Gerard C J, Olsson K, Ramanathan R, Reading C, Hanania E G
SyStemix, Inc., Palo Alto, California 94304, USA.
Cancer Res. 1998 Sep 1;58(17):3957-64.
The complementarity determining region III of the rearranged immunoglobulin heavy chain gene has been the target for tumor-specific PCR assays for the detection and follow-up of B-cell malignancies. Previously, these assays have relied on gel-based end point data collection methods (i.e., band densitometry) and, thus, have provided at best a semiquantitative assessment of tumor levels. We show the development of a novel, real-time TaqMan PCR assay to quantitate residual multiple myeloma cells in clinical samples after high-dose chemotherapy and autologous stem cell transplantation. We provide evidence that real-time PCR is reproducible, sensitive, and quantitative. In a 40-replicate PCR experiment targeting the beta-actin gene, the coefficient of variation for threshold cycle data was 1.6%, whereas it increased to 13.6% and 31%, respectively, for end point fluorescence and gel densitometry. Moreover, in an experiment directly comparing standard curves obtained from band densitometry and threshold cycle data, the standard curve constructed from threshold cycle data had a multiple R2 value of 1.00 and demonstrated a dynamic range >4 logs, compared with the 2-log linear range of gel densitometry. Finally, we show that when a complementarity determining region III-specific PCR primer is used in conjunction with a consensus primer for the immunoglobulin heavy chain joining gene, plasmid DNA can be used as a readily available and effective substitute for clonal plasma-cell genomic DNA when preparing standards. By applying real-time PCR to the analysis of clinical samples, we are able to quantitate levels of tumor involvement with unparalleled reproducibility and statistical confidence. Real-time PCR technology may well provide the accuracy and reliability necessary for minimal residual disease detection to have real prognostic significance.
重排免疫球蛋白重链基因的互补决定区III一直是用于检测和随访B细胞恶性肿瘤的肿瘤特异性PCR检测的靶点。以前，这些检测依赖于基于凝胶的终点数据收集方法（即条带光密度测定），因此，充其量只能对肿瘤水平进行半定量评估。我们展示了一种新型实时TaqMan PCR检测方法的开发，用于定量高剂量化疗和自体干细胞移植后临床样本中残留的多发性骨髓瘤细胞。我们提供的证据表明，实时PCR具有可重复性、敏感性和定量性。在针对β-肌动蛋白基因的40次重复PCR实验中，阈值循环数据的变异系数为1.6%，而终点荧光和凝胶光密度测定的变异系数分别增至13.6%和31%。此外，在一项直接比较从条带光密度测定和阈值循环数据获得的标准曲线的实验中，由阈值循环数据构建的标准曲线的复相关系数R2值为1.00，动态范围>4个对数，而凝胶光密度测定的线性范围为2个对数。最后，我们表明，当将互补决定区III特异性PCR引物与免疫球蛋白重链连接基因的共有引物结合使用时，在制备标准品时，质粒DNA可作为克隆浆细胞基因组DNA的现成且有效的替代品。通过将实时PCR应用于临床样本分析，我们能够以前所未有的可重复性和统计可信度定量肿瘤累及水平。实时PCR技术很可能为微小残留病检测提供具有实际预后意义所需的准确性和可靠性。
