Search

Neuroblastoma is an embryonal tumor of neuroectodermal cells. Patients with metastatic neuroblastoma have a poor survival rate, which has led to numerous efforts to develop prognostic markers. Cancer/testis-specific antigens MAGE-A1 and MAGE-A3 genes were proposed as minimal residual disease (MRD) markers in neuroblastoma, but its usefulness for this purpose is rather limited.We studied 47 primary neuroblastoma tumors. RNA was extracted and cDNA was prepared by reverse transcription. Detection of the MAGE-A1 expression was done by hybridization of the RT-PCR products. We used methylation-specific-PCR to perform the epigenetic studies.We studied the MAGE-A1 and MAGE-A3 expressions, and the MAGE-A1 expression showed significant association with tumor stage, absence of bone marrow infiltration and survival. A multivariate analysis enabled us to conclude that the MAGE-A1 expression represents a new independent predictive factor, which is independent of N-Myc amplification (P value = 0.000), age at diagnosis (P value = 0.002) or tumoral stage (P value = 0.024). Considering the epigenetic regulation of MAGE-A1, we analyzed its methylation profile, and found a significant association with its expression in tumor cells. Moreover, we found tumors that failed to show the MAGE-A1 expression despite the hypomethylated sequence, and corresponded to advanced neuroblastoma that might share another mechanism involved in MAGE-A1 silencing. Given the association described between genome-wide hypomethylation and microsatellite instability, we determined the MSI status of tumor samples, finding a significant correlation with the MAGE-A1 expression and, more specifically, with the hypomethylated status of this gene only in female patients.We conclude that the MAGE-A1 expression is associated with good prognosis in neuroblastoma.

Neuroblastoma and pheochromocytoma have the same embryonal origin. They originate from neural crest cells, and they usually affect suprarenal glands. The SDHB gene encodes the B subunit of succinate dehydrogenase, a protein implicated in the electron transport chain and Krebs cycle. Some mutations have been described in this gene in pheochromocytoma, and this gene could be an appropriate candidate for its study in neuroblastoma given its localization in lp35-36. The aim of this study was to analyze neuroblastoma tumors in order to assess a possible implication of this gene in neuroblastoma development. We studied 28 neuroblastoma tumor samples from different stages. Mutation research in genomic DNA was carried out after individual amplification of each of the eight SDHB exons by SSCP analysis and sequencing of those samples with migration pattern variants. No variant was found except for three polymorphisms in four neuroblastoma samples. The first polymorphism was a synonymous A → C change in the third position of codon 6 (exon 1). The other two polymorphisms were a TTC insert at the 5' flanking intron sequence of exon 5 in a stretch of seven TTC repeats. Upon the basis of posterior microsatellite instability and hyper-methylation promoter studies, which were not significant, we can conclude that the SDHB gene, a positional candidate gene, is unlikely to be related to either initiation or tumoral progression in neuroblastoma.

Childhood myelodysplastic syndromes (MDS) belong to a rare group of disorders of aberrant clonal hematopoiesis manifesting throughout entire childhood and adolescence. We had previously established that GATA2 germline mutations can be considered the most common "first hit" in pediatric MDS seen in 7% of primary MDS. However the secondary somatic aberrations facilitating leukemogenesis are not elucidated in children. Previous sequencing efforts established that most somatic mutations very frequently encountered in adults, i.e. affecting TET2, DNMT3a, and the spliceosome genes, do not play a role in the pathogenesis of childhood MDS. Here we aim to define the global mutational landscape in childhood MDS using targeted next-generation sequencing (NGS) approaches. We investigated children and adolescents enrolled in the prospective studies of the European Working Group of Childhood MDS. Diverse target enrichment and NGS strategies were established including hybridization capture and Ampliseq PCR, Illumina Miseq/Hiseq and Iontorrent PGM. We first examined a pilot cohort of 68 patients for mutations in 138 myeloid leukemia genes. This allowed for the identification of recurrently mutated genes that were selected to be included in a pediatric MDS panel encompassing 28 genes. Targeted NGS using the Iontorrent PGM identified known recurrent mutations. However, the high indel error rate and coverage gaps in homopolymeric regions i.e. in ASXL1 precluded further studies. Using inhouse-adapted Ampliseq-Miseq approach we then sequenced DNA from bone marrow of 586 MDS patients (469 primary and 117 secondary MDS after radio/chemotherapy or inherited bone marrow failure syndromes) at an average depth exceeding 700 reads per amplicon. Somatic mutations were identified in 22% of primary MDS patients, with 1, 2 and 3 genes affected in 16%, 4.5%, and 1.5% of cases, respectively. In secondary MDS twice as many patients (46%) carried mutations; 1, 2, and 3 genes were concurrently mutated in 32.5%, 9.5%, and 4% of patients, respectively. Longitudinal NGS analyses and single CFU colony sequencing confirmed the presence of multiple somatic clones evolving in a hierarchical manner throughout disease course. Most frequent mutations identified in more than 1% of our study cohort of primary MDS were: SETBP1 (7%), ASXL1 (6%), NRAS/KRAS (5%), RUNX1 (3%), PTPN11 (3%) and BCOR/BCORL (1.5%); and in secondary MDS: RUNX1 (14.5%), TP53 (9%), NRAS/KRAS (8.5%), ASXL1 (8%), SETBP1 (6%), PTPN11 (6%), CBL (5%), BCOR/BCORL1 (3.4%). Other genes mutated at very low frequency of In conclusion, we established and cross-platform validated a targeted NGS-panel for pediatric MDS, allowing to identify clonal mutations at a sensitivity of at least 10%. In this, to our knowledge largest systematicaly studied cohort of children with MDS we show that mutational load and clonal complexity differs between primary and secondary MDS, and maintains specific patterns for monosomy 7. Although the biological significance of these genomic changes is currently not understood, the emerging distinctive patterns may already be helpful in establishing therapeutic subgroups. Disclosures No relevant conflicts of interest to declare.

A search of TP53 mutations was undertaken in a series of 51 pediatric brain tumors. The only germ-line mutation was detected in a 9-year-old girl with a PNET. Her family history was unremarkable for neoplastic disease, except for the paternal grandfather, who died of a gallbladder carcinoma at an advanced age. The mutation was a thymine deletion at the first base of codon 241, leading to termination codon at position 246 that has not previously been reported. This mutation was found to be inherited from the proband's father, who was healthy at age 40. In the tumoral sample, loss of heterozygosity in several 17p markers was found, the only TP53 allele preserved in the tumor was the mutated one. The presence of two short tandem repeats and two different palindromic sequences spanning the deletion lead us to propose the predisposition of this region to forming a complex secondary structure during replication. Consequently, it could have facilitated the present deletion. Furthermore, six other short deletions affecting--partially or totally--the region implicated in the folding model that we propose have been described in the literature. These findings confirm that this sequence represents a hotspot of deletion in the TP53 gene.

Cytogenetic and molecular analyses of primitive neuroectodermal tumors (PNETs) of the central nervous system (CNS) have demonstrated material losses of 17p, the region that contains the TP53 gene, as the most frequent abnormality. Mutations in the TP53 gene are, however, very rare in these tumors. These findings strongly suggest that another, as yet unidentified, gene on 17p may be involved. We performed a search for loss of heterozygosity (LOH) on 17p by microsatellite markers on 26 childhood CNS tumors as well as TP53 gene mutations (exons 5-8) by single-strand conformational polymorphism analysis on 41 pediatric brain tumor samples of distinct histologic types. LOH was detected in 10 cases: 7 PNET, 2 astrocytomas, and 1 glioblastoma multiforme. In 4 of the PNETs the losses were limited to more distal markers. On the other hand, TP53 mutations were detected in 6 of 41 samples studied. Our results not only confirm the low penetrance of the TP53 gene on pediatric CNS tumors, but also provide further evidence of a putative tumor suppressor gene distal to TP53, between markers (D17S938, D17S926) and 17pter, specifically taking part in the development of PNET.

This report describes a chromosomal abnormality in an 8-year-old boy with renal adenocarcinoma. All tumor cells had the karyotype 46,XY, t(X;17)(p11.2;q25). This karyotype is compared with the cytogenetic descriptions of renal cell adenocarcinoma in two other pediatric patients and in adults.