Aims. The aim of this study is to determine the predictors of overall survival (OS) and predictive factors of poor prognosis of conventional high-grade osteosarcoma of the limbs in a single-centre in South Africa. Methods. We performed a retrospective cross-sectional analysis to identify the prognostic factors that predict the OS of patients with histologically confirmed high-grade conventional osteosarcoma of the limbs over ten years. We employed the Cox proportional regression model and the Kaplan-Meier method for statistical analysis. Results. This study comprised 77 patients at a three-year minimum follow-up. The predictors of poor OS were: the median age of ≤ 19 years (hazard ratio (HR) 0.96; 95% confidence interval (CI) 0.92 to 0.99; p = 0.021); median duration of symptoms ≥ five months (HR 0.91; 95% CI 0.83 to 0.99; p < 0.037); metastasis at diagnosis (i.e. Enneking stage III) (HR 3.33; 95% CI 1.81 to 6.00; p < 0.001); increased alkaline phosphatase (HR 3.28; 95% CI 1.33 to 8.11; p < 0.010); palliative treatment (HR 7.27; 95% CI 2.69 to 19.70); p < 0.001); and amputation (HR 3.71; 95% CI 1.12 to 12.25; p < 0.032). In contrast, definitive surgery (HR 0.11; 95% CI 0.03 to 0.38; p < 0.001) and curative treatment (HR 0.18; 95% CI 0.10 to 0.33; p < 0.001) were a protective factor. The Kaplan-Meier median survival time was 24 months, with OS of 57.1% at the three years. The projected five-year event-free survival was 10.3% and OS of 29.8% (HR 0.76; 95% CI 0.52 to 1.12; p = 0.128). Conclusion. In this series of high-grade conventional osteosarcoma of the appendicular skeleton from South Africa, 58.4% (n = 45) had detectable metastases at presentation; hence, an impoverished OS of five years was 29.8%. Large-scale future research is needed to validate our results. Cite this article: Bone Jt Open 2024;5(3):210–217

Aims. Low-grade central osteosarcoma (LGCOS), a rare type of osteosarcoma, often has misleading radiological and pathological features that overlap with those of other bone tumours, thereby complicating diagnosis and treatment. We aimed to analyze the clinical, radiological, and pathological features of patients with LGCOS, with a focus on diagnosis, treatment, and outcomes. Methods. We retrospectively analyzed the medical records of 49 patients with LGCOS (Broder’s grade 1 to 2) treated between January 1985 and December 2017 in a single institute. We examined the presence of malignant features on imaging (periosteal reaction, cortical destruction, soft-tissue invasion), the diagnostic accuracy of biopsy, surgical treatment, and oncological outcome. Results. Based on imaging, 35 of 49 patients (71.4%) exhibited malignant features. Overall, 40 of 49 patients (81.6%) had undergone a biopsy before en-bloc resection: 27 of 40 patients (67.5%) were diagnosed on the first biopsy, which was more accurate when carried out by open rather than needle biopsy (91.3% vs 35.3% diagnostic accuracy, respectively; p < 0.001). Of the 40 patients treated by en-bloc resection, surgical margins were wide in 38 (95.0%) and marginal in two (5.0%). Furthermore, nine of 49 patients (18.4%) underwent curettage (intralesional margin) without previous biopsy. All patients with a positive margin developed local recurrence. Distant metastases occurred in five of 49 patients (10.2%). The mean five-year overall survival (OS) and distant relapse-free survival (D-RFS) were 89.3% (SD 5.1%) and 85.7% (SD 5.5%), respectively. Univariate analysis showed that the occurrence of distant metastasis was a poor prognostic factor for OS (hazard ratio 11.54, 95% confidence interval (CI) 1.92 to 69.17; p < 0.001). Local recurrence was a poor prognostic factor for D-RFS (HR 8.72, 95% CI 1.69 to 45.0; p = 0.002). Conclusion. The diagnosis of LGCOS can be challenging because it may present with non-malignant features and has a low diagnostic accuracy on biopsy. If precisely diagnosed, LGCOS can be successfully treated by surgical excision with wide margins. Cite this article: Bone Joint J 2024;106-B(1):99–106

Aims. The purpose of this study was to clarify the clinical behaviour, prognosis, and optimum treatment of dedifferentiated low-grade osteosarcoma (DLOS) diagnosed based on molecular pathology. Patients and Methods. We retrospectively reviewed 13 DLOS patients (six men, seven women; median age 32 years (interquartile range (IQR) 27 to 38)) diagnosed using the following criteria: the histological coexistence of low-grade and high-grade osteosarcoma components in the lesion, and positive immunohistochemistry of mouse double minute 2 homolog (MDM2) and cyclin-dependent kinase 4 (CDK4) associated with MDM2 amplification. These patients were then compared with 51 age-matched consecutive conventional osteosarcoma (COS) patients (33 men, 18 women; median age 25 years (IQR 20 to 38)) regarding their clinicopathological features. Results. The five-year overall survival (OAS) rates in the DLOS and COS patients were 85.7% and 77.1% (p = 0.728), respectively, and the five-year progression-free survival (PFS) rates were 57.7% and 44.9% (p = 0.368), respectively. A total of 12 DLOS patients received chemotherapy largely according to regimens for COS. Among the nine cases with a histological evaluation after chemotherapy, eight showed a poor response, and seven of these had a necrosis rate of < 50%. One DLOS patient developed local recurrence and five developed distant metastases. Conclusion. Based on our study of 13 DLOS cases that were strictly defined by histological and molecular means, DLOS showed a poorer response to a standard chemotherapy regimen than COS, while the clinical outcomes were not markedly different. Cite this article: Bone Joint J 2019;101-B:745–752

Aims. Venous tumour thrombus (VTT) is a rare finding in osteosarcoma. Despite the high rate of VTT in osteosarcoma of the pelvis, there are very few descriptions of VTT associated with extrapelvic primary osteosarcoma. We therefore sought to describe the prevalence and presenting features of VTT in osteosarcoma of both the pelvis and the limbs. Methods. Records from a single institution were retrospectively reviewed for 308 patients with osteosarcoma of the pelvis or limb treated between January 2000 and December 2022. Primary lesions were located in an upper limb (n = 40), lower limb (n = 198), or pelvis (n = 70). Preoperative imaging and operative reports were reviewed to identify patients with thrombi in proximity to their primary lesion. Imaging and histopathology were used to determine presence of tumour within the thrombus. Results. Tumours abutted the blood vessels in 131 patients (43%) and encased the vessels in 30 (10%). Any form of venous thrombus was identified in 31 patients (10%). Overall, 21 of these thrombi were determined to be involved with the tumour based on imaging (n = 9) or histopathology (n = 12). The rate of VTT was 25% for pelvic osteosarcoma and 1.7% for limb osteosarcoma. The most common imaging features associated with histopathologically proven VTT were enhancement with contrast (n = 12; 100%), venous enlargement (n = 10; 83%), vessel encasement (n = 8; 66%), and visible intraluminal osteoid matrix (n = 6; 50%). Disease-specific survival (DSS) for patients with VTT was 95% at 12 months (95% CI 0.87 to 1.00), 50% at three years (95% CI 0.31 to 0.80), and 31% at five years (95% CI 0.14 to 0.71). VTT was associated with worse DSS (hazard ratio 2.3 (95% CI 1.11 to 4.84). Conclusion. VTT is rare with osteosarcoma and occurs more commonly in the pelvis than the limbs. Imaging features suggestive of VTT include enhancement with contrast, venous dilation, and vessel encasement. VTT portends a worse prognosis for patients with osteosarcoma, with a similar survivability to metastatic disease. Cite this article: Bone Joint J 2024;106-B(8):865–870

Osteosarcoma is common in children and adolescents with high mortality due to rapid progression. Therapeutic approaches for osteosarcoma are limited and may cause side effects. Cannabinoid ligands exert antiproliferative, apoptotic effect in cancer cells via CB1/2 or TRPV1 receptors. In this study, we hypothesized that synthetic specific CB2R agonist CB65 might have an antiproliferative and apoptotic effect on osteosarcoma cell lines in vitro. If so, this agent might be a chemotherapeutic candidate for osteosarcoma, with prolonged release, increased stability and bioavailability when loaded into a liposomal system. We first determined CB2 receptor expression in MG63 and Saos-2 osteosarcoma cells by qRT- PCR and FCM. CB65 reduced proliferation in osteosarcoma cells by WST-1 and RTCA. IC50 for MG63 and Saos-2 cells were calculated as 1.11×10-11 and 4.95×10-11 M, respectively. The antiproliferative effect of CB65 on osteosarcoma cells was inhibited by CB2 antagonist AM630. IC50 of CB65 induced late apoptosis of MG63 and Saos-2 cells at 24 and 48 hours, respectively by FCM. CB65 was loaded into the liposomal system by thin film hydration method and particle size, polydispersity index, and zeta potentials were 141.7±0.6 nm, 0.451±0.026, and -10.9±0.3 mV, respectively. The CB65-loaded liposomal formulation reduced MG63 and Saos-2 cell proliferation by RTCA. IC50 of CB65 and CB65-loaded liposomal formulation induced late apoptosis of MG63 and Saos-2 cells at 24 and 48 hours, respectively, by FCM. Scratch width was higher in CB65 and CB65-loaded liposome-treated cells compared to control. In this study, the real-time antiproliferative and apoptotic effect of synthetic specific CB2 agonist CB65 in osteosarcoma cell lines was demonstrated for the first time, and the real time therapeutic window was determined. The CB65-loaded liposomal formulation presents a potential treatment option that can be translated to clinic following its validation within animal models and production under GMP conditions

Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between in vitro/in vivo results and clinical outcomes. Spheroids are a promising in vitro model to mimic osteosarcoma and perform drug-screening tests, as they (i) reproduce the microarchitecture of the tumor, (ii) are characterized by hypoxic regions and necrotic core as the in vivo tumor, (iii) and recapitulate the chemo-resistance phenomena. However, to date, the spheroid model is scarcely used in osteosarcoma research. Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms. To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode

The February 2024 Oncology Roundup. 360. looks at: Does primary tumour resection improve survival for patients with sarcomas of the pelvis with metastasis at diagnosis?; Proximal femur replacements for an oncologic indication offer a durable endoprosthetic reconstruction option: a 40-year experience; The importance of awaiting biopsy results in solitary pathological proximal femoral fractures: do we need to biopsy solitary pathological fractures?; Effect of radiotherapy on local recurrence, distant metastasis, and overall survival in 1,200 extremity soft-tissue sarcoma patients; What to choose in bone tumour resections? Patient-specific instrumentation versus surgical navigation; Optimal timing of re-excision in synovial sarcoma patients: immediate intervention versus waiting for local recurrence; Survival differences of patients with resected extraskeletal osteosarcoma receiving two different (neo) adjuvant chemotherapy regimens; Solitary versus multiple bone metastases in the appendicular skeleton: should the surgical treatment be different?

Macrophages (Mφ) are immune cells that play a crucial role in both innate and adaptive immunity as they are involved in a wide range of physiological and pathological processes. Depending on the microenvironment and signals present, Mφ can polarize into either M1 or M2 phenotypes, with M1 macrophages exhibiting pro-inflammatory and cytotoxic effects, while M2 macrophages having immunosuppressive and tissue repair properties. Macrophages have been shown to play key roles in the development and progression or inhibition of various diseases, including cancer. For example, macrophages can stimulate tumor progression by promoting immunosuppression, angiogenesis, invasion, and metastasis. This work aimed to investigate the effect of extracellular vesicles (EVs)-derived from polarized macrophages on an osteosarcoma cell line. Monocytes were extracted from buffy coats and cultured in RPMI medium with platelet lysate or M-CSF. After 6 days of seeding, Mφ were differentiated into M1 and M2 with INF-γ/LPS and IL-4/IL-13, respectively. The medium with M1 or M2 derived EVs was collected and EVs were isolated by differential centrifugation and size exclusion chromatography and its morphology and size were characterized with SEM and NTA, respectively. The presence of typical EVs markers (CD9, CD63) was assessed by Western Blot. Finally, EVs from M1 or M2-polarized Mφ were added onto osteosarcoma cell cultures and their effect on cell viability and cell cycle, proliferation, and gene expression was assessed. The EVs showed the typical shape, size and surface markers of EVs. Overall, we observed that osteosarcoma cells responded differentially to EVs isolated from the M1 and M2-polarized Mφ. In summary, the use of Mφ-derived EVs for the treatment of osteosarcoma and other cancers deserves further study as it could benefit from interesting traits of EVs such as low immunogenicity, nontoxicity, and ability to pass through tissue barriers. Acknowledgements: Carlos III Health Institute and the European Social Fund for contract CP21/00136 and project PI22/01686

Aims. The aim of this study was to determine the rate of indocyanine green (ICG) staining of bone and soft-tissue tumours, as well as the stability and accuracy of ICG fluorescence imaging in detecting tumour residuals during surgery for bone and soft-tissue tumours. Methods. ICG fluorescence imaging was performed during surgery in 34 patients with bone and soft-tissue tumours. ICG was administered intravenously at a dose of 2 mg/kg over a period of 60 minutes on the day prior to surgery. The tumour stain rate and signal-to-background ratio of each tumour were post hoc analyzed. After tumour resection, the tumour bed was scanned to locate sites with fluorescence residuals, which were subsequently inspected and biopsied. Results. The overall tumour stain rate was 88% (30/34 patients), and specific stain rates included 90% for osteosarcomas and 92% for giant cell tumours. For malignant tumours, the overall stain rate was 94%, while it was 82% for benign tumours. The ICG tumour stain was not influenced by different pathologies, such as malignant versus benign pathology, the reception (or lack thereof) of neoadjuvant chemotherapies, the length of time between drug administration and surgery, the number of doses of denosumab for patients with giant cell tumours, or the tumour response to neoadjuvant chemotherapy. The overall accuracy rate of successfully predicting tumour residuals using fluorescence was 49% (23/47 pieces of tissue). The accuracy rate after en bloc resection was significantly lower than that after piecemeal resection (16% vs 71%; p < 0.001). Conclusion. A high percentage of bone and soft-tissue tumours can be stained by ICG and the tumour staining with ICG was stable. This approach can be used in both benign and malignant tumours, regardless of whether neoadjuvant chemotherapy is adopted. The technique is also useful to detect tumour residuals in the wound, especially in patients undergoing piecemeal resection. Cite this article: Bone Joint J 2023;105-B(5):551–558

Osteosarcoma is the most common primary bone tumour worldwide. This disease presents a formidable challenge to the orthopaedic surgeon, with a mortality rate of 30 per cent, even after surgical clearance. Aberrant Wnt signalling has been implicated in the pathogenesis osteoblastic tumours. The objective of this study is 2 fold- to investigate if osteosarcoma does indeed demonstrate aberrant Wnt signaling, and if so, does osteosarcoma respond to a novel Wnt inhibitor(ETC159). This can potentially lead to the development of a new adjuvant treatment modality for osteosarcoma. A novel Wnt signaling pathway protein antibody (YJ5) was used in immunihistochemistry staining of clinical osteosarcoma samples. A Wnt high osteosarcoma cell line(SJSA-1) was then implanted subcutaneously in a mouse model. These mice were treated with a novel PORCN inhibitor, ETC 159 for a period of 4 weeks in a two-arm randomised control study. The results of treatment were evaulated by clinical outcome parameters as well as immunohstochemistry. 100 per cent of clinical osteosarcoma samples demonstrated increased WLS expression and Wnt protein expression. SJSA-1 showed no significant decrease in tumour volume after 30 days of drug treatment (3070 SD 625 mm3 vs 3480 SD 433 mm3 p= 0.605 and 2060 SD 209 vs 1677 SD 213 mm3 p=0.219 respectively). Significantly, SJSA-1 demonstrated increased tumour necrosis in the treatment arm(30–60 percent increase across all samples p < 0 .005) Treated tumours also demonstrated markedly less angiogenesis compared to the non treatment arm. Osteosarcoma demonstrates aberrant Wnt signaling in a large percentage of cases. The use of a novel PORCN inhibitor ETC 159 for the treatment of Osteosarcoma has a marked effect on tumour necrosis. Our results suggest that ETC159 may cause tumour necrosis by inhibiting angiogenesis within the tumour. Further evaluation and understanding of the mechanism of Wnt singaling in regulating tumour pathogenesis may hold the potential for developing a curative therapeutic drug for this deadly disease

We conducted an epidemiological study of osteosarcoma in Sweden from 1971 to 1984 to investigate whether the typical features of the disease had changed. Of 294 osteosarcoma patients reviewed, 249 had primary skeletal tumours, and for these the mean annual incidence was 2.1 per million, without any clear trend over time. The mean male/female ratio for the period was 1.6 again with no consistent pattern over time; nor was there any significant change in the distribution of tumours according to location. The only feature which showed a significant change over the 14-year period was the mean age of the patients, being at its lowest (19 years) in 1972 and at its highest (40 years) in 1981. Excluding the 22 patients with craniofacial tumours, the remaining 227 also showed a significant increase in mean age. Analysis of the annual age distribution disclosed an increasing fraction of patients older than those in the classical age peak between 10 and 29 years. A large number of trials have shown improved survival in osteosarcoma over the last 15 years, which has been attributed mainly to adjuvant chemotherapy. The change we have observed in age distribution should also be taken into account in the evaluation of the results of treatment; it may be that older patients have a better prognosis

Traditional staging systems for high grade osteosarcoma (Enneking, MSTS) are based largely on gross surgical margins and were developed before the widespread use of neoadjuvant chemotherapy. It is now well known that both microscopic margins and chemotherapy are predictors of local recurrence. However, neither of these variables are used in the traditional surgical staging and the precise safe margin distance is debated. Recently, a novel staging system utilizing a 2mm margin cutoff and incorporating precent necrosis was proposed and demonstrated improved prognostic value for local recurrence free survival (LRFS) when compared to the MSTS staging system. This staging system has not been validated beyond the original patient cohort. We propose to analyze this staging system in a cohort of patients with high-grade osteosarcoma, as well as evaluate the ability of additional variables to predict the risk of local recurrence and overall survival. A retrospective review of a prospectively collected database of all sarcoma patients between 1985 and 2020 at a tertiary sarcoma care center was performed. All patients with high-grade osteosarcoma receiving neo-adjuvant chemotherapy and with no evidence of metastatic disease on presentation were isolated and analyzed. A minimum of two year follow up was used for surviving patients. A total of 225 patients were identified meeting these criteria. Univariate analysis was performed to evaluate variable that were associated with LRFS. Multivariate analysis is used to further analyze factors associated with LRFS on univariate analysis. There were 20 patients (8.9%) who had locally recurrent disease. Five-year LRFS was significantly different for patients with surgical margins 2mm or less (77.6% v. 93.3%; p=0.006) and those with a central tumor location (67.9 v. 94.4; <0.001). A four-tiered staging system using 2mm surgical margins and a percent necrosis of 90% of greater was also a significant predictor of 5-year LRFS (p=0.019) in this cohort. Notably, percent necrosis in isolation was not a predictor of LRFS in this cohort (p=0.875). Tumor size, gender, and type of surgery (amputation v. limb salvage) were also analyzed and not associated with LRFS. The MSTS surgical margin staging system did not significantly stratify groups (0.066). A 2mm surgical margin cutoff was predictive of 5-year LRFS in this cohort of patients with localized high-grade osteosarcoma and a combination of a 2mm margin and percent necrosis outperformed the prognostic value of the traditional MSTS staging system. Utilization of this system may improve the ability of surgeons to stage thier patients. Additional variables may increase the value of this system and further validation is required

The European and American Osteosarcoma study group (EURAMOS) was formed by four multinational study groups (COG, COSS, EOI, SSG) based upon a common understanding that broad international collaboration facilitating randomised trials was important for further progress in the field of osteosarcoma. Representatives from each group reached quick agreement on a study design; to determine whether altering post-operative therapy based on response to pre-operative chemotherapy improves outcome. Additionally, a quality-of-life sub-study was included in the project. After a three-year process to resolve regulatory and organisational issues the study opened for accrual in April 2005. Important for an efficient conduction of the trial a common infrastructure was established with central sponsorship (Medical Research Council, UK), one coordinating data centre (MRC Clinical Trials Unit, UK) and a common Safety Desk and a coordinating Quality-of-life data centre (Münster, DE). As of Dec 2008, a total of 1268 patients from 290 institution in 15 countries have been registered into the trial (AUS 16 patients; B 27; CAN 54; CH 26; D 266; DK 12; FIN 3; H 14; NL 61; NOR 23; NZ 8; OST 7; SWE 25; UK 174; USA 552) and 937 patients participate in the quality-of-life evaluation. 697 patients have been randomized and 53% are assessed as good histological responders. Due to a lower than expected randomisation rate and a higher than expected number of patients with a good histological response the accrual time will be extended by one year to summer 2010. In conclusion, EURAMOS-1 may serve as a model for a successful non-commercial multinational clinical trial in times of increasing economic and regulatory pressure. It is the fastest accruing and largest osteosarcoma trial ever. In addition, to addressing important questions in a randomised setting a common language in osteosarcoma has been established. Supported by the European Science Foundation (ESF) under the EUROCORES Program European Clinical Trials (ECT), through contract No. ERASCT-2003-980409 of the European Commission, DG Research, FP6

Background: The incidence of osteosarcoma varies considerably with age and preschool children are extremely rarely affected. This study was conducted to investigate presentation, treatment, and outcome in very young children with osteosarcoma. Patients and methods: The authors retrospectively analyzed the data of 2706 consecutive COSS patients with newly diagnosed high-grade osteosacroma of bone and identified 28 patients (1.0%) aged less than five years at diagnosis. Demographic, diagnostic, tumor, and treatment related variables; response and survival data of these 28 were analyzed. Results: Of the 28 (male, N=16; female, N=12) toddlers, 27 presented with high-grade central osteosarcoma of an extremity (femur, N= 12; humerus, N=10; tibia, N=5) and one with a secondary osteosarcoma of the orbit. The size of primary extremity tumors was large (≥ 1/3 of the involved bone) in 20/27 evaluable patients. Primary metastases were detected in 4 children. All patients received multiagent chemotherapy, and 13/20 analyzed tumors responded well (> 90% necrosis) to neoadjuvant chemotherapy. Limb sparing surgery was performed in 11, ablative procedures were performed in 14, and no local surgery was performed in two patients with extremity tumors. With a median follow-up of 3.8 years (6.2 years for survivors), 13 patients were alive (CR1, N=12; CR3, N=1). Four patients never achieved a complete remission and 12 developed recurrences (local, N=3; metastatic, N=8; site unknown, N=1); and 15 of these 16 patients died. Five-year overall and event free survival probabilities were 50% (SE 10%) and 46% (SE 10%). Better survival was correlated with good response to chemotherapy. Conclusions: Osteosarcoma is extremely rare in pre-school children. These young patients often have large tumors which may require mutilating resections. Prognosis may be poorer than in older patients

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed. Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging (BLI) and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology. SaOS-2 was more differentiated than 143b, with increased expression of Runx-2 (p = 0.009), Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in partial osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), and did not affect rates of lung metastasis (0% vs. 0%). Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing

Aims. We have previously reported cryoablation-assisted joint-sparing surgery for osteosarcoma with epiphyseal involvement. However, it is not clear whether this is a comparable alternative to conventional joint arthroplasty in terms of oncological and functional outcomes. Methods. A total of 22 patients who had localized osteosarcoma with epiphyseal involvement around the knee and underwent limb salvage surgery were allocated to joint preservation (JP) group and joint arthroplasty (JA) group. Subjects were followed with radiographs, Musculoskeletal Tumor Society (MSTS) score, and clinical evaluations at one, three, and five years postoperatively. Results. Patients in both groups (ten in JP and 12 in JA) did not differ in local recurrence (p ≥ 0.999) and occurrence of metastases (p ≥ 0.999). Overall survival was similar in both groups (p = 0.858). Patients in the JP group had less range of motion (ROM) of the knee (p < 0.001) and lower MSTS scores (p = 0.010) compared with those of the JA group only at one year postoperatively. There was no difference between groups either at three years for ROM (p = 0.185) and MSTS score (p = 0.678) or at five years for ROM (p = 0.687) and MSTS score (p = 0.536), postoperatively. Patients in the JA group tended to have more complications (p = 0.074). Survival of primary reconstruction in the JP group was better than that of the JA group (p = 0.030). Conclusion. Cryoablation-aided joint-sparing surgery offers native joint preservation with comparable functional recovery and more durable reconstruction without jeopardizing oncological outcomes compared with conventional limb salvage surgery. Cite this article: Bone Joint J 2021;103-B(8):1421–1427

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed. Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology. SaOS-2 was more differentiated than 143b, with significantly increased expression of the Osteoblast markers Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), but did not affect rates of lung metastasis (0% vs. 0%). Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing

Aims . Osteosarcoma of the pelvis is a particularly difficult tumour to treat as it often presents late, may be of considerable size and/or associated with metastases when it presents, and is frequently chondroid in origin and resistant to chemotherapy. The aim of this study was to review our experience of managing this group of patients and to identify features predictive of a poor outcome. Patients and Methods. Between 1983 and 2014, 121 patients, (74 females and 47 males) were treated at a single hospital: 74 (61.2%) patients had a primary osteosarcoma and 47 (38.8%) had an osteosarcoma which was secondary either to Paget’s disease (22; 18.2%) or to previous pelvic irradiation (25; 20.7%). . The mean age of those with a primary osteosarcoma was 29.3 years (nine to 76) and their mean follow-up 2.9 years (0 to 29). The mean age of those with a secondary sarcoma was 61.9 years (15 to 85) and their mean follow-up was one year (0 to 14). . A total of 22 patients with a primary sarcoma (52.4%) and 20 of those with a secondary sarcoma (47.6%) had metastases at the time of presentation. . Results. The disease-specific survival at five years for all patients was 27.2%. For those without metastases at the time of diagnosis, the five-year survival was 32.7%. Factors associated with a poor outcome were metastases at diagnosis and secondary tumours. In primary osteosarcoma, sacral location, surgical margin and a diameter > 10 cm were associated with a poor outcome. Conclusion. In this, the largest single series of patients with an osteosarcoma of the pelvis treated in a single hospital, those with secondary tumours and those with metastases at presentation had a particularly poor outcome. For those with a primary sarcoma, sacral location, an intralesional margin and a diameter of > 10 cm were poor prognostic indicators. Cite this article: Bone Joint J 2016;98-B:555–63

Only few patients with osteosarcoma relapse with solitary skeletal lesions as only sign of recurrence. We used the COSS database to learn more about these rare occurrences. This report covers all patients with high-grade osteosarcoma of the limbs or axial skeleton registered into the COSS database between 1980 and 2003 who developed 1st recurrences as solitary osseous lesions distant from the primary tumour before 01/2005. Patient-, tumour-, and treatment-related variables and outcomes were evaluated. 38 patients (27 male, 11 female) developed solitary osseous recurrences a median of 2.1 years (range:.5 – 14.3) from primary diagnosis. Primary sites had been limbs in 36 and axial in 2, relapses involved axial sites (24), limbs (10), or craniofacial bones (4). Treatment for osseous recurrence included surgery in 28 patients, radiotherapy in 10, and chemotherapy in 27. After a median follow-up of 1.9 years (range:.1–21.2) from 1st recurrence for all 38 patients and 5.5 years (.3–21.2) for 16 survivors (10 of these in continuous 2nd surgical remission), 2- & 5-year overall and event-free survival probabilities were 55% & 34% and 34% & 27%, respectively. A long interval to recurrence (> 1.5 years) predicted for better outcomes (p< .01). For those 21 patients achieving a 2nd complete surgical remission, 2- & 5-year overall and event-free survival probabilities were 81% & 61% and 52% & 49%, respectively, while only 1/17 patients failing to achieve a 2nd complete surgical remission survived beyond 5 years (p< .001) after additional radiotherapy. 14/16 survivors had also received 2nd-line chemotherapy. 1st solitary skeletal recurrences of osteosarcoma seem to have a favourable outcome provided treatment includes complete surgery as part of multimodal therapy. Some presumed bone metastases may rather represent second primary osteosarcomas. The COSS studies that form the basis of this report were supported by Deutsche Krebshilfe

Objectives. Eukaryotic translation initiation factor 3 (eIF3) is a multi-subunit complex that plays a critical role in translation initiation. Expression levels of eIF3 subunits are elevated or decreased in various cancers, suggesting a role for eIF3 in tumorigenesis. Recent studies have shown that the expression of the eIF3b subunit is elevated in bladder and prostate cancer, and eIF3b silencing inhibited glioblastoma growth and induced cellular apoptosis. In this study, we investigated the role of eIF3b in the survival of osteosarcoma cells. Methods. To investigate the effect of eIF3b on cell viability and apoptosis in osteosarcoma cells, we first examined the silencing effect of eIF3b in U2OS cells. Cell viability and apoptosis were examined by the Cell Counting Kit-8 (CCK-8) assay and Western blot, respectively. We also performed gene profiling to identify genes affected by eIF3b silencing. Finally, the effect of eIF3b on cell viability and apoptosis was confirmed in multiple osteosarcoma cell lines. Results. eIF3b silencing decreased cell viability and induced apoptosis in U2OS cells, and by using gene profiling we discovered that eIF3b silencing also resulted in the upregulation of tumour necrosis factor receptor superfamily member 21 (TNFRSF21). We found that TNFRSF21 overexpression induced cell death in U2OS cells, and we confirmed that eIF3b silencing completely suppressed cell growth in multiple osteosarcoma cell lines. However, eIF3b silencing failed to suppress cell growth completely in normal fibroblast cells. Conclusion. Our data led us to conclude that eIF3b may be required for osteosarcoma cell proliferation by regulating TNFRSF21 expression. Cite this article: Y. J. Choi, Y. S. Lee, H. W. Lee, D. M. Shim, S. W. Seo. Silencing of translation initiation factor eIF3b promotes apoptosis in osteosarcoma cells. Bone Joint Res 2017;6:186–193. DOI: 10.1302/2046-3758.63.BJR-2016-0151.R2