Competitive polymerase chain reaction as a method to detect the amplification of bcr-abl gene of chronic myeloid leukemia

Abstract

BACKGROUND AND OBJECTIVES: The chimeric product of the bcr-abl rearranged gene is critical in the pathogenesis of chronic myeloid leukemia (CML), yet its role in the progression of the disease remains unclear. There is some evidence that increased bcr-abl expression levels, possibly due to gene amplification, precede the clonal evolution of CML hematopoietic progenitors toward a fully transformed phenotype and might be involved in their resistance to interferon-alpha or tyrosine kinase inhibitors. DESIGN AND METHODS: To quantify the bcr-abl gene both at the genomic and at the transcriptional levels we developed a competitive polymerase chain reaction (PCR) strategy. The competitive PCR technique is based upon the co-amplification of the sample template (target) together with increasing amounts of a DNA fragment (competitor) sharing with the target the primer recognition sites, but differing in size. We constructed a competitor for the quantification of both b2a2 and b3a2 alternative splicing forms of the bcr-abl chimera and established the accuracy and reproducibility of our competitive strategy in a clone of the murine 32DG hematopoietic cell line (32D LG7), which bears a stable integration of a single copy of p210 bcr-abl fusion gene. We utilized this technique to follow, over a period of 200 days, the fusion gene copy numbers and transcription rates in several p210 bcr-abl-transduced 32D cell clones, an experimental condition mimicking the evolution of CML myeloid progenitors in vivo. RESULTS: Our results are consistent with p210 bcr-abl overexpression but not gene amplification associated with their clonal evolution. Increased p210 bcr-abl transcription rate is associated with the abrogation of radiation-induced apoptotic cell death, suggesting a role for the chimeric gene expression level in cell life expectancy after a genotoxic insult. INTERPRETATION AND CONCLUSIONS: We conclude that the assessment of gene amplification and expression might serve to improve prognostic classification and follow-up of CML patients.