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

Authors


Michael Andreeff, M.D., Ph.D., The University of Texas M.D. Anderson Cancer Center, Department of Molecular Hematology and Therapy, 1515 Holcombe Boulevard, Box 81, Houston, TX 77030, USA. E-mail: mandreef@notes.mdacc.tmc.edu

Abstract

We present the results of a novel method developed for evaluation of in situ 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 myelogenous leukaemia (CML). After hybridizing a fluorochrome-labelled probe to the cell-bound RT-PCR product, bcr–abl mRNA-positive cells were determined using image analysis. A dilution series of bcr–abl-positive BV173 into normal cells 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 fluorescence in situ hybridization (FISH) (r = 0·95, P = 0·001) and hypermetaphase FISH (r = 0·81, P < 0·001). The fluorescence intensity was higher in residual CML cells after interferon (IFN) treatment than in newly diagnosed patients (P = 0·004), and was highest in late-stage CML resistant to IFN therapy and lowest in CML blast crisis (P = 0·001). Mean fluorescence values correlated with bcr–abl protein levels, as determined by Western blot analysis (r = 0·62). Laser scanning cytometry allowing automated analysis of large numbers of cells confirmed the results. Thus, fluorescence in situ PCR provides a novel and quantitative approach for monitoring tumour load and bcr–abl transcript levels in CML.

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