Assessment of Circulating Tumor DNA in Cerebrospinal Fluid by Whole Exome Sequencing to Detect Genomic Alterations of Glioblastoma
Glioblastoma (GBM) is the most prevalent and aggressive primary malignant brain tumor in adults, with a dismal prognosis. Despite advances in standard care therapies, including surgical resection, radiotherapy, and chemotherapy, the median survival duration for GBM patients remains only 14.6 months. The diagnosis and classification of GBM have evolved significantly with the incorporation of molecular parameters into the 2016 World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS). These molecular markers not only provide prognostic insights but also serve as predictive biomarkers for therapeutic efficacy, including the extent of surgical resection and sensitivity to chemotherapy. Therefore, early detection of these molecular parameters is crucial for guiding treatment strategies.
Circulating tumor DNA (ctDNA) is tumor-derived fragmented DNA that circulates in body fluids and can reflect the entire tumor genome. For brain tumors, cerebrospinal fluid (CSF) has been identified as a superior source of ctDNA compared to plasma. CSF directly contacts the brain tumor, making it a more representative medium for capturing tumor-specific genomic alterations. Recent studies have demonstrated that ctDNA detected in CSF using targeted deep sequencing can accurately represent the genomic alterations of brain tumors and monitor the evolution of the glioma genome. However, most of these studies have relied on specialized sequencing assays, such as the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), which, although highly effective, are not as accessible or affordable as whole exome sequencing (WES) globally. The feasibility of using WES for ctDNA detection in CSF remains unclear.
This study aimed to determine whether assessment of ctDNA in CSF using WES is a feasible approach to detect genomic alterations in GBM. The study enrolled ten GBM patients who underwent preoperative lumbar puncture as part of their clinical evaluation. CSF samples were collected, and ctDNA was extracted. Tumor tissue samples were also obtained during surgical resection, and genomic DNA was extracted. Both ctDNA from CSF and genomic DNA from tumor tissue were subjected to WES. The identified glioblastoma-associated mutations from ctDNA in CSF and genomic DNA in tumor tissue were compared to assess the feasibility of using WES for ctDNA detection in CSF.
One patient was excluded from the final analysis due to unqualified ctDNA in CSF for exome sequencing, leaving nine patients for the study. The clinicopathological characteristics of these patients were summarized, including age at diagnosis, sex, tumor classification, number of lesions, maximum diameter of lesions, subventricular zone (SVZ) involvement, and molecular markers such as isocitrate dehydrogenase 1 (IDH1) mutation status, O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, 1p/19q chromosomal status, telomerase reverse transcriptase (TERT) promoter mutation status, H3 histone, family 3A (H3F3A) mutation status, and Histone cluster 1, H3b (HIST1H3B) mutation status.
The WES analysis revealed a landscape of genetic alterations in both CSF and tumor tissue samples. The study focused on 27 protein-coding genes that are frequently mutated in GBM, as defined by the Catalogue of Somatic Mutations in Cancer (COSMIC) and The Cancer Genome Atlas (TCGA)-GBM databases. These genes included key players in the receptor tyrosine kinase (RTK)/Ras GTPase/phosphatidylinositol 3-kinase (PI3K) pathway, such as epidermal growth factor receptor (EGFR), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), and fibroblast growth factor receptor 3 (FGFR3), as well as well-known GBM-associated genes like IDH1, ATRX chromatin remodeler (ATRX), and tumor protein P53 (TP53).
The results showed that more glioblastoma-associated mutations tended to be detected in CSF compared to the corresponding tumor tissue samples (3.56 ± 0.75 vs. 2.22 ± 0.32, P = 0.097), although the statistical significance was limited by the small sample size. The average mutation frequencies were similar in CSF and tumor tissue samples (74.1% ± 6.0% vs. 73.8% ± 6.0%, P = 0.924). Notably, the R132H mutation of IDH1 and the G34V mutation of H3F3A, which had been reported in the pathological diagnoses of patients GBM04 and GBM05, respectively, were also detected in ctDNA from CSF by WES. These findings suggest that ctDNA in CSF can effectively capture key molecular alterations in GBM.
The study also observed that patients who had previously received temozolomide (TMZ) chemotherapy or those whose tumors involved the SVZ tended to harbor more mutations in their CSF. For example, patient GBM04, who had received TMZ chemotherapy before CSF collection, exhibited a higher number of mutations in CSF compared to tumor tissue. Similarly, patient GBM08, whose tumor involved the SVZ, also showed a higher mutation burden in CSF. These observations suggest that prior treatment and tumor location may influence the mutation profile detected in CSF ctDNA.
The detection of IDH1 mutations in CSF ctDNA has significant clinical implications. IDH1-mutant GBM is known to be more amenable to surgical resection, and patients with IDH1 mutations have a survival benefit associated with maximal surgical resection. Therefore, preoperative detection of IDH1 mutations in CSF ctDNA could facilitate individualized surgical strategies for GBM patients.
The study also highlighted the limitations of traditional tissue biopsy in capturing the full spectrum of tumor heterogeneity. Tumor heterogeneity within GBM is increasingly recognized, and the detection of more mutations in CSF ctDNA compared to tumor tissue samples suggests that CSF ctDNA may provide a more comprehensive representation of the tumor genome. This is particularly important given the molecular heterogeneity within GBM, which can impact treatment response and prognosis.
In conclusion, this study demonstrates that assessment of ctDNA in CSF using WES is a feasible approach to detect genomic alterations in GBM. The detection of key mutations, such as IDH1 R132H and H3F3A G34V, in CSF ctDNA provides valuable information for guiding treatment strategies. The study also underscores the potential of CSF ctDNA to overcome the limitations of traditional tissue biopsy by capturing a more comprehensive mutation profile. However, the small sample size limits the statistical significance of the findings, and future studies with larger cohorts are needed to confirm these results. Overall, the use of WES for ctDNA detection in CSF represents a promising tool for the molecular characterization of GBM and could play a critical role in the era of precision medicine.
doi.org/10.1097/CM9.0000000000000843
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