Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using an in situ RT-PCR assay

Methods of minimal residual disease (MRD) detection in chronic myelogenous leukemia (CML) include chromosome analysis, reverse transcriptase polymeras e chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) tec hniques. We report a novel method to detect intracellular BCR-ABL messenger on single cells using in situ RT-PCR, which can be performed on blood and marrow slides, without extraction of the nucleic acid. After cellular perme abilization and fixation, the mRNA BCR-ABL was reverse transcribed and ampl ified by PCR using digoxigenin-labelled dUTP. The reaction was revealed wit h the anti-digoxigenin FITC antibody. On the fluorescent microscope, a stro ng positive green fluorescence signal was observed in 98-99% cells in Phl-p ositive cell lines. A faint signal was detected in 1.5% and 2% of negative cell lines. Likewise, a faint signal was found in 1.6-2.8% of the cells in five normal controls (mean 2.2+/-1.1%). The positive threshold for in situ RT-PCR was therefore determined as mean + 2 s.d. = 4.4% cells. We used in s itu RT-PCR by comparison to cytogenetics (at least 30 mitoses examined), an d two-step RT-PCR (10(-6) sensitivity in our hands) in bone marrow samples from 13 CML patients: two patients at diagnosis and 11 patients in hematolo gical remission after alpha interferon (three patients), hydroxyurea (one p atient) autologous bone marrow transplantation (BMT) (one patient) and allo geneic BMT (six patients). In the two diagnostic patients, 90 and 95% cells were respectively strongly positive by in situ RT-PCR. In the six patients treated by allogeneic BMT, the median percentage of positive cells was 2.4 % (range 1.8-3.2). All six patients had normal karyotype and negative two-s tep RT-PCR results. In the five other patients, two were treated by hydroxy urea alone or autologous BMT, and 11 and 13% of the cells were strongly pos itive; three were treated with interferon and 14-62% of the cells were posi tive, generally weakly. All five patients had persistence of Phl (in 9-56% mitoses), and positive RT-PCR results after one round. In conclusion, in si tu RT-PCR can specifically identify cells with BCR-ABL transcript and its r esults are concordant with those of karyotype and RT-PCR. Because of its li mited sensitivity and specificity, however, it appears to have limited valu e in the analysis of MRD. On the other hand, it can evaluate the presence a nd intensity of BCR-ABL fusion transcript at the single cell level, and thi s could he useful in treatment monitoring.