Department of Pathology and Laboratory Medicine, Department of Epidemiology, Brown University, Providence, Rhode Island 02912, USA.
Clin Cancer Res. 2012 Nov 15;18(22):6147-54. doi: 10.1158/1078-0432.CCR-12-1008. Epub 2012 Sep 26.
Natural killer (NK) cells are a key element of the innate immune system implicated in human cancer. To examine NK cell levels in archived bloods from a study of human head and neck squamous cell carcinoma (HNSCC), a new DNA-based quantification method was developed.
NK cell-specific DNA methylation was identified by analyzing DNA methylation and mRNA array data from purified blood leukocyte subtypes (NK, T, B, monocytes, granulocytes), and confirmed via pyrosequencing and quantitative methylation specific PCR (qMSP). NK cell levels in archived whole blood DNA from 122 HNSCC patients and 122 controls were assessed by qMSP.
Pyrosequencing and qMSP confirmed that a demethylated DNA region in NKp46 distinguishes NK cells from other leukocytes, and serves as a quantitative NK cell marker. Demethylation of NKp46 was significantly lower in HNSCC patient bloods compared with controls (P < 0.001). Individuals in the lowest NK tertile had over 5-fold risk of being a HNSCC case, controlling for age, gender, HPV16 status, cigarette smoking, alcohol consumption, and BMI (OR = 5.6, 95% CI, 2.0 to 17.4). Cases did not show differences in NKp46 demethylation based on tumor site or stage.
The results of this study indicate a significant depression in NK cells in HNSCC patients that is unrelated to exposures associated with the disease. DNA methylation biomarkers of NK cells represent an alternative to conventional flow cytometry that can be applied in a wide variety of clinical and epidemiologic settings including archival blood specimens.
自然杀伤 (NK) 细胞是先天免疫系统的关键组成部分,与人类癌症有关。为了研究人类头颈部鳞状细胞癌 (HNSCC) 研究中存档血液中的 NK 细胞水平,开发了一种新的基于 DNA 的定量方法。
通过分析来自纯化血液白细胞亚型(NK、T、B、单核细胞、粒细胞)的 DNA 甲基化和 mRNA 阵列数据,鉴定 NK 细胞特异性 DNA 甲基化,并通过焦磷酸测序和定量甲基化特异性 PCR (qMSP) 进行验证。通过 qMSP 评估了 122 例 HNSCC 患者和 122 例对照者存档全血 DNA 中的 NK 细胞水平。
焦磷酸测序和 qMSP 证实,NKp46 中的一个去甲基化 DNA 区域可区分 NK 细胞与其他白细胞,并作为定量 NK 细胞标志物。与对照组相比,HNSCC 患者血液中的 NKp46 去甲基化明显降低(P < 0.001)。在控制年龄、性别、HPV16 状态、吸烟、饮酒和 BMI 后,NK 细胞最低三分位的个体患 HNSCC 的风险增加了 5 倍以上(OR = 5.6,95%CI,2.0 至 17.4)。病例组根据肿瘤部位或分期在 NKp46 去甲基化方面没有差异。
本研究结果表明,HNSCC 患者的 NK 细胞明显减少,与疾病相关的暴露无关。NK 细胞的 DNA 甲基化生物标志物代表了传统流式细胞术的替代方法,可应用于广泛的临床和流行病学环境,包括存档血液标本。