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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591494/
• 关键词: Colorectal cancer; Prognosis; p16INK4a; Promoter Methylation; 外科; 癌; 遺伝子; 病理学; 大腸癌; 簇出; 遺伝子プロモーター; メチル化; 予後規定因子

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

材料和方法我们研究的受试者完成了一系列151例患者（平均年龄为65.8岁；范围为39-86），并接受了彩色癌的手术。随访的中间持续时间为79个月（范围为60-123）。检查了用苏木精和曙红染色的载玻片，根据UICC/TNM分类完成了PT，PN和病理分期。此外，目前从25例患者中获得了新鲜的肿瘤组织和与肿瘤的正常粘膜相匹配的。从福尔马林固定的石蜡包裹的切片中提取基因组DNA的151个肿瘤和10个正常组织，并从25个肿瘤组织的冷冻切片中进行了额外的提取。从25个肿瘤组织的冷冻切片和4种匹配的正常粘膜中提取总RNA。使用特异性设计的甲基化和未甲基化P16序列的底漆扩增甲硫酸氢盐修饰的DNA。进行了实时定量甲基化特异性PCR，并根据方程计算甲基含量……更多n指数：（甲基化的p16 p16序列/甲基化甲基化的Plus Plus未甲基化p16序列的浓度）x 100。位于外显子Lα中的底漆对和p16基因的内含子1。用单克隆抗P16（E6H4，DAKO）进行免疫染色。此外，对随机选择病例的P16免疫染色载玻片进行了图像分析。正常粘膜样品显示P16甲基化指数从0到2％，肿瘤样品的指数代表0至100％（中位数，14.2％； P = 0.01）。在100/151（66％）肿瘤样品中检测到p16的异常甲基化（甲基化指数> 2％）。在22/151（15％）病例中观察到p16表达的丧失（免疫反应性<5％）。在图像分析中，PL6-免疫标记指数从0到53％不等，与P16甲基化指数无显着相关。正常组织中p16 mRNA的表达水平始终较低，而在某些情况下，肿瘤组织的值在统计学上是高水平的（p = 0.003）。在PL6 mRNA水平和甲基化指数之间未观察到显着相关性，而P16 mRNA水平与具有统计学意义的P16-免疫标记指数正相关（P = 0.043）。 p16甲基化的存在与较小的肿瘤显着相关（p = 0.048），但与其他临床病理学特征无关。在COX的回归模型中，呈现P16甲基化（P = 0.007），高PT（P = 0.007）和晚期肿瘤阶段（P <0.001），是与癌症相关的短期生存的独立预测指标。 Cox的回归多元分析还表明，p16甲基化（P <0.001），晚期PN（P <0.001）和PT阶段（P = 0.014）预测了短期无复发的生存。较少的

项目成果

Tumor budding at the invasive margin can predict prognosis in colorectal cancer

浸润边缘的肿瘤出芽可以预测结直肠癌的预后

• 发表时间: 2007
• 作者: Ohta T.;Wu W;Fukuda M.;H.Kanazawa
• 通讯作者: H.Kanazawa