Molecular diagnosis of diffuse glioma using a chip-based digital PCR system to analyze IDH, TERT, and H3 mutations in the cerebrospinal fluid

Abstract

Purpose Conventional genetic analyzers require surgically obtained tumor tissues to confirm the molecular diagnosis of diffuse glioma. Recent technical breakthroughs have enabled increased utilization of cell-free tumor DNA (ctDNA) in body fluids as a reliable resource for molecular diagnosis in various cancers. Here, we tested the application of a chip-based digital PCR system for the less invasive diagnosis (i.e., liquid biopsy) of diffuse glioma using the cerebrospinal fluid (CSF). Methods CSF samples from 34 patients with diffuse glioma were collected from the surgical field during craniotomy. Preoperative lumbar CSF collection was also performed in 11 patients. Extracted ctDNA was used to analyze diagnostic point mutations in IDH1 R132H, TERT promoter (C228T and C250T), and H3F3A (K27M) on the QuantStudio® 3D Digital PCR System. These results were compared with their corresponding tumor DNA samples. Results We detected either of the diagnostic mutations in tumor DNA samples from 28 of 34 patients. Among them, we achieved precise molecular diagnoses using intracranial CSF in 20 (71%). Univariate analyses revealed that the World Health Organization (WHO) grade (p = 0.0034), radiographic enhancement (p = 0.0006), and Mib1 index (p = 0.01) were significant predictors of precise CSF-based molecular diagnosis. We precisely diagnosed WHO grade III or IV diffuse gliomas using lumbar CSF obtained from 6 (87%) of 7 patients with tumors harboring any mutation. Conclusion We established a novel, non-invasive molecular diagnostic method using a chip-based digital PCR system targeting ctDNA derived from CSF with high sensitivity and specificity, especially for high-grade gliomas.

Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid

“…Mean cfDNA concentrations for intracranial CSF samples are significantly higher than for preoperative lumbar samples (e.g., 73.33 cf. 7.31 pg/μl, respectively [ 22 ]), hence the higher efficacy of genotyping for CSF samples collected intraoperatively .…”

CD206 Expression in Induced Microglia-Like Cells From Peripheral Blood as a Surrogate Biomarker for the Specific Immune Microenvironment of Neurosurgical Diseases Including Glioma

“…Therefore, the development of a tumor-specific marker for GBM would greatly facilitate the presurgical confirmation of diagnosis. There have been several recent attempts of molecular diagnosis of gliomas using cerebrospinal fluid ( 36 , 37 ); however, those techniques have not achieved the detection of glioma-specific mutations in peripheral blood cells. Recently, several reports on serum microRNAs can distinguish patients with gliomas from healthy controls with high sensitivity and specificity ( 38 , 39 ).…”

Pediatric Glioma: An Update of Diagnosis, Biology, and Treatment

“…Liquid biopsy is also one of the current topics as a less invasive method using body fluid, such as plasma or cerebrospinal fluid (CSF), for molecular diagnosis. Recent efforts including ours have developed digital PCR-based liquid biopsy targeting cell-free tumor DNA in CSF for detecting glioma-specific diagnostic mutations [15]. NGS-based liquid biopsy has been attempted.…”