoplastic stromal cells. Here, we studied if cell cycle fractions within the mononuclear cell compartment of GCTB can predict its progression-free survival (PFS)., Methods: 154 cases (100 primaries and 54 recurrent) from 139 patients of 40 progression events, was studied using tissue microarrays. Ploidy and in situ cell cycle progression related proteins including Ki67 and those linked with replication licensing (mcm2), G1-phase (cyclin D1, Cdk4), and S-G2-M-phase (cyclin A; Cdk2) fractions; cell cycle control (p21 waf1 ) and repression (geminin), were tested. The Prentice-Williams-Peterson (PWP) gap-time models with the Akaike information criterion (AIC) were used for PFS analysis., Results: Cluster analysis showed good correlation between functionally related marker positive cell fractions indicating no major cell cycle arrested cell populations in GCTB. Increasing hazard of progression was statistically associated with the elevated post-G1/S-phase cell fractions. Univariate analysis revealed significant negative association of poly-/aneuploidy (p < 0.0001), and elevated cyclin A (p < 0.001), geminin (p = 0.015), mcm2 (p = 0.016), cyclin D1 (p = 0.022) and Ki67 (B56: p = 0.0543; and Mib1: p = 0.0564 -strong trend) positive cell fractions with PFS. The highest-ranked multivariate interaction model (AIC = 269.5) also included ploidy (HR 5.68, 95%CI: 2.62-12.31, p < 0.0001), mcm2 (p = 0.609), cyclin D1 (HR 1.89, 95%CI: 0.88-4.09, p = 0.105) and cyclin A (p < 0.0001). The first and second best prognostic models without interaction (AIC = 271.6) and the sensitivity analysis (AIC = 265.7) further confirmed the prognostic relevance of combining these markers., Conclusion: Ploidy and elevated replication licensing (mcm2), G1-phase (cyclin D1) and post-G1 phase (cyclin A) marker positive cell fractions, indicating enhanced cell cycle progression, can assist in identifying GCTB patients with increased risk for a reduced PFS., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Parosteal osteosarcoma is a low-grade malignant bone tumor that can undergo dedifferentiation. The aim of this study was to analyze clinicopathologic features associated with clinical outcome in a large cohort of patients. Patients consecutively treated for parosteal osteosarcoma at Rizzoli Orthopedic Institute from 1900 to 2018 were reviewed and analyzed. Clincopathologic data of 195 patients with parosteal osteosarcoma were analyzed. Age at diagnosis ranged from 9 to 75 years (median 31). Median follow-up time was 150 months (range, 3-720). The most common tumor locations were femur (61.5%), humerus (15.9%) and tibia (12.8%). Wide surgical margins were achieved in 125 (64.1%) patients. Medullary involvement was present in 69 (35.4%) cases. Dedifferentiation occurred in 48 (24.6%) patients. Forty-five patients developed recurrence (23.1%; median time to recurrence of 36 months). At last follow-up, 155 (79.5%) patients were alive and without evidence of disease, 8 (4.1%) were alive with active disease, 23 (11.8%) died from disease, and 9 (4.6%) from unrelated causes. Patients with dedifferentiated parosteal osteosarcoma had worse 5-year (65% versus 96%) and 10-year survival (60% versus 96%) when compared to conventional tumors (P < .001). Wide surgical margins had positive impact on both disease-free (P < .001) and overall survival (P = .036). Medullary involvement, age at presentation and tumor size had no impact on survival. Dedifferentiation is the most important factor that negatively impacts clinical outcome. Surgical aim is to ensure radical removal with wide surgical margins to improve disease-free survival., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma histotype with uncertain differentiation. EMC is hallmarked by the rearrangement of the NR4A3 gene, which in most cases fuses with EWSR1 or TAF15. TAF15-translocated EMC seem to feature a more aggressive course compared to EWSR1-positive EMCs, but whether the type of NR4A3 chimera impinges upon EMC biology is still largely undefined. To gain insights on this issue, a series of EMC samples (7 EWSR1-NR4A3 and 5 TAF15-NR4A3) were transcriptionally profiled. Our study unveiled that the two EMC variants display a distinct transcriptional profile and that the axon guidance pathway is a major discriminant. In particular, class 4-6 semaphorins and axonal guidance cues endowed with pro-tumorigenic activity were more expressed in TAF15-NR4A3 tumors; vice versa, class 3 semaphorins, considered to convey growth inhibitory signals, were more abundant in EWSR1-NR4A3 EMC. Intriguingly, the dichotomy in axon guidance signaling observed in the two tumor variants was recapitulated in in vitro cell models engineered to ectopically express EWSR1-NR4A3 or TAF15-NR4A3. Moreover, TAF15-NR4A3 cells displayed a more pronounced tumorigenic potential, as assessed by anchorage-independent growth. Overall, our results indicate that the type of NR4A3 chimera dictates an axon guidance switch and impacts on tumor cell biology. These findings may provide a framework for interpretation of the different clinical-pathological features of the two EMC variants and lay down the bases for the development of novel patient stratification criteria and therapeutic approaches. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches.

Ewing sarcoma (EWS) is an aggressive mesenchymal tumor with unmet clinical need and significant social impacts on children, adolescents, and young adults. CD99, a hallmark surface molecule of EWS, participates in crucial biological processes including cell migration, differentiation, and death. EWS cells can release CD99 through exosomes (EXOs), specialized extracellular vesicles with major cell communication roles. Here we show that, as a consequence of CD99 silencing, EWS cells deliver exosomes with oncosuppressive functions that significantly reduce tumor aggressiveness. These CD99-lacking microvesicles modulate gene expression of the EWS-recipient cells, reduce proliferation and migration, in turn inducing a more-differentiated less-malignant phenotype. The most relevant effects were detected on the activator protein-1 signaling pathway whose regulation was found to be dependent on the specific cargo loaded in vesicles after CD99 shutdown. Investigation of the miRNA content of CD99-deprived EXOs identified miR-199a-3p as a key driver able to reverse EWS malignancy in experimental models as well as in clinical specimens. All together, our data provide evidence that the abrogation of CD99 in EWS tumor cells leads to produce and release EXOs capable to transfer their antineoplastic effects into the nearby tumor cells, suggesting a novel atypical role for these microvesicles in reversion of malignancy rather than in priming the soil for progression and metastatic seeding. This conceptually innovative approach might offer a new therapeutic opportunity to treat a tumor still refractory to most treatments.

Background: Ewing sarcoma (ES) is the second most common bone tumor in adolescents and children. Staging workup for ES includes imaging and bone marrow biopsy (BMB). The effective role of BMB is now under discussion., Procedure: A monoinstitutional retrospective analysis reviewed clinical charts, imaging, and histology of patients with diagnosis of ES treated at the Rizzoli Institute between 1998 and 2017., Results: The cohort included 504 cases of ES of bone; 137 (27%) had metastases at diagnosis, while the remaining 367 had localized disease. Twelve patients had a positive BMB (2.4%). Eleven had distant metastases detected at initial workup staging with imaging assessment: six patients presented with bone metastases, five with both bone and lung metastases. Only one patient with ES of the foot (second metatarsus) was found to have bone marrow involvement with negative imaging evaluation (0.3%)., Conclusions: On the basis of our data, we suggest reconsidering the effective role of BMB in initial staging workup for patients with ES with no signs of metastases by modern imaging techniques. In metastatic disease, the assessment of the bone marrow status may remain useful to identify a group of patients at very high risk who could benefit from different treatment strategies., (© 2019 Wiley Periodicals, Inc.)

Bone tissue is critically lagging behind soft tissues and biofluids in our effort to advance precision medicine. The main challenges have been accessibility and the requirement for deleterious decalcification processes that impact the fidelity of diagnostic histomorphology and hinder downstream analyses such as fluorescence in-situ hybridization (FISH). We have developed an alternative fixation chemistry that simultaneously fixes and decalcifies bone tissue. We compared tissue morphology, immunohistochemistry (IHC), cell signal phosphoprotein analysis, and FISH in 50 patient matched primary bone cancer cases that were either formalin fixed and decalcified, or theralin fixed with and without decalcification. Use of theralin improved tissue histomorphology, whereas overall IHC was comparable to formalin fixed, decalcified samples. Theralin-fixed samples showed a significant increase in protein and DNA extractability, supporting technologies such as laser-capture microdissection and reverse phase protein microarrays. Formalin-fixed bone samples suffered from a fixation artifact where protein quantification of β-actin directly correlated with fixation time. Theralin-fixed samples were not affected by this artifact. Moreover, theralin fixation enabled standard FISH staining in bone cancer samples, whereas no FISH staining was observed in formalin-fixed samples. We conclude that the use of theralin fixation unlocks the molecular archive within bone tissue allowing bone to enter the standard tissue analysis pipeline. This will have significant implications for bone cancer patients, in whom personalized medicine has yet to be implemented.

Introduction: DNA damaging drugs are widely used for the chemotherapeutic treatment of high-grade osteosarcoma (HGOS). In HGOS patients, several germline polymorphisms have been reported to impact on the development of adverse toxic events related to DNA damaging drugs treatment. Some of these polymorphisms, when present in tumor cells, may also influence treatment response and prognosis of HGOS patients. Area covered: In this review, the authors have focused on pharmacogenetic markers (mainly germline polymorphisms) described in patients with HGOS, which have proved or indicated to be related to the susceptibility to adverse toxic reactions and/or to influence response to DNA damaging drugs. The concordant and discordant results reported in different studies have also been discussed. Expert opinion: Response and toxicity predisposition to DNA damaging drugs are influenced by genes encoding proteins involved in their uptake, efflux, activation, inactivation, and in DNA repair, activity of which may vary according to specific gene variations. In HGOS, there is a substantial medical need for biomarkers predictive for individual response and toxicity predisposition to DNA-targeting drugs, which may be used to tailor therapy in order to decrease the occurrence of adverse side effects and increase treatment efficacy and safety.

Aims: Osteoblastoma-like osteosarcoma is a rare variant of osteosarcoma (1% of all osteosarcomas), histologically similar to osteoblastoma. In the current World Health Organisation (WHO) classification, osteoblastoma-like osteosarcoma is classified within the group of conventional (high-grade) osteosarcomas. However, several published cases have been actually regarded as low-grade malignant tumours. As strict morphological criteria to distinguish between low- and high-grade lesions are not available, we reviewed our series of osteoblastoma-like osteosarcomas in the attempt to identify clinical and morphological features predictive of aggressiveness., Methods and Results: We retrieved 15 cases of osteoblastoma-like osteosarcoma from the files of the Istituto Ortopedico Rizzoli. Patients received various treatments. Five patients developed metastasis and five patients developed local recurrences (all after incomplete surgery). Eleven patients were alive without disease, while four patients died of their disease. Statistical analysis revealed a statistically significant (P = 0.048) lower disease-free survival in patients with areas of conventional (high-grade) osteosarcoma., Conclusions: With the important limitation of a small cohort of patients, the presence of areas of conventional (high-grade) osteosarcoma is the only parameter to predict the aggressiveness of osteoblastoma-like osteosarcoma., (© 2018 John Wiley & Sons Ltd.)

Synovial sarcoma (SS) is a rare tumour, with dismal survival when metastasis occurs. SS contains a characteristic translocation (X;18)(p11;q11) and the fusion genes appear to be mutually exclusive and concordant in primary and metastatic tumours. Novel prognostic and predictive factors are required. The C‑X‑C motif chemokine ligand 12 (CXCL12)/C‑X‑C chemokine receptor 4 (CXCR4) axis is involved in tumour development and metastatic spread in many types of cancer and previous data have demonstrated a pivotal role of CXCR4 in SS cell migration and invasion. Bioinformatics and biological data indicated CXCR4 is a possible candidate target of miR‑494.3p, known to be involved in tumour progression. In this study, we analysed the expression of miR‑494.3p and its potential target, CXCR4, in a series of SS specimens. A significantly lower miR‑494.3p expression was found in the tumour compared to normal tissue associated with higher levels of CXCR4 both at the gene and protein level. The role of CXCR4 as a potential target of miR‑494.3p was assessed in two SS cell lines (SW982 and SYO‑I). Transfection with miR‑494.3p expression plasmid led to a marked decrease in CXCR4 gene and protein expression, concomitant with a transitory decrease in cell proliferation and migration. The SYO‑I cells also responded with an increased apoptotic fraction. The data of this study also demonstrate that the downregulation of miR‑494.3p in SS surgical specimens, concomitant with an increased expression of its potential target, CXCR4, was more evident in the metastatic subset. In vitro experiments confirmed that miR‑494.3p functioned as a tumour suppressor through the involvement of CXCR4 and ongoing studies are directed to better clarify its role in SS therapeutic strategies.

Background: Primary and recurrent giant cell tumor of bone is typically benign; however, rarely giant cell tumor of bone can undergo malignant transformation. Malignancy in giant cell tumor of bone may be primary (adjacent to benign giant cell tumor of bone at first diagnosis) or secondary (at the site of previously treated giant cell tumor of bone). Malignant giant cell tumor of bone has a poor prognosis; it is important to distinguish malignant from benign lesions to facilitate appropriate management. The true incidence of malignant giant cell tumor of bone is not known, probably owing to inaccurate diagnosis and inconsistent nomenclature. We have analyzed current data to provide a robust estimate of the incidence of malignancy in giant cell tumor of bone., Methods: A literature search was performed to source published reports of primary and secondary cases of malignant giant cell tumor of bone. Studies that reported a denominator were used to estimate the incidence of malignancy., Results: We identified 4 large series of patients with malignant giant cell tumor of bone that provided data on 2315 patients with giant cell tumor of bone. Across these studies, the cumulative incidence of malignancy was 4.0%; the cumulative incidence of primary malignancy was 1.6% compared with 2.4% for secondary malignancy. Our analyses confirmed that most malignant giant cell tumor of bone is secondary and occurs following radiation. In addition, data from 8 small series showed that 4.8% of patients with giant cell tumor of bone who received radiation therapy developed secondary malignancy., Conclusions: Malignant giant cell tumor of bone is rare, and its identification is hindered by a lack of clear diagnostic criteria. For optimal care of patients with giant cell tumor of bone, we recommend: comprehensive histologic sampling to ensure accurate diagnoses; watchful follow-up, particularly for patients treated with radiation; and timely treatment of local recurrence.

Introduction: Genetic characterization of osteosarcoma has evolved during the last decade, thanks to the integrated application of conventional and new candidate-driven and genome-wide technologies. Areas covered: This review provides an overview of the state of art in genetic testing applied to osteosarcoma, with particular regard to novel candidate genetic biomarkers that can be analyzed in tumor tissue and blood samples, which might be used to predict toxicity and prognosis, detect disease relapse, and improve patients' selection criteria for tailoring treatment. Expert commentary: Genetic testing based on modern technologies is expected to indicate new osteosarcoma-related prognostic markers and driver genes, which may highlight novel therapeutic targets and patients stratification biomarkers. The definition of tailored or targeted treatment approaches may improve outcome of patients with localized tumors and, even more, of those with metastatic disease, for whom progress in cure probability is highly warranted.

Malignant peripheral nerve sheath tumor is a rare and aggressive disease with poor treatment response, mainly affecting adolescents and young adults. Few molecular biomarkers are used in the management of this cancer type, and although TP53 is one of few recurrently mutated genes in malignant peripheral nerve sheath tumor, the mutation prevalence and the corresponding clinical value of the TP53 network remains unsettled. We present a multi-level molecular study focused on aberrations in the TP53 network in relation to patient outcome in a series of malignant peripheral nerve sheath tumors from 100 patients and 38 neurofibromas, including TP53 sequencing, high-resolution copy number analyses of TP53 and MDM2, and gene expression profiling. Point mutations in TP53 were accompanied by loss of heterozygosity, resulting in complete loss of protein function in 8.2% of the malignant peripheral nerve sheath tumors. Another 5.5% had MDM2 amplification. TP53 mutation and MDM2 amplification were mutually exclusive and patients with either type of aberration in their tumor had a worse prognosis, compared to those without (hazard ratio for 5-year disease-specific survival 3.5, 95% confidence interval 1.78-6.98). Both aberrations had similar consequences on the gene expression level, as analyzed by a TP53-associated gene signature, a property also shared with the copy number aberrations and/or loss of heterozygosity at the TP53 locus, suggesting a common "TP53-mutated phenotype" in as many as 60% of the tumors. This was a poor prognostic phenotype (hazard ratio = 4.1, confidence interval:1.7-9.8), thus revealing a TP53-non-aberrant patient subgroup with a favorable outcome. The frequency of the "TP53-mutated phenotype" warrants explorative studies of stratified treatment strategies in malignant peripheral nerve sheath tumor.

Purpose: Approximately 5% of giant cell tumors (GCT) of bone develop pulmonary metastases. Although many biomarkers have been proposed, identification of circulating low abundance molecules may be useful to predict malignant progression., Experimental Design: The hydrogel nanoparticle technique followed by MS was used to detect low molecular weight serum proteins or protein fragments in serum of 20 GCT patients with different clinical course and in ten healthy sera used as control. The most representative low-abundant de novo or differentially abundant proteins were submitted to String database that recognized interconnected activated pathways including protein activation cascade, wound healing, cell-substrate adhesion, and response to stress. Statistics were performed for identification of candidate prognostic factors., Results: Proteome cluster analysis separated metastasis-free from metastatic GCT patients in two well-defined groups where serum levels of signaling transduction mediators and regulators of kinase activity presented a high discriminatory power. Increased expression of proteins STAT5B, GRB2, and OXSR1 was related to a higher probability of metastasis. Multivariate analysis demonstrated that tumor grade and STAT5B were independent prognostic factors., Conclusions and Clinical Relevance: By using a noninvasive technique, we identified differentially abundant serum candidate biomarkers, also providing prognostic information in patients with GCT of bone., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Trabectedin is an alkylating drug with a unique mechanism of action causing single-strand and double-strand DNA breaks that activate DNA damage-response pathways. Based on our preclinical data, we hypothesised that poly(ADP-ribose) polymerase 1 (PARP1) inhibitors might be an ideal partner of trabectedin and aimed to assess the safety, identify the recommended phase 2 dose, and explore preliminary signs of activity of trabectedin and olaparib combination treatment in patients with bone and soft-tissue sarcoma., Methods: We did an open-label, multicentre, phase 1b study, recruiting patients from the national Italian sarcoma network aged 18 years and older with histologically confirmed bone and soft-tissue sarcoma progressing after standard treatments with Eastern Cooperative Oncology Group performance status of 1 or less. In a classic 3 + 3 design, patients received a 24 h infusion of trabectedin on day 1 and olaparib orally twice a day in 21-day cycles across six dose levels (trabectedin 0·675-1·3 mg/m 2 every 3 weeks; olaparib 100-300 mg twice a day from day 1 to 21). Intermediate dose levels were permitted to improve safety and tolerability. The primary endpoint was determination of the recommended phase 2 dose (the maximum tolerated dose). Safety and antitumour activity were assessed in all patients who received at least one dose of the study drugs. We report the results of the dose-escalation and dose-expansion cohorts. The trial is still active but closed to enrolment, and follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov, number NCT02398058., Findings: Between Nov 17, 2014, and Jan 30, 2017, of 54 patients assessed for eligibility, we enrolled 50 patients: 28 patients in the dose-escalation cohort and 22 patients in the dose-expansion cohort. Patients received a median of four cycles of treatment (IQR 2-6; range 1-17 [the patients who received the highest number of cycles are still on treatment]) with a median follow-up of 10 months (IQR 5-23). Considering all dose levels, the most common grade 3-4 adverse events were lymphopenia (32 [64%] of 50 patients), neutropenia (31 [62%]), thrombocytopenia (14 [28%]), anaemia (13 [26%]), hypophosphataemia (20 [40%]), and alanine aminotransferase concentration increase (9 [18%]). No treatment-related life-threatening adverse events or deaths occurred. One (2%) patient interrupted treatment without progression without reporting any specific toxicity. Observed dose-limiting toxicities were thrombocytopenia, neutropenia for more than 7 days, and febrile neutropenia. We selected intermediate dose level 4b (trabectedin 1·1 mg/m 2 every 3 weeks plus olaparib 150 mg twice a day) as the recommended phase 2 dose. Seven (14%; 95% CI 6-27) of 50 patients achieved a partial response according to Response Evaluation Criteria In Solid Tumors 1.1., Interpretation: Trabectedin and olaparib in combination showed manageable toxicities at active dose levels for both drugs. Preliminary data on antitumour activity are encouraging. Two dedicated phase 2 studies are planned to assess activity of this combination in both ovarian cancer (EudraCT2018-000230-35) and soft-tissue sarcomas., Funding: Italian Association for Cancer Research, Italian Sarcoma Group, Foundation for Research on Musculoskeletal and Rare Tumors, and Italian Ministry of Health., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose:: Aneurysmal bone cyst (ABC) is a rare skeletal tumor usually treated with surgery/embolization. We hypothesized that owing to similarities with giant cell tumor of bone (GCTB), denosumab was active also in ABC., Methods:: In this observational study, a retrospective analysis of ABC patients treated with denosumab was performed. Patients underwent radiologic disease assessment every 3 months. Symptoms and adverse events were noted., Results:: Nine patients were identified (6 male, 3 female), with a median age of 17 years (range 14-42 years). Primary sites were 6 spine-pelvis, 1 ulna, 1 tibia, and 1 humerus. Patients were followed for a median time of 23 months (range 3-55 months). Patients received a median of 8 denosumab administrations (range 3-61). All symptomatic patients had pain relief and 1 had paresthesia improvement. Signs of denosumab activity were observed after 3 to 6 months of administration: bone formation by computed tomography scan was demonstrated in all patients and magnetic resonance imaging gadolinium contrast media decrease was observed in 7/9 patients. Adverse events were negligible. At last follow-up, all patients were progression-free: 5 still on denosumab treatment, 2 off denosumab were disease-free 11 and 17 months after surgery, and the last 2 patients reported no progression 12 and 24 months after denosumab interruption and no surgery., Conclusions:: Denosumab has substantial activity in ABCs, with favorable toxicity profile. We strongly support the use of surgery and/or embolization for the treatment of ABC, but denosumab could have a role as a therapeutic option in patients with uncontrollable, locally destructive, or recurrent disease.

The identification of new therapeutic strategies against osteosarcoma, the most common primary bone tumor, continues to be a primary goal to improve the outcomes of patients refractory to conventional chemotherapy. Osteosarcoma originates from the transformation of mesenchymal stem cells (MSC) and/or osteoblast progenitors, and the loss of differentiation is a common biological osteosarcoma feature, which has strong significance in predicting tumor aggressiveness. Thus, restoring differentiation through epigenetic reprogramming is potentially exploitable for therapeutic benefits. Here, we demonstrated that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 affected tumor proliferation by blocking osteosarcoma cells in G 1 or G 2 -M phases and induced osteoblastic differentiation through the specific reexpression of genes regulating this physiologic process. Although MC3343 has a similar antiproliferative effect as 5azadC, the conventional FDA-approved nucleoside inhibitor of DNA methylation, its effects on cell differentiation are distinct. Induction of the mature osteoblast phenotype coupled with a sustained cytostatic response was also confirmed in vivo when MC3343 was used against a patient-derived xenograft (PDX). In addition, MC3343 displayed synergistic effects with doxorubicin and cisplatin (CDDP), two major chemotherapeutic agents used to treat osteosarcoma. Specifically, MC3343 increased stable doxorubicin bonds to DNA, and combined treatment resulted in sustained DNA damage and increased cell death. Overall, this nonnucleoside DNMTi is an effective novel agent and is thus a potential therapeutic option for patients with osteosarcoma who respond poorly to preadjuvant chemotherapy. Mol Cancer Ther; 17(9); 1881-92. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Importance: Epithelioid sarcoma (ES) is an exceedingly rare malignant neoplasm with distinctive pathologic, molecular, and clinical features as well as the potential to respond to new targeted drugs. Little is known on the activity of anthracycline-based regimens, gemcitabine-based regimens, and pazopanib in this disease., Objective: To report on the activity of anthracycline-based regimens, gemcitabine-based regimens, and pazopanib in patients with advanced ES., Design, Setting, and Participants: Seventeen sarcoma reference centers in Europe, the United States, and Japan contributed data to this retrospective analysis of patients with locally advanced/metastatic ES diagnosed between 1990 and 2016. Local pathological review was performed in all cases to confirm diagnosis according to most recent criteria., Exposures: All patients included in the study received anthracycline-based regimens, gemcitabine-based regimens, or pazopanib., Main Outcome and Measures: Response was assessed by RECIST. Progression-free survival (PFS) and overall survival (OS) were computed by Kaplan-Meier method. Classic and proximal subtypes were defined based on morphology (according to 2013 World Health Organization guidelines)., Results: Overall, 115 patients were included, 80 (70%) were men and 35 (30%) were women, with a median age of 32 years (range, 15-77 years). Of the 115 patients with ES, 85 were treated with anthracycline-based regimens, 41 with gemcitabine-based regimens, and 18 with pazopanib. Twenty-four received more than 1 treatment. Median follow-up was 34 months. Response rate for anthracycline-based regimens was 22%, with a median PFS of 6 months. One complete response (CR) was reported. A trend toward a higher response rate was noticed in morphological proximal type (26%) vs classic type (19%) and in proximal vs distal primary site (26% vs 18%). The response rate for gemcitabine-based regimens was 27%, with 2 CR and a median PFS of 4 months. In this group, a trend toward a higher response rate was reported in classic vs proximal morphological type (30% vs 22%) and in distal vs proximal primary site (40% vs 14%). In the pazopanib group, no objective responses were seen, and median PFS was 3 months., Conclusions and Relevance: This is the largest retrospective series of systemic therapy in ES. We confirm a moderate activity of anthracycline-based and gemcitabine-based regimens in ES, with a similar response rate and PFS in both groups. The value of pazopanib was low. These data may serve as a benchmark for trials of novel agents in ES.

Ewing sarcoma (EWS) is a pediatric cancer characterized by the EWSR1-FLI1 fusion. We performed a genome-wide association study of 733 EWS cases and 1346 unaffected individuals of European ancestry. Our study replicates previously reported susceptibility loci at 1p36.22, 10q21.3 and 15q15.1, and identifies new loci at 6p25.1, 20p11.22 and 20p11.23. Effect estimates exhibit odds ratios in excess of 1.7, which is high for cancer GWAS, and striking in light of the rarity of EWS cases in familial cancer syndromes. Expression quantitative trait locus (eQTL) analyses identify candidate genes at 6p25.1 (RREB1) and 20p11.23 (KIZ). The 20p11.22 locus is near NKX2-2, a highly overexpressed gene in EWS. Interestingly, most loci reside near GGAA repeat sequences and may disrupt binding of the EWSR1-FLI1 fusion protein. The high locus to case discovery ratio from 733 EWS cases suggests a genetic architecture in which moderate risk SNPs constitute a significant fraction of risk.

Purpose: Large-scale sequencing studies have indicated that besides genomic alterations, the posttranscriptional regulation of gene expression or epigenetic mechanisms largely influences the clinical behavior of Ewing sarcoma. We investigated the significance of the RNA-binding protein IGF2BP3 in the regulation of Ewing sarcoma aggressiveness. Experimental Design: Explorative study was performed in 14 patients with localized Ewing sarcoma using RNA sequencing. Next, 128 patients with localized Ewing sarcoma were divided into two cohorts. In the training set, 29 Ewing sarcoma samples were analyzed using Affymetrix GeneChip arrays. In the validation set, 99 Ewing sarcoma samples were examined using qRT-PCR. Patient-derived cell lines and experimental models were used for functional studies. Results: Univariate and multivariate analyses indicated IGF2BP3 as a potent indicator of poor prognosis. Furthermore, ABCF1 mRNA was identified as a novel partner of IGF2BP3. Functional studies indicated IGF2BP3 as an oncogenic driver and ABCF1 mRNA as a sponge that by binding IGF2BP3, partly repressed its functions. The combined evaluation of IGF2BP3 and ABCF1 could identify different patient outcomes-high IGF2BP3 and low ABCF1 levels indicated poor survival (25%), whereas low IGF2BP3 and high ABCF1 levels indicated favorable survival (85.5%). The bromodomain and extraterminal domain inhibitor (BETi) JQ1 decreased IGF2BP3 expression, modified the expression of its validated targets and inhibited the capability of Ewing sarcoma cells to grow under anchorage-independent conditions. Conclusions: The combined assessment of IGF2BP3 and ABCF1 predicts recurrence in Ewing sarcoma patients. Thus, for patients with high expression of IGF2BP3 and poor probability of survival, the use of BETis should be clinically evaluated. Clin Cancer Res; 24(15); 3704-16. ©2018 AACR ., (©2018 American Association for Cancer Research.)