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

材料与方法本研究对象为151例结直肠癌患者，平均年龄65.8岁，年龄39-86岁。中位随访时间为79个月(范围60-123个月)。切片行苏木精-伊红染色，按UICC/TNM分类进行PT、PN及病理分期。此外，目前从25例患者获得新鲜的肿瘤组织和匹配的远离肿瘤的正常黏膜。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例肿瘤组织冰冻切片中提取基因组DNA。从25例肿瘤组织和4例匹配的正常粘膜冰冻切片中提取总RNA。使用为甲基化和未甲基化的p16序列专门设计的引物来扩增亚硫酸氢盐修饰的DNA。进行实时定量甲基化特异性聚合酶链式反应，甲基化…根据公式计算更多的n指数：(甲基化p16序列浓度/甲基化+未甲基化p16序列浓度)X100。用总RNA合成c DNA，进行实时荧光定量聚合酶链式反应。该引物对位于p16基因α和2外显子，内含子1侧翼。用抗p16单抗(E6H4，DAKO)进行免疫组织化学染色。结果正常黏膜组织中p16甲基化指数为0~2%，肿瘤组织中p16甲基化指数为0~100%(中位数为14.2%；P=0.01)。有100/151(66%)的肿瘤标本检测到p16基因异常甲基化(甲基化指数为2%)。15%(22/151)的病例出现p16表达缺失(免疫反应阳性率为5%)。在图像分析中，p16-免疫阳性指数变化范围为0-53%，与p16甲基化指数无明显相关性。P16mRNA在正常组织中的表达水平较低，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基因表达与p16免疫标记指数呈正相关(P=0.043)。P16甲基化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COX回归模型中，p16甲基化(P=0.007)、高PT(P=0.007)和晚期肿瘤分期(P&lt；0.001)是与癌症相关的短期生存的独立预测因素。COX回归多因素分析还显示，p16甲基化(P&lt；0.001)、晚期PN(P&lt；0.001)和PT分期(P=0.014)预示着短期无复发生存。较少

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