Diagnostic Assay for Thrombocytosis

描述（由申请人提供）：我们的目标是开发一种体外诊断多变量分析（IVDMIA），以区分原发性血小板增多症（ET）和非克隆性反应性血小板增多症（RT）病因。ET是骨髓增生性疾病血小板增多症的一种独特亚型，其特征是巨核细胞增殖增加，从而导致循环血小板水平升高。迄今为止，区分血小板增多症的各种原因的血液学标准在描述克隆性ET和rt的能力方面仍然有限。Jak2基因检测提供了一些诊断价值，但它没有足够的特异性或选择性。因此，通过一系列的临床试验来排除RT和其他疾病。目前ET的诊断基本上是通过排除。我们的PI和合作者率先研究了人类血小板转录组1。我们使用Affymetrix GeneChips发现并首次发表了血小板和血小板特异性mrna，创建定制微阵列，使用定量实时反转录PCR （Q-PCR）调查，发现和验证ET， RT和正常血小板之间基因表达的差异2。一项针对95名受试者（队列I: ET [N=24]; RT [N=23]；健康对照[N=48]）的定制微阵列研究确定了一个11个基因的生物标志物子集，该子集在三组之间的区分准确率为86.3%。双向分类预测（ET vs. RT）准确率为93.6%。采用Q-PCR对一组独立的患者（队列II: ET [N=16]; RT [N=15]）进行判别能力验证，并给出队列II， RT与ET患者准确（87.1%）的表型分类。使用微阵列或Q-PCR分析，一个单独的4-生物标志物基因亚群预测了85%患者样本中的jak2野生型ET。对于诊断测试，初步研究表明，一种新的基于微球的信号放大平台（Panomics和Luminex， Inc.）将是首选。使用简化的程序，它允许同时分析多达33个单独的转录本。这种新型平台还使用体外裂解的完整血小板，从而跳过RNA操作，并允许从少至5 x 107个完整血小板中进行准确的血小板转录物分析，相当于0.1ml全血（其他mRNA分析技术需要20 ml血液或更多，具体取决于血小板水平）。我们建议开发一种简单、敏感、可靠的诊断方法，通过常规静脉切开术和血小板转录谱分析来区分ET和RT。第一阶段将专注于(I)基于微球技术的生成和优化，以测量血小板中生物标志物的表达，以及（ii）将该技术与传统Q-PCR进行全面比较。II期将重点验证该检测（结合分类预测算法）在ET和RT患者中区分大样本血小板的能力。其他因素，如性别和其他疾病等，将在这项大型研究中进行分析。如果成功，该项目将产生一种基于血小板转录谱分析的ET诊断工具。

DESCRIPTION (provided by applicant): Our goal is to develop an In Vitro Diagnostic Multivariate Assay (IVDMIA) to distinguish Essential Thrombocythemia (ET) from non-clonal reactive thrombocytosis (RT) etiologies. ET represents a distinct subtype of myeloproliferative disorders, thrombocytosis, characterized by increased proliferation of megakaryocytes and resultant elevated levels of circulating platelets. To date, hematologic criteria for distinguishing among the various causes of thrombocytosis remains limited in their capacity to delineate clonal ET from RT. Jak2 genetic testing offers some diagnostic value, but it does not have adequate specificity or selectivity. Therefore a long series of clinical testing is utilized to rule-out RT and other disease. Currently ET diagnosis is essentially by exclusion. Our PI and collaborators have pioneered studies of the human blood platelets transcriptome1. We discovered and were the first to publish on platelet and platelet-specific mRNAs using Affymetrix GeneChips, to create custom microarrays, survey, discover and validate, with quantitative real-time reverse-transcription PCR (Q-PCR), differences in gene expression between ET, RT and normal platelets2. A custom microarray study of 95 subjects (Cohort I: ET [N=24]; RT [N=23]; healthy controls [N=48]) identified an 11-gene biomarker subset that discriminated among the three groups with 86.3% accuracy. Two-way class prediction (ET vs. RT) was 93.6% accurate. Discriminant power was validated with an independent group of patients (Cohort II: ET [N=16]; RT [N=15]) using Q-PCR and gave accurate (87.1%) phenotypic classification of Cohort II, RT vs ET patients. A separate 4-biomarker gene subset predicted JAK2-wild type ET in >85% of patient samples using either microarray or Q-PCR profiling. For diagnostic testing, preliminary studies indicate that a novel microsphere-based, signal amplification platform (Panomics and Luminex, Inc.) would be preferred. Using simplified procedures, it allows simultaneous profiling of up to 33 individual transcripts. This novel platform also uses intact platelets lysed in vitro, thus skipping RNA manipulation and allowing accurate platelet transcript profiling from as few as 5 x 107 intact platelets4, equivalent to 0.1ml of whole blood (other mRNA profiling techniques require 20 ml of blood or much more depending upon platelet levels). We propose to develop a simplified, sensitive, and reliable diagnostic assay to discriminate ET from RT using routine phlebotomy followed by platelet transcript profiling. Phase I will focus on (i) generation and optimization of microsphere-based technology to measure expression of biomarkers in platelets, and (ii) comprehensive comparison of this technology to traditional Q-PCR. Phase II will focus on validation of the power of this assay (combined with class prediction algorithms) to discriminate a large sampling of platelets from ET and RT patients. Other factors, such gender and other disease etc, will be analyzed during this large study. If successful, this project will result in a diagnostic tool for ET based on platelet transcript profiling. PUBLIC HEALTH RELEVANCE: This project seeks to create a clinical laboratory diagnostic test for the blood/hematologic disease called essential thrombocytosis (ET). ET is an important subset of all thrombocytoses, high platelet levels in the blood. It is a relatively rare disorder (2-3 per 100,000 people per year), but if left untreated nearly 50% of patients will experience thrombohemorrhagic complications. These include severe neurological, cardiac or peripheral artery manifestations such as stroke or a heart attack. Early definitive, diagnosis will allow early treatment and we have data that supports the potential for a diagnostic test.