A quantitative reverse transcriptase polymerase chain reaction method for the detection of leukaemic cells with t(8;21) in peripheral blood

Abstract

We evaluated the usefulness of a recently developed real-time reverse transcriptase polymerase chain reaction (RT-PCR) system to detect minimal residual diseases (MRD) in patients with acute myelogenous leukaemia (AML) with chromosomal translocation t(8:21). The method was simple, rapid and reproducible for the quantity of chimeric AML1-ETO (MTG8) transcripts. The ratio of the absolute copy number of a target gene (AML1-ETO) to a control gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) was calculated by using a fluorescence curve prepared from amplicons of serially diluted standard RNA. The relative points of MRD in bone marrow (BM) of 8 patients in the acute phase of the disease was from 0.85 to 3.0, whereas those of MRD in complete remission (CR) decreased to below 6.4 x 10(-3). This method was also applied to evaluate chimeric transcripts in peripheral blood (PB) samples. The values in patients with t(8;21) AML were from 0.97 to 2.0 in the acute phase, whereas those in CR showed less than 2.2 x 10(-4). There was 10(-5)-fold difference in AML1-ETO mRNA expression between PB samples in the acute phase and those in CR. The results suggest that we may easily monitor MRD in patients with t(8;21) AML through quantitative analysis of AML1-ETO transcripts in blood samples.

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

“…186 These promising preliminary data suggest that RQ-PCR is likely to be valuable for MRD monitoring in this subset of AML, with the added advantages that the latter technique is less labor intensive, more reproducible and amenable to standardization lending itself to use in large-scale clinical trials. Preliminary studies of RQ-PCR [42][43][44]187,188 have essentially confirmed the results obtained via competitive RT-PCR, revealing a 3.5-to 20-fold variation in AML1-ETO FG expression levels in diagnostic BM that was not related to blast percentage and which needs to be taken into account when assessing response to therapy. Furthermore, variability in kinetics of response to chemotherapy was noted, and interestingly AML1-ETO transcripts were also detected in patients in long-term remission from AML.…”

Monitoring of minimal residual disease (MRD) by real-time quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement

“…In addition, several RQ-RT-PCR studies describe a linear decrease of AML1/ETO for adults in the range of 2-4 log between diagnosis and CR, and some of them remained positive during the ongoing therapy. 23,[33][34][35][36][37] Some children in our study turned from a negative RQ-RT-PCR result to positivity with a low copy number in the next follow-up sample (pts. 2, 5, 12, 13).…”

“…However, the number of patients in all these reports is small, most of them are adults, and in most cases patients are neither analyzed consecutively nor treated uniformly. 19,21,23,[33][34][35][36][37] This study presents 15 children with AML1/ ETO rearrangement, who were analyzed by RQ-RT-PCR at diagnosis and at a minimum of four different time points during follow-up. All of them were treated according the AML-BFM 98 treatment protocol.…”

Stanniocalcin-1 as a Novel Marker to Detect Minimal Residual Disease of Human Leukemia

“…Human STC-1 mRNA is, however, expressed in multiple tissues, suggesting that human STC-1 might have additional functions, for example, neural cell differentiation (Zhang et al 1998), bone formation (Yoshiko et al 1998;Stasko et al 2001). Of note, its expression is observed in malignant tissues such as adrenal tumor, hepatocellular carcinoma and breast cancer, suggesting some roles in transformed cells (Lal et al 2001;Miura et al 2000;Wascher et al 2003).…”