Super-sensitive detection of BCR-ABL mutants driving CML recurrence

Genetag Technology, Inc., Atlanta GA

Investigators

Abstract

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?30ºC) 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.