Use of Denaturing HPLC for Detection of Mutations in the BCR-ABL Kinase Domain in Patients Resistant to Imatinib

Irving, Julie; O’Brien, Stephen G.; Lennard, Anne; Minto, Lynne; Feng, Lin; Hall, Andrew G.

doi:10.1373/clinchem.2004.034801

Cited by 35 publications

(11 citation statements)

References 22 publications

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“…SSCP analysis of genomic DNA gives a reliable estimate of allele frequency, whereas cDNA analysis may be influenced by allele-specific differences in gene expression; if anything using these two methods would tend to increase, not decrease the differences in allele frequencies Table 1 Primers used in this study between the two groups. While our work was in progress, Irving et al 18 reported that one out of 29 healthy controls was heterozygous for the arginine allele, and suspected that K247R may be a rare polymorphism, in accordance with our data. While all five patients with BCR-ABL-K247R achieved a complete hematological response, three failed to achieve a major cytogenetic response to imatinib (median follow-up 15 months, range 6-24 months).…”

Section: Resultssupporting

confidence: 89%

A single nucleotide polymorphism in the coding region of ABL and its effects on sensitivity to imatinib

et al. 2005

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We have identified a gene polymorphism (K247R) within or close to the P-loop of BCR-ABL, which leads to the substitution of arginine for lysine. We investigated the allelic frequency of K247R by screening 157 CML patients and 213 healthy blood donors with conventional sequencing, restriction enzyme digest and single strand conformational polymorphism analysis, and found the arginine allele to be rare. Three out of five CML patients with the arginine allele of K247R failed to achieve a major cytogenetic response to imatinib, suggesting that the arginine allele may have reduced sensitivity. However, despite K247R's position in or near to the P-loop, biochemical and cellular assays of imatinib and dasatinib sensitivity showed no alteration compared to wild type. Clinicians should be aware that possession of the arginine allele of K247R does not reflect a mutation that necessitates a change in the therapeutic strategy, unless there are other signs of inadequate response to drug.

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Section: Resultssupporting

confidence: 89%

A single nucleotide polymorphism in the coding region of ABL and its effects on sensitivity to imatinib

et al. 2005

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“…[39][40][41][42][43][44][45] Its applicability to the analysis of BCR-ABL KD mutations has been well established. 37,38,46,47 In our patients, the elution characteristics indicated predominance of mutations located in the ATP binding pocket (P-loop) in 46% of cases, the E255K and G250E mutations being the most frequent. The T315I gatekeeper mutation, which directly interferes with imatinib binding and thus completely abrogates imatinib activity 19 was detected in 2 patients within 4 weeks of diagnosis.…”

Section: Discussionmentioning

confidence: 58%

Kinase domain mutations of BCR-ABL frequently precede imatinib-based therapy and give rise to relapse in patients with de novo Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL)

Pfeifer

Waßmann

Pavlova

et al. 2007

Blood

227

186

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Acquired imatinib resistance in advanced Philadelphia-positive acute lymphoblastic leukemia (Ph ؉ ALL) has been associated with mutations in the kinase domain (KD) of BCR-ABL. We examined the prevalence of KD mutations in newly diagnosed and imatinib-naive Ph ؉ ALL patients and assessed their clinical relevance in the setting of uniform frontline therapy with imatinib in combination with chemotherapy. Patients enrolled in the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL) trial ADE10 for newly diagnosed elderly Ph ؉ ALL were retrospectively examined for the presence of BCR-ABL KD mutations by denaturing highperformance liquid chromatography (D-HPLC), cDNA sequencing, and allelespecific polymerase chain reaction (PCR). A KD mutation was detected in a minor subpopulation of leukemic cells in 40% of newly diagnosed and imatinib-naive patients. At relapse, the dominant cell clone harbored an identical mutation in 90% of cases, the overall prevalence of mutations at relapse was 80%. P-loop mutations predominated and were not associ- IntroductionIncorporation of the ABL kinase inhibitor imatinib into frontline treatment of Philadelphia-positive acute lymphoblastic leukemia (Ph ϩ ALL) has significantly improved the antileukemic efficacy of induction therapy. Several cooperative ALL study groups have demonstrated complete remission rates consistently above 90%, irrespective of whether imatinib is used alone or combined with multiagent chemotherapy. [1][2][3][4][5][6][7][8][9] These results are superior to previously reported complete remission (CR) rates of 65% to 90% in younger patients [10][11][12][13] and 40% to 60% in Ph ϩ ALL patients older than 60 to 65 years of age. [14][15][16][17] Although accumulating evidence suggests that imatinib-containing therapeutic regimens may also improve long-term outcome in these patients, 3,[6][7][8]14 relapse remains a predominant cause of treatment failure. 3,[7][8][9] Numerous point mutations in the kinase domain (KD) of BCR-ABL that impair imatinib binding to varying degrees have been identified as a major mechanism of acquired resistance in patients with chronic myeloid leukemia (CML). [18][19][20][21][22][23][24][25] Data on BCR-ABL mutations in patients with Ph ϩ ALL or lymphoid blast crisis of CML are more limited. Two studies of patients with advanced Ph ϩ lymphoid leukemias identified 5 different KD mutations in 14 of the 17 evaluated patients with acquired resistance to imatinib. 26,27 Preponderance of the E255K/V P-loop mutation, which occurred in 6 of 9 patients (67%) following their treatment with imatinib was suggested by one of these reports 26 but not by the other. 27 However, all point mutations arose at positions within the KD that are known to be important for drug binding and to confer significant resistance to imatinib in vitro. [18][19][20] This demonstrated that different mutations within the BCR-ABL KD can be responsible for refractoriness of Ph ϩ lymphoid leukemias to imatinib, and also suggested that KD mutations may be a f...

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“…4,5 In the BCR-ABL fusion gene, the amino acid change K247R is based on a rare adenine to guanine SNP at position 58778 (GenBank accession N. U07563) within the ABL kinase domain. 6,7 Despite its juxtaposition to the P-loop, biochemical and cellular assays of imatinib and dasatinib sensitivity showed no significant alteration compared to non-mutated BCR-ABL. 8 This indicates that polymorphisms within the BCR-ABL kinase domain do not necessarily imply a change of treatment -unless there are signs of inadequate response to treatment -and therefore must be distinguished from acquired mutations leading to resistance.…”

Section: Introductionmentioning

confidence: 99%

ABL single nucleotide polymorphisms may masquerade as BCR-ABL mutations associated with resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia

Ernst

Hoffmann²,

Erben³

et al. 2008

Haematologica

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The BCR-ABL K247R change is based on a rare single nucleotide polymorphism occurring likewise in healthy controls and non-hematologic cell types. Despite its juxtaposition to the P-loop, functional analysis showed no alteration compared to non-mutated BCR-ABL. We sought to investigate if other changes in the BCR-ABL kinase domain should be considered as single nucleotide polymorphisms rather than acquired mutations. A total of 911 chronic myeloid leukemia patients after failure or suboptimal response to imatinib were screened for BCR-ABL kinase domain mutations. Single nucleotide polymorphism analysis was based on the search for nucleotide changes in corresponding normal, non-translocated ABL alleles by ABL allele-specific PCR following mutation analysis. In addition to the K247R polymorphism we uncovered five new single nucleotide polymorphisms within the BCR-ABL kinase domain; two of them led to amino acid changes. Single nucleotide polymorphisms could theoretically contribute to primary but not to secondary resistance to tyrosine kinase inhibitors and must therefore be distinguished from acquired mutations. Novel point mutations should be confirmed by analyzing the normal ABL alleles to exclude polymorphisms.

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Use of Denaturing HPLC for Detection of Mutations in the BCR-ABL Kinase Domain in Patients Resistant to Imatinib

Cited by 35 publications

References 22 publications

A single nucleotide polymorphism in the coding region of ABL and its effects on sensitivity to imatinib

A single nucleotide polymorphism in the coding region of ABL and its effects on sensitivity to imatinib

Kinase domain mutations of BCR-ABL frequently precede imatinib-based therapy and give rise to relapse in patients with de novo Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL)

ABL single nucleotide polymorphisms may masquerade as BCR-ABL mutations associated with resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia

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