beta-Glucuronidase is an optimal normalization control gene for molecular monitoring of chronic myelogenous leukemia

Abstract

Quantitative monitoring of breakpoint cluster region (BCR)-Abelson kinase (ABL) transcripts has become indispensable in the clinical care of patients with chronic myelogenous leukemia. Because quantity and quality of RNA in clinical samples are highly variable, a suitable internal normalization control is required for accurate BCR-ABL quantification. However, few studies have examined suitability of the control genes using criteria relevant to residual disease testing. In this study, we evaluated a number of control genes with the application of several novel criteria, including control gene performance on serial patient sample testing and in a residual disease model. We also examined expression of the control genes in BCR-ABL-positive K562 cells in response to Gleevec treatment. We found that beta-glucuronidase is the best control gene among those studied. Importantly, ABL, a widely used control gene, generates misleading BCR-ABL changes that potentially affect the clinical management of chronic myelogenous leukemia patients.

Figure 1

Schematic diagram of the two different sets of primers/probes for ABL quantification (left, ABL1; right, ABL2). Light shaded cylinders represent ABL cDNA, and dark shaded cylinders represent BCR cDNA with exons indicated. Numbers below cDNAs indicate nucleotide positions at exon boundaries. Arrows represent PCR primers and their relative positions to ABL and BCR-ABL cDNAs. Black bars represent the TaqMan probes and their positions. Sequences of primers and probes and their locations are shown under each diagram. Left: The forward primer of ABL1 set hybridizes to the exon 1, and the reverse primer and probe hybridize to exon 2 of the ABL gene. Because the breakpoints mostly occur in the intron between exons 1 and 2, the ABL1 set therefore detects only the wild-type allele of the ABL gene. No PCR products are generated once ABL is fused to BCR. Right: In comparison, the forward primer of ABL2 set hybridizes to exon 2, and the reverse primer and probe hybridize to exon 3 of the ABL gene. It therefore detects both the wild-type ABL and translocated BCR-ABL messages. (Reprinted from J Mol Diagn 2006, 8:231–239 with permission from the American Society for Investigative Pathology and the Association for Molecular Pathology.)

Figure 2

Performance of the control gene in serial sample testing. BCR-ABL was first normalized to each control gene, as indicated in the legend box, to obtain R_first and R_second for the first and second serial samples. Fold reduction from the first to the second samples were then calculated to obtain R = R_first/R_second.

Figure 3

Performance of the control genes in a residual disease model. BCR-ABL was first normalized to each control gene, as indicated on the x axis, for pure and diluted samples to generate R_Pure and R_Diluted. The ratio of the amount of BCR-ABL transcript in pure patient samples and the corresponding 1:16 diluted samples were then calculated as R′ = R_Pure/R_Diluted. The horizontal line intercepts with the y axis at 16.

Figure 4

Performance of the control genes in response to Gleevec treatment. K562 cells (1 × 10⁶/ml) were treated with 1 μmol/L Gleevec. Fresh medium containing 1 μmol/L Gleevec was added to the culture every 48 hours to maintain the cell density and nutrition balance. Cells were collected for RT-PCR analysis before and at indicated times after treatment. Data plotted are mean ± SD of the three independent experiments.