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

描述(申请人提供)：对原发黑色素瘤的分子和基因组分类的关键障碍是要求在福尔马林固定的石蜡包埋(FFPE)生物标本中提供预后测量(即布雷斯洛厚度)和相关的组织学参数，其中与冷冻材料不同，很难或不可能提取和研究未掺杂的mRNA和DNA。与我们的合作者一起，我们成功地应用了Fan等人于2004年首次开发的一项新技术，用于黑色素瘤FFPE生物样本的全球基因表达谱分析。这个平台，命名为DASL，用于DNA介导的退火、选择、扩展和连接)，使福尔马林固定的生物标本以石蜡块的形式嵌入和存档，可用于基因表达分析。我们发现了两个不同的亚类，它们具有两个强大的特征：一个是由一组小眼球和黑素细胞分化基因(“MITF”类)广泛定义的，另一类是由炎症、先天免疫和生长相关基因组成的更不同的组(“IIG”类)。我们的初步数据表明，DASL可以成功地分析和恢复护士健康研究1中FFPE原发黑色素瘤的二级结构。布里格姆妇女医院的钱宁实验室拥有大型队列研究，即护士健康研究1(NHS1)、护士健康研究2(NHS2)和卫生专业人员后续研究(HPFS)。这些队列的进一步优势在于疾病跟踪，每两年更新一次新病例和死亡率，以及队列中黑色素瘤病例的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/死亡率有关。通过获得独特的基于人群的国家队列并与我们的合作者一起开发新技术，这项建议的具体目标如下：具体目标1：使用包含6000多个基因的Illumina表达阵列平台和Sequonome平台来对MC1R和BRAF和NRAS的特定基因座进行测序，以建立NHS、NHS2和HPFS黑色素瘤FFPE样本的分子特征：a.为每个黑色素瘤样本分配MITF或IIG类别，并评估它们与BRAF/NRAS/MC1R状态和黑色素瘤组织学决定因素(例如Breslow厚度、Clark‘s Level、垂直生长阶段)b.评估每个黑色素瘤样本的全基因组(6,000个基因)类别发现，并评估它们与BRAF/NRAS/MC1R状态和黑色素瘤组织学决定因素(例如Breslow，Clark‘s Level，B.评估全基因组(6,000个基因)与BRAF/NRAS/MC1R状态和黑色素瘤复发和死亡率的相关性；b.评估全基因组(6,000个基因)与BRAF/NRAS/MC1R状态和黑色素瘤复发和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命的癌症，对发病率和死亡率有显著影响。临床上的悖论是，黑色素瘤在早期诊断时是可以治愈的，而在晚期疾病中则相当致命。自从20世纪60年代S对克拉克的黑色素瘤侵袭性水平进行验证以来，我们对黑色素瘤的组织学分级与黑色素瘤复发和死亡率之间的重要联系的认识几乎没有改变。我们希望能够识别生物标记物，指出转移潜能的途径，并帮助早期预测。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，显著改善目前黑色素瘤的诊断和治疗模式。

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.