Juhasz Agnes, Frankel Paul, Cheng Catherine, Rivera Hector, Vishwanath Reena, Chiu Alice, Margolin Kim, Yen Yun, Newman Edward M, Synold Tim, Wilczynski Sharon, Lenz Heinz-Josef, Gandara David, Albain Kathy S, Longmate Jeffrey, Doroshow James H
Department of Medical Oncology, City of Hope Comprehensive Cancer Center, Duarte, California 91010, USA.
J Clin Lab Anal. 2003;17(5):184-94. doi: 10.1002/jcla.10091.
The solid tumor mRNA expression of genes related to the mechanism of action of certain antineoplastic agents is often predictive of clinical efficacy. We report here on the development of a rapid and practical real-time RT-PCR method to quantify genetic expression in solid tumors. The genes examined are related to the intracellular pharmacology of gemcitabine and cisplatin, two drugs that are used in the treatment of several types of advanced cancer. We evaluated target gene mRNA levels from breast tumor samples using two quantitative RT-PCR methods: 1) an improved relative RT-PCR method using fluorescence-labeled primers, automated PCR set up, and GeneScan analysis software; and 2) real-time RT-PCR with redesigned primers using an ABI 7900HT instrument, with additional postprocessing of the data to adjust for efficiency differences across the target genes. Using these methods, we quantified mRNA expression levels of deoxycytidine kinase (dCK), deoxycytidylate deaminase (dCDA), the M1 and M2 subunits of ribonucleotide reductase (RRM1, RRM2), and excision cross complementation group 1 (ERCC1) in 35 human "fresh" frozen breast cancer biopsies. While both assay methods were substantially more rapid than traditional RT-PCR, real-time RT-PCR appeared to be superior to the amplification end-point measurement in terms of precision and high throughput, even when a DNA sequencer was used to assess fluorescence-labeled PCR products. This reproducible, highly sensitive real-time RT-PCR method for the detection and quantification of the mRNAs for dCK, dCDA, RRM1, RRM2, and ERCC1 in human breast cancer biopsies appears to be more informative and less time-consuming than either classical radioisotope-dependent RT-PCR or the technique utilizing GeneScan analysis described herein. By allowing the measurement of intratumoral target gene expression, these new methods may prove useful in predicting the clinical utility of gemcitabine- and platinum-containing chemotherapy programs in patients with solid tumors.
某些抗肿瘤药物作用机制相关基因在实体瘤中的mRNA表达通常可预测临床疗效。我们在此报告一种快速实用的实时逆转录聚合酶链反应（RT-PCR）方法的开发，用于定量实体瘤中的基因表达。所检测的基因与吉西他滨和顺铂的细胞内药理学有关，这两种药物用于治疗多种类型的晚期癌症。我们使用两种定量RT-PCR方法评估乳腺肿瘤样本中的靶基因mRNA水平：1）一种改进的相对RT-PCR方法，使用荧光标记引物、自动PCR设置和基因扫描分析软件；2）使用ABI 7900HT仪器，采用重新设计的引物进行实时RT-PCR，并对数据进行额外的后处理，以调整不同靶基因之间的效率差异。使用这些方法，我们在35例人类“新鲜”冷冻乳腺癌活检样本中定量了脱氧胞苷激酶（dCK）、脱氧胞苷酸脱氨酶（dCDA）、核糖核苷酸还原酶的M1和M2亚基（RRM1、RRM2）以及切除修复交叉互补组1（ERCC1）的mRNA表达水平。虽然两种检测方法都比传统RT-PCR快得多，但实时RT-PCR在精度和高通量方面似乎优于扩增终点测量，即使使用DNA测序仪评估荧光标记的PCR产物时也是如此。这种用于检测和定量人类乳腺癌活检样本中dCK、dCDA、RRM1、RRM2和ERCC1的mRNA的可重复、高灵敏度的实时RT-PCR方法，似乎比经典的放射性同位素依赖RT-PCR或本文所述的利用基因扫描分析的技术更具信息量且耗时更少。通过允许测量肿瘤内靶基因表达，这些新方法可能在预测实体瘤患者含吉西他滨和铂类化疗方案的临床效用方面证明是有用的。
