Allele-Specific PCR for Detection of Missense Mutations in the Chimeric BCR::ABL1 Gene Causing Failure of Tyrosine Kinase Inhibitor Therapy in CML Patients

TL;DR

This study developed a fast and cost-effective method to detect specific mutations in CML patients that cause resistance to therapy.

Contribution

A novel allele-specific PCR method for detecting BCR::ABL1 mutations with high sensitivity and potential for clinical use.

Findings

AS-PCR reliably detects mutations at variant allele frequencies as low as 0.01%.

AS-PCR results were comparable to NGS for mutation detection and VAF estimation.

AS-PCR is a sensitive and efficient alternative to NGS for some clinical applications.

Abstract

Missense mutations in the BCR::ABL1 kinase domain are found in approximately 12–80% of patients with chronic myeloid leukemia (CML). Clinically significant mutations include T315I, M244V, Y253H/F, E255K/V, V299L, and F359V. The aim of this study was to create a diagnostic system for rapid and inexpensive detection of the above mutations. We used genomic DNA and RNA from peripheral blood and bone marrow cells of 57 patients with a Ph-positive CML diagnosis established in the chronic phase. We have developed a method to detect mutations in the BCR::ABL1 gene based on allele-specific real-time polymerase chain reaction (AS-PCR). In parallel, we analyzed the RNA sequence of the protein kinase domain of the same samples by next-generation sequencing (NGS) covering the points of putative mutations. In this work, we compared the results obtained by both methods for mutation detection and variant allele frequency (VAF) estimation of mutated vs. normal alleles. The sensitivity and specificity of our diagnostic system were also evaluated. It was found that AS-PCR gives reliable results at VAF up to 0.01%. AS-PCR has high sensitivity and may serve as an alternative for the more time-consuming NGS in some cases, as well as for monitoring CML treatment and for analyzing archival material.

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