Scleroderma Subtyping from Fresh and Archived Biopsies using NextGen Sequencing

描述(申请人提供)：系统性硬化症(SSC)是一种自身免疫性疾病，表现为异质性和复杂的表型。该病的主要表现是皮肤纤维化、血管功能障碍和免疫系统激活。没有经过验证的诊断标记物。目前还没有根治的方法。三分之一的患者在确诊后10年内死亡。一些药物(为其他适应症而开发)目前正在进行临床试验，然而，结果指标(如改良的Rodnan皮肤评分)是不够的，患者群体的异质性阻碍了试验的有效性，因为只有一小部分患者可能对任何给定的药物有反应。Celdara Medical正在通过将基因签名与临床表型相关联来分割这种异质性或“亚型”SSC，以提供患者预后的量化测量、临床试验期间的疗效评估以及对疾病机制的洞察，这些机制将导致为每一种SSC亚型设计药物。由于SSc是一种罕见的疾病，皮肤活检的需求量很大，而用于DNA微阵列分析的新鲜活检样本的纵向收集速度很慢，这严重阻碍了该测试的临床有效性。对单个临床站点存档的福尔马林固定石蜡包埋(FFPE)组织块进行回顾性分析是解决这一问题的一种方法，但这些样本(相对于新鲜冰冻(FF)样本)中的RNA降解程度使其不适合进行DNA微阵列分析。因此，我们建议开发一种稳健的SSC亚型方法，通过从FFPE和FF皮肤活检组织中通过短读超高通量(UHTP)测序来测量基因表达来预测SSC亚型。在第一阶段，我们将完成以下目标：目标1.比较uHTS和NanoStrong技术在FFPE和FF样本基因表达谱测量中的应用。目的2.使用超高温超导技术或纳米串技术提高临床纵向数据的质量和样本量，并分析与内在子集相关的临床协变量。本文描述的工作的成功完成将：1.允许临床验证，2.简化临床实施，3.降低患者亚型的成本，以及4.通过纳入存档样本来显著和快速地扩展基因表达样本数据库，潜在地导致对这种可怕而复杂的疾病的基于基因组学的新见解。 与公共卫生相关：硬皮病是一种人们知之甚少的疾病，影响着30万美国人，并在确诊后10年内导致三分之一的患者死亡。对患者样本的遗传分析揭示了这种疾病的亚型，每种亚型都有不同的预后，每种亚型都提出了不同的潜在治疗方法。该项目的目标是简化目前复杂的样品制备方法，同时提高遗传分析的稳健性并降低成本。

DESCRIPTION (provided by applicant): Systemic sclerosis (SSc) is an autoimmune disease that presents with a heterogeneous and complex phenotype. Major manifestations of the disease are skin fibrosis, vascular dysfunction, and immune system activation. There are no validated diagnostic markers. There are no curative treatments. One in three patients dies within 10 years of diagnosis. Some drugs (developed for other indications) are currently in clinical trials however, outcome measures such as the modified Rodnan skin score are inadequate, and heterogeneity in patient populations hinders trial effectiveness since only a subset of patients is likely to respond to any given drug. Celdara Medical is segmenting this heterogeneity or "subtyping" SSc by associating gene signatures with clinical phenotypes to provide a quantitative measure for patient prognosis, assessment of efficacy during clinical trials, and insight into disease mechanisms which will lead to drugs designed for each SSc subtype. Because SSc is a rare disease, skin biopsies are in high demand and longitudinal accrual of fresh biopsy samples for DNA microarray analysis is slow, which severely hampers clinical validation of the test. A retrospective analysis of archived formalin fixed paraffin embedded (FFPE) tissue blocks from a single clinical site is one solution to this problem, however, the degree of RNA degradation in these samples (vis- `-vis fresh frozen (FF) samples) makes them unsuitable for DNA microarray analysis. We therefore propose to develop a robust SSc subsetting method using measurement of gene expression by short read ultra high-throughput (UHTP) sequencing from FFPE and FF skin biopsies to predict SSc subtype. In Phase I we will accomplish the following Aims: Aim 1. Compare the utility of UHTS to NanoString technology for the measurement of gene expression profiles from FFPE and FF samples. Aim 2. Increase the quality and sample size of clinical longitudinal data using either UHTS or Nanostring and analyze the clinical covariates associated with the intrinsic subsets. Successful completion of the work described herein will: 1. allow for clinical validation, 2. simplify clinical implementation, 3. reduce the cost of patient subtyping, and 4. dramatically and quickly expand the gene expression sample database through inclusion of archived samples, potentially leading to novel genomics- based insights into this terrible and complex disease. PUBLIC HEALTH RELEVANCE: Scleroderma is a poorly-understood disease which affects 300,000 Americans, and kills 1/3 of those afflicted within 10 years of diagnosis. Genetic analyses of patient samples have revealed subtypes of the disease, each with different prognoses, and each suggesting different potential therapeutics. The goal of this project is to simplify the currently complex sample preparation protocol while improving the robustness and decreasing the cost of the genetic analyses.