Siraj Abdul K, Ozbek Ugur, Sazawal Sudha, Sirma Sema, Timson Georgina, Al-Nasser Abdallah, Bhargava Manorama, El Solh Hassan, Bhatia Kishor, Gutiérrez Marina I
Research, King Fahad National Centre for Children's Cancer and Research, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211 Saudi Arabia.
Clin Cancer Res. 2002 Dec;8(12):3832-40.
The purpose is to develop a real-time multiplex reverse transcription-PCR assay for detection and quantification of leukemia-specific chimeric transcripts that identify the genetic subgroups of acute lymphoblastic leukemias (ALLs) proposed by the WHO classification.
Real-time multiplex assay for t(12;21), t(4;11), and t(1;19) with hypoxanthine phosphoribosyltransferase as internal standard used in tandem with a new real time quantitative-RT-PCR assay for the t(9;22). This new strategy was designed to yield an amplicon from each translocation with a distinct melting peak allowing dependable identification using only Sybr green I, without any need for expensive hybridization probes.
We validated this method with 92 primary ALLs and identified 4 E2A-PBX1, 4 mBCR-ABL and 10 TEL-AML1. When compared with conventional RT-PCRs and Southern blot analyses, 100% concordance was obtained. During the course of these studies, we found marked variations in the levels of the TEL-AML1 transcripts in individual patients. We, therefore, extended the study to accurately and reproducibly determine TEL-AML1 mRNA levels in 47 additional patients with t(12;21). The results indicated that the level of expression of TEL-AML1 varied among individual patients, and it was independent of the WBC count.
Our new real-time multiplex assay can be used for rapid, simple, and reliable classification of pediatric ALL. Its reproducible quantification results should also facilitate studies on minimal residual disease. The observed variation in TEL-AML1 transcript levels is of interest because it could reflect biological and/or clinical heterogeneity in the behavior of these leukemias.
开发一种实时多重逆转录 - PCR检测方法，用于检测和定量白血病特异性嵌合转录本，以识别世界卫生组织（WHO）分类提出的急性淋巴细胞白血病（ALL）的基因亚组。
采用次黄嘌呤磷酸核糖转移酶作为内标，对t(12;21)、t(4;11)和t(1;19)进行实时多重检测，并与一种新的t(9;22)实时定量逆转录 - PCR检测方法串联使用。这种新策略旨在从每个易位产生一个具有独特熔解峰的扩增子，仅使用SYBR Green I即可可靠鉴定，无需任何昂贵的杂交探针。
我们用92例原发性ALL验证了该方法，鉴定出4例E2A - PBX1、4例mBCR - ABL和10例TEL - AML1。与传统逆转录 - PCR和Southern印迹分析相比，一致性达100%。在这些研究过程中，我们发现个体患者中TEL - AML1转录本水平存在显著差异。因此，我们将研究扩展至准确且可重复地测定另外47例t(12;21)患者的TEL - AML1 mRNA水平。结果表明，TEL - AML1的表达水平在个体患者中各不相同，且与白细胞计数无关。
我们新的实时多重检测方法可用于小儿ALL的快速、简单且可靠的分类。其可重复的定量结果也应有助于微小残留病的研究。观察到的TEL - AML1转录本水平的差异很有意思，因为它可能反映了这些白血病行为中的生物学和/或临床异质性。
