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(申请人提供)：越来越多的证据表明，如果没有被发现，异质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，它们可能包括重要的生物标记物和“错失的优化治疗机会”，如果突变已知，这些机会就可以应用。我们将针对骨髓增生异常综合征(MDS)的特定病例来检验这一假说。MDS是一组癌前、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，在多因素分析中，TP53、RUNX1、ASLX1、EZH2、ETV6的突变与总生存率的降低有关，是影响预后的独立预后因素。总体而言，MDS患者的突变作为个性化治疗的生物标记物的重要性越来越大，超过了基于临床特征和细胞遗传学的已建立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，但仍不清楚罕见的亚克隆是否会影响临床表型。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流行率，其次确定是否有任何已发现的突变改变了临床表型。我们将采用冷-聚合酶链式反应，这是我们团队开发的一种用于丰富和检测低水平DNA突变的方法，与基于扩增的下一代测序相结合。冷-聚合酶链式反应将基于Illumina的扩增片段测序的灵敏度从目前的~2-5%提高到0.04%，也就是说，使用冷-聚合酶链式反应的“深测序”变成了“超深测序”。来自287名MDS患者的DNA将通过COLD-PCR-Illumina进行筛查，以寻找预后/潜在预后基因的突变。来自两组患者的数据(预后差与预后好，IPSS评分相似)将被分析(A)同时考虑低水平(0.04-5%丰度)和高水平(&gt；5%丰度)突变；以及(B)只考虑高水平突变，就像到目前为止的做法一样。结果将与结果/存活率的相关性进行比较。修订后的申请包含了额外的数据，充分验证了我们对第一个被检查的基因NRAS的假设。识别MDS患者预后低水平突变的能力将使更好地预测结果和对这些患者的治疗进行微调。该方法解决了所有异质肿瘤(如肺、胰腺CA)的共同问题。因此，与公共卫生的相关性很高。

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