Expression of bcr-abl mRNA in individual chronic myelogenous leukaemia cells as determined by in situ amplification

We present the results of a novel method developed for evaluation of in sit u amplification, a molecular genetic method at the cellular level, Reverse transcription polymerase chain reaction (RT-PCR) was used to study bcr-abl transcript levels in individual cells from patients with chronic myelogenou s leukaemia (CML). After hybridizing a fluorochrome-labelled probe to the c ell-bound RT-PCR product, bcr-abl mRNA-positive cells were determined using image analysis, A dilution series of bcr-abl-positive BV173 into normal ce lls showed a good correlation between expected and actual values. In 25 CML samples, the percentage of in situ PCR-positive cells showed an excellent correlation with cytogenetic results (r = 0.94, P < 0.0001), interphase flu orescence in situ hybridization (FISH) (r = 0.95, P = 0.001) and hypermetap hase FISH (r = 0.81, P < 0.001). The fluorescence intensity was higher in r esidual CML cells after interferon (IFN) treatment than in newly diagnosed patients (P = 0.004), and was highest in late-stage CML resistant to IFN th erapy and lowest in CML blast crisis (P = 0.001). Mean fluorescence values correlated with bcr-abl protein levels, as determined by Western blot analy sis (r = 0.62), Laser scanning cytometry allowing automated analysis of lar ge numbers of cells confirmed the results, Thus, fluorescence in situ PCR p rovides a novel and quantitative approach for monitoring tumour load and bc r-abl transcript levels in CML.