The efficacy and safety of vincristine, irinotecan and anlotinib in Epithelioid Sarcoma.

BACKGROUND: Epithelioid sarcoma is a rare soft tissue sarcoma characterized by SMARCB1/INI1 deficiency. Much attention has been paid to the selective EZH2 inhibitor tazemetostat, where other systemic treatments are generally ignored. To explore alternative treatment options, we studied the effects of irinotecan-based chemotherapy in a series of epithelioid sarcoma patients. METHODS: We retrospectively reviewed data from patients with metastatic or unresectable epithelioid sarcoma at the Peking University People's Hospital treated with irinotecan (50 mg/m2/d d1-5 Q3W) in combination with Anlotinib (12 mg Qd, 2 weeks on and 1 week off) from July 2015 to November 2021. RESULTS: A total of 54 courses were administered. With a median follow up of 21.2 months (95% CI, 12.2, 68.1), the 5-year overall survival rate was 83.3%. Five of eight (62.5%) patients presented with unresectable localized lesions, including local tumor thrombosis and lymphatic metastasis. The other patients had unresectable pulmonary metastases. Six of eight (75%) patients had progressed following two lines of systemic therapy. The objective response rate reached 37.5% (three of eight patients) while stabilized disease was observed in 62.5% (five of eight) of patients. No patient had progressed at initial evaluation. At the last follow up, two patients were still using the combination and three patients had ceased the therapy due to toxicities such as diarrhea, nausea, and emesis. One patient changed to tazemetostat for maintenance and one patient stopped treatment due to coronavirus disease 2019 (COVID-19). Another patient stopped therapy as residual lesions had been radiated. CONCLUSIONS: The combination of irinotecan and Anlotinib as a salvage regimen may be considered another effective treatment option for refractory epithelioid sarcoma. TRIAL REGISTRATION: This study was approved in the Medical Ethics Committee of Peking University People's Hospital on October 28, 2022 (No.: 2022PHD015-002). The study was registered in Clinicaltrials.gov with identifier no. NCT05656222.

Exploratory study of an anti-PD-L1/TGF-ß antibody, TQB2858, in patients with refractory or recurrent osteosarcoma and alveolar soft part sarcoma: a report from Chinese sarcoma study group (TQB2858-Ib-02).

BACKGROUND: Novel and effective immunotherapies are required for refractory or recurrent sarcomas. Transforming growth factor-beta (TGF-ß) is a diverse regulatory and fibrogenic protein expressed in multiple sarcoma tumors that promotes epithelial-mesenchymal transition and excessive deposition of extracellular matrix. This study evaluated the efficacy and safety of the anti-PD-L1/TGF-ß antibody TQB2858 in patients with refractory osteosarcoma and alveolar soft part sarcoma (ASPS). METHODS: This single-arm phase 1b exploratory study included patients with refractory osteosarcoma or ASPS who had previously undergone at least two lines of systemic therapy. Patients were administered 1200 mg of TQB2858 once every 3 weeks. The primary endpoint was objective response rate (ORR), with null and alternative hypotheses of ORR ≤5% and ≥20%, respectively. Exploratory biomarker analyses using immunohistochemistry (IHC) staining (for PD-L1 and TGF-ß) were performed on pre-treatment tumor samples. RESULTS: Eleven eligible patients were included in this study. TQB2858 did not demonstrate evidence of efficacy as 0/5 osteosarcomas had any objective response, while 2/6 ASPS showed a partial response. The median progression-free survivals were 1.51 (1.38, Not Evaluable) and 2.86 (1.38, Not Evaluable) months for the osteosarcoma and ASPS groups, respectively. None of the administered cycles met the criteria for unacceptable toxicity. Other Grade 3 toxicities included abnormal liver function and elevation of γ-glutamyl transferase. IHC analysis revealed that functional enrichment in the TGF-ß pathway or PD-L1 was not associated with treatment outcomes. CONCLUSIONS: The combination of PD-L1 and TQB2858 did not significantly improve the ORR in patients with recurrent osteosarcoma. However, it improved immunogenic responses in ASPS, even after progression upon anti-PD-1/PD-L1 therapy, with an acceptable safety profile. IHC profiling with pathway enrichment analysis may not have any predictive value for survival outcomes. TRIAL REGISTRATION: Prospectively registered in the Ethical Review Committee of Peking University People's Hospital. The trial registration number is 2021PHA105-001 and 2021PHA140-001 and the registration date was March 2, 2022. CLINICALTRIALS: gov Identifier CTR20213001 and CTR20220390.

Inhibition of Wnt activity improves peri-implantation development of somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells into totipotency. Although pre-implantation development of SCNT embryos has greatly improved, most SCNT blastocysts are still arrested at the peri-implantation stage, and the underlying mechanism remains elusive. Here, we develop a 3D in vitro culture system for SCNT peri-implantation embryos and discover that persistent Wnt signals block the naïve-to-primed pluripotency transition of epiblasts with aberrant H3K27me3 occupancy, which in turn leads to defects in epiblast transformation events and subsequent implantation failure. Strikingly, manipulating Wnt signals can attenuate the pluripotency transition and H3K27me3 deposition defects in epiblasts and achieve up to a 9-fold increase in cloning efficiency. Finally, single-cell RNA-seq analysis reveals that Wnt inhibition markedly enhances the lineage developmental trajectories of SCNT blastocysts during peri-implantation development. Overall, these findings reveal diminished potentials of SCNT blastocysts for lineage specification and validate a critical peri-implantation barrier for SCNT embryos.

Bilineage embryo-like structure from EPS cells can produce live mice with tetraploid trophectoderm.

Self-organized blastoids from extended pluripotent stem (EPS) cells possess enormous potential for investigating postimplantation embryo development and related diseases. However, the limited ability of postimplantation development of EPS-blastoids hinders its further application. In this study, single-cell transcriptomic analysis indicated that the "trophectoderm (TE)-like structure" of EPS-blastoids was primarily composed of primitive endoderm (PrE)-related cells instead of TE-related cells. We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure. Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation. Furthermore, we demonstrated that blastocyst-like structures reconstituted by combining the EPS-derived bilineage embryo-like structure (BLES) with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses. In summary, our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.

Longer versus Shorter Schedules of Vincristine, Irinotecan, and Temozolomide (VIT) for Relapsed or Refractory Ewing Sarcoma: A Randomized Controlled Phase 2 Trial.

PURPOSE: The optimal dose schedule of vincristine, irinotecan, and temozolomide (VIT) in relapsed or refractory patients with Ewing sarcoma requires clarification. PATIENTS AND METHODS: Patients with relapsed or refractory Ewing sarcoma were randomly assigned (1:1) to either a shorter d × 5 schedule (irinotecan 50 mg/m2/d D1-5, vincristine 1.4 mg/m2 D1) or protracted d × 5×2 schedule (irinotecan 20 mg/m2/d D1-5,8-12, vincristine 1.4 mg/m2 D1,8) together with temozolomide (100 mg/m2/d D1-5). Patients were treated every 3 weeks for up to eight cycles until progression or unacceptable toxic effects occurred. The primary endpoint was objective response rate at 12 weeks (ORR12w). Secondary endpoints were progression-free survival (PFS), overall survival (OS), and safety. RESULT: A total of 46 patients presenting with relapsed or refractory Ewing sarcoma were randomly assigned to the d × 5 (n = 24) or d × 5×2 (n = 22) schedules. Median follow-up was 10.7 months in the d × 5 group and 8.3 months in the d × 5×2 group. ORR12w was lower for d × 5 (5/24; 20.8%) patients than for d × 5×2 (12/22; 54.5%; P = 0.019), but no significant difference was found in PFS (median PFS, 2.3 months for d × 5 vs. 4.3 months for d × 5×2) or OS (median OS, 14.8 months for d × 5 and 12.8 months for d × 5×2). Patients receiving the d × 5 schedule reported more grade 3 and 4 adverse events (AE) than those receiving d × 5×2, including diarrhea/abdominal pain and vomiting/nausea. CONCLUSIONS: The protracted d × 5×2 VIT schedule showed superior efficacy and favorable tolerability compared with the shorter d × 5 VIT schedule in patients with relapsed or refractory Ewing sarcoma.

Aberrant H3K4me3 modification of epiblast genes of extraembryonic tissue causes placental defects and implantation failure in mouse IVF embryos.

Assisted reproductive technology has been widely applied in the treatment of human infertility. However, accumulating evidence indicates that in vitro fertilization (IVF) is associated with a low pregnancy rate, placental defects, and metabolic diseases in offspring. Here, we find that IVF manipulation notably disrupts extraembryonic tissue-specific gene expression, and 334 epiblast (Epi)-specific genes and 24 Epi-specific transcription factors are abnormally expressed in extraembryonic ectoderm (ExE) of IVF embryos at embryonic day 7.5. Combined histone modification analysis reveals that aberrant H3K4me3 modification at the Epi active promoters results in increased expression of these genes in ExE. Importantly, we demonstrate that knockdown of the H3K4me3-recruited regulator Kmt2e, which is highly expressed in IVF embryos, greatly improves the development of IVF embryos and reduces abnormal gene expression in ExE. Our study therefore identifies that abnormal H3K4me3 modification in extraembryonic tissue is a major cause of implantation failure and abnormal placental development of IVF embryos.

Long-term outcome and relapse patterns in Ewing sarcoma patients with extensive lung/pleural metastases after a complete response to systemic therapy.

BACKGROUND: Ewing sarcoma (ES) is sensitive to systemic therapy, including chemotherapy and anti-angiogenesis Tyrosine Kinase Inhibitors(aaTKIs). However, the prognosis of patients with metastatic disease remains poor. Recurrence or distant metastasis after a complete response (CR) or near-CR due to systemic therapy is not rare. METHODS: We reviewed data from 187 ES patients between 2014-2019 treated at a single institute in China. Patients with extensive lung/pleural metastases (L/Pmeta) who had a CR or near-CR after first- or second-line chemotherapy with or without aaTKIs were retrospectively enrolled. Event-free survival (EFS) and overall survival (OS) were determined using the Kaplan-Meier method. For patients who had L/P recurrence, images were reviewed to define the exact location of each recurrent lesion, compared with the primary L/P lesion before chemotherapy and summarized as the relapse pattern. RESULTS: Seventeen patients and 21 cases of CR/nCR (5 by VDC/IE, 3 by VIT, and 13 by AVI) were finally analyzed. Median follow-up for surviving patients was 39.6 (range, 14.5-60.9) months. Median EFS and OS were 9.3 (95% confidence interval [CI], 2.0-16.6) months and 37.5 (95% CI, 21.8-53.1) months, respectively. The 2-year EFS was 19% and the 2-year OS was 70.6%, respectively. Most patients (82.4%) received whole lung irradiation (WLI). Lung/pleural relapse occurred in 71.4% (15/21) of CR/nCR cases. Most notably, all recurrent lesions exactly coincided with the original metastatic lesions before chemotherapy (exactly in situ) in 9 of the 15 recurrent cases, which was thus the major relapse pattern, whereas 42.9% had distant metastases other than L/Pmeta. CONCLUSIONS: Survival of ES patients with extensive L/Pmeta remains poor, even if they have a CR after systemic therapy. Recurrence exactly in situ is the major relapse pattern. WLI is not sufficient to prevent local recurrence in lung or pleura. More aggressive local treatment for metastatic lesions is warranted.

Hydration and Properties of Magnesium Potassium Phosphate Cement Modified by Granulated Blast-Furnace Slag: Influence of Fineness.

Magnesium potassium phosphate cement (MKPC) is an excellent rapid repair material for concrete, and many mineral admixtures have been applied to promote its performance. This study focuses on the quantitative characterization of the physical and chemical contributions of granulated blast-furnace slag with various finenesses to the performance development of MKPC. It was found that the addition of slag could increase the setting time, which is mainly due to the dilution of cement. Fine slag tends to decrease the fluidity of MKPC mortar. The physical contributions of ordinary and ultrafine slag to the early performance of MKPC mortar are 23% and 30%, while the chemical contributions are only 6%~10%. At late ages, the physical contribution is less than 10% and the chemical contribution of slag is even slightly negative. The addition of slag is beneficial to the compact packing of MKPC, which is the main reason for the physical contribution. Slag could react in the MKPC system, and increasing the fineness significantly promotes the reaction kinetics.

Apatinib plus ifosfamide and etoposide for relapsed or refractory osteosarcoma: A retrospective study in two centres.

For osteosarcoma that progresses following first-line chemotherapy, prognosis remains poor although anti-angiogenesis tyrosine kinase inhibitors (TKIs) have been verified to prolong progression-free survival. Apatinib has led to positive responses in the treatment of refractory osteosarcoma. However, it demonstrates only short-lived activity, and the disease control rate of musculoskeletal lesions is worse compared with that of pulmonary lesions. This treatment failure has been partly overcome by the addition of ifosfamide and etoposide (IE). The present study retrospectively compared the activity of apatinib + IE in relapsed or refractory osteosarcoma in two sarcoma centres in China. The included patients had received a combination of apatinib 500 mg (orally) daily and the IE regimen (n=33) between June 3 2017 and July 17 2020. The tumour burden was considerable in these patients: 16/33 (48.5%) Patients had lung and musculoskeletal lesions, and 31/33 (93.9%) patients had progressed to two lines of therapies at baseline. With a median follow-up duration of 28.4 [interquartile range (IQR), 16.1-38.3] months, 21/33 (63.6%) patients had objective responses, and the median event-free survival was 11.4 (IQR, 6.7-18.4) months. The median overall survival time was 19.8 (IQR, 13.1-30.6) months. At the last follow-up, 16/33 patients had tumour downstaging, and all lesions had been completely resected. For osteosarcoma with multiple sites of metastasis, apatinib + IE demonstrated clinically meaningful antitumor activity and delayed disease progression in patients with recurrent or refractory osteosarcoma after failure of chemotherapy. This combination with manageable toxicity deserves further investigation in prospective trials.

Complete remission of metastatic osteosarcoma using combined modality therapy: a retrospective analysis of unselected patients in China.

BACKGROUND: Complete surgical remission (CSR) is the best predictor of overall survival (OS) for patients with metastatic osteosarcoma. However, metastasectomy has not been widely implemented in China in the last decade due to various factors, and instead, most physicians choose hypofractionated radiotherapy to treat pulmonary lesions. This study aimed to retrospectively evaluate the outcomes of different local treatments for pulmonary lesions and identify the best local therapy strategies for these patients. METHODS: We reviewed the clinical courses of osteosarcoma patients with pulmonary metastases who were initially treated in two sarcoma centres in Beijing, China, from June 1st, 2009, to March 26th, 2020. With a median follow-up of 32.4 (95% confidence interval (CI): 30.8, 36.1) months, a total of 127 patients with 605 pulmonary nodules, all of whom had received local therapy and firstly achieved CSR or complete radiated/metabolic remission (CRR), were included in the analysis. A total of 102 patients with 525 nodules were initially diagnosed with resectable lung metastases, while 25 patients had 80 indeterminate nodules at presentation and relapsed with pulmonary metastases within 6 months after the completion of adjuvant chemotherapy. RESULTS: Eighty-eight of 127 (69.3%) patients had fewer than 5 nodules at the time of local therapy, with 48 of 127 (37.8%) located in the unilateral pleura. No patient underwent thoracotomy, and 42 of 127 patients (85 nodules) received video-assisted thoracoscopic surgery (VATS). In addition, 79 of 127 patients (520 nodules) received hypofractionated stereotactic body radiotherapy (RT), such as Gamma Knife radiosurgery or CyberKnife radiosurgery. The twelve-month event-free survival (EFS) (from local therapy to progression) rate of this entire study cohort was 35.6% (95% CI: 26.8, 44.4%), without a significant difference between the two groups (44.7% for VATS vs. 28.4% for RT, P = 0.755). Radiation-induced pneumonitis was observed in 62 of 86 (72.1%) patients, with one patient (1/86, 1.2%) in grade 4. CONCLUSIONS: Our past data showed a similar prognosis with the use of hypofractionated radiotherapy and VATS for the treatment of pulmonary metastasis and no inferiority to thoracotomy regarding historical outcomes. Currently, high-resolution chest computed tomography (CT) provides sufficient information on nodules, and less invasive modalities can thus be considered for treatment.

Anlotinib, Vincristine, and Irinotecan for Advanced Ewing Sarcoma After Failure of Standard Multimodal Therapy: A Two-Cohort, Phase Ib/II Trial.

BACKGROUND: Both protracted irinotecan and antiangiogenesis therapy have shown promising efficacy against Ewing sarcoma (EWS). METHODS: Patients diagnosed with recurrent or refractory EWS were enrolled and further categorized into cohort A (≥16 years) or cohort B (<16 years). In the dose-defining phase Ib portion, anlotinib was given daily at a fixed dose, while a 3+3 design with dose de-escalation was used to determine the dose of irinotecan. The next dose-expanding phase II portion employed a conventional two-stage study design model. The primary endpoint was objective response rate at 12 weeks (ORR12w ). RESULTS: A total of 41 patients finally received the treatment regimen, including 29 in cohort A and 12 in cohort B. For cohort A, the first five patients were treated at the initial level of 20 mg/m2 /d d × 5 × 2, and two of them subsequently a dose-limiting toxicity (DLT). An additional six patients were then treated at 15 mg/m2 without any DLT, and the RP2D was determined. Notably, 23 out of 24 patients in cohort A were available for response evaluation at 12 weeks. ORR12w was determined to be 62.5%. For cohort B, no DLT was observed in the first six patients at the initial dose level. At last, 12 patients were included in cohort B. The ORR12w was 83.3%. The most frequently observed grade 3/4 adverse events were leukopenia (28.5%), neutropenia (24.4%), anemia (8.7%), and diarrhea (3.7%). CONCLUSION: The combination of vincristine, irinotecan, and anlotinib demonstrated an acceptable toxicity profile and promising clinical efficacy in patients with advanced EWS. IMPLICATIONS FOR PRACTICE: This is the first trial to evaluate an irinotecan-based regimen in combination with antiangiogenesis tyrosine kinase inhibitors in Ewing sarcoma (EWS). A 3+3 design with dose de-escalation was used to determine the most appropriate dose of irinotecan in each cohort. The next dose-expanding phase II portion employed a conventional two-stage study design model. The objective response rate was 62.5% for adults and 83.3% for children. Median overall survival was not matured. This study shows that the combination of vincristine, irinotecan, and anlotinib demonstrates an acceptable toxicity profile and promising clinical efficacy in patients with advanced EWS.

Apatinib for Treatment of Inoperable Metastatic or Locally Advanced Chondrosarcoma: What We Can Learn About the Biological Behavior of Chondrosarcoma from a Two-Center Study.

PURPOSE: For patients who have chondrosarcoma in the unresectable setting, antiangiogenic agents are reportedly effective. This multicenter, retrospective study investigated the antitumor activity of apatinib in patients with unresectable chondrosarcoma to gain insight into the biological behavior of this disease. METHODS: All of the patients with unresectable chondrosarcoma who were diagnosed between October 1, 2009, and November 1, 2019, in two sarcoma centers affiliated with Peking University were evaluated. Relevant information was collected from the medical records at both centers, from which patients receiving apatinib for systemic therapy were selected for analysis. RESULTS: In total, efficacy analysis was conducted in 33 patients with a median follow-up time of 22.1 (Q1, Q3, 14.6, 23.0) months. There were 20/33 (60.0%) conventional chondrosarcomas (grades 2-3), 5/33 (15.2%) dedifferentiated chondrosarcomas, 4/33 (12.1%) mesenchymal chondrosarcomas, 3/33 (9.1%) extraskeletal myxoid chondrosarcoma, and 1/33 (3.1%) clear-cell chondrosarcomas with 87.9% in metastatic and 12.1% in locally advanced states. The objective response rate was 6/33 (18.2%). The median progression-free survival (PFS) was 12.4 months (Q1, Q3, 7.0, 21.2), while the median overall survival has not yet been reached. Rare variants of chondrosarcoma tended to have a longer PFS than conventional chondrosarcoma (P=0.06). Based on clinicopathological factors Cox and univariate analysis, only extraskeletal myxoid chondrosarcoma and baseline target lesions <60 mm benefited from the drug apatinib (P=0.14 and P=0.00), respectively. Grade 3 or higher adverse events were frequent in 11/33 (39.3%) of patients who discontinued apatinib due to deterioration of their general condition. CONCLUSION: Apatinib had clinically meaningful activity in patients with inoperable high-grade chondrosarcoma. However, special caution should be made in managing toxicity due to the indolent behavior and slow growth pattern after using this drug. Patients with a smaller tumor size and extraskeletal myxoid chondrosarcoma subtype might benefit from this therapy more. CLINICAL TRIAL REGISTRATION: Registered February 7, 2020, with clinicaltrials.gov: NCT04260113.

Apatinib plus camrelizumab (anti-PD1 therapy, SHR-1210) for advanced osteosarcoma (APFAO) progressing after chemotherapy: a single-arm, open-label, phase 2 trial.

BACKGROUND: Results of our previous study showed high objective response but short-term activity of apatinib in advanced osteosarcoma. We aimed to investigate the activity of apatinib in combination with camrelizumab in patients with inoperable high-grade osteosarcoma progressing after chemotherapy. METHODS: This open-label, phase 2 trial was conducted at Peking University People's Hospital. We enrolled patients with advanced osteosarcoma progressed after chemotherapy. Patients received 500 mg apatinib orally once daily plus 200 mg camrelizumab by intravenous infusion every 2 weeks until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS) and clinical benefit rate at 6 months, which were based on RECIST V.1.1. RESULTS: 43 patients were enrolled between January 25 and September 4, 2018. With median follow-up time of 48.3 (Q1, Q3, 30.6, 66.6) weeks, 13 (30.23%, 95% CI 17.2%, 40.1%) of 43 patients were progression free at 6 months and the 6-month PFS rate was 50.9% (95% CI 34.6%, 65.0%). Until final follow-up, the objective response rate was 20.9% (9/43) and two patients with durable disease control were observed. Patients with programmed cell death 1 ligand-1 (PD-L1) tumor proportion score ≥5% and pulmonary metastases tended to have a longer PFS in comparison to the others (p=0.004 and 0.017, respectively). Toxic effects led to dose reductions, or interruptions, or both in 24 (55.8%) of 43 patients and permanent discontinuation in 4 (9.3%) patients. There were no treatment-related deaths. CONCLUSIONS: Although the combination of apatinib and camrelizumab seemed to prolong PFS in comparison to single agent apatinib in treating advanced osteosarcoma, it did not reach the prespecified target of 6-month PFS of 60% or greater. Overexpression of PD-L1 and the presence of pulmonary metastases only were associated with longer PFS. TRIAL REGISTRATION NUMBER: NCT03359018.

The Clinical Implications of Tumor Mutational Burden in Osteosarcoma.

BACKGROUND: Osteosarcoma (OTS) is aggressive bone malignancy without well-recognized prognosis biomarker. Tumor mutational burden (TMB) has been proved as effective biomarker in predicting clinical outcomes in several cancer types. However, its prognostic value in OTS remains unknown. In this study, we aim to evaluate the implication of TMB in OTS patients. METHODS: To depict the landscape of somatic mutations in OTS, we performed Whole-Exome Sequencing (WES) on 31 OTS tissue samples and corresponding White Blood Cells (WBCs) as matched control. TMB was calculated as the total number of somatic alterations in coding regions normalized to the per sequenced genomic megabase (~30.4Mb in WES). The prognostic values of TMB were evaluated by Kaplan-Meier methods and Cox regression models. RESULTS: The median age was 16.0 years at diagnosis, and 54.8% of patients were male. The most common genetic alterations were mainly involved in cell cycle and DNA damage response and repair, including H3F3A, TP53, MYC, and CDKN2A/B. The median progression-free survival (PFS) was 775.5 days in TMB-High (defined as third quartile of TMB value, <2.565) versus 351 days in TMB-Low (<2.565). All patients with TMB-High are PFS-Long (>400 days), while 36.4% of all patients with TMB-Low were PFS-Long (P=0.003). TMB were significantly greater in PFS-Long than in PFS-Short (<400 days) (P=0.002). Moreover, the median overall survival (OS) was 1,307 days in TMB-High versus 672.5 days in TMB-Low. Furthermore, TMB-High group had significantly improved PFS (P=0.04) and OS (P=0.03). CONCLUSIONS: TMB-High can be used as prognostic marker for OTS. Our findings demonstrate that TMB may be helpful in combination with traditionally clinicopathologic risk factors to optimize risk stratification and guide treatment decisions.

Osteosarcoma cell intrinsic PD-L2 signals promote invasion and metastasis via the RhoA-ROCK-LIMK2 and autophagy pathways.

Known as co-stimulatory molecule, programmed death ligand-2 (PD-L2) contributes to T-cell exhaustion by interaction with programmed death-1 (PD-1) receptor, but its tumor cell-intrinsic signal effects have been little investigated. PD-L2 expression was detected by immunohistochemistry in 18 pairs of primary osteosarcoma tissues and matching lung metastasis tissues. We also investigated the effects of PD-L2 knockdown on osteosarcoma both in vitro and in vivo. In our study, PD-L2 expression was elevated in lung metastases compared with primary osteosarcoma according to an immunohistochemistry assay. Wound-healing and transwell assays revealed that PD-L2 knockdown  leaded to inhibition of migration and invasion of human osteosarcoma cells in vitro. Mechanistically, we demonstrated that PD-L2 knockdown attenuated migration and invasion by inactivating RhoA-ROCK-LIMK2 signaling, suppressing epithelial-mesenchymal transition (EMT), and inhibiting autophagy by decreasing beclin-1 expression. In support of these observations, beclin-1 knockdown also inhibited activation of the RhoA-ROCK-LIMK2 pathway, leading to autophagy inhibition-induced blockade of migration and invasion. Depletion of PD-L2 in KHOS cells markedly weakens pulmonary metastatic potential in vivo by orthotopic transplantation of nude mice. Our study reveals a pro-metastatic functional mechanism for PD-L2 in osteosarcoma. Furthermore, we demonstrate a regulatory role for PD-L2 on autophagy, as well as a relationship between autophagy and metastasis in osteosarcoma, which may represent a potential therapeutic target for osteosarcoma.

Tumor-associated macrophages promote lung metastasis and induce epithelial-mesenchymal transition in osteosarcoma by activating the COX-2/STAT3 axis.

Osteosarcoma (OS) is a common, malignant musculoskeletal tumor in young people. Neoadjuvant chemotherapy has improved the survival of osteosarcoma patients but with limited benefit due to metastasis. Tumor-associated macrophages (TAMs) are involved in various mechanisms of tumor biology, which include oncogenesis, drug resistance, and tumor immune escape, as well as tumor metastasis. In this study, we proved that TAMs possess the ability to promote OS cell migration and invasion by upregulating COX-2, MMP9, and phosphorylated STAT3 and to induce the epithelial-mesenchymal transition (EMT). This evidence has also been verified in a tumor-bearing animal model, and in OS patients. Furthermore, we observed the anti-metastasis effect of COX-2 inhibition by repressing COX-2 expression, EMT-activating transcription factors and the STAT3 pathway, both in vitro and in vivo. We propose that TAMs promote OS metastasis and invasion by activating the COX-2/STAT3 axis and EMT. These findings suggest that TAMs and COX-2 may be potential targets for future anti-metastasis therapy.

Novel oncogene COPS3 interacts with Beclin1 and Raf-1 to regulate metastasis of osteosarcoma through autophagy.

BACKGROUND: Expression of COP9 signalosome subunit 3 (COPS3), an oncogene overexpressed in osteosarcoma, has been demonstrated to be significantly correlated with tumor metastasis. However, the underlying mechanism by which COPS3 promotes metastasis of osteosarcoma and its role in autophagy remain unknown. METHODS: The expression of COPS3 was detected in primary osteosarcoma tissues and matching lung metastasis tissues by immunohistochemistry (IHC). The effect of COPS3 on the metastasis of osteosarcoma cells was investigated by transwell, wound healing assays and animal studies. Indicated proteins was analyzed by western blotting when COPS3 was knockdown or overexpressed. The COPS3 Interacting protein was determined by immunoprecipitation assay. The relationship between COPS3 and autophagy was detected by western blotting and immunofluorescence. RESULTS: We found that knockdown of COPS3 significantly reduced the lung metastasis of osteosarcoma cells in a mouse model, coinciding with downregulation of mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling. The silencing of COPS3 also inhibited the epithelial-mesenchymal transition (EMT) through the 90 kDa ribosomal S6 kinases (RSK), a family of signal transduction proteins downstream of MEK/ERK. Reciprocal immunoprecipitation assays revealed that COPS3 directly interacts with Raf-1, an upstream regulator of MEK/ERK. Surprisingly, Beclin1, an important autophagic protein, appeared in the COPS3-immunoprecipitates, along with the autophagic markers LC3-I and LC3-II. Loss of COPS3 completely inhibited H2O2-induced autophagic flux and reduced Beclin1 expression. Additionally, autophagy inhibitor or silencing of Beclin1 both decreased cell metastasis. CONCLUSIONS: Taken together, these data reveal a novel function of COPS3 in the regulation of autophagy and highlight the relationship between autophagy and metastasis in osteosarcoma cells.

Correction to: PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.

The original article [1] contained an error in Table 1 whereby the 'Positive' column in the 'PD-L1' Tumor type group of columns was mistakenly included at the beginning of the 'PD-L2' Tumor type group of columns.

PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.

BACKGROUND: Immune checkpoint inhibitors have led to a breakthrough in solid tumor immunotherapy, but related studies on musculoskeletal tumors are few, especially for PD-L2. METHODS: We examined expression of three molecular effectors of the PD-1 axis in 234 patients with musculoskeletal tumors, including osteosarcoma, chondrosarcoma, synovial sarcoma, and giant cell tumor. Survival analyses and potential mechanisms were investigated in osteosarcoma per the Gene Expression Omnibus (GEO) and immunohistochemistry analyses. In vivo, humanized mice were used to evaluate the effect of nivolumab on osteosarcoma. RESULTS: PD-L1, PD-L2, and PD-1 expression levels were significantly different between the histologic types of the musculoskeletal tumors. For osteosarcoma, PD-L1 was negatively correlated with prognosis, while PD-1 had a negative correlation tendency with overall survival (OS). Meanwhile, PD-L2 had a positive correlation trend with OS. Nivolumab inhibited osteosarcoma metastasis in humanized mice by increasing CD4+ and CD8+ lymphocytes and the cytolytic activity of CD8 lymphocytes in the lung but did not affect primary osteosarcoma growth. CONCLUSION: We systematically detected the expression patterns of PD-L1, PD-L2, and PD-1 in musculoskeletal tumors for the first time and demonstrated the prognostic roles and underlying mechanisms of PD-1 axis in osteosarcoma. Furthermore, PD-1 blockade could effectively control osteosarcoma pulmonary metastasis in vivo. Therefore, the PD-1 axis may be a potential immunotherapeutic target for metastatic osteosarcoma.