Bcr abl1 transcript level results of the patients were recorded retrospectively. Kinase inhibitors have ushered in an era of targeted therapeutics in oncology. Practical management of patients with chronic myeloid. Atypical myeloproliferative neoplasm with concurrent bcrabl. Nccn guidelines recommend bcr abl1 kinase domain mutation analysis but do not specify what type of testing should be done or how clinicians should weigh these test results when making. Pdf mutational analysis in bcr abl1 positive leukemia by. In all the imatinibresistant patients bcrabl gene was pcr amplified. Bcrabl1 kinase domain mutation analysis dmc university. Therapeutic response to initial treatment is indicated by a drop in the bcr abl1 level. Prevalence of bcr abl1 kd mutations in cml patients. A 57yearold male who had an 11year history of essential thrombocytosis presented to our hospital with leukocytosis and marked splenomegaly for 3 months. The result is that a fusion gene is created by juxtaposing the abl1 gene on chromosome 9 region q34 to a part of the bcr breakpoint cluster region gene on chromosome 22 region q11.
Bcr abl1 translocation in leukemia bcr abl1 transcript levels serve as a marker of tumor burden in leukemia patients by which to monitor efficacy of targeted therapy. To determine the most appropriate time points for mutation analysis, the frequency of bcr abl1 mutation at 3, 6, and 12 months was analyzed. The threshold of bcr abl1 halving time in our analysis was also different from the reports, which may be due to the differences in the number of specimens and the study in different regions. The ratio of bcrablabl expression in post nilotinib treatment was 0.
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. Point mutations within the kinase domain kd of bcrabl, clonal. Nextgeneration sequencing for bcr abl kd mutation screening in philadelphia chromosomepositive leukemias soon after the introduction of tyrosine kinase inhibitors tkis for the treatment of. Nextgeneration sequencing ngs is the only exception. Novel mutations in the kinase domain of bcrabl gene. Mutations in the bcrabl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors tkis in chronic myeloid leukemia patie. After 8 months of nilotinib treatment, mutation analysis of bcr abl kd revealed selection of a second mutation e255v along with the first mutation f317l. Chronic myeloid leukemia, bcr abl1 positive, is a myeloproliferative neoplasm mpn in which granulocytes are the major proliferative component. To explore the clinical features of the patients with bcr abl1 negative chronic myeloproliferative neoplasms mpns in our hospital and to reveal the unique features of bcr abl1 negative mpns patients in our center. Coexistence of jak2 and bcrabl mutation in patient with. The test turned out positive for bcr abl1 e14a2ea2 61. Analysis of the effects of untranslocated abl1 in the sample background on detection and quantification of the bcr abl1 t315i mutation by pyrosequencing.
Bcrabl1 transcript decline ratio combined bcrabl1is as a. Molecular diagnostics lab t315i bcrabl mutation analysis. Conformational control inhibition of the bcrabl1 tyrosine. Bcrabl1 kd mutations and compare the results to a ssbased test in. The quantitative level of bcr abl1 transcript is critical for a successful assay mutation analysis, because the amplification efficiency for a longer mrna template is decreased with a low abundance of target. 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. This test is used to determine if a mutation is present that would interfere. Hyperdiploidy associated with t315i mutation in bcrabl. Chronic myelogenous leukemia chronic myelogenous leukemia cml is characterized by the philadelphia chromosome ph, a shortened chromosome 22 resulting from a t9. 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.
Bcr abl1 testing is ordered to detect the philadelphia ph chromosome and bcr abl1 gene sequence. A pyrosequencingbased test for detection and relative. All calculations were performed using comprehensive meta analysis software, version 2. According to the eln and the esmo recommendations, bcr abl1 kd mutation analysis should be performed during secondline treatment in case of hematologic or cytogenetic failure, whereas based on the nccn guidelines, mutation analysis should also be performed in case of loss of response, inadequate response, or increased bcr abl1 transcript levels. Mutations within the bcrabl1 kinase domain in imatinibtreated chronic myeloid leukemia are the main mechanism of acquired resistance. Order only for patients with an established diagnosis of a bcrabl1 positive leukemia.
The early detection of mutations should provide clinical benefit by allowing early intervention. 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. 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. All calculations were performed with the spss software version 24. But even in the best responders, some bcr abl1 positive cells usually remain. Concurrent calreticulin calr mutation and bcr abl1 fusion are extremely rare in chronic myelogenous leukemia. Bcr abl mutation analysis preparation, procedure, cost. Ahi1 gene expression levels and bcrabl1 t315i mutations in. If the bcr abl1 quantitative pcr level is too low, rtpcr amplification of bcr abl1 may be. Its a qpcrbased in vitro diagnostic test for the quantitation of bcr abl1 and abl1 transcripts in total rna from whole. Molecular analysis through qpcr is will measure levels of bcr abl1 transcripts to determine if nccndefined molecular response milestones are being met. For realtime quantitative pcr analysis of control gene, fusion gene, or mutated gene transcripts.
In addition, newer technologies with greater sensitivity are available, but most of them are limited by their specificity for a definite and limited spectrum of mutations. Monitoring bcr abl1 fusion transcripts by realtime quantitative rtpcr has become an important clinical test for the management of patients with chronic myeloid leukemia. However, mutations may become undetectable after changing therapy, and it is unknown whether they have. Several studies reported that a dnabased assay enhances the sensitivity of detection of the bcr abl1 genomic rearrangement, even if its characterization results difficult.
Overall, response mutation association analysis was conducted using 18 response criteria. Bcrabl1 transcript decline ratio combined bcr abl1 as a. Bcrabl kinase domain mutation analysis in chronic myeloid. Association between bcrabl tyrosine kinase inhibitors for. We developed a dnabased method for detecting and quantifying residual bcr abl1 positive leukemic stem cells in. In chronic myeloid leukemia cml, bcr abl1 tyrosine kinase inhibitors tkis such as imatinib are now standard treatment, but acquired resistance due to kinase domain mutations is a significant problem, particularly the t315i gatekeeper mutation that causes resistance to all approved tkis. Bcr abl1 kd mutation analysis can be conducted by direct sanger sequencing, which has been largely used todate. However, mutations may become undetectable after changing. Incidence and clinical importance of bcrabl1 mutations in. In addition, the total incidence rate of abnormalities in 172 patients was 7. Nextgeneration sequencing for bcrabl1 kinase domain mutation.
The aim was to detect the frequency of bcrabl kd mutations using a sequence analysis method to evaluate the clinical significance of the identified mutation. Bcrabl mutation testing to predict response to tyrosine kinase. T315i bcrabl mutation analysis quantitative indication. This study highlights the need for bcrabl gene sequence analysis to. Ahi1 gene expression levels and bcr abl1 t315i mutations in chronic myeloid leukemia patients tugce bulakbasi balci1, feride iffet sahin1, sema karakus2, hakan ozdogu2 1department of medical genetics, 2department of hematology, faculty of medicine, baskent university, ankara, turkey with the availability of molecular monitoring of bcr abl1 and the use of tyrosine kinase inhibitors, treatment. The clonal evolution of two distinct t315ipositive bcr. Bcrabl1 mutation analysis for tyrosine kinase inhibitor. 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. Discordant results implied the existence of uncommon bcr. Calr exon 9 mutation analysis pcr genotyping of calr exon 9 mutations was performed according to klampfl et al. Several types of tests may be ordered to detect bcr abl1.
In order to quantitate the e19a2 bcr abl1 transcript and monitor response to treatment, a bcr exon 19 forward primer was designed enf007. Chronic myelogenous leukemia cml is a hematopoietic stem cell disorder characterized by the philadelphia chormosome, the result of a 9. Ponatinib, blinatumomab, chemotherapy and transplant are the. Bcrabl1 compound mutations combining key kinase domain. Abl1 transcripts were simultaneously performed in patients undergoing bcr.
Abl1 tyrosine kinase inhibitors sensitivity 1020% mutant allele related assays onestep bcrabl1 quantification p210 and p190 bcrabl1 breakpoint analysis molecularmd has designed and validated bidirectional direct sanger sequencing assays to accurately identify mutations associated with bcrabl1 kinase therapy resistance. Unlike most cancers, the cause of chronic myelogenous leukemia cml and some other leukemias can be traced to a single, specific genetic abnormality in one chromosome. Ahi1 gene expression levels and bcrabl1 t315i mutations. What degree of increase in bcr abl1 transcript suggests secondary resistance.
Chronic myeloid leukemia cml is characterized by the philadelphia chromosome ph and fusion oncogene bcr abl1 and is currently very treatable with tyrosine kinase inhibitors tkis that inhibit the tyrosine kinase activity of the chimeric bcr abl1 protein. Bcrabl kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. Furthermore, the current study intended to emphasize the importance of mutation analysis and support using the asopcr method in drug selection and disease followup. Mutations in the drug binding region of bcrabl lead to imatinib resistance during the management of chronic myeloid leukemia cml. The presence of the gene sequence known as bcrabl1 confirms the diagnosis of cml and a form of acute lymphoblastic lymphoma all. The abl1 gene provides instructions for making a protein involved in many processes in cells throughout the body. Ponatinib induces a sustained deep molecular response in a. Pcrbased pyrosequencing of rna is performed to examine mutational status of codons 315 of the bcrabl fusion transcript. Optimal time points for bcr abl1 tyrosine kinase domain mutation analysis on the basis of european leukemianet recommendations in chronic myeloid leukemia healyun yoo,1 soohyun kim,1 sooyoung choi,1 sungeun lee,2 dongwook kim1,2 abstract to determine appropriate time points for mutation analysis of chronic myeloid leukemia cml patients, the.
In the case of polyclonal mutations, these bcr abl1 mutations green and red stars. Abl1 transcript analysis for the first time at our hospital. Pdf analysis of mutations in the bcrabl1 kinase domain. Our findings demonstrate multiple advantages in using minion approach.
Contractor information contractor name contract typecontract number jurisdictionstates noridian healthcare solutions, llc. A patient with f317l was put on 600mgday nilotinib after this mutation was detected. Improvement in the lower limit of detection could aid clinicians in selecting candidates for discontinuation. The current gold standard for bcr abl1 mutation detection is sanger sequencing ss, which has an analytical sensitivity of approximately 1020%. Bcrabl1 mutations in patients with imatinibresistant. Genetic testing for bcr abl negative myeloproliferative disease l36180 links in pdf documents are not guaranteed to work. The meta analysis was performed using a fixed effects model fem based on the assumption of a similar effect measure between studies. Clinicalandhematologicalrelevanceof jak2 v617fand calr. Full text a novel bcrabl1 mutation in a patient with. Molecular screening and the clinical impacts of bcrabl kd. Mutation analysis was performed when imatinib resistance occurred. They carry information that determines your unique traits, such as height and eye color. What is the international scale for standardization of quantitative bcr abl1 result reporting. Bcrabl expression levels determine the rate of development of resistance to imatinib mesylate in chronic myeloid leukemia.
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. In brief, pcr condition had the following parameters. After 8 months of nilotinib treatment, mutation analysis of bcrabl kd revealed selection of a second mutation e255v along with the first mutation f317l. Effect of lowlevel bcrabl1 kinase domain mutations. Bcrabl1, tyrosine kinase inhibitor resistance, kinase. 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. Novel mutations in the kinase domain of bcrabl gene causing. The study group consisted of newly diagnosed patients,18 months since diagnosis n511, patients who were diagnosed over 18 months ago and negative for bcr abl1 transcript.
The abl1 protein functions as a kinase, which is an enzyme that changes the activity of other proteins by adding a cluster of oxygen and phosphorus atoms a phosphate group at specific positions. Direct sequencing to detect bcr abl1 kd mutations is commonly used, however its sensitivity is limited. Bcrabl1, tyrosine kinase inhibitor resistance, kinase domain. These include chromosome analysis, bcr abl1 molecular genetic test, andor fluorescence in situ hybridization fish. Chromosomes are the parts of your cells that contain your genes. In fact, the prevalence of bcr abl1 kd mutations widely varies from 19% to 90% according to the reports, which used different analysis methods and various time points for sample collections. Bcrabl1 testing detects the presence of the bcrabl1 gene sequence in an abnormal chromosome 22 to help diagnose chronic. The ea2 and e14a2 transcripts formed by breakpoints occurring around exon and exon 14 of the bcr gene respectively are the most common. Aug 04, 2011 bcrabl kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. The statistical analysis using a random effects model is also presented for consistency. Retrospective analysis of routine karyotype analysis results, driver gene mutations and other related clinical parameters of 172 patients with newly diagnosed bcr abl1 negative. We undertook a retrospective audit using local laboratory database and electronic patient care records of 69 cml patients with an ea2 or e14a2. Bcr abl1 mutation analysis is recommended for chronic myeloid leukaemia patients. Several mechanisms may contribute to imatinib resistance in cml cells, including changes in bcr abl1 expression, mutation s in the bcr abl1 gene preventing imatinib binding, deregulation of imatinib cellular exportimport, and switching on bcr abl1 independent prosurvival mechanisms.
Twentytwo patients with cytogenetic or hematologic resistance and 8 patients with optimal response to im were studied for mutational analysis. In a subset of patients who develop clinical resistance to abl1 tkis, more than 1 point mutation in the kinase domain of bcr abl1 is detectable by direct sequencing. The patient was then commenced on imatinib at 400 mg once daily. Mutation analysis of the kinase domain of the bcrabl. In the current study, all uses of the term compound mutation. Bcrabl1 mutation analysis for tyrosine kinase inhibitor resistance by next generation sequencing 2008420 order only for patients with an established diagnosis of a. Our goal was to develop and implement a clinicalgrade bcr abl1 kinase domain mutation detection assay and undertake serial screening in patients with chronic myeloid leukaemia, irrespective of their response to tyrosine kinase inhibitor therapy. Over 100 bcr abl1 kinase domain point mutations have been linked with clinical imatinib resistance apperley, 2007, and resistance profiles for newer bcr abl1 tkis are mainly comprised of subsets of these mutations. Mutational analysis in bcrabl1 positive leukemia by deep. Atypical myeloproliferative neoplasm with concurrent bcr. Assessing complete molecular response requires the highest possible assay sensitivity. Bcr abl1 testing international scale common questions.
Pdf mutational analysis in bcr abl1 positive leukemia. It arises in a hematopoietic stem cell and is characterized by the chromosomal translocation t9. For quantitative detection of the jak2 v617fg1849t mutation. Imatinib resistance mutation analysis prima medgenome. Our study also highlighted that the importance of halving time of early bcr abl1 transcript in the patients with cmlcp on outcome prediction. 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. If the patients tumor burden is low, rtpcr may not generate enough of the bcrabl1 transcript for sequence analysis of the abl kinase. Reactive oxygen species and mitochondrial dna damage and. Direct sequencing is widely available and can utilize the same. The incidence of clonal chromosomal abnormalities in patients with pmf was 25% 624, that in patients with pv was 6. Patients who initially responded well to imatinib have at times relapsed due to the development of resistance caused by changes in the abl1 kinase domain. Molecular analysis showed the presence of an e19a2 bcr abl1 transcript, but no cytogenetic analysis was performed.
Optimal time points for bcrabl1 tyrosine kinase domain. The sequencher program, used for the test and the electropherogram, is produced by gene. Retrospective analysis of the clinical features of 172. Jan 17, 20 in a subset of patients who develop clinical resistance to abl1 tkis, more than 1 point mutation in the kinase domain of bcr abl1 is detectable by direct sequencing. Testing for mutations in bcrabl1 may predict lack of response to.
Between feb 1, 2007, and dec 31, 2014, we screened 121 patients with chronic myeloid leukaemia for bcr abl1 kinase domain mutation. For the quantification of bcr abl1 mbcr b3a2 and b2a2 fusion gene transcripts, results alignment on the international scale, mmr and mr4. A bcrabl genetic test looks for a genetic mutation change on a specific chromosome. Candidates for the bcrabl1 kinase domain mutation analysis include. Although routine mutation analysis of cmlcp patients treated with firstline tkis is not specifically recommended because the bcr abl1 mutation is relatively rare and can appear rapidly, it is important to determine the exact frequency of mutation according to the milestone responses at specific time points. The remaining 22 patients were diagnosed between june 1, 1999, and june 30, 2006, and were screened at the time of resistance to firstline treatment with imatinib.
Rtpcr and sequencing of the bcrabl1 fusion transcript for qualitative detection of mutations associated with resistance to gleevec imatinib and other. Imatinib is still prevalently used in clinical practice as the firstline treatment for newly diagnosed cml patients. Pdf mutational analysis in bcr abl1 positive leukemia by deep. Direct sequencing is widely available and can utilize the same sample. Abl1 kinase domain mutation analysis neogenomics laboratories. Genes are parts of dna passed down from your mother and father. Although the nccn clinical practice guidelines in oncology nccn guidelines do not specify a preferred method for mutational testing, direct sequencing remains the gold standard for bcrabl1 mutational analysis, and is recommended by an international consensus panel. The bcrabl1 mutation analyses performed in this study are represented diagrammatically in supplemental figure 1. These help establish the initial diagnosis of cml or phpositive all. The quantidex qpcr bcrabl is kit takes chronic myeloid leukemia cml monitoring to a new level of sensitivity 0. Among them, 37 patients were excluded in the data analysis because of 4 delaying in molecular monitoring or with the bcr abl1 kinase domain mutation.
In this study, our aim was to detect low level bcr abl1 variants in follow up samples of cml patients with tki resistance using. Bcrabl1 tyrosine kinase inhibitors for the treatment of. Serial screening of both patients who respond to and those who are resistant to tyrosine kinase. Genotypes of slc22a4 and slc22a5 regulatory loci are. The clonal evolution of two distinct t315ipositive bcrabl1. The chromosomal defect in the philadelphia chromosome is a reciprocal translocation, in which parts of two chromosomes, 9 and 22, swap places. Implications of bcrabl1 kinase domainmediated resistance in. Nextgeneration sequencing of the bcrabl1 kinase domain. Theres only one way to detect complete molecular response cmr with a more sensitive assay. The bcr abl1 fusion gene underlying the pathogenesis of cml can arise from a variety of breakpoints. Effective for services performed on or after 12014, coverage requirements for this test is addressed in cgss local coverage determination lcd for molecular diagnostic tests l35394.
Bcrabl1 kd mutation testing by ngs is indicated in cp cml after. Response to imatinib therapy is inferior for ea2 bcr. Analysis includes detection of the common t315i mutation. Majority of the patients were benefited by the dasatinib treatment without selection of a second mutation. However, it has some inherent limitations with regard to its lower limit of detection and limit of quantification. Key bcr abl1 kinase domain positions are frequently represented in clinically reported compound mutants. Rtpcr and sequencing of the bcrabl1 fusion transcript for qualitative detection of mutations associated with resistance to gleevec imatinib and other tyrosine kinase inhibitors. A subsequent 5 to 10fold increase in the % is level suggests developing drug resistance, which is usually due to an abl1 mutation or clonal evolution. Abl1 kinase domain mutations are screened by sequencing of seminested pcr products encompassing the abl1 kinase domain.
Evolution of bcrabl gene mutation in cml is time dependent. Bcrabl1 translocation in leukemia unc medical center. Genomic bcrabl1 breakpoint characterization by a multi. For monitoring of the levels of the thr to ile mutation at codon 315 t315i of the bcrabl kinase seen in cml patients who have developed resistance to imatinib or other kinase inhibitors. We compared a laboratorydeveloped nanoporebased bcr abl1 mutation test to the traditional ss method. Cytogenetic analysis of 172 patients with bcr abl1 negative mpns. Finally, the mutation load allele burden was calculated using allele quantification aq software. If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Bcrabl1 compound mutations in tyrosine kinase inhibitor. Outcomes of 219 chronic myeloid leukaemia patients with. 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.
Genetic testing for bcrabl negative myeloproliferative disease l36180 links in pdf documents are not guaranteed to work. The remaining 22 patients were diagnosed between june 1, 1999, and june 30, 2006, and were screened at the time of. Although the nccn clinical practice guidelines in oncology nccn guidelines do not specify a preferred method for mutational testing, direct sequencing remains the gold standard for bcr abl1 mutational analysis, and is recommended by an international consensus panel. For monitoring of the levels of the thr to ile mutation at codon 315 t315i of the bcrabl kinase seen in cml patients who have developed resistance to imatinib. Molecular screening and the clinical impacts of bcr. Our data indicates that minion is markedly superior to ss in terms of sensitivity, costs and timesaving, and has the.