Malinge M C, Mahon F X, Delfau M H, Daheron L, Kitzis A, Guilhot F, Tanzer J, Grandchamp B
Laboratoire de Génétique Moléculaire, INSERM CJF 8904, Faculté Xavier Bichat, Paris, France.
Br J Haematol. 1992 Dec;82(4):701-7. doi: 10.1111/j.1365-2141.1992.tb06947.x.
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.
Bcr - Abl cDNA的体外扩增已被广泛用于评估慢性粒细胞白血病（CML）患者在达到完全临床和细胞遗传学缓解时微小残留病的存在情况。然而，不同实验室所达到的灵敏度水平在很大程度上仍不明确。此外，结果通常以阳性或阴性表示，这使得对患者进行精确随访变得困难。为了克服这些局限性，我们设计了一种定量方法来测量临床样本中Bcr - Abl mRNA的量。该方法包括一个逆转录步骤，随后分别扩增Bcr - Abl和总Abl mRNA。这两个扩增反应在相同内标不同稀释度的存在下进行。该内标由Abl外显子2缺失八个碱基的Bcr - Abl序列组成。每个单独反应中使用的引物之一用荧光素标记。扩增后，将细胞RNA衍生的PCR产物和内标衍生的PCR产物分离，并使用激光荧光DNA测序仪（ALF，Pharmacia）测定它们的相对荧光。然后计算每个样本中最初存在的Bcr - Abl和总Abl mRNA分子数。通过研究将K562 RNA连续稀释到正常RNA中，评估了该方法的准确性和可重复性。对接受干扰素治疗处于细胞遗传学缓解期的10例患者的血样进行了研究。仅发现一个样本为阴性，而其他样本每1000个Abl mRNA分子中含有0.05至17个杂交Bcr - Abl mRNA分子。这些结果表明，在接受干扰素治疗后处于细胞遗传学缓解期的大多数CML患者中存在数量可变的恶性细胞。
