SBIR-STTR Award

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

The long-term objective of this project is to develop a comprehensive molecular diagnostic assay for the detection of clinically relevant genetic abnormalities in patients with acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), and myeloproliferative disorders (MPDs). An estimated 44,000 new leukemia cases were predicted for 2008, with more than 21,000 deaths. Leukemia and MPDs are a group of similar diseases of the immune system that have different symptoms, treatment targets, and prognoses. Risk stratification for these diseases has shifted from morphology and cytogenetic analysis to molecular markers. The variation in clinical outcome based on disease subtype underscores the very real need to ascertain for each individual patient the combined cytogenetic and mutational events that underlie the disease; thus, these markers are used as an aid in diagnosis. Furthermore, the products of these cytogenetic and mutational alterations are being exploited as molecular targets for novel therapies. Thus, these treatments will require companion diagnostics for comprehensive detection of molecular signatures that are predictive of treatment outcome. During Phase I we established the feasibility of detecting a small set of mutations with clinical relevance to leukemias and MPDs by demonstrating specific detection of a small set of mutations using a multiplex bead array system. Furthermore, we demonstrated simultaneous detection of mutations and translocations in a single-tube assay format. This resulted in the launch of an RUO diagnostic kit for the detection of NPM1 mutations (Signature(R) NPM1 Mutations). The primary goal of this Phase II grant is to expand the assay into a comprehensive system for simultaneous detection of a suite of sequence-level mutations and cytogenetic abnormalities. In addition to expanding the availability of molecular assays for leukemia/MPD testing, our single-tube, bead array format will simplify adoption into diagnostic laboratories in a manner that reduces overall assay time, operator hands-on time, and training time and provides a software interface that simplifies reporting and facilitates reimbursement. We will develop a set of synthetic controls as well as a software interface for test selection and reporting and perform stability testing and other assay performance experiments. We will also work with clinical collaborators to evaluate the test in a diagnostic laboratory setting on clinical patient samples.

Public Health Relevance:
The goal of this Phase II SBIR project is to develop a reliable and cost-effective diagnostic test kit for mutations found in leukemia patients. Detection of mutations using our test will allow physicians and patients to choose the most effective treatment for each individual patient while improving efficiency and driving down costs in the molecular diagnostic laboratory.

Thesaurus Terms:
Aml - Acute Myeloid Leukemia; Aberrant Chromosome; Abnormalities, Chromosomal; Address; Adoption; Assay; Automobile Driving; B23; Bioassay; Biologic Assays; Biological Assay; Blood (Leukemia); C-Ebp Alpha; C-Ebpalpha; C-K-Ras; Ccaat-Enhancer-Binding Protein-Alpha; Cytogen; Cessation Of Life; Chromosomal Aberrations; Chromosomal Alterations; Chromosomal, Gene, Or Protein Abnormality; Chromosome Aberrations; Chromosome Alterations; Chromosome Anomalies; Chromosome Abnormality; Chronic Myeloid Leukemia; Clinical; Clinical Evaluation; Clinical Research; Clinical Study; Clinical Testing; Clinical Trials, Phase Ii; Collaborations; Companions; Computer Programs; Computer Software; Cytogenetic; Cytogenetic Aberrations; Cytogenetic Abnormalities; Cytogenetic Analyses; Cytogenetic Analysis; Cytogenetic Technics; Cytogenetic Techniques; Cytogenetic Or Molecular Genetic Abnormality; Cytogenetics; Death; Detection; Diagnosis; Diagnostic; Diagnostic Tests; Disease; Disorder; Drivings, Automobile; Eight-Twenty-One; Evaluation; Event; Exons; Expression Profiling; Expression Signature; Flk2 Protein, Human; Flt3 Protein, Human; Flt-3 Receptor; Fms-Related Tyrosine Kinase 3; Forecast Of Outcome; Gud; Gene Products, Rna; Genehomolog; Genes; Genetic Abnormality; Genetic Alteration; Genetic Change; Genetic Defect; Goals; Grant; Hand; Healthcare Systems; Homolog; Homologous Gene; Homologue; Hour; Immune Diseases; Immune Disorders; Immune Dysfunction; Immune System Diseases; Immune System Disorder; Immunologic Diseases; Immunological Diseases; Individual; Investigators; Jak2; Jak2 Gene; K-Ras2a; K-Ras2b; K-Ras 2a; Kras; Kras2; Kras2 Gene; Ki-Ras; Laboratories; Leukemia, Granulocytic, Chronic; Leukemia, Myelocytic, Acute; Leukemias, General; Molecular; Molecular Abnormality; Molecular Cytogenetic Technics; Molecular Cytogenetic Techniques; Molecular Fingerprinting; Molecular Profiling; Molecular Target; Morphology; Mutation; Mutation Detection; Myeloblastic Leukemia, Acute; Myelocytic Leukemia, Chronic; Myelogenous Leukemia, Acute; Myelogenous Leukemia, Chronic; Myeloid Disease; Myeloid Leukemia, Chronic; Myeloid Malignancy; Myeloid Neoplasm; Myeloid Tumor; Myeloproliferative Disorders; Myeloproliferative Tumors; Myeloproliferative Disease; Notch1; Notch1 Gene; Npm; Npm1; Npm1 Gene; Numatrin; Neuroblastoma; Neuroblastoma (Schwannian Stroma-Poor); Oncogene K-Ras; Oncogene, K-Ras-2; Outcome; Output; Prov; Patients; Performance; Phase; Phase 2 Clinical Trials; Phase Ii Clinical Trials; Physicians; Point Mutation; Prognosis; Provider; Rask2; Rna; Rna, Non-Polyadenylated; Reading; Reporting; Research Personnel; Researchers; Ribonucleic Acid; Risk; Sbir; Sbirs (R43/44); Stk-1 Protein, Human; Sampling; Schools, Medical; Small Business Innovation Research; Small Business Innovation Research Grant; Software; Stratification; Symptoms; System; System, Loinc Axis 4; Systems, Health Care; Tan1; Testing; Time; Training; Transcript; Treatment Outcome; Tube; Variant; Variation; Viral Oncogene; Wagr; Wt1; Wt1 Gene; Wt33; Wilms Tumor 1; Work; Acute Granulocytic Leukemia; Acute Myeloid Leukemia; Acute Nonlymphocytic Leukemia; Base; Clinical Relevance; Clinical Test; Clinically Relevant; Computer Program/Software; Cost; Disease Subtype; Disease/Disorder; Disorder Subtype; Driving; Effective Therapy; Experiment; Experimental Research; Experimental Study; Fetal Liver Kinase-2 Protein, Human; Fms-Related Tyrosine Kinase 3 Protein, Human; Genome Mutation; Hn1; Human Flt3 Protein; Improved; Leukemia; Medical Schools; Molecuar Profile; Molecular Marker; Molecular Signature; Multiplex Detection; Myeloproliferative Neoplasm; Novel; Outcome Forecast; Phase 2 Study; Phase 2 Trial; Phase Ii Trial; Protocol, Phase Ii; Prototype; Public Health Relevance; Research Clinical Testing; Research Study; Stability Testing; Stem Cell Tyrosine Kinase 1; Stem Cell Tyrosine Kinase 1 Protein, Human; Study, Phase Ii; T(8;21); T(8;21)(Q22;Q22); V-Ki-Ras2 Kirsten Rat Sarcoma 2 Viral Oncogene Homolog