High-Throughput DNA-Methylation Profiling from Fixed Melanocytic Tissues

项目概述/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。由于局部和转移性黑色素瘤的生存率存在显著差异，因此在早期诊断黑色素瘤是必要的；然而，即使是专业的病理学家也很难区分黑色素瘤和良性痣（痣），尤其是非典型痣。DNA甲基化有望作为一种工具，与组织病理学相结合，通过提供分子信息来增强黑色素瘤的诊断。高通量dna甲基化阵列分析有可能发现对诊断有用的候选dna甲基化位点，但必须对福尔马林固定石蜡包埋（FFPE）组织有效，这是黑色素细胞病变通常唯一可用的诊断材料。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，将该技术应用于FFPE组织仍存在许多挑战，包括处理小样本的检测能力和验证肿瘤百分比的方法。我们的长期目标是开发一种实用的临床检测方法，在尽可能早的阶段进行黑色素瘤的分子诊断，同时避免假阳性，并最大限度地降低诊断的总体成本。本应用程序的目的是鉴定内部标记以评估肿瘤百分比，确定小FFPE组织高通量dna甲基化谱的有效条件；并通过其他检测确认dna甲基化差异。我们建议的中心假设是FFPE组织的高通量DNA甲基化阵列分析可以进一步发展以处理小样本，并且可以确定内部标准以评估肿瘤百分比。所提议的工作的基本原理是，允许大多数样本进行分析将减少选择候选dna甲基化差异的偏差，而内部标准将排除不会给出有效结果的样本。在强有力的初步数据的指导下，这一假设将通过追求三个特定目标来验证：1)确定区分黑素细胞（痣或黑色素瘤）与非黑素细胞（周围皮肤或淋巴细胞浸润）细胞的候选dna甲基化差异，作为内部质量控制标准来量化样本肿瘤百分比；2)确定小型FFPE黑素细胞组织高通量dna甲基化阵列分析的有效条件；3)利用更多的定量分析，从高通量分析中确认候选dna甲基化差异。该方法具有创新性，因为它将新兴的高通量DNA甲基化技术与生物标本库和FFPE黑色素细胞标本结合在一起，因为它们通常是在医院和社区皮肤科实践中制备的。该研究具有重要意义，因为对大多数FFPE标本进行有效的高通量dna甲基化分析以及内部质量控制措施将为黑色素瘤和其他恶性肿瘤的诊断开辟新的机会。

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. PUBLIC HEALTH RELEVANCE: Project Narrative Melanoma has a predilection to metastasize when only a few millimeters in depth; however, early detection and diagnosis are difficult due to the overlap in clinical and histologic appearances of melanomas with highly prevalent benign moles. High-throughput DNA-methylation array technology holds the promise of discovery of candidate DNA-methylation markers useful for improving melanoma diagnosis but must be valid on small formalin-fixed tissues embedded in paraffin blocks, which is typically the only diagnostic tissue available for primary melanomas and moles. The goal for this R33 is validation and advanced development of methodology to overcome the challenges when this novel technology is directly implemented for analysis of small formalin- fixed paraffin-embedded specimens, along with identification of criteria for quality assessment.