Evaluation of the Cepheid GeneXpert BCR-ABL assay

Abstract

Patients with chronic myeloid leukemia harbor the chromosomal translocation t(9;22), which corresponds to fusion of the BCR and ABL genes at the DNA level. The translated fusion product is an oncogenic protein with increased ABL tyrosine kinase activity causing cell transformation. To date, reverse transcriptase-polymerase chain reaction is considered the most sensitive method available for detecting low copy numbers of the BCR-ABL gene fusion. Recently, Cepheid introduced its GeneXpert-based assay for the identification of the BCR-ABL gene fusion in cells from blood samples. This system comprises a walk-away self-contained instrument that combines cartridge-based microfluidic sample preparation with reverse transcriptase-polymerase chain reaction-based fluorescent signal detection and BCR-ABL and ABL Ct (threshold cycle) determination. The difference between the BCR-ABL Ct and ABL Ct (DeltaCt) is expected to represent the ratio of the two populations of mRNAs and ultimately the percentage of neoplastic cells present. We tested whether this BCR-ABL fusion detection system could be used as a clinical diagnostic tool for monitoring patients with minimal residual disease of chronic myelogenous leukemia. We report similar performance characteristics, including limit of detection, specificity, sensitivity, and precision, of this automated BCR-ABL fusion detection system to those of a manual TaqMan reverse transcriptase-polymerase chain reaction-based test.

Figure 1

FusionQuant plasmid copy number-based standard curves. A and B: Relationship of average Ct values to FusionQuant Standard plasmid copy numbers obtained by the GeneXpert (dashed line) and clinical ASR (solid line) methods. C: Correlation between the GeneXpert and the clinical ASR assays using average Ct values obtained for ABL (solid line) and BCR-ABL (dashed line) FusionQuant plasmid standards. D: Comparison of the efficiencies of the BCR-ABL and ABL reactions within the same GeneXpert multiplex PCR assays. For the GeneXpert method, each data point represents the average of three independent runs (except for the 10-copy data point shown on graph B, where two positive and three invalid results were obtained out of five runs). For the reference method, each data point represents the average of two independent assays as described in our standard operating protocol for the clinical assay.

Figure 2

K562 cell number-based standard curves. A: Relationship between K562 cell numbers and calculated ΔCt values obtained by the two assays. For the GeneXpert assay (diamonds and dashed line), each data point represents the average of two to four independent runs obtained in two separate experiments except for the lowest K562 cell number, which represents the single positive result out of three valid runs. For the FusionQuant assay (squares and solid line), each data point represents the average of two independent runs obtained from two separate experiments except for K562 cell numbers 5, 50, 500, and 5000, where only single measurements were possible due to limited samples. B: Linear regression plots of the calculated ΔCt values within 12 to 12,000 K562 cell number range for the GeneXpert (dashed line) and FusionQuant (solid line) assays. The outlier at 50 K562 cell number was omitted for the FusionQuant assay plot. C: Linear regression plot for comparison of the GeneXpert and the clinical ASR methods. One outlier related to the FusionQuant assay was omitted.

Figure 3

Comparison of the GeneXpert and the reference assay for the quantification of relative ratios of BCR-ABL and ABL gene products from clinical samples. A: Deming regression plot. The solid line corresponds to the regression line and the dashed line represents the identity line. B: Bland-Altman percent difference plot.