Interrogating malignant gliomas using released tumor DNA in cerebrospinal fluid

项目总结/摘要 在美国，每年约有17，000人被诊断患有恶性神经胶质瘤， 这些病人中的绝大多数将死于他们的疾病。由于缺乏临床可用的 CNS恶性肿瘤的生物标志物，在这些患者中进行疾病监测的常规方法是 放射学的。不幸的是，MRI和CT扫描检测到的解剖学变化往往是非特异性的和滞后的 疾病进展或消退的背后此外，很难区分治疗 影响和肿瘤生长的因素。因此，患者必须接受额外的手术， 组织诊断或不适当地等待放射学检查结果随着疾病进展而改变 有增无减因此，在神经系统肿瘤中，对更敏感和更特异的肿瘤生物标志物的需求令人难以置信。 肿瘤学 我们和其他人已经表明，大多数癌症都将肿瘤衍生DNA的无细胞分子脱落到肿瘤细胞中。 这些分子可以量化为疾病负担的量度。脑肿瘤是 这是一个例外，很少有可检测到的DNA进入血液。但我们 具有挑衅性的数据表明，恶性胶质瘤脱落的肿瘤衍生DNA的细胞游离分子进入 脑脊液（CSF-tDNA）。CSF-tDNA可以通过以下方式与来源于正常细胞的DNA区分开： 存在定义体细胞突变的疾病。CSF-tDNA的水平可以使用灵敏的数字化技术来定量。 基于测序的测定，例如“Safe-SeqS”。在目标1中，我们将使用Safe-SeqS来定量CSF-tDNA水平。 纵向脊髓液样本来自20例恶性胶质瘤患者。我们将决定如何 CSF-tDNA水平与通过临床、放射学和病理学测量的疾病状态密切相关 调查结果。如果成功，这种方法将准确评估肿瘤反应，并确定这些患者在 复发风险最高，从而使临床医生能够改变治疗方案。 也有新兴的数据表明，所有的癌症，包括恶性胶质瘤，随着时间的推移， 对各种选择压力的反应，包括治疗。这些基因变化具有重要的临床意义。 影响，但目前还没有能够提供见解的微创临床测定 神经胶质瘤基因型。因此，在目标2中，我们将直接对CSF进行全外显子组测序。 并与来自匹配的肿瘤/正常样品的外显子组测序结果进行比较。这将使我们能够 了解通过直接分析CSF可以检测到肿瘤基因型的比例。为了 为了确定CSF中的背景突变率，在目标3中，我们将直接对50 没有任何癌症史的人。在这项资助完成后，我们希望验证CSF-tDNA作为一种 恶性神经胶质瘤个体的候选生物标志物，并为大规模临床试验奠定了基础， 可用于证明临床实用性。

PROJECT SUMMARY/ABSTRACT Approximately 17,000 individuals each year are diagnosed with a malignant glioma in the United States and the vast majority of these patients will succumb to their disease. Given the lack of clinically available biomarkers for CNS malignancies, the conventional method for disease monitoring in these patients is radiographic. Unfortunately, anatomic changes detected by MRI and CT scans are often non-specific and lag behind progressing or regressing disease. Moreover, it can be difficult to discriminate between treatment effect and cancer growth with imaging alone. Patients must, therefore, have additional surgeries for definitive tissue diagnosis or inappropriately wait for radiographic findings to change as their disease progresses unabated. As a result, there is an incredible need for more sensitive and specific tumor biomarkers in neuro- oncology. We and others have shown that most cancers shed cell free molecules of tumor derived DNA into the circulation and that these molecules can be quantified as a measure of disease burden. Brain tumors are the exception to the rule and rarely shed detectable levels DNA into the bloodstream. However, we have provocative data to suggest that malignant gliomas shed cell free molecules of tumor derived DNA into the cerebrospinal fluid (CSF-tDNA). CSF-tDNA can be distinguished from DNA derived from normal cells by the presence of disease defining somatic mutations. Levels of CSF-tDNA can be quantified using sensitive digital sequencing based assays, such “Safe-SeqS”. In Aim 1, we will use Safe-SeqS to quantify CSF-tDNA levels in longitudinal spinal fluid samples derived from 20 patients with malignant gliomas. We will determine how closely CSF-tDNA levels correlate with disease status as measured by clinical, radiographic and pathological findings. If successful, this approach will accurately assess tumor response and identify those patients at highest risk for recurrence, thus enabling the clinician to alter treatment regimens. There are also burgeoning data to suggest that all cancers, including malignant gliomas, evolve over time in response to various selection pressures, including treatment. These genetic changes have important clinical implications but currently there are no minimally invasive clinical assays that are capable of providing insights into the glioma genotype. As a result, in Aim 2, we will perform whole exome sequencing directly on the CSF and compare to exomic sequencing results from matched tumor/normal samples. This will allow us to understand what fraction of tumor genotype can be detected by analyzing the CSF directly. In order to determine the background mutation rate in CSF, in Aim 3 we will perform Safe-SeqS directly on the CSF of 50 individuals without any history of cancer. At the completion of this grant, we hope to validate CSF-tDNA as a candidate biomarker for individuals with malignant glioma and set the stage for large scale clinical trials that can be conducted to demonstrate clinical utility.