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

Wongboonma W, Thongnoppakhun W, Auewarakul CU - J Hematol Oncol (2011)

Bottom Line: T315I mutation, as one of the most frequent KD mutations, has been shown to be strongly associated with TKI resistance and subsequent therapeutic failure.T315I mutant bands were observed in the mixtures containing as low as 0.5-1% of mutant alleles by AS-PCR.Detection of the most resistant leukemic clone in CML patients undergoing TKI therapy should be feasible with this simple and inexpensive method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

ABSTRACT

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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Related in: MedlinePlus

Sensitivity of T315I mutation detection by AS-PCR method . Serial dilutions of T315I mutants with wild-type cells demonstrates 158-bp mutant bands in 100%, 25%, 10%, 1%, and 0.5% mixtures (Figure 1A); Representative samples of T315I mutated cell lines (Lane 1), T315I mutated CML case, (Lane 2), seven non-mutated non-leukemic cases (Lanes 3-9), and BA/F3 cell lines (Lane 10) are shown in Figure 1B; Lane 11, blank; Lane M, a 100-bp DNA marker.
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Figure 1: Sensitivity of T315I mutation detection by AS-PCR method . Serial dilutions of T315I mutants with wild-type cells demonstrates 158-bp mutant bands in 100%, 25%, 10%, 1%, and 0.5% mixtures (Figure 1A); Representative samples of T315I mutated cell lines (Lane 1), T315I mutated CML case, (Lane 2), seven non-mutated non-leukemic cases (Lanes 3-9), and BA/F3 cell lines (Lane 10) are shown in Figure 1B; Lane 11, blank; Lane M, a 100-bp DNA marker.

Mentions: AS-PCR was designed to specifically detect T315I mutations using the cDNA templates synthesized from RNA with known percentages of the mutant allele. Mutants, WT, and internal controls could be detected in a single reaction. We first optimized the annealing temperature and the ratio of each primer pair and the results of our triplicate independent experiments demonstrated that the optimal annealing temperature was 57°C with the primers ratio of 7:3:0.5 of mutants, WT, and internal control primer pairs, respectively. A 158-bp PCR product was derived from the mutant allele whereas a 374-bp PCR product could represent a heterozygous allele or no mutant allele and a 540-bp product was an internal control. In three independent experiments, a strong T315I mutant band was detected in as low as 1% dilution and a faint band was observed in 0.5% dilution (Figure 1). In addition, 30 samples from non-leukemic patients and WT BA/F3 cell lines were also tested to ensure our AS-PCR's specificity; all of which were found negative for T315I, therefore, BA/F3 cell lines were subsequently utilized as a T315I negative control.


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

Wongboonma W, Thongnoppakhun W, Auewarakul CU - J Hematol Oncol (2011)

Sensitivity of T315I mutation detection by AS-PCR method . Serial dilutions of T315I mutants with wild-type cells demonstrates 158-bp mutant bands in 100%, 25%, 10%, 1%, and 0.5% mixtures (Figure 1A); Representative samples of T315I mutated cell lines (Lane 1), T315I mutated CML case, (Lane 2), seven non-mutated non-leukemic cases (Lanes 3-9), and BA/F3 cell lines (Lane 10) are shown in Figure 1B; Lane 11, blank; Lane M, a 100-bp DNA marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045387&req=5

Figure 1: Sensitivity of T315I mutation detection by AS-PCR method . Serial dilutions of T315I mutants with wild-type cells demonstrates 158-bp mutant bands in 100%, 25%, 10%, 1%, and 0.5% mixtures (Figure 1A); Representative samples of T315I mutated cell lines (Lane 1), T315I mutated CML case, (Lane 2), seven non-mutated non-leukemic cases (Lanes 3-9), and BA/F3 cell lines (Lane 10) are shown in Figure 1B; Lane 11, blank; Lane M, a 100-bp DNA marker.
Mentions: AS-PCR was designed to specifically detect T315I mutations using the cDNA templates synthesized from RNA with known percentages of the mutant allele. Mutants, WT, and internal controls could be detected in a single reaction. We first optimized the annealing temperature and the ratio of each primer pair and the results of our triplicate independent experiments demonstrated that the optimal annealing temperature was 57°C with the primers ratio of 7:3:0.5 of mutants, WT, and internal control primer pairs, respectively. A 158-bp PCR product was derived from the mutant allele whereas a 374-bp PCR product could represent a heterozygous allele or no mutant allele and a 540-bp product was an internal control. In three independent experiments, a strong T315I mutant band was detected in as low as 1% dilution and a faint band was observed in 0.5% dilution (Figure 1). In addition, 30 samples from non-leukemic patients and WT BA/F3 cell lines were also tested to ensure our AS-PCR's specificity; all of which were found negative for T315I, therefore, BA/F3 cell lines were subsequently utilized as a T315I negative control.

Bottom Line: T315I mutation, as one of the most frequent KD mutations, has been shown to be strongly associated with TKI resistance and subsequent therapeutic failure.T315I mutant bands were observed in the mixtures containing as low as 0.5-1% of mutant alleles by AS-PCR.Detection of the most resistant leukemic clone in CML patients undergoing TKI therapy should be feasible with this simple and inexpensive method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus