多重PCR法检测初诊成人急性淋巴细胞白血病患者克隆性免疫球蛋白和T细胞受体基因重排

Detection of clonal immunoglobulin and T-cell receptor gene rearrangements in newly diagnosed adult patients with acute lymphoblastic leukemia by using multiplex PCR protocols

目的应用多重PCR方法检测初诊成人急性淋巴细胞白血病（ALL）患者的克隆性免疫球蛋白（Ig）和T细胞受体（TCR）基因重排，为实时定量RT—PCR（RQ—PCR）法监测ALL患者体内的微量残留病（MRD）奠定基础。方法参照BIOMED－2协作组制定的Ig和（或）TCR检测方法，设计96条不同的PCR引物，分成14个混合管，通过多重PCR，分别检测患者骨髓单个核细胞的IgH、IgK、TCRB、TCRG、TCRD克隆性基因重排。结果在22例成人B系ALL患者中，Ig克隆性重排检出率为96％，其中IgH为86％，IgK为14％。在18例成人T系ALL患者中，TCR克隆性重排检出率为100％，其中TCRB为83％，TCRG为78％，TCRD为39％。两个及两个以上克隆性标志物的检出率在B和T系ALL中分别为91％（22例中20例）和89％（18例中16例）。结论BIOMED－2协作组设计的14管多重PCR引物和方法，几乎可检测到淋巴细胞白血病患者体内所有占优势的克隆性T、B细胞增殖群体，方法简便、可靠、覆盖面广，适用于成人ALL患者基凶重排检测和MRD监测。

Objective To provide the evidence of RQ-PCR-based assessment of minimal residual disease（MRD）, the clonal immunoglobulin and T-cell receptor （Ig/TCR） gene rearrangements were identified in newly diagnosed adult patients with acute lymphoblastic leukemia （ALL） by multiplex PCR protocols. Methods Forty newly diagnosed adult patients with B-lineage（B-） and T cell （T-） ALL were involvled in this study. All DNA samples were obtained from the bone marrow （BM） mononuclear ceils （MNC）. IgH, IgK,TCRB,TCRG and TCRD gene rearrangements were detected by BIOMED-2 multiplex PCR protocols, which included 96 different primers and 14 muhiplex PCR tubes. Results The clonal immunoglobulin（Ig） rearrangements were found in 96% of B- ALL, 86% being IgH and 14% IgK. While in T-ALL, clonal TCR rearrangements were found in all of the patients, 83% being TCRB,78% TCRG and 39% TCRD. More than two clonal markers were found in 91% of B- ALL and 89% of T- ALL patients. Conclusions The detection rate of clonal rearrangements using the BIOMED-2 14 nmhiplex PCR tubes is unprecedentedly high, which can detect virtually all clonal B and T-cell proliferations. It can be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.