Nanoscale analysis of proteomic response to novel targeted therapies for lymphoma

淋巴瘤新型靶向疗法的蛋白质组反应的纳米级分析

• 批准号: 8517030
• 负责人: ALICE Chen FAN
• 金额: $ 17.34万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-19 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517030
• 关键词: Affect; Antibodies; Apoptosis; Appointment; BCL-2 Protein; BCL2 gene; Bioinformatics; Biological Assay; Biological Markers; Biometry; Blood; Burkitt Lymphoma; Cancer Center; Cell Count; Cells; Chronic Lymphocytic Leukemia; Chronic Myeloid Leukemia; Clinical; Clinical Sciences; Clinical Trials; Clinical Trials Design; Commit; Data Analyses; Data Base Management; Development; Diagnostic; Education; Equipment; Family; Financial Support; Fine needle aspiration biopsy; Flow Cytometry; Follicular Lymphoma; Foundations; Funding; Glean; Goals; Grant; Health; Health Sciences; Immune; Immunoassay; Individual; Institutes; Investigation; Isoelectric Focusing; Isomerism; Knowledge; Laboratories; Leadership; Lymphoma; MAPK1 gene; MEKs; MYC Family Protein; Malignant Neoplasms; Measurable; Measurement; Measures; Mentors; Methods; Mitogen-Activated Protein Kinases; Molecular; Monitor; Neoplasm Circulating Cells; Non-Hodgkin' s Lymphoma; Oncogene Proteins; Oncogenic; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phosphorylation; Policy Research; Process; Protein Isoforms; Proteins; Proteomics; Protocols documentation; Refractory; Research; Research Design; Research Personnel; Research Support; Resources; STAT3 gene; STAT5A gene; Sampling; Science; Signal Transduction; Specimen; Techniques; Technology; Therapeutic; Therapeutic Agents; Time; Transgenic Model; Translational Research; Tyrosine Kinase Inhibitor; Work; atorvastatin; base; cancer therapy; career; career development; clinical decision-making; clinical effect; data integration; design; education evaluation; experience; inhibitor/antagonist; innovative technologies; instructor; instrument; meetings; minimally invasive; nanofluidic; nanolitre; nanoscale; neoplastic cell; new technology; novel; novel therapeutics; oncogene addiction; patient oriented research; phase 1 study; phase 2 study; population health; programs; protein activation; protein profiling; research study; response; skills; square foot; standard care; therapy design; translational clinical trial; tumor

The focus of my research is to develop novel technologies to measure the proteomic response of individual patients to targeted therapies for lymphoma. I have established a strong foundation in the use of transgenic models to study cancer. I now aim to gain the skills needed to become an independent translational researcher, including rigorous knowledge of biostatistics and clinical trial design and experience as a co- investigator in ongoing studies, with the goal to successfully incorporate potential new biomarkers into patient oriented research to validate biomarkers for clinical decision making. At the bench, I have developed a novel nanofluidic proteomic immunoassay (NIA) for the analysis of oncoproteins in cells from patient specimens collected using minimally invasive techniques such as fine needle aspirate (FNA) or blood draw, in as few as 25 cells in 4 nanoliters of lysate. NIA can be used to simultaneously identify multiple phosphorylated and non- phosphorylated isoforms of individual proteins. I have developed the use of NIA to measure total oncoproteins, including BCL2, MYC, ERK and MEK in lymphoma, to measure percent phosphorylation of ERK and MEK proteins in the MAP Kinase (MAPK) pathway; and identify decreases of a specific phosphorylated ERK2 isomer early during CML treatment that could distinguish patients who would respond to tyrosine kinase inhibitors from those that were treatment refractory. Finally, I have used NIA together with flow cytometry (FACS) to identify changes in RAS/MAPK/STAT signaling in cells from patients with low grade NHL treated on a clinical trial of single agent atorvastatin. We discovered that patients with Chronic Lymphocytic Leukemia (CLL) and Marginal Zone Lymphoma (MZL) showed an increase in tumor cell apoptosis and a decrease in circulating tumor cells upon treatment with atorvastatin, indicating that atorvastatin may have a measurable clinical effect associated with apoptosis and signaling inhibition. Comparison of proteomic signatures in individual patients before and early during treatment can provide valuable information that can not be gleaned from a single timepoint. I hypothesize that nanoscale analysis of proteomic changes in blood and FNA's can reveal the molecular activity of novel therapeutics in individual patients. I also hypothesize that NIA can be used to identify when a drug preferentially effects a specific protein phospho-isoform and FACS analysis can resolve drug effects on tumor cells versus non-tumor cells. I will develop complementary NIA and FACS technologies for analysis of minimally invasive blood or fine needle aspirate samples from individual patients before and during treatment. I will determine if changes in BCL2 and RAS pathway profiles can be used to monitor the molecular activity of a BCL2 family inhibitor (ABT-263) and atorvastatin, respectively. My proposal will be amply supported by state-of-the art facilities, equipment, including a CB1000 instrument to perform NIA, and technical support. The Stanford Cancer Center and Stanford Clinical Trials Office provide resources for clinical trial support, including support for Oncore, our online research management database. Biomarker studies will be performed in the laboratory of Dr. Felsher in the Center for Clinical Sciences Research building. Finally, our new Population Health Sciences Institute (PHSI) is a newly opened patient-based research center that occupies 40,000 square feet containing a Clinical Investigation Unit to assist investigators with the development, implementation, and conduct of patient-based research studies, examination rooms and a core laboratory to perform specialized sample processing and storage in support of research protocols. The PHSI also has a Quantitative Sciences Unit and a Bioinformatics Unit to provide biostatistical support in research design and data analysis and to facilitate integration of data from multiple platforms. I will have access to all these facilities, and equipment. A key component to my career development is an ambitious plan to complete 31 units of biostatistical and clinical design coursework through the Stanford Center for Clinical and Translational Education and Research, Department of Health Research and Policy. I will gain clinical experience by becoming a co-investigator on an ongoing Phase I study of ABT-263, and as co-protocol director for our investigator-initiated study of atorvastatin in lymphoma. My instructor appointment has 100% protected time for research-related activities in order to accomplish these goals, guided my Primary Mentor, Dr. Dean Felsher and Co-Mentor, Dr. Ronald Levy. I am fortunate to have assembled this well-matched team committed to my career development. The three of us will meet together monthly. Dr. Felsher, Director of the Stanford Cancer Center Molecular Therapeutics Program, has established a superb and highly funded research program, including 5 different federal grants, studying the mechanism of "oncogene addiction". Dr. Felsher will assure that I receive the education, evaluation and guidance, financial support and resources for my studies, leadership opportunities and protected time, and provide specific guidance for career development. Dr. Levy, a world leader in translational research for lymphoma, will provide guidance in performing translational clinical trials and in achieving academic advancement. My career is dedicated to translational investigation for realizing the goal of individualized cancer treatment.

我的研究重点是开发新的技术来测量单个患者对淋巴瘤靶向治疗的蛋白质组反应。我已经在使用转基因模型研究癌症方面奠定了坚实的基础。我现在的目标是获得成为一名独立翻译研究员所需的技能，包括生物统计学和临床试验设计的严谨知识，以及在正在进行的研究中作为联合研究员的经验，目标是成功地将潜在的新生物标记物纳入以患者为导向的研究，以验证临床决策的生物标记物。在工作台上，我开发了一种新的纳米流体蛋白质组免疫分析(NIA)，用于分析使用细针抽吸(FNA)或抽血等微创技术收集的患者标本中的细胞中的癌蛋白，在4纳升裂解物中仅有25个细胞。NIA可用于同时鉴定单个蛋白质的多种磷酸化和非磷酸化异构体。我已经开发出使用NIA来测量淋巴瘤中的总癌蛋白，包括BCL2，MYC，ERK和MEK，以测量ERK和MEK蛋白在MAP Kinase(MAPK)通路中的磷酸化百分比；并确定在CML治疗早期，特定的磷酸化ERK2异构体的减少，可以区分对酪氨酸激酶抑制剂有反应的患者和那些治疗难治的患者。最后，我将NIA与流式细胞术(FACS)结合使用，以确定在单剂阿托伐他汀临床试验中接受低级别NHL治疗的患者细胞中RAS/MAPK/STAT信号的变化。我们发现，阿托伐他汀治疗慢性淋巴细胞性白血病(CLL)和边缘带淋巴瘤(MZL)后，肿瘤细胞凋亡率增加，循环中肿瘤细胞减少，提示阿托伐他汀可能通过抑制细胞凋亡和信号转导而具有一定的临床疗效。比较个别患者在治疗前和治疗早期的蛋白质组特征可以提供不能从单一时间点收集的有价值的信息。我假设，对血液和FNA中蛋白质组变化的纳米级分析可以揭示新疗法在个别患者中的分子活性。我还假设，NIA可以用来确定药物何时优先作用于特定的蛋白磷酸异构体，而FACS分析可以区分药物对肿瘤细胞和非肿瘤细胞的影响。我将开发补充的NIA和FACS技术，用于在治疗前和治疗期间对个别患者的微创血液或细针抽吸样本进行分析。我将确定BCL2和RAS通路的变化是否可以分别用于监测BCL2家族抑制剂(ABT-263)和阿托伐他汀的分子活性。我的建议将得到最先进的设施、设备和技术支持，包括一台用于执行NIA的CB1000仪器。斯坦福癌症中心和斯坦福临床试验办公室提供临床试验支持资源，包括对我们的在线研究管理数据库OnCore的支持。生物标记物研究将在位于临床科学研究中心大楼的费尔舍博士的实验室进行。最后，我们的新人口健康科学研究所(PHSI)是一个新开设的以患者为基础的研究中心，占地40,000平方英尺，其中包括一个临床调查单位，以协助研究人员开发、实施和进行以患者为基础的研究研究，检查室和核心实验室，以执行专门的样本处理和存储，以支持研究方案。生物科学研究所还设有一个定量科学股和一个生物信息学股，以在研究设计和数据分析方面提供生物统计支助，并促进整合来自多个平台的数据。我将可以使用所有这些设施和设备。我职业发展的一个关键组成部分是一个雄心勃勃的计划，通过卫生研究和政策系斯坦福临床和翻译教育与研究中心完成31个单元的生物统计学和临床设计课程。我将通过成为正在进行的ABT-263第一阶段研究的联合研究员和我们研究员发起的阿托伐他汀在淋巴瘤研究的联合方案主任来获得临床经验。我的主要导师迪恩·费尔舍博士和共同导师罗纳德·利维博士指导我的导师安排了100%受保护的时间用于与研究相关的活动，以实现这些目标。我很幸运地组建了这支致力于我职业发展的匹配团队。我们三个每个月都会见面。作为斯坦福大学癌症中心分子治疗项目的主任，费尔谢尔博士建立了一个卓越且资金雄厚的研究项目，其中包括5项不同的联邦拨款，研究“癌基因成瘾”的机制。费尔谢尔博士将确保我获得教育、评估和指导，为我的学习提供财政支持和资源，提供领导机会和受保护的时间，并为职业发展提供具体指导。利维博士是淋巴瘤翻译研究的世界领先者，他将在进行翻译临床试验和实现学术进步方面提供指导。我的职业生涯致力于翻译研究，以实现癌症个体化治疗的目标。