Quantitative determination of the hybrid Bcr‐Abl RNA in patients with chronic myelogenous leukaemia under interferon therapy

Abstract

In vitro amplification of the Bcr-Abl cDNA has been widely used to assess for the presence of minimal residual disease in patients with chronic myelogenous leukaemia (CML) presenting with complete clinical and cytogenetic remission. However, the level of sensitivity achieved in different laboratories remains largely unknown. Moreover, the results are usually expressed as positive or negative, making a precise follow-up of the patients difficult. In an attempt to overcome these limitations, we devised a quantitative method to measure the amount of Bcr-Abl mRNA in clinical samples. This methodology involves a single reverse transcription step, followed by separate amplifications of Bcr-Abl and total Abl mRNA. These two amplifications are performed in the presence of different dilutions of a same internal standard. This standard consists of Bcr-Abl sequences with an eight bases deletion in exon 2 of Abl. One of the primers used in each separate reaction is labelled with fluorescein. Following amplification, PCR products derived from cellular RNA and those from the internal standard are separated and their relative fluorescence is determined using a laser fluorescent DNA sequencer (ALF, Pharmacia). The number of Bcr-Abl and total Abl mRNA molecules initially present in each sample is then calculated. The accuracy and reproducibility of this method was assessed by studying serial dilutions of K562 RNA into normal RNA. Blood samples from 10 patients in cytogenetic remission under interferon therapy were studied. Only one sample was found negative while the others contained between 0.05 and 17 hybrid Bcr-Abl mRNA molecules per 1000 molecules of Abl mRNA. These results suggest that a variable number of malignant cells are present in the majority of CML patients in cytogenetic remission following interferon therapy.

Quantification of BCR-ABL transcripts in CML patients in cytogenetic remission after interferon-α-based therapy

“…In fact, our results confirm those of Lee et al 7 and Hochhaus et al 12 who reported that all complete cytogenetic responders on IFN-␣ have detectable residual disease by two-step RT-PCR independent of the duration of CCR. Although at least 23 cases have been reported 12,13,[16][17][18][19][20][21][22][23] with intermittent or persistent PCR negativity on IFN-␣ using different PCR techniques, these observations may be due to the considerable variability in the sensitivity of PCR techniques performed in different laboratories. Consequently, the PCR negativity reported in CCR patients could be due more to technical discrepancies among different laboratories than to a real cure of the patients.…”

“…Taking advantage of a linear relationship between the input template (usually cDNA obtained after RT from extracted total RNA) and amplification product within the exponential range of PCR, several authors have also reported a competitive PCR assay for quantification of bcrabl amplified transcript. [10][11][12][13] We have developed a similar competitive quantitative RT-PCR assay associated with amplified bcr-abl transcript quantification based on a capillary electrophoresis spectrophotometric analysis (CE). 8 We applied this method to evaluate the level of bcr-abl transcript at diagnosis and at the time of major (Ͼ66% Ph negativity) or complete cytogenetic remission (100% Ph negativity) in a restricted group of CML patients after receiving IFN-␣-based therapy, sometimes followed by autologous bone marrow transplantation.…”

Simple competitive two-step RT-PCR assay to monitor minimal residual disease in CML patients after bone marrow transplantation

“…In CML the fusion BCR/ABL gene is tested in parallel with a control ABL gene and the amount of BCR/ABL transcript is related to the total mount of ABL transcript. 19,33,34 However, as the tested ABL fragment is present not only in the normal ABL gene but also in the BCR/ABL fusion gene, some objection can be raised.…”

Chronic myelogenous leukemia: Laboratory diagnosis and monitoring

“…In a typical noncommercial Q-RT-PCR assay, for which many variations exist (Lion et al, 1992;Malinge et al, 1992;Thompson et al, 1992;Cross et al, 1993), cDNA of an internal reference, such as ␤2 microglobulin or ABL, is co-amplified in the same tube as cDNA of BCR-ABL to control for variations in the procedures such as sample preparation and loading. PCR products are then analyzed by gel electrophoresis and densitometry.…”