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中华结直肠疾病电子杂志

中华结直肠疾病电子杂志 ›› 2016, Vol. 05 ›› Issue (05) : 398 -404. doi: 10.3877/cma.j.issn.2095-3224.2016.05.006

所属专题： 文献；

论著

结直肠癌错配修复蛋白和微卫星不稳定检测的对比分析
陈琼荣1, 王满香1, 郭芳1, 况晶1, 方娜1, 王明伟1, 金苏1, 吴德1, 邓云特1, 魏少忠2,†()   
  1. 1. 430079　湖北省肿瘤医院病理科
    2. 湖北省肿瘤医院胃肠肿瘤外科
  • 收稿日期:2016-05-15 出版日期:2016-10-25
  • 通信作者: 魏少忠
  • 基金资助:
    湖北省卫生厅重点资助项目(No.JX6A06); 湖北省自然科学基金重点资助项目(No.2013CFA078); 湖北省自然科学基金资助项目(No.2013CFC022)

Testing mismatch repair proteins versus microsatellite instability in colorectal carcinoma

Qiongrong Chen1, Manxiang Wang1, Fang Guo1, Jing Kuang1, Na Fang1, Mingwei Wang1, Su Jin1, De Wu1, Yunte Deng1, Shaozhong Wei2,†()   

  1. 1. Department of Pathology, Hubei Cancer Hospital, Wuhan 430079, China
    2. Department of Gastrointestinal Surgical Oncology, Hubei Cancer Hospital, Wuhan 430079, China
  • Received:2016-05-15 Published:2016-10-25
  • Corresponding author: Shaozhong Wei
  • About author:
    Corresponding author: Wei Shaozhong, Email:
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引用本文：

陈琼荣, 王满香, 郭芳, 况晶, 方娜, 王明伟, 金苏, 吴德, 邓云特, 魏少忠. 结直肠癌错配修复蛋白和微卫星不稳定检测的对比分析[J]. 中华结直肠疾病电子杂志, 2016, 05(05): 398-404.

Qiongrong Chen, Manxiang Wang, Fang Guo, Jing Kuang, Na Fang, Mingwei Wang, Su Jin, De Wu, Yunte Deng, Shaozhong Wei. Testing mismatch repair proteins versus microsatellite instability in colorectal carcinoma[J]. Chinese Journal of Colorectal Diseases(Electronic Edition), 2016, 05(05): 398-404.

目的

目前最常用的筛查结直肠癌DNA错配修复基因缺失的方法是免疫组化检测错配修复（MMR）基因相关蛋白的表达，以及基于PCR检测多个微卫星位点判断有否微卫星不稳定（MSI）这2种方法，本研究主要目的是比较这二种检测之间的一致性，并对分子病理室作室内质量控制。

方法

收集2014年8月至2015年10月湖北省肿瘤医院结直肠癌368例手术切除标本，免疫组化常规检测癌组织MLH1，PMS2，MSH2及MSH6这4种蛋白的表达。免疫组化显示任一蛋白完全缺失，判读为MMR蛋白缺失（dMMR）；如癌细胞4个MMR有多少不等的核着色，判读为MMR无缺失（pMMR）。选取其中的65例行PCR-毛细管电泳法检测MSI，其中28例为pMMR，37例为dMMR。然后对这65例组织用PCR毛细管电泳法检测Bethesda推荐的5个微卫星位点。比对这65例患者上述二种检测结果之间的一致性，并分析、整理其临床病理特征。

结果

368例结直肠癌中有37例免疫组化结果为dMMR中，其余331例为pMMR。37例中剔除2例后对其中35例行毛细管PCR法检测，显示高频MSI者32例，微卫星稳定（MSS）者3例。选取331例中的28例行PCR检测，显示MSS者27例，MSI-H者1例。免疫组化法检测的敏感度和特异性分别为97.0%和90.0%，PCR检测结果的敏感度和特异性分别为91.4%和96.4%；二者总的一致性为93.7%。伴MSI的结直肠癌原发灶以右半结肠最多见（占48.6%），病理形态以低分化腺癌伴淋巴细胞浸润和粘液分泌最常见，病理TNM分期以Ⅱ期和Ⅲ期为主。

结论

免疫组化检测MMR蛋白和基于PCR的毛细管电泳法检测MSI二者的一致性高，其中免疫组化法可以作为临床初筛结直肠癌微卫星不稳定性的一种经济而便捷的方法，值得推广。

关键词: 结直肠肿瘤, 免疫组织化学, 微卫星不稳定, 错配修复蛋白
Objective

Immunohistochemical (IHC) staining for mismatch repair (MMR) proteins and PCR-based detection for microsatellite status are routinely performed on colorectal carcinoma (CRC) surgical samples. However, the concordance of the two detections, which is related to the quality control of our molecular pathology laboratory, is unknown so far. So the main aim of this study is to compare the differences between the two analyses and to improve our work.

Methods

IHC analyzed the expression of MLH1, PMS2, MSH2 and MSH6 which was performed on 368 cases of formalin-fixed paraffin-embedded (FFPE) CRC tissues. If any one protein is negative in all of cancer cells but positive in normal colorectal mucosa, the IHC staining was reported as mismatch repair defective (dMMR). If the four MMR proteins are expressed in the nucleus of one or more cancer cells, the IHC result was interpreted as mismatch repair proficient (pMMR). All of the 37 cases of dMMR and selected 28 cases of pMMR were tested by PCR-based MSI analysis. Paired normal and cancer DNA samples isolated from the FFPE tissues were tested for MSI using Bethesda recommended 5 markers (BAT25, BAT26, D2S123, D5S346, D17S250). At last the results of IHC and PCR were compared and their concordance were analyzed.

Results

IHC analyses were performed on 368 cases of CRC, among which 37 cases were dMMR and 331 cases were pMMR. After excluding 2 cases from the 37 samples, the remained 35 samples were tested for MSI, among which 32 samples were high-level microsatellite instability (MSI-H) and 3 samples were microsatellite stable (MSS). In addition, 28 cases of pMMR samples were selected to be tested by PCR for MSI, among which 27 cases were MSS but one case was MSI-H. The sensitivity and specificity of immunohistochemistry was 97.0% and 90.0%, separately, the sensitivity and specificity of PCR was 91.4% and 96.4%, separately, and the total concordance of the two detections achieved 93.7%. The most common original site of dMMR CRC was right hemicolon (occupying 48.6%), and the most common pathological features included mucous adenocarcinoma, poor differentiated adenocarcinoma with lymphocytes infiltration, and pathologic TNM stage Ⅱ and stage Ⅲ.

Conclusions

The total concordance of the immunohistochemistry for MMR proteins and PCR-based MSI testing achieved 93.7%, and the former is an economic and quick screening method which is deserved to be popularized in China. Moreover, we have to emphasize the important role of intra and external laboratory quality control of the two methods and then to improve the process, so as to increase the testing accuracy.

Key words: Colorectal neoplasms, Immunohistochemistry, Microsatellite instability, Mismatch repair proteins
图1 A、B、C显示1例84岁女性患者右半结肠癌病理特征：A图显示低分化腺癌（HE染色，×20倍）；B图免疫组化判读为PMS2缺失（IHC染色，×40倍）；C图免疫组化MSH6表达正常（IHC染色，×40倍）。D、E、F显示另1例27岁男性患者右半结肠癌病理特征：D图显示中分化腺癌伴粘液成分（HE染色，×100倍）；E图免疫组化判读为MSH6缺失（IHC染色，×100倍）；F图免疫组化MLH1表达正常（IHC染色，×40倍）
图2 显示1例57岁男性患者乙状结肠癌组织和相应的正常远癌切断组织的PCR毛细管电泳法检测Bethesda推荐的5个微卫星位点的检测结果，红色箭头显示癌组织的4个位点（D5S346，Bat-25，D17S250和Bat-26）相较于正常切端组织发生了位点偏移，判读为MSI-H
表1 免疫组化检测MMR蛋白比对PCR检测MSI的分析（例）
PCR检测MSI 例数 IHC检测MMR蛋白
dMMR pMMR
MSI-H 33 32 1
MSS 30 3 27
表2 PCR检测MSI比对免疫组化检测MMR蛋白的分析（例）
IHC检测MMR蛋白 例数 PCR检测MSI
MSI-H MSS
dMMR 35 32 3
pMMR 28 1 27
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