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

材料与方法我们的研究对象包括一系列的151例患者（平均年龄，65.8岁;范围，39-86）诊断和接受手术的结直肠癌。中位随访时间为79个月（范围：60-123）。行HE染色，按UICC/TNM分期标准进行pT、pN及病理分期。此外，目前从25名患者中获得了新鲜肿瘤组织和远离肿瘤的匹配正常粘膜。从151例肿瘤组织和10例正常组织的福尔马林固定石蜡包埋切片中提取基因组DNA，并从25例肿瘤组织的冷冻切片中提取基因组DNA。从25例肿瘤组织和4例正常粘膜的冰冻切片中提取总RNA。亚硫酸氢盐修饰的DNA扩增使用专门设计的引物的甲基化和未甲基化的p16序列。采用实时定量甲基化特异性PCR技术， ...更多信息 根据以下公式计算n指数：（甲基化p16序列的浓度/甲基化加未甲基化p16序列的浓度）× 100。用总RNA合成cDNA，用于实时定量PCR。引物对位于p16基因的外显子1 α和2，侧翼内含子1。用单克隆抗p16（E6 H4，Dako）进行免疫染色。结果正常粘膜组织p16甲基化指数为0 ~ 2%，肿瘤组织p16甲基化指数为0 ~ 100%（中位数为14.2%，P=0.01）。在100/151（66%）肿瘤样本中检测到p16异常甲基化（甲基化指数>2%）。151例中有22例（15%）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到53%变化，与p16甲基化指数没有显著相关性。p16 mRNA在正常组织中的表达水平一直较低，而在肿瘤组织中的表达水平较高，差异有统计学意义（P=0.003）。p16 mRNA水平与p16免疫标记指数呈正相关（P=0.043），而与甲基化指数无显著相关（P> 0.05）。p16甲基化的存在与较小的肿瘤显著相关（P=0.048），但与其他临床病理特征无关。在考克斯回归模型中，p16甲基化（P=0.007）、高pT（P=0.007）和晚期肿瘤（P<0.001）被证明是短的癌症相关生存期的独立预测因子。考克斯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）预测短期无复发生存。少

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