Spondin-2 (SPON2), a More Prostate-Cancer-Specific Diagnostic Biomarker

BackgroundProstate-specific antigen (PSA) screening, although common, has recently been called into question. To find prostate cancer (PCa) diagnostic biomarkers that can make up for the defects of PSA, we compared the secretomes of several benign and PCa cell lines, selected candidate molecules, and then confirmed their clinical value. Methodology/Principal FindingsWe first identified extracellular proteins by two-dimensional gel electrophoresis (2-DE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification. We then validated the secreted proteins on a cellular level, and finally determined whether they could be used as PCa diagnostic biomarkers using prostate tissue and serum specimens of Chinese volunteers by immunohistostaining and sandwich ELISA. We obtained credible extracellular protein 2-DE graphs of prostate cell lines. The 5 spots that showed superior repeatability were selected for LC-MS/MS analysis, which identified seven candidate molecules. One of the candidate molecules, spondin-2 (SPON2), was only expressed in the conditioned media (CM) of androgen receptor (AR) positive PCa cell lines. Using tissue microarray by immunohistostaining, we found SPON2 to be over-expressed in PCa. SPON2 staining was more intense in Gleason score sum 7–8 and in PCa patients with metastasis. By receiver operator characteristic (ROC) curve analysis, we found that the serum SPON2 level was elevated in PCa patients, showing sensitivity and specificity suitable for diagnostic use. We also found that SPON2 could be used to identify PCa patients with serum PSA levels no higher than 10 ng/ml from healthy elderly men. Conclusion/SignificanceSPON2 is a new serum and histological diagnostic biomarker for PCa. It can avoid some of the problems of PSA testing and was here found to offer relatively high sensitivity and specificity relative to PSA.

背景
前列腺特异性抗原（Prostate-specific antigen, PSA）筛查虽为临床常规手段，但近年来其应用价值饱受争议。为发掘可弥补PSA筛查缺陷的前列腺癌（Prostate cancer, PCa）诊断生物标志物，本研究对比了多株良性与前列腺癌细胞系的分泌组，筛选候选分子并验证其临床应用价值。

方法与主要研究结果
本研究首先通过二维凝胶电泳（two-dimensional gel electrophoresis, 2-DE）结合液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）鉴定细胞外蛋白；随后在细胞水平验证候选分泌蛋白；最终借助免疫组化染色与夹心酶联免疫吸附试验（sandwich ELISA），利用中国志愿者的前列腺组织与血清样本，评估这些蛋白作为前列腺癌诊断生物标志物的可行性。本研究获得了可信度较高的前列腺细胞系细胞外蛋白2-DE图谱，选取5个重复性优异的蛋白斑点进行LC-MS/MS分析，共鉴定出7个候选分子。其中候选分子之一——黏附蛋白-2（spondin-2, SPON2）——仅在雄激素受体（androgen receptor, AR）阳性前列腺癌细胞系的条件培养基（conditioned media, CM）中表达。通过组织微阵列免疫组化分析，本研究发现SPON2在前列腺癌组织中呈高表达；其染色强度在格里森评分7~8分的肿瘤样本及发生转移的前列腺癌患者中更为显著。受试者工作特征（receiver operator characteristic, ROC）曲线分析显示，前列腺癌患者血清SPON2水平显著升高，其诊断灵敏度与特异性均符合临床诊断要求。此外，本研究发现SPON2可用于区分血清PSA水平不高于10 ng/ml的前列腺癌患者与健康老年男性。

结论与意义
SPON2是一种新型的血清与组织学前列腺癌诊断生物标志物，可规避PSA筛查的部分局限性，且相较于PSA，本研究证实其具备更优异的诊断灵敏度与特异性。