Diagnostic Assay for Thrombocytosis

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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.