Abstract
目的
研究微卫星不稳定性(MSI)结直肠癌病人错配修复(MMR)基因突变的类型和临床病理特征。
方法
收集南方医科大学南方医院2016~2018年1394例结直肠癌病人石蜡标本，采用免疫组化染色技术检测MMR蛋白; MSI基因检测106例dMMR病例，46例肿瘤细胞呈异质性表达的pMMR病例和147例随机选择的pMMR病例; 分析错配修复蛋白缺失与结直肠癌临床病理特征参数的关系; 比较两种方法检测结果的一致性。
结果
MMR蛋白缺失发生率占总结直肠癌病人的7.6%，缺失类型主要为MLH1、PMS2双缺失，占总缺失的55.7%;MMR蛋白缺失与结直肠癌患者的年龄、发生部位、大体分型、肿瘤大小、组织学分型、分化程度和临床TNM分期均有关(P < 0.05)，而与患者的性别无关(P=0.47);dMMR结直肠癌病人具有典型的临床病理特征：MMR蛋白缺失在年龄 < 50岁、右半结肠、肿瘤直径 > 6 cm、腺癌伴粘液腺癌或粘液分泌的混合型癌、低分化腺癌和TNM分期Ⅰ/Ⅱ期组的发生率高; 免疫组化和PCR-毛细管电泳法在结直肠癌微卫星不稳定性检测结果一致性为98.7%。
结论
微卫星不稳定性错配修复蛋白表达缺失(dMMR)在结直肠癌的主要类型为MLH1、PMS2双缺失，具有典型的临床病理特征，其发生率低于西方报道; 免疫组化和PCR毛细管电泳法的检测结果显示高度的一致性。
Keywords: 结直肠癌, 微卫星不稳定, 错配修复, 免疫组化, PCR-毛细管电泳
Abstract
Objective
To explore the clinicopathological features and types of genic mutations in DNA mismatch repair (MMR) in colorectal cancer (CRC).
Methods
Immunohistochemistry was used to determine the expression of MMR proteins in 1394 patients with CRC, and PCR-capillary electrophoresis (PCR-CE) was used to detect microsatellite instability (MSI) in 106 cases of defective MMR (dMMR), 46 cases of proficient MMR (pMMR) with heterogeneous expression and 147 randomly selected cases of pMMR. The relationship between the expressions of MMR proteins and the clinicopathological features of the patients was evaluated. The consistency between the results of immunohistochemistry and PCR-CE was assessed.
Results
Immunohistochemical staining showed an incidence of dMMR of 7.6% in the patients. The main type of dMMR was co-deletion of MLH1 and PMS2, accounting for 55.7% of the total dMMR cases. The deletion of MMR proteins was significantly correlated with the patients' age, tumor location, tumor size, gross type, histological type, degree of differentiation, lymph node status and TNM stage (P < 0.05), but not with gender (P > 0.05). The total accordance rate of immunohistochemistry and PCR-CE was 98.7% in these patients.
Conclusion
The main type of dMMR is co-deletion of MLH1 and PMS2 in patients with colorectal cancer. dMMR colorectal cancer has typical clinicopathological features and a lower incidence in China than in Western countries. The results of immunohistochemistry and PCR-CE are highly consistent for detecting dMMR in colorectal cancer patients.
Keywords: colorectal cancer, microsatellite instability, mismatch repair, immunohistochemistry, polymerase chain reaction-capillary electrophoresis
据2018年最新的全球肿瘤统计，结直肠癌位居全球总人口最新癌症发病率排行榜第四位[1]，2017年《中国肿瘤的现状和趋势》报告结直肠癌位居我国癌症发病率的第五位，占癌症发病人口的6.79%。针对结直肠癌的研究是近年的热点。
微卫星不稳定性(MSI)是由DNA错配修复(MMR)蛋白功能缺失所导致，其分子特征被认为是重要的基因标记物[2-3]，为公认的结直肠癌的重要致癌途径之一，其在所有结直肠癌病例中占10%~20%，包括12%散发性结直肠癌(SCRC)和3%遗传性非息肉病性结直肠癌(HNPCC，即Lynch综合征)[4-5]。因而，开展对MSI相关结直肠癌(MSI型结直肠癌)筛查、识别、治疗和预后的研究具有重要的临床应用意义，越来越受到重视。
近年来，国内外多项研究对MSI与结直肠癌的临床病理特征进行分析，大多数研究显示MSI-H结直肠癌患者具有明显且独特的特征[6-8]。有研究发现不同地区及不同人种发生突变的微卫星基因类型有差异。国内关于结直肠癌微卫星不稳定和临床病理特征的相关性，缺少大样本数据分析，得出的结论不完全相同[9-11]。结直肠癌错配修复蛋白缺失主要发生在MLH1，PMS2，MSH2和PMS2蛋白，但对于微卫星不稳定性结直肠癌错配修复蛋白各种缺失类型组合的数据总结，国内未见报道。同时对于MMR蛋白和MSI基因检测结果不一致的原因分析，国内文献也缺少数据分析。因此，本研究拟对我科室2016~2018年确诊为结直肠癌病人1394例手术标本进行MMR蛋白免疫组化染色检测，并对其中299例进行MSI基因检测，探讨微卫星不稳定结直肠癌错配修复蛋白缺失的类型和临床病理特征，分析免疫组化与PCR-毛细管电泳检测结果的一致性，并对结果不一致的数据进行详细分析，为结直肠癌临床诊断、治疗和预后判断提供科学依据。
1. 资料和方法
1.1. 标本来源
收集2016~2018年在南方医科大学南方医院行根治性手术切除且已进行MMR蛋白检测的结直肠癌标本1394例，其中男性病人862例，女性病人532例，年龄17~93岁，中位年龄61岁，高分化癌121例，中分化癌1065例，低分化癌117例，粘液腺癌91例。根据国际抗癌联盟(UICC)2017年颁布的第八版TNM分期，Ⅰ和Ⅱ期785例，Ⅲ和Ⅳ期609例。患者术前未行放、化疗及免疫治疗。
1.2. MMR蛋白检测
MMR蛋白抗体MLH1、PMS2、MSH2和MSH6工作液均购自DAKO公司，二抗和显色系统使用DAKO K8002系统试剂，采用DAKAO Link48自动免疫组化机进行免疫组化染色。四种抗体均为细胞核着色，只要发现有肿瘤细胞核着色即判定为阳性，无肿瘤细胞核着色则判定为阴性。MLHl、PMS2、MSH2、MSH6这4种抗体中≥1种以上染色结果为阴性即判定为MMR蛋白表达缺失，简称dMMR，表现为高频度微卫星不稳定(MSI-H)。4种抗体都为阳性表达者判定为MMR蛋白表达正常，简称pMMR，表现为低频度微卫星不稳定(MSI-L)或微卫星稳定(MSS)。
1.3. MSI检测
对MMR蛋白缺失组、肿瘤细胞呈异质性表达组和部分MMR蛋白未缺失组的病例共299例，采用上海普洛麦格生物产品有限公司生产的微卫星不稳定性(MSI)检测试剂盒(荧光PCR-毛细管电泳法)进行MSI检测。检测位点包括8个微卫星位点(NR-21、BAT-25、BAT-26、Mono-27、BAT-52、BAT-56、BAT-59、BAT-60)，通过荧光PCR-毛细管电泳的方法对8个微卫星位点进行扩增以及比对正常组织与癌症组织中每个位点片段大小的变化来确定微卫星不稳定性。当癌组织微卫星片段大小与正常组织比较有≥3 pb的差异，该微卫星位点不稳定。统计所有微卫星位点结果，当有≥2个微卫星位点不稳定，该癌组织样本判读为MSI-H; 当只有1个微卫星位点不稳定，该癌组织样本判读为MSI-L; 当没有微卫星位点不稳定，该癌组织样本判读为MSS[12]。
1.4. 统计学分析
应用SPSS13.0分析软件，计数资料用百分率进行统计描述，组间率的比较采用卡方检验，P < 0.05表示差异有统计学意义。
2. 结果
2.1. MMR蛋白在结直肠癌组织中的表达
MMR蛋白阳性表达为肿瘤细胞核着色，呈棕色，阴性表达为肿瘤细胞无着色(图 1)。在1394例结直肠癌组织中，MMR蛋白有缺失的病例有106，占总数的7.6%。MMR蛋白无缺失的病例有1288例，其中46例病例至少有一种蛋白在肿瘤细胞中呈异质性表达。106例MMR蛋白表达缺失的类型分析：结直肠癌患者MMR蛋白缺失的类型主要为MLH1和PMS2同时缺失，占比为55.7%;其次为MSH2和MSH6同时缺失，占比为21.7%(表 1)。
1.
MMR蛋白在结直肠癌组织中的表达
Expression of MMR proteins in colorectal cancer tissue (Immunohistochemistry, original magnification: × 200). A: Positive MSH2 expression; B: Positive MSH6 expression; C: Negative MLH1 expression; D: Negative PMS2 expression.
1.
106例结直肠癌病人MMR蛋白表达缺失的类型分析
Analysis of the patterns of dMMR protein expression in 106 patients with colorectal cancer
| DMMR type | n | Percentage |
| MLH1(-), PMS2(-) | 59 | 55.7% |
| MSH2(-), MSH6(-) | 23 | 21.7% |
| PMS2(-) | 9 | 8.5% |
| MSH2(-) | 8 | 7.5% |
| MSH6(-) | 7 | 6.6% |
| Total | 106 | 100% |
2.2. MMR蛋白表达与结直肠癌临床病理特征的关系
dMMR和pMMR病人在年龄、部位、大体分型、肿瘤大小、组织学分型、分化程度、有无淋巴结转移和TNM分期组，差异具有统计学意义(P < 0.05)，而在性别组间没有统计学意义(表 2)。
2.
MMR蛋白表达与结直肠癌临床病理特征的关系
Correlation between clinicopathological characteristics and MMR protein expression in colorectal cancer [n(%)]
| Parameters | n (%) | MMR proteins expression | P | |
| dMMR | pMMR | |||
| Gender | ||||
| Male | 862(61.8) | 69(65.1) | 793(61.6) | 0.473 |
| Female | 532(38.2) | 37(34.9) | 495(38.4) | |
| Age (year) | ||||
| < 50 | 282(20.2) | 42(39.6) | 240(18.6) | < 0.001 |
| ≥50 | 1112 (79.8) | 64(60.4) | 1048 (81.4) | |
| Location | ||||
| Right colon | 367(26.3) | 68(64.1) | 299(23.2) | < 0.001 |
| Left colon | 489(35.1) | 29(27.4) | 460(35.7) | |
| Rectum | 538(38.6) | 9(8.5) | 529(41.1) | |
| Gross type | ||||
| Ulcerative | 779(55.9) | 52 (49.1) | 727(56.4) | 0.032 |
| Protuberant | 575(41.2) | 54 (50.9) | 521(40.5) | |
| Infiltrating | 40(2.9) | 0 (0) | 40(3.1) | |
| Tumor size (cm) | ||||
| < 3 cm | 285(20.4) | 7(6.6) | 278(21.6) | < 0.001 |
| 3-6 cm | 872(62.6) | 47(44.3) | 825(64.1) | |
| > 6 cm | 237(17.0) | 52(49.1) | 185(14.3) | |
| Histological classification | ||||
| Adenocarcinoma | 1147 (82.2) | 63(59.4) | 1084 (84.2) | < 0.001 |
| Adenocarcinoma with mucous adenocarcinoma | 139(10.0) | 28(26.4) | 111 (8.6) | |
| Mucous adenocarcinoma | 93(6.7) | 14(13.2) | 79(6.1) | |
| Signet ring cell Carcinoma | 15(1.1) | 1(1.0) | 14(1.1) | |
| Differentiation | ||||
| Well | 121 (8.7) | 12(11.3) | 109 (8.4) | < 0.001 |
| Moderate | 1065 (76.4) | 53(50.0) | 1012 (78.5) | |
| Poor | 117 (8.4) | 27(25.5) | 90(7.0) | |
| Mucinous adenocarcinoma | 91(6.5) | 14(13.2) | 79(6.1) | |
| Lymph node metastasis | ||||
| With | 599(43.0) | 29(27.4) | 570(44.3) | 0.001 |
| Without | 795(57.0) | 77(72.6) | 718(55.7) | |
| TNM stage | ||||
| I and II | 785(56.3) | 76(71.7) | 709(55.0) | 0.001 |
| III and IV | 609(43.7) | 30(28.3) | 579(45.0) | |
2.3. PCR-毛细管电泳法和免疫组化法检测结果比较
我们对106例dMMR病例和193例pMMR病例(包含46例肿瘤细胞呈异质性表达的病例)进行了PCR-毛细管电泳法检测，发现两种方法结果不一致的病例有14例，我们对这14例标本选用了另一家公司抗体重做了4项MMR蛋白免疫组化并重新检测了其MSI状态，最终确定两种方法不一致的病例为4例(表 3)。我们分析了两种方法第一次检测结果有误的10例样本，造成检测有误的原因主要有4种(表 4)：免疫组化染色弱导致误判为dMMR，免疫组化背景高导致误判为pMMR，标本污染导致PCR-CE结果为MSI-H，肿瘤细胞少导致PCR-CE结果为MSS。最终结果证实为MSI-H的有106例，MSI-L的有22例，MSS的有171例。最后通过对免疫组化和PCR-毛细管电泳法检测结果的比较，发现两者差异没有统计学意义(P=1.000)，295例检测结果一致，两种方法检测结果的符合率为98.7%(表 5);46例肿瘤细胞呈异质性表达的pMMR病例中，PCR-毛细管电泳法检测结果为MSS或者MSI-L的有44例，检测结果为MSI-H的有2例。
3.
两种方法检测结果不一致病例重新检测结果比较
Comparison between pre-retested and post-retested detection results in the two methods
| Inconsistent results | Pre-retested | Post-retested | |
| The result of IHC was pMMR, while it was MSI-H of PCR-CE; The result of IHC was dMMR, while it was MSI-L/MSS of PCR-CE. | |||
| IHC: pMMR | PCR-CE: MSI-H | 9 | 2 |
| IHC：dMMR | PCR-CE: MSI-L/MSS | 6 | 2 |
4.
两种方法检测结果错误诊断的原因分析
Analysis of causes of misdiagnosis by the two methods
| Cause | Misdiagnosis | n |
| Immunohistochemical weak staining led to dMMR misdiagnosis; High background staining of IHC led to pMMR misdiagnosis; Specimen contamination led to MSI-H false positives; The lack of tumor cells led to MSS false negatives. | ||
| Immunohistochemical weak staining | dMMR misdiagnosis | 2 |
| IHC high background staining | pMMR misdiagnosis | 1 |
| Specimen contamination | MSI-H false positives | 5 |
| Tumor cells lack | MSS false negatives | 2 |
5.
免疫组化法和PCR-毛细管电泳法检测结果比较
Comparison of detection results between immuno-histochemistry and PCR-capillary electrophoresis
| IHC | PCR-CE | Total | |
| MSI-L and MSS | MSI-H | ||
| pMMR | 191 | 2 | 193 |
| dMMR | 2 | 104 | 106 |
| Total | 193 | 106 | 299 |
3. 讨论
微卫星(MS)是指散布在整个基因组中以少数几个核苷酸(1~6个碱基对)为单位串联重复的DNA序列，约占人类基因组的3%。由于MS的重复结构，它特别容易发生复制错误，而正常由DNA错配修复系统修复[13]。MMR功能缺陷(dMMR)时，MS出现的复制错误得不到纠正并不断累积，使得MS序列长度或碱基组发生改变，称为微卫星不稳定性(MSI)。错配修复蛋白包括MutS和MutL两大家族，前者包括MSH2、MSH3和MSH6等，后者包括MLHl、MLH3、PMS1和PMS2[14-15]。目前研究发现并非所有错配修复基因均发生突变，常见突变基因为hMSH2、hMSH6和hLMH1，不同地区及不同人种发生突变的基因也有所不同。研究发现在欧美国家hMSH2突变多见[16]，而在日本、韩国一些研究者发现在亚洲人中hMLH1基因突变多见[17]。有研究发现MSI-H结直肠癌占散发性结直肠癌的15%，多见于右半结肠，组织学类型以黏液腺癌和低分化腺癌为常见，而且超过90%的Lynch综合征相关性结直肠癌均通过MSI途径发生[18-20]。
本研究中dMMR在结直肠癌的发生率为7.6%，与韩国的报道相近(5.5%~9.4%)[21-27]，低于西方的报道(约15%)[28-30]，这可能与中西方人种的差别有关。错配修复蛋白缺失的类型以MLH1和PMS2双突变为主，占所有突变病例的55.7%，其次为MSH2和MSH6双突变，占所有突变病例的21.7%。本研究中，dMMR结直肠癌病人具有明显且独特的临床病理特征：dMMR在年龄 < 50岁的结直肠癌病人发生率较高，达到14.9%，而50岁以上结直肠病人发生率仅为5.8%;不同部位的结直肠癌dMMR发生率有差异，右半结肠癌最高(18.5%)，左半结肠癌次之(5.9%)，而直肠癌最低(1.7%); 通过对肿瘤大小的分析，最大直径 > 6 cm的结直肠癌dMMR发生率最高(21.9%)，最大直径为4~6 cm的次之(5.4%)，最大直径 < 3 cm的最低(2.5%); 对比不同组织学类型结直肠癌dMMR的发生率，我们发现腺癌伴粘液腺癌或粘液分泌的混合型结直肠癌dMMR的发生率最高(20.1%)，单纯粘液腺癌次之(15.1%)，单纯腺癌最低(5.9%); 而腺癌中以低分化腺癌的dMMR发生率最高，达到23.1%。通过对临床TNM分期的比较，我们发现在Ⅰ和Ⅱ期结直肠癌dMMR发生率(9.7%)高于Ⅲ和Ⅳ期发生率(4.9%)。
研究证实MSI多由MMR蛋白表达缺失所致。因此，可通过检测MMR蛋白或MSI来反映微卫星状态。通过IHC法检测MMR蛋白和通过PCR法检测MSI是评估相同生物学效应的不同检测方法。IHC法检测MMR蛋白是一种间接评估微卫星状态的方法。直接检测MSI状态的常用方法是多重荧光PCR毛细血管电泳法，这也是目前认可的金标准。我们通过对免疫组化和PCR-毛细管电泳法检测结果的分析发现两种方法对MSI的检出率显示高度的一致性，符合率达到98.7%。这稍高于国内报道的95%左右[31]。本实验采用的ProDx®微卫星不稳定性(MSI)检测试剂盒含长单核苷酸重复位点, 长单核苷酸重复位点有更长的位移, 具有高突变频率和高检测灵敏度[32]。我们对4例结果不相符的病例进行分析：有2例均是MSH6呈小局灶性表达，肿瘤组织有异质性，微卫星不稳定性检测为MSI-H，这表明散发性结直肠癌中存在异质性表达现象[33]，提示对MMR蛋白呈小灶状表达的病例，有必要加做MSI检测，以最终确认其MSI状态; 另外2例为MLH1和PMS2均为阴性表达，而微卫星不稳定性检测为MSS，造成这种结果的原因可能与错配修复系统的蛋白存在功能重叠有关(PMS2和PMS1、MSH6和MSH3)，因此虽然蛋白表达缺失，IHC表现为dMMR，而PCR却显示MSS。
通过对第一次两种检测方法结果不一致的病例进行分析，我们认为影响免疫组化结果可靠性的最重要原因是标本的固定和抗体的质量，染色弱是造成误判为MMR蛋白表达缺失的最主要原因，提示保证标本的规范固定和选择质量好的一抗至关重要; 而造成PCR-毛细管电泳结果有误的最重要原因是标本的污染而造成的假阳性，其次为肿瘤细胞少而造成的假阴性，提示必须要严格按照PCR守则规范操作，避免标本污染，而且要保证肿瘤细胞量的足够。对于免疫组化和PCR-毛细管电泳两种方法检测结果不一致的病例，必须重新进行免疫组化和PCR-毛细管电泳检测，以保证结果的可靠性。
综上所述，本研究发现：dMMR在结直肠癌病人的发生率较西方报道的低; MMR蛋白缺失的主要类型为MLH1、PMS2双缺失; dMMR结直肠癌具有典型的临床病理特征，MMR蛋白缺失在年龄 < 50岁、右半结肠、肿瘤直径 > 6 cm、腺癌伴粘液腺癌或粘液分泌的混合型癌、低分化腺癌、TNM分期Ⅰ/Ⅱ期病人的发生率高; 免疫组化和PCR毛细管电泳法的检测结果显示高度的一致性。本研究这些发现有助于为结直肠癌临床诊断、治疗和预后判断提供科学依据。
Biography
黎相照，硕士，主管技师，E-mail: 45537212@qq.com
Funding Statement
国家重点研发计划(2017YFC1309002)；国家自然科学基金(81872041，81672821)；广东省自然科学基金重点项目(2018B0303110017)
Supported by National Natural Science Foundation of China(81872041, 81672821)
Contributor Information
黎 相照 (Xiangzhao LI), Email: 45537212@qq.com.
梁 莉 (Li LIANG), Email: 2159878@qq.com.
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