Circulating Cell-Free DNA as a Personalized Biomarker to Diagnose and Monitor Glioblastoma

项目总结/摘要 胶质母细胞瘤（GBM）是最常见和最具侵袭性的成人原发性脑肿瘤。无论 治疗中，中位生存时间小于15个月，因为GBM几乎总是复发。诊断GBM 目前依赖于获取肿瘤组织进行组织学检查，以区分GBM与其他类型的肿瘤。 脑部病变有相似的影像学表现检测GBM复发也具有挑战性， 诸如假进展和放射性坏死的效应模拟复发性疾病。因此，风险相关 诊断性侵入性程序仍然是GBM患者护理的关键方面， 神经系统缺陷可能会缩短已经很短的生存时间。因此，一种非侵入性的诊断方法 GBM和确定真正的复发有可能直接影响整个疾病的患者护理 当然了在程序性细胞死亡过程中从细胞中释放的DNA，称为无细胞DNA，是一种新兴的 用于诊断人类癌症和监测对治疗的反应的生物标志物。肿瘤衍生细胞的检测- 血浆中的游离DNA，也称为循环肿瘤DNA（ctDNA），以前在很大程度上是不成功的 在患有GBM的人中，由于不能在正常的GBM的丰富背景中找到ctDNA， 出现无细胞DNA巨大的肿瘤内和肿瘤间GBM遗传异质性阻碍了研究 对于无细胞DNA中的特定肿瘤变体，而GBM转移到中枢神经系统以外的罕见性， 系统已经大大限制了ctDNA存在的量。然而，我们发现的ctDNA在 使用人GBM干细胞样细胞的异种移植脑肿瘤模型强烈支持检测的可行性， GBM衍生的ctDNA。此外，我们先前在动物模型中的成功为 人类翻译-减少或消除与下一代测序（NGS）相关的噪音， 干扰检测非常低频率的变异是必要的，以允许搜索ctDNA， 不依赖于实体瘤变体的先验知识。在这个建议中，我们衰减NGS相关的噪声 并证明在初始诊断时和复发时GBM衍生的ctDNA的无偏检测。 我们还表明，通过减少错误，ctDNA中GBM实体瘤DNA变体的鉴定得到了增强。 与NGS相关，以改善GBM典型的实体瘤遗传异质性的采样。因此，我们认为， 该提案寻求将血浆游离DNA作为生物标志物来检测人类GBM， 与NGS相关的错误，以改善在非常低的等位基因频率下的变异检测。成功 应用ctDNA生物标志物非侵入性评估GBM将直接影响患者护理， 在风险相关的侵入性诊断程序之前或代替风险相关的侵入性诊断程序优化临床管理。

PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is the most common and most aggressive adult primary brain tumor. Regardless of therapy, the median survival time is less than 15 months as GBM nearly always recurs. Diagnosing GBM currently depends on acquiring tumor tissue for histologic examination to differentiate GBM from other types of brain lesions with a similar imaging appearance. Detecting GBM recurrence is also challenging as treatment effects such as pseudoprogression and radiation necrosis mimic recurrent disease. Thus, risk-associated diagnostic invasive procedures remain a critical aspect of GBM patient care even though surgically acquired neurologic deficits may reduce an already short survival time. Therefore, a non-invasive approach to diagnose GBM and identify true recurrence has the potential to directly impact patient care throughout the entire disease course. DNA released from cells during programmed cell death, termed cell-free DNA, is an emerging biomarker for diagnosing human cancers and monitoring response to therapy. Detection of tumor-derived cell- free DNA in plasma, also known as circulating tumor DNA (ctDNA), has previously been largely unsuccessful in humans with GBM due to an inability to find the ctDNA amongst the abundant background of normally occurring cell-free DNA. The large intra- and inter-tumor genetic heterogeneity of GBM has hindered searches for specific tumor variants in cell-free DNA, while the rarity of GBM to metastasize beyond the central nervous system has considerably restricted the quantity of ctDNA present. However, our uncovering of ctDNA in a xenograft brain tumor model using human GBM stem-like cells strongly supports the feasibility of detecting GBM-derived ctDNA in humans. Moreover, our previous success in the animal model provides direction for the human translation – reducing or eliminating noise associated with next-generation sequencing (NGS) that interferes with the detection of very low frequency variants is necessary to allow searches for ctDNA that are not dependent on a priori knowledge of solid tumor variants. In this proposal, we attenuate NGS-related noise and demonstrate the unbiased detection of GBM-derived ctDNA at time of initial diagnosis and at recurrence. We also show that identification of GBM solid tumor DNA variants in ctDNA is enhanced by reducing errors associated with NGS to improve sampling of the solid tumor genetic heterogeneity typical of GBM. Therefore, this proposal seeks to translate plasma cell-free DNA as a biomarker to detect GBM in humans by suppressing errors associated with NGS to improve variant detection at very low allele frequencies. The successful application of ctDNA biomarkers to non-invasively assess GBM will directly affect patient care by enabling the optimization of clinical management prior to or instead of risk-associated invasive diagnostic procedures.