Koga Yasuo, Kitajima Yoshihiko, Miyoshi Atsushi, Sato Ken, Kitahara Kenji, Soejima Hidenobu, Miyazaki Kohji
Department of Surgery, Saga University Faculty of Medicine, Nabeshima 5-1-1, Saga 849-8501, Japan.
Ann Surg Oncol. 2005 May;12(5):354-63. doi: 10.1245/ASO.2005.07.020. Epub 2005 Mar 31.
We previously demonstrated in an immunohistochemical study that reduced expression of O(6)-methylguanine-DNA methyltransferase (MGMT) correlated with a poorer prognosis in patients with biliary tract cancers. The purpose of this study was to clarify how MGMT deficiency leads to a poor outcome in biliary tract cancer. Thus, we examined epigenetic (promoter methylation) and genetic (gene mutation) alterations in biliary tract cancer.
We examined 37 biliary tract cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by one-step or two-step methylation-specific polymerase chain reaction. Gene mutation was identified by direct sequencing. The expression of MGMT protein in paraffin-embedded tissue was examined by immunohistochemistry.
Frequencies of promoter methylation were 70% for p16/INK4a, 49% for MGMT, 46% for hMLH1, 41% for E-cadherin, and 32% for DAPK genes. MGMT methylation status was closely correlated with the MGMT protein expression determined by immunohistochemistry (P < .001). Although this was not statistically significant, biliary tract cancer tumors with MGMT methylation expressed multigene methylation more frequently than tumors without MGMT methylation (P = .071). A total of 33 mutations were identified in 4 cancer-related genes: p53, K-ras, beta-catenin, and p16/INK4a genes. The most common mutation was GC to AT transitions (58%), which were significantly associated with MGMT promoter methylation (P = .011). These findings suggest that loss of MGMT expression by promoter methylation results in accumulation of GC to AT gene mutations.
Reduced MGMT expression may increase the malignant potential of biliary tract cancer through both epigenetic and genetic mechanisms.
我们先前在一项免疫组织化学研究中证明，O(6)-甲基鸟嘌呤-DNA甲基转移酶（MGMT）表达降低与胆道癌患者的预后较差相关。本研究的目的是阐明MGMT缺陷如何导致胆道癌预后不良。因此，我们研究了胆道癌中的表观遗传（启动子甲基化）和遗传（基因突变）改变。
我们检查了37例接受手术切除患者的胆道癌标本。通过一步或两步甲基化特异性聚合酶链反应确定启动子甲基化。通过直接测序鉴定基因突变。通过免疫组织化学检查石蜡包埋组织中MGMT蛋白的表达。
p16/INK4a启动子甲基化频率为70%，MGMT为49%，hMLH1为46%，E-钙黏蛋白为41%，DAPK基因为32%。MGMT甲基化状态与免疫组织化学测定的MGMT蛋白表达密切相关（P <.001）。虽然这没有统计学意义，但与未发生MGMT甲基化的肿瘤相比，发生MGMT甲基化的胆道癌肿瘤更频繁地表达多基因甲基化（P =.071）。在4个癌症相关基因（p53、K-ras、β-连环蛋白和p16/INK4a基因）中总共鉴定出33个突变。最常见的突变是GC到AT的转换（58%），这与MGMT启动子甲基化显著相关（P =.011）。这些发现表明，启动子甲基化导致的MGMT表达缺失会导致GC到AT基因突变的积累。
MGMT表达降低可能通过表观遗传和遗传机制增加胆道癌的恶性潜能。
