Biomarker significance of plasma and tumor miR-21, miR-221, and miR-106a in osteosarcoma.

Osteosarcoma is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, osteosarcoma patients who have a poor response to chemotherapy or develop relapses have a dismal outcome. Identification of biomarkers for active disease may help to monitor tumor burden, detect early relapses, and predict prognosis in these patients. In this study, we examined whether circulating miRNAs can be used as biomarkers in osteosarcoma patients. We performed genome-wide miRNA profiling on a discovery cohort of osteosarcoma and control plasma samples. A total of 56 miRNAs were upregulated and 164 miRNAs were downregulated in osteosarcoma samples when compared to control plasma samples. miR-21, miR-221 and miR-106a were selected for further validation based on their known biological importance. We showed that all three circulating miRNAs were expressed significantly higher in osteosarcoma samples than normal samples in an independent cohort obtained from the Children's Oncology Group. Furthermore, we demonstrated that miR-21 was expressed significantly higher in osteosarcoma tumors compared with normal bone controls. More importantly, lower expressions of miR-21 and miR-221, but not miR-106a, significantly correlated with a poor outcome. In conclusion, our results indicate that miR-21, miR-221 and miR-106a were elevated in the circulation of osteosarcoma patients, whereas tumor expressions of miR-21 and miR-221 are prognostically significant. Further investigation of these miRNAs may lead to a better prognostic method and potential miRNA therapeutics for osteosarcoma.

The prognostic significance of circulating serum amyloid A and CXC chemokine ligand 4 in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common pediatric bone cancer.  Despite advances in treatment regimens, the survival rate remains 60-70%.  There is an urgent need to identify prognostic biomarkers, so that targeted therapies can be developed to improve the outcome. PROCEDURE: Our laboratory has previously identified that circulating serum amyloid A (SAA) and CXC chemokine ligand 4 (CXCL4) are upregulated in patients with OS.  In this study, we tested if they could be used as prognostic biomarkers.  We used enzyme-linked immunosorbent assays to measure their concentrations in serum samples (n = 233) and immunohistochemistry to examine their expressions in primary tumors (n = 37).  Prognostic significance of the serum concentrations and tumor expressions of the biomarkers was then evaluated. RESULTS: Patients with "high SAA" and "low CXCL4" circulating levels at diagnosis significantly correlated with a worse outcome (HR = 1.68, P = 0.014), which was independent of the metastatic status.  These patients also exhibited a significantly higher rate of poor histologic response to chemotherapy.  Furthermore, low tumor expression of CXCL4 correlated with poor survival (HR = 3.57, P = 0.005). CONCLUSIONS: Our results demonstrate that circulating SAA and CXCL4 may serve as prognostic biomarkers in OS.  Targeting CXCL4 has been reported, suggesting that it may be exploited as a therapeutic target in OS.

A novel prognostic model for osteosarcoma using circulating CXCL10 and FLT3LG.

BACKGROUND: Osteosarcoma (OS) is the most common malignant pediatric bone tumor. The identification of novel biomarkers for early prognostication will facilitate risk-based stratification and therapy. This study investigated the significance of circulating cytokines/chemokines for predicting the prognosis at the initial diagnosis. METHODS: Luminex assays were used to measure cytokine/chemokine concentrations in blood samples from a discovery cohort of OS patients from Texas Children's Hospital (n = 37) and an independent validation cohort obtained from the Children's Oncology Group (n = 233). After the validation of the biomarkers, a multivariate model was constructed to stratify the patients into risk groups. RESULTS: The circulating concentrations of C-X-C motif chemokine ligand 10 (CXCL10), Fms-related tyrosine kinase 3 ligand (FLT3LG), interferon γ (IFNG), and C-C motif chemokine ligand 4 (CCL4) were significantly associated with overall survival in both cohorts. Among these candidates, CXCL10 and FLT3LG were independent of the existing prognostic factor, metastasis at diagnosis, and CCL4 further discriminated cancer cases from controls. CXCL10, FLT3LG, and the metastatic status at diagnosis were combined to develop a multivariate model that significantly stratified the patients into 4 distinct risk groups (P = 1.6 × 10-8 ). The survival analysis showed that the 5-year overall survival rates for the low-, intermediate-, high-, and very high-risk groups were 77%, 54%, 47%, and 10%, respectively, whereas the 5-year event-free survival rates were 64%, 47%, 27%, and 0%, respectively. Neither CXCL10 nor FLT3LG tumor expression was significantly associated with survival. CONCLUSIONS: High circulating levels of CXCL10 and FLT3LG predicted worse survival for patients with OS. Because both CXCL10 and FL3LG axes are potentially targetable, further study may lead to novel risk-based stratification and therapy for OS. Cancer 2017;144-154. © 2016 American Cancer Society.

p27 Is a Candidate Prognostic Biomarker and Metastatic Promoter in Osteosarcoma.

Metastatic progression is the major cause of death in osteosarcoma, the most common bone malignancy in children and young adults. However, prognostic biomarkers and efficacious targeted treatments for metastatic disease remain lacking. Using an immunoproteomic approach, we discovered that autoantibodies against the cell-cycle kinase inhibitor p27 (KIP1, CDKN1B) were elevated in plasma of high-risk osteosarcoma patients. Using a large cohort of serum samples from osteosarcoma patients (n = 233), we validated that a higher level of the p27 autoantibody significantly correlated with poor overall and event-free survival (P < 0.05). Immunohistochemical analysis also showed that p27 was mislocalized to the cytoplasm in the majority of osteosarcoma cases and in highly metastatic osteosarcoma cell lines. We demonstrated that ectopic expression of cytoplasmic p27 promoted migration and invasion of osteosarcoma cells, whereas shRNA-mediated gene silencing suppressed these effects. In addition, mutations at the p27 phosphorylation sites S10 or T198, but not T157, abolished the migratory and invasive phenotypes. Furthermore, the development of pulmonary metastases increased in mice injected with cells expressing cytoplasmic p27 compared with an empty vector control. Collectively, our findings support further investigation of p27 as a potential prognostic biomarker and therapeutic target in osteosarcoma cases exhibiting aberrant p27 subcellular localization. Cancer Res; 76(13); 4002-11. ©2016 AACR.