|Year : 2017 | Volume
| Issue : 1 | Page : 7-11
Spectrum of BCR-ABL1 kinase domain mutations: A cohort study from Saudi Arabia
Halah Abalkhail, Salem Khalil
Department of Pathology and Laboratory Medicine, MBC 10 King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia
|Date of Web Publication||12-Apr-2017|
Department of Pathology and Laboratory Medicine, MBC 10King Faisal Specialist Hospital and Research Centre, Riyadh 11211
Kingdom of Saudi Arabia
Source of Support: None, Conflict of Interest: None
Background: The BCR-ABL1 tyrosine kinase domain mutation constitutes a major cause of resistance to the tyrosine kinase inhibitors in patients with chronic myeloid leukemia (CML). In this retrospective study, we assessed the ABL kinase domain mutation in 123 patients (61 females and 62 males) aged 10–79 years (median age of 50 years). These patients were referred to our clinics at King Faisal Specialist Hospital and Research Center (General Organization), Riyadh, Kingdom of Saudi Arabia during period (2011–2014). These patients had Philadelphia-positive CML displaying either failure to tyrosine kinase inhibitor (TKI) or suboptimal response with increased BCR-ABL1 levels through serial monitoring by using quantitative Real time PCR.
Methods: The mutation analysis was performed on RNA extracted from Peripheral blood samples after the amplification of the BCR-ABL1 transcript by nested PCR followed by direct sequencing of the BCR-ABL1 kinase domain including the residues (243–487).
Results: Of 123 patients, 119 adults and four pediatrics were analyzed. From the total, 25 (20%) were tested positive for 11 different mutations in the ABL1 kinase domain (11 patients with T315I, 3 with Y253H, 2 with E255K, 2 F317L, and 1 patient having each of the following mutations: F359I, E355G, V299L, L248V, L298, M244V, and Y326H). The duration from the diagnosis to mutation detection ranged between 3 and 144 months with a median duration of four years.
Conclusion: Despite the retrospective nature of the study and relatively small sample size of a single center analysis, the mutation frequency is in line with similar reported studies from other parts of the world.
Keywords: BCR-ABL1, chronic myeloid leukemia, philadelphia, sequencing, tyrosine kinase, tyrosine kinase inhibitor
|How to cite this article:|
Abalkhail H, Khalil S. Spectrum of BCR-ABL1 kinase domain mutations: A cohort study from Saudi Arabia. J Appl Hematol 2017;8:7-11
| Introduction|| |
Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm associated with a characteristic translocation between chromosomes 9 and 22, which results in the formation of Philadelphia (Ph) chromosome. The introduction of tyrosine kinase inhibitors (TKIs) imatinib mesylate for CML marked a new era for the targeted therapy. In general, the patients with CML in the chronic phase (CP) showed a good response to imatinib mesylate with an ∼85% projected overall rate of survival. However, a significant proportion of the general population harbored the molecular residual disease and developed acquired resistance after the initial response through a number of cellular and molecular mechanisms; this resistance may be due to the mutations in the ABL kinase domain. Mutations were detected in 35% of the patients with resistance to TKI in CP and up to 80% of the patients with accelerated phase (AP) and blast crisis (BC).
Given the clinical importance of the mutation status, the mutation analyses were established as a clinical service to aid in the clinical decision and identify the subset of the patients who would benefit from the alternative therapies.
Imatinib mesylate (STI571; Gleevic; Glivic; Novartis) is the first TKI approved by the US Food and Drug Administration (FDA) for the treatment of Ph-positive CML in the CP (Ph+ CML-CP). The results obtained from the 6-year follow-up study of the International Randomized Study of Interferon Plus Ara-C versus STI571 (IRIS) demonstrated an event-free survival rate of 83% and estimated the rate of freedom from progression to AP or BC to be 93%, associated with imatinib therapy.
In 2010, the second-generation TKIs, nilotinip (Tasigna, Novartis), and dasatinib (Sprycel; Bristol-Myers Squibb) were approved by the US FDA initially for the patients who developed resistance or intolerance to imatinib and also as first-line treatment for the patients newly diagnosed with CML-CP.,,,
According to the IRIS study, 24% of the patients treated with imatinib failed to achieve complete cytogenetic response within 18 months of the treatment. The treatment responses to imatinib at various time points showed variation among the patients with CML. The European LeukemiaNet (ELN) and National Comprehensive Cancer Network published the definitions of the treatment failure by the first-generation TKI with recommendations at specified time points, which are now being widely used. The ELN guidelines, also, defined suboptimal responses at specified time points.
The aim of this retrospective study was to identify the spectrum of BCR-ABL1 kinase domain mutations among the patients with CML in CP (CML/CP), who developed resistance to the first-generation TKI (imatinib) at our institution.
| Materials and Methods|| |
A total of 123 patients with CML, who were on imatinib therapy with signs of failure or suboptimal response to the treatment based on ELN guidelines, were tested for a possible resistance to the mutation in the ABL kinase domain. These patients were followed up at the King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia (General Organization). The duration from the diagnosis to the detection of the mutation ranged between 3 and 144 months with a median duration of 4 years.
This retrospective review study was approved by the Research Advisory Council and the hospital ethical committee under the RAC# 2131152.
RNA extraction and nested reverse transcription-PCR amplification
The mRNA was extracted from peripheral blood samples in EDTA using MagNa Pure 96 automated nucleic acid extraction system (Roche Diagnostics, Germany) according to the manufacturer’s instructions.
The extracted mRNA was subjected to one-step reverse transcription-PCR (using SuperScript III) using the following amplification primers: BCR F 5′-TGA CCA ACT CGT GTG TGA AAC TC-3′, BCR P 5′-CCT CGC AGA ACT CGC AAC A-3′, and ABL 5′-TCC ACT TCG TCT GAG ATA CTG GAT T-3′ followed by nested PCR using a forward 5′-CGC AAC AAG CCC ACT GTC T-3′ and a reverse 5′-TCC ACT TCG TCT GAG ATA CTG GAT T-3′ primers that anneal to the breakpoint cluster region (BCR) exon b2 and junction of ABL exons, 9 and 10, respectively.
The PCR amplification was confirmed by 1.5% agarose gel electrophoresis to check for an 863 base pair encompassing the ABL kinase domain. The amplified product was then excised from the gel and purified using the gel extraction kit (Qiagen) as per the manufacturer’s instructions.
The sequencing was performed on the eluted gel product using forward and reverse primers specific for the kinase domain of BCR/ABL fusion gene with a BigDye Terminator v3.1 cycle sequencing ready reaction kit (Applied Biosystems, CA, USA). The purification was performed by using Agencourt® CleanSEQ® kit (Beckman Coulter, CA, USA) according to the manufacturer’s instructions.
The direct sequencing was performed on ABI 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA), and the sequencing data were base-called, assembled, and analyzed by using SeqScape software by making a comparison with the Gene Bank accession NM_005157.4.,
| Results|| |
The BCR-ABL1 kinase domain mutations were detected in the peripheral blood samples of the 25 (20%) patients of the analyzed population (of the 123 patients) using direct sequencing of the entire domain proceeded by the nested RT-PCR. [Table 1] shows the types and frequencies of the BCR-ABL1 kinase domain mutations among the studied, 123 patients with CML at our institution. Eleven different point mutations were detected in the 25 patients (20%); among them, eleven patients were detected to have T315I, three patients had Y253H, two patients had E255K, and two more patients had F317L. Additionally, the following mutations were observed in one patient each: F359I, E355G, V299L, L248V, L298, M244V, and Y326H. [Figure 1] shows the spectrum and distribution of different mutations in the ABL-kinase domain regions among the cohort patients.
|Figure 1: Schematic diagram of ABL kinase domain mutations. *HSCT or investigational drugs, e.g. Ponatinib (Iclusig®). Nilotinib, Dasatinib, High-dose Imatinib, Dasatinib or Nilotinib.|
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The figure also highlights the most appropriate alternative therapeutic options based on the BCR-ABL1 kinase domain mutation status recommended by the ELN guidelines.
| Discussion|| |
During the last decade, intensive efforts have been made in the characterization of the biological and clinical significance of the ABL kinase domain mutations and the development of novel inhibitors retaining the efficacy against as many BCR-ABL1 mutant forms as possible.
The list of amino acid substitutions detected in patients, who are imatinib-resistant, has steadily grown to include more than 100 different mutations. Although some of these mutations have been found to be more frequent than the others, different mutations have shown to confer variable degrees of resistance to imatinib.,,,,,,
Apart from the quantitative BCR-ABL transcript analysis that provides essential data on the efficacy of TKI treatment, it is also important to detect the mutations. Of all the available molecular methods, the direct sequencing method is considered to be the most common and reliable approach in detecting the ABL kinase domain mutations.
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 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.
In the GIMEMA study, BCR-ABL mutations were found in 127 of the 297 evaluated patients (43%). The defects were found in 27% of the patients with CP (14% treated with imatinib frontline and 31% treated with imatinib after interferon failure), 52% with AP, 75% with myeloid BC, and 83% with lymphoid BC/Ph-positive acute lymphoblastic leukemia. The BCR-ABL mutations were documented in 30% of the patients with primary resistance (44% hematologic and 28% cytogenetic) and 57% of those with acquired resistance, including 23% who lost complete hematologic response, 55% who lost hematologic response, and 87% who progressed to AP or BC.
Despite a large spectrum of detected mutations, amino acid substitutions at seven residues (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) accounted for 85% of all the resistance-associated aberrations.
In this retrospective study of the 123 patients with CML in the CP on imatinib therapy, 11 different mutations across the ABL1 kinase domain mutations were detected, including in the order of frequency T315I, Y253H, E255K, F317L, F359I, E355G, V299L, L248V, L298, M244V, and Y326H.
The T315I mutation occurred in approximately 15% of the patients, who are imatinib-resistant, and these mutations mostly depended on the stage of the disease. The findings of our study concerning ABL kinase domain mutations in CML were in line with what had been described in the literature stating that the mutations found in 25 out of the 123 patients with CML/CP (20%) treated with imatinib, with T315I (9%) [Table 1], were more frequently seen.
A similar study from Jordan reported a detection rate of 11.35% (21/185) of the patients with CML in CP with T315I mutation being most frequently detected.
In one series, 87% of the patients with CP along with the T315I mutation were alive at 2 years, and many showed an indolent disease course. However, a more recent study suggested that the presence of T315I mutation in the patients with CP-CML compromises the OS and PFS rates.
| Conclusion|| |
Because of the ineffectiveness of imatinib, nilotinib, dasatinib, and bosutinib against the T315I mutation, there are limited treatment options for the patients with T315I mutations. Although an allogeneic stem-cell transplantation is potentially curative for these patients, ponatinib (AP24534), an orally available TKI, is a potent pan-Bcr-Abl inhibitor with activity against all the tested imatinib-resistant mutants, including T315I.,, Despite the regular follow-up of the patients from the time of CML diagnosis, further follow-up is also needed to identify the course of the ABL kinase mutations and the outcomes faced by these patients.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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