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Development of a novel real-time quantitative polymerase chain reaction assay for detecting minimal residual disease in coloreactal cancer

开发一种用于检测结直肠癌微小残留病的新型实时定量聚合酶链反应测定法

基本信息

批准号：
14571222
负责人：
HIGUCHI Taro
金额：
$ 1.92万
依托单位：
Iwate Medical University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571222/
关键词：
MGB RQ-PCR colorectal cancer MRD CEA lymph node metastasis ASO k-ras p53 APC

项目摘要

Tumor-specific point mutations are stable biomarkers compared with tumor-specific mRNA expression, and are therfore useful to detect occult tumor cells. These mutations have never been used for real-time quantitative polymerase chain reaction (RQ-PCR) assays, because the ability of conventional probes to discriminate between wild-type and mutant alleles is poor. Recently, DNA probes with conjugated minor groove binder (MGB) have been developed. Because of their high melting temperature, these probes achieve high performance in detecting single nucleotide mismatches. Using the MOB technology, we developed a new RQ-PCR system for detecting occult tumor cells in patients with colorectal cancer (CRC), targeting K-ras point mutations. Sixteen MGB-conjugated DNA probes were designed for all previously reported K-ras mutations. The performance of these probes was examined with plasmid DNAs into which K-ras point mutations had been inserted, 32 cancer cell lines and 338 lymph nodes obtained from 15 CRC patients. Fifteen of the 16 MOB probes designed were useful for accurate quantitative assessment, and achieved high sensitivity (1/10^4-10^5 background cells) and high reproducibility (coefficients of variation < 10%). Performance in discriminating single nucleotide mismatches was superior for MGB probes compared with non-MOB probes. We detected a micrometastasis (5.85/l0~ cells equivalent) in one (0.9%) of 110 lymph nodes obtained from 6 patients with K-ras mutations. There was no true false-positive result in 209 lymph nodes obtained from 9 patients without K-ras mutations. The MOB RQ-PCR assay targeting K-ras mutations is an accurate quantitative method for detecting occult tumor cells in CRC.

与肿瘤特异性mRNA表达相比，肿瘤特异性点突变是稳定的生物标志物，因此可用于检测隐匿性肿瘤细胞。这些突变从未用于实时定量聚合酶链反应（RQ-PCR）测定，因为常规探针区分野生型和突变等位基因的能力很差。最近，已经开发了具有缀合的小沟结合剂（MGB）的DNA探针。由于它们的高解链温度，这些探针在检测单核苷酸错配方面实现了高性能。利用MOB技术，我们开发了一种新的RQ-PCR系统，用于检测结直肠癌（CRC）患者中的隐匿性肿瘤细胞，靶向K-ras点突变。设计了16个MG-缀合的DNA探针用于所有先前报道的K-ras突变。这些探针的性能进行了检查与质粒DNA，其中K-ras点突变已插入，32个癌细胞系和338个淋巴结从15个CRC患者。16种MOB探针中有15种可用于准确定量检测，并具有高灵敏度（1/10^4-10^5背景细胞）和高重复性（变异系数< 10%）。与非MOB探针相比，MGB探针在区分单核苷酸错配方面的性能是上级的。我们在6例K-ras突变患者的110个淋巴结中检测到一个微转移（5.85/10 ~细胞当量）（0.9%）。9例无K-ras基因突变患者的209枚淋巴结无真假阳性。针对K-ras突变的MOB RQ-PCR检测是检测CRC中隐匿性肿瘤细胞的准确定量方法。