Integrated Technology for microRNA-based Diagnostic Assays

描述（由申请人提供）：本I期申请的目标是利用现成的临床样本，建立基于检测microRNA （miRNA）的定量分子诊断技术的可行性。分子诊断分析提供了更敏感、更准确和更客观的临床判断的潜力。对于前列腺癌（PrCa），病理诊断的金标准是活检组织的细胞病理学分析。然而，这种方法无法检测到约20%的癌症患者。由于每年进行近100万次前列腺活组织检查，每年有近20万名男性患有无法检测的肿瘤。癌症的病理过程伴随着基因组和转录组的大量变化。因此，有重要的机会发展核酸测定，将生物标志物特征的存在与疾病诊断、预后或分期相关联。microrna是一类令人兴奋的新型小调控生物分子。迄今为止的数据表明，多达一半的基因可能受到miRNA的调节。在过去的几年里，科学家们已经证明mirna在早期发育、细胞增殖和分化、细胞凋亡和脂肪代谢以及肿瘤发生等多种过程中发挥着重要作用。事实上，肿瘤相关的、差异表达的mirna，被称为“肿瘤细胞”，已经在超过6种不同的癌症中被发现，而且肿瘤细胞的数量正在迅速增长。在这个I期应用中，我们将展示从PrCa诊断样本中分离和定量miRNA的集成技术的可行性。我们将优化从两种不同的临床样品基质中提取和检测miRNA的程序，这两种样品基质都可以从当前的PrCa患者工作流程中获得：1)固定和包埋活检组织，以及2)前列腺活检的硝化纤维素指纹（“组织指纹”）。我们将比较两种样品与相应的冷冻组织对照的miRNA的产量和代表性，以确定哪种样品提供了灵敏度和鲁棒性的最佳组合。最后，我们将整合miRNA的提取和定量程序，以实现miRNA检测的简化方法。在第二阶段，我们将使用新兴的miRNA生物标志物来完善和应用这项技术，开发一种对标准前列腺细胞病理学活检具有卓越敏感性的PrCa分子诊断方法。该项目的目标是通过开发一种集成技术来改善癌症诊断，该技术将增强当前的病理实践，并导致更准确的癌症诊断。我们将把这项技术应用于前列腺癌，为医生提供额外的分子信息，这可能会克服病理学家直接观察癌细胞的局限性。这将导致更少的漏诊肿瘤，并可能减少模糊诊断的数量。

DESCRIPTION (provided by applicant): The goal of this phase I application is to establish feasibility for a quantitative molecular diagnostics technology based on the detection of microRNA (miRNA) using readily available clinical samples. Molecular diagnostics assays offer the potential for more sensitive, more accurate, and more objective clinical judgments. For prostate cancer (PrCa), the gold standard for pathological diagnosis is cytopathological analysis of biopsy tissue. However, this approach fails to detect ~20% of patients with cancer. Since nearly 1,000,000 prostate biopsies are performed each year, nearly 200,000 men each year harbor tumors that escape detection. The pathology of cancer is accompanied by numerous changes in the genome and transcriptome. Thus, there is significant opportunity to develop nucleic acid assays that correlate the presence of biomarker signatures with disease diagnosis, prognosis, or staging. MicroRNAs comprise an exciting new class of small, regulatory biomolecules. Data to date suggests that as many as half of all genes may be regulated by miRNA. Over the past few years, scientists have demonstrated that miRNAs play important roles in processes as diverse as early development, cell proliferation and differentiation, apoptosis and fat metabolism, and oncogenesis. Indeed, tumor-associated, differentially expressed miRNAs, termed "oncomirs," have been found in more than a half dozen different cancers and the number of oncomirs is growing rapidly. In this phase I application, we will demonstrate the feasibility for an integrated technology for the isolation and quantification of miRNA from PrCa diagnostic samples. We will optimize procedures for the extraction and detection of miRNA from two different clinical sample matrices, both of which are accessible from the current PrCa patient workflow: 1) fixed and embedded biopsy tissue, and 2) nitrocellulose prints ("tissue prints") of prostate biopsies. We will compare the yields and representation of miRNA for both samples with corresponding frozen tissue controls to determine which sample offers the best combination of sensitivity and robustness. Last, we will integrate procedures for the extraction and quantification of miRNA to enable a streamlined method for miRNA detection. In phase II, we will refine and apply this technology using emerging miRNA biomarkers to develop a PrCa molecular diagnostics assay with superior sensitivity to the standard cytopathological prostate biopsy. The goal of this project is to improve cancer diagnostics by developing an integrated technology that will augment current pathology practices and result in more accurate cancer diagnoses. We will apply this technology to prostate cancer to give physicians additional molecular information that may overcome limitations of the direct observation of cancer cells by a pathologist. This should result in fewer missed tumors and may be able to reduce the number of ambiguous diagnoses.