Exhaled Breath DNA Methylation in Lung Carcinogenesis

描述（由申请方提供）：在无症状吸烟者的非恶性和癌前肺上皮细胞中也检测到了在临床上明显的肺恶性肿瘤中常见的甲基化肿瘤抑制基因启动子。这些早期癌发生的甲基化标志物，如果前瞻性地预测临床疾病的出现，可以作为癌症风险的生物标志物。该实验室已经开发并发表了一种简化的方法，通过亚硫酸氢盐基因组测序（tBGS）的标签适应，在基因启动子DNA的双链酶片段上以单碱基分辨率生成启动子甲基化图谱，而不需要克隆。我们的试点研究已经可靠地确定了呼出气冷凝液（EBC）中的DNA。将tBGS甲基化图谱与EBC样品结合，产生了呼出气DNA甲基化测定的第一个例子。该实验室现在能够对一组六个基因启动子进行多重检测。该R21提案旨在测试呼出的DNA提供以下信息的潜力：（i）气道起源部位;（ii）烟草烟雾暴露;和（iii）肺癌病例对照状态。为了确定呼出气DNA的来源，将直接比较可能有助于呼出气DNA甲基化模式（口腔、支气管、肺泡）的气道水平与来自相同75名患者的EBC，以确定这些水平中每一个的6种癌症相关肿瘤抑制基因的甲基化模式。还将在150名受试者中进行烟草烟雾暴露、肺癌病例对照状态和肿瘤特征的多变量比较，以初步测试这种非侵入性生物标志物的潜在临床效用。公共卫生相关性：该项目将初步测试呼出气DNA甲基化作为一类新的非侵入性肺癌生物标志物的有效性。如果得到验证，这种测试将能够识别出肺癌风险特别高的个人，以便将预防和早期检测工作集中在最有可能受益的人身上。

DESCRIPTION (provided by applicant): Methylated tumor suppressor gene promoters that are commonly observed in clinically-apparent lung malignancies have also been detected in non-malignant and pre-malignant lung epithelia in asymptomatic tobacco smokers. These methylation markers of early carcinogenesis, if prospectively predictive of the emergence of clinical disease, could be applied as biomarkers of cancer risk. The laboratory has developed and published a simplified method of generating promoter methylation maps at single-base resolution across kilobase stretches of gene promoter DNA, without the need for cloning, by a tag-adaptation of bisulfite genomic sequencing (tBGS). Our pilot studies have reliably identified DNA in exhaled breath condensate (EBC). The coupling of tBGS methylation maps to EBC samples has yielded the first examples of DNA methylation assays from exhaled breath. The laboratory is now able to multiplex this assay for a panel of six gene promoters. This R21 proposal is designed to test the potential of that exhaled DNA to be informative for: (i) airway site of origin; (ii) tobacco smoke exposure; and (iii) lung cancer case-control status. In order to determine the origin of exhaled breath DNA, those levels of the airway that may plausibly contribute to exhaled breath DNA methylation patterns (buccal, bronchial, alveolar) will be directly compared to EBC from the same 75 patients, for methylation patterns for six cancer-relevant tumor suppressor genes at each of these levels. Multivariate comparisons to tobacco smoke exposure, to lung cancer case-control status, and to tumor characteristics, will also be made in 150 subjects, to pilot test the potential clinical utility of this non-invasive biomarker. PUBLIC HEALTH RELEVANCE: The project will pilot test the validity of exhaled breath DNA methylation as a new class of non-invasive lung carcinogenesis biomarker. If validated, this test would enable the identification of individuals at particularly high risk for lung cancer, for the purpose of focusing prevention and early detection efforts on those most likely to benefit.