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

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.

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.

Figure 2

Sensitivity of T315I mutation detection by DHPLC and sequencing analysis . DHPLC chromatogram patterns generated by each mutant allele concentration are shown in Figure 2A and sequencing results corresponding to each DHPLC-generated chromatogram are shown in Figure 2B; Red arrow indicates c.947 C > T mutation; C, Wild-type; T, Mutant.

Figure 3

Detection of T315I in CML patient samples by DHPLC, sequencing and AS-PCR; Figure 3A shows DHPLC patterns followed by sequencing analysis if a suspicious peak was observed; T315I cell lines and wild types are also shown; Figure 3B demonstrates representative AS-PCR results of four CML cases with abnormal DHPLC and sequencing results (patients no.360, no.461, no.504, and no. 509) .