A pilot study of multi-antigen stimulated cell therapy-I plus camrelizumab and apatinib in patients with advanced bone and soft-tissue sarcomas.

BACKGROUND: Cell-based  immunotherapy shows the therapeutic potential in sarcomas, in addition to angiogenesis-targeted tyrosine kinase inhibitor (TKI) and immune checkpoint inhibitor (ICI). Multi-antigen stimulated cell therapy-I (MASCT-I) technology is a sequential immune cell therapy for cancer, which composes of multiple antigen-loaded dendritic cell (DC) vaccines followed by the adoptive transfer of anti-tumor effector T-cells. METHODS: In this phase 1 study, we assessed MASCT-I plus camrelizumab (an ICI against PD-1) and apatinib (a highly selective TKI targeting VEGFR2) in patients with unresectable recurrent or metastatic bone and soft-tissue sarcoma after at least one line of prior systemic therapy. One MASCT-I course consisted of 3 DC subcutaneous injections, followed by 3 active T cell infusions administered 18-27 days after each DC injection. In schedule-I group, 3 DC injections were administered with a 28-day interval in all courses; in schedule-II group, 3 DC injections were administered with a 7-day interval in the first course and with a 28-day interval thereafter. All patients received intravenous camrelizumab 200 mg every 3 weeks and oral apatinib 250 mg daily. RESULTS: From October 30, 2019, to August 12, 2021, 19 patients were enrolled and randomly assigned to schedule-I group (n = 9) and schedule-II group (n = 10). Of the 19 patients, 11 (57.9%) experienced grade 3 or 4 treatment-related adverse events. No treatment-related deaths occurred. Patients in schedule-II group showed similar objective response rate (ORR) with those in schedule-I group (30.0% versus 33.3%) but had higher disease control rate (DCR; 90.0% versus 44.4%) and longer median progression-free survival (PFS; 7.7 versus 4.0 months). For the 13 patients with soft-tissue sarcomas, the ORR was 30.8%, DCR was 76.9%, and median PFS was 12.9 months; for the 6 patients with osteosarcomas, the ORR was 33.3%, the DCR was 50.0%, and median PFS was 5.7 months. CONCLUSIONS: Overall, MASCT-I plus camrelizumab and apatinib was safe and showed encouraging efficacy in advanced bone and soft-tissue sarcoma, and schedule-II administration method was recommended. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04074564.

D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment.

The effect of immunotherapy is limited by oncometabolite D-2-hydroxyglutarate (D2HG). D2HGDH is an inducible enzyme that converts D2HG into the endogenous metabolite 2-oxoglutarate. We aimed to evaluate the impairment of CD8 T lymphocyte function in the high-D2HG environment and to explore the phenotypic features and anti-tumor effect of D2HGDH-modified CAR-T cells. D2HG treatment inhibited the expansion of human CD8 T lymphocytes and CAR-T cells, increased their glucose uptake, suppressed effector cytokine production, and decreased the central memory cell proportion. D2HGDH-modified CAR-T cells displayed distinct phenotypes, as D2HGDH knock-out (KO) CAR-T cells exhibited a significant decrease in central memory cell differentiation and intracellular cytokine production, while D2HGDH over-expression (OE) CAR-T cells showed predominant killing efficacy against NALM6 cancer cells in high-D2HG medium. In vivo xenograft experiments confirmed that D2HGDH-OE CAR-T cells decreased serum D2HG and improved the overall survival of mice bearing NALM6 cancer cells with mutation IDH1. Our findings demonstrated that the immunosuppressive effect of D2HG and distinct phenotype of D2HGDH modified CAR-T cells. D2HGDH-OE CAR-T cells can take advantage of the catabolism of D2HG to foster T cell expansion, function, and anti-tumor effectiveness.

Impact of Secondary Aneurysmal Bone Cysts on Survival of Patients with Enneking Stage IIB Extremity Osteosarcoma: A Propensity Score Matching Analysis.

BACKGROUND: The clinicopathology of aneurysmal bone cysts (ABCs) secondary to osteosarcoma has not yet been reported. We conduct a retrospective review of ABCs secondary to osteosarcoma to characterize clinicopathology and influence on the survival of patients with Enneking stage IIB extremity osteosarcoma. PATIENTS AND METHODS: A total of 300 patients with Enneking stage IIB extremity osteosarcoma were eligible for analysis. These cases were divided, according to the pathology of biopsy and magnetic resonance imaging (MRI), into ABCs group and no ABCs group. Patients (ABCs versus no ABCs) were compared using a 1:2 propensity score analysis to best match between groups. Clinicopathology and survival data were analyzed. RESULTS: The total occurrence rate of secondary ABCs was 10.3%. A higher prevalence of pathological fractures was observed in the ABCs group (22.6%) compared with the no ABCs group (8.6%) (p = 0.032). Patients with ABCs were more likely to undergo amputation compared with patients without ABCs (p = 0.007). Those with secondary ABCs had poorer response to chemotherapy before and after propensity score matching (p = 0.006 and p = 0.048, respectively). Kaplan-Meier survival analysis showed that EFS and OS distributions were not significantly different between the two patient groups. ABCs were not significantly different in terms of EFS or OS in the multivariate analysis model (p > 0.05). CONCLUSIONS: The presence of secondary ABCs was associated with increased occurrence rate of pathological fracture and high percentage of amputation. Moreover, patients with secondary ABCs had poorer response to chemotherapy. However, the presence of secondary ABCs did not influence survival of patients with Enneking stage IIB extremity osteosarcoma.

Clinical significance of indeterminate pulmonary nodules on the survival of 364 patients with nonmetastatic, high-grade, localized osteosarcoma: A 12-year retrospective cohort study.

BACKGROUND AND OBJECTIVES: We investigated the clinical significance of indeterminate pulmonary nodules (IPNs) in patients diagnosed with nonmetastatic, high-grade localized osteosarcoma. METHODS: We retrospectively analyzed the clinical data of 364 patients with nonmetastatic, high-grade localized osteosarcoma. Based on pulmonary computed tomography findings at presentation, the patients were categorized into the no-nodules and the IPNs group and were further categorized into subgroups based on age (<18 and ≥18 years). We performed an intergroup comparison of event-free survival (EFS) and overall survival (OS). RESULTS: At presentation, 276 (75.8%) patients showed no nodules, and 88 (24.2%) patients showed IPNs. The EFS and OS were similar between adults with IPNs (n = 54 [30.5%]) and without nodules (n = 123 [69.5%]) (p = .200 and p = .609, respectively). No significant intergroup difference in OS was observed in pediatric patients (p = .093). However, pediatric patients with IPNs (n = 34 [18.2%]) had poorer EFS than those without nodules (n = 153 [81.8%]) (p = .016). Multivariate analyses confirmed that IPNs were independently associated with poorer EFS in pediatric patients (hazard ratio 1.788, 95% confidence interval 1.092-2.926, p = .021). CONCLUSIONS: This study showed that IPNs at presentation did not affect the survival of adults with nonmetastatic, high-grade localized osteosarcoma but were associated with poorer EFS in pediatric patients.

Safety and efficacy of fruquintinib-based therapy in patients with advanced or metastatic sarcoma.

BACKGROUND: The purpose of this study was to evaluate the efficacy and safety of fruquintinib-based therapy as a salvage therapy for patients with advanced or metastatic sarcoma, including soft tissue sarcoma (STS) and bone sarcoma. METHODS: Patients with advanced or metastatic sarcoma were divided into two groups. One group received fruquintinib monotherapy, while the other received fruquintinib combined therapy. Safety and efficacy of fruquintinib-based therapy were recorded and reviewed retrospectively, including progression-free survival (PFS), overall response rate (ORR), and adverse events (AEs). RESULTS: Between August 2021 and December 2022, 38 sarcoma patients were retrospectively included. A total of 14 patients received fruquintinib alone (including 6 STS and 8 bone sarcoma), while 24 were treated with fruquintinib combined therapy (including 2 STS and 22 bone sarcoma). The median follow-up was 10.2 months (95% CI, 6.4-11.5). For the entire population, the median PFS was 8.0 months (95% CI, 5.5-13.0). The ORR was 13.1%, while the disease control rate (DCR) was 86.8%. The univariate analysis showed that radiotherapy history (HR, 4.56; 95% CI, 1.70-12.24; p = 0.003), bone sarcoma (HR, 0.34; 95% CI, 0.14-0.87; p = 0.024), and treatment method of fruquintinib (HR, 0.36; 95% CI, 0.15-0.85; p = 0.021) were significantly associated with PFS. The multivariate analysis showed that patients without radiotherapy history were associated with a better PFS (HR, 3.71; 95% CI: 1.31-10.55; p = 0.014) than patients with radiotherapy history. Patients in combination group reported pneumothorax (8.3%), leukopenia (33.3%), thrombocytopenia (12.5%), diarrhea (4.2%), and anemia (4.2%) as the most frequent grade 3 or higher treatment-emergent AEs (TEAEs), while there was no severe TEAEs occurred in the monotherapy group. CONCLUSIONS: Fruquintinib-based therapy displayed an optimal tumor control and an acceptable safety profile in patients with advanced or metastatic sarcoma.

Oncometabolite D-2-hydroxyglutarate-dependent metabolic reprogramming induces skeletal muscle atrophy during cancer cachexia.

Cancer cachexia is characterized by weight loss and skeletal muscle wasting. Based on the up-regulation of catabolism and down-regulation of anabolism, here we showed genetic mutation-mediated metabolic reprogramming in the progression of cancer cachexia by screening for metabolites and investigating their direct effect on muscle atrophy. Treatment with 93 µM D-2-hydroxyglutarate (D2HG) resulted in reduced myotube width and increased expression of E3 ubiquitin ligases. Isocitrate Dehydrogenase 1 (IDH1) mutant patients had higher D2HG than non-mutant patients. In the in vivo murine cancer cachexia model, mutant IDH1 in CT26 cancer cells accelerated cachexia progression and worsened overall survival. Transcriptomics and metabolomics revealed a distinct D2HG-induced metabolic imbalance. Treatment with the IDH1 inhibitor ivosidenib delayed the progression of cancer cachexia in murine GL261 glioma model and CT26 colorectal carcinoma models. These data demonstrate the contribution of IDH1 mutation mediated D2HG accumulation to the progression of cancer cachexia and highlight the individualized treatment of IDH1 mutation associated cancer cachexia.

First-in-Maintenance Therapy for Localized High-Grade Osteosarcoma: An Open-Label Phase I/II Trial of the Anti-PD-L1 Antibody ZKAB001.

PURPOSE: We investigated the safety and preliminary efficacy of anti-PD-L1 antibody (ZKAB001) as maintenance therapy for localized patients with high-grade osteosarcoma to reduce the risk of recurrence and metastasis. PATIENTS AND METHODS: This open-label Phase I/II study was divided into dose-escalation Phase I and expansion Phase II. Phase I used a 3+3 design with ZKAB001 at three escalating doses ranging: 5, 10, 15 mg/kg every 2 weeks in 9 patients with localized high-grade osteosarcoma and Phase II tested 10 mg/kg in 12 patients for up to 24 cycles. Primary endpoints were safety and tolerability assessed using CTCAE4.0.3. RESULTS: Between October 2018 and 2019, 21 eligible patients were enrolled and accepted ZKAB001 treatment: 9 in the dose-escalation phase, and 12 in expansion phase. Six patients with disease progression withdrew from this study and follow-up is ongoing. The MTD was not defined in Phase I. All doses had a manageable safety profile. The recommended dose in Phase II was set at 10 mg/kg. Most frequent immune-related adverse events were thyroiditis (76.2%) and dermatitis (42.9%). Only 1 (4.8%) of 21 patients had a Grade 3 skin rash. The median 3-year event-free survival (EFS) and overall survival (OS) were not established; however, 24-month EFS was 71.4% (95% confidence interval, 47.2-86.0) and 2-year OS was 100%. Preliminary efficacy data showed EFS benefits in patients with PD-L1 positive or an MSI-H sub-population. CONCLUSIONS: Switching to maintenance using ZKAB001 showed an acceptable safety profile and provided preliminary evidence of clinical activity in localized patients with osteosarcoma.

Pirarubicin inhibits multidrug-resistant osteosarcoma cell proliferation through induction of G2/M phase cell cycle arrest.

AIM: Pirarubicin (THP) is recently found to be effective in treating patients with advanced, relapsed or recurrent high-grade osteosarcoma. In this study, the effects of THP on the multidrug-resistant (MDR) osteosarcoma cells were assessed, and the underlying mechanisms for the disruption of cell cycle kinetics by THP were explored. METHODS: Human osteosarcoma cell line MG63 and human MDR osteosarcoma cell line MG63/DOX were tested. The cytotoxicity of drugs was examined using a cell proliferation assay with the Cell Counting Kit-8 (CCK-8). The distribution of cells across the cell cycle was determined with flow cytometry. The expression of cell cycle-regulated genes cyclin B1 and Cdc2 (CDK1), and the phosphorylated Cdc2 and Cdc25C was examined using Western blot analyses. RESULTS: MG63/DOX cells were highly resistant to doxorubicin (ADM) and gemcitabine (GEM), but were sensitive or lowly resistant to THP, methotrexate (MTX) and cisplatin (DDP). Treatment of MG63/DOX cells with THP (200-1000 ng/mL) inhibited the cell proliferation in time- and concentration-dependent manners. THP (50-500 ng/mL) induced MG63/DOX cell cycle arrest at the G(2)/M phase in time- and concentration-dependent manners. Furthermore, the treatment of MG63/DOX cells with THP (200-1000 ng/mL) downregulated cyclin B1 expression, and decreased the phosphorylated Cdc2 at Thr(161). Conversely, the treatment increased the phosphorylated Cdc2 at Thr(14)/Tyr(15) and Cdc25C at Ser(216), which led to a decrease in Cdc2-cyclin B1 activity. CONCLUSION: The cytotoxicity of THP to MG63/DOX cells may be in part due to its ability to arrest cell cycle progression at the G(2)/M phase, which supports the use of THP for managing patients with MDR osteosarcoma.

Surgical Management of Cardiac Masses in Right Atrium Among Bone Sarcoma Pediatric Patients With Totally Implanted Ports.

Introduction: Totally implanted ports (PORTs) have been widely used among patients with malignancy. Cardiac metastasis secondary to bone sarcoma and catheter-related right atrial thrombosis (CRAT) can be both present as cardiac masses. However, these two cardiac masses share very similar imaging characteristics. Methods: The features, treatments, and outcomes of 5 bone sarcoma pediatric patients with PORTs who suffered from cardiac masses in the right atrium were analyzed. Clinical data and histological characteristics of cardiac masses were also recorded. Results: Among 928 patients with malignancy and PORTs, 5 bone sarcoma pediatric patients were found to have cardiac masses in the right atrium. The catheter tips were located in the right atrium of 4 patients and the superior vena cava-right atrium junction (CAJ) of 1 patient. Four patients with good response to anti-tumor treatment had received surgical lumpectomies for pathologic identification and mass excision, with cardiac metastases among 1 patient and thromboses among 3 patients. The median time from venous access port implantation to cardiac mass detection for CRAT was 6.3 months (range: 4.7-6.8 months) and to diagnosis of or possible cardiac metastasis was 13.3 months (range: 11.2-15.4 months). Conclusion: The placement of a catheter tip into the right atrium should be avoided. The time from PORTs implantation to cardiac mass detection might serve as a potential tool to differentiate cardiac metastasis from CRAT. Surgical management may be an effective treatment for bone sarcoma pediatric patients who had good response to anti-tumor treatment and suffered from cardiac masses in the right atrium.

FAT1 and MSH2 Are Predictive Prognostic Markers for Chinese Osteosarcoma Patients Following Chemotherapeutic Treatment.

Osteosarcoma is characterized by diverse genetic mutations, including single-nucleotide variants (SNVs), which can complicate clinical outcomes of the treatment. This study identified key mutations or polymorphisms in genes that correlate with osteosarcoma prognoses. A total of 110 patients with osteosarcoma were assigned to "good" or "poor" cohorts depending on their 5-year disease-free survival (DFS) after surgery and chemotherapeutic treatment. We performed next-generation sequencing analysis of tumor tissues for prognosis-associated SNVs in 315 tumorigenesis-related genes, followed by modeling of clinical outcomes for these patients using random forest classification via a support vector machine (SVM). Data from the Chinese Millionome Database were used to compare SNV frequency in osteosarcoma patients and healthy people. SVM screening identified 17 nonsynonymous SNVs located in 15 genes, of which rs17224367 and rs3733406 (located in MSH2 and FAT1, respectively) were strongly correlated with osteosarcoma prognosis. These results were verified in a 26-patient validation cohort, confirming that these SNVs could be used to predict prognosis. These results demonstrated that two SNVs located in MSH2 and FAT1 are associated with prognosis of osteosarcoma patients. © 2022 American Society for Bone and Mineral Research (ASBMR).

Case Report: Sequential Chemotherapy and Immunotherapy Produce Sustained Response in Osteosarcoma With High Tumor Mutational Burden.

Background: Immunotherapy has provided an effective method for the treatment of many cancers. However, its efficacy in osteosarcoma is not satisfactory so far. Case Presentation: Here, we presented a case of osteosarcoma treated with sequential chemotherapy and immunotherapy and showed promising therapeutic potential. The 29-year-old female patient presented 9th rib osteosarcoma with suspected right lung lower lobe metastasis. Surgery was performed to remove the primary lesion, and a series of chemotherapies were given afterward in consideration of the response and tolerance. The right lung lower lobe metastasis was under control first but progressed (PD) 9 months after the initiation of therapy. The lesion was surgically removed and subsequent chemotherapy was implemented. The patient had good tolerance with chemotherapy and maintained well for approximately 11 months before the discovery of 11th rib and right lung upper lobe metastases. Surgery was then performed on both lesions and achieved complete response. Post-surgical brief chemotherapy and subsequent long-term immunotherapy (pembrolizumab) maintained continuous remission for 33 months. The patient survived for 60 months with well-controlled disease from the time of confirmed diagnosis. Genetic alterations of all primary and metastatic lesions were investigated by whole-exome sequencing (WES). Substantial similarity in mutational landscape between the primary lesion and 11th rib metastasis and between the two lung metastases were revealed, while substantial heterogeneity was found between the rib lesions and lung metastases. The tumor mutational burden (TMB) for the 9th rib primary lesion, the metastatic 11th rib lesion, and the metastatic right upper and lower lobe nodule tissues was 8.02, 2.38, 4.61, and 0.14 mutations/Mb, respectively. The primary lesion exhibited the most diverse copy number variation (CNV) changes among all lesions. Furthermore, pathway enrichment analysis also suggested significant heterogeneity among the lesions. Conclusions: Surgery with sequential chemotherapy and maintenance immunotherapy was shown to have good response for the first time on osteosarcoma patient who had high TMB tumor lesions and good tolerance for chemotherapy and immunotherapy.

P4HA2-induced prolyl hydroxylation suppresses YAP1-mediated prostate cancer cell migration, invasion, and metastasis.

Yes-associated protein 1 (YAP1), a key player in the Hippo pathway, has been shown to play a critical role in tumor progression. However, the role of YAP1 in prostate cancer cell invasion, migration, and metastasis is not well defined. Through functional, transcriptomic, epigenomic, and proteomic analyses, we showed that prolyl hydroxylation of YAP1 plays a critical role in the suppression of cell migration, invasion, and metastasis in prostate cancer. Knockdown (KD) or knockout (KO) of YAP1 led to an increase in cell migration, invasion, and metastasis in prostate cancer cells. Microarray analysis showed that the EMT pathway was activated in Yap1-KD cells. ChIP-seq analysis showed that YAP1 target genes are enriched in pathways regulating cell migration. Mass spectrometry analysis identified P4H prolyl hydroxylase in the YAP1 complex and YAP1 was hydroxylated at multiple proline residues. Proline-to-alanine mutations of YAP1 isoform 3 identified proline 174 as a critical residue, and its hydroxylation suppressed cell migration, invasion, and metastasis. KO of P4ha2 led to an increase in cell migration and invasion, which was reversed upon Yap1 KD. Our study identified a novel regulatory mechanism of YAP1 by which P4HA2-dependent prolyl hydroxylation of YAP1 determines its transcriptional activities and its function in prostate cancer metastasis.

TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1.

BACKGROUND: Tripartite motif-containing proteins (TRIM) play a crucial role in carcinogenesis. Little attention has been focused on the possible functions of TRIM6 on carcinogenesis. METHODS: The expression levels of TRIM6 were assessed in colorectal cancer (CRC) samples. TRIM6 expression was knocked down in CRC cell lines, and subjected to Cell counting kit-8 (CCK-8), bromodeoxyuridine (BrdU) incorporation and cell cycle assays. Immunoprecipitation and proteomics analysis was performed to identify potential associated proteins of TRIM6. RESULTS: TRIM6 expression was up-regulated in CRC samples and TRIM6 expression may be an independent prognostic marker for CRC. Knocking down TRIM6 expression suppressed CRC cell proliferation, induced cell cycle arrested at G2/M phase and increased sensitivity to 5-fluorouracil and oxaliplatin. TIS21, an anti-proliferative protein involved in the regulation of G2/M arrest, was identified as an interaction partner of TRIM6. Moreover, CRC cells with TRIM6 overexpression showed decreased TIS21 protein stability. TIS21 ubiquitination was increased in CRC cells overexpressing TRIM6, but not in those overexpressing TRIM6 E3 catalytic mutant (C15A). Further, Lys5 was essential for TRIM6 mediated TIS21 ubiquitination. TIS21 overexpression reversed the induced effects of TRIM6 overexpression on CRC cell proliferation, and the levels of forkhead box M1 (FoxM1), phosphorylated FoxM1, Cyclin B1 and c-Myc. Thiostrepton, a specific inhibitor for FoxM1, was less effective in anti-proliferative activity against CRC cells with lower level of TRIM6 in vitro and in vivo. CONCLUSIONS: Our study suggests that TRIM6 promotes the progression of CRC via TIS21/FoxM1.

N6-Methyladenosine modification of the TRIM7 positively regulates tumorigenesis and chemoresistance in osteosarcoma through ubiquitination of BRMS1.

BACKGROUND: Metastasis is the leading cause of death in patients with osteosarcoma. Some of these patients fail to respond to chemotherapy and die of metastasis within a short period. Therefore, it is important to identify novel biomarkers to improve the diagnosis and treatment of osteosarcoma. TRIM7 is a member of the tripartite motif (TRIM) family protein that is involved in various pathological conditions including cancer; however, its role in osteosarcoma remains elusive. METHODS: Cell proliferation, invasion and migration were measured by CCK-8 and Transwell. Immunoprecipitation and mass spectrometry analysis were used to identify candidate proteins associated with TRIM7. Immunoprecipitation, immunofluorescence, pull down and ubiquitination assay were performed to examine the regulation between TRIM7 and its candidate protein. m6A modification of TRIM7 was measured by RNA immunoprecipitation. FINDINGS: TRIM7 expression was upregulated in osteosarcoma tissues and was an independent risk factor in predicting poor prognosis. TRIM7 regulates osteosarcoma cell migration and invasion through ubiquitination of breast cancer metastasis suppressor 1 (BRMS1). Moreover, chemoresistance was readily observed in osteosarcoma cells and in patient-derived xenograft (PDX) mice with higher TRIM7 levels. Loss of TRIM7 m6A modification was observed in osteosarcoma tissues. METTL3 and YTHDF2 were the main factors involved in the aberrant m6A modification of TRIM7. INTERPRETATION: Overall, our findings show that TRIM7 plays a key role in regulating metastasis and chemoresistance in osteosarcoma through ubiquitination of BRMS1. FUNDING: This work was financially supported by grants of NSFC (81001192, 81672658 and 81972521) and National Key Research Project of Science and Technology Ministry (2016YFC0106204).

The expression of CRM1 is associated with prognosis in human osteosarcoma.

The nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 (CRM1) is involved in the nuclear export of proteins and messenger RNAs and, thus, mediates the subcellular distribution of important molecules. Osteosarcoma is a ubiquitous and highly aggressive malignant bone tumor. The expression of CRM1 protein in human osteosarcoma has not been reported to date. We investigated the expression of CRM1 in 57 human osteosarcoma and 5 normal cartilage tissues. Western blot investigation revealed expression of CRM1 was significantly increased in osteosarcoma compared with normal tissues. High expression of CRM1 was significantly associated with increased serum level of alkaline phosphatase (ALP, P=0.001) but did not associate with that of lactate dehydrogenase (LDH, P=0.06). In univariate analysis, a significant association between CRM1 expression and tumor size (P=0.014) as well as histological grade (P=0.003) was observed, while high CRM1 expression was not correlated with the other clinicopathological parameters. In Kaplan-Meier survival analysis, high CRM1 expression was a significant prognostic indicator for progression-free survival (P=0.016) as well as overall survival (P=0.008). Multivariate analysis demonstrated that expression of CRM1 was an independent prognostic parameter for longer overall survival (95% CI, 1.27-5.39). Additional prospective studies are required to investigate the prognostic role of high expression of CRM1.

Relationship of serum methotrexate concentration in high-dose methotrexate chemotherapy to prognosis and tolerability: A prospective cohort study in chinese adults with osteosarcoma.

BACKGROUND: Cancer that originates in the bone, termed primary bone cancer, is rare. Osteosarcoma (OS) occurs primarily in growing bone tissue and is more prevalent in children and adolescents. OS in adults is rare, with 3 to 5 cases per million population per year worldwide. There are limited data on treatment-related prognosis and adverse reactions in adults reported in the literature. OBJECTIVES: The aims of this study were to investigate factors that influence serum methotrexate (MTX) concentrations used in chemotherapy in Chinese adult patients with OS, and to determine the correlations (based on age, sex, and dosage), if any, between MTX and prognosis, in terms of disease-free survival (DFS) and overall survival (OAS), and tolerability. METHODS: Adult patients aged ≥30 years with OS received ≥3 courses (2 courses before surgery and 3-4 courses postsurgery) of high-dose MTX (6 or 8 g/m(2)) combined chemotherapy. The regimen consisted of day 1: MTX + folinic acid (herein referred to as citrovorum factor rescue); day 8: cisplatin; days 21 to 25: ifosfamide + mesna; and day 21: doxorubicin. Serum MTX concentrations were assessed immediately after the end of infusion (baseline) and at 24 and 48 hours using high-performance liquid chromatography. Changes in serum MTX concentrations, factors that influence serum MTX concentrations, and the relationship between serum MTX concentrations and prognosis and tolerability (determined by adverse reactions) were analyzed. Patients received a second course of treatment after a 3-week period. RESULTS: Ninety patients (58 men, 32 women; age range, 30-67 years) with OS were included in the study. A total of 532 courses of combined chemotherapy were administered. The serum MTX concentrations ranged widely at baseline (244.31-929.68 mol/L, Cmin and Cmax, respectively) and at 24 hours (0.73-28.24 mol/L, respectively), suggesting that the serum MTX concentrations varied significantly between different individuals and within the same individual at different time points. The serum MTX concentrations in ~23% of cases (122/532) determined at 24 and/or 48 hours were numerically higher than the safety values (according to Nirenberg's reference: irreversible damage if MTX concentration was >10 umol/L and > 1 umol/L at 24 and 48 hours, respectively). No correlation was found between high serum MTX concentration at baseline and high serum MTX concentration at 24 hours (r = 0.401). The prevalences of the 3 most common adverse reactions in these patients were depressed white blood cell count (44.03%), dental ulcer (23.0%), and rash (18.0%). However, in the remaining 410 courses in which serum MTX concentrations were lower than the safety values, these prevalences were 14.6%, 3-9%, and 2.4%, respectively. Neither age nor sex was significantly associated with MTX Cmax, but dosage was (P < 0.05). Patients with a serum MTX Cmax concentration >500 µmol/L at baseline had a significantly longer DFS rate than those with ≤500 umol/L (P = 0.040). There were no significant between-group differences in the OAS rates. conclusions: In these Chinese patients with OS, serum MTX concentrations measured at different time points were varied. The findings suggest that adverse reactions occurred in patients whose serum MTX concentrations at 24 and/or 48 hours were higher than the safety values. The dosage appeared to have influenced MTX Cmax, while sex and age did not, and the Cmax was significantly related to DFS but not OAS.

SKA1 induces de novo MTX-resistance in osteosarcoma through inhibiting FPGS transcription.

De novo methotrexate (MTX)-resistance, whose underlying mechanism remains largely unknown, usually leads to very poor prognosis in patients with osteosarcoma (OS). In this study, we established the de novo MTX-resistant OS cell line SF-86 and identified the candidate gene spindle and kinetochore associated complex subunit 1 (SKA1) as potentially related to de novo MTX-resistance. Analysis of a cohort of 95 OS patients demonstrated that SKA1 overexpression significantly correlated with de novo MTX-resistance and poor 5-year survival. Mechanistically, SKA1 overexpression lead to a downregulation of folylpoly-γ-glutamate synthetase (FPGS), a key enzyme that converts MTX into its active form, MTX-PG. We further demonstrated that SKA1 interacts with DNA-directed RNA polymerase II subunit RPB3. ChIP analysis revealed that RPB3 binds the promoter region of the FPGS gene and triggers FPGS transcription upon MTX treatment in SW1353, a MTX-sensitive OS cell line lacking endogenous SKA1 expression. On the contrary, this process is blocked in SF-86 cells due to the formation of an inhibitory SKA1-RPB3 complex. Furthermore, downregulation of SKA1 levels restores MTX sensitivity in SF-86. Collectively, our study has established the de novo MTX-resistant cell line SF-86 and identified SKA1 as a novel regulator of FPGS, playing a key role in the development of de novo MTX-resistance in OS.

[Effects of ST1571 on the development of dendritic cells derived from bone marrow mononuclear cells in patients with chronic myeloid leukemia].

OBJECTIVE: To investigate the effects of ST1571 on the development of dendritic cells (DC) derived from bone marrow mononuclear cells of patients with chronic myeloid leukemia (CML). METHODS: Bone marrow mononuclear cells (BMMNC) from CML patients and healthy volunteers were cultured initially using multiple cytokine combinations as follows: recombinant human granulocyte/ macrophage colony-stimulating-factor (rhGM-CSF) plus recombinant human interleukin-4 (rhIL-4) as CML and normal control groups, rhGM-CSF plus rhIL-4 and ST1571 as CML experimental groups, and from day 8 recombinant human tumor necrosis factor-alpha ( rhTNF-alpha) was added to stimulate DC maturation. The morphologic features of cells were observed by Wright's staining and phenotypes were assessed by flow cytometry. Cytogenetic analysis was performed by fluorescence in-situ hybridization (FISH), and the antigen-presenting function was assayed by mixed lymphocyte reaction (MLR). The concentration of VEGF was detected by ELISA. RESULTS: CML experimental groups treated with STI571 displayed morphological features similar to those of control groups with delicate membrane projections. However, in comparison with the CML control groups, the CML experimental groups showed an increased expression of CD80, CD86, CD83 and HLA-DR and showed more intense abilities of allogeneic antigen presentation, which were similar to those of normal control groups. FISH confirmed that DCs of both CML, groups were of leukemic origin. The concentration of VEGF was dramatically reduced in CML experimental groups. CONCLUSION: In vitro, STI571 promotes the activation/maturation of DCs derived from BMMNCs of patients with CMI, and decreases VEGF production by the leukemic cells. The promotion of DC maturation may be partially due to decreased inhibitory effect of VEGF.

[Effects of Imatinib mesylate on the development of dendritic cells derived from bone marrow mononuclear cells of patients with chronic myeloid leukemia].

OBJECTIVE: To investigate the effects of Imatinib mesylate (STI571) on the development of dendritic cells (DC) derived from the bone marrow mononuclear cells of patients with chronic myeloid leukemia (CML). METHODS: Bone marrow mononuclear cells (BMMNC) from CML patients were cultured initially using multiple cytokine combinations as follows: recombined human granulocyte/macrophage colony-stimulating-factor (rhGM-CSF) plus recombined human interleukin-4 (rhIL-4) as control groups, rhGM-CSF plus rhIL-4 and STI571 as experimental groups, and from day 8 added recombined human tumor necrosis factor-alpha (rhTNF-alpha) for stimulating maturation. The morphologic features of cells were observed by Wright's staining, Cytogenetic analysis was performed by Fluorescence in-situ hybridization (FISH), phenotypes were assessed by flow cytometry, and the functions of antigen-presenting were assayed by mixed lymphocyte reaction (MLR). The concentration of VEGF was detected by enzyme-linked immunosorbent assay (ELISA). NF-kappaB activation was evaluated by TransAM(TM) ELISA kit. RESULTS: CML experimental groups treated with STI571 displayed features in morphology which were similar to those of control groups with delicate membrane projections. FISH confirmed the DC of both CML groups were leukemic origin. In comparison with the CML control groups, the CML experimental groups showed an increased expression of CD80, CD86, CD83 and HLA-DR and showed more intense abilities of allogeneic antigen presentation. The concentration of VEGF was dramatically reduced, and yet NF-kappaB activation was increased in experimental groups. CONCLUSION: STI571 could promote the activation/maturation of DC derived from BMMNCs of patients with CML in vitro, which might be partially responsible for the fact that the inhibitory effect of VEGF on DC NF-kappaB activation was relieved through STI571 inhibiting the overproduction of VEGF in CML.