QUANTITATION OF MINIMAL RESIDUAL DISEASE IN PHILADELPHIA-CHROMOSOME-POSITIVE CHRONIC MYELOID-LEUKEMIA PATIENTS USING REAL-TIME QUANTITATIVERT-PCR

We used a recently developed system for real-time quantitative polymer ase chain reaction (PCR) to determine residual disease in patients wit h chronic myeloid leukaemia. The expression of the Bcr-Abl hybrid onco gene was determined and normalized by using the PBGD housekeeping gene product as endogenous reference. The sensitivity and reproducibility of the assay was tested on cell line K562. A dilution of Bcr-Abl-posit ive cell line K562 remained positive at up to 250 fg of RNA. 10 copies of Bcr-Abl DNA in water could still be detected. The dynamic range of the method spanned six orders of magnitude. Analysis of 10 identical assays on K562 RNA resulted in a variation of 15%. To test the feasibi lity of normalization of Bcr-Abl dosage by the PBGD product, we compar ed the efficiencies of the RT-PCRs in 150 patient analyses, We conclud ed that PBGD was a suitable and stringent quality control standard. Th ree patients who were treated with donor leucocyte infusions for chron ic myeloid leukaemia who had relapsed after bone marrow transplantatio n were followed over time, The normalized Bcr-Abl dosage was compared to the results of cytogenetics. Cytogenetic analysis was negative belo w a normalized Bcr-Abl dose of about 3x10(-2). This semiautomated meth od is fast, sensitive and accurate and enables a high throughput of sa mples.