Ota Naohiro, Kawakami Kazuyuki, Okuda Toshiyuki, Takehara Akira, Hiranuma Chikashi, Oyama Kaeko, Ota Yasuhiko, Oda Makoto, Watanabe Go
Department of Surgery, Kanazawa University School of Medicine, Ishikawa 920-8641, Japan.
Anticancer Res. 2006 Sep-Oct;26(5B):3729-32.
p16(INK4a) is a tumor suppressor gene frequently inactivated by aberrant promoter hypermethylation. In the present study, p16(INK4a) methylation was evaluated in non-small cell lung cancer (NSCLC) using a quantitative assay and the clinical significance of the methylation was explored.
A total of 244 tumor samples from formalin-fixed paraffin-embedded archives were examined in this study. p16(INK4a) methylation was analyzed by the fluorescence-based, real-time methylation-specific PCR assay, MethyLight. The quantitative methylation value was expressed as the percentage of methylated reference (PMR).
The median level of p16(INK4) methylation was 0.55 PMR (range 0.00-503.4). The p16(INK4) methylation value was significantly higher in males (p = 0.005) and in squamous cell carcinoma (p = 0.018). Prognostic analysis using the Cox proportional hazard model showed that the p16(INK4a) methylation value was a significant prognostic factor (odds ratio, 1.005; 95% CI, 1.003 to 1.008; p < 0.0001). The p16(INK4a) methylation value remained a significant prognostic factor (p = 0.0004) in multivariate analysis including age, gender, histological type and clinical stage. Specimens were then classified into hypermethylated or non-hypermethylated groups based on the p16(INK4a) methylation value using various cut-offs from 1 to 100 PMR. There was no significant difference in prognosis between the two groups using a cut-off value of 1 PMR. On the other hand, there was a significant difference using 6 PMR or more as the cut-off value (p < 0.01).
These results provide clear evidence for the prognostic significance of p16(INK4a) methylation in NSCLC using quantitative DNA methylation analysis. Careful assessment of DNA methylation is needed because qualitative methylation analysis may overestimate low levels of methylation, which have less clinical significance.
p16(INK4a)是一种肿瘤抑制基因，常因启动子异常高甲基化而失活。在本研究中，采用定量分析方法评估非小细胞肺癌（NSCLC）中p16(INK4a)的甲基化情况，并探讨其甲基化的临床意义。
本研究共检测了244份来自福尔马林固定石蜡包埋存档的肿瘤样本。采用基于荧光的实时甲基化特异性PCR检测方法（MethyLight）分析p16(INK4a)的甲基化情况。定量甲基化值以甲基化参考百分比（PMR）表示。
p16(INK4)甲基化的中位水平为0.55 PMR（范围0.00 - 503.4）。p16(INK4)甲基化值在男性（p = 0.005）和鳞状细胞癌中（p = 0.018）显著更高。使用Cox比例风险模型进行的预后分析表明，p16(INK4a)甲基化值是一个显著的预后因素（比值比，1.005；95%置信区间，1.003至1.008；p < 0.0001）。在包括年龄、性别、组织学类型和临床分期的多因素分析中，p16(INK4a)甲基化值仍然是一个显著的预后因素（p = 0.0004）。然后根据p16(INK4a)甲基化值，使用1至100 PMR的不同临界值将样本分为高甲基化组或非高甲基化组。使用1 PMR作为临界值时，两组预后无显著差异。另一方面，使用6 PMR及以上作为临界值时存在显著差异（p < 0.01）。
这些结果为使用定量DNA甲基化分析评估p16(INK4a)甲基化在NSCLC中的预后意义提供了明确证据。由于定性甲基化分析可能高估临床意义较小的低水平甲基化，因此需要仔细评估DNA甲基化情况。
