Quantification of the DHFR gene in blast cells of leukaemia patients by fluorescence in situ hybridisation.

The fluorescence signal intensity of the DHFR gene was analysed in lymphocytes from 15 normal donors, in MTX-resistant HeLa cells (carrying DHFR gene amplification) and in bone marrow blasts from 16 patients with acute leukaemia (AL) by in situ hybridisation. Our aim was to verify if DHFR gene amplification may be responsible for increased enzyme activity in leukemic cells. The results obtained with a fluorescence in situ hybridisation method were quantified using the Scion image software program and compared with cytochemical and cytophotometric data relating to DHFR activity. In AL a heterogeneous hybridisation pattern was generally observed at the single cell level. However, leukemic lymphoblasts showed higher fluorescence signal intensity of the DHFR gene as compared with normal lymphocytes, and leukemic myeloblasts a much higher signal than lymphoblasts. HeLa cells showed the highest fluorescence signal intensity. In all samples enzyme activity behaved in parallel. These results indicate that the increased expression of DHFR in leukemic blasts is due to a gene amplification. The high levels in AML can explain the MTX natural resistance.