Szczepański T, Beishuizen A, Pongers-Willemse M J, Hählen K, Van Wering E R, Wijkhuijs A J, Tibbe G J, De Bruijn M A, Van Dongen J J
Department of Immunology, University Hospital Dijkzigt/Eramus University, Rotterdam, The Netherlands.
Leukemia. 1999 Feb;13(2):196-205. doi: 10.1038/sj.leu.2401277.
A large series of 202 childhood precursor-B cell acute lymphoblastic leukemia (ALL) patients was analyzed by Southern blotting (SB) for cross-lineage rearrangements and/or deletions in the T cell receptor TCRB, TCRG and TCRD loci. In 93% (187/201) of the precursor-B-ALL patients one or more genes were rearranged and/or deleted. TCRB gene rearrangements were found in 35% (69/196), TCRG gene rearrangements in 59% (113/192), TCRD gene rearrangements in 55% (112/202), and isolated monoallelic or biallelic deletions of TCRD loci in 34% (68/202) of the cases. TCRB gene rearrangements involved exclusively the Jbeta2 locus with complete V(D)Jbeta2 joinings in 53% of gene rearrangements and incomplete Dbeta-Jbeta2 gene rearrangements in 33%. TCRG gene rearrangements frequently occurred on both alleles (65% of cases) and in approximately 70% concerned rearrangements to Jgamma1 gene segments. Most rearranged TCRD alleles (80%) represented incomplete Vdelta2-Ddelta3 or Ddelta2-Ddelta3 gene rearrangements, while the remaining TCRD gene rearrangements remained unidentified. Subsequently, we evaluated, whether heteroduplex PCR analysis of rearranged TCRG and TCRD genes can be used for reliable identification of PCR targets for detection of minimal residual disease (MRD). The concordance between SB and heteroduplex PCR analysis for detection of the various types of clonal TCRG and TCRD gene rearrangements ranged between 78% and 87%. The discrepancies could be assigned to the presence of 'atypical' TCRD gene rearrangements or translocations only detectable by SB, but also to efficient PCR-based detection of rearrangements derived from small subclones, which are difficult to detect with SB. Indications for oligoclonality were observed in 38% and 30% of patients with TCRG and TCRD gene rearrangements, respectively, which is comparable to the frequency of oligoclonality in IGH locus. Based on the combined data it was possible to reduce the broad panel of six TCRD and 12 TCRG primer combinations for MRD studies to two TCRD combinations (Vdelta2-Ddelta3 and Ddelta2-Ddelta3) and six TCRG combinations (VgammaI, VgammaII, VgammaIV family-specific primers with Jgamma1.1/2.1 and Jgamma1.3/2.3 primers) resulting in the detection of 80% and 97% of all TCRD and TCRG gene rearrangements, respectively. Finally, the heteroduplex PCR data indicate that MRD monitoring with TCRG and/or TCRD targets is possible in approximately 80% of childhood precursor-B-ALL patients; approximately 55% of patients even have two TCRG and/or TCRD targets.
采用Southern印迹法（SB）对202例儿童前体B细胞急性淋巴细胞白血病（ALL）患者进行分析，以检测T细胞受体TCRB、TCRG和TCRD基因座的跨谱系重排和/或缺失。在93%（187/201）的前体B-ALL患者中，一个或多个基因发生了重排和/或缺失。TCRB基因重排在35%（69/196）的患者中被发现，TCRG基因重排在59%（113/192）的患者中被发现，TCRD基因重排在55%（112/202）的患者中被发现，34%（68/202）的病例存在TCRD基因座的孤立单等位基因或双等位基因缺失。TCRB基因重排仅涉及Jbeta2基因座，53%的基因重排为完整的V（D）Jbeta2连接，33%为不完整的Dbeta-Jbeta2基因重排。TCRG基因重排经常发生在两个等位基因上（65%的病例），约70%涉及与Jgamma1基因片段的重排。大多数重排的TCRD等位基因（80%）代表不完整的Vdelta2-Ddelta3或Ddelta2-Ddelta3基因重排，而其余的TCRD基因重排仍未明确。随后，我们评估了重排的TCRG和TCRD基因的异源双链PCR分析是否可用于可靠鉴定检测微小残留病（MRD）的PCR靶点。SB与异源双链PCR分析检测各种类型的克隆TCRG和TCRD基因重排的一致性在78%至87%之间。差异可能归因于仅通过SB可检测到的“非典型”TCRD基因重排或易位的存在，也归因于基于PCR对源自小亚克隆的重排的有效检测，而这些小亚克隆难以用SB检测到。在分别有38%和30%的TCRG和TCRD基因重排患者中观察到寡克隆性迹象，这与IGH基因座的寡克隆性频率相当。基于综合数据，有可能将用于MRD研究的六种TCRD和12种TCRG引物组合的广泛面板减少到两种TCRD组合（Vdelta2-Ddelta3和Ddelta2-Ddelta3）和六种TCRG组合（VgammaI、VgammaII、VgammaIV家族特异性引物与Jgamma1.1/2.1和Jgamma1.3/2.3引物），分别导致检测到所有TCRD和TCRG基因重排的80%和97%。最后，异源双链PCR数据表明，在大约80%的儿童前体B-ALL患者中可以用TCRG和/或TCRD靶点进行MRD监测；大约55%的患者甚至有两个TCRG和/或TCRD靶点。
