Semczuk Andrzej, Boltze Carsten, Marzec Barbara, Szczygielska Anna, Roessner Albert, Schneider-Stock Regine
2nd Department of Gynecology, Lublin University School of Medicine, 8 Jaczewski Street, 20-954, Lublin, Poland.
J Cancer Res Clin Oncol. 2003 Oct;129(10):589-96. doi: 10.1007/s00432-003-0482-2. Epub 2003 Aug 14.
To date, the significance of p16INK4A tumor suppressor gene inactivation in sporadic endometrial cancer (EC) has only rarely been described. In this study, we examined the alteration type and frequency of gene alterations [point mutations, aberrant promoter methylation and loss of heterozygosity (LOH)] in 50 sporadic ECs, and correlated the genetic findings with the immunohistochemical expression of the p16INK4A protein and the classical clinicopathological features.
Gene mutations were detected by PCR-SSCP-sequencing analysis, promoter hypermethylation by methylation-specific PCR (MSP), and LOH by PCR of the STS-marker c5.1.
In total, p16INK4A alterations were found in 14 of 50 (28%) sporadic ECs. In six (12%) cases, two alterations occurred simultaneously. Partial p16INK4A deletions were found in four of 50 (8%) samples. There was one missense mutation (codon 70; CCC-->GCC) and one frameshift mutation (1-bp deletion in exon 2). Only 2 of 47 (4.2%) tumors exhibited aberrant promoter methylation. An allelic loss was detected in 12 of 50 (24%) carcinomas with a higher incidence in advanced endometrial carcinomas than in early-stage uterine tumors. p16INK4A alterations were generally accompanied by gene silencing, confirmed by aberrant protein immunostaining ( r=-0.442; P=0.001). There was a significant difference in the frequency of p16INK4A alterations between early (stage I; 18%) and advanced (stages II-IV; 58%) ECs ( P=0.022). One case showed complete protein loss, but absence of genetic alterations.
Our data indicate that p16INK4A inactivation plays a role in the tumorigenesis of the subset of sporadic ECs, particularly in cases exhibiting an aggressive clinical behavior. We demonstrate that p16INK4A methylation can act efficiently and similarly to other genetic alterations as one of the two necessary hits according to the Knudson two-hit hypothesis of tumor suppressor gene inactivation.
迄今为止，p16INK4A肿瘤抑制基因失活在散发性子宫内膜癌（EC）中的意义鲜有报道。在本研究中，我们检测了50例散发性EC中基因改变（点突变、异常启动子甲基化和杂合性缺失[LOH]）的类型及频率，并将这些遗传学发现与p16INK4A蛋白的免疫组化表达及经典临床病理特征进行关联分析。
通过PCR-SSCP测序分析检测基因突变，通过甲基化特异性PCR（MSP）检测启动子高甲基化，通过STS标记c5.1的PCR检测LOH。
50例散发性EC中，共有14例（28%）存在p16INK4A改变。6例（12%）同时出现两种改变。50例样本中有4例（8%）发现部分p16INK4A缺失。有1个错义突变（密码子70；CCC→GCC）和1个移码突变（外显子2中1个碱基缺失）。47例肿瘤中仅2例（4.2%）表现为异常启动子甲基化。50例癌中有12例（24%）检测到等位基因缺失，晚期子宫内膜癌的发生率高于早期子宫肿瘤。p16INK4A改变通常伴有基因沉默，异常蛋白免疫染色证实二者存在相关性（r=-0.442；P=0.001）。早期（I期；18%）和晚期（II-IV期；58%）EC中p16INK4A改变的频率存在显著差异（P=0.022）。1例显示蛋白完全缺失，但无基因改变。
我们的数据表明，p16INK4A失活在散发性EC亚组的肿瘤发生中起作用，特别是在表现出侵袭性临床行为的病例中。我们证明，根据肿瘤抑制基因失活的Knudson双击假说，p16INK4A甲基化可作为两个必要打击之一，与其他基因改变一样有效发挥作用。
