Rapid and sensitive allele-specific (AS)-RT-PCR assay for detection of T315I mutation in chronic myeloid leukemia patients treated with tyrosine-kinase inhibitors

Arechavaleta, Gonzalo Manrique; Scholl, Vanesa; Bittencourt, Roberta; Moellmann, Arthur; Hassan, Rocı́o; Seuánez, Héctor N.; Dobbin, Jane; Martinez, Lem; Renault, Ilana Zalcberg; Uriarte, Rosario

doi:10.1007/s10238-010-0101-x

Cited by 10 publications

(7 citation statements)

References 12 publications

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“…Also, using the same technique and sets of primers that used in this study, Kang et al (2006) were reviled that 2.63% (1/38) of patients included in their study were positive to T315I mutation. Arechavaleta et al (2011) study referred to that 4.6% (3/65) of IM resistance CML patients were positive to this mutation using Allele Specific-Reverse Transcriptase-PCR technique. In Mir et al (2009) 12 IM resistance CML patients but they detected F317L mutation in 4/12 after treatment with dasatinib for 10ms.…”

Section: Discussionmentioning

confidence: 99%

Molecular Screening for T315I and F317L Resistance Mutations in Iraqi Chronic Myeloid Leukemia Non-Responders Patients to Imatinib

Dhahi¹,

Matti²,

Fadel³

2013

CCO

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The development of Imatinib Mesylate (IM), the first generation specific tyrosine kinase inhibitor (TKI) of BCR-ABL, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TKIs (SGTKIs), which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. The aim of this prospective study was to determine prevalence of T315I, which confers full resistance to all available tyrosine-kinase inhibitors, and F317l mutations, which classified as resistant mutation to IM and dasatinib but sensitive to nilotinb, in IM non-responder CML patients from Iraq with highly sensitive Allele-Specific Oligonucleotide-PCR assay (ASO-PCR). Forty four CML patients were recruited from Baghdad Teaching Hospitalet-Hematology Unite from Feb. 2009 to Feb. 2013. Those patients were followed up (hematological, cytogenetically, and molecularly). At the time of sampling, thirty four CML patients in advance phase(accelerated phase(AP) or blast crisis(BC)) or loss their cytogenetic response were IM non-responders while ten CML patients in CP were IM responder, considered as T315I and F317 L mutations negative control group. Ten IM non-responders patients out of those 34 patients were recruited to SGTKIs (Dasatinib and/or Nilotinib). The results shown that only T315I mutation was detected in one IM non-responder CML patient (3%). Although relation between response to SGTKIs and mutation status was non-significance (P= 0.100), mutation is limited to only one patient who did not respond to either treatment forms. In conclusion, the uses of bcr-abl mutation screening test for T315I and F317L mutations in CML patients may influences the choice of specific SGTKIs after treatment with IM has failed.

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Section: Discussionmentioning

confidence: 99%

Molecular Screening for T315I and F317L Resistance Mutations in Iraqi Chronic Myeloid Leukemia Non-Responders Patients to Imatinib

Dhahi¹,

Matti²,

Fadel³

2013

CCO

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“…Several methods have been utilized to detect the existence of BCR-ABL KD mutations such as direct sequencing, DHPLC, restriction fragment length polymorphism (RFLP), pyrosequencing, double-gradient denaturing electrophoresis, AS-PCR, AS real-time PCR, array based assays, and high-resolution melt curve analysis (HRM), with varying sensitivity and specificity [25,26,29-35]. In this study, we established an AS-PCR-based method to detect T315I which is the most resistant genotype associated with the highest impact on clinical outcome of CML patients.…”

Section: Discussionmentioning

confidence: 99%

A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients

2011

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Background BCR-ABL kinase domain (KD) mutation is the major mechanism contributing to suboptimal response to tyrosine kinase inhibitors (TKI) in BCR-ABL -positive chronic myeloid leukemia (CML) patients. T315I mutation, as one of the most frequent KD mutations, has been shown to be strongly associated with TKI resistance and subsequent therapeutic failure. A simple and sensitive method is thus required to detect T315I mutation at the earliest stage. Methods A single-tube allele specific-polymerase chain reaction (AS-PCR) method was developed to detect T315I mutation in a mixture of normal and mutant alleles of varying dilutions. Denaturing high performance liquid chromatography (DHPLC) and direct sequencing were performed as a comparison to AS-PCR. Results T315I mutant bands were observed in the mixtures containing as low as 0.5-1% of mutant alleles by AS-PCR. The detection sensitivity of DHPLC was around 1.5-3% dilution whereas sequencing analysis was unable to detect below 6.25% dilution. Conclusion A single-tube AS-PCR is a rapid and sensitive screening method for T315I mutation. Detection of the most resistant leukemic clone in CML patients undergoing TKI therapy should be feasible with this simple and inexpensive method.

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“…There may be a role for more sensitive mutation analysis, particularly when considering the lack of TKI effectiveness against T315I mutated clones (27) and when considering therapeutic options after IM resistance. However this has not yet been established.…”

Section: What Is the Meaning Of Fluctuating Bcr-abl Levels?mentioning

confidence: 99%

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

Renault¹,

Scholl²,

Hassan³

et al. 2011

Rev. Bras. Hematol. Hemoter.

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Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging.

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Rapid and sensitive allele-specific (AS)-RT-PCR assay for detection of T315I mutation in chronic myeloid leukemia patients treated with tyrosine-kinase inhibitors

Cited by 10 publications

References 12 publications

Molecular Screening for T315I and F317L Resistance Mutations in Iraqi Chronic Myeloid Leukemia Non-Responders Patients to Imatinib

Molecular Screening for T315I and F317L Resistance Mutations in Iraqi Chronic Myeloid Leukemia Non-Responders Patients to Imatinib

A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

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