BACKGROUND:Sarcomas are rare, diverse malignancies with limited therapeutic options and poor clinical outcomes. Preclinical models that preserve tumour biology are urgently needed to advance mechanistic understanding and functional precision oncology. We established and comprehensively characterized 29 early-passage patient-derived sarcoma cell (PDC) cultures from 19 patients, representing 11 sarcoma subtypes. Multi-region and multi-site sampling enabled generation of PDCs from spatially distinct areas of individual tumours and from matched primary, recurrent and metastatic lesions. METHODS:PDCs underwent genomic, transcriptomic and proteomic profiling alongside extracellular vesicle (EV) biomarker evaluation and phenotypic and drug-response assays. RESULTS:Copy-number variant (CNV) analysis revealed recurrent alterations affecting key regulators of cell cycle control and growth signalling. Bulk RNA-sequencing captured substantial inter- and intra-subtype heterogeneity. Proteomic and EV analyses recapitulated subtype- and site-specific differences. A functional drug screen of 38 clinically relevant and investigational agents identified both shared and divergent therapeutic vulnerabilities in the different PDCs. DISCUSSION:These results demonstrate that early-passage sarcoma PDCs can be regarded as biologically faithful and experimentally tractable models that capture lineage identity, tumour evolution and functional heterogeneity. Integrated multi-omic and functional profiling reveals therapeutic vulnerabilities not evident from genomic data only, supporting PDCs as valuable platforms for translational sarcoma research. KEY POINTS:A PDC biobank that addresses a critical gap in sarcoma research. Investigation of intra-patient and inter-patient heterogeneity. Comprehensive characterization shows truthful recapitulation of sarcoma features. Functional assays highlight the importance of functional models in cancer research.

The chick chorioallantoic membrane (CAM) assay is an in vivo metastasis model, yet traditional Alu-qPCR quantification suffers from non-specific background noise in avian tissue. To improve analytical specificity, we developed a species-specific digital PCR (dPCR) assay targeting the human mitochondrial locus MT-ND4. While Alu-qPCR generated false positives in chicken-only DNA, MT-ND4 dPCR provided high species specificity and robust relative quantification of human mitochondrial DNA across various embryonic organs. As a proof of concept in five embryos, organ-dependent differences in metastatic burden were detected (p < 0.001) with high technical precision. This MT-ND4 dPCR assay avoids false-positive overestimations, providing a specific tool for quantifying human metastatic burden in the CAM model.

BACKGROUND:Sarcomas are rare and heterogeneous malignancies arising from mesenchymal tissue, often requiring wide surgical resection that may result in large and complex soft and bony tissue defects needing complex reconstruction. Growing evidence indicates the added value of plastic surgery participation in multidisciplinary sarcoma care, yet evidence remains limited. METHODS:This retrospective single-center study included patients who underwent resection for soft-tissue or bone sarcoma at Ghent University Hospital between 2010 and 2022. The patients were categorized according to closure strategy during the index procedure (oncologic-only closure vs combined oncologic-plastic closure). Three multivariable logistic regression models were fitted. One model identified preoperative predictors of combined oncologic-plastic surgery, and two models assessed postoperative outcomes (seroma formation and R0 margin status by closure strategy). RESULTS:Of the 530 patients included, 409 (77.2 %) underwent primary tumor resection by the oncologic surgeon only, whereas 121 (22.8 %) had primary surgery performed by a combined oncologic-plastic team. Closure by the oncologic surgeon was independently predicted by upper leg location, whereas the combined oncologic-plastic operation was associated with myxofibrosarcoma histology, trunk location, high-grade tumors, and location superficial to the fascia. The combined oncologic-plastic approach reduced the formation of seroma. The R0 resection rates did not differ significantly between groups. CONCLUSION:This study may help define clearer indications for early reconstructive input during sarcoma surgery. Early integration of plastic surgery within a multidisciplinary sarcoma team enables more comprehensive surgical planning and may both facilitate radical resections and improve postoperative wound outcomes.

Aims:Achieving tumour-free margins and precise prosthetic alignment remains an important challenge in limb-salvage surgery (LSS). Malalignment can compromise limb function and implant longevity, whereas inadequate margins increase the risk of local recurrence. Therefore, this study aims to develop a novel patient-specific guide (PSG) concept that integrates both osteotomy and prosthetic alignment into a single, user-friendly platform. Methods:A multifunctional PSG was developed, composed of two primary components: a patient-specific insert that is precisely contoured to the patient's bone anatomy, and a reusable universal frame that envelops the patient-specific element. The universal frame serves as a central platform to attach auxiliary instrumentation guides that facilitate osteotomy, reaming, and prosthetic alignment. A proof-of-concept study was conducted on ten cadaveric femora. Following CT imaging, virtual preoperative planning was performed in a 3D environment. The patient-specific parts of the guide were 3D-printed based on the virtual osteotomy and prosthetic alignment planes. The guide system was sequentially applied to perform the osteotomy and prosthesis placement on each specimen. Accuracy was assessed by comparing postoperative CT scans to the preoperative virtual plans, quantifying alignment and resection errors across all planes. Results:The PSG achieved sub-degree accuracy in coronal and sagittal alignment, with median deviations of 0.4° in both planes and a median rotational deviation of 1.2° (IQR 0.2° to 2.2°). Resection height deviation was 2.4 mm (IQR 1.9 to 3.6), with 90% of cases within a 6 mm margin. No significant correlation was observed between angular and translational errors, indicating no cross-dimensional propagation of error. Conclusion:This cadaveric proof-of-concept study demonstrates that this innovative multifunctional PSG enables precise intraoperative execution of virtual preoperative plans. By integrating both resection and prosthetic alignment into a single, user-friendly, and cost-effective platform, it addresses an unmet need in limb salvage surgery and could support precision-driven standardization in oncological limb reconstruction.

Aims:The aim of this study was to achieve consensus for important topics related to periprosthetic infection (PJI) in orthopaedic oncology, and to identify areas for future research. Methods:In January 2024, the Birmingham Orthopaedic Oncology Meeting (BOOM) held in Birmingham, UK, gathered 309 delegates from 53 countries to debate 20 consensus statements on PJI in orthopaedic oncology using a modified Delphi process. Results:Of 20 questions and statements on PJI in orthopaedic oncology, none achieved unanimous consensus, 18 achieved strong consensus, one achieved moderate consensus, and one achieved weak consensus. The statements that reached consensus with notable agreement were on the prophylaxis of infection, management of leaking wounds, and surgical strategies for the treatment of PJI. Short-duration antibiotic prophylaxis was deemed as effective as longer courses for lower-risk reconstructions, and aggressive management was recommended for wounds draining beyond five to seven days to prevent deep infection. Furthermore, single-stage, two-stage, and 1.5-stage revision were recognized as valid strategies, with two-stage revision remaining the most reliable. The statements that did not achieve consensus were on the role of debridement, antibiotics, and implant retention and prolonged antibiotic use post-revision. Conclusion:The BOOM meeting achieved consensus for important topics on periprosthetic infection in orthopaedic oncology, but highlighted the low quality of the underlying evidence. This study has provided recommendations for the treatment of leaky wounds, duration of postoperative antibiotic prophylaxis, and choice of revision strategy.

OBJECTIVE:To identify aggressive regions in high-grade and dedifferentiated chondrosarcomas on MRI by obtaining an exact correlation between radiology and histopathology. MATERIALS AND METHODS:One chondrosarcoma grade II (CSII), chondrosarcoma grade III (CSIII) and dedifferentiated chondrosarcoma (DDCS) were segmented on (DCE-)MRI images. Around the segmentations, a patient specific mold was constructed and 3D-printed with cutting grooves perfectly aligned with selected MRI slices. In this way, resection specimens could be cut at the same locations as the MRI slices. Histopathology slides were stained with hematoxylin-eosin and CD31 (vascularization) and correlated with the (DCE-)MRI. Histopathologically, atypical cartilaginous tumor (ACT), CSII, CSIII and DDCS regions were delineated and vascular hotspots were selected by an experienced pathologist. Exact point-to-point correlation was performed. RESULTS:ACT, CSII, CSIII and DDCS regions all had similar mean signal intensity on T1-weighted images. On fat-saturated T2-weigthed images, CSII and CSIII regions had a higher mean signal intensity. On fat-saturated T1-weighted images after gadolinium contrast injection and on wash-in parametric maps DDCS regions had the highest mean signal intensity and highest wash-in values. There was a higher microvessel density in CSII and CSIII regions. This corresponded with a thick enhancing rim on fat-saturated T1-weighted images after gadolinium contrast injection and with a higher wash-in. CONCLUSION:This new 3D mold helps to identify high-grade chondrosarcoma regions with myxoid. This may be useful for targeted biopsy and histopathological investigation.

AIM:Worldwide, of all cancers, colorectal cancer has the fourth highest rate of mortality. Curative treatment for locally advanced or recurrent rectal cancer can require extended resections with sacrectomy, but this sugery can have major consequences for patients. The aim of this study was to investigate the oncological and functional outcomes after sacrectomy in patients with colorectal cancer. METHOD:The protocol was registered in (PROSPERO), the international prospective register of systematic reviews. PubMed, Embase, The Cochrane Library, Scopus and Google Scholar were searched using a predetermined search strategy. Article selection, quality of evidence [Grading of Recommendation, Assessment, Development and Evaluation (GRADE)] and risk of bias [The Risk Of Bias In Non-randomized Studies-of Interventions (ROBINS-I)] were assessed by two independent reviewers. Studies reporting on sacrectomy for colorectal cancer were included. Oncological and functional outcomes after sacrectomy for colorectal cancer were the primary outcomes. RESULTS:Forty-six articles with 1687 patients (1115 men; 506 women) were included. Median follow-up was 31.4 months. Mean 30-day mortality was 1.1%. After R0 resection, overall survival was achieved in 86.2%, 68.0% and 42.1% patients, and disease-free survival was achieved in 75.0%, 51.0% and 43.0% patients, respectively, after 1, 3 and 5 years. Survival rates were lower after R1 or R2 resection. Most patients reported elimination of or significantly reduced pain after surgery, and 82.2% were able to walk independently, without the use of assistive devices. More patients were dependent on walking-assist devices after high sacrectomy than after low sacrectomy. The most commonly reported bowel dysfunctions were bowel obstruction (22.3%) and fistula (4.6%). Bladder dysfunction was mainly reported as incontinence (8.3%) and neurogenic bladder (23.3%). No study included quality of life as an outcome. CONCLUSIONS:The mortality was limited, and the morbidity rates were in concordance with published literature. The results suggest that R0 resection has higher survival rates than R1/R2 resections. Both short- and long-term functional outcomes could have major impact on a patient's quality of life. Heterogeneity was high, and neither comparative analyses nor meta-analysis could be performed.

In the original publication [...].

BACKGROUND:Sarcoma is a rare type of cancer, of which over 70 distinct molecular subtypes are known. Because these tumours are so rare and complex, treatment outcomes have remained similar over the past decades and research is progressing slowly. For these rare cancers, personalised medicine and patient-derived models might pose solutions for therapeutic problems, but researchers depend on clinicians to access fresh, viable tumour tissue. METHODS:Over the past five years, a bedside-to-bench flow was implemented in Ghent University Hospital for a living biobank of sarcoma tissue to support translational research. All tumour tissue was cryopreserved in cryopreservation medium (90 % foetal bovine serum and 10 % dimethyl sulfoxide) maintaining viability of the tissue. RESULTS:As of July 2024, this biobank houses 217 sarcoma samples available for model development, biomarker discovery and precision medicine initiatives. The samples were collected from 167 patients, with 40 patients of whom we collected several samples throughout the disease course. DISCUSSION:With this article, we aim to incentivise surgeons to engage in biobanking initiatives, as they have a unique position with direct access to fresh tumour tissue. We present our biobank and clinical flow that is implemented in daily clinical practice. The primary aim of the biobank is to advance sarcoma research by creating models and sharing data with fellow researchers.

Aims:Following resection of a primary bone tumour, reconstruction is commonly performed using either a megaprosthesis or biological reconstruction. Periprosthetic joint infection (PJI) remains one of the most frequent complications. Various treatment strategies exist for PJI, including debridement, antibiotics, and implant retention (DAIR), and single- and two-stage revision, although consensus on optimal management remains elusive. This study aimed to investigate the global practices regarding DAIR in tumour cases through an electronic survey among orthopaedic oncology surgeons. Methods:A global cross-sectional observational survey study was distributed to 272 orthopaedic oncology surgeons who attended the BOOM Consensus Meeting in January 2024. The survey contained 19 multiple choice questions focusing on DAIR practices. Responses were collected anonymously and analyzed using descriptive statistics. Results:The survey was completed by 173/272 surgeons (64%) from 44 countries. While 62% (169/272) routinely performed radical soft-tissue debridement in DAIR, only 39% exchanged all modular components, indicating variability in surgical approaches. DAIR was more commonly performed in acute rather than chronic infections, with 55% finding it very useful in acute cases. The use of local antibiotic delivery was supported by 56%, although only 49% found antibiotic cement coatings beneficial. Systemic antibiotic duration post-DAIR varied, with 39% favouring six weeks and 35% preferring three months. Conclusion:The study highlights global inconsistencies in DAIR practices for PJI in orthopaedic oncology, with financial disparities impacting modular component exchange. Standardized definitions are lacking, and we propose that if only polyethylene is changed, then the procedure is referred to as 'poly exchange'; we recommend defining the procedure as DAIR when extensive debridement, lavage, and removal, wash, and reimplanting of all modular components is done while retaining stable stems, followed by suppressive antibiotic therapy; and finally, we recommend that if all the modular components are changed for new ones, the procedure is referred to as 'DAIR plus'.

Objectives: To evaluate the value of (dynamic) contrast-enhanced MRI for the diagnosis and follow-up of central cartilage tumors (CCT) of the proximal humerus, distal femur and proximal tibia. Materials & methods: 97 patients (44 +/- 11 years, 31men) with a CCT (histopathologically proven and/or > 2 years follow-up (5 +/- 3 years)) were retrospectively/consecutively included at the Ghent University Hospital (Belgium, 2003-2021). Thickness of the enhancing rim and dynamic contrast-enhanced MRI parameters of the entire tumor were calculated. Tumor volumes were calculated to assess tumor growth. Significant differences between enchondromas, atypical cartilaginous tumors, high-grade chondrosarcomas, tumors with and without growth at follow-up were evaluated and ROC-curves were analyzed. Results: A significant difference (p = 0.015) existed in enhancing rim thickness between high-grade chondrosarcomas and other groups. A ROC-curve with an AUC of 0.89 and cut-off value of 1.2 mm had a sensitivity of 100 % and a specificity of 64 % to identify high-grade chondrosarcomas. A significant correlation was found between the relative maximal enhancement (Rel-Emax) of the whole tumor compared to muscle and the absolute growth rate (rho = 0.75, p < 0.001). Lesions with a Rel-Emax < 1 remained stable or showed regression during follow-up (mean-0.1 +/- 0.3 ml/year). Lesions with a Rel-Emax between 1 and 2 showed little growth (mean + 0.2 +/- 0.2 ml/year), and lesions with a Rel-Emax > 2 had the highest growth rate (mean 0.4 +/- 0.2 ml/year). Conclusion: A thick enhancing rim > 1.2 mm has a high sensitivity to detect high-grade chondrosarcomas. The higher the Rel-Emax of the whole tumor compared to muscle, the higher the growth rate of a CCT.

Despite optimal multimodal treatment including surgical resection, 50%-80% of high-grade soft tissue sarcoma (STS) patients metastasize. Here, we present a protocol for the generation and use of post -surgical minimal residual disease models to investigate metastatic relapse in STS patient -derived xenografts. We describe steps for orthotopic engraftment of high-grade STS patient -derived tumor tissue. We then detail procedures for primary tumor resection with broad, negative resection margins and follow-up until metastases using MRI. For complete details on the use and execution of this protocol, please refer to Fischer et al. (2023).1

Osteosarcoma and Ewing sarcoma are bone tumors mostly diagnosed in children, adolescents, and young adults. Despite multimodal therapy, morbidity is high and survival rates remain low, especially in the metastatic disease setting. Trials investigating targeted therapies and immunotherapies have not been groundbreaking. Better understanding of biological subgroups, the role of the tumor immune microenvironment, factors that promote metastasis, and clinical biomarkers of prognosis and drug response are required to make progress. A prerequisite to achieve desired success is a thorough, systematic, and clinically linked biological analysis of patient samples, but disease rarity and tissue processing challenges such as logistics and infrastructure have contributed to a lack of relevant samples for clinical care and research. There is a need for a Europe-wide framework to be implemented for the adequate and minimal sampling, processing, storage, and analysis of patient samples. Two international panels of scientists, clinicians, and patient and parent advocates have formed the Fight Osteosarcoma Through European Research consortium and the Euro Ewing Consortium. The consortia shared their expertise and institutional practices to formulate new guidelines. We report new reference standards for adequate and minimally required sampling (time points, diagnostic samples, and liquid biopsy tubes), handling, and biobanking to enable advanced biological studies in bone sarcoma. We describe standards for analysis and annotation to drive collaboration and data harmonization with practical, legal, and ethical considerations. This position paper provides comprehensive guidelines that should become the new standards of care that will accelerate scientific progress, promote collaboration, and improve outcomes.

To determine the value of CT and dynamic contrast-enhanced (DCE-)MRI for monitoring denosumab therapy of giant cell tumors of bone (GCTB) by correlating it to histopathology. Patients with GCTB under denosumab treatment and monitored with CT and (DCE-)MRI (2012-2021) were retrospectively included. Imaging and (semi-)quantitative measurements were used to assess response/relapse. Tissue samples were analyzed using computerized segmentation for vascularization and number of neoplastic and giant cells. Pearson’s correlation/Spearman’s rank coefficient and Kruskal-Wallis tests were used to assess correlations between histopathology and radiology. Six patients (28 ± 8years; five men) were evaluated. On CT, good responders showed progressive re-ossification (+7.8HU/month) and cortical remodeling (woven bone). MRI showed an SI decrease relative to muscle on T1-weighted (−0.01 A.U./month) and on fat-saturated T2-weighted sequences (−0.03 A.U./month). Time-intensity-curves evolved from a type IV with high first pass, high amplitude, and steep wash-out to a slow type II. An increase in time-to-peak (+100

Background: The decreased perfusion of osteosarcoma in dynamic contrast-enhanced (DCE) MRI, reflecting a good histological response to neoadjuvant chemotherapy, has been described. Purpose: In this study, we aim to explore the potential of the relative wash-in rate as a prognostic factor for event-free survival (EFS). Methods: Skeletal high-grade osteosarcoma patients, treated in two tertiary referral centers between 2005 and 2022, were retrospectively included. The relative wash-in rate (rWIR) was determined with DCE-MRI before, after, or during the second cycle of chemotherapy (pre-resection). A previously determined cut-off was used to categorize patients, where rWIR < 2.3 was considered poor and rWIR ≥ 2.3 a good radiological response. EFS was defined as the time from resection to the first event: local recurrence, new metastases, or tumor-related death. EFS was estimated using Kaplan–Meier’s methodology. Multivariate Cox proportional hazard model was used to estimate the effect of histological response and rWIR on EFS, adjusted for traditional prognostic factors. Results: Eighty-two patients (median age: 17 years; IQR: 14–28) were included. The median follow-up duration was 11.8 years (95% CI: 11.0–12.7). During follow-up, 33 events occurred. Poor histological response was not significantly associated with EFS (HR: 1.8; 95% CI: 0.9–3.8), whereas a poor radiological response was associated with a worse EFS (HR: 2.4; 95% CI: 1.1–5.0). In a subpopulation without initial metastases, the binary assessment of rWIR approached statistical significance (HR: 2.3; 95% CI: 1.0–5.2), whereas its continuous evaluation demonstrated a significant association between higher rWIR and improved EFS (HR: 0.7; 95% CI: 0.5–0.9), underlining the effect of response to chemotherapy. The 2- and 5-year EFS for patients with a rWIR ≥ 2.3 were 85% and 75% versus 55% and 50% for patients with a rWIR < 2.3. Conclusion: The predicted poor chemo response with MRI (rWIR < 2.3) is associated with shorter EFS even when adjusted for known clinical covariates and shows similar results to histological response evaluation. rWIR is a potential tool for future response-based individualized healthcare in osteosarcoma patients before surgical resection.

Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications.

Taxanes, such as paclitaxel (PTX), stabilize microtubules and are used as a first-line therapy in multiple cancer types. Disruption of microtubule equilibrium, which plays an essential role in mitosis and cell homeostasis, ultimately results in cell death. Even though PTX is a very potent chemotherapy, its use is associated with major side effects and therapy resistance. Pelophen B (PPH), a synthetic analog of peloruside A, stabilizes microtubules through interaction with a non-taxoid binding site of β-tubulin. We evaluated the anticancer effect of PPH in a variety of tumor types by using established cell lines, early-passage cultures and ex vivo tumor-derived cultures that preserve the 3D architecture of the tumor microenvironment. PPH significantly blocks colony formation capacity, reduces viability and exerts additivity with PTX. Interestingly, PPH overcomes resistance to PTX. Mechanistically, PPH induces a G2/M cell cycle arrest and increases the presence of tubulin polymerization promoting protein (TPPP), inducing lysine 40 acetylation of α-tubulin. Although, results induced by paclitaxel or PPH are concordant, PPH’s unique microtubule binding mechanism enables PTX additivity and ensures overcoming PTX-induced resistance. In conclusion, PPH results in remarkable anti-cancer activity in a range of preclinical models supporting further clinical investigation of PPH as a therapeutic anticancer agent.

OBJECTIVE:To identify which dynamic contrast-enhanced (DCE-)MRI features best predict histological response to neoadjuvant chemotherapy in patients with an osteosarcoma.METHODS:Patients with osteosarcoma who underwent DCE-MRI before and after neoadjuvant chemotherapy prior to resection were retrospectively included at two different centers. Data from the center with the larger cohort (training cohort) was used to identify which method for region-of-interest selection (whole slab or focal area method) and which change in DCE-MRI features (time to enhancement, wash-in rate, maximum relative enhancement and area under the curve) gave the most accurate prediction of histological response. Models were created using logistic regression and cross-validated. The most accurate model was then externally validated using data from the other center (test cohort).RESULTS:Fifty-five (27 poor response) and 30 (19 poor response) patients were included in training and test cohorts, respectively. Intraclass correlation coefficient of relative DCE-MRI features ranged 0.81-0.97 with the whole slab and 0.57-0.85 with the focal area segmentation method. Poor histological response was best predicted with the whole slab segmentation method using a single feature threshold, relative wash-in rate <2.3. Mean accuracy was 0.85 (95%CI: 0.75-0.95), and area under the receiver operating characteristic curve (AUC-index) was 0.93 (95%CI: 0.86-1.00). In external validation, accuracy and AUC-index were 0.80 and 0.80.CONCLUSION:In this study, a relative wash-in rate of <2.3 determined with the whole slab segmentation method predicted histological response to neoadjuvant chemotherapy in osteosarcoma. Consistent performance was observed in an external test cohort.