Luo Daya, Zhang Baozhen, Lv Lingbo, Xiang Shengyan, Liu Yahang, Ji Jiafu, Deng Dajun
Peking University School of Oncology, Beijing Cancer Hospital and Beijing Institute for Cancer Research, Beijing, China.
Lab Invest. 2006 Jun;86(6):591-8. doi: 10.1038/labinvest.3700415.
Inactivation of p16 by methylation of CpG islands is a frequent early event in gastric carcinogenesis. The positive relationship between p16 methylation and the clinical characteristics of gastric carcinomas (GC) has not been reported to date. In the present study, a DHPLC assay to quantify p16 methylation was established (detection limit by fluorescence detector: 1:255 (Methlyated vs Unmethylated)). The proportion of methylated p16 in the representative samples was confirmed and standardized by clone sequencing. Then, the DHPLC and two regular methylation-specific PCR (MSP) assays were used to detect p16 methylation in 82 paired, resected GCs and their adjacent normal tissues. Results showed that the average proportion of methylated p16 in GCs was significantly higher than that in their adjacent samples (12.90 vs 0.63%; t-test P=0.005). A much higher proportion of methylated p16 was detected in GCs with metastases (local or distant) than without metastases (14.76 vs 2.61%; t-test P=0.014). A proportional relationship was observed between clinical stages and positive rates of p16 methylation in GCs and/or adjacent tissues: 27.3, 37.5, and 58.8% (by DHPLC) for stage-I, -II, -III-IV of GCs, respectively (two-sided Fisher's exact test P=0.016). To confirm the data obtained by DHPLC, two MSP primer sets (p16-M and p16-M2) were also used to analyze p16 methylation in the same set of samples simultaneously. Data of MSP assay using the primer set p16-M2, but not p16-M, correlated with that of DHPLC. These results imply that the primer set p16-M2 might be more suitable than p16-M to detect p16 methylation in gastric tissues. In conclusion, the present data indicates that p16 methylation correlates with progression of GCs significantly.
CpG岛甲基化导致p16失活是胃癌发生过程中常见的早期事件。迄今为止，尚未见p16甲基化与胃癌（GC）临床特征之间存在正相关关系的报道。在本研究中，建立了一种用于定量p16甲基化的变性高效液相色谱（DHPLC）检测方法（荧光检测器检测限：1:255（甲基化与未甲基化））。通过克隆测序确认并标准化了代表性样本中甲基化p16的比例。然后，采用DHPLC和两种常规甲基化特异性PCR（MSP）检测方法，对82对手术切除的GC及其癌旁正常组织中的p16甲基化进行检测。结果显示，GC中甲基化p16的平均比例显著高于其癌旁样本（12.90%对0.63%；t检验P = 0.005）。与无转移的GC相比，有转移（局部或远处）的GC中检测到的甲基化p16比例更高（14.76%对2.61%；t检验P = 0.014）。观察到GC和/或癌旁组织的临床分期与p16甲基化阳性率之间存在比例关系：GC的I期、II期、III-IV期p16甲基化阳性率分别为27.3%、37.5%和58.8%（采用DHPLC检测）（双侧Fisher精确检验P = 0.016）。为了证实DHPLC获得的数据，还使用了两套MSP引物（p16-M和p16-M2）同时分析同一组样本中的p16甲基化情况。使用引物组p16-M2而非p16-M进行MSP检测的数据与DHPLC数据相关。这些结果表明，引物组p16-M2可能比p16-M更适合检测胃组织中的p16甲基化。总之，目前的数据表明p16甲基化与GC的进展显著相关。
