Glycan Biomarkers of Prostate Cancer in Prostatic Fluids

描述(申请人提供)：前列腺特异性抗原(PSA)和前列腺酸性磷酸酶(PAP)是由前列腺上皮细胞分泌的糖蛋白，作为前列腺癌的血清生物标志物有很长的临床历史。这两种蛋白和许多其他前列腺源性蛋白在精浆中的浓度明显较高，在指诊直肠检查前列腺按摩后的尿液中也有表达。质谱仪和技术的新进展促使人们重新强调与癌症相关的蛋白质上的糖链结构变化的特征。之前发表的对少数样本的研究表明，PSA和PAP上存在不同的N-连接糖链结构可以区分良性前列腺疾病和前列腺癌。我们对来自大型精浆队列的PSA和PAP多糖的初步数据以及对单个样本点的比较证实了这一点。提出了一种结合多重质谱学和基于ELISA法的方法来鉴定前列腺液中来源于PSA和PAP的N-连接多聚糖。这种方法特别测试了一种假设，即随着前列腺癌疾病严重程度的进展，可以检测到PSA和PAP等分泌的前列腺蛋白上可检测到并持续发生多种糖链结构变化的假设。我们的方法代表了前列腺癌生物标记物战略的范式转变，远离了传统的血清或组织来源。它还强调了基于与癌症表型相关的表面和分泌糖蛋白的碳水化合物表达变化的诊断分析的发展。为了实现这些目标，我们组建了一个协同合作团队，拥有前列腺癌翻译研究和蛋白质组学方面的专业知识，并结合多糖分析专家。必要的临床样品、仪器和葡聚糖分析工作流程可用于实现以下特定目标。1)鉴定前列腺癌近端前列腺液中PAP和PSA上的N-连接糖链生物标志物，以反映前列腺癌的病理和疾病状态。2)对直肠指检后获得的大量EPS尿液中前列腺特异性PAP和PSA糖形式的检测和预验证。将开发特定的唾液酸和岩藻糖靶向凝集素ELISA。3)使用iTRAQ比较，确定EPS样本队列中的其他候选前列腺病糖蛋白生物标记物，包括不同疾病州的糖蛋白变化的全面概况。我们期望PSA和PAP上的葡聚糖的特征将确定改善前列腺癌检测和风险分层的分子标记物。与公共卫生相关：目前PSA血清检测在前列腺癌早期诊断和治疗中的优势和局限性都有很好的文献记载，显然需要新的诊断生物标记物来诊断这种疾病。我们建议描述前列腺癌分泌液中蛋白质的糖链成分的癌症特异性变化。这些分子标志物的识别将改善前列腺癌的检测和前列腺癌活检和前列腺切除术前的风险分层。

DESCRIPTION (provided by applicant): Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) are glycoproteins secreted by prostate epithelial cells, and have a long clinical history of use as serum biomarkers of prostate cancers. These two proteins and many other prostatic-derived proteins are present at significantly higher concentrations in seminal plasma and expressed prostatic secretions (EPS) in urine following a digital rectal exam prostate massage. New advances in mass spectrometry instrumentation and techniques have fueled a renewed emphasis on characterizing alterations in glycan structures on proteins associated with cancers. Previous published studies on a handful of samples have suggested that the presence of different N-linked glycan structures on PSA and PAP could distinguish benign prostate diseases from prostate cancer. Our preliminary data on PSA and PAP glycans derived from large seminal plasma cohorts and comparisons of individual sample spots has confirmed this. A combination of multiple mass spectrometry and ELISA- based approaches to characterizing N-linked glycans derived from PSA and PAP in prostatic fluids are proposed. This approach specifically tests the hypothesis that multiple glycan structural changes are detectable and consistently occur on secreted prostatic proteins like PSA and PAP as prostate cancers progress in disease severity. Our approach represents a paradigm shift for prostate cancer biomarker strategies away from the traditional serum or tissue based sources. It also emphasizes the development of diagnostic assays based on the well described changes in carbohydrate expression of surface and secreted glycoproteins associated with the cancer phenotype. To accomplish these goals, we have assembled a synergistic collaborative team with expertise in prostate cancer translational research and proteomics combined with glycan analysis specialists. The requisite clinical samples, instrumentation and glycan analysis workflows are available to accomplish the following specific aims. 1) Identify N-linked glycan biomarkers on PAP and PSA in proximal prostatic fluids reflective of prostate cancer pathology and disease state. 2) The detection and pre-validation of prostate-disease specific PAP and PSA glycoforms in a large-scale cohort of EPS urines obtained after digital rectal exams. Specific sialic acid and fucose targeted lectin ELISAs will be developed. 3) Identify additional candidate prostatic disease glycoprotein biomarkers in the EPS sample cohort using iTRAQ comparisons, including a comprehensive profile of the glycan changes across disease states. We expect that characterization of the glycans on PSA and PAP will identify molecular markers that improve prostate cancer detection and risk stratification. PUBLIC HEALTH RELEVANCE: The current strengths and limitations of the PSA serum test for early detection and treatment of prostate cancers are well documented, and it is clear new diagnostic biomarkers are needed for this disease. We propose to characterize cancer specific changes in the glycan components of proteins in fluids secreted by the prostate. Identification of these molecular markers will improve prostate cancer detection and risk stratification prior to biopsy and prostatectomy procedures.