Discovery of New DNA Methylation Biomarkers for Predicting the Malignant Outcome of Low-Grade Oral Dysplasia

口腔鳞状细胞癌（OSCC）是一种毁灭性的恶性肿瘤，可能源于前体粘膜上皮细胞， 口腔癌前病变（oral premalignant lesions，OPL）。OPL包括低度和高度发育不良（OD）。 尽管大多数低度OD数年保持稳定，有些甚至自发消退，但 其中少数（4 - 11%）迅速发展为OSCC。目前，没有生物标志物可用于筛查低- 分级ODs，以识别那些处于发展OSCC的高风险的人，以实施循证癌症预防 管理几项研究报道了口腔癌发生过程中DNA甲基化的变化，但未能解决 进行性与静态低级别OD之间的差异，或产生全基因组覆盖率， 有足够的宽度和深度来进行最大的新发现。我们的长期目标是了解 导致癌前病变恶性进展的机制。R21的目标是 发现与OPL进展相关的新的差异甲基化区域（DMR）， 基因组单核苷酸解析方法，创建新的基于DMR的模型，并验证其在 预测低度ODs向OSCC的进展。R21的核心假设是， OPL向OSCC的进展是由DNA甲基化变化驱动的，DNA甲基化变化可以通过全基因组检测来发现， 进行性与静态OPL的DMR分析，用于预测OPL的癌症风险以进行管理 决定这一假设是基于以下科学前提制定的：1）表观遗传修饰是一种 关键的疾病驱动机制，捕获早期致癌环境-基因相互作用事件， 在整个疾病进展过程中忠实地保留，2）DNA甲基化变化已被证明是 与正常口腔上皮相比，ODs和OSCC中发生。进行拟议研究的理由 一旦基于高质量识别出渐进式OD和静态OD之间的DMR谱， 通过数据和强大的分析，我们将发现驱动和预测乳腺癌恶性进展的关键DMR位点。 低级别的药物过量否则会被漏掉目标1将发现基因组区域差异 使用全基因组亚硫酸氢盐测序（WGBS）， 纵向随访样本。目标2将创建基于DMR的模型，并验证其在以下方面的能力： 预测低级别ODs进展为OSCC的风险。如果成功完成， 新的DMR能够区分渐进与静态OD，并创建新的风险预测DMR模型。 该结果将满足对一种敏感可靠的筛查方法的迫切需求，以预测OPL的风险， 在早期阶段成为OSCC，此时医疗干预更有效，损害更小， 将直接影响减少口腔鳞状细胞癌的发病率和相关的健康问题，以及发现 新的DNA甲基化机制驱动口腔鳞状细胞癌的恶性进展，可能是药物。可能 也对推进其他烟草相关HNSCC的研究产生广泛影响。

Oral squamous cell carcinoma (OSCC) is a devastating malignancy that may arise from precursor mucosal lesions, called oral premalignant lesions (OPLs). OPLs consist of low- and high-grade dysplasia (ODs). Although most low-grade ODs remain stable for years and some even regress spontaneously, a significant minority of them (4-11%) rapidly develop OSCC. Currently, there is no biomarker available for screening low- grade ODs to identify those at high risk of developing OSCC to implement evidence-based cancer prevention management. Several studies reported DNA methylation changes in oral carcinogenesis, but failed to address the differences between progressive vs. static low-grade ODs or generate whole genome coverage with sufficient width and depth for maximal new discoveries. Our long-term goal is to understand the epigenetic mechanisms that drive the malignant progression of precancerous lesions. The objective of this R21 is to discover new differentially methylated regions (DMRs) associated with OPL progression using a whole- genome single-nucleotide-resolution approach, create novel DMR-based models, and validate their power in predicting the progression of low-grade ODs to OSCC. The central hypothesis of this R21 states that the progression of OPLs to OSCC is driven by DNA methylation changes that can be discovered by whole-genome DMR analysis of progressive vs. static OPLs and used to predict the cancer risk of OPLs for management decision. This hypothesis is formulated based on the scientific premises that: 1) epigenetic modification is a key disease-driving mechanism that captures early carcinogenic environment-gene interaction events and is faithfully preserved throughout disease progression, and 2) DNA methylation changes have been shown to occur in ODs and OSCC compared to normal oral epithelium. The rationale for pursuing the proposed research is that once the spectrum of DMRs between the progressive and static ODs are identified based on high quality data and powerful analysis, we will discover key DMR sites that drive and predict the malignant progression of low-grade ODs, which would otherwise be missed. Aim 1 will discover genomic regions differentially methylated in progressive vs. static low-grade ODs using whole genome bisulfite sequencing (WGBS) on samples with longitudinal follow-ups. Aim 2 will create DMR-based models and validate their power in predicting the risk of low-grade ODs in progression to OSCC. A successful completion is expected to discover new DMRs capable of differentiating progressive vs. static ODs and create novel risk-prediction DMR models. The outcome will fill the critical need for a sensitive and reliable screening method to predict the risk of OPLs in becoming OSCCs at an early stage, when medical interventions are more effective and less damaging, which will lead to a direct impact on reducing the incidence of OSCC and related health issues, as well as finding novel DNA methylation mechanisms driving the malignant progression of OSCC that may be druggable. It may also have a broad impact on advancing research on other tobacco-related HNSCC.