Early Detection Research Network (EDRN) BiomarkerReferenceResourceCenter (BRRC)

早期检测研究网络 (EDRN) 生物标记参考资源中心 (BRRC)

• 批准号: 8565842
• 金额: $ 76.35万
• 依托单位: NATIONAL CANCER INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8565842
• 关键词: Affinity; Alternative Splicing; Antibodies; Biological Assay; Biological Markers; Blood; Breast; Case-Control Studies; Categories; Clinical; Collaborations; Contracts; Coupled; Detection; Development; Digestion; Early Detection Research Network; Early Diagnosis; Electrospray Ionization; Enzyme-Linked Immunosorbent Assay; Evaluation; Funding; Gene Fusion; Human; Individual; Ions; Laboratories; Malignant Neoplasms; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Mass Spectrum Analysis; Measures; Pentas; Peptides; Phase; Plasma; Prostate; Prostatic Tissue; Protein Isoforms; Proteolytic Processing; Protocols documentation; Relative (related person); Research Personnel; Running; Sampling; Sensitivity and Specificity; Source; Specificity; Specimen; Stable Isotope Labeling; Staging; Technology; Tissues; Training; Urine; Validation; Work; base; cancer diagnosis; case control; data management; ionization; mass spectrometer; multiplex detection; nano-electrospray; novel; pressure; validation studies

3.1.1. Task 1: Demonstrate the sensitivity of quantitative SRM-MS assays in detection of target biomarkers. The initial detection of the biomarkers will focus on human prostate tissue. The Agency will search for the same markers in blood and urine only if they are successfully detected in the prostate cancer tissue or the normal prostate tissue. A) Demonstrate the ability to quantitatively measure approximately 50 prostate cancer biomarkers at a concentration as low as 1-50 ng/ml first, in human prostate and prostate cancer tissue and then, only for detectable biomarkers also in plasma and urine using LC-SRM-MS platform using: 1. Triple quadrupole mass spectrometer with a novel dual ion-funnel interface 2. Tandem IgY14-SuperMix immunoaffinity depletion. B) Comparison of the limit of detection (LOD), limit of quantification (LOQ) and absolute concentration estimates between SRM and commercial ELISA for 5 selected markers. C) Evaluation of the multiplex power of SRM assays. D) Improvement of the limit of detection of the biomarkers to a range of 100- 1000 pg/ml ¿in human plasma and urine through the application of technologies, but are not limited to, as listed below: 1. SPIN (sub-ambient pressure ionization with nanoelectrospray) source. 2. Multi-ESI (electrospray ionization) emitters. 3. Synchronized ion-funnel trap for trap and release ions to increase sensitivity. 4. Penta-quadrupoles for further enhance specificity of the assay. E) Development of alternative approach (a backup plan) for multiplex detection of biomarkers based on affinity enrichment of specific peptides coupled with mass spectrometry. Overall, the approach would be based on antibody capture of individual tryptic peptides from a digest of the target tissue (e.g., prostate and prostate cancer and plasma from a case and control). Antipeptide antibodies will be developed against a selected signature of tryptic peptides of a chosen biomarker. After the tryptic digestion of the target tissue and addition of known amounts of stable isotope¿labeled calibrator peptide, both added and sample-derived versions will be enriched, and the relative amounts of the biomarker will be measured by SRM-MS. Task 1 Requirement: 1. Identification of biomarkers detectable at concentrations as low as 100-1000 pg/ml in prostate cancer tissue and in normal prostatic tissue. 2. Based on the tissue detectable biomarker, detection of candidate prostate cancer biomarkers at concentrations of 100 ¿ 1000 pg/ml in human plasma and urine. 3. Demonstration of multiplex ability for at least 25 distinct biomarkers in the same run, using either platform, and achieving a sensitivity of at least 1 ng/ml. 4. Development of an alternative approach (a backup approach) based on affinity enrichment of specific peptides coupled with SRM-MS to detect biomarkers at concentration of 100-1000pg/m. 3.1.2. Task 2: Develop isoform-specific SRM assays for quantifying proteolytic processing products (e.g. multiple products of PSA), alternative splicing isoforms and gene fusion products in human urine or plasma. Task 2 Requirement: Demonstrated ability to use SRM to quantitatively distinguish between two distinct isoforms in at least two of the three categories (proteolytic processing isoforms, alternative splicing isoforms, gene fusion isoforms). 3.1.3. Task 3: Quantitative detection of approximately 50 selected prostate cancer markers in blood and urine samples using LC-SRM-MS. The initial verification will focus on cancer biomarkers which were detected in the prostate cancer (tissue). Validation of the selected biomarkers should be carried in a case control study in two steps; the first should be a pre-Validation (~200 samples). Samples will be provided through collaboration with the EDRN GU Collaborative group and if necessary supported by EDRN statisticians from the EDRN Data Management and Coordination Center (DMCC). In addition, it is anticipated that at this stage of the work there will be additional EDRN Biomarkers Reference Laboratory, which will be trained to reproduce the PNNL protocol for the selected markers, which will independently analyze least 10%-15% of selected specimens. The EDRN laboratory will not be funded by this contract. This step will allow the NCI to evaluate whether the sensitivity and specificity of the best panel of biomarkers. Task 3 Requirement: pre-Validation of a panel of biomarkers and establishment of their concentration in blood and urine of cases and controls. 3.1.4. Task 4: Clinical validation of the biomarkers (in collaboration with the EDRN). Validation study based on the best panel/s (for subtypes of the cancer) using (~1000 samples. Samples will be provided through collaboration with the EDRN GU Collaborative group and if necessary supported by EDRN statisticians from the Data Management and Coordination Center (DMCC). In addition, it is anticipated that at this stage of the work there will be additional EDRN Biomarkers Reference Laboratory, which will be trained to reproduce the PNNL protocol for the selected markers, which will independently analyze least 10%-15% of selected specimens. The EDRN laboratory will not be funded by this contract. Task 4 Requirements: Clinical validation of quantitatively detectable panel or panels of biomarkers for early and diagnosis of prostate cancer/s in collaboration with EDRN and the EDRN DMCC. 3.2. Phase 2: Development of additional multiplex detection of biomarkers by SRM-MS for early detection and diagnosis of cancers of the pancreas and breast and validation of their clinical utility. The list of required tasks for phase 2 is similar to that for prostate cancer. Phase 2 Tasks' Requirements: Analytical and Clinical validations of quantitatively detectable panel or panels of biomarkers for early detection and diagnosis of cancers of the pancreas and breast in collaboration with the NCI, the EDRN investigators and the EDRN DMCC.

3.1.1。任务1：证明定量SRM-MS测定在检测目标生物标志物时的敏感性。生物标志物的最初检测将集中在人类前列腺组织上。只有在前列腺癌组织或正常的前列腺组织中成功检测到血液和尿液中相同的标记。 a）证明能够以低至1-50 ng/ml的浓度定量测量大约50个前列腺癌生物标志物，首先在人类前列腺和前列腺癌组织中，然后仅在LC-SRM-MS平台中使用：1。tripleQuadrupole质量谱仪在血浆和尿液中可检测到的生物标志物，也可以检测到可检测的生物标志物。 Igy14-Supermix免疫亲和力耗竭。 b）对于5个选定标记的SRM和商业ELISA之间的检测极限（LOD），定量极限（LOQ）和绝对浓度估计值的比较。 c）评估SRM分析的多重功率。 d）通过应用技术，在人血浆和尿液中，生物标志物检测到100-1000 pg/mL的限制的限制不限于下面列出：1。旋转（Spin（Spin）（纳米电源）的spin（spar-ambient压力电离）。 2。多ESI（电喷雾电离）发射器。 3。用于陷阱和释放离子的同步离子孔陷阱，以提高灵敏度。 4。五局部，以进一步提高测定法的特异性。 e）开发基于与质谱法结合的特定肽的亲和力富集生物标志物多路复用检测的替代方法（备用计划）。总体而言，该方法将基于从靶组织的摘要（例如，前列腺和前列腺癌和血浆中的抗体捕获单个胰蛋白酶肽的抗体捕获）。抗肽抗体将针对所选生物标志物的胰蛋白酶肽的选定特征开发。靶组织的胰蛋白酶消化并添加已知量的同位素稳定量标记的校准pepperoni之后，添加和样品衍生的版本将富集，生物标志物的相对量将通过SRM-MS测量。任务1要求：1。在前列腺癌组织和正常前列腺组织中可检测到的生物标志物在低至100-1000 pg/ml的浓度下可检测到的生物标志物。 2。基于可检测的生物标志物，在人血浆和尿液中以100 pg/ml的浓度检测候选前列腺癌生物标志物。 3。在同一运行中，使用任何一个平台，在同一运行中至少25种不同的生物标志物的多重能力演示，并达到至少1 ng/ml的灵敏度。 4。基于特定宠物的亲和力富集与SRM-MS的亲和力富集以以100-1000pg/m的浓度检测生物标志物，开发了一种替代方法（一种备份方法）。 3.1.2。任务2：开发用于量化蛋白水解加工产物（例如PSA的多种产物）的同工型特异性SRM分析，在人类尿液或血浆中的替代剪接同工型和基因融合产物。任务2要求：证明使用SRM定量区分这三个类别中至少两个（蛋白水解的同工型，替代剪接同工型，基因融合同工型）的两个不同的同工型的能力。 3.1.3。任务3：使用LC-SRM-MS在血液和尿液样品中大约50个选定的前列腺癌标志物进行定量检测。最初的验证将集中于在前列腺癌（组织）中检测到的癌症生物标志物。应在案例控制研究中以两个步骤进行验证。第一个应该是预验证（约200个样本）。样本将通过与EDRN GU合作小组的合作提供，并在必要时得到EDRN数据管理与协调中心（DMCC）的EDRN统计学家的支持。此外，预计在工作的这一阶段，将有其他EDRN生物标志物参考实验室，该实验室将接受培训以重现所选标记的PNNL方案，该协议将独立分析至少10％-15％的选定标本。 EDRN实验室将不会由本合同资助。此步骤将允许NCI评估最佳生物标志物面板的灵敏度和特异性。任务3要求：验证一组生物标志物，并建立其在病例和对照组的血液和尿液中的集中度。 3.1.4。任务4：生物标志物的临床验证（与EDRN合作）。 Validation study based on the best panel/s (for subtypes of the cancer) using (~1000 samples. Samples will be provided through collaboration with the EDRN GU Collaborative group and if necessary supported by EDRN statistics from the Data Management and Coordination Center (DMCC). In addition, it is anticipated that at this stage of the work there will be additional EDRN Biomarkers Reference Laboratory, which will be trained to reproduce the PNNL protocol for选定的标记将分析选定的标本的10％-15％，而EDRN实验室将由该合同提供的临床验证。 SRM-MS用于胰腺和乳房的早期检测和诊断癌症以及临床实用性的验证。第2阶段所需任务的列表与前列腺癌相似。第2阶段任务的要求：与NCI，EDRN研究人员和EDRN DMCC合作，对可检测到的可检测到的可检测到的可检测到的生物标志物的分析和临床验证。