Novel Methods for Molecular Analysis of Colorectal Cancer Tissue

描述（由申请人提供）：该项目的一个关键理由是对改进的低成本、高灵敏度的肿瘤多基因突变谱分析方法的需求日益增长。一个重要的例子是国家综合癌症网络（NCCN）最近建议筛查所有诊断为转移性结直肠癌的患者原发肿瘤或转移部位的KRAS突变。已经发现KRAS和EGFR下游的其他基因的突变高度预测对抗EGFR疗法的抗性。第二个例子是癌症复发的情况，其中发现几个基因的突变与转移性复发高度相关，尽管在建立特定多基因突变组的功效之前仍需要进一步研究。一般来说，需要一种比常规DNA测序更低成本、更高灵敏度的方法，能够扫描来自肿瘤（包括来自FFPE块）的多个基因中的突变，以改善当前CRC肿瘤筛查并加速新筛查测定的开发。在第一阶段，一种新的方法，在体外表达的蛋白质（MASSIVE-PRO）的基础上质谱为此目的而开发。初步研究表明，在三个测试基因中可以检测到低至1- 2%的突变群体：APC、KRAS和P53。由于MASSIVE-PRO是一种扫描技术，因此只需知道基因的WT序列，即可发现添加到基因签名中的新突变。在第二阶段，研究将集中在MASSIVE-PRO的改进及其在CRC组织分子谱分析中的应用，特别是开发一种更准确地预测mCRC患者对抗EGFR药物耐药性的检测方法。将引入先进的无细胞蛋白表达和质谱分析技术，以显著提高检测和表征突变的能力，包括使用重组提取物、引物设计、MS-MS测序、同位素耗尽、多重处理以及检测和自动化。我们将与波士顿医学中心胃肠科临床研究主任Paul Schroy博士和安大略癌症研究所肿瘤库副总裁布伦特赞克博士合作，从CRC组织档案沿着相关患者病史中获取并分析200份以FFPE组织块形式储存的肿瘤样本。靶基因和扫描区域的选择基于最近的研究，这些研究表明KRAS、BRAF、PTEN和PIK 3的突变导致抗EGFR药物治疗的耐药性。为了最终临床实施、监管（FDA）批准和基于MASSIVE-PRO AmberGen的分子谱分析试剂盒的上市，Genzyme Corporation和Quest Diagnostics，Inc.将密切合作，所有结果将与波士顿大学公共卫生学院生物统计学副教授Josie Dupuis教授合作进行统计分析。公共卫生相关性：开发一种低成本、高灵敏度的方法来扫描来自肿瘤的DNA中的突变的需求日益增加。在结直肠癌的情况下，一个例子是需要确定那些对抗EGFR药物耐药的患者。目前用于突变检测的方法，例如常规DNA测序，对于在临床实践中常规使用来说不够敏感。该项目旨在开发一种新的低成本，高通量的方法，用于基于无细胞蛋白翻译和质谱分析CRC肿瘤，如果成功，将降低每年诊断为CRC的15万多名美国公民的高死亡率。

DESCRIPTION (provided by applicant): A key rationale of this project is the growing need for an improved low-cost, high sensitivity multigene mutation profiling method for tumors. An important example is the recent recommendation of the National Comprehensive Cancer Network (NCCN) to screen all patients diagnosed with metastatic colorectal cancer for KRAS mutations in the primary tumor or a site of metastasis. Mutations in KRAS and other genes downstream of EGFR have been found to be highly predictive of resistance to anti-EGFR therapies. A second example is the case of cancer recurrence, where mutations in several genes have been found to be highly correlated with metastatic relapse, although further research is still needed before the efficacy of particular multigene mutation panels are established. In general, a lower-cost, higher sensitivity method than conventional DNA sequencing capable of scanning for mutation in multiple genes from tumors including from FFPE blocks is needed to improve current CRC tumor screening and accelerate the development of new screening assays. During Phase I, a new method based on mass spectrometry of in vitro expressed proteins (MASSIVE-PRO) was developed for such a purpose. Initial studies demonstrated that as low as 1- 2% mutant population could be detected in three test genes: APC, KRAS and P53. Since MASSIVE-PRO is a scanning technique, only the WT sequence of a gene needs to be known in order to discover a new mutation which is added to the gene signature. During Phase II, research will focus on the improvement of MASSIVE-PRO and its application to molecular profiling of CRC tissue with particular focus on developing an assay to more accurately predict mCRC patient's resistance to anti-EGFR drugs. Advanced techniques for cell-free protein expression and mass spectrometry will be introduced to significantly improve the ability to detect and characterize mutations including the use of reconstituted extracts, primer design, MS-MS sequencing, isotope depletion, multiplex processing and detection and automation. We will obtain and analyze 200 tumor samples stored as FFPE blocks from CRC tissue archives along with correlated patient history in collaboration with Dr. Paul Schroy, Director of Clinical Research for the Section of Gastroenterology at the Boston Medical Center and Dr. Brent Zanke, Vice-President of the Ontario Institute for Cancer Research Tumor Bank. Choice of target genes and regions scanned is based on recent studies showing that mutations in KRAS, BRAF, PTEN and PIK3 result in resistance to anti-EGFR drug therapy. For the purpose of ultimate clinical implementation, regulatory (FDA) approval, and marketing of a molecular profiling assay based on MASSIVE-PRO AmberGen will work closely with both Genzyme Corporation and Quest Diagnostics, Inc., two of the largest diagnostic companies in the U.S. All results will be statistically analyzed in collaboration with Prof. Josie Dupuis, Associate Professor of Biostatistics at Boston University School of Public Health. PUBLIC HEALTH RELEVANCE: There exists an increasing need to develop a low-cost, high sensitivity method for scanning for mutations in the DNA from tumors. In the case of colorectal cancer, one example is the need to identify those patients who are resistant to anti-EGFR drugs. Current methods for mutation detection, such as conventional DNA sequencing are not sufficiently sensitive to be used routinely in clinical practice. This project aims to develop a novel low-cost, high-throughput method for profiling CRC tumors based on cell-free protein translation and mass spectrometry, which if successful will reduce the high mortality rate of the over 150,000 U.S. citizens diagnosed each year with CRC.