Light microscopic detection of BCR-ABL transcripts after in-cell RT-PCR: Fusion gene expression might correlate with clinical evolution of chronic myeloid leukemia

A procedure for in-cell amplification of the hybrid BCR-ABL mRNA by reverse transcription and polymerase chain reaction (RT-PCR) without extraction of the nucleic acids was performed directly in fixed and permeabilized cells of leukemia patients (22 patients with Ph'-positive chronic myeloid leukaem ia-CML and 1 with Ph'-positive acute leukaemia- AL, as well as 7 Ph'-negati ve cases) and Ph'-positive human leukaemia cell lines (K562, LAMA-84, BV173 ). The labelling of the amplified sequences was done employing biotinylated primers and a second PCR in a semi-nested fashion with a low number of cyc les. An enzymatic system based on biotin-streptavidin-chromogen reaction wa s used for the detection of labeled PCR product, thus producing a coloured product, visible to the eye under a standard light microscope. All samples from patients with cytogenetic and molecular evidence of BCR-ABL rearrangem ent showed specific cytoplasmic staining at the site of the amplified hybri d transcripts. It allowed definite distinction between positive and negativ e cells. K562, LAMA-84, BV173 cells were characterized with strong diffuse staining while an interesting finding of the present study was the presence of variable quantities of colour product in patients' samples which might be due to different mRNA expression. Early and intermediate stages of myelo id maturation showed more intense reactivity. Cases with an aggressive cour se of accelerated or blast phase CML and AL were found to have a considerab le subset of cells with strongly expressed signal while cases in chronic ph ase were characterised with uniform weak to moderate reaction. Our observat ions support the hypothesis that the amount of BCR-ABL transcript expressio n within neoplastic cells may play a role in dictating the eventual behavio ur of the leukaemic clone. Future studies at a single cell level of larger series of consecutive cases with a follow up might be able to identify thos e patients who are prone to transformation-and provide certain indications for further therapeutic decisions.