High-Throughput DNA-Methylation Profiling from Fixed Melanocytic Tissues

描述（由申请人提供）：项目摘要/摘要黑色素瘤具有早期转移的能力，其病程很少受到医疗干预的影响。由于局限性和转移性疾病之间的生存率存在显著差异，因此必须在最早期诊断黑色素瘤;然而，即使是专家病理学家也很难区分黑色素瘤和良性痣（痣），特别是非典型痣。DNA甲基化有望作为一种工具，结合组织病理学，通过提供分子信息来增强黑色素瘤诊断。高通量DNA甲基化阵列分析具有发现可用于诊断的候选DNA甲基化位点的潜力，但必须在福尔马林固定石蜡包埋（FFPE）组织上有效，其通常是可用于黑素细胞病变的唯一诊断材料。在R21阶段，我们证明了使用FFPE组织进行DNA甲基化分析的技术可行性，并确定了区分黑色素瘤和痣的“原理证明”特征。然而，在将该技术应用于FFPE组织方面仍然存在许多挑战，包括该测定处理小样本的能力和用于验证肿瘤百分比的方法。我们的长期目标是在尽可能早的阶段开发一种用于黑色素瘤分子诊断的实用临床检测方法，同时避免假阳性并最大限度地降低诊断的总成本。本申请的目的是鉴定内部标志物以评估肿瘤百分比，确定小FFPE组织高通量DNA甲基化谱分析的有效条件;并使用其他测定法确认DNA甲基化差异。我们的建议的中心假设是FFPE组织的高通量DNA甲基化阵列分析可以进一步开发以处理小样品，并且可以鉴定内标物以评估肿瘤百分比。拟议工作的基本原理是，允许对大多数样品进行分析将减少候选DNA甲基化差异选择的偏倚，而内标物将排除不会给出有效结果的样品。在强有力的初步数据的指导下，该假设将通过追求三个具体目标来测试：1）识别区分黑素细胞的候选DNA甲基化差异，（痣或黑色素瘤）与非黑色素细胞（周围皮肤或淋巴细胞浸润）细胞用作内部质量控制标准以量化样品肿瘤百分比; 2）鉴定小尺寸FFPE黑素细胞组织的高通量DNA甲基化阵列分析的有效条件;和3）使用更多定量测定从高通量分析确认候选DNA甲基化差异。该方法是创新的，因为它将新兴的高通量DNA甲基化技术与生物标本库和FFPE黑素细胞标本相结合，因为它们通常在医院和社区皮肤科实践中制备。拟议的研究是重要的，因为有效的高通量DNA甲基化分析的大多数FFPE标本沿着与内部质量控制措施将打开新的诊断机会黑色素瘤和其他恶性肿瘤。

DESCRIPTION (provided by applicant): Project Summary/Abstract Melanoma 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, even expert pathologists can have difficulty distinguishing melanomas from benign nevi (moles), especially atypical nevi. DNA methylation holds promise as a tool, in conjunction with histopathology, for enhancing melanoma diagnosis by providing molecular information. High- throughput DNA-methylation array profiling has the potential for discovery of candidate DNA-methylation sites useful for diagnosis but must be valid on formalin-fixed paraffin-embedded (FFPE) tissue, which is typically the only diagnostic material available for melanocytic lesions. In an R21 phase, we demonstrated technical feasibility of DNA-methylation profiling using FFPE tissues and identified a "proof-of-principle" signature for discriminating melanomas from nevi. However, many challenges remain in the application of this technology to FFPE tissues, including the ability of the assay to handle small specimens and methods for verifying percent tumor. Our long-term goal is to develop a practical clinical assay for molecular diagnosis of melanoma at the earliest possible stage, while avoiding false-positives and minimizing the overall cost of diagnosis. The objectives in this application are to identify internal markers to assess percent tumor, determine valid conditions for high-throughput DNA-methylation profiling of small FFPE tissues; and confirm DNA-methylation differences using additional assays. The central hypothesis of our proposal is that high-throughput DNA- methylation array profiling of FFPE tissues can be further developed to handle small samples and internal standards can be identified to assess percent tumor. The rationale for the proposed work is that allowing the majority of samples to be profiled will decrease bias in selection of candidate DNA-methylation differences, while the internal standards will exclude samples that will not give valid results. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identify candidate DNA-methylation differences that distinguish melanocytic (nevus or melanoma) vs. non-melanocytic (surrounding skin or lymphocytic infiltrate) cells for use as internal quality control standards to quantify sample percent tumor; 2) Identify valid conditions for high-throughput DNA-methylation array profiling of small-sized FFPE melanocytic tissues; and 3) Confirm candidate DNA-methylation differences from high-throughput profiling using more quantitative assays. The approach is innovative because it couples emerging high-throughput DNA- methylation technology to a biospecimen repository and FFPE melanocytic specimens, as they are typically prepared in hospital and community-based dermatologic practices. The proposed research is significant because valid high-throughput DNA-methylation profiling of the majority of FFPE specimens along with internal quality control measures will open new diagnostic opportunities for melanoma and other malignancies.