DNA-Methylation Profiling from Fixed Melanocytic Tissues

描述（由申请人提供）：黑色素瘤的发病率正在增加，具有早期转移的能力，其病程很少受到医疗干预的影响。由于局部疾病和转移性疾病之间的生存率存在显着差异，因此必须在最早期诊断黑色素瘤;然而，黑色素瘤的临床和组织病理学表现与高度流行的良性痣（痣）的重叠使早期诊断变得混乱。分子病理学已被证明是有用的辅助诊断提供的组织病理学家，以提高早期癌症检测。肿瘤DNA甲基化有望成为分子病理学的一种工具，因为异常的启动子甲基化通常导致肿瘤抑制基因的异常沉默，已被证明广泛发生在人类黑色素瘤中。高通量甲基化阵列技术是一种能够同时检测多个肿瘤相关基因启动子甲基化的新技术，有望发现可用于黑色素瘤诊断的候选DNA甲基化位点。然而，这些阵列已经被开发用于未固定的组织，并且它们的有效性和再现性尚未在福尔马林固定的石蜡包埋（FFPE）组织上确定，FFPE组织通常是可用于原发性黑素瘤和痣的唯一诊断组织。我们的建议的中心假设是，DNA甲基化模式的存在，可以区分黑色素瘤与良性痣具有高灵敏度，特异性和可重复性;和高通量DNA甲基化检测是一种可行的方法，发现这些模式在诊断FFPE组织。该R21的目标是通过评估匹配的福尔马林固定和冷冻黑色素瘤标本之间以及福尔马林固定重复样本之间的结果重现性，评估福尔马林固定组织是否是高通量甲基化阵列分析的合适DNA来源。重要的是，作为第二个目标，将确定“剂量反应曲线”，以评估使用高通量阵列进行肿瘤DNA甲基化检测所需的黑素细胞肿瘤与周围非黑素细胞组织的比例，并且这些结果将建立肿瘤比例，低于该比例，将前瞻性地使用激光捕获显微切割进行选择性采购。此外，本申请提出鉴定将区分黑素瘤与良性痣的“原理证明”甲基化签名算法。这项研究将是开发诊断甲基化检测的第一步，该检测可用于标准化黑色素瘤诊断，从而减少黑色素细胞病变的治疗不足和过度。

DESCRIPTION (provided by applicant): Melanoma, which is increasing in incidence, has the capacity to metastasize early and its course is rarely impacted by medical intervention. Because of the pronounced difference in survival between localized and metastatic disease, it is imperative to diagnose melanoma in its earliest form; however, early diagnosis is confounded by the overlap of the clinical and histopathological appearances of melanomas with highly prevalent benign nevi (moles). Molecular pathology has proven useful as an adjunct to diagnosis rendered by histopathologists for enhancing early cancer detection. Tumor DNA-methylation holds promise as a tool for molecular pathology because aberrant promoter methylation, which often results in the abnormal silencing of tumor suppressor genes, has been shown to occur widely in human melanomas. High-throughput methylation arrays, a new technology which can simultaneously evaluate promoter methylation in many cancer-related genes, has potential for discovery of candidate DNA-methylation sites useful for melanoma diagnosis. However, these arrays have been developed for use on unfixed tissues, and their validity and reproducibility has not been determined on formalin-fixed paraffin-embedded (FFPE) tissue, which is typically the only diagnostic tissue available for primary melanomas and nevi. The central hypothesis of our proposal is that DNA methylation patterns exist that can discriminate melanomas from benign moles with high sensitivity, specificity, and reproducibility; and high-throughput DNA-methylation assays are a feasible method for discovery of these patterns in diagnostic FFPE tissues. A goal for this R21 is to assess whether formalin-fixed tissues are a suitable source of DNA for high-throughput methylation array profiling by assessing the reproducibility of results between matched formalin-fixed and frozen melanoma specimens and between formalin-fixed duplicates. Importantly, as a second aim, a 'dose response curve' will be determined to assess the proportion of melanocytic tumor to surrounding non-melanocytic tissue necessary for tumor DNAmethylation detection using high-throughput arrays, and these results will establish the proportion tumor below which selective procurement using laser capture microdissection will be done prospectively. Furthermore, this application proposes to identify a 'proof-of-principle' methylation-signature algorithm which will differentiate melanomas from benign moles. This study will be a first step toward the development of diagnostic methylation assays that could be used to standardize melanoma diagnosis, thereby decreasing under- and over-treatment of melanocytic lesions.