Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Research Institute Children's Cancer Center Hamburg, Hamburg, Germany.
Clin Chem. 2024 Jan 4;70(1):250-260. doi: 10.1093/clinchem/hvad115.
Molecular brain tumor diagnosis is usually dependent on tissue biopsies or resections. This can pose several risks associated with anesthesia or neurosurgery, especially for lesions in the brain stem or other difficult-to-reach anatomical sites. Apart from initial diagnosis, tumor progression, recurrence, or the acquisition of novel genetic alterations can only be proven by re-biopsies.
We employed Nanopore sequencing on cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) and analyzed copy number variations (CNV) and global DNA methylation using a random forest classifier. We sequenced 129 samples with sufficient DNA. These samples came from 99 patients and encompassed 22 entities. Results were compared to clinical diagnosis and molecular analysis of tumor tissue, if available.
110/129 samples were technically successful, and 50 of these contained detectable circulating tumor DNA (ctDNA) by CNV or methylation profiling. ctDNA was detected in samples from patients with progressive disease but also from patients without known residual disease. CNV plots showed diagnostic and prognostic alterations, such as C19MC amplifications in embryonal tumors with multilayered rosettes or Chr.1q gains and Chr.6q losses in posterior fossa group A ependymoma, respectively. Most CNV profiles mirrored the profiles of the respective tumor tissue. DNA methylation allowed exact classification of the tumor in 22/110 cases and led to incorrect classification in 2/110 cases. Only 5/50 samples with detected ctDNA contained tumor cells detectable through microscopy.
Our results suggest that Nanopore sequencing data of cfDNA from CSF samples may be a promising approach for initial brain tumor diagnostics and an important tool for disease monitoring.
分子脑肿瘤诊断通常依赖于组织活检或切除。这可能会带来与麻醉或神经外科相关的一些风险，尤其是对于脑干或其他难以到达的解剖部位的病变。除了初始诊断外，肿瘤进展、复发或获得新的遗传改变只能通过重新活检来证实。
我们使用 Nanopore 测序对来自脑脊液 (CSF) 的游离 DNA (cfDNA) 进行测序，并使用随机森林分类器分析拷贝数变异 (CNV) 和全基因组 DNA 甲基化。我们对 129 个具有足够 DNA 的样本进行了测序。这些样本来自 99 名患者，涵盖了 22 种实体。结果与临床诊断和肿瘤组织的分子分析进行了比较，如果有可用的结果。
129 个样本中有 110 个在技术上是成功的，其中 50 个样本通过 CNV 或甲基化分析检测到了可检测的循环肿瘤 DNA (ctDNA)。ctDNA 不仅在进展性疾病的患者样本中检测到，也在无已知残留疾病的患者样本中检测到。CNV 图谱显示了诊断和预后改变，例如具有多层玫瑰花结的胚胎性肿瘤中的 C19MC 扩增，或在后颅窝 A 组室管膜瘤中分别出现的 Chr.1q 增益和 Chr.6q 缺失。大多数 CNV 图谱反映了相应肿瘤组织的图谱。DNA 甲基化在 22/110 例肿瘤中能够准确分类，并导致 2/110 例错误分类。在 50 个检测到 ctDNA 的样本中，只有 5/50 个样本含有可通过显微镜检测到的肿瘤细胞。
我们的研究结果表明，CSF 样本 cfDNA 的 Nanopore 测序数据可能是一种有前途的初始脑肿瘤诊断方法，也是疾病监测的重要工具。
