Development of a fluorescent multiplex assay for detection of MSI-High tumors.
Development of a fluorescent multiplex assay for detection of MSI-High tumors.
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DOI：
10.1155/2004/136734
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
Thibodeau SN
中科院分区：
文献类型：
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作者：
Bacher JW;Flanagan LA;Smalley RL;Nassif NA;Burgart LJ;Halberg RB;Megid WM;Thibodeau SN
确定肿瘤是否表现出微卫星不稳定性（MSI）有助于识别遗传性非息肉病性结直肠癌和散发性胃肠道癌伴DNA错配修复缺陷（MMR）的患者。MSI状态的评估有助于建立临床预后，并可预测肿瘤对化疗的反应。1997年，美国国家癌症研究所（NCI）的一个研讨会提出了一个由五个标志物组成的参考小组用于MSI分析，这有助于标准化检测。但这组标记物具有由于包含二核苷酸标记物而导致的局限性，与目前可用的其他类型的标记物相比，二核苷酸标记物对于检测具有MMR缺陷的肿瘤的灵敏度和特异性较低。这项研究表明，单核苷酸是最敏感和特异性的标志物，用于检测MMR缺陷的肿瘤，并确定了一个最佳的面板的标志物检测MSI-H肿瘤。一组266单，双，四和五核苷酸重复微卫星标记被用来筛选结直肠肿瘤的MSI。为MSI多重系统选择了检测MSI-H肿瘤的最佳标记物，其中包括五个单核苷酸标记物：BAT-25、BAT-26、NR-21、NR-24和MONO-27。此外，还添加了两种五核苷酸标记物以鉴定样品混合和/或污染。我们使用新的MSI多重系统对153例结直肠肿瘤进行了分类，并将结果与10个微卫星标记结合免疫组化（IHC）分析的结果进行了比较。我们观察到两种方法之间的一致性为99%，在MSI-H肿瘤检测中的准确率接近100%。大约5%的MSI-H肿瘤具有正常水平的四种MMR蛋白，因此仅基于IHC分析就会被错误分类，强调了进行MSI测试的重要性。新的MSI多重系统提供了几个明显的优势，比其他方法的MSI测试，因为它是非常敏感和具体的，并服从高通量分析。MSI多重系统符合最近2002年NCI HNPCC和MSI测试研讨会上提出的新建议，并克服了原始五标记面板固有的问题。使用单一多重荧光MSI检测减少了MSI检测所涉及的时间和成本，提高了可靠性和准确性，因此应有助于广泛筛查胃肠道癌患者肿瘤中的微卫星不稳定性。
Determining whether a tumor exhibits microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer and sporadic gastrointestinal cancers with defective DNA mismatch repair (MMR). The assessment of MSI status aids in establishing a clinical prognosis and may be predictive of tumor response to chemotherapy. A reference panel of five markers was suggested for MSI analysis by a National Cancer Institute (NCI) workshop in 1997 that has helped to standardize testing. But this panel of markers has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumors with MMR deficiencies compared to other types of markers that are currently available. This study demonstrates that mononucleotides are the most sensitive and specific markers for detection of tumors with defects in MMR and identifies an optimal panel of markers for detection of MSI-H tumors. A set of 266 mono-, di-, tetra- and penta-nucleotide repeat microsatellite markers were used to screen for MSI in colorectal tumors. The best markers for detection of MSI-H tumors were selected for a MSI Multiplex System, which included five mononucleotide markers: BAT-25, BAT-26, NR-21, NR-24 and MONO-27. In addition, two pentanucleotide markers were added to identify sample mix-ups and/or contamination. We classified 153 colorectal tumors using the new MSI Multiplex System and compared the results to those obtained with a panel of 10 microsatellite markers combined with immunohistochemical (IHC) analysis. We observed 99% concordance between the two methods with nearly 100% accuracy in detection of MSI-H tumors. Approximately 5% of the MSI-H tumors had normal levels of four MMR proteins and as a result would have been misclassified based solely on IHC analysis, emphasizing the importance of performing MSI testing. The new MSI Multiplex System offers several distinct advantages over other methods of MSI testing in that it is both extremely sensitive and specific and amenable to high-throughput analysis. The MSI Multiplex System meets the new recommendations proposed at the recent 2002 NCI workshop on HNPCC and MSI testing and overcomes problems inherent to the original five-marker panel. The use of a single multiplex fluorescent MSI assay reduces the time and costs involved in MSI testing with increased reliability and accuracy and thus should facilitate widespread screening for microsatellite instability in tumors of patients with gastrointestinal cancers.