Prognostic potential of low-level mutations in meylodysplastic syndrome

描述（由申请人提供）：越来越多的证据表明，如果异质性肿瘤或癌前综合征中的体细胞低水平突变未被发现，可能会产生深远的临床后果。此外，它们可能包含重要的生物标志物和“错失的机会”，如果已知突变，可以应用于优化治疗。我们将在骨髓发育不良综合征（MDS）的具体病例中检验这一假设。MDS是一组癌前、克隆性骨髓疾病，其进展为急性髓性白血病（AML）的风险增加。合作者Ebert和Bejar证明TP53、RUNX1、ASLX1、EZH2、ETV6的突变与总生存期降低有关，并且在多变量分析中是结果的独立预后因素。总的来说，MDS患者的突变作为“个性化”治疗的生物标志物越来越重要，超越了基于临床特征和细胞遗传学的国际预后评分系统（IPSS）。虽然MDS显然是一种遗传异质性疾病，但尚不清楚罕见的亚克隆是否会影响临床表型。本研究的目的首先是确定现有技术无法检测到的特定突变的患病率，其次是确定任何已识别的突变是否会改变临床表型。我们将采用COLD-PCR，这是我们小组开发的一种富集和检测低水平DNA突变的方法，与基于扩增子的下一代测序相结合。COLD-PCR将illumina扩增子测序的灵敏度从目前的~2-5%提高到0.04%。使用COLD-PCR，“深度测序”变成了“超深度测序”。来自287名MDS患者的DNA将通过COLD-PCR-Illumina筛选预后/潜在预后基因突变。将分析两组患者的数据（结果差vs结果好，IPSS评分相似）(a)考虑低水平（0.04- 5%丰度）和高水平（0.05 5%丰度）突变；(b)只考虑高水平的突变，就像迄今为止的做法一样。将比较结果与预后/生存率的相关性。修订后的应用程序包含了额外的数据，完全验证了我们对第一个检测基因NRAS的假设。识别MDS患者预后低水平突变的能力将能够更好地预测结果，并对这些患者的治疗进行微调。该方法解决了所有异质性肿瘤（如肺、胰腺CA）的共同问题。因此，与公共卫生的相关性很高。

DESCRIPTION (provided by applicant): Accumulating evidence suggests that, if they go undetected, somatic low-level mutations in heterogeneous tumors or pre-cancerous syndromes can have profound clinical consequences. Furthermore, they may comprise important biomarkers and 'missed opportunities' for optimized therapy that could be applied had the mutation been known. We shall examine this hypothesis for the specific case of myelo-dysplastic syndromes (MDS). MDS are a collection of pre-cancerous, clonal bone marrow disorders with an increased risk of progression to acute myeloid leukemia (AML). Collaborators Ebert and Bejar demonstrated that mutations in TP53, RUNX1, ASLX1, EZH2, ETV6, are associated with decreased overall survival and are independent prognostic factors of outcome in multivariate analysis. Overall, mutations in MDS patients are of growing significance as biomarkers for 'personalization' of therapy beyond the established International Prognostic Scoring System (IPSS) which is based on clinical features and cytogenetics. While MDS is clearly a genetically heterogeneous disease, it is still not clear whether rare sub-clones influence clinical phenotype. This study aims first, to determine the prevalence of specific mutations that are below detection by existing technologies, and second to determine whether any identified mutations alter clinical phenotype. We shall employ COLD-PCR, a method developed by our group for enriching and detecting low-level DNA mutations, in conjunction with amplicon-based next-generation-sequencing. COLD-PCR increases the sensitivity of Illumina-based amplicon sequencing from the current ~2-5% down to 0.04% abundance, i.e. 'deep-sequencing' becomes 'ultra-deep-sequencing' using COLD-PCR. DNA from a group of 287 MDS patients will be screened via COLD-PCR-Illumina for mutations in the prognostic/potentially prognostic genes. Data from two groups of patients (poor outcome vs. favorable outcome, similar IPSS score) will be analyzed (a) accounting for both, low-level (0.04- 5% abundance) and high level (>5% abundance) mutations; and (b) accounting only for high level mutations, as practice has been until now. Results will be compared for their correlation with outcome/survival. The revised application contains additional data that fully validate our hypothesis for the first gene examined, NRAS. The ability to identify prognostic low-level mutations in MDS patients will enable better prediction of outcome and the fine-tuning of treatment for these patients. The approach addresses a problem common to all heterogeneous tumors (e.g. lung, pancreatic CA). Therefore relevance to Public Health is high.