Establishing the clinical utility of cell-free tumor DNA methylation profiling as a reliable liquid biopsy approach in brain tumors

项目总结 中枢神经系统肿瘤是最致命的恶性肿瘤。确定最佳治疗方案 依赖于准确的诊断，目前只能通过组织病理学分析来实现 通过侵入性脑外科手术获得的组织。大脑中的手术会给患者带来巨大的焦虑和 对神经系统的发病率甚至死亡率构成极大的风险。此外，精准的纵向监测 对治疗的反应，并区分复发与治疗相关的影响(假性进展)， 依靠在放射成像上获得的有限信息作为侵入性获取组织的选项 由于重复手术的发病率，纵向上超过病程是令人望而却步的。考虑到这些 局限性，人们对识别用于诊断的非侵入性或“液体”生物标志物一直很感兴趣 中枢神经系统肿瘤。大多数研究小组都观察了血浆或血浆中循环中无细胞肿瘤DNA的突变 脑脊液。不幸的是，中枢神经系统肿瘤通常不会释放足够的DNA进入体循环。 这样的方法在预期的临床基础上是可靠的。此外，搜索单个突变 不允许区分包括在差异中的许多不同类型的脑瘤 诊断。我们关注的不是突变，而是高度突变的DNA甲基化 丰富的和癌症特异性的。我们开发、优化和验证了一种名为无细胞的新方法 甲基化DNA免疫沉淀和测序(cfMeDIP-seq)，涉及分离和浓缩 使用甲基化特异性抗体对无细胞肿瘤DNA进行甲基化处理，然后测序。 我们小组公布的数据提供了血浆cfMeDIP-seq可以可靠和准确的原理证明 诊断全身的肿瘤，包括中枢神经系统的肿瘤。根据这些数据，我们假设 通过cfMeDIP-seq对循环DNA进行甲基化分析，确定了可靠的生物标志物 中枢神经系统肿瘤的无创性诊断、预后和监测。我们将建立在现有数据的基础上 CfMeDIP-seq作为建立和验证全面、非侵袭性中枢神经系统肿瘤的新技术 分类器(AIM 1)；建立脑膜瘤无创预测的准确预测模型(AIM 2)；确定胶质瘤对治疗、复发和恶变的反应的血浆生物标志物 (目标3)；并确定系统性脑转移瘤发生的甲基化和血浆生物标记物 癌症(目标4)。这一提议的成功将真正有助于在该领域形成亟需的范式转变 通过将cfMeDIP-seq建立为用于非侵入性诊断的可靠液体生物标志物， 原发和转移性中枢神经系统肿瘤患者的预后和监测。

PROJECT SUMMARY Tumors of the central nervous system (CNS) are the most fatal malignancies. Determining the optimal treatment for patients relies on accurate diagnoses, which at present is only achievable by histopathological analyses of the tissue obtained by invasive brain surgery. Operations in the brain instill tremendous anxiety in patients and pose a significant risk for neurological morbidity and even mortality. In addition, accurate longitudinal monitoring of response to treatment, and distinguishing recurrence from treatment-related effects (pseudoprogression), relies on limited information obtained on radiologic imaging as the option to invasively obtain tissue longitudinally over the course of disease is prohibitive due to the morbidity of repeat procedures. Given these limitations, there has been significant interest in identifying non-invasive or “liquid” biomarkers for the diagnosis of CNS tumors. Most groups have looked at mutations in circulating cell-free tumor DNA in the plasma or cerebrospinal fluid. Unfortunately, CNS tumors usually do not release enough DNA into the systemic circulation for such an approach to be reliable on a prospective clinical basis. Furthermore, searching for single mutations does not allow for the discrimination of the many different types of brain tumors included in a differential diagnosis. Instead of focusing on mutations, we focused on DNA methylation alterations that are highly abundant and cancer specific. We developed, optimized, and validated a novel approach termed cell-free methylated DNA immuno-precipitation and sequencing (cfMeDIP-seq) that involves isolating and enriching for methylated fragments of tumor cell-free DNA using a methylation-specific antibody and then sequencing. Published data from our group provides proof-of-principle that plasma cfMeDIP-seq can reliably and accurately diagnose tumors throughout the body, including those in the CNS. Based on these data, we hypothesize that methylation profiling of circulating DNA, via cfMeDIP-seq, identifies reliable biomarkers for the diagnosis, prognosis, and monitoring of CNS tumors non-invasively. We will build upon our existing data to use cfMeDIP-seq as a novel technique to establish and validate a comprehensive, non-invasive CNS tumor classifier (Aim 1); build an accurate predictive model for the non-invasive prognostication of meningiomas (Aim 2); identify plasma biomarkers of response to treatment, recurrence, and malignant transformation in gliomas (Aim 3); and identify methylation and plasma biomarkers of brain metastases development from systemic cancers (Aim 4). Success in this proposal will genuinely help shape a much-needed paradigm-shift in the field of neuro-oncology by establishing cfMeDIP-seq as a reliable liquid biomarker for the non-invasive diagnosis, prognostication, and monitoring of patients with primary and metastatic CNS tumors.