Arnold Christian N, Goel Ajay, Compton Carolyn, Marcus Victoria, Niedzwiecki Donna, Dowell Jeannette M, Wasserman Linda, Inoue Toru, Mayer Robert J, Bertagnolli Monica M, Boland C Richard
Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California, USA.
Cancer Biol Ther. 2004 Jan;3(1):73-8. doi: 10.4161/cbt.3.1.590. Epub 2004 Jan 5.
About 15% of all colorectal cancers (CRCs) demonstrate high levels of microsatellite instability (MSI-H) and are currently best identified by molecular analysis of microsatellite markers. Most sporadic CRCs with MSI-H are known to be associated with the methylation of the hMLH1 promoter. Promoter methylation coincided with lack of hMLH1 expression. We aimed to investigate the association between MSI status, hMLH1 protein expression and methylation status of the hMLH1 promoter, and to determine the usefulness of each method in defining the MSI phenotype in sporadic CRCs.
CRCs from 173 patients from the Cancer and Leukemia Group B (CALGB) were assessed for their MSI status. An additional cohort of 18 MSI-H tumors from the University of California San Diego (UCSD) was included in the analysis of the MSI-H subgroup. MSI testing was performed by PCR using five standard MSI markers. hMLH1 promoter analysis was investigated by methylation specific PCR (MSP), and expression of the MMR genes hMLH1 and hMSH2 was examined by immunohistochemistry (IHC).
Of the 173 CALGB tumors, 111 (64%) were MSS, 35 (20%) were MSI-L and 27 (16%) MSI-H, respectively. Data on hMLH1 protein expression, hMSH2 protein expression and hMLH1 methylation are available on 128, 173 and 81 of these tumors, respectively. Presence of hMLH1 and hMSH2 protein expression was significantly associated with MSI status. Four of 45 (8.9%) MSI-H tumors and 0 of 146 (0%) MSS/MSI-L tumors did not express hMSH2 (p = 0.0028). hMLH1 protein expression was present in 107 of 108 (99%) MSS and MSI-L tumors versus 11 of 20 (55%) MSI-H tumors (p < 0.0001). Of 61 MSS and MSI-L cancers studied for methylation, 11 (18%) were methylated at the hMLH1 promoter whereas 14 of 20 (70%) MSI-H cancers were methylated (p = 0.0001). In 27 MSI-H tumors studied for hMLH1 protein expression and methylation, 93% of tumors with loss of expression (93%) were also methylated while 42% (5/12) with positive immunostaining for hMLH1 were methylated at the hMLH1 promoter (p = 0.009).
Promoter methylation and hMLH1 expression are significantly associated with the MSI-H phenotype in CRC. Promoter methylation analysis provides a useful means to screen for MSI-H tumors. Our data further suggests that hMLH1 promoter methylation analysis alone cannot replace MSI testing, as a significant number of MSI-H tumors could be potentially overseen by such an approach. We suggest that phenotypic evaluation of CRC is performed most reliably with MSI testing, although expression analysis and investigation of the promoter methylation status may complement the screening process.
约15%的结直肠癌（CRC）表现出高水平的微卫星不稳定性（MSI-H），目前通过微卫星标记的分子分析能最好地识别此类癌症。大多数散发性MSI-H CRC已知与hMLH1启动子的甲基化有关。启动子甲基化与hMLH1表达缺失同时出现。我们旨在研究MSI状态、hMLH1蛋白表达与hMLH1启动子甲基化状态之间的关联，并确定每种方法在定义散发性CRC的MSI表型中的实用性。
对来自癌症与白血病B组（CALGB）的173例患者的CRC进行MSI状态评估。另外18例来自加利福尼亚大学圣地亚哥分校（UCSD）的MSI-H肿瘤纳入MSI-H亚组分析。使用五个标准MSI标记通过PCR进行MSI检测。通过甲基化特异性PCR（MSP）研究hMLH1启动子分析，并通过免疫组织化学（IHC）检测错配修复基因hMLH1和hMSH2的表达。
在173例CALGB肿瘤中，分别有111例（64%）为微卫星稳定（MSS）、35例（20%）为低度微卫星不稳定（MSI-L）和27例（16%）为高度微卫星不稳定（MSI-H）。这些肿瘤中分别有128例、173例和81例可获得hMLH1蛋白表达、hMSH2蛋白表达和hMLH1甲基化的数据。hMLH1和hMSH2蛋白表达的存在与MSI状态显著相关。45例MSI-H肿瘤中有4例（8.9%）不表达hMSH2，而146例MSS/MSI-L肿瘤中无1例（0%）不表达hMSH2（p = 0.0028）。108例MSS和MSI-L肿瘤中有107例（99%）存在hMLH1蛋白表达，而20例MSI-H肿瘤中有11例（55%）存在hMLH1蛋白表达（p < 0.0001）。在研究甲基化的61例MSS和MSI-L癌症中，11例（18%）hMLH1启动子发生甲基化，而20例MSI-H癌症中有14例（70%）发生甲基化（p = 0.0001）。在研究hMLH1蛋白表达和甲基化的27例MSI-H肿瘤中，93%表达缺失的肿瘤（93%）也发生甲基化，而hMLH1免疫染色阳性的肿瘤中有42%（5/12）hMLH1启动子发生甲基化（p = 0.009）。
启动子甲基化和hMLH1表达与CRC中的MSI-H表型显著相关。启动子甲基化分析为筛选MSI-H肿瘤提供了一种有用方法。我们的数据进一步表明，仅hMLH1启动子甲基化分析不能替代MSI检测，因为大量MSI-H肿瘤可能会被这种方法遗漏。我们建议，尽管表达分析和启动子甲基化状态研究可能补充筛选过程，但CRC的表型评估通过MSI检测进行最为可靠。
