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，用于跨组织样本的遗传异质性分析。这项技术涉及使用光刻技术将数百万个微孔的矩阵直接构建到组织切片上，在这些孔中进行单独但同时的PCR反应，然后通过荧光检测读出结果。与目前的高通量方法(免疫组织化学、原位杂交)相比，该技术将提供更高的灵敏度，而相对于高灵敏度方法(激光捕获显微切割)，该技术将提供更高的通量。拟议的项目将为这项技术提供原则性证明。选择的模型系统是结直肠癌的KRAS突变，这是因为它在检测耐药性方面的直接临床用途，以及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，组织DNA在微孔基质内成功地原位扩增，但我们仍然需要组装突变图谱并优化技术。这些目标的实现将证明该技术适用于结直肠癌，并为扩展到其他应用奠定了基础。例如，它可以通过提供两全其美的(高灵敏度和高通量)同时保留形态信息来革命性地改变病理学。它将在已知突变(结直肠癌中的KRAS和肺癌中的T790/MET)的临床耐药测试中立即应用，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进治疗。这项技术也将是研究前列腺癌中遗传异质性与癌症侵袭性/致命性之间关系的一种很好的工具。如果扩展到RT-PCR，它也可以准确地分层 乳腺癌患者基于HER2的表达，从而改善治疗和预后。因此，这项技术将成为一种在疾病细胞基础研究中具有高影响力和广泛适用性的变革性工具。 公共卫生相关性：我们提出了一种新的技术，用于高通量和高灵敏度地分析组织样本中的遗传异质性，该技术使用并行PCR和来自数百万个独立微孔的光学检测，同时保留了组织的上下文信息。这项技术将在疾病细胞基础的基础研究以及相关的临床实践中产生广泛的变革性影响，例如在了解和测试结直肠癌、乳腺癌和肺癌的耐药性方面，这将挽救生命和1亿美元的成本。

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.