Quantitative RT-PCR (RQ-PCR) of BCR-ABL Fusion Transcripts

Quantitative RT-PCR (RQ-PCR) of BCR-ABL Fusion Transcripts: A Comparison of the Cepheid GeneXpert and the Roche LightCycler Platforms. Kathryn Ramey and Mary Lowery Nordberg. Department of Pathology and Feist-Weiller Cancer Center.

Introduction

CML patients have a distinct translocation between chromosome 9 and 22(t(9;22)(q34;q11)) which results in the BCR-ABL gene. Quantitative RT-PCR is a highly sensitive method for detecting the BCR-ABL fusion gene. Cepheid GeneXpert BCR-ABL assay detects the BCR-ABL transcripts and ABL endogenous control gene in peripheral blood. Cepheid GeneXpert automates and integrates sample purification, nuclei acid amplification and detection of fusion transcripts with results available within a two-hour window. This study compared the sensitivity and accuracy of the RT-PCR values obtained from the GeneXpert System to the LightCycler platform (Roche).

Materials and Methods

The GeneXpert BCR-ABL assay monitors the p210 protein produced from the BCR-ABL translocation in peripheral blood. 200 microliters of peripheral blood from 37 CML patients, including 17 negatives and 20 positives were assayed using the GeneXpert platform. The blood is lysed and transferred to the designated chamber in the cartridge. Subsequently, the wash reagent, rinse reagent and elution reagent are transferred into designated chambers in the cartridge. The cartridges are loaded into the GeneXpert machine which automates the RT-PCR followed by nested RT-PCR. The same 37 specimens also had RNA extracted, amplified and analyzed using the LightCycler platform with GAPDH as the internal control.

Results

Qualitative BCR/ABL results were identical for all patients tested: patients positive for BCR-ABL were positive on both platforms (18 patients); patients negative for BCR-ABL were negative on both platforms (17 patients). Two patients showed discrepant results due to the complexity of the BCR-ABL translocation. These patients produced invalid results on the GeneXpert, however were successfully analyzed on the LightCycler. One patient had a complex, three-way translocation involving the ABL locus at 22q11.2, thereby preventing the use of ABL as the endogenous control. A second patient was a BCR-ABL-positive B-ALL sample. Although the reaction was invalid using the GeneXpert, review of the graphic data from the PCR reaction clearly showed dysfunctional ABL and accumulation of BCR-ABL product. One of the positive samples gave an initial aberrant quantitative result using the GeneXpert system due to high WBC in the sample.

Conclusion

The GeneXpert assay is a rapid way to perform quantitative BCR-ABL testing for CML patients. The Gene Xpert cartridge based system decreases the risk for cross contamination of specimens and minimizes hands on technical time. This assay can detect approximately 1 in 100,000 normal cells and therefore suitable for monitoring minimal residual disease.