Susan Pfeifer

The Risk-Associated Long Noncoding RNA NBAT-1 Controls Neuroblastoma Progression by Regulating Cell Proliferation and Neuronal Differentiation

Neuroblastoma is an embryonal tumor of the sympathetic nervous system and the most common extracranial tumor of childhood. By sequencing transcriptomes of low- and high-risk neuroblastomas, we detected differentially expressed annotated and nonannotated long noncoding RNAs (lncRNAs). We identified a lncRNA neuroblastoma associated transcript-1 (NBAT-1) as a biomarker significantly predicting clinical outcome of neuroblastoma. CpG methylation and a high-risk neuroblastoma associated SNP on chromosome 6p22 functionally contribute to NBAT-1 differential expression. Loss of NBAT-1 increases cellular proliferation and invasion. It controls these processes via epigenetic silencing of target genes. NBAT-1 loss affects neuronal differentiation through activation of the neuronal-specific transcription factor NRSF/REST. Thus, loss of NBAT-1 contributes to aggressive neuroblastoma by increasing proliferation and impairing differentiation of neuronal precursors.

The H19 Transcript Is Associated with Polysomes and May Regulate IGF2 Expression in trans

The imprinted H19 gene produces a fully processed transcript that does not exhibit any conserved open reading frame between mouse and man. Although transcriptional control elements associated with the mouse H19 locus have been shown to control the neighboring Igf2 gene incis, the prevailing view is that the cytoplasmicH19 transcript does not display any function. In contrast to earlier reports, we show here that the H19 transcript is associated with polysomes in a variety of cell types, in both mouse and man. A possible trans-function of the H19 gene is suggested by a reciprocal correlation in trans between cytoplasmic H19 and IGF2 mRNA levels, as well as IGF2 mRNA translatability. We discuss these results in terms of their challenge to the prevailing dogma on the function of the enigmatic H19 gene, as well as with respect to the ontogeny of the Beckwith-Wiedemann syndrome, and propose that the human H19 gene is an antagonist of IGF2expressivity in trans.

Classification, incidence and survival analyses of children with CNS tumours diagnosed in Sweden 1984-2005

Aim:  Primary tumours in the central nervous system (CNS) are the second most common malignancy in childhood after leukaemia. Sweden has a high incidence and a high‐survival rate in international comparative studies. This has raised the question about the type of tumours included in the Swedish Cancer registry. We therefore compared international data to the Swedish Childhood Cancer registry.

Epigenetic variability and the evolution of human cancer

Although the leading dogma for the origin of the diversity in cancer cell subpopulations is based on a stepwise selection and accumulation of genetic changes that allow uncontrollable malignant growth, there is an emerging understanding that the variability of heritable phenotypes in cancer and cancer-prone cells may also involve epigenetic mechanisms. We discuss here experimental data that allow us to postulate that the genome is organized into epigenetic territories with lineage-specific differences in the stringencies of the active and inactive states. Low-stringency epigenetic states are predicted to be closer to mosaicism, or chaos, than high-stringency states. In pathological situations, the result is an epigenetic variability upon which selection mechanisms can act during tumor progression. This view may have significant implications on clinical assessment and prognosis, and also suggests that major factors involved in the resetting and/or maintenance of epigenetic states may serve as new attractive targets for therapeutic interventions.

Characterization of novel and complex genomic aberrations in glioblastoma using a 32K BAC array.

Glioblastomas (GBs) are malignant CNS tumors often associated with devastating symptoms. Patients with GB have a very poor prognosis, and despite treatment, most of them die within 12 months from diagnosis. Several pathways, such as the RAS, tumor protein 53 (TP53), and phosphoinositide kinase 3 (PIK3) pathways, as well as the cell cycle control pathway, have been identified to be disrupted in this tumor. However, emerging data suggest that these aberrations represent only a fraction of the genetic changes involved in gliomagenesis. In this study, we have applied a 32K clone-based genomic array, covering 99% of the current assembly of the human genome, to the detailed genetic profiling of a set of 78 GBs. Complex patterns of aberrations, including high and narrow copy number amplicons, as well as a number of homozygously deleted loci, were identified. Amplicons that varied both in number (three on average) and in size (1.4 Mb on average) were frequently detected (81% of the samples). The loci encompassed not only previously reported oncogenes (EGFR, PDGFRA, MDM2, and CDK4) but also numerous novel oncogenes as GRB10, MKLN1, PPARGC1A, HGF, NAV3, CNTN1, SYT1, and ADAMTSL3. BNC2, PTPLAD2, and PTPRE, on the other hand, represent novel candidate tumor suppressor genes encompassed within homozygously deleted loci. Many of these genes are already linked to several forms of cancer; others represent new candidate genes that may serve as prognostic markers or even as therapeutic targets in the future. The large individual variation observed between the samples demonstrates the underlying complexity of the disease and strengthens the demand for an individualized therapy based on the genetic profile of the patient.

Human Cytomegalovirus Tegument Protein pp65 Is Detected in All Intra- and Extra-Axial Brain Tumours Independent of the Tumour Type or Grade

Human cytomegalovirus (HCMV) has been indicated being a significant oncomodulator. Recent reports have suggested that an antiviral treatment alters the outcome of a glioblastoma. We analysed the performance of commercial HCMV-antibodies applying the immunohistochemical (IHC) methods on brain sample obtained from a subject with a verified HCMV infection, on samples obtained from 14 control subjects, and on a tissue microarray block containing cores of various brain tumours. Based on these trials, we selected the best performing antibody and analysed a cohort of 417 extra- and intra-axial brain tumours such as gliomas, medulloblastomas, primary diffuse large B-cell lymphomas, and meningiomas. HCMV protein pp65 immunoreactivity was observed in all types of tumours analysed, and the IHC expression did not depend on the patients age, gender, tumour type, or grade. The labelling pattern observed in the tumours differed from the labelling pattern observed in the tissue with an active HCMV infection. The HCMV protein was expressed in up to 90% of all the tumours investigated. Our results are in accordance with previous reports regarding the HCMV protein expression in glioblastomas and medulloblastomas. In addition, the HCMV protein expression was seen in primary brain lymphomas, low-grade gliomas, and in meningiomas. Our results indicate that the HCMV protein pp65 expression is common in intra- and extra-axial brain tumours. Thus, the assessment of the HCMV expression in tumours of various origins and pathologically altered tissue in conditions such as inflammation, infection, and even degeneration should certainly be facilitated.

RANDOM MONOALLELIC EXPRESSION OF THE IMPRINTED IGF2 AND H19 GENES IN THE ABSENCE OF DISCRIMINATIVE PARENTAL MARKS

Abstract The IGF2 and H19 genes are genomically imprinted and expressed preferentially from the paternal and maternal alleles, respectively, during human prenatal development. The exact role of the parental imprint(s), however, is not known. To explore this issue in some detail, we have examined human androgenetic cells which by definition should be incapable of allelic discrimination given the paternal origin of both genomes. Allele-specific in situ hybridisation analysis of dispermic complete hydatidiform moles shows that IGF2 and H19 can be found to be transcriptionally active in a variegated manner, which results in the generation of random monoallelic expression patterns. This data shows that imprinted genes can be expressed monoallelically in the absence of discriminating parental marks and raises the question whether or not mechanisms underlying monoallelic expression preceded the acquisition of parental imprints during evolution.

The genotype and epigenotype synergize to diversify the spatial pattern of expression of the imprinted H19 gene.

Little is known of how the genetic background effects the phenomenon of genomic imprinting. The H19 gene belongs to a cluster of imprinted genes on human chromosome 11. Here we show that the alternative splicing of a human H19 transcript is genotype-specific. Moreover, this variant transcript, which lacks exon 4, is either not found at all, is widely expressed or is confined to extra-villous cytotrophoblasts in first trimester placenta, depending on a combination of the genotype and the sex of the transmitting parent.

Abstract 3175: Discoveries from whole exome sequencing of medulloblastomas.

Medulloblastoma is the most common malignant brain tumour in children and survivors suffer from serious long-term effects due to current treatment. More advanced molecular markers are desirable to aid in diagnosis/prognosis as well as to guide treatment. Moreover, identification of new targets for development of more efficient therapeutic agents is highly needed. As part of a comprehensive program to detect candidate genes and signaling pathways disrupted in medulloblastoma and brain tumour stem cells (BTSC) we are performing whole exome sequencing on paired tumour and blood samples. So far samples from 21 patients have been analyzed and the results show that damaging somatic small scale mutations in the coding regions are relatively rare in medulloblastoma genomes, in average 12 per patient. Few genes are recurrently mutated in the data set but interestingly, despite the low number, several candidate genes have been found to converge in the same developmental and oncogenic pathways. WNT, SHH and Notch as well as mTOR and AKT are examples of affected pathways already known to be implicated in medulloblastoma. Furthermore, we find damaging mutations among components of the BMP/TGF-beta and RAC/Rho signaling cascades in different subset of tumours. These are important neuronal developmental, differentiation and motility pathways not yet well studied in this pediatric malignancy. Significant findings are also a few mutations in DNA and histone methylation associated genes. The success of generating DNA copy number profiles from the sequencing data have further increased our understanding of the studied samples and several tumour suppressor genes have shown mutation of one allele and loss of the other, as PTEN, KDM6A and DULLARD. The relatively few events per sample may add support to the proposal that medulloblastomas could arise from stem/progenitor cells that are present in normal neuronal development and importantly, already possess the capacity of self renewal and proliferation and therefore might need fewer hits for malignant transformation. Overall these results contain several interesting candidate genes as well as describe the heterogeneity of the disease but also the possibility of being able to pinpoint aberrant pathways in the tumors depending on the genetic status. Additional analyses will be done, including integrating the mutation data with results from RNA-seq, Methyl-seq and ChIP-seq. Comparing genetic and epigenetic status of established BTSC with primary tumours and perform functional studies and evaluation of selected drugs on BTSC is also planned. As targeted therapies are being developed based on new genetic and biology knowledge it is likely that (epi)genomic/genetic analysis will be increasingly implemented in the clinic to aid in diagnosis and treatment choice. This personalized medicine approach should be of great benefit for children with brain tumours as increased survival and quality of life will follow. Citation Format: Johanna Sandgren, Susan Pfeifer, Monica Nister, Teresita Diaz de Stahl. Discoveries from whole exome sequencing of medulloblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3175. doi:10.1158/1538-7445.AM2013-3175