DNA methylation-based assays for detecting disease spread in rhabdomyosarcoma

基于 DNA 甲基化的检测用于检测横纹肌肉瘤疾病传播

• 批准号: 7875543
• 负责人: FREDERIC G BARR
• 金额: $ 17.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7875543
• 关键词: Alveolar; Aspirate substance; Biological Assay; Blood Cells; Blood specimen; Bone Marrow; Cells; Characteristics; Childhood; Children' s Oncology Group; Clinical; Clinical Research; Clinical Trials; Collection; Controlled Clinical Trials; Controlled Study; DNA; DNA Methylation; DNA Modification Process; Data; Detection; Diagnosis; Disease; Distal; Failure; Family; Freezing; Frequencies; Gel; Gene Fusion; Genes; Genomics; Goals; Histologic; Hypermethylation; Institution; Investigation; Laboratory Study; Malignant Childhood Neoplasm; Malignant Neoplasms; Methodology; Methods; Methylation; Mutation; Neoplasm Metastasis; Newly Diagnosed; Normal Cell; Oncology Group; Operative Surgical Procedures; Outcome; Patients; Performance; Predictive Value; Primary Neoplasm; Protocols documentation; Publishing; RNA; RNA Degradation; Research Project Grants; Rhabdomyosarcoma; Risk; Risk Assessment; Sampling; Series; Site; Skeletal Muscle; Soft Tissue Neoplasms; Specimen; Staging; System; Testing; Time; Tissues; base; bisulfite; cancer cell; cancer type; design; molecular marker; neoplastic cell; peripheral blood; prognostic; public health relevance; sarcoma; soft tissue; tumor

DESCRIPTION (provided by applicant): Rhabdomyosarcoma (RMS) is a family of pediatric soft tissue tumors related to the skeletal muscle lineage. There have been incremental improvements in outcome over the last three decades, such that cure is now possible for ~70% of newly diagnosed RMS patients. A risk-adapted therapy system has been developed for RMS with risk assessment based on pre-surgical stage, post-surgical group, presence of distal metastases, and histologic subtype. Beyond these clinical indicators of cancer status, molecular markers are now being evaluated to provide further improvement in risk prediction. This application focuses on analysis of minimal disseminated disease in which PCR-based assays of tumor-specific molecular markers permit sensitive detection of tumor cell spread to sites such as bone marrow. For RMS, several small studies used RNA-based PCR methods to demonstrate the feasibility of detecting RMS cells in bone marrow in patients without clinical evidence of metastatic disease. Furthermore, there is evidence from these and other studies that detection of occult tumor cells in the bone marrow is predictive of a worse outcome. To fully understand the utility of this approach in RMS management, these minimal disseminated disease assays must be conducted as part of large well-controlled clinical trials. In an effort to pursue this goal, bone marrow and peripheral blood samples were collected in the recently completed Children's Oncology Group D9803 trial of intermediate risk RMS. Due to the time required for the centralized collection protocol, the RNA in these specimens was often partially degraded. However, the DNA in these samples is still intact and this project will develop DNA-based assays applicable to RMS. DNA hypermethylation is a DNA modification that is often tumor-specific at numerous genomic loci. It can be detected by a PCR-based methodology that has been used to screen for occult cancer cells in multiple cancer types. Based on preliminary evidence that DNA hypermethylation occurs at specific loci in both RMS subtypes, a microarray strategy will be used to screen for genomic loci that are frequently hypermethylated in RMS tumors but not in normal blood cells. These findings will be confirmed and refined by PCR-based assays. To extend these findings, a small panel of quantitative methylation-specific PCR assays will be designed that detect at least one methylation change in most RMS tumors. This panel of quantitative PCR assays will be applied to DNA from the D9803 bone marrow samples as well as a panel of paired primary tumors. The results of these assays will be compared with clinical and pathological characteristics as well as patient outcome to explore the significance of minimal disseminated disease in the bone marrow at diagnosis. This application thus creates a valuable opportunity to identify, develop and test new assays, and then apply these assays to perform correlative laboratory studies on an existing set of annotated biospecimens that are part of an important clinical initiative of the Soft Tissue Sarcoma Committee of the Children's Oncology Group. PUBLIC HEALTH RELEVANCE: This research project directly focuses on the pediatric cancer rhabdomyosarcoma, and proposes an approach to identify patients with early spread of this cancer to distal sites. In this approach, DNA will be isolated from a tissue to which cancer cells often spread, such as bone marrow, and will be screened for the presence of cancer cells by assaying for genetic changes that commonly occur in the cancer but not in the normal version of that tissue. The value of this approach will then be investigated by using these assays on a series of bone marrow samples from a clinical trial of the Children's Oncology Group, and then comparing the assay results with the patients' outcome data.

描述（申请人提供）：横纹肌肉瘤（RMS）是一个与骨骼肌谱系相关的儿科软组织肿瘤家族。在过去的三十年里，结果已经有了逐步的改善，现在大约70%的新诊断的RMS患者可以治愈。已经开发了一种风险适应性治疗系统，用于RMS，并根据术前阶段、术后组、远端转移的存在和组织学亚型进行风险评估。除了这些癌症状态的临床指标外，目前正在评估分子标记物，以进一步改善风险预测。该应用集中于微小播散性疾病的分析，其中基于PCR的肿瘤特异性分子标记物的测定允许灵敏地检测肿瘤细胞扩散到诸如骨髓的部位。对于RMS，几项小型研究使用基于RNA的PCR方法来证明在没有转移性疾病临床证据的患者中检测骨髓中RMS细胞的可行性。此外，从这些和其他研究中有证据表明，在骨髓中检测到隐匿性肿瘤细胞预示着更糟糕的结局。为了充分了解这种方法在RMS管理中的效用，这些最小播散性疾病测定必须作为大型对照良好的临床试验的一部分进行。为了实现这一目标，在最近完成的儿童肿瘤组D9803中等风险RMS试验中收集了骨髓和外周血样本。由于集中采集方案所需的时间，这些标本中的RNA通常会部分降解。然而，这些样本中的DNA仍然是完整的，本项目将开发适用于RMS的基于DNA的检测方法。DNA超甲基化是一种DNA修饰，通常在许多基因组位点具有肿瘤特异性。它可以通过基于PCR的方法检测，该方法已用于筛选多种癌症类型中的隐匿性癌细胞。基于初步证据表明，DNA超甲基化发生在特定的位点在两个RMS亚型，微阵列策略将被用来筛选基因组位点，经常在RMS肿瘤，但不是在正常血细胞的超甲基化。这些结果将通过基于PCR的检测进行确认和完善。为了扩展这些发现，将设计一小组定量甲基化特异性PCR检测，以检测大多数RMS肿瘤中的至少一种甲基化变化。该组定量PCR试验将应用于D9803骨髓样本以及一组配对原发性肿瘤的DNA。将这些测定的结果与临床和病理学特征以及患者结局进行比较，以探索诊断时骨髓中微小播散性疾病的意义。因此，该应用程序创造了一个宝贵的机会，以识别，开发和测试新的测定，然后应用这些测定对现有的一组注释的生物标本进行相关的实验室研究，这些生物标本是儿童肿瘤组软组织肉瘤委员会的重要临床倡议的一部分。 公共卫生关系：该研究项目直接关注儿科癌症横纹肌肉瘤，并提出了一种方法来识别这种癌症早期扩散到远端部位的患者。在这种方法中，DNA将从癌细胞经常扩散的组织中分离出来，例如骨髓，并通过分析癌症中通常发生的遗传变化来筛选癌细胞的存在，而不是正常组织。然后将通过对来自儿童肿瘤学小组的临床试验的一系列骨髓样本使用这些测定来研究这种方法的价值，然后将测定结果与患者的结果数据进行比较。