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Neuroblastoma (NB) is a tumor of the sympathetic nervous system, and the most common extracranial tumor of childhood. The prognosis for high-stage NBs is still poor, with survival rates of about 35%. Side-effects of treatment in these young children can also be severe. It is therefore important to develop better tools for improved patient stratification as well as to identify new targets for therapy. Aims: Using genetic and epigenetic approaches, this thesis aimed to analyze candidate genes with potential involvment in the initation/progression of NB and to identify genes that can be used for improved patient stratification. Results: The six candidate genes located in chromosome region 1p36.22 were down-regulated in tumors from patients with an unfavorable outcome compared with a favorable. DNA methylation was shown not to be involved in the down-regulation of gene transcripts. In a more comprehensive analysis of 1p36, four genes, ERRFI1, PIK3CD, RBP7 and CASZ1, were up-regulated by epigenetic treatment. Bisulfite sequencing revealed that DNA methylation most likely was not involved, suggesting for the potential involvement of other epigenetic mechanisms such as histone deacetylation. Missense mutations were identified in PIK3CD and ERRFI1 and the down-regulated mRNA expression of PIK3CD and CASZ1 was detected in high-stage NB. CASZ1 plays a role in neural development and is therefore an interesting candidate for further study. In a genome-wide analysis of DNA methylation, a group of methylated genes for which we showed gene expression was affected by epigenetic treatment was selected for further analysis. A selected group, e.g. SCNN1A, PRKCDBP and KRT19 could be used to distinguish between patients with an unfavorable outcome from those with a favorable one. Whole-genome copy number analysis of NB tumors identified homozygous deletions in the CDKN2A and RBMS3 genes. Moreover, copy neutral loss of heterozygosity was rare, but could be detected in three chromosomal regions. Tumors with MYCN amplification and those with 11q deletion displayed very different genomic profiles. The 11q-deletion group had significantly more chromosomal breaks than the other group, indicative of an 11q localized chromosomal instability gene (CIN). This group also had a significantly higher age at diagnosis. The groups defined by 11q deletion, MYCN amplification and 17q gain were the only groups associated with poor patient outcome. Conclusions: Whole-genome profiles add valuable information about genomic aberrations, which are important prognostic factors in NB. Aberrant DNA methylation can be a very early event in tumor development as well as in tumor progression. It is therefore of great importance to learn more about both the genetic and epigenetic profiles of NB. This thesis has added to the current knowledge in these regards and has also identified important genetic aberrations, as well as aberrantly methylated genes. In the future, these aberrations could possibly be used in patient stratification, as biomarkers or as targets for therapy.

Aim: Neuroblastoma (NB) is the most common tumor during infancy. It arises from undifferentiated cells in the sympathetic nervous system and is characterized by both clinical and genetic heterogeneity. One of the features of NBs with unfavorable outcome is loss on distal chromosome 1p. The aim of this thesis was to identify genetic factors with relevance for NB tumorigenesis, with special emphasis on the tumor suppressor candidate region on chromosome 1p36.2.Results: All known genes (UBE4B, KIF1B, PGD, CORT, DFFA and PEX14) in the hotspot NB tumor suppressor gene region on 1p36.22 were screened for mutations by capillary-based sequencing of DNA from a large series of NB tumors. The potential tumor suppressor properties of the genes ENO1, ICAT and CASP9, located close to this region, were also analyzed. A few mutations with predicted deleterious effects on protein function were found in the apoptotic regulator DFFA and in the UBE4B gene, which is involved in selective protein degradation. We were also able to show that expression of ENO1, DFFA, UBE4B and CASP9, critical for induction of the apoptotic cascade, is down-regulated in tumors with unfavorable outcome. We identified a novel gene, denoted APITD1, in 1p36.22; two alternative transcripts of this gene were abundantly expressed in normal adult and fetal tissues. Primary NB tumors showed either very weak or no measurable APITD1 expression and the lowest levels were detected in tumors with unfavorable outcome. The coding sequence of APITD1 was well-conserved in different species and no coding variations were found in primary NB tumors. Anti-proliferative and apoptotic effects were observed after over-expression of APITD1 and ENO1 in NB cell lines and in embryonal kidney- derived cells. A mutated ENO1 construct was used to show that the glycolytic enzyme á-enolase had as strong an effect on cell proliferation as the other translational product, MBP1, which is a negative regulator of the c-Myc oncogene. Biallelic inactivation of the transcription factor PHOX2B on chromosome 4p was found in a patient with multifocal NB, emphasizing the importance of early embryologic differentiation pathways in tumors of neural crest origin. Conclusions: A few rare mutations were found in genes with possible tumor suppressor functions. Several genes on 1p36.2 were also shown to be down-regulated in aggressive tumors. We therefore propose that the poor prognosis for NB patients with 1p deletions is a syngenic effect of mutations or transcriptional silencing of several genes in this region.

Neuroblastoma is characterised by a lack of TP53 mutations and no other tumour suppressor gene consistently inactivated has yet been identified in this childhood cancer form. Characterisation of a new gene, denoted APITD1, in the neuroblastoma tumour suppressor candidate region in chromosome 1p36.22 reveals that APITD1 contains a predicted TFIID-31 domain, representing the TATA box-binding protein-associated factor, TAF(II)31, which is required for p53-mediated transcription activation. Two different transcripts of this gene were shown to be ubiquitously expressed, one of them with an elevated expression in foetal tissues. Primary neuroblastoma tumours of all different stages showed either very weak or no measurable APITD1 expression, contrary to the level of expression observed in neuroblastoma cell lines. A reduced pattern of expression was also observed in a set of various tumour types. APITD1 was functionally tested by adding APITD1 mRNA to neuroblastoma cells, leading to the cell growth to be reduced up to 90% compared to control cells, suggesting APITD1 to have a role in a cell death pathway. Furthermore, we determined the genomic organisation of APITD1. Automated genomic DNA sequencing of the coding region of the gene as well as the promoter sequence in 44 neuroblastoma tumours did not reveal any loss-of-function mutations, indicating that mutations in APITD1 is not a common abnormality of neuroblastoma tumours. We suggest that low expression of this gene might interfere with the ability for apoptosis through the p53 pathway.