Quantitative nested reverse transcriptase PCR vs. real-time PCR for measuring AML1/ETO (MTG8) transcripts

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Abstract
A quantitative nested reverse transcriptase polymerase chain reaction (QN‐RT‐PCR) method was developed using a plasmid cDNA containing the AML1/ETO (MTG8) fusion transcript from Kasumi‐1 cells, an acute‐myelogenous leukemia cell line with the t(8;21) translocation. In this method, the plasmid was detectable at a concentration of 10−17 m. The fusion transcript in a mixture of 107 Rice94 (Burkitt lymphoma cell line) cells containing two Kasumi‐1 cells was detectable at 10−17 m. In a previously published real‐time PCR method, the plasmid containing the fusion transcript was detectable at 10−16 m or higher, and 20 or more Kasumi‐1 cells were detectable in 107 Rice94 cells. Thus, this QN‐RT‐PCR method is more sensitive than the real‐time PCR. When the same samples were examined by real‐time PCR and our QN‐RT‐PCR method, in one patient in clinical remission after chemotherapy and allogeneic‐bone marrow transplantation (BMT), the transcript was detected by QN‐RT‐PCR 60 days prior to hematological relapse, in contrast to 10 days before hematological relapse by real‐time PCR. The transcript level was below 10−17 m (undetectable) with this QN‐RT‐PCR in patients in clinical remission after chemotherapy and BMT, while it was 10−15–10−16 m in patients in clinical remission after chemotherapy alone. The quantitative difference of the transcript level in minimal residual disease (MRD) between these two different types of clinical remission was estimated to be at least 102‐fold. This QN‐RT‐PCR method is useful for predicting hematological relapse and for quantitatively estimating MRD in different types of clinical remission.

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