Real-time quantification of different types of bcr-abl transcript in chronic myeloid leukemia

Background and Objectives. The most common translocation in chronic myeloid leukemia (cml) t(9;22) (q34;q22) produces the Bcr/abl fusion gene. We set up and evaluated a rapid and reliable real-time reverse-transcription-polymerase chain reaction (rt-pcr) approach using Taqman technology for detection and quantification of bcr-abl transcripts in Cml patients at diagnosis and during therapy. Design and Methods. A pair of primers and probe complementary to Abl exon 2 were designed, enabling detection of the most frequent bcr-abl transcripts, and also of the normal Abl-la transcript as an internal control. Conditions were established to amplify less than 1^-10 target molecules/reaction and detect one Cml cell in 10⁶ cells from healthy donors. To determine the utility of the assay, we quantified the bcr-abl/abl-la ratio in 59 bone marrow samples (45 samples with evidence of different Ph⁺ chromosome percentages and 14 samples in complete cytogenetic remission) from 48 Cml patients, 34 of them at diagnosis and 14 in clinical remission (cr). In 14 cases, this ratio was compared with results obtained by a competitive-quantitative Rt-pcr/capillary electrophoresis method from contemporary specimens. Results. By real-time Rt-pcr, the median value of bcrabl/abl-la ratio at diagnosis was 15.334 (range 3.3-28.81) and fell to 0.9 (range 0.003-26.1) in CR. The median value of bcr-abl/ABL-la ratio at cytogenetic remission was 0.7 (range 0.003-2.83). The real-time bcrabl/ABL-la ratios correlated with those obtained by competitive RT-PCR (p