Spatial multiplexed immunofluorescence analysis reveals coordinated cellular networks associated with overall survival in metastatic osteosarcoma.

Patients diagnosed with advanced osteosarcoma, often in the form of lung metastases, have abysmal five-year overall survival rates. The complexity of the osteosarcoma immune tumor microenvironment has been implicated in clinical trial failures of various immunotherapies. The purpose of this exploratory study was to spatially characterize the immune tumor microenvironment of metastatic osteosarcoma lung specimens. Knowledge of the coordinating cellular networks within these tissues could then lead to improved outcomes when utilizing immunotherapy for treatment of this disease. Importantly, various cell types, interactions, and cellular neighborhoods were associated with five-year survival status. Of note, increases in cellular interactions between T lymphocytes, positive for programmed cell death protein 1, and myeloid-derived suppressor cells were observed in the 5-year deceased cohort. Additionally, cellular neighborhood analysis identified an Immune-Cold Parenchyma cellular neighborhood, also associated with worse 5-year survival. Finally, the Osteosarcoma Spatial Score, which approximates effector immune activity in the immune tumor microenvironment through the spatial proximity of immune and tumor cells, was increased within 5-year survivors, suggesting improved effector signaling in this patient cohort. Ultimately, these data represent a robust spatial multiplexed immunofluorescence analysis of the metastatic osteosarcoma immune tumor microenvironment. Various communication networks, and their association with survival, were described. In the future, identification of these networks may suggest the use of specific, combinatory immunotherapeutic strategies for improved anti-tumor immune responses and outcomes in osteosarcoma.

Predictors of pulmonary metastases on chest computed tomography in children and adolescents with osteosarcoma-tips for qualifying patients for thoracotomy.

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumour in children and adolescents. Lungs are the most frequent and often the only site of metastatic disease. The presence of pulmonary metastases is a significant unfavourable prognostic factor. Thoracotomy is strongly recommended in these patients, while computed tomography (CT) remains the gold imaging standard. The purpose of our study was to create tools for the CT-based qualification for thoracotomy in osteosarcoma patients in order to reduce the rate of useless thoracotomies. METHODS: Sixty-four osteosarcoma paediatric patients suspected of lung metastases on CT and their first-time thoracotomies (n = 100) were included in this retrospective analysis. All CT scans were analysed using a compartmental evaluation method based on the number and size of nodules. Calcification and location of lung lesions were also analysed. Inter-observer reliability between two experienced radiologists was assessed. The CT findings were then correlated with the histopathological results of thoracotomies. Various multivariate predictive models (logistic regression, classification tree and random forest) were built and predictors of lung metastases were identified. RESULTS: All applied models proved that calcified nodules on the preoperative CT scan best predict the presence of pulmonary metastases. The rating of the operated lung on the preoperative CT scan, dependent on the number and size of nodules, and the total number of nodules on this scan were also found to be important predictors. All three models achieved a relatively high sensitivity (72-92%), positive predictive value (81-90%) and accuracy (74-79%). The positive predictive value of each model was higher than of the qualification for thoracotomy performed at the time of treatment. Inter-observer reliability was at least substantial for qualitative variables and excellent for quantitative variables. CONCLUSIONS: The multivariate models built and tested in our study may be useful in the qualification of osteosarcoma patients for metastasectomy through thoracotomy and may contribute to reducing the rate of unnecessary invasive procedures in the future.

A Rare Case of Renal Sarcoma Mimicking Metastatic Osteosarcoma.

Pediatric renal sarcomas are exceedingly rare entities that present diagnostic challenges. We report a remarkable case of a 14-month-old female with an 8 cm right renal mass, accompanied by disseminated bone metastases, posing intricate clinical and histopathological dilemmas. Initial suspicion leaned towards clear cell sarcoma of the kidney (CCSK), however, subsequent histological examination post-chemotherapy revealed high-grade osteosarcoma and further differential considerations arose, including primary renal osteosarcoma and osseous osteosarcoma with secondary renal involvement. Despite inconclusive histology, treatment proceeded with the UH1 chemotherapy protocol for CCSK, incorporating high-dose Methotrexate for potential osteosarcoma, and the patient demonstrated a favorable response to therapy.

Pediatric Leukemia Roadmaps Are a Guide for Positive Metastatic Bone Sarcoma Trials.

ALL cures require many MRD therapies. This strategy should drive experiments and trials in metastatic bone sarcomas.

Head and Neck Osteosarcoma: Perineural Invasion is Associated With Disease-Free Survival and Tumor Metastasis.

BACKGROUND: Head and neck osteosarcoma (HNOS) is the most common bone malignancy in the head and neck region, accounting for 10% of all osteosarcoma cases. Perineural invasion (PNI) is a notable indication of aggressive tumor behavior, which includes the phenomenon of tumor cells invading any of the 3 layers of the nerve sheath or tumor cells gathering, encircling one-third of the nerve circumference, and infiltrating and metastasizing along the nerve. PNI has been reported in various malignant tumors and is considered to be linked to poor prognosis. PURPOSE: The study's purpose is to measure the association between PNI and survival outcomes in patients with HNOS. STUDY DESIGN, SETTING, SAMPLE: This retrospective cohort study focused on HNOS patients who underwent surgery at the Department of Oral and Maxillofacial Head and Neck Oncology, Shanghai Ninth People's Hospital School of Medicine, Shanghai Jiao Tong University, from January 1, 2019 to December 31, 2021. Patients who did not undergo complete surgical resection of the tumor, did not receive a conventional osteosarcoma diagnosis, and had positive surgical margins were eliminated. PREDICTOR VARIABLE: The predictor variable is PNI status. The pathological section of the tumor was consistent with any of the PNI features, which was considered PNI-positive. MAIN OUTCOME VARIABLE(S): The primary outcome variables were 3-year disease-free survival (DFS) and 3-year overall survival. Secondary outcomes were 3-year tumor local recurrence and 3-year metastasis (MT). COVARIATES: Covariates were categorized into the following categories: demographic variables (age, sex), clinical variables (tumor region, primary tumor), and treatment variables (chemotherapy, radiotherapy). ANALYSES: Analytic statistical methods were used for the data analysis. Pearson χ2 or Fisher's exact test was used to describe the baseline data. Kaplan-Meier is used to calculate survival rates. The Cox regression model was adapted for univariate and multivariate analysis. A P value less than .05 indicated statistical significance. RESULTS: The study sample comprised 70 patients; 33 (47.1%) were male, and the mean age was 42.2 (standard deviation: 16.7) years. There were 15 (21.4%) cases of PNI. The 3-year DSF rate and OS rate were 67.3% and 82.0%, respectively. PNI-positive resulted in higher risk for MT (P ＜ .01, hazard ratio: 5.95, 95% confidence interval: 1.62-21.86) and negative impact on DFS (P < .01, hazard ratio: 6.35, 95% confidence interval: 2.11-19.17) for HNOS patients. CONCLUSION AND RELEVANCE: Positive PNI status was associated with decreased DFS and increased risk of MT.

Extracellular Vesicles from Highly Metastatic Osteosarcoma Cells Induce Pro-Tumorigenic Macrophage Phenotypes.

Metastasis is the principal factor in poor prognosis for individuals with osteosarcoma (OS). Understanding the events that lead to metastasis is critical to develop better interventions for this disease. Alveolar macrophages are potentially involved in priming the lung microenvironment for OS metastasis, yet the mechanisms involved in this process remain unclear. Since extracellular vesicles (EVs) are a known actor in primary tumor development, their potential role in OS metastagenesis through macrophage modulation is explored here. The interaction of EVs isolated from highly metastatic (K7M2) and less metastatic (K12) osteosarcoma cell lines is compared with a peritoneal macrophage cell line. An EV concentration that reproducibly induced macrophage migration is identified first, then used for later experiments. By confocal microscopy, both EV types associated with M0 or M1 macrophages; however, only K7M2-EVs are associated with M2 macrophages, an interaction that is abrogated by EV pre-treatment with anti-CD47 antibody. Interestingly, all interactions appeared to be surface binding, not internalized. In functional studies, K7M2-EVs polarized fewer macrophages to M1. Together, these data suggest that K7M2-EVs have unique interactions with macrophages that can contribute to the production of a higher proportion of pro-tumor type macrophages, thereby accelerating metastasis.

METTL3 Promotes Osteosarcoma Metastasis via an m6A-dependent Epigenetic Activity of CBX4.

BACKGROUND: Osteosarcoma cells are prone to metastasis, and the mechanism of N6-methyladenosine (m6A) methylation modification in this process is still unclear. Methylation modification of m6A plays an important role in the development of osteosarcoma, which is mainly due to abnormal expression of enzymes related to methylation modification of m6A, which in turn leads to changes in the methylation level of downstream target genes messenger RNA (mRNA) leading to tumor development. METHODS: We analyzed the expression levels of m6A methylation modification-related enzyme genes in GSE12865 whole-genome sequencing data. And we used shRNA (short hairpin RNA) lentiviral interference to interfere with METTL3 (Methyltransferase 3) expression in osteosarcoma cells. We studied the cytological function of METTL3 by Cell Counting Kit-8 (CCK8), flow cytometry, migration and other experiments, and the molecular mechanism of METTL3 by RIP (RNA binding protein immunoprecipitation), Western blot and other experiments. RESULTS: We found that METTL3 is abnormally highly expressed in osteosarcoma and interferes with METTL3 expression in osteosarcoma cells to inhibit metastasis, proliferation, and apoptosis of osteosarcoma cells. We subsequently found that METTL3 binds to the mRNA of CBX4 (chromobox homolog 4), a very important regulatory protein in osteosarcoma metastasis, and METTL3 regulates the mRNA and protein expression of CBX4. Further studies revealed that METTL3 inhibited metastasis of osteosarcoma cells by regulating CBX4. METTL3 has been found to be involved in osteosarcoma cells metastasis by CBX4 affecting the protein expression of matrix metalloproteinase 2 (MMP2), MMP9, E-Cadherin and N-Cadherin associated with osteosarcoma cells metastasis. CONCLUSIONS: These results suggest that the combined action of METTL3 and CBX4 plays an important role in the regulation of metastasis of osteosarcoma, and therefore, the METTL3-CBX4 axis pathway may be a new potential therapeutic target for osteosarcoma.

Do Children With Osteosarcoma Benefit From Pulmonary Metastasectomy?: A Systematic Review of Published Studies and "Real World" Outcomes.

OBJECTIVE: To critically examine the evidence-base for survival benefit of pulmonary metastasectomy (PM) for osteosarcoma (OS) in the pediatric population. BACKGROUND: PM for OS is recommended as the standard of care in both pediatric and adult treatment protocols. Recent results from the "Pulmonary Metastasectomy in Colorectal Cancer" trial demonstrate no survival benefit from PM in colorectal cancer in adults. METHODS: A systematic review was undertaken according to "Preferred Reporting Items for Systematic Reviews and Meta-Analysis" guidelines. Medline, Embase, and 2 clinical trial registers were searched for all studies detailing pediatric patients with OS (<18 years) undergoing PM with a comparison cohort group that did not receive PM. RESULTS: Eleven studies met inclusion criteria dating from 1984 to 2017. All studies were retrospective and none directly compared PM versus no PM in pediatric patients as its main objective(s). Three-year survival rates ranged from 0% to 54% for PM and 0% to 16% for no PM. No patients receiving PM were usually those with unresectable disease and/or considered to have a poor prognosis. All studies were at high risk of bias and there was marked heterogeneity in the patient selection. CONCLUSIONS: There is a weak evidence base (level IV) for a survival benefit of PM for OS in pediatric patients likely due to selection bias of "favorable cases." The included studies many of which detailed outdated treatment protocols were not designed in their reporting to specifically address the questions directly. A randomized controlled trial-while ethically challenging in a pediatric population-incorporating modern OS chemotherapy protocols is needed to crucially address any "survival benefit."

Massage manipulation and progression of osteosarcoma, does it really correlate: a combination of prospective and retrospective cohort study.

In Indonesia, the challenge of osteosarcoma progression is further worsened by patients' dependence on traditional massage therapy, low socio-economy, and educational status. This study aims to analyze the differences in the characteristics, laboratory findings, surgery techniques, degree of histopathological necrosis, and metastasis between osteosarcoma patients with and without prior massage manipulation therapy. This research is an analytical observational study with a prospective and retrospective cohort design. Patients were treated and followed for one year to evaluate the occurrence of metastasis. Prospective data was collected through interviews, and secondary data was collected from the patient's medical record. Of 84 subjects analyzed, 69% had a history of massage. There was an increase in LDH and ALP in patients with massage manipulation (p = 0.026). The median time to metastasis from baseline in the massage group (4 months) was statistically significant compared to the non-manipulation group (12 months) (p < 0.0001). This research found that massage therapy significantly increases LDH and ALP levels, making amputations more likely to be performed and a higher risk of metastasis that lowered the survival rate. The onset of metastasis was three times faster in patients with prior massage therapy. Therefore, we strongly recommend against massage manipulation therapy in osteosarcoma patients.

CT features of osteosarcoma lung metastasis: a retrospective study of 127 patients.

OBJECTIVE: Osteosarcoma lung metastases have a wide variety of CT presentations, representing a challenge for radiologists. Knowledge of atypical CT patterns of lung metastasis is important to differentiate it from benign lung disease and synchronous lung cancer, as well as to determine the extent of primary disease. The objective of this study was to analyze CT features of osteosarcoma lung metastasis before and during chemotherapy. METHODS: Two radiologists independently reviewed chest CT images of 127 patients with histopathologically confirmed osteosarcoma treated between May 10, 2012 and November 13, 2020. The images were divided into two groups for analysis: images obtained before chemotherapy and images obtained during chemotherapy (initial CT examination). RESULTS: Seventy-five patients were diagnosed with synchronous or metachronous lung metastases. The most common CT findings were nodules (in 95% of the patients), distributed bilaterally (in 86%), with no predominance regarding craniocaudal distribution (in 71%). Calcification was observed in 47%. Less common findings included intravascular lesions (in 16%), cavitation (in 7%), and the halo sign (in 5%). The primary tumor size was significantly greater (i.e., > 10 cm) in patients with lung metastasis. CONCLUSIONS: On CT scans, osteosarcoma lung metastases typically appear as bilateral solid nodules. However, they can have atypical presentations, with calcification being the most common. Knowledge of the typical and atypical CT features of osteosarcoma lung metastasis could play a key role in improving image interpretation in these cases.

[Synchronic multicentric osteosarcoma. A case in a 10 year old boy and review of the literature]. / Osteosarcoma multicéntrico sincrónico. Un caso en niño de 10 años y revisión de la literatura.

The osteosarcoma is a malignant neoplasm originating in bone-producing cells whose usual presentation is predominantly monostotic in long bones. The polyostotic expression is rare, variable and controversial, predominantly high-grade cellular lesions in children; low-grade lesions that affect long bones in adults and others that show a dominant lesion in long bones from which metastases arise. We present the case of a ten-year-old patient with painful tumors in metaphasis and diaphysis of the femur, tibia, fibula, clavicle, humerus, as well as in pelvis, vertebrae and base of the skull of synchronous appearance with blindness. Diagnosis was made through X-ray images, CT scan, magnetic resonance imaging (MRI), and incisional biopsy of the femur. Evidenced of absence of neoplastic manifestation in lung imaging, at the time of pathological diagnosis. Histological findings revealed a high-grade osteosarcoma.

[CircRNA circTNPO1 promotes the proliferation and metastasis of osteosarcoma by sponging miR-338-3p].

Objective: To explore the effects of circTNPO1 on the proliferation and metastasis of osteosarcoma (OS) by sponging miR-338-3p. Methods: The expression of circTNPO1 on osteoblasts and multiple OS cell lines were detected by qRT-PCR. CircTNPO1 stable knockdown 143B cell line was constructed by sh-circTNPO1. Cell count kit 8 (CCK-8) assay and wound healing assay were applied to evaluate the proliferation and metastasis of this cell. Luciferase reporter assay was used to explore the binding between circTNPO1 and miR-338-3p. In xenograft tumor model, miR-338-3p inhibitor or its control was injected into the circTNPO1 knockdown tumors. The weight and size of the tumors were evaluated and Ki-67 expression was detected by immunohistochemistry. Results: The RNA expression of circTNPO1 in OS cell lines U2OS, HOS, MG63, 143B, ZOS and ZOSM were 2.73±0.27, 3.18±0.54, 4.33±0.52, 5.75±0.65, 4.50±0.49 and 3.96±0.35, respectively, higher than 1.00±0.09 in hFOB1.19 (P<0.001). CCK-8 assay revealed that after 48 h and 72 h, the absorbance of sh-circTNPO1 #1 was 0.81±0.05 and 1.09±0.06, while sh-circTNPO1 #2 143B cells was 0.84±0.04 and 1.2±0.04, which were sharply reduced compared with the control (1.00±0.06 and 1.49±0.06, P<0.001); after 48 h and 72 h, the absorbance of 143B cells transfected with circTNPO1 #1 and miR-338-3p (0.92±0.06 and 1.32±0.07) were higher than those of cells transfected with sh-circTNPO1 cells and miR NC (0.92±0.06 and 1.32±0.07, P<0.050). Wound healing assay demonstrated that the 24 hour-migration rates of sh-circTNPO1 #1 and sh-circTNPO1 #2 cells were (24.43±2.15)% and (39.70±4.20)% respectively, which were significantly lower than that of the control [(56.51±3.27)%, P<0.010]; the migration rates of sh-circTNPO1 #1+ miR NC and sh-circTNPO1 #1+ miR-338-3p inhibitor were (26.70±2.21)% and (46.10±5.71)%, with a significant difference (P<0.005). In xenograft tumor model, the weight and size of tumors in control, sh-circTNPO1 #1+ miR NC and sh-circTNPO1 #1+ miR-338-3p inhibitor mice were (458.80±158.10) mg, (262.50±82.09) mg, (395.40±137.60) mg and (593.00±228.40) mm(2,) (203.30±144.20) mm(2,) (488.60±208.60) mm(2,) respectively. Compared with control, sh-circTNPO1 tumors were significantly smaller (P<0.01). Injection with miR-338-3p inhibitor significantly reversed both the weight and size of tumors (P<0.05). Conclusion: CircTNPO1 promotes the proliferation and metastasis of OS by sponging miR-338-3p, which could be a new target for OS treatments.

Prognostic significance of the ratio of surgically resected to radiologically detected lung nodules in patients with metastatic osteosarcoma.

The factors that affect the prognosis of patients' metastatic osteosarcoma are still poorly understood. In this study, we investigated a new prognostic factor, the ratio of surgically resected to radiologically detected osteosarcoma lung nodules (SR/RD), which may have predictive value. PATIENTS AND METHODS: Data from patients with metastatic osteosarcoma who underwent metastasectomy between January 2009 and December 2020, in a single center, were reviewed. The relationships between survival and the SR/RD ratio, timing of lung metastases, number of nodules, laterality, and presence of tumor necrosis at first metastasectomy were investigated. RESULTS: Among the 125 metastatic osteosarcoma patients, 80 patients had an SR/RD ratio ≤1. The median duration of follow-up was 72 months, ranging from 6 to 118 months. The five-year overall survival (OS) and postmetastasectomy event-free survival (EFS) for all patients were 36.5% and 18.1%, respectively. The five-year OS of patients with a low SR/RD ratio was 49.6% and that of patients with a high SR/RD ratio was 11.8 (P = 0.001). The two-year postmetastasectomy EFS rates of the high and low ratio groups were 24.1% and 9.4%, respectively (P = 0.001). The SR/RD ratio, number of nodules, and tumor necrosis had significant effects on OS and postmetastasectomy EFS in univariate analysis. A Cox proportional hazard model demonstrated that tumor necrosis and an SR/RD ratio >1 were associated with OS (HR = 1.8 and 2.01) and postmetastasectomy EFS (HR = 1,69 and 1.97). CONCLUSIONS: A high SR/RD ratio of greater than 1 and poor tumor necrosis were significantly associated with poor survival among patients with metastatic osteosarcoma who had lung metastasectomy. The high SR/RD ratio may be a surrogate outcome for incomplete metastatic tumor resection.

Potential Anti-Metastatic Role of the Novel miR-CT3 in Tumor Angiogenesis and Osteosarcoma Invasion.

Osteosarcoma (OS) is the most common primary bone tumor mainly occurring in young adults and derived from primitive bone-forming mesenchyme. OS develops in an intricate tumor microenvironment (TME) where cellular function regulated by microRNAs (miRNAs) may affect communication between OS cells and the surrounding TME. Therefore, miRNAs are considered potential therapeutic targets in cancer and one of the goals of research is to accurately define a specific signature of a miRNAs, which could reflect the phenotype of a particular tumor, such as OS. Through NGS approach, we previously found a specific molecular profile of miRNAs in OS and discovered 8 novel miRNAs. Among these, we deepen our knowledge on the fifth candidate renamed now miR-CT3. MiR-CT3 expression was low in OS cells when compared with human primary osteoblasts and healthy bone. Through TargetScan, VEGF-A was predicted as a potential biological target of miR-CT3 and luciferase assay confirmed it. We showed that enforced expression of miR-CT3 in two OS cell lines, SAOS-2 and MG-63, reduced expression of VEGF-A mRNA and protein, inhibiting tumor angiogenesis. Enforced expression of miR-CT3 also reduced OS cell migration and invasion as confirmed by soft agar colony formation assay. Interestingly, we found that miR-CT3 behaves inducing the activation of p38 MAP kinase pathway and modulating the epithelial-mesenchymal transition (EMT) proteins, in particular reducing Vimentin expression. Overall, our study highlights the novel role of miR-CT3 in regulating tumor angiogenesis and progression in OS cells, linking also to the modulation of EMT proteins.

LncRNA MATN1-AS1 for Prediction of Prognosis in Osteosarcoma Patients and Its Cellular Function.

Long non-coding RNAs show essential roles in various cancer processes. This study aimed at the expression features, prognosis significance, and biological effect of lnc MATN1-AS1 in osteosarcoma (OS). Five kinds of cell lines and 117 pairs of tissues were analyzed by qRT-PCR for quantification of lnc MATN1-AS1 and miR-1299 level. Clinical data were analyzed using Chi-Square Tests to show the association with lnc MATN1-AS1 level. Kaplan-Meier analysis and Cox regression were used to judge the prognostic value. Cell counting kit-8 and Transwell assay were conducted, respectively, to analyze the effect of lnc MATN1-AS1 on cell proliferation and metastasis. The target miRNA was predicted. lnc MATN1-AS1 level was significantly elevated in OS cells and tissues and related to Enneking staging, lung metastasis, and histologic type. Patients with high lnc MATN1-AS1 level showed a shorter overall survival and recurrence-free survival. Lnc MATN1-AS1 knockdown inhibited OS cell proliferation, migration, and invasion by sponging miR-1299. Lnc MATN1-AS1 has oncogenic features and prognostic significance in OS and is a novel therapeutic strategy for OS.

Survival after pulmonary metastasectomy for relapsed osteosarcoma.

OBJECTIVES: The purpose of this study was to evaluate the postrelapse survival of relapsed osteosarcoma with pulmonary metastases in patients who received pulmonary metastasectomy using intent to treat and propensity score analysis. METHODS: Patients with osteosarcoma who relapsed with pulmonary metastases between 2004 and 2018 who were treated in a hospital affiliated with a medical school were included. All the enrolled patients were evaluated as operable with assessment algorithm at the time of diagnosis of pulmonary relapse and intent to treat analysis was done. Multiple propensity score methods (eg, matching, stratification, covariate adjustment, and inverse probability of treatment weighting) were performed to balance confounding bias. Cox proportional hazards regression and the Kaplan-Meier method were used to evaluate patient survival. RESULTS: A total of 125 patients met the study criteria. Of these, 59 (47.2%) patients received pulmonary metastasectomy combined with chemotherapy and 66 (52.8%) received chemotherapy alone. The 2-year and 5-year postrelapse survival rate of metastasectomy group and nonmetastasectomy group were 68.4% versus 25.0% and 41.0% versus 0%, respectively. The median postrelapse survival was 24.9 versus 13.5 months, respectively. Pulmonary metastasectomy was independently associated with improved survival (hazard ratio, 0.185; 95% confidence interval, 0.103-0.330; P < .001). These results were confirmed by multiple propensity score analyses. Further stratified analysis revealed that the survival advantage associated with metastasectomy was not significant in patients with metastases involving ≥3 lung lobes and patients with very high pretreatment serum alkaline phosphatase (more than twice the upper limit). CONCLUSIONS: Pulmonary metastasectomy is associated with improved survival in patients with recurrent osteosarcoma.