Multiplex Detection of Mutations in Myeloproliferative Disorder and Leukemia Pati

描述(申请人提供)：该项目的总体目标是开发一种快速检测急性髓系白血病(AML)、非bcr/abl慢性粒细胞白血病(CML)和骨髓增生性疾病(MPDS)患者主要突变的诊断方法。我们建议建立一种单管多重检测方法，它使用高保真的聚合酶链式反应来扩增目标突变，并用Luminex珠状标记的探针杂交来检测特定的序列。我们已经成功地利用这项技术开发了高度多元化的基于DNA和RNA的分析方法。在野生型转录本的背景下检测点突变将为这些疾病的诊断和预后检测以及临床处理提供实质性的好处。根据美国癌症协会的数据，2005年美国报告了34,800例所有类型的白血病新病例。白血病是20岁以下儿童和年轻人癌症死亡的头号原因。尽管髓系恶性肿瘤传统上是根据其形态和细胞遗传学特征进行分类的，但在染色体水平上无法检测到的突变已被证明与白血病和MPDS的不同预后有关。随着这些疾病的分子发病机制变得越来越明显，以及突变特异性抑制剂的出现，分类将需要包括分子鉴定。我们的目标是开发一种全面的检测系统，该系统1)快速检测异常序列，以解决AML、非BCR/ABL CML和MPDS的模糊诊断；2)预测治疗结果，从而在不同的治疗方案中进行选择。这种检测白血病和MPD突变的方法的可用性将极大地改善有效治疗白血病和MPD患者所需的临床决策，同时改善临床实验室工作流程并降低成本。具体目的是：1)建立同时检测AML和CML/MPDS患者主要突变的快速单管检测方法。2)为该分析的临床评价制定全面的对照。在第二阶段，我们将在诊断实验室环境中针对大量临床样本评估该分析的诊断价值。我们将开发分析化验结果的软件，并协助临床对结果进行解释。最终结果是推出突变特异性分析特异性试剂(引物、探针)，并申请FDA批准作为体外诊断试验。这种检测白血病和MPD突变的方法的可用性将极大地改善有效治疗白血病和MPD患者所需的临床决策，同时改善临床实验室工作流程并降低成本。这项建议的总体目标是开发一种检测突变的方法，这些突变对确定骨髓增生性疾病和白血病的治疗至关重要。这种检测的液体微珠阵列形式将为诊断实验室提供效率和成本效益，并为医生和患者提供关键信息，用于制定治疗决策。

DESCRIPTION (provided by applicant): The overall objective for this project is to develop a diagnostic assay for rapid detection of major mutations in patients with acute myeloid leukemia (AML), non-BCR/ABL chronic myelogenous leukemia (CML) and myeloproliferative disorders (MPDs). We propose to develop a single tube multiplex assay which uses high fidelity PCR for amplification of target mutations and hybridization of Luminex bead-tagged probes for detection of specific sequences. We have successfully developed highly multiplexed DNA- and RNA-based assays using this technology. The detection of point mutations in a background of wild-type transcripts will provide substantial benefits for diagnostic and prognostic testing and clinical management of these diseases. According to the American Cancer Society, 34,800 new cases of all types of leukemia were reported in the US in 2005. Leukemia is the number one cause of deaths from cancer in children and young adults under age 20. Although myeloid malignancies have been traditionally classified by their morphological and cytogenetic features, mutations that are undetectable at a chromosomal level have been shown to be correlated with distinct prognosis in leukemia and MPDs. As the molecular pathogenesis for these diseases is becoming evident, and mutation-specific inhibitors become available, classification will need to include molecular identification. It is our goal to develop a comprehensive assay system which 1) rapidly detects aberrant sequences for resolving ambiguous diagnosis of AML, non-BCR/ABL CML and MPDs, and 2) is predictive of therapeutic outcomes and thus useful in selecting between different treatment options. The availability of this assay for leukemia and MPD mutations should greatly improve the clinical decisions necessary for effective treatment of leukemia and MPD patients while improving clinical laboratory workflow and reducing costs. The Specific Aims are: 1) Develop a rapid single-tube assay for the simultaneous detection of major mutations in patients with AML and CML/MPDs. 2) Develop comprehensive controls for the clinical evaluation of the assay. In Phase II, we will evaluate the diagnostic value of the assay against a large number of clinical samples in a diagnostic laboratory setting. We will develop software for analyzing the assay results and to assist in clinical interpretation of the results. The final outcome is to launch mutation-specific Analyte Specific Reagents (primers, probes) and apply for FDA approval as an In Vitro Diagnostic Assay. The availability of this assay for leukemia and MPD mutations should greatly improve the clinical decisions necessary for effective treatment of leukemia and MPD patients while improving clinical laboratory workflow and reducing costs. The overall goal of this proposal is to develop an assay to detect mutations that are important for determining treatment of myeloproliferative disorders and leukemia. This liquid bead array format of this assay will provide efficiency and cost benefits to diagnostic laboratories and provide crucial information to physicians and patients for use in making treatment decisions.