PO.CL01.22 · 临床研究
通过液体活检对弥漫性胶质瘤进行分子分型：来自循环肿瘤细胞与ctDNA分析的见解
Molecular profiling of diffuse gliomas via liquid biopsy: Insights from circulating tumor cells and ctDNA analysis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：弥漫性胶质瘤是侵袭性的原发性脑肿瘤，其特征为预后不良及显著的分子异质性。尽管由于血脑屏障的存在，全身播散并不常见，但肿瘤细胞及核酸仍可到达外周循环。液体活检，包括循环肿瘤细胞(CTCs)及循环肿瘤DNA(ctDNA)，为弥漫性胶质瘤的肿瘤特征分析及监测提供了一种微创方法。
方法：在使用U87-MG细胞系进行加标实验验证我们的CTC检测流程后，我们分析了接受弥漫性胶质瘤根治性切除患者的外周血样本。样本于术前采集于Cell-Free DNA BCT®管(Streck公司)中。CTCs使用CytoTrack CT11™(2/C)半自动免疫荧光显微镜平台鉴定，采用GFAP、vimentin及CD45免疫染色，并以DAPI进行核复染。在ctDNA分析中，使用QIAamp Circulating Nucleic Acid Kit(Qiagen)从2 ml血浆中分离cfDNA，并通过数字微滴PCR(Bio-Rad Laboratories公司)评估IDH1 R132H突变。
结果：在首次手术时采集的110份血液样本中，29.1%检测到CTCs。在19份复发患者样本中，CTC阳性率升至54.5%。对67例IDH野生型胶质母细胞瘤且随访至少两年的患者队列进行的Kaplan-Meier生存分析显示，CTC状态与总生存期之间无显著相关性。cfDNA分析在14%的弥漫性胶质瘤患者中鉴定出IDH1 R132H突变，包括肿瘤指导下阳性及阴性状态的病例。
结论：我们的结果表明，CTCs与ctDNA均可在弥漫性胶质瘤患者中可靠地检测到，强调了液体活检补充传统基于组织的诊断的潜力。在血浆中检测到IDH1 R132H突变——即使在肿瘤指导下阴性的病例中——可能提示肿瘤内异质性、区室化肿瘤生物学或采样局限性。缺乏生存相关性可能反映了GBM总体较短的预期寿命和/或CNS外受限的肿瘤增殖。患者招募及数据收集仍在进行中。致谢：本研究由欧盟-下一代欧盟(LX22NPO5102)、奥洛穆茨帕拉茨基大学(IGA LF 2025_006)及SALVAGE-CZ.02.01.01/00/22_008/0004644资助。
查看英文原文 English abstract
Introduction: Diffuse gliomas are the aggressive primary brain tumor, characterized by poor prognosis and pronounced molecular heterogeneity. Although systemic spread is uncommon due to the blood-brain barrier, tumor cells and nucleic acids can still reach the peripheral circulation. Liquid biopsy, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides a minimally invasive approach for tumor characterization and monitoring in diffuse gliomas.
Methods: After validating our CTC detection workflow using spiking experiments with the U87-MG cell line, we analyzed peripheral blood samples from patients undergoing curative resection for diffuse gliomas. Samples were collected prior to surgery in Cell-Free DNA BCT® tubes (Streck, Inc.). CTCs were identified using the CytoTrack CT11™ (2/C) semi-automated immunofluorescence microscopy platform, with immunostaining for GFAP, vimentin, and CD45, and DAPI nuclear counterstaining. For ctDNA analysis, cfDNA was isolated from 2 ml of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen), and the IDH1 R132H mutation was assessed by digital droplet PCR (Bio-Rad Laboratories, Inc.).
Results: CTCs were detected in 29.1% of 110 blood samples collected from patients at the time of their first surgery. Among 19 samples from relapsing patients, CTC positivity increased to 54.5%. Kaplan-Meier survival analysis in a cohort of 67 patients with IDH wildtype glioblastoma and at least two years of follow-up revealed no significant association between CTC status and overall survival.Analysis of cfDNA identified IDH1 R132H mutations in 14% of patients with diffuse gliomas, including cases with both tumor-informed positive and negative status.
Conclusion: Our results demonstrate that both CTCs and ctDNA can be reliably detected in patients with diffuse gliomas, underscoring the potential of liquid biopsy to complement traditional tissue-based diagnostics. Detection of IDH1 R132H mutations in plasma- even in tumor-informed negative cases-may indicate intratumoral heterogeneity, compartmentalized tumor biology, or sampling limitations. The lack of survival association may reflect the overall short life expectancy in GBM and/or restricted tumor proliferation outside the CNS. Patient recruitment and data collection remain ongoing. Acknowledgment: This study was supported by European Union - Next Generation EU (LX22NPO5102), Palacky University Olomouc (IGA LF 2025_006), and SALVAGE - CZ.02.01.01/00/22_008/0004644.
利益披露 Disclosure
J. Srovnal, None..
P. Stejskal, None..
O. Kalita, None..
M. Slachta, None..
L. Hrabalek, None..
A. Rehulkova, None..
M. Vidlarova, None..
M. Hajduch, None.