Design, Develop, Validate, and Implement Biomarkers for Clinical Investigations

背景资料：靶向抗癌治疗的开发需要证明研究药物对预期靶点和途径的影响，并发现可能与反应相关的标志物。因此，我们致力于检测试剂对途径特异性生物标志物的影响，并发现相关研究中要实施的反应生物标志物。 I.技术、试验设计、开发和验证。我们使用基于电化学发光（ECL）的免疫分析开发、验证和实施临床标本的分析。这是当今最灵敏的定量免疫分析技术平台。ECL平台非常适合这项持续的任务，因为它提供了高度的灵活性，稳定性和可靠性。它能够进行多重分析，以使用有限量的临床标本在单个测定孔中测定总蛋白和磷蛋白的水平。由于临床样本可能差异很大，因此在总蛋白浓度之外对这些样本进行标准化的能力对于生成患者样本的统计学显著数据至关重要。 目前，我们开发、验证和利用一系列生物标志物测定来解决临床研究中的以下领域：1.血浆中的血管生成因子和缺氧组织中HIF 1a的上调用于抗血管生成剂2.细胞因子和生长因子的各种靶向药物3.细胞表面受体由于靶向治疗性抗体的施用而改变4。细胞内磷酸化蛋白激酶抑制剂5。靶向和细胞毒性药物的凋亡生物标志物II。当前和最近完成的生物标志物研究。目前，我们在NCI-CCR参与了10项临床方案。对于其中的许多临床试验，我们帮助设计、开发、验证和实施了相关分析研究的定制生物标志物检测。以下是一些实例：1）对于索拉非尼在雄激素非依赖性前列腺癌中的II期临床试验（PI，William Dahut），我们的实验室用研究药物索拉非尼进行测定验证，并用骨髓活检标本进行测定以确定其对靶向MAP激酶途径的作用（Dahut WL，et al. Clin. Cancer Res.14：209-14，2008）。2)对于另一项研究针对卵巢癌的组合靶向疗法的临床试验（PI，Elise Kohn），我们的小组开发了测定法并进行了对血管生成因子血管内皮生长因子（VEGF）和细胞因子的分析，所述细胞因子包括来自在整个药物试验期间从患者获得的血浆的白细胞介素-6（IL 6）和白细胞介素-8（IL 8）（Azad NS等人，J. Clin. Oncol. 26：3709-14，2008）。3)在另一项雷帕霉素试验（PI，Chand卡纳）中，我们测量了雷帕霉素给药后骨肉瘤活检组织和外周血单核细胞（PBMC）中S6 RP信号传导的相对变化。我们的数据显示PBMC中p-S6 RP的高度统计学显著降低。p/t-S6 RP比值从给药前25%变为给药后0.5%，p <0.001，从第2天至研究结束采集样本。我们的研究结果进一步表明，肿瘤活检中存在高度变异。雷帕霉素对S6 RP途径的统计学显著影响只能使用我们的能够在同一测定孔中定量测量p-S6 RP和t-S6 RP的双重测定法以p/t-S6 RP比率来证明（Paoloni MC等人，PLoS One，5：e11013，2010）。

Background: The development of targeted anti-cancer therapies requires the demonstration of the effects of the investigation agents on the intended target and pathway and the discovery of the markers that can be associated with the response. Thus, we are engaged in detecting the effects of agents on pathway-specific biomarkers and in discovering response biomarkers to be implemented in correlative studies. I. Technology, Assay Design, Development, and Validation. We develop, validate, and implement assays for clinical specimens using electrochemiluminescence (ECL)-based immunoassays. This is the most sensitive and quantitative immunoassay technology platform today. The ECL platform is well suited for this ongoing task because it offers a high degree of flexibility, stability and reliability. It is capable of multiplex analysis to determine the levels of total and phospho-proteins in a single assay well using a limited amount of clinical specimens. Because clinical samples may vary dramatically, the ability to normalize these samples beyond total protein concentration is critical in generating statistically significant data with patient specimens. At the present, we developed, validated and utilized a range of biomarker assays to address the following areas in clinical investigations: 1. Angiogenic factors in plasma and up-regulation of HIF1a in hypoxic tissues for Anti-angegenic agents 2. Cytokines and growth factors for varies targeted agents 3. Cell surface receptors changes as a results of the administration of targeted therapeutic antibodies 4. Intracellular phosphoproteins for a varies kinase inhibitors 5. Apoptotic biomarkers for targeted and cytotoxic agents II. Current and Recently Completed Biomarker Studies. Currently, we are engaged with 10 clinical protocols at NCI-CCR. For many of these clinical trials, we helped to design, develop, validate, and implement customized biomarker assays for correlative analytical studies. The following are some examples: 1) For a phase II clinical trial of sorafenib in androgen-independent prostate cancer (PI, William Dahut), our laboratory performed assay validation with the investigational agent sorafenib and implemented assays with bone marrow biopsy specimens to determine its effects on the targeted MAP kinase pathway (Dahut WL, et al. Clin. Cancer Res. 14: 209-14, 2008). 2) For another clinical trial that investigated a combination targeted therapy against ovarian cancer (PI, Elise Kohn), our group developed assays and performed the analysis of angiogenic factor vascular endothelial growth factor (VEGF) and cytokines, including interleukin-6 (IL6) and interleukin-8 (IL8) from plasma obtained from the patients throughout the drug trial period (Azad NS, et al. J. Clin. Oncol. 26: 3709-14, 2008). 3) In another rapamycin trial (PI, Chand Khanna), we measured the changes in the relative changes of S6RP signaling from both osteosarcoma biopsies and peripheral blood mononuclear cells (PBMC) following the administration of rapamycin. Our data showed a highly statistically significant reduction of p-S6RP in PBMC. The p/t-S6RP ratio changed from 25% pre-drug to 0.5% post-drug with p less than 0.001, with samples obtained from day 2 to the end of the study. Our results further showed that there is a high degree of variation in tumor biopsies. The statistically significant effect of rapamycin on the S6RP pathway can only be demonstrated as p/t-S6RP ratio using our duplex assay capable of quantitatively measuring both p-S6RP and t-S6RP in the same assay well (Paoloni MC et al., PLoS One, 5:e11013, 2010).