Elia Loredana, Mancini Marco, Moleti Luisa, Meloni Giovanna, Buffolino Sonia, Krampera Mauro, De Rossi Giulio, Foà Robin, Cimino Giuseppe
Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Ematologia, University La Sapienza, via Benevento 6, 00161 Rome, Italy.
Haematologica. 2003 Mar;88(3):275-9.
In the last few years molecular methods have allowed the identification of leukemia-associated genetic lesions, which may represent the most accurate predictors of clinical outcome. These considerations strengthen the need for rapid identification of the abnormalities. Our aim was to demonstrate whether a modified multiplex reverse transcription polymerase chain reaction (RT-PCR) system might be successfully used to screen a large number of patients with acute lymphoblastic leukemia (ALL).
In this study we adapted the multiplex RT-PCR assay, previously described by Pallisgaard et al., to detect all the most frequent genetic lesions with their characteristic splicing variants occurring in acute lymphoblastic leukemia, such as the MLL/AF4, MLL/ENL, BCR/ABL p190 (e1a2) and p210 (b2a2,b3a2) isoforms, E2A/PBX1, TEL/AML1, SIL/TAL1 and the novel NUP98/RAP1GDS1 transcript, recently described in a T-ALL leukemic subtype.
We used the multiplex RT-PCR assay to screen 170 ALL patients (70 children and 100 adults). PCR positivity was detected in 67 (39%) of the 170 ALL patients studied. The comparison between cytogenetic and molecular analyses showed complete correspondence between the two assays in all patients with an evaluable karyotype. Finally, the observed incidence of genetic lesions in our ALL patients was similar to the frequency usually reported both in children and in adults with ALL.
These results show that, compared to single RT-PCR reactions, our multiplex RT-PCR system allows rapid, specific, simultaneous as well as a less expensive, laborious and time-consuming detection of the most frequent fusion transcripts in ALL patients. Therefore, it might be recommended for rapid diagnostic molecular screening of large numbers of patients, such as those enrolled in multicenter, co-operative studies. Furthermore, we have shown that multiplex RT-PCR is an open system that can easily be adapted to detect new leukemic genes.
在过去几年中，分子方法已能够识别与白血病相关的基因损伤，这些损伤可能是临床结果最准确的预测指标。这些考虑因素强化了快速识别异常情况的必要性。我们的目的是证明改良的多重逆转录聚合酶链反应（RT-PCR）系统是否可成功用于筛查大量急性淋巴细胞白血病（ALL）患者。
在本研究中，我们采用了先前由帕利斯gaard等人描述的多重RT-PCR检测方法，以检测急性淋巴细胞白血病中所有最常见的基因损伤及其特征性剪接变体。例如MLL/AF4、MLL/ENL、BCR/ABL p190（e1a2）和p210（b2a2、b3a2）异构体、E2A/PBX1、TEL/AML1、SIL/TAL1以及最近在一种T-ALL白血病亚型中描述的新型NUP98/RAP1GDS1转录本。
我们使用多重RT-PCR检测方法对170例ALL患者（70例儿童和100例成人）进行了筛查。在研究的170例ALL患者中，有67例（39%）检测到PCR阳性。细胞遗传学分析与分子分析之间的比较显示，在所有可评估核型的患者中，两种检测方法完全一致。最后，我们ALL患者中观察到的基因损伤发生率与儿童和成人ALL患者通常报道的频率相似。
这些结果表明，与单个RT-PCR反应相比，我们的多重RT-PCR系统能够快速、特异性、同时地检测ALL患者中最常见的融合转录本，且成本更低、操作更简便、耗时更少。因此，它可能推荐用于对大量患者进行快速诊断分子筛查，例如参与多中心合作研究的患者。此外，我们已经表明多重RT-PCR是一个开放系统，可以很容易地进行调整以检测新的白血病基因。
