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Franz X Gruber,
Tuija Lundán,
Rasmus Goll,
Aleksandra Silye, Ingvild Mikkola,
Ole Petter Rekvig,
Sakari Knuutila,
Kari Remes,
Tobias Gedde-Dahl,
Kimmo Porkka,
Henrik Hjorth-Hansen
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ABSTRACT: Imatinib, a small molecule inhibitor of ABL, PDGFR and C-KIT, has revolutionized treatment of chronic myeloid leukaemia (CML). However, resistance to treatment is of increasing importance and often is due to point mutations in the Abl kinase domain (Abl KD). Here, we analysed clinical outcome and mutation status in two independent Nordic populations (n = 77) of imatinib-resistant CML patients. We detected BCR-ABL transcripts containing point mutations of residues in the P-loop, A-loop and other kinase domain residues in 32 patients (42%). In contrast to previous data, mutations in BCR-ABL were as frequently found in patients with primary resistance (56%) as with secondary resistance (53%). No T315I mutations were found in the study cohort. BCR-ABL splice variants were identified in a significant number of our cases (19%): BCR-ABL transcripts of variable length; a variant fusion transcript joining BCR exon 14 sequences to ABL exon 4; partial, in-frame-deletion of exon 4 due to induction of a cryptic splice site by the L248V and finally, alternative splicing of ABL exon 7 sequences. Though the majority of splice variants observed in this study do not encode functional proteins, alternative splicing appears to represent a common phenomenon in the biology of CML. We conclude that Abl KD point mutations represent a major mechanism of imatinib resistance. Other sequence irregularities were also detected, but their significance in conferring resistance is unclear. Diagnostic strategies looking for imatinib-resistant clones should be designed to detect a broader profile of BCR-ABL variants than just point mutations.
Medical Oncology 01/2011; 29(1):219-26. · 2.14 Impact Factor
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ABSTRACT: Imatinib effectively inhibits the tyrosine kinase activity conferred by the BCR-ABL gene [fusion gene of BCR (breakpoint cluster region) and ABL1 (c-abl oncogene 1, receptor tyrosine kinase)] and thereby appreciably improves outcomes for chronic myelogenous leukemia (CML). A small percentage of patients relapse because of the proliferation of escape clones; such relapses can be treated with second-generation drugs. Early detection and monitoring of resistant clones may provide clinical benefit. We describe the development and testing of a new approach for quantitative monitoring of CML resistance.
We designed mutation-specific assays that use hydrolysis probes and an array of allele-specific primers containing nucleotides mismatched at various positions. All assays were tested with plasmids containing corresponding mutant or wild-type sequences, allowing identification of optimal assays for specific and effective amplification of the target template. Clinical samples were then used to compare the results of selected assays with those of standard genotyping.
We used a modified amplification refractory mutational system approach and testing with plasmid constructs to design assays that allowed highly selective detection of resistance for all target mutations. By taking advantage of single-step performance and high PCR efficiency, we were able to quantitatively track the absolute amount of resistance conferred by a specific mutation over 4 orders of magnitude. Moreover, we designed an integrated test for dasatinib resistance that uses multiple primers simultaneously.
These single-step, closed-tube assays specifically target mutations associated with resistance to dasatinib or nilotinib. Compared with standard genotyping, such biased genotyping improves the detection of resistance or alternative features via quantitative analysis of the absolute amount of resistance.
Clinical Chemistry 03/2010; 56(3):469-73. · 7.91 Impact Factor
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Thomas Ernst,
Franz X Gruber,
Oliver Pelz-Ackermann,
Jacqueline Maier,
Markus Pfirrmann,
Martin C Müller, Ingvild Mikkola,
Kimmo Porkka,
Dietger Niederwieser,
Andreas Hochhaus,
Thoralf Lange
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ABSTRACT: Various techniques have been employed to detect BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia who are resistant to imatinib. This has led to different reported frequencies of mutations and the finding of a heterogeneous pattern of individual mutations.
We compared direct sequencing alone and in combination with denaturing high-performance liquid chromatography and two high-sensitivity allele-specific oligonucleotide polymerase chain reaction approaches for analysis of BCR-ABL mutations in 200 blinded cDNA samples prior to and during second-line dasatinib or nilotinib therapy in patients with chronic myeloid leukemia in whom imatinib treatment had failed.
One hundred and fourteen mutations were detected by both direct sequencing alone or in combination with high performance liquid chromatography and 13 mutations were additionally detected by the combined technique. Eighty of 83 mutations (96%) within a selected panel of 11 key mutations were confirmed by both allele-specific oligonucleotide polymerase chain reaction techniques and 62 mutations were identified in addition to those detected by combined liquid chromatography and direct sequencing, indicating the presence and a high prevalence of low-level mutations in this cohort of patients. Furthermore, 125 mutations were detected by only one allele-specific oligonucleotide polymerase chain reaction technique. Pre-existing mutations were traceable 4.5 months longer and emerging clones were detectable 3.0 months earlier by allele-specific oligonucleotide polymerase chain reaction than by direct sequencing together with liquid chromatography.
Our results suggest that denaturing high performance liquid chromatography combined with direct sequencing is a reliable screening technique for the detection of BCR-ABL kinase domain mutations. Allele-specific oligonucleotide polymerase chain reaction further increases the number of detected mutations and indicates a high prevalence of mutations at a low level. The clinical impact of such low-level mutations remains uncertain and requires further investigation. Allele-specific oligonucleotide polymerase chain reaction allows detection of defined mutations at a lower level than does denaturing high performance liquid chromatography combined with direct sequencing and may, therefore, provide clinical benefit by permitting early reconsideration of therapeutic strategies.
Haematologica 08/2009; 94(9):1227-35. · 6.42 Impact Factor
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ABSTRACT: The transcription factor Pax6 is essential for the development of the eyes and the central nervous system of vertebrates and invertebrates. Pax6 contains two DNA-binding domains; an N-terminal paired domain and a centrally located homeodomain. We have previously shown that the vertebrate paired-less isoform of Pax6 (Pax6DeltaPD), and several other homeodomain proteins, interact with the full-length isoform of Pax6 enhancing Pax6-mediated transactivation from paired domain-DNA binding sites. By mutation analyses and molecular modeling we now demonstrate that, surprisingly, the recognition helix for specific DNA binding of the homeodomains of Pax6 and Chx10 interacts with the C-terminal RED subdomain of the paired domain of Pax6. Basic residues in the recognition helix and the N-terminal arm of the homeodomain form an interaction surface that binds to an acidic patch involving residues in helices 1 and 2 of the RED subdomain. We used fluorescence resonance energy transfer assays to demonstrate such interactions between Pax6 molecules in the nuclei of living cells. Interestingly, two mutations in the homeodomain recognition helix, R57A and R58A, reduced protein-protein interactions, but not DNA binding of Pax6DeltaPD. These findings suggest a critical role for the recognition helix and N-terminal arm of the paired class homeodomain in protein-protein interactions.
Nucleic Acids Research 02/2005; 33(8):2661-75. · 8.03 Impact Factor
