A multiplex reverse transcriptase-polymerase chain reaction strategy for the diagnostic molecular screening of chimeric genes: a clinical evaluation on 170 patients with acute lymphoblastic leukemia.
A multiplex reverse transcriptase-polymerase chain reaction strategy for the diagnostic molecular screening of chimeric genes: a clinical evaluation on 170 patients with acute lymphoblastic leukemia.
复制标题
用于嵌合基因诊断分子筛选的多重逆转录酶-聚合酶链反应策略：对 170 例急性淋巴细胞白血病患者的临床评估。
作者：
L. Elia;M. Mancini;L. Moleti;G. Meloni;S. Buffolino;M. Krampera;G. de Rossi;R. Foà;G. Cimino
背景和目标
在过去的几年里，分子方法已经能够识别白血病相关的遗传损伤，这可能是临床结果最准确的预测因素。这些考虑加强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式反应(RT-PCR)系统是否可以成功地用于筛查大量的急性淋巴细胞白血病(ALL)患者。
设计与方法
在这项研究中，我们采用Pallisgaard等人先前描述的多重RT-PCR方法来检测发生在急性淋巴细胞白血病中的所有最常见的基因损伤及其特征剪接变体，例如MLL/AF4、MLL/en1、bcr/abl p190(E1a2)和p210(b2a2，b3a2)亚型、E2a/Pbx1、tel/AML1、sIL/TAL1和最近在T-ALL亚型中描述的新的NUP98/RAP1GDS1转录本。
结果
我们使用多重RT-PCR方法对170名ALL患者(70名儿童和100名成人)进行了筛查。在研究的170例ALL患者中，67例(39%)检测到PCR阳性。细胞遗传学和分子分析的比较表明，在所有核型可评估的患者中，两种分析结果完全一致。最后，在我们的ALL患者中观察到的遗传损害的发生率与通常在儿童和成人ALL患者中报告的频率相似。
解读和结论
这些结果表明，与单一的RT-PCR反应相比，我们的多重RT-PCR系统可以快速、特异、同时检测所有患者中最常见的融合转录本，而且成本更低、费力和耗时。因此，它可能被推荐用于大量患者的快速诊断分子筛查，例如那些参加多中心合作研究的患者。此外，我们已经证明多重RT-PCR是一个开放的系统，可以很容易地适应于检测新的白血病基因。
BACKGROUND AND OBJECTIVES
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).
DESIGN AND METHODS
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.
RESULTS
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.
INTERPRETATION AND CONCLUSIONS
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.