Rapid Intraoperative Genetic Analysis of Adult-type Diffuse Gliomas Using a Microfluidic Real-Time Polymerase Chain Reaction Device.

Background: The 5th edition of the World Health Organization Classification of Tumors of the Central Nervous System introduced a subclassification of tumors based on key molecular markers. In adult-type diffuse gliomas, isocitrate dehydrogenase (IDH) and telomerase reverse transcriptase (TERT) promoter mutations play pivotal roles in the molecular classification. This study developed a rapid genotyping system using GeneSoC®, a real-time polymerase chain reaction (PCR) platform with microfluidic thermal cycling capable of completing 50 cycles of PCR within 20 min.

Methods: To establish optimal analytical conditions, frozen tumor tissues from 67 patients and artificial DNA vectors were analyzed using this system. This system demonstrated a detection limit of at least 5% variant allele frequency for the IDH1 R132H and TERT promoter C228T/C250T mutations. Subsequently, intraoperative testing was performed in 120 cases using this system.

Results: The sensitivity and specificity of IDH1 R132H mutation were 0.985 and 0.982, respectively, whereas those of TERT promoter C228T/C250T mutation were 1.000 and 1.000, respectively. These mutations were detected intraoperatively within approximately 25 min after tumor tissue collection. Furthermore, this assay identified tumor boundaries in an IDH-mutated glioma case, where IDH1 R132H mutations could not be detected.

Conclusions: The GeneSoC®-based rapid genotyping system may be effective not only for intraoperative diagnosis of diffuse glioma but also for detecting tumor boundaries.