Light Microscopic Detection ofBCR-ABLTranscripts afterIn-CellRT-PCR: Fusion Gene Expression Might Correlate with Clinical Evolution of Chronic Myeloid Leukemia

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Abstract
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 leukaemia- CML and 1 with Ph'-positive acute leukaemia- AL, as well as 7 Ph'-negative cases) and Ph'-positive human leukaemia cell lines (K562, LAMA-84, BV173). The labelling of the amplified sequences was done employing biotiny-lated primers and a second PCR in a semi-nested fashion with a low number of cycles. An enzymatic system based on biotin-streptavidin-chromogen reaction was 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 rearrangement showed specific cytoplasmic staining at the site of the amplified hybrid transcripts. It allowed definite distinction between positive and negative 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 myeloid maturation showed more intense reactivity. Cases with an aggressive course of accelerated or blast phase CML and AL were found to have a considerable subset of cells with strongly expressed signal while cases in chronic phase were characterised with uniform weak to moderate reaction. Our observations support the hypothesis that the amount of BCR-ABL transcript expression within neoplastic cells may play a role in dictating the eventual behaviour 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 those patients who are prone to transformation and provide certain indications for further therapeutic decisions.