Apostle 技术在脑脊液科研、临床领域的应用案例

72. Liquid Biopsy for Evaluating Mutations and Chromosomal Aberrations in Cerebrospinal Fluid from Patients with Primary or Metastatic Central Tumors. Ahmad Charifa, Sally Agersborg,  Arash Mohtashamian, Andrew Ip, Andre Goy, Maher Albitar, The Journal of Liquid Biopsy (2024), https://doi.org/10.1016/j.jlb.2024.100281

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Background

Cytopathology analysis of cerebrospinal fluid (CSF) is limited in detecting tumors in patients with suspected primary or metastatic central nervous system (CNS) malignancy. We investigated the use of CSF liquid biopsy (LBx) to detect neoplastic processes in the CNS. Methods: Cell-free DNA (cfDNA) from the CSF of patients with suspected metastatic (N=106) or primary CNS (N=23) tumors was deep sequenced using a 302-gene panel.

Results

Four samples (3%) (3 metastatic and 1 primary) failed sequencing quality control criteria. Metastatic tumor was confirmed in 84 (82%) of the 103 patients suspected of metastatic tumor. Primary CNS tumor was confirmed in 11 of 22 (50%) patients suspected of CNS tumor. Chromosomal abnormalities were detected in 55 samples (54%). Germline mutations were detected in 23 (22%) patients with metastatic tumors and in 1 (5%) with a primary CNS tumor. Of the 29 patients with metastatic breast cancers, 2 (7%) had mutations in ESR1 and 9 (31%) had mutations in PIK3CA. Of the 21 patients with metastatic lung cancer, 9 (43%) had EGFR mutations and 5 (24%) had KRAS mutations. Upon comparing CSF LBx with peripheral blood LBx in 14 patients, 13 (93%) showed only CHIP and one patient showed CNS primary tumor mutation. Serial samples from 14 patients demonstrate that CSF LBx can be used for monitoring therapy efficacy.

Conclusions

LBx using CSF is clinically reliable and provides informative results in a substantial proportion of patients with metastatic CNS tumors and to a lesser degree in patients with primary CNS tumors.

(Materials and Methods section)

cfDNA and cfRNA Extraction

We used the Apostle MiniMax High-Efficiency total nucleic acid isolation Kit (Beckman Coulter, Brea, CA, USA) and followed the protocol recommended by the manufacturer as previously described in detail. After extraction, half of the cell-free total nucleic acid was treated with DNase to obtain cfRNA, and the other half was used for cfDNA analysis.

61. Sequencing of cerebrospinal fluid cell-free DNA facilitated early differential diagnosis of intramedullary spinal cord tumors. Chai, et al. npj Precision Oncology volume 8, Article number: 43 (2024)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Pre-surgery differential diagnosis is valuable for personalized treatment planning in intramedullary spinal cord tumors. This study assessed the performance of sequencing cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) for differential diagnosis of these tumors. Prospectively enrolling 45 patients with intramedullary spinal cord lesions, including diffuse midline glioma (DMG), H3K27-altered (14/45), glioblastoma (1/45), H3-wildtype-astrocytoma (10/45), ependymoma (11/45), and other lesions (9/45), CSF samples were collected via lumbar puncture (41/45), intraoperative extraction (3/45), and Ommaya reservoir (1/45). Then, these samples underwent targeted sequencing along with paired tissue DNA. DMG, H3K27-altered patients exhibited a higher ctDNA positivity (85.7%, 12/14) compared to patients with H3-wildtype-astrocytoma (0/8, P = 0.0003), ependymoma (2/10, P = 0.003), and glioneuronal tumor (0/3, P = 0.009). The histological-grade-IV (P = 0.0027), Ki-67 index ≥10% (P = 0.014), and tumor reaching spinal cord surface (P = 0.012) are also associated with higher ctDNA positivity. Interestingly, for patients with TERT promoter mutant tumors, TERT mutation was detectable in the CSF cfDNA of one DMG case, but not other five cases with histological-grade-II tumors. Shared copy number variants were exclusively observed in DMG, H3K27-altered, and showed a strong correlation (Correlation = 0.95) between CSF and tissue. Finally, H3K27M mutations in CSF exhibited high diagnostic efficiency for DMG, H3K27-altered (Sensitivity = 85.7%, Specificity = 100.0%, AUC = 0.929). Notably, H3K27M was detectable in CSF from patients with recurrent tumors, making it easily applicable for postoperative monitoring. In conclusion, the molecular profile from ctDNA released into CSF of malignant tumors was more frequently detected compared to relatively benign ones. Sequencing of ctDNA in CSF exhibited high efficiency for the differential diagnosis of DMG, H3K27-altered.

(Methods section)  Circulating cell-free DNA (cfDNA) isolation from cerebrospinal fluid (CSF)

Then, circulating nucleic acid was extracted from CSF using the Apostle MiniMax High-Efficiency cfDNA Isolation Kit (Apostle, USA) following the manufacturer's instructions. 

54. Concordance analysis of cerebrospinal fluid with the tumor tissue for integrated diagnosis in gliomas based on next-generation sequencing. Wang, et al. Pathology and Oncology Research, 26 September 2023 https://doi.org/10.3389/pore.2023.1611391

(Note: Apostle MiniMax technology is used in this study.) 

Abstract 

Purpose: The driver mutations of gliomas have been identified in cerebrospinal fluid (CSF). Here we compared the concordance between CSF and tumor tissue for integrated diagnosis in gliomas using next-generation sequencing (NGS) to evaluate the feasibility of CSF detection in gliomas.

Patients and methods: 27 paired CSF/tumor tissues of glioma patients were sequenced by a customized gene panel based on NGS. All CSF samples were collected through lumbar puncture before surgery. Integrated diagnosis was made by analysis of histology and tumor DNA molecular pathology according to the 2021 WHO classification of the central nervous system tumors.

Results: A total of 24 patients had detectable circulating tumor DNA (ctDNA) and 22 had at least one somatic mutation or chromosome alteration in CSF. The ctDNA levels varied significantly across different ages, Ki-67 index, magnetic resonance imaging signal and glioma subtypes (p < 0.05). The concordance between integrated ctDNA diagnosis and the final diagnosis came up to 91.6% (Kappa, 0.800). We reclassified the clinical diagnosis of 3 patients based on the results of CSF ctDNA sequencing, and 4 patients were reassessed depending on tumor DNA. Interestingly, a rare IDH1 R132C was identified in CSF ctDNA, but not in the corresponding tumor sample.

Conclusion: This study demonstrates a high concordance between integrated ctDNA diagnosis and the final diagnosis of gliomas, highlighting the practicability of NGS based detection of mutations of CSF in assisting integrated diagnosis of gliomas, especially glioblastoma.

(Methods section)  DNA extraction and quantification

Cell-free DNA (cfDNA) was extracted using an Apostle MiniMax High Efficiency cfDNA Isolation Kit (APOSTLE) according to the manufacturer’s instructions.