The amount of BCR-ABL fusion transcripts detected by the real-time quantitative polymerase chain reaction method in patients with Philadelphia chromosome positive chronic myeloid leukemia correlates with the disease stage

Abstract

 The use of the real-time reverse-transcription polymerase-chain reaction (RT-PCR) method to quantify BCR-ABL transcripts before and after allogeneic transplant was prospectively studied in 65 patients with chronic myeloid leukemia (CML). The expression of the BCR-ABL transcript was determined and normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene product as an endogenous reference. In the single step real-time PCR assay, tenfold serial dilutions of cDNA of the K5652 cell line remained positive down to 100 pg cDNA only. However, molecular relapses of CML after transplant were only safely detectable when a nested real-time PCR assay was performed, which was able to detect 1–10 pg cDNA from a tenfold serial dilution. The median normalized BCR-ABL transcript level was measured as 0.004% in 17 patients with a molecular relapse, 0.4% in 7 patients with a cytogenetic relapse, 2.6% in 36 patients with a stable phase of CML, and 36% in 5 patients with a relapse in a blast crisis. The analyzed median normalized amount of BCR-ABL transcript differed significantly (P<0.001) between the various disease stages. In ten CML patients with relapse, the real-time PCR method was used to monitor the response of various immunotherapies as donor leukocyte infusions, withdrawal of immunosuppression, or interferon-α application. The results of the quantitative evaluation of BCR-ABL transcripts reflected very well the clinical effect of the different applied immunotherapies. The new real-time PCR method seems to be a suitable technique for the early detection of relapse after allogeneic transplant in patients with the BCR-ABL transcript. Its ability to distinguish between molecular and cytogenetic relapse (P<0.001) allows early therapeutic decisions.