Detection of Tumor DNA in Plasma from Carolina Breast Cancer Study Patients

描述（由申请人提供）：乳腺癌是一种异质性疾病，表现为多种临床、组织病理学和分子亚型，具有不同的治疗反应和预后。异常DNA甲基化通过调控基因转录和基因组稳定性导致乳腺癌，基因启动子CpG岛胞嘧啶残基甲基化可以沉默对乳腺癌发生和进展至关重要的肿瘤抑制基因。在乳腺癌患者的血液循环中可以检测到高甲基化的肿瘤DNA，特别是那些疾病晚期的患者；因此，循环肿瘤DNA可作为癌症诊断、临床分期、监测治疗效果、全身性疾病活动和复发的生物标志物。测量DNA甲基化的灵敏方法可以检测生物样品（如血浆）中隐藏或隐藏的癌细胞。在我们最近对来自卡罗莱纳乳腺癌研究（CBCS）（1期）病例的519例侵袭性，主要是早期乳腺肿瘤的基于阵列的DNA甲基化分析研究中，我们发现高度甲基化的基因与更晚期的临床阶段或预测疾病特异性生存、总体或特定内在肿瘤亚型显著相关。本研究的中心假设是DNA甲基化模式在区分乳腺癌与正常乳腺组织和确定乳腺肿瘤表型方面很重要，敏感的甲基化检测可用于检测乳腺癌患者血浆中异常的肿瘤相关DNA。利用在CBCS（一期）乳腺癌肿瘤中发现的10个高甲基化预后或分期相关的CpG标记物，我们建议进行一项试点研究，以确定是否可以从CBCS（二期）受试者的血浆样本中回收足够数量和质量的DNA进行甲基化研究，以优化用于血浆甲基化检测的敏感定量甲基化特异性PCR分析。并比较CBCS乳腺癌病例和对照组标本中的这10种甲基化标记谱。将对100例具有不同临床分期和内在亚型的乳腺癌患者进行原发性乳腺肿瘤、血浆和外周血白细胞（pbl）的评估，这些患者在接受化疗前都进行了抽血。将从年龄和种族匹配的对照（n=50）中评估血浆和pbl。比较病例和对照组血浆中标记物甲基化将提供其对肿瘤DNA特异性的指示。此外，我们将确定血浆甲基化是否与匹配肿瘤的甲基化状态相关，或者与包括分期、淋巴结阳性、年龄、种族或亚型在内的病例特征相关。最后，分析PBL甲基化将检查作为血浆样品污染的可能来源。我们期望这项工作的结果将有助于开发改进的生物标志物，用于乳腺癌的癌症诊断、治疗效果、预后和疾病复发。

DESCRIPTION (provided by applicant): Breast cancer is a heterogeneous disease, manifesting as multiple clinical, histopathologic and molecular subtypes with different therapeutic responses and prognoses. Aberrant DNA methylation contributes to breast cancer through regulation of gene transcription and genome stability, and methylation of cytosine residues in CpG islands of gene promoters can silence tumor suppressor genes critical to breast cancer development and progression. Hypermethylated tumor DNA can be detected in the circulation of breast cancer patients, particularly those with more advanced disease; thus, circulating tumor DNA could be useful as a biomarker for cancer diagnosis, clinical staging, and to monitor therapeutic effects, systemic disease activity and recurrences. Sensitive methods for measuring DNA methylation could permit detection of occult or hidden cancer cells in biological samples such as plasma. In our recent study of array-based DNA methylation profiling of 519 invasive, mainly early-stage breast tumors from cases in the Carolina Breast Cancer Study (CBCS) (phase 1), we identified highly methylated genes significantly correlated with more advanced clinical stage or that predicted disease-specific survival, overall or among specific intrinsic tumor subtypes. The central hypothesis of this study is that DNA methylation patterns are important in distinguishing breast cancers from normal breast tissue and in defining breast tumor phenotypes, and sensitive methylation assays can be used to detect aberrant tumor-associated DNA in the plasma from breast cancer patients. Using 10 hypermethylated prognostic or stage-related CpG markers identified in breast tumors from CBCS (phase 1), we propose to conduct a pilot study to determine whether sufficient quantity and quality of DNA can be recovered from banked plasma samples from CBCS (phase 2) subjects for methylation studies, to optimize sensitive quantitative methylation-specific PCR assays for plasma methylation detection, and to compare these 10 methylation marker profiles in specimens from CBCS breast cancer cases and controls. Primary breast tumor, plasma and peripheral blood leukocytes (PBLs) will be evaluated in 100 breast cancer cases having varying clinical stages and intrinsic subtypes and who had their blood drawn prior to receiving chemotherapy. Plasma and PBLs will be evaluated from age and race-matched controls (n=50). Comparisons of marker methylation in plasma from cases and controls will provide an indication of its specificity for tumor DNA. In addition, we will determine if plasma methylation is correlated with methylation state in the matched tumor, or with characteristics of the cases including stage, node-positivity, age, race or subtype. Finally, analysis of PBL methylation will be examined as a possible source of contamination of plasma samples. We expect the results of this work will facilitate development of improved biomarkers for cancer diagnosis, treatment effects, and prognosis and disease recurrence in breast cancer.