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

项目摘要 中枢神经系统（CNS）肿瘤是最致命的恶性肿瘤。确定最佳治疗 对患者的治疗依赖于准确的诊断，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。脑部手术会给病人带来巨大的焦虑， 对神经系统发病率甚至死亡率构成重大风险。此外，精确的纵向监测 对治疗的反应，并区分复发与治疗相关效应（假进展）， 依赖于在放射成像上获得的有限信息作为侵入性获得组织的选择 由于重复手术的发病率，在疾病过程中纵向地进行手术是禁止的。鉴于这些 由于这些局限性，人们对鉴定用于诊断的非侵入性或“液体”生物标志物有很大的兴趣。 中枢神经系统肿瘤大多数研究小组已经研究了血浆或血浆中循环无细胞肿瘤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，寻找单一突变 不允许区分包括在鉴别诊断中的许多不同类型的脑肿瘤 诊断.我们没有关注突变，而是专注于DNA甲基化改变， 丰富和癌症特异性。我们开发、优化并验证了一种称为无细胞的新方法， 甲基化DNA免疫沉淀和测序（cfMeDIP-seq），包括分离和富集 使用甲基化特异性抗体检测肿瘤无细胞DNA的甲基化片段，然后测序。 我们小组发表的数据提供了原理证明，血浆cfMeDIP-seq可以可靠和准确地 诊断全身肿瘤，包括中枢神经系统肿瘤。根据这些数据，我们假设， 通过cfMeDIP-seq对循环DNA的甲基化谱分析，确定了用于治疗癌症的可靠生物标志物。 非侵入性地诊断、预后和监测CNS肿瘤。我们将根据现有的数据 使用cfMeDIP-seq作为一种新技术来建立和验证一种全面的、非侵入性的CNS肿瘤， 分类器（目标1）;建立一个准确的预测模型，用于脑膜瘤的非侵入性诊断（目标 2）;鉴定胶质瘤中对治疗、复发和恶性转化的反应的血浆生物标志物 (Aim 3）;并从全身性脑转移瘤中鉴定脑转移瘤发展的甲基化和血浆生物标志物。 癌症（目标4）。这一建议的成功将真正有助于在该领域形成急需的范式转变 通过建立cfMeDIP-seq作为非侵入性诊断的可靠液体生物标志物， 原发性和转移性CNS肿瘤患者的诊断和监测。

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.