DNA methylation-based assays for detecting disease spread in rhabdomyosarcoma

描述（由申请人提供）：横纹肌肉瘤（Rhabdomyosarcoma， 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，治疗结果有了渐进式的改善，例如现在大约70%的新诊断的RMS患者有可能治愈。一种针对RMS的风险适应治疗系统已经被开发出来，其风险评估基于术前分期、术后分组、远端转移的存在和组织学亚型。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。该应用侧重于微小弥散性疾病的分析，其中基于肿瘤特异性分子标记的pcr检测可以灵敏地检测肿瘤细胞扩散到骨髓等部位。对于RMS，一些小型研究使用基于rna的PCR方法来证明在没有临床转移性疾病证据的患者骨髓中检测RMS细胞的可行性。此外，这些研究和其他研究的证据表明，在骨髓中检测到隐匿的肿瘤细胞预示着更糟糕的结果。为了充分了解这种方法在RMS管理中的效用，这些最小弥散性疾病检测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。由于集中收集方案需要时间，这些标本中的RNA通常部分降解。然而，这些样品中的DNA仍然是完整的，本项目将开发适用于RMS的基于DNA的分析。DNA超甲基化是一种DNA修饰，通常在许多基因组位点上具有肿瘤特异性。它可以通过基于聚合酶链反应的方法检测，该方法已用于筛选多种癌症类型中的隐性癌细胞。基于DNA高甲基化发生在两种RMS亚型的特定位点的初步证据，微阵列策略将用于筛选在RMS肿瘤中经常高甲基化而在正常血细胞中不高甲基化的基因组位点。这些发现将通过基于聚合酶链反应的分析得到证实和完善。为了扩展这些发现，将设计一组定量甲基化特异性PCR检测，以检测大多数RMS肿瘤中至少一种甲基化变化。这组定量PCR检测将应用于D9803骨髓样本的DNA以及配对原发性肿瘤的一组。这些检测结果将与临床和病理特征以及患者预后进行比较，以探讨骨髓中微小弥散性疾病在诊断中的意义。因此，该应用程序创造了一个宝贵的机会来识别、开发和测试新的分析方法，然后将这些分析方法应用于对现有的一组注释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小组软组织肉瘤委员会重要临床倡议的一部分。

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.