Gliomas in children and adolescents: investigation of molecular alterations with a potential prognostic and therapeutic impact.

PURPOSE: Gliomas represent the most frequent central nervous system (CNS) tumors in children and adolescents. However, therapeutic strategies for these patients, based on tumor molecular profile, are still limited compared to the wide range of treatment options for the adult population. We investigated molecular alterations, with a potential prognostic marker and therapeutic target in gliomas of childhood and adolescence using the next-generation sequencing (NGS) strategy. METHODS: We selected 95 samples with initial diagnosis of glioma from patients treated at Pediatric Oncology Institute-GRAACC/UNIFESP. All samples were categorized according to the 2021 World Health Organization Classification of Tumors of the CNS, which included 39 low-grade gliomas (LGGs) and 56 high-grade gliomas (HGGs). Four HGG samples were classified as congenital glioblastoma (cGBM). NGS was performed to identify somatic genetic variants in tumor samples using the Oncomine Childhood Cancer Research Assay® (OCCRA®) panel, from Thermo Fisher Scientific®. RESULTS: Genetic variants were identified in 76 of 95 (80%) tumors. In HGGs, the most common molecular alteration detected was H3F3A c.83A > T variant (H3.3 K27M) and co-occurring mutations in ATRX, TP53, PDGFRA, MET, and MYC genes were also frequently observed. One HGG sample was reclassified as supratentorial ependymoma ZFTA-fusion positive after NGS was performed. In LGGs, four KIAA1549-BRAF fusion transcripts were detected and this alteration was the most recurrent genetic event and favorable prognostic factor identified. Additionally, genetic variants in ALK and NTRK genes, which provide potential targets for therapy with Food and Drug Administration-approved drugs, were identified in two different cases of cGBM that were classified as infant-type hemispheric glioma, a newly recognized subgroup of pediatric HGG. CONCLUSION: Molecular profiling by the OCCRA® panel comprehensively addressed the most relevant genetic variants in gliomas of childhood and adolescence, as these tumors have specific patterns of molecular alterations, outcomes, and effectiveness to therapies.

Molecular profiling of pediatric and adolescent ependymomas: identification of genetic variants using a next-generation sequencing panel.

PURPOSE: Ependymoma (EPN) accounts for approximately 10% of all primary central nervous system (CNS) tumors in children and in most cases, chemotherapy is ineffective and treatment remains challenging. We investigated molecular alterations, with a potential prognostic marker and therapeutic target in EPNs of childhood and adolescence, using a next-generation sequencing (NGS) panel specific for pediatric neoplasms. METHODS: We selected 61 samples with initial diagnosis of EPN from patients treated at Pediatric Oncology Institute-GRAACC/UNIFESP. All samples were divided according to the anatomical compartment of the CNS - 42 posterior fossa (PF), 14 supratentorial (ST), and five spinal (SP). NGS was performed to identify somatic genetic variants in tumor samples using the Oncomine Childhood Cancer Research Assay® (OCCRA®) panel, from Thermo Fisher Scientific®. RESULTS: Genetic variants were identified in 24 of 61 (39.3%) tumors and over 90% of all variants were pathogenic or likely pathogenic. The most commonly variants detected were in CIC, ASXL1, and JAK2 genes and have not been reported in EPN yet. MN1-BEND2 fusion, alteration recently described in a new CNS tumor type, was identified in one ST sample that was reclassified as astroblastoma. Additionally, YAP1-MAMLD1 fusion, a rare event associated with good outcome in ST-EPN, was observed in two patients diagnosed under 2 years old. CONCLUSIONS: Molecular profiling by the OCCRA® panel showed novel alterations in pediatric and adolescent EPNs, which highlights the clinical importance in identifying genetic variants for patients' prognosis and therapeutic orientation.

Abnormal spindle-like microcephaly-associated (ASPM) gene expression in posterior fossa brain tumors of childhood and adolescence.

PURPOSE: In neurogenesis, ASPM (abnormal spindle-like microcephaly-associated) gene is expressed mainly in the ventricular zone of posterior fossa and is the major determinant in the cerebral cortex. Besides its role in embryonic development, ASPM overexpression promotes tumor growth, including central nervous system (CNS) tumors. This study aims to investigate ASPM expression levels in most frequent posterior fossa brain tumors of childhood and adolescence: medulloblastoma (MB), ependymoma (EPN), and astrocytoma (AS), correlating them with clinicopathological characteristics and tumor solid portion size. METHODS: Quantitative reverse transcription (qRT-PCR) is used to quantify ASPM mRNA levels in 80 pre-treatment tumor samples: 28 MB, 22 EPN, and 30 AS. The tumor solid portion size was determined by IOP-GRAACC Diagnostic Imaging Center. We correlated these findings with clinicopathological characteristics and tumor solid portion size. RESULTS: Our results demonstrated that ASPM gene was overexpressed in MB (p = 0.007) and EPN (p = 0.0260) samples. ASPM high expression was significantly associated to MB samples from patients with worse overall survival (p = 0.0123) and death due to disease progression (p = 0.0039). Interestingly, two patients with AS progressed toward higher grade showed ASPM overexpression (p = 0.0046). No correlation was found between the tumor solid portion size and ASPM expression levels in MB (p = 0.1154 and r = - 0.4825) and EPN (p = 0.1108 and r = - 0.3495) samples. CONCLUSION: Taking in account that ASPM gene has several functions to support cell proliferation, as mitotic defects and premature differentiation, we suggest that its overexpression, presumably, plays a critical role in disease progression of posterior fossa brain tumors of childhood and adolescence.

CYP Genotypes Are Associated with Toxicity and Survival in Osteosarcoma Patients.

Purpose: Osteosarcoma is the malignant bone tumor most common in children and adolescents. Many cytochrome P-450 (CYP) members detoxify anticancer drugs used in osteosarcoma treatment, and thus, the aim of the present study was to investigate CYP polymorphisms in osteosarcoma patients. Methods: The present study investigated DNA from peripheral blood from 70 osteosarcoma patients treated with high doses of cisplatin, doxorubicin, and methotrexate. CYP1A2*1F (163C>A; rs762551); CYP2C9*3 (1075A>C; rs1057910); and CYP3A5*3 (6986A>G; rs776746) polymorphisms were investigated through real-time PCR using TaqMan probes. Results: The CYP2C9*3 allele did not present any association with clinical events. The CYP1A2 CC/AC genotypes were associated with ototoxicity occurrence (p = 0.041, odds ratio [OR] = 8.4) and high grades of ototoxicity (p = 0.039, OR = 10.7), when compared with patients carrying the CYP1A2 AA genotype. The CYP1A2 CC genotype was associated with high grades of diarrhea (p = 0.043, OR = 4.6) and fever (p = 0.041, OR = 7.1) in comparison with the CYP1A2 AA/AC genotypes. The CYP3A5 CC genotype was associated with weight loss (p = 0.009, OR = 3.8) and high grades of hepatotoxicity (p = 0.010, OR = 4.3) when compared with the CYP3A5 TT/CT genotypes. The CYP3A5 CC/CT genotypes were associated with high grades of vomit (p = 0.013, OR = 10.8), pulmonary relapse absence (p = 0.029, OR = 9.5), and better overall and event-free survivals (p = 0.017, hazard ratio [HR] = 3.1; p = 0.044, HR = 2.5; respectively) when compared with the CYP3A5 AA genotype. Conclusion:CYP1A2*1A and CYP3A5*3 alleles were associated with toxicity events. CYP3A5*3 allele was associated with better survival. Thus, CYP genotypes might be promising markers to tailoring treatment in osteosarcoma patients.

Analysis of KIAA1549-BRAF fusion gene expression and IDH1/IDH2 mutations in low grade pediatric astrocytomas.

Low-grade astrocytomas comprise about 30 % of the central nervous system tumors in children. Several investigations have searched a correlation between the BRAF gene fusions alterations and mutations at IDH1 and IDH2 genes in low grade pediatric astrocytomas. This study identified the expression of KIAA1549-BRAF fusion gene and BRAF V600E mutation, mutations at exon 4 of the IDH1 and IDH2 genes in samples of pilocytic astrocytomas (PA) and grade-II astrocytomas (A-II) pediatric patients. The correlation between these alterations and the clinical profile of the patients was also evaluated. Eighty-two samples of low-grade astrocytomas (65 PA and 17 A-II) were analyzed by PCR and sequencing for each of the targets identified. We identified the KIAA1549-BRAF fusion transcript in 45 % of the samples. BRAF V600E and BRAFins598T mutations were detected in 7 and 1 % of the samples, respectively. Mutations in the R132/R172 residues of the IDH1/IDH2 genes were detected in only two samples, and the G105G polymorphism (rs11554137:C>T) was identified in ten patients. Additionally, we observed two mutations out of the usual hotspots at IDH1 and IDH2 genes. We observed a smaller frequency of mutations in IDHs genes than previously described, but since the prior studies were composed of adult or mixed (adults and children) samples, we believe that our results represent a relevant contribution to the growing knowledge in low grade childhood astrocytomas.