Identification of a Plasma Four-microRNA Panel as Potential Noninvasive Biomarker for Osteosarcoma

Background
Circulating microRNAs (miRNAs) are emerging as promising biomarkers for human cancer. Osteosarcoma is the most common human primary malignant bone tumor in children and young adults. The objective of this study was to investigate whether circulating miRNAs in plasma could be a useful biomarker for detecting osteosarcoma and monitoring tumor removal dynamics.
Methods
Plasma samples were obtained from 90 patients before surgery, 50 patients after one month of surgery, and 90 healthy individuals. The study was divided into three steps: First, initial screening of the profiles of circulating miRNAs in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients using a TaqMan low density array (TLDA). Second, evaluation of miRNA concentration in individual plasma samples from 90 pre-operative osteosarcoma patients and 90 healthy controls by a quantitative real time PCR (qRT-PCR) assay. Third, evaluation of miRNA concentration in paired plasma samples from 50 pre- and post-operative osteosarcoma patients by qRT-PCR assay.
Results
Four plasma miRNAs including miR-195-5p, miR-199a-3p, miR-320a, and miR-374a-5p were significantly increased in the osteosarcoma patients. Receiver operating characteristics curve analysis of the combined populations demonstrated that the four-miRNA signature could discriminate cases from controls with an area under the curve of 0.9608 (95% CI 0.9307-0.9912). These 4 miRNAs were markedly decreased in the plasma after operation. In addition, circulating miR-195-5p and miR-199a-3p were correlated with metastasis status, while miR-199a-3p and miR-320a were correlated with histological subtype.
Conclusions
Our data suggest that altered levels of circulating miRNAs might have great potential to serve as novel, non-invasive biomarkers for osteosarcoma.

背景
循环微RNA（microRNAs, miRNAs）正逐渐成为人类癌症极具潜力的生物标志物。骨肉瘤是儿童及年轻成人中最常见的原发性恶性骨肿瘤。本研究旨在探讨血浆中的循环微RNA能否作为检测骨肉瘤及监测肿瘤切除后动态变化的有效生物标志物。

方法
血浆样本取自90例术前骨肉瘤患者、50例术后1个月的骨肉瘤患者及90名健康个体。本研究分为三个步骤：其一，采用TaqMan低密度芯片（TaqMan low density array, TLDA）对健康对照者与术前骨肉瘤患者的混合血浆样本中的循环微RNA表达谱进行初步筛选；其二，通过实时定量聚合酶链反应（quantitative real time PCR, qRT-PCR）检测90例术前骨肉瘤患者与90名健康对照者的单个血浆样本中的微RNA浓度；其三，采用qRT-PCR检测50例骨肉瘤患者术前与术后配对血浆样本中的微RNA浓度。

结果
骨肉瘤患者血浆中miR-195-5p、miR-199a-3p、miR-320a及miR-374a-5p这4种循环微RNA的水平显著升高。对合并人群的受试者工作特征曲线（Receiver operating characteristics curve, ROC）分析显示，该4种微RNA标志物可有效区分患者与健康对照个体，曲线下面积达0.9608（95%置信区间 0.9307-0.9912）。术后患者血浆中这4种微RNA的水平显著降低。此外，循环miR-195-5p与miR-199a-3p的水平与肿瘤转移状态相关，而miR-199a-3p与miR-320a的水平与组织学亚型相关。

结论
本研究数据表明，循环微RNA水平的异常改变有望成为骨肉瘤新型无创性生物标志物。