Integrated Technology for microRNA-based Diagnostic Assays

描述(由申请人提供)：这一第一阶段申请的目标是建立一种基于使用现成的临床样本检测microRNA(MiRNA)的定量分子诊断技术的可行性。分子诊断分析为更灵敏、更准确、更客观的临床判断提供了可能性。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，这种方法未能检测到约20%的癌症患者。由于每年进行近100万次前列腺活检，每年有近20万男性患有逃脱检测的肿瘤。癌症的病理学伴随着基因组和转录组的大量变化。因此，有很大的机会发展核酸分析，将生物标志物特征的存在与疾病诊断、预后或分期相关联。MicroRNAs构成了一类令人兴奋的新的小的、可调控的生物分子。到目前为止的数据表明，多达一半的基因可能受到miRNA的调控。在过去的几年里，科学家们已经证明了miRNAs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过程中发挥着重要作用。事实上，在六种不同的癌症中已经发现了与肿瘤相关的、差异表达的miRNAs，称为“oncomir”，而且oncomir的数量正在迅速增长。在这个第一阶段的应用中，我们将展示一种从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本矩阵中提取和检测miRNA的程序，这两种方法都可以从当前PrCA患者的工作流程中获得：1)固定和包埋的活检组织，以及2)前列腺活检的硝酸纤维素印迹(“组织印迹”)。我们将比较两个样本与相应冰冻组织对照的miRNA产量和表达情况，以确定哪个样本提供了灵敏度和稳健性的最佳组合。最后，我们将整合miRNA的提取和定量程序，以实现简化的miRNA检测方法。在第二阶段，我们将使用新兴的miRNA生物标记物来改进和应用这项技术，以开发一种对标准细胞病理前列腺活检具有更高灵敏度的PrCa分子诊断方法。该项目的目标是通过开发一种综合技术来改进癌症诊断，该技术将增强当前的病理学实践，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，这些信息可能会克服病理学家直接观察癌细胞的局限性。这应该会导致更少的肿瘤遗漏，并可能能够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.