Preudhomme C, Chams-Eddine L, Roumier C, Duflos-Grardel N, Denis C, Cosson A, Fenaux P
Laboratoire d'Hématologie, CHU Lille, France.
Leukemia. 1999 May;13(5):818-23. doi: 10.1038/sj.leu.2401393.
Methods of minimal residual disease (MRD) detection in chronic myelogenous leukemia (CML) include chromosome analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) techniques. We report a novel method to detect intracellular BCR-ABL messenger on single cells using in situ RT-PCR, which can be performed on blood and marrow slides, without extraction of the nucleic acid. After cellular permeabilization and fixation, the mRNA BCR-ABL was reverse transcribed and amplified by PCR using digoxigenin-labelled dUTP. The reaction was revealed with the anti-digoxigenin FITC antibody. On the fluorescent microscope, a strong positive green fluorescence signal was observed in 98-99% cells in Ph1-positive cell lines. A faint signal was detected in 1.5% and 2% of negative cell lines. Likewise, a faint signal was found in 1.6-2.8% of the cells in five normal controls (mean 2.2 +/- 1.1%). The positive threshold for in situ RT-PCR was therefore determined as mean + 2 s.d. = 4.4% cells. We used in situ RT-PCR by comparison to cytogenetics (at least 30 mitoses examined), and two-step RT-PCR (10(-6) sensitivity in our hands) in bone marrow samples from 13 CML patients: two patients at diagnosis and 11 patients in hematological remission after alpha interferon (three patients), hydroxyurea (one patient) autologous bone marrow transplantation (BMT) (one patient) and allogeneic BMT (six patients). In the two diagnostic patients, 90 and 95% cells were respectively strongly positive by in situ RT-PCR. In the six patients treated by allogeneic BMT, the median percentage of positive cells was 2.4% (range 1.8-3.2). All six patients had normal karyotype and negative two-step RT-PCR results. In the five other patients, two were treated by hydroxyurea alone or autologous BMT, and 11 and 13% of the cells were strongly positive; three were treated with interferon and 14-62% of the cells were positive, generally weakly. All five patients had persistence of Ph1 (in 9-56% mitoses), and positive RT-PCR results after one round. In conclusion, in situ RT-PCR can specifically identify cells with BCR-ABL transcript and its results are concordant with those of karyotype and RT-PCR. Because of its limited sensitivity and specificity, however, it appears to have limited value in the analysis of MRD. On the other hand, it can evaluate the presence and intensity of BCR-ABL fusion transcript at the single cell level, and this could be useful in treatment monitoring.
慢性粒细胞白血病（CML）微小残留病（MRD）的检测方法包括染色体分析、逆转录聚合酶链反应（RT-PCR）和荧光原位杂交（FISH）技术。我们报告了一种使用原位RT-PCR检测单细胞内BCR-ABL信使的新方法，该方法可在血液和骨髓涂片上进行，无需提取核酸。细胞通透和固定后，使用地高辛标记的dUTP通过PCR对mRNA BCR-ABL进行逆转录和扩增。用抗地高辛FITC抗体显示反应。在荧光显微镜下，在Ph1阳性细胞系中98 - 99%的细胞中观察到强阳性绿色荧光信号。在1.5%和2%的阴性细胞系中检测到微弱信号。同样，在五个正常对照的1.6 - 2.8%的细胞中发现微弱信号（平均2.2 +/- 1.1%）。因此，原位RT-PCR的阳性阈值确定为平均 + 2标准差 = 4.4%细胞。我们将原位RT-PCR与细胞遗传学（至少检查30个有丝分裂）以及两步RT-PCR（在我们手中灵敏度为10^(-6)）进行比较，用于检测13例CML患者骨髓样本中的情况：2例诊断时的患者和11例血液学缓解后的患者，后者分别接受了α干扰素（3例）、羟基脲（1例）、自体骨髓移植（BMT）（1例）和异基因BMT（6例）治疗。在2例诊断患者中，原位RT-PCR分别显示90%和95%的细胞为强阳性。在6例接受异基因BMT治疗的患者中，阳性细胞的中位数百分比为2.4%（范围1.8 - 3.2）。所有6例患者的核型正常，两步RT-PCR结果为阴性。在其他5例患者中，2例仅接受羟基脲或自体BMT治疗，11%和13%的细胞为强阳性；3例接受干扰素治疗，14 - 62%的细胞为阳性，通常较弱。所有5例患者均存在Ph1（在9 - 56%的有丝分裂中），一轮RT-PCR结果为阳性。总之，原位RT-PCR可特异性鉴定具有BCR-ABL转录本的细胞，其结果与核型和RT-PCR结果一致。然而，由于其有限的灵敏度和特异性，它在MRD分析中的价值似乎有限。另一方面，它可以在单细胞水平评估BCR-ABL融合转录本的存在和强度，这在治疗监测中可能有用。
