Prognostic potential of low-level mutations in meylodysplastic syndrome

骨髓增生异常综合征低水平突变的预后潜力

• 批准号: 8787719
• 负责人: G. Mike Makrigiorgos
• 金额: $ 22.84万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8787719
• 关键词: Accounting; Acute Myelocytic Leukemia; Address; Biological; Biological Assay; Biological Markers; Blood; Bone Marrow Diseases; CBL gene; Cancerous; Clinical; Clinical Data; Collection; Cytogenetics; DNA; DNA Sequence Alteration; Data; Detection; Disease; ETV6 gene; EZH2 gene; Event; Future; Genes; Genetic Fingerprintings; Genotype; Health; Hematopoiesis; International; Lung; Malignant Neoplasms; Malignant neoplasm of pancreas; Methods; Modeling; Multivariate Analysis; Mutation; Mutation Detection; NRAS gene; Outcome; Pancreas; Patients; Prevalence; Process; Prognostic Factor; Prognostic Marker; Public Health; RUNX1 gene; Risk; Sampling; Solid; Somatic Mutation; Syndrome; System; TP53 gene; Technology; Testing; Time; Validation; Work; base; cancer therapy; clinical phenotype; clinically significant; deep sequencing; digital; falls; improved; new technology; next generation sequencing; prevent; prognostic; prognostic value; tumor

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.

描述（由申请人提供）：越来越多的证据表明，如果未被发现，异质性肿瘤或癌前综合征中的体细胞低水平突变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”，可以在已知突变的情况下应用优化疗法。我们将针对骨髓增生异常综合征（MDS）的具体病例检验这一假设。 MDS 是一系列癌前克隆性骨髓疾病，进展为急性髓系白血病 (AML) 的风险增加。合作者 Ebert 和 Bejar 证明，TP53、RUNX1、ASLX1、EZH2、ETV6 的突变与总体生存率降低相关，并且是多变量分析结果的独立预后因素。总体而言，MDS 患者的突变作为超越基于临床特征和细胞遗传学的既定国际预后评分系统 (IPSS) 的“个性化”治疗的生物标志物越来越重要。虽然MDS显然是一种遗传异质性疾病，但仍不清楚罕见的亚克隆是否影响临床表型。这项研究的目的首先是确定现有技术检测不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用于富集和检测低水平DNA突变的方法，与基于扩增子的下一代测序相结合。 COLD-PCR 将基于 Illumina 的扩增子测序的灵敏度从目前的约 2-5% 丰度降低至 0.04%，即使用 COLD-PCR 的“深度测序”变为“超深度测序”。来自 287 名 MDS 患者的 DNA 将通过 COLD-PCR-Illumina 进行预后/潜在预后基因突变的筛查。将分析两组患者的数据（不良结果与有利结果，相似的 IPSS 评分）：(a) 考虑低水平（0.04-5% 丰度）和高水平（>5% 丰度）突变； (b) 按照迄今为止的实践，仅考虑高水平突变。将比较结果与结果/生存的相关性。修订后的应用程序包含额外的数据，完全验证了我们对第一个检查基因 NRAS 的假设。识别MDS患者的预后低水平突变的能力将能够更好地预测这些患者的结果并微调治疗。该方法解决了所有异质性肿瘤（例如肺、胰腺 CA）所共有的问题。因此，与公共卫生的相关性很高。

项目成果

DMSO Increases Mutation Scanning Detection Sensitivity of High-Resolution Melting in Clinical Samples.

• DOI: 10.1373/clinchem.2015.245357
• 发表时间: 2015-11
• 期刊: Clinical chemistry
• 影响因子: 9.3
• 作者: Song C;Castellanos-Rizaldos E;Bejar R;Ebert BL;Makrigiorgos GM
• 通讯作者: Makrigiorgos GM

Enriching mutant sequences by modulating the denaturation time during PCR.

通过调节 PCR 过程中的变性时间来富集突变序列。

• DOI: 10.1373/clinchem.2014.221465
• 发表时间: 2014
• 期刊: Clinical chemistry
• 影响因子: 9.3
• 作者: Murphy,DerekM;Castellanos-Rizaldos,Elena;Makrigiorgos,GMike
• 通讯作者: Makrigiorgos,GMike

Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment.

• DOI: 10.1093/nar/gkw650
• 发表时间: 2016-11-02
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Song C;Liu Y;Fontana R;Makrigiorgos A;Mamon H;Kulke MH;Makrigiorgos GM
• 通讯作者: Makrigiorgos GM

Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR.

• DOI: 10.1373/clinchem.2014.228361
• 发表时间: 2015-01
• 期刊: Clinical chemistry
• 影响因子: 9.3
• 作者: Castellanos-Rizaldos E;Richardson K;Lin R;Wu G;Makrigiorgos MG
• 通讯作者: Makrigiorgos MG

COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions.

• DOI: 10.1371/journal.pone.0094103
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Castellanos-Rizaldos E;Milbury CA;Karatza E;Chen CC;Makrigiorgos GM;Merewood A
• 通讯作者: Merewood A