DECODING CANCER SIGNATURE GLYCOFORMS OF SERUM TUMOR MARK

测量血清前列腺特异性抗原（PSA）水平，并在监测已知前列腺癌患者中发挥良好作用。广泛使用PSA作为前列腺癌的筛查，虽然存在争议，但已导致癌症检测的增加和早期。广泛使用PSA作为前列腺癌的筛查，虽然存在争议，但已导致癌症检测的增加和早期。目前，迫切需要一种测试，可以区分治疗的益处，以及哪些患者可以避免不必要的积极治疗。癌症糖生物学基础研究中有希望的新进展提供了一种合理的机制，通过这种机制，前列腺癌细胞表面粘附分子的特异性聚糖改变调节癌症的侵袭性。此外，已经报道了来自人前列腺癌细胞系的PSA上存在这些聚糖结构。本申请的目标是开发和完善PSA的糖型测定，该测定能够使用来自个体患者的血清进行高通量分析。成功开发该检测试剂盒将实现三个特定目标：1）测定PSA糖型含量的患者间差异，2）得出肿瘤标志物糖基化模式与患者临床状态之间的初步相关性，3）阐明特定目标1和2中鉴定的PSA聚糖结构的初始结构细节。具体的目标将通过应用一种新的毛细管电泳（CE）方法来检测PSA糖变体使用纳克或亚纳克量的糖蛋白来实现。与传统的基于分辨率的糖型分析方法不同，该试点项目将使用毛细管电泳峰模式迁移率测定来鉴定具有变异聚糖的PSA分子。将进行CE分析，以使组分糖型不分离，但使每个个体对峰模式的迁移率的贡献与其量和迁移率成比例。重复的移动性测定允许统计分析。确定流动性方面的统计学显著差异表明存在潜在的结构差异。通过特异性重组糖苷酶减少这种迁移率差异将提供统计学确认的迁移率差异的结构基础的证据。这种方法优于更标准方法的优点在于，对迁移率值精度的优化没有理论限制。不同PSA糖型之间可分辨的结构细节程度取决于迁移率值测定的精度。将优化的对迁移率值精密度重要的参数包括：毛细管壁平衡、样品基质控制、缓冲液稳定性、离子耗尽、样品蒸发、时间、时间分辨率、样品稳定性、PSA-丝氨酸蛋白酶抑制剂络合、温度控制、样品进样、相同的塞长度和样品浓度。优化的检测试剂盒将用于分析一系列前瞻性血清PSA标本的唾液酸化和/或N-聚糖分支的显著变化。这些发现将与患者的临床状态相关。

Measurement of the serum level of prostate specific antigen (PSA) and a well-established role in monitoring patients with known prostate cancer. Widespread use of PSA as a screen for prostate cancer, while controversial, has resulted in increased and earlier cancer detection. Widespread use of PSA as a screen for prostate cancer, while controversial, has resulted in increased and earlier cancer detection. Currently, there is an urgent need for a test that will distinguish benefit from treatment and which patients can avoid unnecessary aggressive therapies. Promising new developments in basic research on the glycobiology of cancer have provided a plausible mechanism by which specific glycan alterations to adhesion molecules on the surface of prostate cancer cells modulate the aggressiveness of the cancer. Furthermore, the presence of these glycan structures on PSA from a human prostate cancer cell line has been reported. The goal of this application is the development and refinement of a glycoform assay for PSA that is capable of high throughput analysis using serum from individual patients. Successful development of this assay will accomplishment of three specific aims: 1) determination of the patient-to- patient variation in glycoform content of PSA, 2) draw preliminary correlations between tumor marker glycosylation pattern and patient clinical status, and 3) elucidation of the initial structural detail of the PSA glycan structures identified in specific aims 1 and 2. The specific aims will be accomplished by applying a novel capillary electroporesis (CE) approach to detect PSA glycovariants using nanogram or subnanogram amounts of glycoprotein. In contrast to the traditional resolution based approach to glycoform analysis, this pilot project will use capillary electrophoresis peak mode mobility determinations to identify PSA molecules with variant glycans. CE analysis will be performed so that the component glycoforms are not resolved but so that each individual contributes to the mobility of the peak mode in proportion to its amount and mobility. Replicate mobility determinations allows statistical analysis. The identification of a statistically significant difference in mobility indicates an underlying structural difference. Reduction of that mobility difference by specific recombinant glycosidases will provide evidence of the structural basis of statistically confirmed mobility differences. The strength of this approach over more standard methodologies is that there is no theoretical limit to optimization of mobility value precision. The degree of structural detail that can be resolved between different PSA glycoforms is dependent on the precision of mobility value determinations. The parameters important to mobility value precision that will be optimized include:: capillary wall equilibration, sample matrix control, buffer stability, ion depletion, sample evaporation, temporal, temporal resolution, sample stability, PSA- serpin complexation, temperature control, sample injection, same plug length, and sample concentration. The optimized assay will be used to analyze a prospective series of serum PSA specimens for significant changes in sialylation and/or N-glycan branching. These findings will be correlated with the patient's clinical status.