Real-world longitudinal data documenting the diagnostic impact of DNA methylation array (MA) profiling in pediatric ependymoma remain sparse. We report a single-center retrospective analysis evaluating MA-driven reclassification, molecular subgroup distribution, and long-term survival outcomes in a national pediatric referral cohort. Sixty-three pediatric patients with a histological ependymoma diagnosis treated at Motol and Homolka University Hospital (2010–2025) were included. DNA methylation profiling, RNA sequencing, copy number variation and t-SNE analysis were performed. Survival was estimated by the Kaplan–Meier method. MA confirmed ependymoma in 48 patients (76.2

CIC-rearranged sarcomas represent a distinct rare pathological entity within the spectrum of undifferentiated small round cell sarcomas. This single institution retrospective study investigates the histopathological, molecular, and clinical characteristics of a cohort of 14 CIC-rearranged sarcomas. These tumors were diagnosed in eight male and six female patients, with the age at initial diagnosis ranging from 8 to 67 years and a mean age of 23.7 years. Among the patients, three novel locations of CIC::DUX4 sarcomas were detected in the mesentery, pancreas, and pterygopalatine fossa. Genetically, we identified 12 cases as CIC::DUX4 sarcomas and two as sarcomas with CIC gene rearrangement detected by break-apart fluorescence in situ hybridization only. The most common gene translocation had a breakpoint located in exon 20 of the CIC gene and exon 1 of the DUX4 gene; this alteration was found in 83

MYCN functions as a developmental oncogene, but its role in pediatric high-grade gliomas (pHGGs) remains unclear. In co-operation with Trp53 and Pten loss, MYCN initiates tumorigenesis and establishes an origin for MYCN-driven pHGGs. This transformation creates a vulnerability to PI3K and mTOR inhibition. However, prolonged treatment drives adaptive resistance through MYCN protein rebound, mediated by the attenuation of IGFBP5 and the induction of insulin-like growth factor 2. Although insulin pathway feedback has been implicated in resistance to PI3K targeted therapies, MYCN emerges as the central node of this adaptive program. Resistance can be overcame by sustained MYCN suppression using PI3K and mTOR inhibitors, combined with insulin-like growth factor 1 receptor and insulin receptor inhibitors or dietary intervention. A degradation-resistant MYCN isoform abolishes this response, establishing MYCN as both an initiating oncogene and a resistance driver and revealing a mechanistically defined therapeutic vulnerability.

Pediatric low-grade gliomas (pLGG) are the most common group of childhood brain tumors. Genetic alterations in the RAS-RAF-mitogen-activated protein kinase (MAPK) pathway are the molecular drivers in the vast majority of pLGG. A large proportion of pediatric pLGG are characterized by the presence of fusion genes. An institutional molecular analysis together with an RNA-NGS study was performed to reveal LGG-associated molecular alterations. In our cohort of pLGG patients, molecular alterations were identified in 318 out of 342 cases (92.9%) through a combination of RT-PCR, Sanger sequencing, and NGS methodologies. Fusion events were independently called using three fusion callers: Archer Analysis 6.0 and/or 7.0, Arriba version 2.4, and STAR-Fusion 24. Among these, STAR-Fusion had the lowest sensitivity, detecting rearrangements in only 67% of fusion-positive cases. In contrast, Arriba detected rearrangements in 97.77% of cases, while Archer detected rearrangements in 88.6% of cases. These findings highlight differences in detection efficiency among fusion callers, emphasizing the importance of tool selection in molecular diagnostics. The detection of fusion genes is very important for correct diagnosis, prognosis, and adequate targeted treatment.

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors. The development of these tumors is associated with more than 20 genes. The aforementioned genes are subdivided into three clusters. The pseudohypoxic, kinase-signaling and Wnt clusters. The pseudohypoxic cluster is the only one that has been demonstrated to be associated with DNA methylation changes, including alterations in the 11p15.5 region. The objective of this study was to identify alterations in the 11p15.5 region, ascertain their prevalence in PPGLs, and subsequently compare them with the genomic and somatic mutations that cluster PPGLs. One hundred and fifty tumor samples were subjected to analysis. A total of 90 cases (60%) exhibited no alterations in the 11p15.5 region. The most prevalent alterations were maternal allele loss, observed in 45 cases (30%), pUPD (paternal uniparental disomy) in five cases (3.33%), and paternal allele gain in four cases (2.67%). The data presented here suggest that two mechanisms may be involved in the formation of PPGLs. These are reduced expression of CDKN1C (maternal allele deletion) and overexpression of IGF2 (pUPD, paternal allele gain). A statistically significant difference was observed in the frequency of alterations in the 11p15.5 region when comparing cluster 1 and cluster 2 (P-value <0.0001). This study is the first to describe pUPD and paternal allele gain as somatic alterations in PPGLs. In addition, our findings indicate that alterations in the 11p15.5 region are not exclusive to cluster 1. Consequently, the alterations in the 11p15.5 region cannot be regarded as a marker for cluster 1.

Neuroblastomas are the most common solid tumours in early childhood. Currently, the prognosis of patients is evaluated almost exclusively on the basis of molecular-genetic markers from a biopsy sample of tumour tissue. This study is focused on evaluating the potential use of 3‑methoxytyramine as an alternative non-invasive prognostic marker for patients with neuroblastoma. For this purpose, a method was developed to determine free 3‑methoxytyramine in urine using liquid chromatography coupled with tandem mass spectrometric detection. The method was fully validated and subsequently tested on 25 pediatric urine samples. In the group of patients with a poor prognosis, the median value of free 3-methoxy­tyramine was 666.7 nmol mmol−1Krea, while in the group with a favourable prognosis, it was 168.4 nmol mmol−1Krea. The analysis of pediatric urine samples from neuroblastoma patients revealed statistically significant differences in the concentration of free 3‑methoxytyramine (P < 0,05) between the low/medium-risk group and the high-risk group.

Next-generation sequencing methods have opened a door to a new era of genotype testing. As this method has become more affordable and available we are now routinely able to look into patients’ DNA in detail, searching for new and known gene variants when suspected. Primary aldosteronism is the most common and underdiagnosed cause of secondary hypertension in adults. One of the uncommon causes of primary aldosteronism is familial hyperaldosteronism, found frequently in the pediatric population, with its five most common and well-described forms. We present a review and a case of severe arterial hypertension and familial hyperaldosteronism diagnosed in a 15-year-old girl who has been treated with central precocious puberty since the age of five. After establishing the proper diagnosis and beginning the appropriate treatment, whole-exome sequencing (WES) revealed several genetic variants that in combination are likely the underlying cause of the primary aldosteronism and central precocious puberty phenotype. Next-generation sequencing should be considered in patients where a rare genetic syndrome is suspected.

Epigenetic mechanisms are of pivotal importance in the normal development and maintenance of cell and tissue-specific gene expression patterns, and are fundamental to the genesis of cancer. One significant category of epigenetic modifications is histone methylation. Histone methylation plays a crucial role in the regulation of gene expression, and its dysregulation has been observed in various diseases, including cancer. The maintenance of the histone methylation state is dependent on two classes of enzymes: histone methyltransferases, which add methyl groups to arginine and lysine residues, and lysine demethylases, which remove methyl groups from lysine residues of histones. To date, eight subfamilies have been identified, comprising approximately 30 lysine demethylases. These enzymes are expressed differently across cells and tissues and exert a substantial impact on the development and progression of cancer. The diverse range of lysine demethylases influence a multitude of oncogenic pathways, either by promoting or inhibiting their activity. However, comprehensive data on the full spectrum expression of lysine demethylases in distinct cancer types remain scarce. Lysine demethylases have been demonstrated to play a role in drug resistance in numerous cancers. This is achieved by modulating the metabolic profile of cancer cells, enhancing the ratio of cancer stem cells, and elevating the expression of drug-tolerant genes. Additionally, they facilitate epithelial-mesenchymal transition and metastatic potential. The objective of this review is to synthesize recent data on the relationship between lysine demethylases and cancer, with a particular focus on cancer cell drug resistance.

Paragangliomas are neuroendocrine tumors arising from chromaffin cells within the parasympathetic or sympathetic nervous system. These types of tumors are significantly attributed to genetic mutations. Among these, mutations in the SDHC (succinate dehydrogenase complex subunit C) gene emerge as tumor suppressors, predisposing an individual to tumor development. This study examines a family with an SDHC mutation (p.Arg133Ter), where three members presented with rare mediastinal paragangliomas. Our analysis reveals that the tumor manifestation related to this genetic mutation varies greatly in location and age of onset, displaying a broad range of clinical presentations within the same family. This variability aligns with previous research showing different levels of tumor occurrence among individuals with SDHC mutations. The rarity of SDHC mutations and the resulting tumors, along with the variability in clinical manifestations, make it challenging to define a clear protocol for screening and ongoing monitoring of individuals with these genetic changes. By employing genetic sequencing and clinical assessments, we identified a link between the specific SDHC mutation and the appearance of mediastinal paragangliomas in the family, highlighting the genetic and clinical diversity associated with SDHC mutations. This emphasizes the need for tailored monitoring and treatment plans. These findings contribute to a more comprehensive understanding of the genetic and clinical characteristics of SDHC-associated paragangliomas, highlighting the importance of genetic counseling for at-risk families and emphasizing the complexities involved in the management of affected patients.

In high-risk neuroblastoma, identification of ALK activating genetic alterations is considered for clinical decision-making at relapse or more recently in frontline treatment. The accurate diagnosis of genetic alterations requires harmonization of molecular techniques and reporting, especially when these concern inclusion criteria for clinical trials. Analysis and validation of 14 DNA samples harboring distinct ALK alterations were performed across the 21 SIOPEN (International Society of Paediatric Oncology Europe Neuroblastoma) molecular diagnostic laboratories. These included ALK mutations at or outside hotspots in the tyrosine kinase domain with variant allele frequencies (VAFs) of 1% to 91% or ALK genomic amplification. Each laboratory used their own techniques: ALK amplifications were detected by pan-genomic copy number techniques or fluorescence in situ hybridization, and ALK mutations were characterized by next-generation sequencing techniques. All laboratories correctly identified high-level ALK amplification and ALK mutations within the known hotspots with VAF >5%, with the exception of two cases. Differences in interpretation and reporting were apparent for samples harboring mutations with a VAF <5% or outside known hotspots. These results highlight the importance of standard operating procedures, standardized reporting, and the robustness of ALK genetic testing in the SIOPEN laboratories, and the need for expert discussions regarding atypical ALK alterations, to validate eligibility for ALK targeted treatment in clinical trials.

Abstract BACKGROUND The etiology of pediatric cancer remains largely unclear, with only a small proportion of patients (8-10%) harboring cancer predisposition syndromes (CPS). However, prospective studies evaluating CPS in pediatric neuro-oncology patients are scarce. We aimed to investigate the incidence and spectrum of CPS in this patient population. METHODS Newly diagnosed pediatric brain tumor patients from 2020 to 2023 were consented, and their germline DNA was sequenced using a DNA panel for next-generation sequencing (NGS) or whole-exome sequencing. Patients with previously known NF1, NF2, or TSC were excluded from the study. RESULTS A total of 164 consecutive patients from our center were enrolled over four years. Pathogenic germline variants (UV4 or UV5) were detected in 24 cases (14.6%). Half of these mutated genes are known to be associated with pediatric tumors. Examples include variants in TP53 (two patients with high-grade glioma), biallelic FANCD1 (medulloblastoma), SMARCE1 (meningioma), NF1 (high-grade glioma), NF2 (meningioma), MSH2 (medulloblastoma), and others. The second group of patients harbored pathogenic variants without confirmed associations with pediatric cancer, including heterozygous variants in MUTYH (n=3), FANC genes (n=2), NBN (n=2), PALB2, XPC, and pathogenic variants in WRN or CHEK2 (n=3). In addition to pathogenic variants in CHEK2, variants of uncertain significance, such as c.470T>C, were also identified (total n=8). The majority of patients with a CHEK2 variant were diagnosed with glial tumors. Notably, 8.3% of patients with pediatric low-grade gliomas (LGG) from our cohort harbored a CHEK2 germline variant. CONCLUSIONS The incidence of CPS in our cohort (14.6%) is higher than what is reported in studies evaluating all pediatric cancer patients. This percentage might be underestimated due to the exclusion of known phacomatoses. The role of some variants, including those in CHEK2, warrants further investigation. This study was supported by the Ministry of Health of the Czech Republic, grant number NU21-07-00419.

BACKGROUND The etiology of pediatric cancer remains largely unclear, with only a small proportion of patients (8-10%) harboring cancer predisposition syndromes (CPS). However, prospective studies evaluating CPS in pediatric neuro-oncology patients are scarce. We aimed to investigate the incidence and spectrum of CPS in this patient population. METHODS Newly diagnosed pediatric brain tumor patients from 2020 to 2023 were consented, and their germline DNA was sequenced using a DNA panel for next-generation sequencing (NGS) or whole-exome sequencing. Patients with previously known NF1, NF2, or TSC were excluded from the study. RESULTS A total of 164 consecutive patients from our center were enrolled over four years. Pathogenic germline variants (UV4 or UV5) were detected in 24 cases (14.6%). Half of these mutated genes are known to be associated with pediatric tumors. Examples include variants in TP53 (two patients with high-grade glioma), biallelic FANCD1 (medulloblastoma), SMARCE1 (meningioma), NF1 (high-grade glioma), NF2 (meningioma), MSH2 (medulloblastoma), and others. The second group of patients harbored pathogenic variants without confirmed associations with pediatric cancer, including heterozygous variants in MUTYH (n=3), FANC genes (n=2), NBN (n=2), PALB2, XPC, and pathogenic variants in WRN or CHEK2 (n=3). In addition to pathogenic variants in CHEK2, variants of uncertain significance, such as c.470T>C, were also identified (total n=8). The majority of patients with a CHEK2 variant were diagnosed with glial tumors. Notably, 8.3% of patients with pediatric low-grade gliomas (LGG) from our cohort harbored a CHEK2 germline variant. CONCLUSIONS The incidence of CPS in our cohort (14.6%) is higher than what is reported in studies evaluating all pediatric cancer patients. This percentage might be underestimated due to the exclusion of known phacomatoses. The role of some variants, including those in CHEK2, warrants further investigation. This study was supported by the Ministry of Health of the Czech Republic, grant number NU21-07-00419.

Pulmonary atresia with ventricular septal defect and major aortopulmonary collateral arteries, paragangliomas, and syringomyelia are uncommon diseases. Furthermore, in the absence of any genetic link and with less than five reported adult patients surviving unrepaired rare form of Tetralogy of Fallot, our case shows noteworthiness. The possibility of definitive treatment of these conditions is rendered unsafe due to this persistent defect. Thus, management and ongoing survival of this patient remains complex and challenging.