Juliana Silveira Ruas

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Paediatric central nervous system (CNS) tumours are the second most common childhood malignancy and the leading cause of cancer‐related mortality in this age group. Histopathological diagnosis can be challenging, particularly for rare or ambiguous tumours, and may result in misclassification. To evaluate the utility of DNA methylation profiling in a middle‐income country, we performed the Infinium MethylationEPIC BeadChip (Illumina) on tumours from 182 paediatric patients treated at a reference centre for paediatric oncology in Campinas, state of São Paulo, Brazil. Data were analysed using the DKFZ/Heidelberg CNS tumour classifier (v12.8). After excluding control tissue, 163 samples (89.6%) were suitable for classification; 135 (74.2%) achieved a calibrated score ≥ 0.9 and were assigned to a methylation class family. Methylation profiling resulted in a tumour subtype for 88 cases (65.7%) and changed the diagnosis in 28 cases (20.9%), identifying several rare tumour subtypes that were identified solely through methylation analysis, confirming the value of this method in improving diagnostic accuracy. This study highlights the utility of DNA methylation profiling for paediatric CNS tumours in a resource‐limited setting and provides a cohort from an underrepresented middle‐income population to international molecular databases.

Developing advanced preclinical models and targeted therapies is essential for reducing cancer-related deaths in children with solid tumors. Patient-derived xenografts (PDX) have the potential to replicate key elements of the original tumor, including morphology, genetic alterations, and microenvironment, making them valuable tools for studying tumor biology and drug response. We implanted 124 pediatric solid tumor samples, collected for 1 y, into NOD/SCID/IL2Rg (NSG) mice. Tumor fragments were placed subcutaneously, and the animals were monitored for up to 1 y. Histopathology, Short Tandem Repeat (STR) profiling, RT-PCR and/or RNA-sequencing were performed to confirm tumor identity and detect driver fusions. Fifty-five xenografts were successfully established (44.35% of implanted samples), representing 19 tumor types. Sarcomas, notably osteosarcoma, Ewing sarcoma, synovial sarcoma, and rhabdomyosarcoma, displayed first-generation engraftment rates above 55%. Central nervous system tumors had lower success, reflecting unique microenvironmental requirements. Histopathology and STR concordances were 85.45% and 81.1%, respectively, while 92.6% of sarcoma PDXs retained original fusion genes. Second-generation xenografts showed faster growth, suggesting adaptation to the murine host. Sporadic discrepancies, such as new fusions or lymphoproliferative expansions, indicated the need for ongoing molecular validation parallel to other techniques. A pediatric PDX biobank can effectively capture key tumor features while facilitating the study of therapeutic responses and tumor evolution. Our models confirm the feasibility of achieving stable histological and molecular profiles, offering a valuable resource for precision oncology research. Ultimately, these pediatric PDXs could accelerate the discovery of targeted therapy and significantly improve treatment outcomes.

Pediatric sarcomas present heterogeneous morphology, genetics and clinical behavior posing a challenge for an accurate diagnosis. DNA methylation is an epigenetic modification that coordinates chromatin structure and regulates gene expression, determining cell type and function. DNA methylation-based tumor profiling classifier for sarcomas (known as sarcoma classifier) from the German Cancer Research Center (Deutsches Krebsforschungszentrum) was applied to 122 pediatric sarcomas referred to a reference pediatric oncology hospital. The classifiers reported 88.5% of agreement between histopathological and molecular classification confirming the initial diagnosis of all osteosarcomas and Ewing sarcomas. The Ewing-like sarcomas were reclassified into sarcomas with BCOR or CIC alterations, later confirmed by orthogonal diagnostic techniques. Regarding the CNAs profile, osteosarcomas had several chromosomal gains and losses as well as chromothripsis, whereas Ewing sarcomas had few large events, such as amplifications of chromosomes 8 and 12. The molecular classification together with clinical and histopathological assessment could improve the diagnosis of pediatric sarcomas although there are limitations to deal with more rare classes. This study provides an increase in the number of sarcomas evaluated for DNA methylation profiling in the pediatric population.

Embryonic tumors share few recurrent mutations, suggesting that other mechanisms, such as aberrant DNA methylation, play a prominent role in their development. The loss of imprinting (LOI) at the chromosome region 11p15 is the germline alteration behind Beckwith–Wiedemann syndrome that results in an increased risk of developing several embryonic tumors. This study analyzed the methylome, using EPIC Beadchip arrays from 99 sporadic embryonic tumors. Among these tumors, 46.5% and 14.6% presented alterations at imprinted control regions (ICRs) 1 and 2, respectively. Based on the methylation levels of ICR1 and ICR2, four clusters formed with distinct methylation patterns, mostly for medulloblastomas (ICR1 loss of methylation (LOM)), Wilms tumors, and hepatoblastomas (ICR1 gain of methylation (GOM), with or without ICR2 LOM). To validate the results, the methylation status of 29 cases was assessed with MS-MLPA, and a high level of agreement was found between both methodologies: 93% for ICR1 and 79% for ICR2. The MS-MLPA results indicate that 15 (51.7%) had ICR1 GOM and 11 (37.9%) had ICR2 LOM. To further validate our findings, the ICR1 methylation status was characterized via digital PCR (dPCR) in cell-free DNA (cfDNA) extracted from peripheral blood. At diagnosis, we detected alterations in the methylation levels of ICR1 in 62% of the cases, with an agreement of 76% between the tumor tissue (MS-MLPA) and cfDNA methods. Among the disagreements, the dPCR was able to detect ICR1 methylation level changes presented at heterogeneous levels in the tumor tissue, which were detected only in the methylome analysis. This study highlights the prevalence of 11p15 methylation status in sporadic embryonic tumors, with differences relating to methylation levels (gain or loss), location (ICR1 or ICR2), and tumor types (medulloblastomas, Wilms tumors, and hepatoblastomas).

Increased glycolytic metabolism plays an important role in B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL). We previously showed that IGFBP7 exerts mitogenic and prosuvival effects in ALL by promoting IGF1 receptor (IGF1R) permanence on the cell surface, thus prolonging Akt activation upon IGFs/insulin stimulation. Here, we show that sustained activation of the IGF1R–PI3K–Akt axis concurs with GLUT1 upregulation, which enhances energy metabolism and increases glycolytic metabolism in BCP-ALL. IGFBP7 neutralization with a monoclonal antibody or the pharmacological inhibition of the PI3K–Akt pathway was shown to abrogate this effect, restoring the physiological levels of GLUT1 on the cell surface. The metabolic effect described here may offer an additional mechanistic explanation for the strong negative impact seen in ALL cells in vitro and in vivo after the knockdown or antibody neutralization of IGFBP7, while reinforcing the notion that it is a valid target for future therapeutic interventions.

Pediatric tumors share few recurrent mutations and are instead characterized by copy number alterations (CNAs). The cell-free DNA (cfDNA) is a prominent source for the detection of cancer-specific biomarkers in plasma. We profiled CNAs in the tumor tissues for further evaluation of alterations in 1q, MYCN and 17p in the circulating tumor DNA (ctDNA) in the peripheral blood at diagnosis and follow-up using digital PCR. We report that among the different kinds of tumors (neuroblastoma, Wilms tumor, Ewing sarcoma, rhabdomyosarcoma, leiomyosarcoma, osteosarcoma and benign teratoma), neuroblastoma presented the greatest amount of cfDNA, in correlation with tumor volume. Considering all tumors, cfDNA levels correlated with tumor stage, metastasis at diagnosis and metastasis developed during therapy. In the tumor tissue, at least one CNA (at CRABP2, TP53, surrogate markers for 1q and 17p, respectively, and MYCN) was observed in 89% of patients. At diagnosis, CNAs levels were concordant between tumor and ctDNA in 56% of the cases, and for the remaining 44%, 91.4% of the CNAs were present only in cfDNA and 8.6% only in the tumor. Within the cfDNA, we observed that 46% and 23% of the patients had MYCN and 1q gain, respectively. The use of specific CNAs as targets for liquid biopsy in pediatric patients with cancer can improve diagnosis and should be considered for monitoring of the disease response.

Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism of proliferating astrocytes. The electron flux through mitochondrial respiratory complexes and oxidative phosphorylation may impact the growth and viability of these astrocytes. Here, we aimed at assessing to which extent mitochondrial oxidative metabolism is required for astrocyte survival and proliferation. Primary astrocytes from the neonatal mouse cortex were cultured in a physiologically relevant medium with the addition of piericidin A or oligomycin at concentrations that fully inhibit complex I-linked respiration and ATP synthase, respectively. The presence of these mitochondrial inhibitors for up to 6 days in a culture medium elicited only minor effects on astrocyte growth. Moreover, neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by piericidin A or oligomycin. Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity. Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.

Infection with HPV virus and exposure to extrinsic carcinogens are the main causative factors for oropharyngeal squamous cell carcinoma (OPSCC). While HPV-related OPSCC typically shows a better prognosis and may be a candidate for de-intensification therapy, there is a subset of HPV-related cancers that show aggressive phenotype with frequent metastatic spread. The identification and refinement of molecular markers can better serve for prediction of prognosis and thus improve treatment decisions and outcome. We conducted a systematic review according to the PRISMA guidelines of all relevant studies addressing novel biomarkers in publications prior to July 2021. We identified studies that evaluated the association between molecular markers and prognosis in HPV-positive OPSCC. Full-text publications were entirely reviewed, classified, and selected if a clear predictive/prognostic value was seen in patients with HPV-positive OPSCC. Furthermore, a functional analysis of the target genes was conducted to understand biological processes and molecular pathways impacting on HPV-positive OPSCC outcomes. The systematic review yielded a total of 14 studies that matched the inclusion and exclusion criteria. Differential expression was identified for 31 different biomarkers. The first common pattern identified was the association of HPV-related circulating antibodies to activated immune function. Second, gene–gene interaction analysis further identified interacting gene networks tightly implicated in hypoxia tumor metabolism including the Warburg effect. Survival in HPV-positive OPSCC can be predicted by distinct selective biomarkers mainly indicative of immune host response and oxidative metabolism. Among these markers, some were identified to be unsuitable for HPV-positive de-escalation trials aimed at improving patients’ quality of life.