Fredrik Hedborg

Differentiation and Survival Influences of Growth Factors in Human Neuroblastoma

Human neuroblastoma cell lines are established from high-stage, highly malignant tumours. Despite this and the fact that these tumours are arrested at an early, immature stage, many cell lines have the capacity to undergo neuronal differentiation under proper growth conditions. One such cell line is the noradrenergic SH-SY5Y cell line. These cells can be induced to mature by a variety of modalities, resulting in different mature phenotypes. The use of this cell system as a model to study the stem cell character of neuroblastoma is reviewed and discussed. In particular, we focus on growth factor dependencies in the SH-SY5Y system, and compare that to the normal situation, i.e. growth factor control of sympathetic neuronal and neuroendocrine differentiation during human and rat embryogenesis.

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.

Emergence of New ALK Mutations at Relapse of Neuroblastoma

PURPOSE In neuroblastoma, the ALK receptor tyrosine kinase is activated by point mutations. We investigated the potential role of ALK mutations in neuroblastoma clonal evolution. METHODS We analyzed ALK mutations in 54 paired diagnosis-relapse neuroblastoma samples using Sanger sequencing. When an ALK mutation was observed in one paired sample, a minor mutated component in the other sample was searched for by more than 100,000× deep sequencing of the relevant hotspot, with a sensitivity of 0.17%. RESULTS All nine ALK-mutated cases at diagnosis demonstrated the same mutation at relapse, in one case in only one of several relapse nodules. In five additional cases, the mutation seemed to be relapse specific, four of which were investigated by deep sequencing. In two cases, no mutation evidence was observed at diagnosis. In one case, the mutation was present at a subclonal level (0.798%) at diagnosis, whereas in another case, two different mutations resulting in identical amino acid changes were detected, one only at diagnosis and the other only at relapse. Further evidence of clonal evolution of ALK-mutated cells was provided by establishment of a fully ALK-mutated cell line from a primary sample with an ALK-mutated cell population at subclonal level (6.6%). CONCLUSION In neuroblastoma, subclonal ALK mutations can be present at diagnosis with subsequent clonal expansion at relapse. Given the potential of ALK-targeted therapy, the significant spatiotemporal variation of ALK mutations is of utmost importance, highlighting the potential of deep sequencing for detection of subclonal mutations with a sensitivity 100-fold that of Sanger sequencing and the importance of serial samplings for therapeutic decisions.

Inhibition of angiogenesis induces chromaffin differentiation and apoptosis in neuroblastoma.

Inhibition of angiogenesis has been shown to reduce tumor growth, metastasis, and tumor microvascular density in experimental models. To these effects we would now like to add induction of differentiation, based on biological analysis of xenografted human neuroblastoma (SH-SY5Y, WAG rnu/rnu) treated with the angiogenesis inhibitor TNP-470. Treatment with TNP-470 (10 mg/kg s.c., n = 15) reduced the tumor growth by 66% and stereological vascular parameters (Lv, Vv, Sv) by 36-45%. The tumor cell apoptotic fraction increased more than threefold, resulting in a decrease in viable tumor cells by 33%. In contrast, the mean vascular diameter (29 microm) and the mean tumor cell proliferative index (49%) were unaffected. TNP-470-treated tumors exhibited striking chromaffin differentiation of neuroblastoma cells, observed as increased expression of insulin-like growth factor II gene (+88%), tyrosine hydroxylase (+96%), chromogranin A, and cellular processes. Statistical analysis revealed an inverse correlation between differentiation and angiogenesis. It is suggested that by inhibiting angiogenesis, TNP-470 induces metabolic stress, resulting in chromaffin differentiation and apoptosis in neuroblastoma. Such agonal differentiation may be the link between angiostatic therapy and tumor cell apoptosis.

Delimitation of a critical tumour suppressor region at distal 1p in neuroblastoma tumours

We analysed DNA from 68 neuroblastoma tumours for loss of heterozygosity (LOH) on the distal chromosome 1p (1p-LOH) using PCR-based DNA polymorphisms. Fifteen tumours (22%) displayed 1p-LOH. The shortest region of overlap (SRO) for the deletions was defined proximally by marker D1S244 and distally by marker D1S80. The CDC2L1 locus, located on chromosome 1p36, has been put forward as a neuroblastoma tumour suppressor. We analysed coding regions of the CDC2L1 gene in a subset of aggressive neuroblastoma tumours with known allelic loss for different 1p-markers. Single-stranded conformation polymorphism, heteroduplex and sequencing analysis of tumour DNA did not reveal any significant changes in the coding region. Using a DNA sequence polymorphism, we showed, in a primary tumour with an interstitial allelic deletion, that this tumour had both alleles of the CDC2L1 locus retained in the tumour. Thus, we showed that the neuroblastoma tumour suppressor critical region on 1p in our material is defined by loci D1S244 and D1S80 and that the CDC2L1 locus is distal to the critical region.

Homozygous deletion of the neurofibromatosis-1 gene in the tumor of a patient with neuroblastoma

The neurofibromatosis type 1 (von Recklinghausen, NF1) gene has been proposed as a suppressor gene in tumors associated with neurofibromatosis. Recent publications have indicated that the NF1 gene can be rearranged in neuroblastoma cell lines. We analyzed DNA from a neuroblastoma patient with NF1 inherited as a familial trait on the paternal side. Using PCR and Southern techniques we showed that the patient had a constitutional deletion of several exons of the paternally derived NF1 gene and that the maternal copy of the gene had been deleted in the tumor of the patient. This is the first instance of a homozygous deletion reported in a primary neuroblastoma tumor. This suggests that NF1 inactivation in involved in the development or progression of some neuroblastomas in agreement with the hypothesized two hit model of inactivation for a tumor suppressor. These results are concordant with other groups that have detected unbalanced translocations t(1;17) in neuroblastoma tumors, with a breakpoint in chromosome 17 that may coincide with the location of the NF1 gene.

Biochemical evidence for a mature phenotype in morphologically poorly differentiated neuroblastomas with a favourable outcome

Neuroblastoma is an embryonal tumour of the sympathetic nervous system with marked heterogeneity in terms of histological maturity and clinical course. A previous study revealed that high tumour levels of the csrc protein, particularly its neuronal isoform (pp60csrcN), correlated with favourable outcome. To test whether this feature reflects a higher degree of neuronal maturation in these tumours, an extended series (47 consecutive neuroblastomas and 10 ganglioneuromas) were analysed for levels of csrc protein isoforms, neuron-specific enolase, and synaptophysin. Immunoblotting and radioimmunoassay techniques were employed. The results were compared with conventional histological signs of neuronal maturation. High pp60csrcN levels were specific for prognostically favourable neuroblastomas and correlated with high neuronal marker levels. However, signs of histological maturation correlated poorly with these parameters. It is therefore concluded that low stage tumours are highly differentiated in biochemical terms despite their frequently immature histology. Furthermore, the clinical usefulness of these biochemical parameters as prognostic markers was compared with established parameters in a multivariate analysis. Stage 4 disease, MYCN amplification, and age above 18 months at diagnosis was the most powerful combination of variables found for predicting a poor outcome. As expected, none of the neuronal differentiation markers investigated could add to the prediction of aggressive disease when compared with this model. However, high expression of pp60csrcN appeared to be useful in predicting long-term survival in high stage infant neuroblastoma.

Evidence for hypoxia-induced neuronal-to-chromaffin metaplasia in neuroblastoma

We present evidence that in neuroblastoma, a pediatric malignancy of embryonal sympathetic origin, hypoxia, underlies a phenotypic switch from a primitive neuronal to a chromaffin cell type. This conclusion is based on morphological and molecular data on 116 clinical tumors and is supported by data on the phenotypic effects of hypoxia on neuroblastoma cell lines when studied in monolayer culture and as tumor xenografts. In the clinical material, extensive chromaffin features were seen in regions of chronic tumor hypoxia. This was the exclusive form of intratumoral maturation of stroma‐poor tumors and was also seen in stroma‐rich tumors, either exclusively or in combination with ganglion‐like cells. In neuroblastoma cell lines, hypoxia induced changes in gene expression associated with the chromaffin features observed in vivo. We therefore propose tumor hypoxia as a major cue determining phenotype in sympathetic tumors of neuroblastic origin. Because it appears to be reversible upon reoxygenation in monolayer culture, we suggest the term metaplasia for the phenomenon.

Nuclear receptor binding protein 2 is induced during neural progenitor differentiation and affects cell survival.

We previously identified nuclear receptor binding protein 2 (NRBP2) in a screen for genes induced by differentiation of neural stem/progenitor cells. Here we show that during embryonic mouse brain development NRBP2 was expressed in the walls of the third and fourth ventricles, and in the hippocampus. In the adult brain, Purkinje cells of the cerebellum and neurons in the CA3 region of the hippocampus were main sites of NRBP2 expression. Analysis of a pediatric medulloblastoma showed that clusters of NRBP2 positive tumor cells co-expressed neurofilament, but not GFAP. Thus, NRBP2 was associated with neuronal differentiation both in normal and malignant brain tissue. We report that NRBP2 is a 55-60 kDa protein with mainly cytoplasmic location. In vitro, NRBP2 protein levels increased as neural stem/progenitor cells differentiated, and its down regulation by siRNA rendered neural progenitor cells more vulnerable to apoptosis. NRBP2 has no previously assigned function and our studies suggest a role for NRBP2 in neural progenitor cell survival.

Age dependence of tumor genetics in unfavorable neuroblastoma: arrayCGH profiles of 34 consecutive cases, using a Swedish 25-year neuroblastoma cohort for validation.

BackgroundAggressive neuroblastoma remains a significant cause of childhood cancer death despite current intensive multimodal treatment protocols. The purpose of the present work was to characterize the genetic and clinical diversity of such tumors by high resolution arrayCGH profiling.MethodsBased on a 32K BAC whole-genome tiling path array and using 50-250K Affymetrix SNP array platforms for verification, DNA copy number profiles were generated for 34 consecutive high-risk or lethal outcome neuroblastomas. In addition, age and MYCN amplification (MNA) status were retrieved for 112 unfavorable neuroblastomas of the Swedish Childhood Cancer Registry, representing a 25-year neuroblastoma cohort of Sweden, here used for validation of the findings. Statistical tests used were: Fisher’s exact test, Bayes moderated t-test, independent samples t-test, and correlation analysis.ResultsMNA or segmental 11q loss (11q-) was found in 28/34 tumors. With two exceptions, these aberrations were mutually exclusive. Children with MNA tumors were diagnosed at significantly younger ages than those with 11q- tumors (mean: 27.4 vs. 69.5 months; p=0.008; n=14/12), and MNA tumors had significantly fewer segmental chromosomal aberrations (mean: 5.5 vs. 12.0; p<0.001). Furthermore, in the 11q- tumor group a positive correlation was seen between the number of segmental aberrations and the age at diagnosis (Pearson Correlation 0.606; p=0.037). Among nonMNA/non11q- tumors (n=6), one tumor displayed amplicons on 11q and 12q and three others bore evidence of progression from low-risk tumors due to retrospective evidence of disease six years before diagnosis, or due to tumor profiles with high proportions of numerical chromosomal aberrations. An early age at diagnosis of MNA neuroblastomas was verified by registry data, with an average of 29.2 months for 43 cases that were not included in the present study.ConclusionMNA and segmental 11q loss define two major genetic variants of unfavorable neuroblastoma with apparent differences in their pace of tumor evolution and in genomic integrity. Other possible, but less common, routes in the development of aggressive tumors are progression of low-risk infant-type lesions, and gene amplifications other than MYCN. Knowledge on such nosological diversity of aggressive neuroblastoma might influence future strategies for therapy.