Interrogating malignant gliomas using released tumor DNA in cerebrospinal fluid

项目摘要/摘要 在美国，每年约有17,000人被诊断出患有恶性胶质瘤， 这些患者中的绝大多数会死于他们的疾病。鉴于缺乏临床上可用的 作为中枢神经系统恶性肿瘤的生物标志物，对这些患者进行疾病监测的传统方法是 放射照片。不幸的是，MRI和CT扫描检测到的解剖变化通常是非特异性的和滞后的 疾病进展或退化的背后。此外，很难区分不同的治疗方法 仅用成像就能有效地控制肿瘤的生长。因此，患者必须进行额外的手术才能确诊 组织诊断或不适当地等待放射检查结果随着疾病的进展而改变 有增无减。因此，在神经细胞中存在对更敏感和更特异的肿瘤生物标记物的难以置信的需求。 肿瘤学。 我们和其他人已经证明，大多数癌症将肿瘤来源的DNA的细胞自由分子释放到 这些分子可以被量化为疾病负担的一种衡量标准。脑瘤是 这是规则的例外，很少会将可检测到的DNA水平送入血液。然而，我们有 具有挑衅性的数据表明，恶性胶质瘤将肿瘤来源的DNA的细胞自由分子释放到 脑脊液(CSF-tDNA)。Csf-tDNA与来自正常细胞的DNA可以通过 定义体细胞突变的疾病的存在。脑脊液tDNA水平可以用灵敏的数字进行量化 基于测序的分析，如“安全序列分析”。在目标1中，我们将使用SAFE-SeqS来量化 20例恶性胶质瘤患者的纵向脊液标本。我们将决定如何 Csf-tDNA水平与临床、放射学和病理学所测量的疾病状态密切相关。 调查结果。如果成功，这种方法将准确地评估肿瘤反应，并在 复发风险最高，从而使临床医生能够改变治疗方案。 也有迅速增长的数据表明，所有的癌症，包括恶性胶质瘤，都会随着时间的推移在 应对各种选择压力，包括治疗。这些基因变化具有重要的临床意义。 暗示，但目前还没有能够提供洞察力的微创临床测试 变成了神经胶质瘤的基因。因此，在目标2中，我们将直接对脑脊液进行完整的外显子组测序 并与来自匹配的肿瘤/正常样本的外周基因组测序结果进行比较。这将使我们能够 了解通过直接分析脑脊液可以检测出肿瘤基因的哪一部分。为了 测定脑脊液中的背景突变率，在目标3中，我们将直接对50例脑脊液进行安全序列分析 没有任何癌症病史的个人。在这笔赠款完成后，我们希望验证csf-tdna作为一种 恶性胶质瘤患者的候选生物标记物，并为大规模临床试验奠定了基础 可以进行，以证明临床实用。

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.