Among the 94 imatinib resistant cml patients, 37 39% patients had mutations in the abl kinase domain of the bcr abl gene. Bcrabl tyrosine kinase domain mutations are the most important factor. Effect of lowlevel bcrabl1 kinase domain mutations. Bcrabl1 compound mutations combining key kinase domain. Soverini s1, hochhaus a, nicolini fe, gruber f, lange t, saglio g, pane f, muller mc, ernst t, rosti g, porkka k, baccarani m, cross nc, martinelli g. The early detection of mutations should provide clinical benefit by allowing early intervention. To overcome imatinib resistance, more potent tyrosine kinase inhibitors tkis such as nilotinib, dasatinib, and bosutinib have been developed with demonstrable activity against most of the bcrabl.
Bcr abl1 kinase domain mutation, 35nucleotide insertion chronic myelogenous leukemia cml is a hematopoietic stem cell disorder characterized by the philadelphia chromosome, the result of a 9. Abl1 kinase domain mutations are screened by sequencing of seminested pcr products encompassing the abl1 kinase domain. Point mutations within the kinase domain kd of bcrabl, clonal. All basic analyses were performed using r software version 3. The result will include the codon number, amino acid changes and relative abundance of the mutation s found.
The sequence is compared to an abl kinase domain reference sequence using sequencing analysis software, which aligns the sequences and highlights single or multiple mutations. Structure of the kinase domain of an imatinibresistant abl. Clinical resistance to sti571 cancer therapy caused by bcr. Use of direct sequencing for detection of mutations in the. The abl kinase domain mutation test uses reverse transcriptionpolymerase chain reaction rtprc to amplify the bcr1abl fusion transcript before sequence analysis of the abl kinase domain. Key words chronic myeloid leukemia bcrabl imatinib mesylate mutation kinase domain realtime quantitative pcr direct sequencing. Abl gene kinase domain mutation scanning by denaturing. Bcrabl1 mutation analysis for tyrosine kinase inhibitor. Novel mutations in the kinase domain of bcrabl gene.
Indications for bcrabl kinase domain mutational analy. Bcrabl kinase domain mutations represented the major cause of resistance to tki therapy 5. Analysis includes detection of the common t315i mutation. Rtpcr and sequencing of the bcrabl1 fusion transcript for qualitative detection of mutations associated with resistance to gleevec imatinib and other tyrosine kinase inhibitors. To overcome imatinib resistance, more potent tyrosine kinase inhibitors tkis such as nilotinib, dasatinib, and bosutinib have been developed with demonstrable activity against most of the bcr abl kinase domain mutations seen in patients treated with imatinib, with the notable exception of the t315i mutation. Aug 04, 2011 bcrabl kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. Pdf computational analysis of abl kinase mutations. Methods we investigated bcrabl kinase domain mutations in. All patients with a mutation in abl kinase domain showed treatment failure based on 20 eln recommendations for management of cml. Monitoring cml patients responding to treatment with tyrosine kinase inhibitors.
The quantitative bcr abl gene expression and kinase domain of bcr abl gene mutation analysis was done in 4162 cml patients and 21 patients declined to participate in the study. Bcrabl1 kinase domain kd mutation status is considered to be an important. Hence, it is important to contact the testing lab for exact specimen requirements, before initiating the testing process. We show that this compound inhibits bcrabl activity in cells derived from patients carrying the t315i mutation in the kinase domain of bcrabl, and that it retains activity against purified. Dysregulation of bdnf signaling via its tropomyosin receptor kinase b trkb receptor has a wellcharacterized role in many aspects of hd pathogenesis 3,8 and more recent evidence suggests that. Analysis of mutations in the bcrabl1 kinase domain, using. Bcrabl v280g mutation, potential role in imatinib resistance. Bcrabl kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine. Primary resistance relapse progression early chronic phase late. Imatinib was the first bcrabl1 tyrosine kinase inhibitor tki approved by fda for the treatment of chronic myeloid leukemia cml. In cml, the gene is activated by being translocated within the bcr breakpoint cluster region gene on chromosome 22. Drug resistance has mostly arisen as a result of point mutations in the bcrabl gene that reduce drug binding within the kinase domain or due to overexpression of bcrabl protein 1518.
Bcrabl kinase domain mutations represented the major. Dynamics of bcrabl kinase domain mutations in chronic. Recommendations aimed to rationalize the use of bcrabl mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the european leukemianet eln and european treatment and outcome study and are here reported. The metaanalysis of rdws diagnostic value was performed using stata software version 11. More than 90 various mutations occurred in patients with imatinib resistance. Abl gene kinase domain mutation scanning by denaturing high. Compound mutations have been reported to often cause stronger resistance to tkis 27. Characterizing of four common bcrabl kinase domain. The most common mechanisms of acquired resistance to imatinib are bcr abl amplification at the genomic or transcript level and point mutations in the kinase domain.
Chronic myeloid leukemia cml is initiated in hematopoietic stem cells by the bcrabl oncogene generated from the t9. Imatinib was the first bcr abl1 tyrosine kinase inhibitor tki approved by fda for the treatment of chronic myeloid leukemia cml. Ponatinib is the only currently approved tyrosine kinase inhibitor tki that can inhibit all clinical bcr abl1 single mutations. In patients without t315i the range of bcr abl abl was 0. Bcrabl kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. Other than the atpbinding site, mutation of the sh3sh2. The sequencher program, used for the test and the electropherogram. Gleevec is a selective bcrabl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia cml. The presence of point mutations in the abl1 kinase domain of the bcr abl1 fusion gene codons 221500 is assessed by direct sanger sequencing of the bcr abl1 fusion transcripts following nested pcr. Nextgeneration sequencing for bcrabl1 kinase domain mutation. Mutations within the bcrabl1 kinase domain in imatinibtreated chronic myeloid leukemia are the main mechanism of acquired resistance. Ellwood k, hsu n, paquette r, rao pn and sawyers cl 2001 clinical resis5tance to sti571 cancer therapy caused by bcrabl gene mutation or amplification. Rtpcr and sequencing of the bcr abl1 fusion transcript for qualitative detection of mutations associated with resistance to gleevec imatinib and other tyrosine kinase inhibitors.
Bcr abl1 kinase domain kd mutation status is considered to be an important element of clinical decision algorithms for chronic myeloid leukemia cml patients who do not achieve an optimal response to tyrosine kinase inhibitors tkis. The inhibitors such as imatinib, dasatinib and nilotinib are effective drugs but are resistant to some bcrabl mutations. Bcrabl1 mutation analysis for tyrosine kinase inhibitor resistance by next generation sequencing feedback i want to provide feedback regarding select test content or test information pricing and availability general usability of test directory look and feel of test directory request a new feature in test directory. Characterizing of four common bcrabl kinase domain mutations. The optimal cutoff value for each factor was decided based on the highest youden index. Molecular screening and the clinical impacts of bcrabl kd. Phasing analysis of tki resistance mutations in the bcr. Most patients in chronic phase maintain durable responses. Following is the specimen collection process for abl1 kinase domain mutation test. Jul 10, 2008 realtime quantitative pcr analysis can be used as a primary screen to identify patients with cml treated with imatinib who have bcrabl kinase domain mutations. Point mutations can cause amino acid substitutions inside the kinase domain of the bcr abl protein and disrupt the binding site of imatinib on the tyrosine kinase, resulting in a loss of sensitivity to the drug. Clinical resistance to sti571 cancer therapy caused by. Diagnostic use indications patients philadephia chromosome positive.
Bcrabl1 kd mutation testing by ngs is indicated in cp cml after. We show that this compound inhibits bcr abl activity in cells derived from patients carrying the t315i mutation in the kinase domain of bcr abl, and that it retains activity against purified t315i in vitro. Bcr abl abl levels median and range and t315i mutation status in three studied groups with inadequate im response. Abl1 kinase domain blue in complex with the secondgeneration bcrabl tyrosinekinase inhibitor nilotinib red mutations in the abl1 gene are associated with chronic myelogenous leukemia cml. The y253, e255, t315, and m351 mutations account for approximately 60% of those detected at the time of relapse. Labcorp and its specialty testing group, a fully integrated portfolio of specialty and esoteric testing laboratories. Abl1 kinase domain and neiggghboring domains using next. Frequency and clinical significance of bcrabl mutations. Bcrabl1 kinase domain mutation, 35nucleotide insertion. Rtpcr and sequencing of the bcrabl1 fusion transcript for qualitative. Molecular analysis of the bcrabl1 kinase domain in chronic. Detection of bcrabl mutations and resistance to imatinib. Rtpcr and sequencing of the bcrabl1 fusion transcript for qualitative detection of mutations associated with resistance to gleevec imatinib and other.
Drug resistance has mostly arisen as a result of point mutations in the bcr abl gene that reduce drug binding within the kinase domain or due to overexpression of bcr abl protein 1518. Bcr abl kinase domain inhibition can be used to treat chronic myeloid leukemia. Abl1 kinase domain mutation analysis md anderson cancer. The specimen sample requirements may vary from lab to lab. Evolution of bcrabl gene mutation in cml is time dependent. Bcrabl1, tyrosine kinase inhibitor resistance, kinase domain. Recommendations from an expert panel on behalf of european leukemia net. Abl1 kinase domain blue in complex with the secondgeneration bcr abl tyrosine kinase inhibitor nilotinib red mutations in the abl1 gene are associated with chronic myelogenous leukemia cml. Bcrabl kinase domain mutation analysis in chronic myeloid.
These mutations normally affect the structure of the bcr abl protein, leading either to interruption of critical. This assay is comprehensive for detecting bcr abl1 kd mutations, but does not detect all possible mutations in abl1. Tyrosine kinase inhibitor drug resistance required patient information. Our results suggest a structural explanation for how vx680 retains activity to mutant proteins, which no longer are sufficiently inhibited. The quantitative bcrabl gene expression and kinase domain of bcrabl gene mutation analysis was done in 4162 cml patients and 21 patients declined to participate in the study. Abl1 kinase domain mutation analysis neogenomics laboratories. Since the first imatinibresistant cases, point mutations in the kinase domain kd of bcr abl were identified 16 that could impair or even totally abrogate. Mutation analysis of the kinase domain coding region of the bcrabl fusion protein. This assay is comprehensive for detecting bcrabl1 kd mutations, but does not detect all possible mutations in abl1. Clinical studies with the abl tyrosine kinase inhibitor sti571 in chronic myeloid leukemia demonstrate that many patients with advanced stage disease respond initially but then relapse. Bcrabl1 translocation in leukemia unc medical center. Mutations of the bcrabl kinase domain are a common mechanism of resistance to imatinib in chronic myeloid leukemia. Testing for the acquired mutations in the abl1 kinase domain will be performed by rtpcr of the bcr abl1 translocation followed by nested pcr of the abl1 kinase domain region and bidirectional sequencing to identify mutations associated with drug resistance.
The pan bcr abl kinase inhibitor ponatinib exhibits potent activity against native, t315i, and all other clinically relevant mutants, and showed better inhibition than the previously known. Among the 94 imatinib resistant cml patients, 37 39% patients had mutations in the abl kinase domain of the bcrabl gene. Mutations of the bcrabl tyrosine kinase domain constitute a major cause of. Based on the presence of mutation at tyrosine kinase domain of bcrabl1 gene, mutation free patients showed significantly higher overall survival compared to patients who showed the presence of m351. Pdf analysis of mutations in the bcrabl1 kinase domain. A dna sample of gum mucosa was negative for this type of mutation, which allowed us to conclude that acquired v280g variant was. Bcrabl1, tyrosine kinase inhibitor resistance, kinase. Mutation analysis of the kinase domain coding region of the bcrabl fusion protein using sanger fluorescent sequencing. Molecular screening and the clinical impacts of bcr. Novel mutations in the kinase domain of bcrabl gene causing. The frequency of mutations was higher in the p loop accounting to 43% of the total mutations in the kinase domain. Clinical significance of t315i abl kinase domain mutation. Mutations in the drug binding region of bcrabl lead to imatinib resistance during the management of chronic myeloid leukemia cml.
Optimal time points for bcrabl1 tyrosine kinase domain. In clonal selection, bcr abl mutated cells have a higher survival rate due to the selective pressure of imatinib therapy. The frequency of bcr abl1 gene mutations in the patients resistant to imatinib ranges from 40 to 90% depending on the definition of resistance, methodology of detection, and cml phase. Bcrabl point mutations and tki treatment in cml patients. Chronic myeloid leukemia cml is a myeloproliferative disease characterized by the presence of bcrabl fusion gene and consequently bcrabl fusion protein. Pdf computational analysis of abl kinase mutations allows. Mutations in the bcrabl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors tkis in chronic myeloid leukemia patients. The presence of point mutations in the abl1 kinase domain of the bcrabl1 fusion gene codons 221500 is assessed by direct sanger sequencing of the bcrabl1 fusion transcripts following nested. However, sanger sequencing has limited sensitivity and. Abl1 kinase domain mutation analysis md anderson cancer center. Sensitive detection of preexisting bcrabl kinase domain.
Through biochemical and molecular analysis of clinical material, we find that drug resistance is associated with the reactivation of bcr abl signal transduction in all cases examined. It has been shown that the occurrence of the bcrabl1 t315i mutation leads to a very poor therapeutic outcome in chronic myelogenous leukemia cml patients treated with. Sequences were analyzed with sequence analysis software v3. This is the preferred initial test to identify the presence of acquired bcrabl1 mutations associated with tyrosine kinase inhibitor tkiresistance shipping instructions. The most common mechanisms of acquired resistance to imatinib are bcrabl amplification at the genomic or transcript level and point mutations in the kinase domain.
We screened for mutations 171 patients failing imatinib therapy. This study highlights the need for bcrabl gene sequence analysis to detect the. Soverini s, hochhaus a, nicolini fe, gruber f, lange t, saglio g, pane f, muller mc, ernst t, rosti g, et al. Abl kinase domain mutations in patients with chronic myeloid. Candidates for the bcrabl1 kinase domain mutation analysis include. Absolute quantitative detection of abl tyrosine kinase domain. The mutation presented in this case report causes a substitution of a valine by a glycine at amino acid 280, in the kinase active domain, being the only alteration found in this patient after bcr. Mutations in the bcrabl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors tkis in chronic myeloid leukemia patie. Bcrabl kinase domain inhibition can be used to treat chronic myeloid leukemia. Soverini s, hochhaus a, nicolini fe, gruber f, lange t, saglio g, et al. Nextgeneration sequencing for bcrabl1 kinase domain. Cml patients with these four abl kinase domain mutations cannot achieve major.
Bcrabl1 kinase domain mutation analysis dmc university. Bcr abl kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. Conventional sanger sequencing is the method currently recommended to test bcr abl1 kd mutations. Comparison of mutation analysis in peripheral blood and bone marrow was performed in 39 pts. Mutation analysis of the kinase domain of the bcrabl. The metaanalysis of rdws diagnostic value was performed using stata software. The mutation presented in this case report causes a substitution of a valine by a glycine at amino acid 280, in the kinase active domain, being the only alteration found in this patient after bcr abl mutation analysis. On the basis of the eln guidelines, mutation analysis for abl kinase domain is recommended both in the case of failure and suboptimal response to imatinib. The inhibitors such as imatinib, dasatinib and nilotinib are effective drugs but are resistant to some bcr abl mutations. If the patients tumor burden is low, rtpcr may not generate enough of the bcrabl1 transcript for sequence analysis of the abl kinase domain. Diagnostic use indications patients philadephia chromosome positive displaying a lack of response to tryosine kinase inhibitor therapy, or a rising bcr abl1 transcript level or loss of a major molecular response 0.
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