An optimized pentaplex PCR for detecting DNA mismatch repair-deficient colorectal cancers.
An optimized pentaplex PCR for detecting DNA mismatch repair-deficient colorectal cancers.
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DOI：
10.1371/journal.pone.0009393
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发表时间：
2010-02-24
期刊：
影响因子：
3.7
通讯作者：
Boland CR
中科院分区：
文献类型：
--
作者：
Goel A;Nagasaka T;Hamelin R;Boland CR
微卫星不稳定性 (MSI) 用于筛查结直肠癌 (CRC) 中的林奇综合征，并预测结果和治疗反应。目前测量 MSI 的技术需要来自正常组织和肿瘤组织的 DNA，并且无法识别具有特定 DNA 错配修复 (MMR) 缺陷的肿瘤。我们测试了一组在单一多重 PCR 反应（五重 PCR）中扩增的五个准单态单核苷酸重复标记，以检测 MSI。我们研究了 213 名 CRC 患者的队列，其中包括 114 名 MMR 缺陷型肿瘤和 99 名 MMR 丰富型肿瘤。免疫组织化学 (IHC) 分析评估了 MLH1、MSH2、PMS2 和 MSH6 的表达。 MSI 状态是通过正常 DNA 样本库的准单态变异范围 (QMVR) 的差异以及测量肿瘤 DNA 中等位基因长度的差异来定义的。 426 个正常等位基因的扩增允许优化每个标记的 QMVR，并且无需匹配参考 DNA 来定义每个样本中的 MSI。使用≥2/5不稳定标志物作为MSI标准，敏感性为95.6% (95% CI = 90.1–98.1%)，阳性预测值为100% (95% CI = 96.6%–100%)。使用所有技术检测 MSH6 缺陷都受到限制。三标记物组（BAT26、NR21 和 NR27）的数据分析在敏感性 (97.4%) 和阳性预测值 (96.5%) 方面与五标记物组相当。这两种方法均优于测量 MSI 的标准方法。优化的五重（或三重）PCR 为 CRC 中的 MSI 鉴定提供了一种简便、稳定、非常便宜、高度灵敏且特异的检测方法。
Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI. We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA. Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI. An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.
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影响因子：
64.8
作者：
BRONNER, CE;BAKER, SM;LISKAY, RM
通讯作者：
LISKAY, RM
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Ribic, CM;Sargent, DJ;Gallinger, S
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Gallinger, S
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作者：
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Gassull, Miquel A.
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通讯作者：
Hamelin, R
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作者：
THIBODEAU, SN;BREN, G;SCHAID, D
通讯作者：
SCHAID, D