A novel, high-throughput assay for detection of ABL T315I mutations

B68

Mutations in the kinase domain of BCR-ABL result in impaired drug binding and are the leading cause of acquired resistance to the tyrosine kinase inhibitor imatinib (Gleevec®). While imatinib is a highly effective therapy in all stages of chronic myelogenous leukemia (CML), relapse after an initial response is common in patients with advanced disease. The T315I point mutation is one of the most common imatinib-resistant mutations and patients with this mutation are also resistant to two second generation tyrosine kinase inhibitors, dasatinib and nilotinib. Thus, appropriate detection of this mutation is essential to optimal management of patients with imatinib resistance and may be useful for clinical trials of agents that target patients with the T315I mutation. Current methods for mutation detection, such as direct DNA sequencing, are not sensitive enough for detection of point mutations at low levels of BCR-ABL transcript. Conversely, ultrasensitive detection methods such as allele specific PCR (AS-PCR) may be too sensitive and can display a propensity for false positives. In addition the clinical relevance/significance of mutation detection at ultra sensitive levels (<1% mutant) is questionable. We developed a novel T315I mutation detection assay, using Fluorescent Resonance Energy Transfer (FRET)-based hybridization probes and melting curve analysis. BCR-ABL amplicons generated from a first round of PCR are amplified using primers flanking the ABL kinase region encoding for codon 315. The resultant amplicon is hybridized with fluorescein-labeled anchor probe and a LC Red 640-labeled T315I mutation specific probe. Wild-type and T315I mutant amplicons are distinguished by melting curve analysis (Roche Light Cycler 480™). Using a series of plasmid and cell line dilutions we determined that the sensitivity of this assay for detection of T315I mutations was 5-10%. Using patient-derived samples we were able to successfully genotype samples containing as few as 20-50 BCR-ABL transcripts. To date, the assay sensitivity and specificity are 100%. The assay is performed in a plate based format (96 or 384 wells) and commercially available software allows automated genotype assignment. This approach is suitable for high-throughput detection of T315I mutations for clinical management of CML patients and/or screening of patients to determine eligibility for clinical studies.