High-Throughput Single-Cell-PCR Technique for Tissue Analysis of Genetic Heteroge

描述（由申请人提供）：我们提出了一种新的单细胞高灵敏度高通量技术，用于分析组织样本的遗传异质性。该技术包括使用光刻技术在组织切片上直接建立数百万个微孔的基质，在孔中进行分离但同时进行的PCR反应，然后通过荧光检测读出结果。相对于目前的高通量方法（免疫组织化学、原位杂交），该技术将提供更高的灵敏度，相对于高灵敏度方法（激光捕获显微解剖），该技术将提供更高的灵敏度。拟议的项目将为该技术提供原理证明。选择的模型系统是结直肠癌中的KRAS突变，因为它在耐药测试中具有直接的临床实用性，也因为我们在当地的专业知识和组织样本的可用性。我们的初步结果显示，在原位微孔基质中成功扩增了组织DNA，但我们仍然需要组装突变图和优化技术。这些目标的实现将证明该技术可用于结直肠癌，并为扩展到其他应用奠定基础。例如，它可以在保留形态学信息的同时提供两全其美（高灵敏度和高通量），从而彻底改变病理学。它将在已知突变（结直肠癌中的KRAS和肺癌中的T790/MET）的耐药临床试验中得到非常重要和直接的应用，节省1亿美元，并通过避免对耐药患者使用昂贵的靶向药物来改善治疗。这项技术也将成为研究前列腺癌遗传异质性与癌症侵袭性/致死率之间关系的绝佳工具。如果扩展到RT-PCR，也可以准确分层

DESCRIPTION (provided by applicant): We propose a novel single-cell high-sensitivity high-throughput technique for analysis of genetic heterogeneity across tissue samples. The technique involves building matrices of millions of microwells directly onto tissue slices using photolithography, performing separate but simultaneous PCR reactions in the wells, and then reading out the results by fluorescence detection. The technique would offer improved sensitivity with respect to current high-throughput methods (immunohistochemistry, in-situ hybridization) and improved throughput with respect to high-sensitivity methods (laser capture microdissection). The proposed project would produce proof of principle for the technique. The chosen model system is KRAS mutation in colorectal cancer, due to its immediate clinical usefulness in testing for drug resistance, as well as due to our local expertise and availability o tissue samples. Our preliminary results show successful amplification of tissue DNA inside the microwell matrix in- situ, but we still need to assemble mutation maps and optimize the technique. The accomplishment of these goals will prove the technique for use with colorectal cancer and serve as a basis for expansions into other applications. For example, it could revolutionize pathology by providing the best of both worlds (high sensitivity and high throughput) while preserving the morphological information. It would have very important and immediate application in clinical tests for drug resistance in cases of known mutations (KRAS in colorectal cancer and T790/MET in lung cancer), saving $100M's and improving treatment by avoiding the use of expensive targeted drugs on resistant patients. The technique would also be a wonderful tool to study the relationship between genetic heterogeneity and cancer invasiveness/lethality in prostate cancer. If extended to RT-PCR, it could also accurately stratify patients in breast cancer based on HER2 expression, thereby improving treatments and outcomes. Thus the technique would be a transformative tool of high impact and broad applicability in fundamental research of the cellular basis of disease. PUBLIC HEALTH RELEVANCE: We propose a new technique for high-throughput and high-sensitivity analysis of genetic heterogeneity in tissue samples, which uses parallelized PCR and optical detection from millions of independent tiny wells, while retaining the contextual information of the tissue. The technique would have a broad transformative impact in the fundamental research of the cellular basis of disease, as well as in related clinical practice, e.g in understanding and testing for drug resistance in colorectal, breast, and lung cancers, which would save lives and $100M's in costs.