Project 2: Primary melanoma DNA Methylation profiling for evaluating subtypes and survival

尽管已经确定黑色素瘤经常有异常的DNA甲基化模式，但它是 尚不清楚原发性黑色素瘤的DNA甲基化是否定义了生物学上相关的亚类或预测了疾病特异性 生死存亡。该项目的目标是识别和表征基于DNA甲基化的初级 黑色素瘤亚类，并训练、测试和验证CpG签名对疾病特异性生存的预后 原发黑色素瘤。我们的假设是，原发黑色素瘤的DNA甲基化将定义亚群 (包括预后不佳的CIMP-‘CpG岛甲基化表型’亚型)，CpG签名将 对黑色素瘤的存活率有预测作用。将执行Illumina Infinium甲基EPIC(850K)阵列评测 在整个方案项目中使用相同的1,000个原发黑色素瘤。这些黑色素瘤会 来自AJCC IIA/IIB/IIC/IIIA/IIIB期的患者，包括500名死于黑色素瘤的患者 5年和那些活了至少5年的人的500人在舞台上是匹配的。在目标1中，使用来自1,000个 原发黑色素瘤，我们将确定基于甲基化的黑色素瘤亚类，其特征是5年生存率 和其他结果、人口因素、集中审查的组织病理学特征、CD3/CD8/FOXP3 免疫谱、PD1和PDL1蛋白表达、体细胞突变、拷贝数改变和mRNA 和miRNA表达数据。进一步的表征将确定这些子类是否与 我们的团队和其他人之前已经从 独立的数据集。在目标2中，使用统计建模，我们将训练一个原发黑色素瘤CpG签名 使用660例原发性黑色素瘤患者预测5年内死于黑色素瘤的预后(主要结果) 并用340例原发黑色素瘤进行检测。我们将确定这个CpG生存签名是否增加了 向AJCC分期提供信息。作为次要结果，我们还将开发5个内复发的签名 前哨淋巴结(SLN)阳性，以及SLN活检阴性后的远处复发。在《目标3》中，我们将 用定量方法从技术上验证生存信号中CPGS/基因的DNA甲基化水平 甲基化特异性聚合酶链式反应(PCR)。对于选定的标记，我们将定量测量其 用多重免疫组织化学方法比较原发黑色素瘤患者 活着的患者和5年内死亡的患者诊断和评估预后最好的功能 项目3中的目标：我们的目标是识别和表征基于DNA甲基化的原发黑色素瘤 将CpG签名的黑色素瘤生存预后亚类划分并发现和验证 向AJCC分期提供信息。我们将启动该签名在临床可行的检测预后中的应用 为了生存。该项目应该能够识别出预计存活率较低的黑色素瘤患者 他们可以从更密切的随访和辅助治疗中受益。我们的结果也应该表明哪些目标 预后不良的黑色素瘤存在治疗反应的预测性。

Although it has been established that melanomas frequently have aberrant DNA methylation patterns, it is unknown if DNA methylation of primary melanoma defines biologically relevant subclasses or predicts diseasespecific survival. This project’s goals are to identify and characterize DNA methylation-based primary melanoma subclasses, and train, test, and validate a CpG signature prognostic for disease-specific survival in primary melanoma. Our hypothesis is that DNA methylation in primary melanoma will define subgroups (including a poor-prognostic CIMP - ‘CpG island methylator phenotype’ subtype) and that a CpG signature will be prognostic for melanoma survival. Illumina Infinium MethylationEPIC (850K) array profiling will be performed on the same 1,000 primary melanomas to be utilized throughout this program project. These melanomas will be from AJCC stage IIA/IIB/IIC/IIIA/IIIB patients, including 500 from patients who died due to melanoma within 5 years and 500 from those who lived at least 5 years matched on stage. In Aim 1, using data from the 1,000 primary melanomas, we will identify methylation-based melanoma subclasses, characterized by 5-year survival and other outcomes, demographic factors, centrally reviewed histopathological features, a CD3/CD8/FOXP3 immune profile, PD1 and PDL1 protein expression, somatic mutations, copy number alterations, and mRNA and miRNA expression data. Further characterization will determine if the subclasses are associated with biologically relevant DNA methylation signatures that our group and others have previously produced from independent datasets. In Aim 2 using statistical modeling, we will train a primary melanoma CpG signature prognostic for death from melanoma within 5 years (the primary outcome) using 660 of the primary melanomas and test it using 340 of the primary melanomas. We will determine whether this CpG survival signature adds information to AJCC staging. As secondary outcomes, we will also develop signatures for recurrence within 5 years, sentinel lymph node (SLN) positivity, and distant recurrence after negative SLN biopsy. In Aim 3, we will technically validate DNA methylation levels of CpGs/genes in the survival signature using quantitative methylation-specific polymerase chain reaction (PCR). For select markers, we will quantitatively measure their protein expression differences using multiplexed immunohistochemistry, comparing primary melanomas from patients alive and those who died within 5 years of diagnosis and assess the function of the most prognostic targets in Project 3. Our goal is to identify and characterize DNA methylation-based primary melanoma subclasses and discover and validate a CpG signature prognostic for survival from melanoma that adds information to AJCC staging. We will initiate application of the signature to a clinically viable assay prognostic for survival. This project should allow identification of melanoma patients expected to have worse survival and who could benefit from closer follow-up and adjuvant treatments. Our results should also indicate which targets predictive of treatment responses are present in poor prognostic melanomas.