Cell-free DNA (cfDNA) in body fluids is invaluable for cancer diagnostics. Despite the impressive potential of liquid biopsies for the diagnostics of central nervous system (CNS) tumors, a number of challenges prevent introducing this approach into routine laboratory practice. In this study, we adopt a protocol for sensitive detection of the H3 K27M somatic variant in cerebrospinal fluid (CSF) by using digital polymerase chain reaction (dPCR). Optimization of the protocol was carried out stepwise, including preamplification of the H3 target region and adjustment of dPCR conditions. The optimized protocol allowed detection of the mutant allele starting from DNA quantities as low as 9 picograms. Analytical specificity was tested using a representative group of tumor tissue samples with known H3 K27M status, and no false-positive cases were detected. The protocol was applied to a series of CSF samples collected from patients with CNS tumors (n = 18) using two alternative dPCR platforms, QX200 Droplet Digital PCR system (Bio-Rad) and QIAcuity Digital PCR System (Qiagen). In three out of four CSF specimens collected from patients with H3 K27M-positive diffuse midline glioma, both platforms allowed detection of the mutant allele. The use of ventricular access for CSF collection appears preferential, as lumbar CSF samples may produce ambiguous results. All CSF samples collected from patients with H3 wild-type tumors were qualified as H3 K27M-negative. High agreement of the quantitative data obtained with the two platforms demonstrates universality of the approach.

Ependymomas are among the most enigmatic tumors of the central nervous system, posing enormous challenges for pathologists and clinicians. Despite the efforts made, the treatment options are still limited to surgical resection and radiation therapy, while none of conventional chemotherapies is beneficial. While being histologically similar, ependymomas show considerable clinical and molecular diversity. Their histopathological evaluation alone is not sufficient for reliable diagnostics, prognosis, and choice of treatment strategy. The importance of integrated diagnosis for ependymomas is underscored in the recommendations of Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy. These updated recommendations were adopted and implemented by WHO experts. This minireview highlights recent advances in comprehensive molecular-genetic characterization of ependymomas. Strong emphasis is made on the use of molecular approaches for verification and specification of histological diagnoses, as well as identification of prognostic markers for ependymomas in children.

Infant hemispheric high-grade gliomas (iHGGs) constitute a biologically and clinically distinct subgroup of pediatric HGGs. One defining hallmark of iHGG is rearrangement of receptor tyrosine kinase either ALK, ROS1, NTRK1,2,3, or MET. The ROS1-positive iHGGs are an exceptionally rare tumor subtype with a relatively poor prognosis.¹ The most typical ROS1 fusion partner for such tumors is GOPC.² Here we describe two extremely rare cases of iHGG with ROS1 fused with the Zinc Finger CCHC-Type Containing 8 (ZCCHC8) gene. Although these patients had major differences in clinical presentation, MRI characteristics, and histopathology, both children were successfully treated with the NTRK/ROS1/ALK inhibitor entrectinib. To date, only two ZCCHC8:ROS1-positive gliomas were described; one of the patients received targeted therapy.^3,4

Choroid plexus carcinomas (CPCs) are rare aggressive pediatric tumors of the brain with no treatment standards. Genetic profiling of CPCs is often confined to possible association with Li–Fraumeni syndrome, though only about a half of CPCs develop from syndromic predispositions. Whole-chromosome gains and losses typical of CPCs reflect genomic instability of these tumors, but only partially explain the aggressive clinical course. This retrospective study enrolled 25 pediatric patients with CPC, receiving treatment between January 2009 and June 2022. Molecular genetic testing was performed for 20 cases with available tumor tissue and encompassed mutational status, chromosomal aberrations and gene expression profiles. We analyzed several factors presumably influencing the outcomes, including molecular profiles and clinical parameters. The median follow-up constituted 5.2 years (absolute range 2.8–12.6 years). All studied CPCs had smooth mutational profiles with the only recurrent event being TP53 variants, either germline or somatic, encountered in 13 cases. Unbalanced whole-chromosome aberrations, notably multiple monosomies, were highly typical. In seven tumors, chromosome losses were combined with complex genomic rearrangements: segmental gains and losses or signs of chromothripsis. This phenomenon was associated with extremely low 5-year survival: 20.0±17.9% vs. 85.7±13.2%; p=0.009. Transcriptomically, the cohort split into two polar clusters Ped_CPC1 and Ped_CPC2 differing by survival: 31.3±17.8% vs. 100%; p=0.012. CPCs split into at least two molecular subtypes distinguished both genomically and transcriptomically. Clusterization of the tumors into Ped_CPC1 and Ped_CPC2 significantly correlates with survival. The distinction may prove relevant in clinical trials for dedicated and patient-oriented optimization of clinical protocols for these rare tumors.

PURPOSE: Midline low-grade gliomas (mLGGs) of early childhood have a poorer prognosis compared with tumors of other localizations and in older patients. LGGs are associated with aberrant activation of RAS-RAF-MEK pathway, and pharmacological inhibition of the pathway has therapeutic promise. The aim of this study was clinical and molecular characterization of infantile mLGGs, with emphasis on the efficacy of targeted kinase inhibition. PATIENTS AND METHODS: This study enrolled 40 patients with mLGG age <3 years. The majority of the patients (30/40) received first-line chemotherapy (CT) as per International Society of Paediatric Oncology LGG 2004 guidelines. In all patients, molecular genetic investigation of tumor tissue by polymerase chain reaction and RNA sequencing was performed. The median follow-up was 3.5 years. RESULTS: First-line CT failed in 24 of 30 recipients. The identified molecular profiles included KIAA1549::BRAF fusions in 26 patients, BRAF V600E in six patients, FGFR1::TACC1 fusions in two patients, and rare fusion transcripts in four patients. At disease progression, targeted therapy (TT) was initiated in 27 patients (22 patients received trametinib) on the basis of molecular findings. TT was administered for a median of 16 months, with partial response achieved in 12 of 26 (46%) patients in which response was evaluated. Severe adverse events were detected only on trametinib monotherapy: acute damage of GI or urinary mucosa complicated by hemorrhage and development of transfusion-dependent anemia in four patients and grade 3 skin toxicity in three patients. CONCLUSION: mLGGs of early childhood are often aggressive tumors, resistant to CT, and frequently require alternative treatment. The majority of patients harbor druggable molecular targets and respond to molecular TT.