Prognosis value of aberrant p16^<INK4a> gene methylation in colorectal cancer

p16^<INK4a>基因甲基化异常在结直肠癌中的预后价值

基本信息

批准号：
18591494
负责人：
KANAZAWA Hideki
金额：
$ 0.65万
依托单位：
Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara National Hospital
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

Materials and MethodsThe subjects of our study comprised a series of 151 patients(mean age, 65.8 years ; range, 39-86) diagnosed and undergoing surgery for colorectal carcinoma. The median duration of follow-up was 79 months(range, 60-123). Slides stained with hematoxylin and eosin were examined, and pT, pN and pathological staging was completed according to the UICC/TNM classification. In addition, fresh tumor tissues and matched normal mucosa away from the tumor were currently obtained from 25 patients. Genomic DNA was extracted from formalin-fixed paraffin-embedded sections of 151 tumor and 10 normal tissues, with additional extraction from the frozen sections of 25 tumor tissues. Total RNA was extracted from the frozen sections of 25 tumor tissues and 4 matched normal mucosas. Bisulfite-modified DNA was amplified using specifically designed primers for the methylated and unmethylated p16 sequence. The real-time quantitative methylation specific PCR was performed, and the methylatio … More n index was calculated according to the equation : (concentration of methylated p16 sequence/concentrations of methylated plus unmethylated p16 sequence) X 100. Total RNA was employed to synthesize cDNA which was then used for real-time quantitative PCR. The primer pairs located in exons lα and 2 with flanking intron 1 of the p16 gene. Immunostaining was performed with monoclonal anti-p16(E6H4, Dako). In addition, image analysis was performed on p16 immunostained slides from randomly selected cases.Results Normal mucosa samples showed p16 methylation indices varying from 0 to 2%, and the indices of tumor samples represented 0 to 100%(median, 14.2% ; P=0.01) . Aberrant methylation of p16(methylation indices >2%) was detected in 100/151(66%) tumor samples. Loss of p16 expression(immunoreactivity <5%) was observed in 22/151(15%) cases. In image analyses, pl6-immunolabehng indices varied from 0 to 53% with no significant correlation to p16 methylation index. The expression level of p16 mRNA in normal tissues was consistently low whereas the value in tumor tissue was, in some cases, high level, which was statistically different(P=0.003). No significant correlation was observed between pl6 mRNA levels and methylation indiceswhereas p16 mRNA levels were positively correlated with p16-immunolabeling indices with statistically significant(P=0.043). Presence of p16 methylation was significantly associated with smaller tumor(P=0.048), but not with other clinicopathological features. In the Cox's regression model, present p16 methylation(P=0.007), high pT(P=0.007) and advanced tumor stage(P<0.001), proved to be independent predictors of short cancer-related survival. Cox's regression multivariate analysis also showed that p16 methylation(P<0.001), and advanced pN(P<0.001) and pT stages(P=0.014) predicted short recurrence-free survival. Less

材料和方法我们的研究对象包括 151 名被诊断为结直肠癌并接受手术的患者（平均年龄，65.8 岁；范围，39-86 岁），中位随访时间为 79 个月（范围，60-123 个月）。）））检查用苏木精和伊红染色的载玻片，并根据UICC/TNM分类完成pT、pN和病理分期。目前，从 25 名患者的福尔马林固定石蜡包埋切片和 10 名正常组织中提取了基因组 DNA，并从 25 名肿瘤组织的冷冻切片中提取了总 RNA。使用针对甲基化和非甲基化 p16 专门设计的引物扩增从 25 个肿瘤组织和 4 个匹配的正常粘膜的冷冻切片中提取的 DNA。进行实时定量甲基化特异性PCR，并根据以下公式计算甲基化指数：（甲基化p16序列浓度/甲基化加非甲基化p16序列浓度）X 100。总RNA用于合成 cDNA，然后将其用于实时定量 PCR，引物对位于 p16 基因的外显子 1α 和 2 以及侧翼内含子 1 中。使用单克隆抗p16（E6H4，Dako）进行此外，对随机选择的病例的p16免疫染色切片进行图像分析。结果正常粘膜样本显示p16甲基化指数在0至2%之间变化，而肿瘤样本的指数则不同。代表 0 至 100%（中位数，14.2%；P=0.01） p16 的异常甲基化（甲基化指数）。在 100/151（66%）个肿瘤样本中检测到 p16 表达缺失（免疫反应性 <5%）。在图像分析中，p16 免疫标记指数从 0 变化。至 53%，与 p16 甲基化指数无显着相关性正常组织中 p16 mRNA 的表达水平始终较低，而肿瘤组织中的值在某些情况下较高。 p16 mRNA水平与甲基化指数之间无显着相关性(P=0.003)，而p16 mRNA水平与p16免疫标记指数呈正相关，且具有统计学意义(P=0.043)。与较小的肿瘤相关（P=0.048），但与其他临床病理特征无关。在 Cox 回归模型中，呈现 p16。甲基化（P=0.007）、高pT（P=0.007）和晚期肿瘤分期（P<0.001）被证明是癌症相关生存期短的独立预测因子，Cox回归多变量分析也显示p16甲基化（P<0.001）。，晚期 pN（P<0.001）和 pT 分期（P=0.014）预测较短的无复发生存期。