Chronic myeloid leukemia (CML) is caused by a translocation between chromosomes 9 and 22, where the BCR gene promoter drives overexpression of ABL kinase. This rearrangement causes transformation of blood- forming cells of the bone marrow and accounts for ~15% of all new leukemia cases in adults. Three generations of tyrosine kinase inhibitors (TKIs) have been developed that are used clinically to inhibit the ABL kinase in CML. The first-generation TKI, imatinib, can be highly effective against CML patients, although many patients do not respond to the drug or acquire one or more of >100 different mutations that confer resistance. Some of these mutations also confer resistance to the second-generation TKIs, bosutinib, dasatinib, and nilotinib. However, the second-generation TKIs have slightly different drug-resistance profiles, which enables appropriate targeted therapy in many cases, provided that the mutation status is known. The BCR-ABL T315I mutation renders second-generation TKIs inactive. The third-generation inhibitor, ponatinib, is active against all clinically relevant BCR-ABL1 mutants and is the only approved TKI capable of inhibiting BCR-ABL T315I. However, ponatinib is only approved for patients with a T315I mutation or that have failed on two or more second-generation inhibitors. The overall goal of this research is to develop a quantitative real-time PCR test to detect emerging drug- resistance mutations in BCR-ABL to help physicians determine if/when treatment should be changed. GeneTAG Technology develops molecular diagnostic assays for cancer and infectious diseases. Our primary system, the internal DNA-Detection Switch (iDDS) probe system, comprises two interacting components: a fluor-labeled probe, and a quencher-labeled antiprobe that is nearly complementary to the probe. In the absence of the intended target, the paired probes and antiprobes bind together, quenching fluorescence and preventing off- target detection. This unique probe system shows superior single-base discrimination over a much wider annealing-temperature range (10?30C) than common methods. Recently, we merged our iDDS probe technology with our Wild Terminator (WTx) methods that enable detection of rare mutants (0.1?0.01% frequency) by blocking amplification of the wild-type sequence. The combined method showed greater sensitivity in detecting circulating EGFR variants from lung cancer patients than the FDA-approved cobas EGFR Mutation Test, v2. This enhanced qPCR sensitivity meets or exceeds the sensitivity of specialized platforms without the high instrumentation cost. Our higher sensitivity and specificity will enable development of a platform- independent, liquid biopsy assay.
Our Specific Aims are: 1) to develop XNA-enhanced WTx assays to detect BCR-ABL drug-resistance mutations, and 2) to demonstrate the feasibility of detecting drug-resistant BCR-ABL mutations in plasma from CML patients. Success in Phase I will justify expanded Phase II assay-validation studies in preparation for filing for FDA approval. Currently, no FDA-approved NAAT is available for detecting drug-resistance mutations in ABL. Success in this project will help address an unmet need in patient care.
Cancer is the leading cause of death worldwide, and point mutations in the BCR-ABL fusion gene are known to drive drug-resistance against targeted therapeutics for chronic myeloid leukemia (CML). We have invented highly precise probe systems for detecting small DNA mutations and we have developed companion methods that greatly enhance probe sensitivity. We expect to further develop and apply these methods to detect drug- resistance mutations (associated with recurrence) in blood samples from CML patients. These advances will be particularly valuable for CML diagnostics since they offer the potential to detect emerging drug-resistance mutations at an early stage and to expedite appropriate therapy.
