First Transcriptome Analysis of Hepatoblastoma in Brazil: Unraveling the Pivotal Role of Noncoding RNAs and Metabolic Pathways.

Hepatoblastoma stands as the most prevalent liver cancer in the pediatric population. Characterized by a low mutational burden, chromosomal and epigenetic alterations are key drivers of its tumorigenesis. Transcriptome analysis is a powerful tool for unraveling the molecular intricacies of hepatoblastoma, shedding light on the effects of genetic and epigenetic changes on gene expression. In this study conducted in Brazilian patients, an in-depth whole transcriptome analysis was performed on 14 primary hepatoblastomas, compared to control liver tissues. The analysis unveiled 1,492 differentially expressed genes (1,031 upregulated and 461 downregulated), including 920 protein-coding genes (62%). Upregulated biological processes were linked to cell differentiation, signaling, morphogenesis, and development, involving known hepatoblastoma-associated genes (DLK1, MEG3, HDAC2, TET1, HMGA2, DKK1, DKK4), alongside with novel findings (GYNG4, CDH3, and TNFRSF19). Downregulated processes predominantly centered around oxidation and metabolism, affecting amines, nicotinamides, and lipids, featuring novel discoveries like the repression of SYT7, TTC36, THRSP, CCND1, GCK and CAMK2B. Two genes, which displayed a concordant pattern of DNA methylation alteration in their promoter regions and dysregulation in the transcriptome, were further validated by RT-qPCR: the upregulated TNFRSF19, a key gene in the embryonic development, and the repressed THRSP, connected to lipid metabolism. Furthermore, based on protein-protein interaction analysis, we identified genes holding central positions in the network, such as HDAC2, CCND1, GCK, and CAMK2B, among others, that emerged as prime candidates warranting functional validation in future studies. Notably, a significant dysregulation of non-coding RNAs (ncRNAs), predominantly upregulated transcripts, was observed, with 42% of the top 50 highly expressed genes being ncRNAs. An integrative miRNA-mRNA analysis revealed crucial biological processes associated with metabolism, oxidation reactions of lipids and carbohydrates, and methylation-dependent chromatin silencing. In particular, four upregulated miRNAs (miR-186, miR-214, miR-377, and miR-494) played a pivotal role in the network, potentially targeting multiple protein-coding transcripts, including CCND1 and CAMK2B. In summary, our transcriptome analysis highlighted disrupted embryonic development as well as metabolic pathways, particularly those involving lipids, emphasizing the emerging role of ncRNAs as epigenetic regulators in hepatoblastomas. These findings provide insights into the complexity of the hepatoblastoma transcriptome and identify potential targets for future therapeutic interventions.

High-Resolution Magic-Angle-Spinning NMR in Revealing Hepatoblastoma Hallmarks.

Cancer is one of the leading causes of death in children and adolescents worldwide; among the types of liver cancer, hepatoblastoma (HBL) is the most common in childhood. Although it affects only two to three individuals in a million, it is mostly asymptomatic at diagnosis, so by the time it is detected it has already advanced. There are specific recommendations regarding HBL treatment, and ongoing studies to stratify the risks of HBL, understand the pathology, and predict prognostics and survival rates. Although magnetic resonance imaging spectroscopy is frequently used in diagnostics of HBL, high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy of HBL tissues is scarce. Using this technique, we studied the alterations among tissue metabolites of ex vivo samples from (a) HBL and non-cancer liver tissues (NCL), (b) HBL and adjacent non-tumor samples, and (c) two regions of the same HBL samples, one more centralized and the other at the edge of the tumor. It was possible to identify metabolites in HBL, then metabolites from the HBL center and the border samples, and link them to altered metabolisms in tumor tissues, highlighting their potential as biochemical markers. Metabolites closely related to liver metabolisms such as some phospholipids, triacylglycerides, fatty acids, glucose, and amino acids showed differences between the tissues.

Unraveling the Genetic Architecture of Hepatoblastoma Risk: Birth Defects and Increased Burden of Germline Damaging Variants in Gastrointestinal/Renal Cancer Predisposition and DNA Repair Genes.

The ultrarare hepatoblastoma (HB) is the most common pediatric liver cancer. HB risk is related to a few rare syndromes, and the molecular bases remain elusive for most cases. We investigated the burden of rare damaging germline variants in 30 Brazilian patients with HB and the presence of additional clinical signs. A high frequency of prematurity (20%) and birth defects (37%), especially craniofacial (17%, including craniosynostosis) and kidney (7%) anomalies, was observed. Putative pathogenic or likely pathogenic monoallelic germline variants mapped to 10 cancer predisposition genes (CPGs: APC, CHEK2, DROSHA, ERCC5, FAH, MSH2, MUTYH, RPS19, TGFBR2 and VHL) were detected in 33% of the patients, only 40% of them with a family history of cancer. These findings showed a predominance of CPGs with a known link to gastrointestinal/colorectal and renal cancer risk. A remarkable feature was an enrichment of rare damaging variants affecting different classes of DNA repair genes, particularly those known as Fanconi anemia genes. Moreover, several potentially deleterious variants mapped to genes impacting liver functions were disclosed. To our knowledge, this is the largest assessment of rare germline variants in HB patients to date, contributing to elucidate the genetic architecture of HB risk.

Preserved executive functioning and low stress symptoms in children treated for acute lymphoblastic leukemia.

Pediatric cancer treatment can negatively impact cognitive and psychosocial development, although it has been suggested that these adverse effects may be minimized when children have higher resilience and better executive functioning. We aimed to evaluate the impact of pediatric Acute Lymphoblastic Leukemia (ALL) treatment on executive function, resilience and stress in survivors and to investigate correlations between executive functioning and resilience and between executive functioning and stress. The neuropsychological assessment was performed in 32 ALL survivors aged 7-17 years and 28 age-, sex- and socioeconomic status matched controls. Executive functioning was assessed by inhibitory control, mental flexibility and working memory tasks. Children's self-report scales were used to assess stress symptoms and resilience. Results revealed no executive function impairment nor stress symptom differences between ALL survivors and control group. In the ALL group, executive function and resilience were positively correlated, whereas executive function and stress were negatively correlated. We concluded that ALL treatment was not associated with impairment in executive functioning nor to increased stress symptoms in our sample. ALL survivors with better performance in mental flexibility and inhibition tasks reported fewer stress symptoms and more resilience, indicating a possible relationship between these variables.

Hepatoblastomas exhibit marked NNMT downregulation driven by promoter DNA hypermethylation.

Hepatoblastomas exhibit the lowest mutational burden among pediatric tumors. We previously showed that epigenetic disruption is crucial for hepatoblastoma carcinogenesis. Our data revealed hypermethylation of nicotinamide N-methyltransferase, a highly expressed gene in adipocytes and hepatocytes. The expression pattern and the role of nicotinamide N-methyltransferase in pediatric liver tumors have not yet been explored, and this study aimed to evaluate the effect of nicotinamide N-methyltransferase hypermethylation in hepatoblastomas. We evaluated 45 hepatoblastomas and 26 non-tumoral liver samples. We examined in hepatoblastomas if the observed nicotinamide N-methyltransferase promoter hypermethylation could lead to dysregulation of expression by measuring mRNA and protein levels by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays. The potential impact of nicotinamide N-methyltransferase changes was evaluated on the metabolic profile by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Significant nicotinamide N-methyltransferase downregulation was revealed in hepatoblastomas, with two orders of magnitude lower nicotinamide N-methyltransferase expression in tumor samples and hepatoblastoma cell lines than in hepatocellular carcinoma cell lines. A specific TSS1500 CpG site (cg02094283) of nicotinamide N-methyltransferase was hypermethylated in tumors, with an inverse correlation between its methylation level and nicotinamide N-methyltransferase expression. A marked global reduction of the nicotinamide N-methyltransferase protein was validated in tumors, with strong correlation between gene and protein expression. Of note, higher nicotinamide N-methyltransferase expression was statistically associated with late hepatoblastoma diagnosis, a known clinical variable of worse prognosis. In addition, untargeted metabolomics analysis detected aberrant lipid metabolism in hepatoblastomas. Data presented here showed the first evidence that nicotinamide N-methyltransferase reduction occurs in hepatoblastomas, providing further support that the nicotinamide N-methyltransferase downregulation is a wide phenomenon in liver cancer. Furthermore, this study unraveled the role of DNA methylation in the regulation of nicotinamide N-methyltransferase expression in hepatoblastomas, in addition to evaluate the potential effect of nicotinamide N-methyltransferase reduction in the metabolism of these tumors. These preliminary findings also suggested that nicotinamide N-methyltransferase level may be a potential prognostic biomarker for hepatoblastoma.

Evaluation of whole-body MRI with diffusion-weighted sequences in the staging of pediatric cancer patients.

BACKGROUND: The purpose of this study was to determine whether whole-body MRI (WBMRI) with diffusion-weighted sequences, which is free of ionizing radiation, can perform as well as traditional methods when used alone for staging or follow-up of pediatric cancer patients. METHODS: After obtaining approval from our institutional research ethics committee and appropriate informed consent, we performed 34 examinations in 32 pediatric patients. The examinations were anonymized and analyzed by two radiologists with at least 10 years' experience. RESULTS: The sensitivity and specificity findings, respectively, were as follows: 100% and 100% for primary tumor; 100% and 86% for bone metastasis; 33% and 100% for lung metastasis; 85% and 100% for lymph node metastasis; and 100% and 62% for global investigation of primary or secondary neoplasias. We observed excellent interobserver agreement for WBMRI and excellent agreement with standard staging examination results. CONCLUSIONS: Our results suggest that pediatric patients can be safely imaged with WBMRI, although not as the only tool but in association with low-dose chest CT (for subcentimeter pulmonary nodules). However, additional exams with ionizing radiation may be necessary for patients who tested positive to correctly quantify and locate the lesions.

Assessment of somatic mutations in urine and plasma of Wilms tumor patients.

Tumor DNA has been detected in body fluids of cancer patients. Somatic tumor mutations are being used as biomarkers in body fluids to monitor chemotherapy response as a minimally invasive tool. In this study, we evaluated the potential of tracking somatic mutations in free DNA of plasma and urine collected from Wilms tumor (WT) patients for monitoring treatment response. Wilms tumor is a pediatric renal tumor resulting from cell differentiation errors during nephrogenesis. Its mutational repertoire is not completely defined. Thus, for identifying somatic mutations from tumor tissue DNA, we screened matched tumor/leukocyte DNAs using either a panel containing 16 WT-associated genes or whole-exome sequencing (WES). The identified somatic tumor mutations were tracked in urine and plasma DNA collected before, during and after treatment. At least one somatic mutation was identified in five out of six WT tissue samples analyzed. Somatic mutations were detected in body fluids before treatment in all five patients (three patients in urine, three in plasma, and one in both body fluids). In all patients, a decrease of the variant allele fraction of somatic mutations was observed in body fluids during neoadjuvant chemotherapy. Interestingly, the persistence of somatic mutations in body fluids was in accordance with clinical parameters. For one patient who progressed to death, it persisted in high levels in serial body fluid samples during treatment. For three patients without disease progression, somatic mutations were not consistently detected in samples throughout monitoring. For one patient with bilateral disease, a somatic mutation was detected at low levels with no support of clinical manifestation. Our results demonstrated the potential of tracking somatic mutations in urine and plasma DNA as a minimally invasive tool for monitoring WT patients. Additional investigation is needed to check the clinical value of insistent somatic mutations in body fluids.

Insights Into the Somatic Mutation Burden of Hepatoblastomas From Brazilian Patients.

Hepatoblastoma is a very rare embryonal liver cancer supposed to arise from the impairment of hepatocyte differentiation during embryogenesis. In this study, we investigated by exome sequencing the burden of somatic mutations in a cohort of 10 hepatoblastomas, including a congenital case. Our data disclosed a low mutational background and pointed out to a novel set of candidate genes for hepatoblastoma biology, which were shown to impact gene expression levels. Only three recurrently mutated genes were detected: CTNNB1 and two novel candidates, CX3CL1 and CEP164. A relevant finding was the identification of a recurrent mutation (A235G) in two hepatoblastomas at the CX3CL1 gene; evaluation of RNA and protein expression revealed upregulation of CX3CL1 in tumors. The analysis was replicated in two independents cohorts, substantiating that an activation of the CX3CL1/CX3CR1 pathway occurs in hepatoblastomas. In inflammatory regions of hepatoblastomas, CX3CL1/CX3CR1 were not detected in the infiltrated lymphocytes, in which they should be expressed in normal conditions, whereas necrotic regions exhibited negative labeling in tumor cells, but strongly positive infiltrated lymphocytes. Altogether, these data suggested that CX3CL1/CX3CR1 upregulation may be a common feature of hepatoblastomas, potentially related to chemotherapy response and progression. In addition, three mutational signatures were identified in hepatoblastomas, two of them with predominance of either the COSMIC signatures 1 and 6, found in all cancer types, or the COSMIC signature 29, mostly related to tobacco chewing habit; a third novel mutational signature presented an unspecific pattern with an increase of C>A mutations. Overall, we present here novel candidate genes for hepatoblastoma, with evidence that CX3CL1/CX3CR1 chemokine signaling pathway is likely involved with progression, besides reporting specific mutational signatures.

TET Upregulation Leads to 5-Hydroxymethylation Enrichment in Hepatoblastoma.

Hepatoblastoma is an embryonal liver tumor carrying few genetic alterations. We previously disclosed in hepatoblastomas a genome-wide methylation dysfunction, characterized by hypermethylation at specific CpG islands, in addition to a low-level hypomethylation pattern in non-repetitive intergenic sequences, in comparison to non-tumoral liver tissues, shedding light into a crucial role for epigenetic dysregulation in this type of cancer. To explore the underlying mechanisms possibly related to aberrant epigenetic modifications, we evaluated the expression profile of a set of genes engaged in the epigenetic machinery related to DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L, UHRF1, TET1, TET2, and TET3), as well as the 5-hydroxymethylcytosine (5hmC) global level. We observed in hepatoblastomas a general disrupted expression of these genes from the epigenetic machinery, mainly UHRF1, TET1, and TET2 upregulation, in association with an enrichment of 5hmC content. Our findings support a model of active demethylation by TETs in hepatoblastoma, probably during early stages of liver development, which in combination with UHRF1 overexpression would lead to DNA hypomethylation and an increase in overall 5hmC content. Furthermore, our data suggest that decreased 5hmC content might be associated with poor survival rate, highlighting a pivotal role of epigenetics in hepatoblastoma development and progression.

DNA methylation landscape of hepatoblastomas reveals arrest at early stages of liver differentiation and cancer-related alterations.

Hepatoblastomas are uncommon embryonal liver tumors accounting for approximately 80% of childhood hepatic cancer. We hypothesized that epigenetic changes, including DNA methylation, could be relevant to hepatoblastoma onset. The methylomes of eight matched hepatoblastomas and non-tumoral liver tissues were characterized, and data were validated in an independent group (11 hepatoblastomas). In comparison to differentiated livers, hepatoblastomas exhibited a widespread and non-stochastic pattern of global low-level hypomethylation. The analysis revealed 1,359 differentially methylated CpG sites (DMSs) between hepatoblastomas and control livers, which are associated with 765 genes. Hypomethylation was detected in hepatoblastomas for ~58% of the DMSs with enrichment at intergenic sites, and most of the hypermethylated CpGs were located in CpG islands. Functional analyses revealed enrichment in signaling pathways involved in metabolism, negative regulation of cell differentiation, liver development, cancer, and Wnt signaling pathway. Strikingly, an important overlap was observed between the 1,359 DMSs and the CpG sites reported to exhibit methylation changes through liver development (p<0.0001), with similar patterns of methylation in both hepatoblastomas and fetal livers compared to adult livers. Overall, our results suggest an arrest at early stages of liver cell differentiation, in line with the hypothesis that hepatoblastoma ontogeny involves the disruption of liver development. This genome-wide methylation dysfunction, taken together with a relatively small number of driver genetic mutations reported for both adult and pediatric liver cancers, shed light on the relevance of epigenetic mechanisms for hepatic tumorigenesis.

Osteosarcoma in patients younger than 12 years old without metastases have similar prognosis as adolescent and young adults.

BACKGROUND: Childhood cancer is relatively rare and tends to present specific age distribution, as a prognostic factor for some of these diseases. Information on how young age affects prognosis, response to chemotherapy, and local control options in children versus AYA with osteosarcoma (OST) is minimal. METHODS: In order to identify the main differences in clinical pathologic features, surgical approaches and survival rates of primary high grade OST of the extremity between children (n = 156; <12 years old) and AYA (n = 397; 12-30 years old), the institutional database with 553 patients treated by BOTG studies over 15 years were reviewed. RESULTS: There were no differences in metastases at diagnosis, tumor size, and grade of necrosis between the two age groups. The rate of amputation was 30% higher in the children group (P = 0.018). The rate of limb salvage surgery using reconstruction with allograft or autograft was 70% higher in the children group (P = 0.018) while endoprosthesis rate was 40% higher in the AYA group (P = 0.018). The log rank test revealed that survival is similar between the two age groups for non-metastatic patients (P = 0.424 for OS and P = 0.393 for EFS). Metastatic patients of both ages group had higher risk of dying compared to non-metastatic (HR 3.283 95% CI 2.581-4.177; P < 0.001). Children with metastases at diagnosis had less OS time (P = 0.049) and EFS time (P = 0.032) than adolescents. CONCLUSION: Non-metastatic OST in preadolescent patients does not appear to be significantly different from those seen in AYA patients, but has local control challenges. Children presenting with metastases should be considered an ultra-high-risk group.

Upregulated genes at 2q24 gains as candidate oncogenes in hepatoblastomas.

AIM: Cytogenetic data of hepatoblastomas, a rare embryonal tumor of the liver, mostly consist of descriptions of whole-chromosome aneuploidies and large chromosome alterations. High-resolution cytogenetics may provide clues to hepatoblastoma tumorigenesis and indicate markers with clinical significance. PATIENTS & METHODS: We used array-CGH (180K) to screen for genomic imbalances in nine hepatoblastomas. Additionally, we investigated the expression pattern of selected genes exhibiting copy number changes. RESULTS: Analysis showed mainly whole-chromosome or chromosome-arm aneuploidies, but some focal aberrations were also mapped. Expression analysis of 48 genes mapped at one 10 Mb amplification at 2q24 revealed upregulation of DAPL1, ERMN, GALNT5, SCN1A and SCN3A in the set of tumors compared with differentiated livers. CONCLUSION: These genes appear as candidates for hepatoblastoma tumorigenesis.

Large germline copy number variations as predisposing factor in childhood neoplasms.

AIMS: Constitutive genetic factors are believed to predispose to cancer in children. This study investigated the role of rare germline copy number variations (CNVs) in pediatric cancer predisposition. PATIENTS & METHODS: A total of 54 patients who developed cancer in infancy were screened by array-CGH for germline CNVs. RESULTS: In total, 12 rare CNVs were detected, including a Xq27.2 triplication, and two >1.8 Mb deletions: one of them at 13q31, containing only RNA genes, and another at 3q26.33-q27.1, in a patient with congenital malformations. Detected rare CNVs are significantly larger than those identified in controls, and encompass genes never implicated in cancer predisposition. CONCLUSION: Our results suggest that constitutive CNVs contribute to the etiology of pediatric neoplasms, revealing new candidate genes for tumorigenesis.