Network pharmacology and experimental verification-based strategy for exploring the mechanisms of luteolin in the treatment of osteosarcoma.

BACKGROUND: Luteolin is an active ingredient in various traditional Chinese medicines for the treatment of multiple tumors. However, the mechanisms of its inhibitory effect on osteosarcoma proliferation and metastasis remain unclear. PURPOSE: To elucidate the anti-osteosarcoma mechanisms of luteolin based on network pharmacology and experimental verification. STUDY DESIGN: Integrate network pharmacology predictions, scRNA-seq analysis, molecular docking, and experimental validation. METHODS: Luteolin-related targets and osteosarcoma-associated targets were collected from several public databases. The luteolin against osteosarcoma targets were screened and a PPI network was constructed to identify the hub targets. The GO and KEGG enrichment of osteosarcoma-associated targets and luteolin against osteosarcoma targets were performed. And scRNA-seq analysis was performed to determine the distribution of the core target expression in OS tissues. Molecular docking, cell biological assays, and osteosarcoma orthotopic mouse model was performed to validate the inhibitory effect and mechanisms of luteolin on osteosarcoma proliferation and metastasis. RESULTS: Network pharmacology showed that 251 luteolin against osteosarcoma targets and 8 hub targets including AKT1, ALB, CASP3, IL6, JUN, STAT3, TNF, and VEGFA, and the PI3K-AKT signaling pathway might play an important role in anti-osteosarcoma of luteolin. Analysis of public data revealed that AKT1, IL6, JUN, STAT3, TNF, and VEGFA expression in OS tissue was significantly higher than that in normal bones, and the diagnostic value of VEGFA for overall survival and metastasis was increased over time. scRNA-seq analysis revealed significantly higher expression of AKT1, STAT3, and VEGFA in MYC+ osteoblastic OS cells, especially in primary samples. Moreover, the docking activity between luteolin and the hub targets was excellent, as verified by molecular docking. Experimental results showed that luteolin could inhibit cell viability and significantly decrease the expression of AKT1, STAT3, IL6, TNF, and VEGFA, and luteolin could also inhibit osteosarcoma proliferation and metastasis in osteosarcoma orthotopic mouse model. CONCLUSION: This study shows that luteolin may regulate multiple signaling pathways by targeting various genes like AKT1, STAT3, IL6, TNF, and VEGFA to inhibit osteosarcoma proliferation and metastasis.

A novel limb-salvage reconstruction strategy with a custom hemipelvic endoprosthesis and preserved femoral head following the resection of periacetabular tumors: A preliminary study.

The treatment of periacetabular malignancy frequently challenges surgeons. To simplify the surgical procedure, we performed a novel reconstruction strategy preserving the femoral head for patients with periacetabular malignancies. We retrospectively reviewed 14 patients who underwent total en bloc resection of a periacetabular tumor and reconstruction of the hip joint with an individualized hemipelvic endoprosthesis and remaining femoral head from July 2015 to January 2019 at our center. Regions of pelvic resection: region II-4 (28.6%), region I + II-5 (35.7%), region II + III-2 (14.3%) and region I + II + III-3 (21.4%). The oncological outcomes were that 13 patients survived without disease and one patient survived with lung metastasis. None of the patients experienced local recurrence (range: 20-62 months; mean: 32 months). The incidence of postoperative complications was 35.7%, including delayed wound healing and deep venous thrombosis. No prosthesis-related complications occurred until the last follow-up in this study (range: 20-62 months; mean: 32 months). The mean Musculoskeletal Tumor Society functional outcome score was 23.2. The mean Toronto Extremity Salvage Score of the patients was 75.7 points, with a mean limb discrepancy of 1.51 cm (range: 0.5-3.2 cm). Reconstruction with preservation of the femoral head showed acceptable early functional and oncological outcomes, and it had an acceptable complication rate.

Minimally invasive direct anterior approach versus standard lateral approach in the management of tumors of the femoral neck.

BACKGROUND: To compare the clinical outcomes of patients with benign or aggressive tumors of the femoral neck who underwent surgical curettage with the use of the direct anterior approach (DAA) and a standard lateral approach. METHODS: Those patients from 2010 to 2017 were retrospectively enrolled. The patients were divided into two groups: group A, consisting of patients who had undergone surgery via the lateral approach; and group B, consisting of patients who had undergone the same procedure via the DAA. RESULTS: Fifty-eight patients were divided into group A (n = 46) and group B (n = 12). The median follow-up was 43 months (15-97 months). There was no significant difference in the 1-year and 3-year recurrence rates (p = 0.74). Group B had comparable operation time and a significantly shorter incision length, less intraoperative blood loss, less postoperative drainage, a shorter hospital stay and less pain on the first postoperative day. Group B also had better hip function as assessed by the Harris Hip Score one month and one year postoperatively. One patient in group B experienced intraoperative incomplete fracture of the femoral neck, which was treated conservatively. CONCLUSIONS: Surgical curettage for patients with benign or aggressive tumors of the femoral neck via the DAA had a comparable local control rate and a better perioperative and functional outcome than via the lateral approach. Certain quality of the femoral neck should be required to avoid pathological fracture, which is difficult to treat by internal fixation in the DAA.

Plastic lengthening amputation with vascularized bone grafts in children with bone sarcoma: a preliminary report.

BACKGROUND: At present, amputation was widely adopted for young patients when limb salvage was deemed risky with several surgical strategy such as rotationplasty. However, leg length discrepancies and unfavorable cosmetic results were indispensable complication of this strategy. The purpose of this study was to propose a novel reconstruction strategy and evaluate the early clinical and functional outcomes of the strategy. METHODS: Plastic lengthening amputation (PLA) has been developed by lengthening the stump to preserve one additional distal joint for fixing the artificial limb well. The surgical technique and postoperative management were documented, and the functional outcomes were compared with those of traditional amputation (TA). Six pairs of patients matched for age, sex, location, pathological type, and final prosthesis underwent individually designed plastic lengthening amputation with vascularized autografts or traditional amputation between January 2005 and December 2007. All patients were followed, and the locomotor index and the musculoskeletal tumor society score (MSTS) were used to describe and quantitatively grade limb functional outcomes after amputation. The complications and functional outcomes of the patients taken two kinds of procedures were compared. RESULTS: Twelve patients with osteosarcoma or Ewing's sarcoma of either the femur or tibia were included in the study. Six patients underwent plastic lengthening amputations, three of whom also underwent vascular anastomosis. Patients were followed for an average of 48.17 months; bone healing required an average of 3.3 months. No local recurrence was found. The average postoperative locomotor index functional score of the affected limb was 32.67 ± 5.89 in the plastic lengthening amputation group while was 19.50 ± 7.87 in the traditional amputation group. The MSTS functional scores were 22.67 ± 1.33 and 24.17 ± 1.45 at 6 and 12 months for patients in PLA group while 17.00 ± 1.549 and 17.83 ± 1.64 at 6 and 12 months for patients in TA group. CONCLUSIONS: Plastic lengthening amputations with vascularized autografts could preserve the knee joint to improve the function of the amputated limb in selected bone sarcoma patients.

Reconstruction with a novel combined hemipelvic endoprosthesis after resection of periacetabular tumors involving the sacroiliac joint: a report of 25 consecutive cases.

BACKGROUND: Our purpose was to examine the outcomes of patients who underwent extensive resection of periacetabular tumors involving the sacroiliac joint and joint reconstruction with a hemipelvic endoprosthesis. METHODS: The records of 25 consecutive patients diagnosed with Enneking type I/II/IV pelvic tumors from 2010 to 2016 who received resection and hemipelvic endoprosthesis reconstruction were retrospectively reviewed. RESULTS: The median follow-up period was 48 months. At the most recent follow-up, 11 patients were alive, with estimated 3- and 5-year survival rates of 45.6 and 38.0%, respectively. Fourteen patients died, with a mean survival of 20.8 months, and 8 patients had local recurrence at an average of 9.3 months after surgery. Distal metastases were detected in 11 patients at an average of 11.0 months after surgery. The total complication rate was 56.0%, and the most common complications were wound healing disturbances (28.0%) and deep infections (16.0%). The prosthesis-related complication rate was 24.0%; periprosthetic infections and aseptic loosening were most common. The estimated 1- and 3-year prosthesis survival rates were 81.2 and 63.2%, respectively. The mean Musculoskeletal Tumor Society score was 48.0%. Function and prosthesis-related complications did not differ significantly after adding an extra screw fixation to the first sacral vertebra. CONCLUSIONS: Reconstruction with the hemipelvic endoprosthesis described herein provides satisfactory function with a relatively low complication rate. Adding an extra screw fixation to the first sacral vertebra was not associated with any improvement in the clinical results after short-term follow-up. Improvement and further studies of this endoprosthesis are needed.

Genetic and clonal dissection of osteosarcoma progression and lung metastasis.

Osteosarcoma is a primary malignant bone tumor that has a high potential to metastasize to lungs. Little is known about the mechanisms underlying the dissemination of OS cancer cells to lungs. We performed whole exome sequencing of 13 OS primary tumors, with matched lung metastases and normal tissues. Phylogenetic analyses revealed that lung metastatic tumors often harbor clones that are nonexistent or rare in the matched primary OS tumors. Spatially and temporally separated lung metastases were from parallel seeding events with a polyphyletic pattern. Loss of TP53 or RB1 is among the early events during OS tumorigenesis, while loss of PTEN is involved at the later stages associated with lung metastases. Finally, KEAP1 was identified as a novel biomarker for increased metastatic risk. Patients whose primary tumors harbored KEAP1 amplification have significantly poorer lung-metastasis free survival. This finding was validated in two independent datasets. Further, in vitro experiments exhibited that KEAP1 depletion suppressed the invasion of OS cells. Our findings uncover the patterns of clonal evolution during OS progression and highlight KEAP1 as a novel candidate associated with the risk of lung metastasis in OS patients.

Comparison of clinical features, management and outcomes of osteosarcoma located in proximal fibula and proximal tibia: a propensity score matching analysis.

BACKGROUND: The aim of this study was to compare proximal fibular and proximal tibial sites regarding osteosarcoma in the proximal crus. Furthermore, we proposed a hypothesis explaining the differences. METHODS: From Jaunary 2000 to February 2015, 28 patients with non-metastatic proximal fibular osteosarcoma and 214 patients with non-metastatic proximal tibial osteosarcoma underwent surgery were identified in our center. All clinical data were analyzed retrospectively. Propensity score matching of patients in a 1:2 ratio was conducted based on age, gender and Enneking stage. To analyze possible factors resulting in amputation, we investigated extraosseous tumor volumes (ETVS), the nearest of the blood vessel to reactive zone (NBR) and the nearest of the blood vessel to tumor (NBT). RESULTS: Amputation rates were higher in the proximal fibula cohort (35.7%) than in the proximal tibia cohort (14.3%; p = 0.046). Comparing possible clinical characteristics related with amputation between two cohorts, the proximal fibula cohort had larger ETVS (p = 0.000). Moreover, the proximal fibula cohort had a smaller NBT for anterior tibial vessels (p = 0.025), a smaller NBR for posterior tibial vessels (p = 0.013) and a smaller NBT for posterior tibial vessels (p = 0.007) than the proximal tibia cohort. Univarite and multivariable analyses showed that NBT for posterior tibial vessels was the only independent factor associated with amputation. The 3-year event-free survival (EFS) and overall survival (OS) rates for the proximal fibula cohort vs. the proximal tibia cohort were 52.6% vs. 78.0% (p = 0.045) and 63.7% vs. 81.2% (p = 0.177), respectively. The MSTS scores for the functional evaluation of limb-salvaging surgery were similar in both groups (p = 0.212). CONCLUSIONS: Amputation rates among patients were higher when osteosarcoma was located in proximal fibula than in proximal tibia. A smaller NBT for posterior tibial vessels was associated with higher amputation rates. Prognosis of the proximal fibula cohort was poorer than that of the proximal tibia cohort of osteosarcoma patients.

Robot-assisted sacral tumor resection: a preliminary study.

BACKGROUND: Few studies have been done on robot-assisted sacral surgery. This study aims to evaluate the outcomes of seven patients with benign sacral or presacral tumors treated with a robotic surgical system at a single center. METHODS: All patients with benign sacral or presacral tumors who underwent transperitoneal resection (between June 2015 and June 2016) using the da Vinci Si HD robotic surgical system (Intuitive Surgical Inc.) were included in this retrospective study. RESULTS: Seven patients with a mean age of 43.8 years (range: 22- 62 years) were included in this study. The operation time ranged from 60 to 335 min. Five out of these seven patients with presacral tumor underwent complete tumor resection by the da Vinci robotic surgical system, with a median blood loss of 52 ml. The other patients underwent excision of the presacral tumor by the da Vinci robotic surgical system, followed by a posterior approach, with a median blood loss of 675 ml. The histological diagnosis was schwannoma of the sacral nerve in five cases (71.5%). The other two cases were chordoma and solitary fibroma of the sacrum, respectively. No perioperative or postoperative complications were encountered. The average hospitalization stay was 5.7 days. No recurrences were found at follow-up 24 to 31 months later. CONCLUSION: Robot-assisted minimally invasive sacral surgery can provide precise dissection of the tissue under a perfect view. It is a technically feasible procedure that is associated with minimal blood loss, fewer injuries and short hospitalization. It is particularly suitable for presacral benign tumors.

Microarray Expression Profile and Functional Analysis of Circular RNAs in Osteosarcoma.

BACKGROUND/AIMS: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. However, the molecular mechanisms regulating osteosarcoma tumorigenesis and progression are still poorly understood. Circular RNAs (circRNAs) have been identified as microRNA sponges and are involved in many important biological processes. This study aims to investigate the global changes in the expression pattern of circRNAs in osteosarcoma and provide a comprehensive understanding of differentially expressed circRNAs. METHODS: Microarray based circRNA expression was determined in osteosarcoma cell lines and compared with hFOB1.19, which was used as the normal control. We confirmed the microarray data by real time-qPCR in both osteosarcoma cell lines and tissues. The circRNA/microRNA/mRNA interaction network was predicted using bioinformatics. Gene Ontology analysis and 4 annotation tools for pathway analysis (KEGG, Biocarta, PANTHER and Reactome) were used to predict the functions of differentially expressed circRNAs. RESULTS: We revealed a number of differentially expressed circRNAs and 12 of them were confirmed, which suggests a potential role of circRNAs in OS. Among these differentially expressed circRNAs, hsa_circRNA_103801 was up-regulated in both osteosarcoma cell lines and tissues, while hsa_circRNA_104980 was down-regulated. The most likely potential target miRNAs for hsa_circRNA_103801 include hsa-miR-370-3p, hsa-miR-338-3p and hsa-miR-877-3p, while the most potential target miRNAs of hsa_circRNA_104980 consist of hsa-miR-1298-3p and hsa-miR-660-3p. Functional analysis found that hsa_circRNA_103801 was involved in pathways in cancer, such as the HIF-1, VEGF and angiogenesis pathway, the Rap1 signaling pathway and the PI3K-Akt signaling pathway, while hsa_circRNA_104980 was related to some pathways such as the tight junction pathway. CONCLUSIONS: This study has identified the comprehensive expression profile of circRNAs in osteosarcoma for the first time. And the ceRNA network prediction and bioinformatics functional analysis could provide a comprehensive understanding of hsa_circRNA_103801 and hsa_circRNA_104980, which may be involved in the initiation and progression of osteosarcoma. The present study indicates that circRNAs may play important roles in osteosarcoma and thus serve as biomarkers of osteosarcoma diagnosis and treatment.

Local control of giant cell tumors of the long bone after aggressive curettage with and without bone cement.

BACKGROUND: Aggressive curettage has been well established for the treatment of giant cell tumors (GCTs) of the bone. The purpose of this study was to review our experience and evaluate the role of different implant materials in patients with GCTs of the extremities after aggressive curettage. METHODS: A total of 119 patients with GCTs of the long bone were treated at the First Affiliated Hospital of Sun Yat-Sen University between 2004 and 2009. We excluded patients presenting metastases, recurrent tumors, and soft tissue involvement and those with Jaffe pathological grade III. The remaining 65 patients were treated with aggressive curettage using a bone graft or bone cement to fill the cavity. The recurrence rates and functional scores associated with the different fillings were analyzed. RESULTS: Aggressive curettage and bone grafting was performed in 34 cases (52.3%), and aggressive curettage with bone cement was performed in 31 cases (47.7%). The overall recurrence rate after the aggressive intralesional procedures was 35.3% with bone grafting and 12.9% when bone cement was used as an adjuvant filling. The recurrence rate following aggressive curettage and bone grafting was higher than that following aggressive curettage with cement (p = 0.038). The Musculoskeletal Tumor Society (MSTS) score for bone graft patients was 91.1%, which was significantly lower than that for patients treated with bone cement (94.7%). CONCLUSIONS: The use of bone cement was associated with a significantly lower recurrence rate than bone grafting following aggressive intralesional curettage to treat benign giant cell tumors of the long bone. Better MSTS functional results were also observed in the bone cement group compared to the bone graft group.

[Proteomics research of bufalin-induced apoptosis in osteosarcoma cell lines].

OBJECTIVE: To study the apoptosis inducing effects of bufalin on various human osteosarcoma cells and the concerning molecular mechanisms. METHOD: MTT assay was used to detect the growth inhibition rates of osteosarcoma cells U-20S, U-20S/MTX300, SaOS-2, IOR/OS9 treated with bufalin in different concentrations and times. The apoptosis of cells was observed flow cytometry 48 h following bufalin treatment. The proteomic techniques were used to separate and compare the treated and control groups 48 h after bufalin-incubation. Then, the proteomic results were validated by western blot. RESULT: Bufalin inhibited the growth of human osteosarcoma cells U20S, U20S/MTX300 (methotrexate resistant cells), SAOS2, IOR/OS9 in a dose- and time-dependent manner. The 72 h IC50 were (37.43 +/- 4.1), (32.24 +/- 5.3) nmol x L(-1) in U20S,U20S/MTX300 cells,respectivly. Flow cytometry showed that the apoptosis cells were increased following bufalin treatment. The protein expression profile showed 24 differentiated expression proteins. Among these proteins, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27) decreased significantly and the result was then validated by western blot. Ectopic expression of Hsp27 could reduce the bufalin-induced apoptosis remarkably in U20S and U20S/MTX300 cells. CONCLUSION: Bufalin could inhibit the cell growth and induce apoptosis on human osteosarcoma cells. The effect of bufalin may be related to the joint intervention with multiple protein targets. Among them, downregulation of Hsp27 plays a critical role in the bufalin-induced apoptosis in human osteosarcoma cells.

Comparison of oncological and functional outcomes in Lower-limb osteosarcoma pediatric patients: a large single-center retrospective cohort study.

OBJECTIVE: Treating pediatric osteosarcoma in long bones is challenging due to skeletal immaturity, which restricts the generalizability of insights derived from adult patients. Are there disparities in outcomes? How should surgical protocols be tailored for children of varying ages? What are the specific postoperative complications? A large single-center retrospective cohort study of 345 patients under 14 years old with lower-limb osteosarcoma treated in our department since 2000 was conducted to address these inquiries. METHODS: A retrospective analysis of 345 pediatric patients with lower-limb osteosarcoma admitted to our department between 2000 and 2019 was conducted. Clinical and functional outcomes were compared based on age groups, surgical methods, type of prosthesis, and primary tumor location. Patients were divided into the low-age group (≤10 y old) and the high-age group (>10 y old). Overall survival rate (OS), progressionfree survival rate (PFS), and prosthesis survival rate were assessed using Kaplan-Meier curves, nonparametric survival analysis (log-rank test), and Univariate cox regression were used for comparison. The incidence of complications, local relapse rate (LRR), metastasis rate, final limb-salvage, and amputation rate, and Musculoskeletal Tumor Society (MSTS) score of different independent groups were further evaluated using χ2 test or Fisher's exact test, and t -test was employed to evaluate the measurement data. RESULTS: The average age of the patients was 11.10±2.32 years ranging from 4 to 14 y, with an average follow-up duration of 48.17 months. The 5, 10, and 15-year OS rates were 50.3%, 43.8%, and 37.9%, respectively. The progression-free survival rate was 44.8% at 5 years and 41.1% at 10 years. The final limb salvage rate was 61.45%, while the final amputation rate was 38.55%. The low-age group had a higher amputation rate compared with the high-age group (48.00% vs. 33.18%, P =0.009). The overall LRR was 9.28%, and the incidence of metastasis was 28.99%. The LRR of the limb-salvage group was higher than the amputation group ( P =0.004). The low-age group experienced more prosthesis-related complications than the high-age group ( P =0.001). The most common prosthesis-related complication in the low-age group was soft-tissue failure, while the periprosthetic infection was most frequent in the high-age group. The high-age group had a higher cumulative prosthesis survival compared with the low-age group ( P =0.0097). Modular prosthesis showed better MSTS scores and higher cumulative prosthetic survival than expandable prosthesis in pediatric patients ( P <0.05). CONCLUSION: Limb preservation in pediatric patients becomes increasingly efficacious with advancing age, while consideration of amputation is warranted for younger patients. The prevailing postoperative complications associated with prosthesis encompass soft tissue failure and periprosthetic infection. Younger patients diagnosed with lower limb osteosarcoma exhibit a heightened amputation rate and a greater incidence of prosthesis-related complications.

The survival and complication profiles of the Compress® Endoprosthesis: A systematic review and meta-analysis.

Background/purpose: This study aimed to summarize the survival and complication profiles of the compress® endoprosthesis (CPS) through a systematic review and meta-analysis. Methods: Online databases (PubMed, EMBASE and Web of Science) were searched from inception to November 2023. Trials were included that involved the use of CPS for endoprosthetic replacement in patients with massive segmental bone defects. Patients' clinical characteristics and demographic data were extracted using a standardized form. The methodological quality of included 13 non-comparative studies was assessed on basis of the Methodological Index for Non-Randomized Studies (MINORS). All the available Kaplan-Meier curves in the included studies were digitized and combined using Engauge-Digitizer software and the R Project for Statistical Computing. Results: The meta-analysis of thirteen included studies indicated: the all-cause failure rates of CPS were 26.3 % after surgery, in which the occurrence rates of aseptic loosening were 5.8 %. And the incidences of other complications were as follows: soft tissue failure (1.8 %), structure failure (8.2 %), infection (9.5 %), tumor progression (1.1 %). The 1-, 4-, and 8-year overall survival rates for all-cause failure with 95 % CI were 89 % (86 %-92 %), 75 % (71 %-79 %) and 65 % (60 %-70 %), respectively. The estimated mean survival time of all-cause failure was 145 months (95 % CI, 127-148 months), and the estimated median survival time of all-cause failure was 187 months (95 % CI, 135-198 months). The 1-, 4-, and 8-year overall survival rates of aseptic loosening with 95 % CI were 96 % (94 %-98 %), 91 % (87 %-95 %) and 88 % (83 %-93 %), respectively. The estimated mean survival time of aseptic loosening was 148 months (95 % CI, 137-153 months). Conclusion: CPS's innovative spring system promotes bone ingrowth by providing immediate and high-compression fixation, thereby reducing the risk of aseptic loosening caused by stress shielding and particle-induced osteolysis. CPS requires less residual bone mass for reconstructing massive segmental bone defects and facilitates easier revision due to its non-cemented fixation. In addition, the survival rate, estimated mean survival time, and complication rates of CPS are not inferior to those of common endoprosthesis.

Tumor-informed deep sequencing of ctDNA detects minimal residual disease and predicts relapse in osteosarcoma.

Background: Current surveillance modalities of osteosarcoma relapse exhibit limited sensitivity and specificity. Although circulating tumor DNA (ctDNA) has been established as a biomarker of minimal residual disease (MRD) in many solid tumors, a sensitive ctDNA detection technique has not been thoroughly explored for longitudinal MRD detection in osteosarcoma. Methods: From August 2019 to June 2023, 59 patients diagnosed with osteosarcoma at the First Affiliated Hospital of Sun Yat-sen University were evaluated in this study. Tumor-informed MRD panels were developed through whole exome sequencing (WES) of tumor tissues. Longitudinal blood samples were collected during treatment and subjected to multiplex PCR-based next-generation sequencing (NGS). Kaplan-Meier curves and Log-rank tests were used to compare outcomes, and Cox regression analysis was performed to identify prognostic factors. Findings: WES analysis of 83 patients revealed substantial mutational heterogeneity, with non-recurrent mutated genes accounting for 58.1%. Tumor-informed MRD panels were successfully obtained for 85.5% of patients (71/83). Among 59 patients with successful MRD panel customization and available blood samples, 13 patients exhibited positive ctDNA detection after surgery. Patients with negative post-operative ctDNA had better event-free survival (EFS) compared to those with positive ctDNA, at 1-6 months after surgery, after adjuvant chemotherapy, and more than 6 months after surgery (p < 0.05). In both univariate and multivariate Cox regression analysis, ctDNA results emerged as a significant predictor of EFS (p < 0.05). ctDNA detection preceded positive imaging in 5 patients, with an average lead time of 92.6 days. Thirty-nine patients remained disease-free, with ctDNA results consistently negative or turning negative during follow-up. Interpretation: Our study underscores the applicability of tumor-informed deep sequencing of ctDNA in osteosarcoma MRD surveillance and, to our knowledge, represents the largest cohort to date. ctDNA detection is a significant prognostic factor, enabling the early identification of tumor relapse and progression compared to standard imaging, thus offering valuable insights in guiding osteosarcoma patient management. Funding: The Grants of National Natural Science Foundation of China (No. 82072964, 82072965, 82203798, 82203026), the Natural Science Foundation of Guangdong (No. 2023A1515012659, 2023A1515010302), and the Regional Combination Project of Basic and Applied Basic Research Foundation of Guangdong (No. 2020A1515110010).

Age-dependent molecular variations in osteosarcoma: implications for precision oncology across pediatric, adolescent, and adult patients.

Background: Osteosarcoma is a leading subtype of bone tumor affecting adolescents and adults. Comparative molecular characterization among different age groups, especially in pediatric, adolescents and adults, is scarce. Methods: We collected samples from 194 osteosarcoma patients, encompassing pediatric, adolescent, and adult cohorts. Genomic analyses were conducted to reveal prevalent mutations and compare molecular features in pediatric, adolescent, and adult patients. Results: Samples from 194 osteosarcoma patients across pediatric to adult ages were analyzed, revealing key mutations such as TP53, FLCN, NCOR1, and others. Children and adolescents showed more gene amplifications and HRD mutations, while adults had a greater Tumor Mutational Burden (TMB). Mutations in those over 15 were mainly in cell cycle and PI3K/mTOR pathways, while under 15s had more in cell cycle and angiogenesis with higher VEGFA, CCND3, TFEB mutations. CNV patterns varied with age: VEGFA and XPO5 amplifications more in under 25s, and CDKN2A/B deletions in over 25s. Genetic alterations in genes like MCL1 and MYC were associated with poor prognosis, with VEGFA mutations also indicating worse outcomes. 58% of patients had actionable mutations, suggesting opportunities for targeted therapies. Age-specific patterns were observed, with Multi-TKI mutations more common in younger patients and CDK4/6 inhibitor mutations in adults, highlighting the need for personalized treatment approaches in osteosarcoma. In a small group of patients with VEGFR amplification, postoperative treatment with multi-kinase inhibitors resulted in a PR in 3 of 13 cases, especially in patients under 15. A significant case involved a 13-year-old with a notable tumor size reduction achieving PR, even with other genetic alterations present in some patients with PD. Conclusion: This study delineates the molecular differences among pediatric, adolescent, and adult osteosarcoma patients at the genomic level, emphasizing the necessity for precision diagnostics and treatment strategies, and may offer novel prognostic biomarkers for patients with osteosarcoma. These findings provide a significant scientific foundation for the development of individualized treatment approaches tailored to patients of different age groups.

Super-enhancer-driven MLX mediates redox balance maintenance via SLC7A11 in osteosarcoma.

Osteosarcoma (OS) is a common type of bone tumor for which there has been limited therapeutic progress over the past three decades. The prevalence of transcriptional addiction in cancer cells emphasizes the biological significance and clinical relevance of super-enhancers. In this study, we found that Max-like protein X (MLX), a member of the Myc-MLX network, is driven by super-enhancers. Upregulation of MLX predicts a poor prognosis in osteosarcoma. Knockdown of MLX impairs growth and metastasis of osteosarcoma in vivo and in vitro. Transcriptomic sequencing has revealed that MLX is involved in various metabolic pathways (e.g., lipid metabolism) and can induce metabolic reprogramming. Furthermore, knockdown of MLX results in disturbed transport and storage of ferrous iron, leading to an increase in the level of cellular ferrous iron and subsequent induction of ferroptosis. Mechanistically, MLX regulates the glutamate/cystine antiporter SLC7A11 to promote extracellular cysteine uptake required for the biosynthesis of the essential antioxidant GSH, thereby detoxifying reactive oxygen species (ROS) and maintaining the redox balance of osteosarcoma cells. Importantly, sulfasalazine, an FDA-approved anti-inflammatory drug, can inhibit SLC7A11, disrupt redox balance, and induce massive ferroptosis, leading to impaired tumor growth in vivo. Taken together, this study reveals a novel mechanism in which super-enhancer-driven MLX positively regulates SLC7A11 to meet the alleviated demand for cystine and maintain the redox balance, highlighting the feasibility and clinical promise of targeting SLC7A11 in osteosarcoma.

Autologous menisci-cruciate ligament composite as a flap for soft tissue reconstruction following malignant bone tumor resection around the knee.

BACKGROUND: Despite significant improvements in oncological treatment, the management of soft tissue defects following malignant tumor resection remains challenging. We investigated whether autologous menisci and cruciate ligament, which are traditionally discarded, can be recycled as a supplemental flap in repairing soft tissue defects following malignant bone tumor resection and endoprosthetic reconstruction around the knee. METHODS: Four knee specimens were dissected to provide a basis for the design of the menisci-cruciate ligament composite. Then, 40 patients with bone malignancies around the knee were enrolled and underwent reconstruction with free or vascularized composite following malignant tumor resection. The clinical, radiographic, and functional outcomes of this technique were evaluated in >1-year follow-up in each patient and compared with 87 patients who suffered from bone malignancies around the knee and were treated by limb salvage but without composite at our center over the same period. During the follow-up, a composite from one patient who underwent secondary amputation was retrieved and examined for in vivo remodeling. RESULTS: Fourteen patients were treated with vascularized composite transfer (10 distal femurs and 4 proximal tibias) and 26 patients with free composite transfer (19 distal femurs and 7 proximal tibias). The composite can be used to cover the area of soft tissue defect from 22 to 48.38 cm2 (34.67 ± 6.48 cm2 ). With contrast-enhanced ultrasound, peripheral rim healing and dotted blood flow signal at the side of anastomosis were detected on a patient 16 months after free composite transfer. Gross macroscopic remodeling and histopathologic analysis of a retrieved composite also indicated good healing with surrounding tissues and living cells in the composite. The complications and oncologic outcomes were comparable between study and control cohorts, but better Musculoskeletal Tumor Society (MSTS) score for patients reconstructed with composite (26.68 vs. 25.66, p  = 0.004). Of note, MSTS score was higher for patients reconstructed with composite at distal femur subdivision compared with the same subdivision in the control cohort (26.97 vs. 25.90, p  = 0.009). No statically significant difference was noted in complications, oncologic, and functional outcomes for patients reconstructed with free or vascularized composite. CONCLUSION: Autogenous menisci-cruciate ligament composite is an alternative option for soft tissue reconstruction. Either vascularized or free composite can be applied, depending on the size and localization of the defect.

Preoperative denosumab treatment in patients with giant cell bone tumors in limbs: A retrospective study using propensity score matching.

BACKGROUND AND OBJECTIVES: Denosumab is recommended for advanced giant cell tumor of bone (GCTB) that is unresectable or resectable with unacceptable morbidity. But the effect of preoperative denosumab treatment on the local control GCTB remains controversial. METHODS: We conducted a study of 49 patients with GCTB in the limbs treated with denosumab before surgery and 125 patients without in our hospital from 2010 to 2017. Propensity-score matching (PSM) at a 1:1 ratio between the denosumab and control groups was performed to minimize possible selection bias, and compared the recurrence rate, limb function, and surgical degradation between the two groups. RESULTS: The 3-year recurrence rates in the denosumab group and the control group were 20.4% and 22.9% after PSM, respectively (p = 0.702). In the denosumab group, 75.5% (n = 37/49) of patients experienced surgical downgrading. Limb joint preservation rates were 92.1% (35) for 38 patients treated with denosumab and 60.2% (71) for 118 control subjects. (p ≺ 0.001). Postoperative MSTS were higher in patients in the denosumab group than in the control group (24.1 vs. 22.6, p = 0.034). CONCLUSIONS: Preoperative denosumab treatment did not result in an increased risk of local recurrence of GCTB. Patients with advanced GCTB may benefit from preoperative denosumab treatment for surgical downgrading and the preservation of the joint.

Cancer-testis antigens expressed in osteosarcoma identified by gene microarray correlate with a poor patient prognosis.

BACKGROUND: From 30% to 40% patients with osteosarcoma eventually experience medical failure; and few biomarkers of prognostic significance have been established. High-throughput methods like gene microarray analysis can help to identify molecular biomarkers that are useful for diagnosing osteosarcoma and targeting its treatment. METHODS: Oligonucleotide microarrays were used to compare expression profiles of osteosarcoma cell lines and osteoblasts. Differentially expressed genes were confirmed by real-time polymerase chain reaction (PCR) analysis. Corresponding proteins were evaluated by flow cytometry and Western blot analysis in osteosarcoma cell lines and by immunohistochemistry in osteosarcoma tissues. The association between staining intensity and clinical outcome was analyzed further. RESULTS: Cancer-testis antigens, including melanoma antigen family A (MAGEA), chondrosarcoma-associated gene family, member 2 (CSAG2), and preferentially expressed antigen in melanoma (PRAME), were increased significantly in all osteosarcoma cell lines that were analyzed. Real-time PCR examinations indicated that cancer-testis antigen expression was frequent and coordinated in patients with osteosarcoma. The expression of MAGEA was confirmed by Western blot and flow cytometry analyses in osteosarcoma cell lines. Furthermore, immunohistochemical staining analysis suggested that MAGEA expression may be used to predict distant metastasis and poor survival. The adjusted relative risk for lung metastasis was 2.79 (95% confidence interval, 1.12-6.93; P = .028) for MAGEA-positive patients. Five-year survival rates for patients with and without MAGEA expression were 39.6% ± 8.4% and 80% ± 8.9%, respectively (log-rank test; P = .01). CONCLUSIONS: The combined use of an oligonucleotide microarray, a clinical database, and a tissue bank was useful for identifying molecular tumor markers. The frequent expression of MAGEA and other cancer-testis antigens in osteosarcoma indicates that they may be useful as diagnostic markers and targets of immunotherapy that warrant further investigation.

Expression and prognostic relevance of PRAME in primary osteosarcoma.

The preferentially expressed antigen of melanoma (PRAME), a cancer-testis antigen with unknown function, is expressed in many human malignancies and is considered an attractive potential target for tumor immunotherapy. However, studies of its expression and function in osteosarcoma have rarely been reported. In this study, we found that PRAME is expressed in five osteosarcoma cell lines and in more than 70% of osteosarcoma patient specimens. In addition, an immunohistochemical analysis showed that high PRAME expression was associated with poor prognosis and lung metastasis. Furthermore, PRAME siRNA knockdown significantly suppressed the proliferation, colony formation, and G1 cell cycle arrest in U-2OS cells. Our results suggest that PRAME plays an important role in cell proliferation and disease progression in osteosarcoma. However, the detail mechanisms of PRAME function in osteosarcoma require further investigation.