Molecular signatures of melanoma histology and progression: A Population Based A

描述（由申请方提供）：原发性黑色素瘤分子和基因组分类的关键障碍是要求提供福尔马林固定、石蜡包埋（FFPE）生物标本中的预后指标（即Breslow厚度）和相关组织学参数，与冷冻材料不同，纯mRNA和DNA难以或不可能提取和研究。与我们的合作者一起，我们成功地应用了Fan等人于2004年首次开发的新技术，用于黑色素瘤FFPE生物标本的全球基因表达谱分析。该平台（指定用于DNA介导退火、选择、延伸和连接的DASL）使福尔马林固定的生物标本以石蜡块的形式包埋和存档，可用于基因表达分析。我们发现了两个不同的亚类，其特征在于两个强大的签名：一个广泛定义的小眼球和黑素细胞分化基因（“MITF”类）和一个更异质的炎症，先天免疫和生长相关基因（“IIG”类）的集群。我们的初步数据表明，在护士健康研究1中，DASL可以成功地描述和恢复FFPE原发性黑色素瘤的2类结构。布里格姆妇女医院的钱宁实验室是大型队列研究的所在地，即护士健康研究1（NHS 1），护士健康研究2（NHS 2）和卫生专业人员随访研究（HPFS）。这些队列的进一步优势在于疾病随访，每两年更新一次队列中黑色素瘤病例的新病例和死亡率以及原发肿瘤部位组织块的可用性。我们假设黑色素瘤的侵袭性是由MITF/IIG分类决定的，我们建议评估这种2类结构是否与已知的组织学参数（如Breslow厚度）和黑色素瘤复发/死亡率相关。具体目标1：使用具有超过6000个基因的Illumina表达阵列平台和Sequonome平台开发NHS、NHS 2和HPFS黑色素瘤FFPE样品的分子特征，以对BRAF和NRAS的MC 1 R和基因型特异性基因座进行测序，以：a.为每个黑色素瘤样本分配MITF或IIG类别，并评价其与BRAF/NRAS/MC 1 R状态和黑色素瘤组织学决定因素（例如Breslow厚度、Clark水平、垂直生长期）的相关性B。评估每个黑素瘤样品的全基因组（6，000个基因）类别发现，并评估它们与BRAF/ NRAS/MC 1 R状态和黑素瘤组织学决定因素（例如Breslow、Clark水平、垂直生长期）的关联。分配MITF或IIG分类与BRAF/NRAS/MC 1 R状态和黑色素瘤复发和黑色素瘤致死率的相关性B。评估全基因组（6，000个基因）与BRAF/NRAS/MC 1 R状态和黑色素瘤复发和黑色素瘤所致死亡率的相关性公共卫生相关性：皮肤恶性黑色素瘤是一种致死性癌症，对发病率和死亡率有显著影响。临床上的矛盾是，黑色素瘤在早期诊断时是可以治愈的，并且在晚期疾病中相当致命。自20世纪60年代Clark's黑色素瘤侵袭性水平的验证以来，我们对黑色素瘤组织学分级与黑色素瘤复发和死亡率之间显著相关性的理解几乎没有变化。我们希望能够确定指向转移潜力途径的生物标志物，并有助于早期诊断。因此，我们的目标是通过最终降低这种致命皮肤癌的死亡率来显著改善目前诊断和治疗黑色素瘤的范例。

DESCRIPTION (provided by applicant): critical obstacle to molecular and genomic classification of primary melanoma has been the requirement to provide prognostic measures (i.e. Breslow thickness) and related histologic parameters in formalin-fixed, paraffin-embedded (FFPE) biospecimens, where unlike frozen material, unadulterated mRNA and DNA have been difficult or impossible to extract and study. With our collaborators, we have successfully applied a novel technology first developed by Fan et al in 2004 for global gene expression profiling of melanoma FFPE biospecimens. This platform, designated DASL for DNA-mediated Annealing, Selection, extension, and Ligation) renders formalin-fixed biospecimens embedded and archived in the form of paraffin blocks usable for gene expression analysis. We found two distinct subclasses characterized by two robust signatures: one broadly defined by a cluster of microphthalmia and melanocyte differentiation genes ("MITF" class) and a more heterogeneous group of inflammation, innate immune, and growth-related genes ("IIG" class). Our preliminary data indicate that DASL can successfully profile and recover the 2-class structure from FFPE primary melanomas in the Nurses Health Study 1. The Channing Laboratory at Brigham and Women's Hospital is home to large cohort studies, i.e. the Nurses' Health Study 1 (NHS1), Nurses' Health Study-2 (NHS2) and the Health Professionals Follow-up Study (HPFS). The further strength of these cohorts lies in disease follow-up with bi-annual updates on new cases and mortality and availability of tissue blocks from the primary tumor site for melanoma cases in the cohorts. We hypothesize that melanoma aggressiveness is determined by MITF/IIG classes and we propose to evaluate whether this 2-class structure is associated with known histological parameters (e.g. Breslow thickness), and melanoma recurrence/mortality. With access to unique population-based national cohorts and development of new technologies with our collaborators, the specific aims of this proposal are as follows: Specific Aim 1: Develop molecular signatures of NHS, NHS2 and HPFS melanoma FFPE samples using an Illumina expression array platform with over 6000 genes and a Sequonome platform to sequence MC1R and genotype specific loci for BRAF and NRAS to: a. Assign MITF or IIG class for each melanoma sample and evaluate their association with BRAF/NRAS/MC1R status and determinants of melanoma histology (e.g. Breslow thickness, Clark's level, vertical growth phase) b. Evaluate genome-wide (6,000 genes) class discovery for each melanoma sample and evaluate their association with BRAF/ NRAS/MC1R status and determinants of melanoma histology (e.g. Breslow, Clark's level, vertical growth phase) Specific Aim 2: Using the transcriptional and genomic signatures from Aim 1 to: a. Assign MITF or IIG class association with BRAF/NRAS/MC1R status and melanoma recurrence and mortality due to melanoma b. Evaluate genome-wide (6,000 genes) association with BRAF/NRAS/MC1R status and melanoma recurrence and mortality due to melanoma PUBLIC HEALTH RELEVANCE: Malignant melanoma of the skin is a lethal cancer with significant impact on morbidity and mortality. The clinical paradox is that melanoma is curable when diagnosed early and quite lethal with advanced disease. Since validation of the Clark's level for melanoma invasiveness in the 1960's, there has been little change in our understanding of the significant association between histologic grading of melanoma and melanoma recurrence and mortality. We hope to be able to identify biomarkers that point to pathways of metastatic potential and assist in earlier prognostication. Thus we aim to significantly improve the current paradigm for diagnosis and therapy for melanoma by eventually reducing mortality from this deadly skin cancer.