Development of a BCR/ABL Real-Time Quantitative RT-PCR Assay and Correlation with an Existing Qualitative Test.

Introduction: With the recent introduction of imatinib (Gleevec) as a therapeutic agent for chronic myeloid leukemia (CML), the need for quantitation of the level of tumor cells after treatment has become essential. Our lab has for a number years tested for CML using a sensitive qualitative nested RT-PCR assay for BCR/ABL. We wanted to develop a quantitative test that retained the sensitivity of our qualitative test and contained an initial screen to prevent unnecessary quantitative testing as well as develop quantitative tests for the various breakpoints.

Methods: The new test is divided into three parts. A real-time multiplex qualitative RT-PCR screen containing specific primers for the p190, p210 and p230 BCR/ABL forms and a common labeled probe. If a patient sample is positive in the screen, the original nested RT-PCR test is performed to determine which BCR/ABL form is present. This is followed by a real-time quantitative RT-PCR test for the identified BCR/ABL translocation. Subsequent follow up tests require only the specific quantitative test.

Results: Comparing negative results from 82 patient (various clinical presentations) samples tested by the previous qualitative test with the new qualitative screen showed completely correlation. Out of 31 CML patients studied, both test strategies gave the same result for 20 positive and 13 negative samples. Patients with multiple samples collected over time showed complete correlation between the two assays.

Conclusions: The new quantitative BCR/ABL testing strategy yielded results that correlated with the previous qualitative BCR/ABL assay.