Greenberg S J, Choi Y, Ballow M, Du T L, Ward P M, Rickert M H, Frankel S, Bernstein S H, Brecher M L
Department of Neurology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
J Leukoc Biol. 1995 Jun;57(6):856-64. doi: 10.1002/jlb.57.6.856.
The predominant B cell immunoglobulin heavy chain variable gene (IgH-V) usage and the uniquely rearranged, clonotype-specific variable-diversity-joining region gene (VDJ) sequences were identified in patients with B cell acute lymphoblastic leukemia (B-ALL) using a novel DNA-based gene amplification strategy. The approach allows a thorough and sensitive determination of the number of clonal leukemic IgH rearrangements and their precise V gene usage. This strategy may be applied in the detection of minimal residual disease, in surveillance after induction of disease-free states, and in analyzing the effectiveness of purging autologous bone marrow of malignant clones. An initial primary polymerase chain reaction (PCR), directed by an IgH-J generic primer and a complement of family-specific IgH-V primers, defined the major B cell IgH-V gene usage. Use of an IgH-J generic primer supplanted the use of a constant region primer anchor and thus eliminated the need to target mRNA by the traditional RNA reverse transcription-PCR amplification method. Monoclonality of rearranged VDJ bands was further substantiated by high-resolution denaturant gel electrophoretic analysis. The predominant amplified bands were subcloned and sequenced. By sequencing through VDJ juxtaposed regions, that is, the third complementarity-determining region, clonotype-specific primers were developed and used in a secondary clonotype primer-directed PCR (CPD-PCR) to detect, with extreme sensitivity and specificity, a unique B cell clone. Analysis of the products of the CPD-PCR permitted the detection of a single malignant cell among 1 million polyclonal cells and superseded the constraints of prior studies that have provided a limited evaluation of family variable gene repertoire usage. Leukemic clonal rearrangements were detected in 100% of the eight cases of pediatric and two cases of adult B-ALL studied. Two or more clonal IgH-VDJ amplified sequences were observed in 50% of the B-ALL bone marrows analyzed. In two cases, clonotype-specific oligodeoxynucleotide primers, derived from B-ALL VDJ sequences, directed the secondary CPD-PCR, and disease activity was monitored after chemotherapy and allogeneic bone marrow transplantation.
采用一种基于DNA的新型基因扩增策略，在B细胞急性淋巴细胞白血病（B-ALL）患者中确定了主要的B细胞免疫球蛋白重链可变基因（IgH-V）使用情况以及独特重排的、克隆型特异性可变-多样-连接区基因（VDJ）序列。该方法能够全面且灵敏地测定克隆性白血病IgH重排的数量及其精确的V基因使用情况。此策略可应用于微小残留病的检测、诱导无病状态后的监测以及分析清除恶性克隆的自体骨髓的有效性。最初的一次聚合酶链反应（PCR）由一个IgH-J通用引物和一组家族特异性IgH-V引物进行引导，确定了主要的B细胞IgH-V基因使用情况。使用IgH-J通用引物取代了恒定区引物锚的使用，从而无需通过传统的RNA逆转录-PCR扩增方法靶向mRNA。通过高分辨率变性凝胶电泳分析进一步证实了重排VDJ条带的单克隆性。对主要的扩增条带进行亚克隆并测序。通过对VDJ并列区域（即第三个互补决定区）进行测序，开发了克隆型特异性引物，并用于二次克隆型引物引导的PCR（CPD-PCR），以极高的灵敏度和特异性检测独特的B细胞克隆。对CPD-PCR产物的分析能够在100万个多克隆细胞中检测到单个恶性细胞，克服了以往研究在评估家族可变基因库使用情况方面的局限性。在所研究的8例儿童和2例成人B-ALL病例中，100%检测到白血病克隆重排。在50%分析的B-ALL骨髓中观察到两个或更多克隆性IgH-VDJ扩增序列。在两例病例中，源自B-ALL VDJ序列的克隆型特异性寡脱氧核苷酸引物引导二次CPD-PCR，并在化疗和异基因骨髓移植后监测疾病活性。
