Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR

We took advantage of a recently developed system allowing performance of re al-time quantitation of polymerase chain reaction to develop a quantitative method of measurement of PML-RAR alpha transcripts which are hallmarks of acute promyelocytic leukemia (APL) with t(15;17) translocation. Indeed, alt hough quantitation of minimal residual disease has proved to be useful in p redicting clinical outcome in other leukemias such as chronic myeloid leuke mia or acute lymphoblastic leukemia, no quantitative data have been provide d in the case of APL. We present here a method for quantitation of the most frequent subtypes of t(15;17) transcripts (namely bcr1 and bcr3). One spec ific forward primer is used for each subtype in order to keep amplicon leng th under 200 bp. The expression of PML-RAR alpha transcripts is normalized using the housekeeping porphobilinogen deaminase (PBGD) gene. This techniqu e allows detection of 10 copies of PML-RARa or PBGD plasmids, and quantitat ion was efficient up to 100 copies. One t(15;17)-positive NB4 cell could be detected among 10(6) HL60 cells, although quantitation was efficient up to one cell among 10(5). Repeatability and reproducibility of the method were satisfying as intra- and inter-assay variation coefficients were not highe r than 15%. The efficiency of the method was finally tested in patient samp les, showing a decrease of the PML-RAR alpha copy number during therapy, an d an increase at the time of relapse.