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

用于遗传异质性组织分析的高通量单细胞 PCR 技术

• 批准号: 8414065
• 负责人: Emil P Kartalov
• 金额: $ 24.6万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-13 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8414065
• 关键词: Biological; Biological Models; Cancer Detection; Cancer Diagnostics; Cells; Chemistry; Clinical; Colorectal Cancer; Complex; DNA; Detection; Disease; Drug Delivery Systems; Drug resistance; ERBB2 gene; Fluorescence; Genetic Heterogeneity; Goals; Heterogeneity; Immunohistochemistry; In Situ; In Situ Hybridization; Individual; KRAS2 gene; Location; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Maps; Measurement; Methods; Mutation; Mutation Detection; Noise; Optics; Pathologist; Pathology; Patients; Performance; Process; Reaction; Reading; Research; Resistance; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Roche brand of trastuzumab; Signal Transduction; Slice; Stratification; System; Techniques; Testing; Tissue Sample; Tissues; Training; Treatment outcome; Validation; Work; base; cancer invasiveness; cancer site; clinical practice; cost; fluorescence imaging; fundamental research; genetic analysis; improved; laser capture microdissection; malignant breast neoplasm; novel; outcome forecast; tool

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.

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法在组织切片上直接构建数百万个微孔威尔斯的矩阵，在威尔斯孔中进行单独但同时的PCR反应，然后通过荧光检测阅读结果。该技术将提供相对于目前的高通量方法（免疫组织化学，原位杂交）和相对于高灵敏度的方法（激光捕获显微切割）提高吞吐量的灵敏度。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，这是由于其在测试耐药性方面的直接临床实用性，以及由于我们的本地专业知识和组织样本的可用性。我们的初步结果显示在微孔基质内原位成功扩增了组织DNA，但我们仍需要组装突变图谱并优化该技术。这些目标的实现将证明该技术可用于结直肠癌，并作为扩展到其他应用的基础。例如，它可以通过提供最好的两个世界（高灵敏度和高通量），同时保留形态信息，从而彻底改变病理学。它将在已知突变的情况下（结直肠癌中的KRAS和肺癌中的T790/MET）的耐药性临床测试中具有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。该技术也将是研究遗传异质性与前列腺癌侵袭性/致死性之间关系的极好工具。如果将其扩展到RT-PCR， 基于HER 2表达的乳腺癌患者，从而改善治疗和结果。因此，该技术将是一种具有高度影响力和广泛适用性的变革性工具，用于疾病细胞基础的基础研究。 公共卫生相关性：我们提出了一种高通量和高灵敏度分析组织样本中遗传异质性的新技术，该技术使用来自数百万个独立的微小威尔斯孔的并行PCR和光学检测，同时保留组织的背景信息。该技术将在疾病的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，例如在理解和测试结直肠癌，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。