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

背景：靶向抗癌治疗的发展需要证明研究药物对预期靶点和途径的作用，并发现与反应相关的标记物。因此，我们致力于检测药物对途径特异性生物标志物的影响，并在相关研究中发现反应性生物标志物。技术、试验设计、开发和验证。我们开发，验证，并实施分析临床标本使用电化学发光（ECL）为基础的免疫分析。这是当今最灵敏、最定量的免疫分析技术平台。ECL平台非常适合这项正在进行的任务，因为它提供了高度的灵活性、稳定性和可靠性。它能够多重分析，以确定总蛋白和磷酸化蛋白的水平，在一个单一的分析井使用有限数量的临床标本。由于临床样本可能会有很大的差异，因此对这些超过总蛋白浓度的样本进行标准化处理的能力对于产生具有统计意义的患者样本数据至关重要。我们已经在NCI的临床方案中成功地利用该技术进行免疫分析，检测ERK、AKT通路、mTOR抑制、血管生成因子以及各种细胞因子的变化。目前，我们是NCI唯一一家在这些平台上提供检测服务和合作的实验室。我们不断挑战极限，提出能够满足临床试验中未满足需求的新检测方法。2。当前和最近完成的生物标志物研究。目前，我们正在参与NCI-CCR的10个临床方案。对于许多此类临床试验，我们帮助设计、开发、验证和实施相关分析研究的定制生物标志物测定。以下是一些例子：1)对于索拉非尼治疗雄激素非依赖型前列腺癌的II期临床试验（PI, William Dahut），我们的实验室对研究药物索拉非尼进行了分析验证，并对骨髓活检标本进行了分析，以确定其对靶向MAP激酶途径的影响（Dahut WL, et al）。中国。癌症，14:209-14,2008)。2)在另一项研究卵巢癌联合靶向治疗的临床试验中（PI, Elise Kohn），我们的团队开发了检测方法并分析了血管生成因子血管内皮生长因子（VEGF）和细胞因子，包括白细胞介素-6 （IL6）和白细胞介素-8 (IL8)，这些细胞因子来自于整个药物试验期间患者的血浆（Azad NS, et al）。j .中国。中国生物医学工程学报，26(3):389 - 394,2008)。3)在另一项雷帕霉素试验（PI, Chand Khanna）中，我们测量了雷帕霉素给药后骨肉瘤活检和外周血单个核细胞（PBMC）中S6RP信号的相对变化。我们的数据显示PBMC中p-S6RP的降低具有高度统计学意义。从第2天到研究结束，p/t-S6RP比率从用药前的25%变为用药后的0.5%,p<0.001。我们的结果进一步表明，肿瘤活检存在高度差异。雷帕霉素对S6RP通路的统计学显著影响只能通过p/t-S6RP比值来证明，我们的双相分析能够在同一分析井中定量测量p-S6RP和t-S6RP（手稿提交）。3。支持药物开发的临床前研究。我们参与了广泛的临床前研究，以支持NCI的临床研究。例如：1)与Dr. Lee Helman合作发现针对rhabdomyosarcoma的抗igf1r抗体的预测性生物标志物，2)与Dr. Giuseppe Giaccone合作研究抗igf1r治疗胸腺癌的可行性，3)与Dr. Stephen Yoo合作评估TORC1/2抑制剂Palomid 529联合放疗治疗头颈癌。4)开发和评估高灵敏度的HIF1a检测方法作为HIF1a抑制剂的生物标志物。总之，药效学生物标志物和预测性生物标志物的生物标志物分析是早期癌症药物开发的重要组成部分。我们在分析开发，验证和实施方面具有独特的能力，并且我们积极参与NCI的临床试验。

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. We have successfully utilized the technology for immunoassays that detect changes in ERK, AKT pathways, mTOR inhibition, angiogenic factors, as well as a variety of cytokines in clinical protocols at NCI. We are currently the only lab at NCI provides services and collaboration with assays on these platform. We are constantly pushing the envelope to come up with new assays capable of meeting the unmet needs in clinical trials. 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<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 (manuscript submitted). III. Preclinical investigations in support of drug development. We are engaged with a wide range of preclinical investigations in support of clinical studies at NCI. Some examples are: 1) Discovering the predictive biomarker for anti-IGF1R antibodies against rhabdomyosarcoma in collaboration with Dr. Lee Helman. 2) Feasibility studies for anti-IGF1R therapy in thymic cancers with Dr. Giuseppe Giaccone. 3) Evaluation of TORC1/2 inhibitor Palomid 529 in combination with radiation against head and neck cancers in collaboration with Dr. Stephen Yoo of DCTD. 4) Development and evaluation of highly sensitive assay HIF1a assay as a biomarker for HIF1a inhibitors. In summary, biomarker analysis for pharmacodynamic biomarkers and predictive biomarkers is an essential part of early stage cancer drug development. We have unique capabilities in assay development, validation and implementation, and we actively engage clinical trials at NCI.