Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data.

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.

Generation of post-surgical minimal residual disease models to investigate metastasis in soft tissue sarcoma patient-derived orthotopic xenografts.

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.

RAF1-rearranged Spindle Cell Mesenchymal Tumor With Calcification and Heterotopic Ossification: A Case Report and Review of Literature.

We report an exceptional case of a spindle cell mesenchymal tumor with S100 and CD34 co-reactivity, which harbored a SLMAP::RAF1 fusion. To the best of our knowledge, this is the second case of a spindle cell mesenchymal tumor with S100 and CD34 co-reactivity with this specific fusion. Remarkable is the presence of calcification and heterotopic ossification in the center of our lesion, a feature that, to our knowledge, has not been described yet in RAF1-rearranged spindle cell mesenchymal tumors.

Evaluation of response to neoadjuvant chemotherapy in osteosarcoma using dynamic contrast-enhanced MRI: development and external validation of a model.

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.

Diagnosis and monitoring denosumab therapy of giant cell tumors of bone: radiologic-pathologic correlation.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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%) and a decrease in Ktrans (-71%) were observed. This is consistent with microscopic examination, showing a decrease of giant cells (-76%), neoplastic cells (-63%), and blood vessels (-28%). There was a strong statistical significant inverse correlation between time-to-peak and microvessel density (ρ = -0.9, p = 0.01). Significantly less neoplastic (p = 0.03) and giant cells (p = 0.04) were found with a time-intensity curve type II, compared to a type IV. Two patients showed relapse after initial good response when stopping denosumab. Inverse imaging and pathological findings were observed. CONCLUSION: CT and (DCE-)MRI show a good correlation with pathology and allow adequate evaluation of response to denosumab and detection of therapy failure.

Diagnosis of epithelioid hemangioendothelioma eight days postpartum: Is there a link with pregnancy? A case report and review of the literature.

Epithelioid hemangioendothelioma (EHE) is a rare and heterogeneous malignant vascular tumor. Decision making on a treatment strategy is difficult and a standard of care does not exist. EHE shows a wide age distribution but is rare in children. It can appear anywhere in the body, although lung and liver involvement are most common. There is a female predominance for visceral lesions and several case reports in which EHE developed during or after pregnancy are described in literature, hinting towards a putative role of sex hormones in the course of the disease. We present a case of a 32-year-old woman diagnosed with symptomatic pulmonary metastatic hepatic EHE (HEHE) 8 days postpartum, while the patient was completely asymptomatic before. A wait and see policy was chosen and the patient became asymptomatic in the months following the diagnosis. Although no expression of estrogen and progesterone receptors was found in the diagnostic liver biopsy specimen, we presume that the increased level of sex hormones during pregnancy may have triggered disease progression. The clinical behaviour of the disease in this case report reinforces the suspicion of female hormonal involvement in this type of malignancy and hints toward the potential role of other pregnancy-related factors, e.g. placental growth factor (PlGF), in the development of the disease.

Undifferentiated sarcoma of bone with a round to epithelioid cell phenotype harboring a novel EWSR1-SSX2 fusion identified by RNA-based next-generation sequencing.

Due to the increased application of RNA-based next-generation sequencing techniques on bone and soft tissue round cell sarcomas new fusions are frequently found, thereby expanding the molecular landscape of these tumors. In this report, we describe and discuss the finding of an undifferentiated sarcoma of the bone with a round to epithelioid cell phenotype harboring a novel EWSR1-SSX2 fusion. Treatment of this new bone tumor entity according to the Euro Ewing 2012 protocol led to complete pathologic response.

Late Lung Metastasis in a Patient with a Clear Cell Chondrosarcoma: An Indication for a Life-Long Follow-Up?

BACKGROUND: Clear cell chondrosarcoma (CCCS) is a rare subtype of chondrosarcoma and comprises between 1.6% and 2.5% of all chondrosarcoma. They are known to be chemo- and radiotherapy resistant; surgical resection is therefore the therapy of choice. METHODS: We present a 63-year-old woman with a progressive lung nodule 20 years after initial diagnosis and treatment of a clear cell chondrosarcoma of the right os naviculare. RESULTS: On serial CT scans of the chest, an asymptomatic, slowly growing nodule in the left upper lung lobe was detected. CT-guided transthoracic biopsy of this nodule confirmed the diagnosis of a chondrosarcoma lung metastasis. Video-assisted thoracoscopic wedge resection was performed with complete removal of the nodule. The patient recovered well from surgery and remains in good health during further follow-up. CONCLUSION: Given the tendency of clear cell chondrosarcoma to recur and metastasize after extended periods of time, a long-term, possibly life-long follow-up and clinical surveillance is advisable in these patients.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

NGS-analysis to the rescue: dual checkpoint inhibition in metastatic osteosarcoma - a case report and review of the literature.

Background: The prognosis and treatment of metastatic osteosarcoma have not changed in the last decades and the responses to chemotherapy in this setting are disappointing. In the past years, immunotherapy has found its place in the treatment of different tumor types. Its role in the treatment of sarcomas, and in particular osteosarcoma, is less clear. Next-generation sequencing (NGS) can help identify patients who could benefit from immunotherapy.Methods: Single case study and review of the literatureCase summary and intervention: We discuss a case of a 26-year-old man with a metastatic osteosarcoma not responsive to several lines of standard chemotherapy. NGS-analysis of resected tumor tissue revealed amplification of PD-L1 and PD-L2, which is associated in literature with response to anti-PD-1/PD-L1 blockade in other tumor types and in osteosarcoma mouse models. Treatment of our patient with the combination of an anti-PD-1 antibody (Nivolumab) and an anti-CTLA4 antibody (Ipilimumab) showed a stabilization of life-threatening retrocardiac tumoral masses that had previous significantly progressed despite radiotherapy, while one bone lesion in the right os ilium was growing and needed treatment with concomitant radiotherapy.Conclusion: To our knowledge, this is the first case report of a metastasized osteosarcoma patient with no further standard treatment options being treated with dual checkpoint inhibition. A complete stabilization of life-threatening retrocardiac lesions could be achieved and with the addition of radiotherapy to a growing bone lesion, the patient is still doing well. NGS-analysis can help identify druggable targets in patients with rare tumors with limited treatment options.Abbreviations: PD-L1: Programmed death-ligand 1; anti-PD-L1: anti-programmed death-ligand 1; PD-1: programmed cell death protein 1; anti-PD-1: anti-programmed cell death protein 1; anti-CTLA4: anti-cytotoxic T-lymphocyte-associated protein 4; AUC: area under the curve; NGS: next-generation sequencing; Gy: Gray; CT scan: computed tomography scan; RCC: renal cell carcinoma; IHC: immunohistochemistry; TMB: tumor mutational burden; NSCLC: Non-small cell lung cancer.

Enhancing rotational placement of reconstruction prostheses of the distal femur after sarcoma resection.

INTRODUCTION: Currently there are no accepted international guidelines for the correct placement of reconstruction prostheses in the axial plane of the femur after en bloc resection. The most accepted method is based on the linea aspera as an intraoperative landmark, indicating posterior. This study was conducted to address the reliability of the linea aspera as a landmark for rotational alignment. MATERIAL AND METHODS: 50 CT angiographies of the right limb were used for this purpose. These 2D images were reconstructed into 3D models using proprietary software (materialize NV, Leuven, Belgium). The posterior condylar line was used as a reference axis. The orientation of the linea aspera was described as the angle between the perpendicular line to the PCL, through the center of the diaphysis, and the lateral (α) and medial labium (ß). RESULTS: The linear mixed model shows that the α- and ß-angles are significantly associated with the distance from the joint line (p<0.001) and vary significantly between subjects (p<0.001). The α-angle has the lowest variance and approximates more closely true posterior, while the median ß-angle never overlaps true posterior. DISCUSSION: When a surgeon would blindly rely on the linea aspera as a posterior landmark roughly 78% of the femoral implants would exceed the accepted ±3° deviation around the surgical transepicondylar axis (sTEA) as defined in total knee replacement. The linea aspera is not a reliable landmark for axial rotation of femoral implants. The position is highly dependent on the osteotomy height and in addition differs between individual patients. Preoperative assessment of the linea aspera is advocated in order to reduce the risk of malrotation. As the height of the osteotomy cannot always be determined correctly preoperatively, a table was designed as a guideline for how much a deviation from the planned resection height will affect the rotation of the implant.

Expandable distal femur megaprosthesis: A European Musculoskeletal Oncology Society study on 299 cases.

BACKGROUND AND OBJECTIVES: Expandable distal femur prostheses have become more popular over the last decades, but scientific data is limited. METHODS: A retrospective study was performed, including cases treated between 1986 and 2019 in 15 European referral centers for bone sarcomas. RESULTS: A total of 299 cases were included. Average follow-up was 80 months (range, 8-287 months). Mean patient age was 10 years. Most (80%) of the implants were noninvasive growers and a fixed hinge knee was used more often (64%) than a rotating hinge. Most prosthetic designs showed good (>80%) implant survival at 10 years, but repeat surgery was required for 63% of the patients. The most frequent reason for revision procedure was the completion of lengthening potential. Noninvasive expandable implants showed less risk of infection compared to invasive growers (11.8% vs 22.9% at 10 years). No difference in aseptic loosening was found between cemented and uncemented stems. CONCLUSIONS: This study shows the increasing popularity of expandable distal femur prostheses, with overall good results for function and implant survival. However, repeat surgery is frequently required, especially in patients under the age of 10 years old. Infection is less frequent in noninvasive growers compared to implants that require invasive lengthening procedures.

Accuracy assessment of surgical planning and three-dimensional-printed patient-specific guides for orthopaedic osteotomies.

This study analyses the accuracy of three-dimensional pre-operative planning and patient-specific guides for orthopaedic osteotomies. To this end, patient-specific guides were compared to the classical freehand method in an experimental setup with saw bones in two phases. In the first phase, the effect of guide design and oscillating versus reciprocating saws was analysed. The difference between target and performed cuts was quantified by the average distance deviation and average angular deviations in the sagittal and coronal planes for the different osteotomies. The results indicated that for one model osteotomy, the use of guides resulted in a more accurate cut when compared to the freehand technique. Reciprocating saws and slot guides improved accuracy in all planes, while oscillating saws and open guides lead to larger deviations from the planned cut. In the second phase, the accuracy of transfer of the planning to the surgical field with slot guides and a reciprocating saw was assessed and compared to the classical planning and freehand cutting method. The pre-operative plan was transferred with high accuracy. Three-dimensional-printed patient-specific guides improve the accuracy of osteotomies and bony resections in an experimental setup compared to conventional freehand methods. The improved accuracy is related to (1) a detailed and qualitative pre-operative plan and (2) an accurate transfer of the planning to the operation room with patient-specific guides by an accurate guidance of the surgical tools to perform the desired cuts.

Long-term follow-up of the anterior lumbar interbody fusion procedure.

PURPOSE: To evaluate the long-term clinical results and the effectiveness of the anterior lumbar interbody fusion procedure. METHODS: Between 1999 and 2005, 60 ALIFs were performed in 59 patients. Mean age was 41.1 years. Clinically, patients were evaluated at a mean follow-up of 9.5 years using the Visual Analogue Scale grading scale, the Oswestry Disability score and the SF-36 questionnaire. RESULTS: Preoperative and postoperative clinical evaluation scores of 38 patients were available. Nineteen patients were lost to follow-up, and 2 patients died during the follow-up. The fusion rate was 84%. Mean preoperative VAS-score for back pain was 6.69 (±2.15) ; in the long term, the mean VAS-score was 4.95 (±2.95), which was a significant improvement. (p<0.01). The postoperative ODI-score was 36.11 (±22.32), while the preoperative ODI-score was 59.31 (±17.16), which demonstrates a significant improvement. According to the SF-36, mild to good results were observed. CONCLUSIONS: The ALIF procedure can offer significant pain relief and improved function if a strict indication policy is followed.

Radiologic Evaluation of Compressive Osseointegration for the Fixation of Reconstruction Prostheses after Tumor Resection.

OBJECTIVE: In pursuance of thoroughly understanding and facilitating the evaluation of the radiological changes in the preloaded bone by Compliant Pre-Stress osseointegration (Compress Biomet, Warsaw, Indiana) a new staging method was created depicting four stages. METHODS: Two cohorts (10 and 17 patients resp., not-receiving and receiving chemotherapy) were compared in terms of progression of osseointegration. Based on the changes at the bone-metal interface seen on röntgenorgrams four stages were defined: stage 0: immediate postoperative status, no ingrowth, or noncalcified callus; stage 1: early mineralization, calcified callus; stage 2: mature mineralization; and stage 3: hypertrophy at the level of the pins. RESULTS: There were no significant differences between the two cohorts. Group 2, which was significantly younger than group 1 (p < 0.001), presented a delayed initial rate of bone formation and reached stage 1 at 6 months instead of 3 months like group 1. The children from the group 2 demonstrated a visible rebound ingrowth. CONCLUSION: Despite the fact that the staging fails to demonstrate a statistical difference, it is rather simple and can be used for future studies.

The in ovo CAM-assay as a xenograft model for sarcoma.

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions. This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.