The Mutation Spectrum of CML Patients Resistant to Imatinib: More Heterogeneous Than Just BCR-ABL Kinase Domain Point Mutations?

Cells harboring BCR-ABL kinase domain (KD) mutations are the major source for resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML). A second and third generation of TKIs are in development for inhibition of mutated forms of BCR-ABL. At present, clinical treatment strategies are based on molecular response and mutational profiling. Profiling is usually performed using unbiased methods (sequencing and D-HPLC of PCR products), but mutation specific methods are upcoming (ARMS-PCR, ASO-PCR, ligation-PCR). Here, we describe molecular findings resulting from a collaborative Nordic approach for molecular characterization of imatinib resistant Norwegian (N) and Finnish (F) CML patients. Complementary DNA derived from a total of 87 subjects (60 N and 27 F) with suboptimal or failed response to imatinib (less than 3 log reduction in tumor load after 12 months of treatment or loss of any response) were subjected to BCR-ABL KD sequencing of amino acid residues 240 to 420 (N) and 209 to 498 (F). In addition, single samples were analyzed by multiplex PCR for detection of transcript variants as well as cloning and sequencing of single clones. BCR-ABL KD mutations were found in 35 patients (20 N, 15 F, 40% of the total material). The most frequent findings were ABL KD point mutations with M351T and G250E representing the single most frequent mutations, 25 and 16%, respectively. The most frequently mutated cluster was in the catalytic loop (40% of all mutations), followed by P-loop (38%) and other regions (22%). Mutations in the gatekeeper cluster were extremely rare in our material (one case of a F317L). Additional sequence variations were observed in 6 patients: the L248V associated deletion of exon 5 (n=1), smaller insertion or deletion variants (2-4 basepairs) resulting in truncated proteins (n=2) and ABL exon 7 splicing (n=3). Finally, three cases with imatinib resistance expressed rare BCR-ABL transcript types without any evidence for KD mutations (two cases of e6a2 and one with e19a2 transcript). Imatinib resistance due to T315I BCR-ABL is extremely rare in Nordic CML patients. In addition to known hotspot mutations we identified novel sequence irregularities in association to imatinib resistance. We hypothesize that resistance-associated BCR-ABL isoforms are more heterogeneous than previously thought. Consequently, diagnostic procedures aiming in comprehensive molecular verification of TKI resistance should be designed as unbiased, since methods solely targeting known mutated BCR-ABL variants will miss many patients. However, an early detection of low-level mutated clones will require sensitive, mutation-specific analyses.