Background: High grade gliomas (HGG), including diffuse midline gliomas (DMG), are the most malignant tumors of the central nervous system (CNS); one in five identified CNS tumors in children is DMG. The treatment of DMG represents a «dark page» in pediatric oncology, since over many decades of studying the disease, no therapeutic options have been found that would improve overall and disease-free survival. In modern neurooncology, the importance of molecular genetic examination is steadily increasing, and thus more and more patients go through the morphological verification procedures, but, unfortunately, their results are still only prognostic, which makes it necessary to search for alternative methods of getting material for more comprehensive assessment of the mutation status in these patients. We believe that liquid biopsy may help to solve this problem. 

Aim: To demonstrate the mutational heterogeneity of HGG and DMG, in children, detected by cerebrospinal fluid analysis using droplet digital PCR (ddPCR).

Methods: To assess the presence of somatic mutations in cerebrospinal fluid, a cohort of 96 patients with HGG was formed; 57 of them (59.37%) were diagnosed with DMG. Prior to radiotherapy or chemoradiotherapy, all the patients underwent procedures of sampling cerebrospinal fluid for subsequent quantification of mutant and wild type free circulating DNA (fcDNA) by ddPCR method. 

Results: The analysis of cerebrospinal fluid of the entire group showed that in 78 cases (81.25%), mutant forms of the studied genes (BRAF [V600E], IDH1 [R132H], H3F3A [K27M]) were detected in the cerebrospinal fluid material; the mutant form of the H3F3A [K27M] gene was found with the highest frequency (34 cases [35.42%]). Also, using the ddPCR method, it was determined that in the cerebrospinal fluid of 13 patients (13.5% of cases) there were various combinations of mutations in the studied genes: several variants of double mutations in 12 cases and a triple combination of mutations in 1 case. The most common mutation in the combined forms was H3F3A (K27M), which was detected in 8 cases of multiple mutations (66.70%), and the most common combination was IDH1 (R132H)/H3F3A (K27M), detected in 5 cases (41.67%) of the multiple mutations subgroup. 

Conclusion: Currently, the determination of the mutation status of the IDH1 and H3F3A genes is necessary for the final verification of histological variants of gliomas in adult patients, and the detection of the K27M mutation in the H3F3A gene has become mandatory to diagnose the mutant type of pediatric diffuse midline gliomas characterized by an extremely aggressive clinical course and an unfavorable prognosis. This study shows the potential of liquid biopsy in the detection of the HGG mutational heterogeneity. The presented results confirm the previously published data by demonstrating the high prevalence of those mutational changes. However, the clinical significance of the identified combinations of mutations requires further research.