靶向深度测序循环肿瘤DNA在胶质母细胞瘤复发及疗效监测中的作用

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain malignancy with recurrence and poor prognosis. Intratumor heterogeneity is likely the key to understand treatment failure. Genomics in single tissue biopsy may underestimate the whole tumor genomics information. Circulating tumor DNA (ctDNA) is single or double strand DNA released by tumor cell in circulatory system. Plasma ctDNA possessed many cancer-associated molecular characteristics, is a specific tumor biomarker. The Blood-Brain Barrier is disturbed in glioblastoma multiforme, ctDNA in cerebrospinal fluid derived can flow to plasma. Half-life of CtDNA in plasma is very short, often less than 2 hours. ctDNA could reflect tumor dynamics and evolution. We sereially collect plasma ctDNA and detect ctDNA concentration before and after surgery, before and after radio-and chemo-therapy and every reexamination after treatment, to explore the assocaition between ctDNA concentration and tumor size, clinical stages, recurrence and prognosis. We performed deep sequencing on plasma ctDNA before surgery, after radio-and chemo-therapy and recurrence to characterize changes of mutation spectrum and mutation frequency, to select radio-and chemo-therapy sensitive genes for GBM, as well as recurrence related ctDNA driver gene mutation, to explore the relationship between ctDNA driver gene mutation and GBM prognosis, provide chemotherapy target gene for GBM precise treatment. We also optimize and validate target gene panel to provide target gene for GBM screen in clinic.

胶质母细胞瘤（GBM）易复发、预后差, 肿瘤内部异质性是复发和治疗耐药性的主要原因。单个活检组织样本的基因组结果可能低估了整个肿瘤基因组学信息。ctDNA携带有与原发肿瘤组织一致的分子遗传学改变，是一种特征性的肿瘤生物标志物。GBM血脑屏障功能紊乱或缺失，脑脊液ctDNA可以通过紊乱的血脑屏障循环到血液中。血浆中ctDNA半衰期很短，一般小于两小时，能清晰的反映肿瘤的实时动态及演化。本研究连续收集GBM患者手术前后、放化疗前后及每次复查时血浆ctDNA，探索GBM患者不同时间点血浆ctDNA含量与肿瘤大小、临床分期、复发及预后的关系；深度测序术前、放化疗后及复发时血浆ctDNA突变谱及突变频率的改变，探索ctDNA基因突变与GBM预后的关系；筛选放化疗敏感基因及复发相关基因突变，为GBM精准治疗提供药物靶点。优化并验证靶向测序基因panel，为临床动态监测疗效提供监测基因panel。

研究背景：探索临床病理资料、基因突变、甲基化、LncRNA和mRNA与胶质母细胞瘤复发及预后的关系。cfDNA浓度、基因突变及其动态变化与胶质母细胞瘤复发和预后的关系。.研究方法：应用前瞻性队列研究靶向深度测序不同时间点ctDNA基因突变谱，筛选胶质母细胞瘤复发相关基因突变谱，探索相关基因突变与胶质母细胞瘤复发及预后的关系，探索ctDNA含量与肿瘤大小、临床分期、复发及预后的关系。通过LncRNA测序和RRBS甲基化测序筛选胶质母细胞瘤复发及预后相关的标志物。.研究结果：低级别胶质瘤预后的影响因素包括：性别、年龄、语言障碍、术前KPS评分、是否对抗生素过敏，肿瘤位置，术前外周血的淋巴细胞、中性细胞和嗜酸性粒细胞计数，术前外周血NLR和MLR、化疗和放疗。高级别胶质瘤预后影响因素包括：性别、BMI、是否对抗生素过敏，术前外周血NLR、是否化疗、是否放疗、是否同时接受放化疗。高水平术前外周血NLR是低级别胶质瘤和高级别胶质瘤的预后的独立危险因素。术前外周血嗜酸性粒细胞计数和MLR是低级别胶质瘤预后的独立影响因素。对抗生素过敏是高级别胶质瘤得到独立保护因素。WES测序数据分析发现基因突变与胶质瘤预后无关，而NUTM1-PAIP2B融合基因与胶质瘤预后有关。cfDNA浓度中位数为0.709，cfDNA浓度与肿瘤大小、病理分期及复发、预后均无统计学意义。手术后血浆cfDNA浓度明显高于手术前，并持续很长一段时间，放化疗并没有降低cfDNA浓度。脑脊液中的cfDNA浓度明显高于血浆。LncRNA测序数据联合CGGA数据库分析发现26个mRNA表达水平与胶质母细胞瘤预后的关系有统计学意义，其中CTC-297N7.1、LCE1E和RP11-106M7.1基因的表达与胶质母细胞瘤较好的预后有关，其余基因的表达与胶质母细胞瘤较差的预后有关。RRBS甲基化测序联合TCGA和GEO数据库分析发现甲基化驱动基因PIPOX和PCDHB4基因高甲基化与胶质母细胞瘤预后的关系有统计学意义，PCDHB4高低表达水平与T辅助细胞和巨噬细胞M0浸润细胞、NK细胞和单核浸润细胞具有明显的差异。具有高浸润B细胞和CD4T细胞的胶质母细胞瘤患者PCDHB4高甲基化具有良好的生存优势。