Detection and Quantification of the Abelson Tyrosine Kinase Domains of the bcr-abl Gene Translocation in Chronic Myeloid Leukaemia Using Genomic Quantitative Real-time Polymerase Chain Reaction

ABSTRACT

Introduction: Since undetectable BCR-ABL mRNA transcription does not always indicate eradication of the Ph⁺ CML clone and since transcriptionally silent Ph⁺ CML cells exist, quantitation by genomic PCR of bcr-abl genes can be clinically useful. Furthermore, hotspot mutations in the Abelson tyrosine kinase (ABLK) domain of the bcr-abl gene translocation in Philadelphia chromosome-positive (Ph⁺) chronic myeloid leukaemia (CML) cells confer resistance on the specific kinase blocking agent, STI571.

Materials and Methods: Genomic DNA from K562, CESS and patient CML cells were amplified using rapid cycle quantitative real-time polymerase chain reaction for the gene regions spanning the mutation hotspots. In assays for ABLK exons 4 or 6, exonic or intronic PCR primers were used.

Results: We show that separation of cycle threshold (CT) values for log-fold amplicon quantification was 2.9 cycles for ABLK exon 4, and 3.8 cycles for exon 6 with rapid amplification times. K562 CML cells were found to have a ~2 log-fold ABLK gene amplification. In contrast, patient CML cells had CT differences of 2.2 for both exon, suggesting that there was no significant ABLK gene amplification. DNA sequencing confirmed that neither K562 nor patient CML cells contained ABLK hotspot mutations. Messenger RNA transcription analysis permitted the assessment of BCR-ABL transcription, which was qualitatively correlated to genomic amplification.

Conclusions: This novel Q-PCR assay was found to have high fidelity and legitimacy, and potentially useful for monitoring minimal residual disease, transcriptionally silent Ph⁺ CML cells, and bcr-abl gene amplification.

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