Quantification of PML‐RARα transcripts in acute promyelocytic leukaemia: explanation for the lack of sensitivity of RT‐PCR for the detection of minimal residual disease and induction of the leukaemia‐specific mRNA by alpha interferon

Abstract

The RT-PCR technique for the identification of the PML-RAR alpha fusion mRNA is widely used for the detection of minimal residual in acute promyelocytic leukaemia (APL). A positive result after remission induction is highly predictive of early relapse, but the vast majority of patients have no detectable disease by this technique after chemotherapy consolidation, despite the fact that many later relapse. We report a quantitative PCR technique for the PML-RAR alpha cDNA which was used to show that less than 1000 PML-RAR alpha molecules are obtained from 1 microgram of diagnostic bone marrow RNA derived from approximately 1 million APL blasts. The lack of sensitivity of currently employed RT-PCR methods may therefore be explained by their poor yield of PML-RAR alpha cDNA. Minor modifications to the reverse transcription procedure improved this yield 3 fold. Furthermore, expression of the leukaemia-specific transcript increased by approximately one order of magnitude after incubation of the patient's cells for 24 h in vitro with 100 iu/ml alpha interferon.