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

项目摘要/摘要 胶质母细胞瘤(GBM)是最常见和最具侵袭性的成人原发脑肿瘤。不管 治疗后，由于GBM几乎总是复发，中位生存期不到15个月。诊断GBM 目前依靠获取肿瘤组织进行组织学检查来区分GBM与其他类型的 脑部病变具有相似的影像表现。检测基底膜复发也是一项具有挑战性的治疗 假性进展和辐射坏死等效应与复发性疾病相似。因此，与风险相关的 诊断性侵入性程序仍然是GBM患者护理的关键方面，即使是通过手术获得的 神经缺陷可能会缩短本已很短的生存时间。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。在细胞程序性死亡过程中从细胞中释放的DNA，被称为无细胞DNA，是一种新兴的 用于诊断人类癌症和监测治疗反应的生物标志物。检测肿瘤来源的细胞- 血浆中的游离dna，也被称为循环肿瘤dna(Ctdna)，以前在很大程度上没有成功。 在患有GBM的人类中，由于无法在正常的丰富背景中找到ctDNA 产生无细胞的DNA。GBM在肿瘤内和肿瘤间的巨大遗传异质性阻碍了研究 对于特定的肿瘤变异的无细胞DNA，而基底膜罕见地转移到中枢神经系统以外 系统在很大程度上限制了存在的ctDNA的数量。然而，我们发现的ctDNA在一个 使用人GBM干细胞的异种移植脑瘤模型有力地支持了检测 人类中的GBM来源的ctDNA。此外，我们之前在动物模型上的成功为我们的 人工翻译-减少或消除与下一代测序(NGS)相关的噪音 干扰极低频率变异的检测是允许搜索ctDNA所必需的 不依赖于对实体肿瘤变体的先验知识。在这个方案中，我们减弱了与NGS相关的噪声 并证明了在初诊时和复发时无偏倚地检测GBM来源的ctDNA。 我们还表明，通过减少错误，ctdna中的gbm实体瘤dna变异体的识别得到了增强。 与NGS相关，以改善对典型的GBM实体瘤遗传异质性的采样。因此， 这项提议寻求将无浆细胞DNA翻译为生物标记物，通过抑制 与NGS相关的错误，以改进在非常低的等位基因频率下的变异检测。成功者 将ctDNA生物标志物应用于无创评估GBM将直接影响患者的护理，因为 在风险相关的侵入性诊断程序之前或替代风险相关侵入性诊断程序之前优化临床管理。

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.