A domain of DNA designated N-myc is amplified 20- to 140-fold in human neuroblastoma cell lines but not in cell lines from other tumor types. N-myc has now been found to be amplified in neuroblastoma tissue from 24 of 63 untreated patients (38 percent). The extent of amplification appears to be bimodal, with amplification of 100- to 300-fold in 12 cases and 3- to 10-fold in 10 others. Amplification was found in 0 of 15 patients with stage 1 or 2 disease, whereas 24 of 48 cases (50 percent) with stage 3 or 4 had evidence of N-myc amplification. These data indicate that N-myc amplification is a common event in untreated human neuroblastomas. Furthermore, N-myc amplification is highly correlated with advanced stages of disease (P less than 0.001) and with the ability to grow in vitro as an established cell line, both of which are associated with a poor prognosis.

https://science.sciencemag.org/content/sci/224/4653/1121.full.pdf

Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage

Neuroblastoma is a tumour derived from primitive cells of the sympathetic nervous system and is the most common solid tumour in childhood. Interestingly, most infants experience complete regression of their disease with minimal therapy, even with metastatic disease. However, older patients frequently have metastatic disease that grows relentlessly, despite even the most intensive multimodality therapy. Recent advances in understanding the biology and genetics of neuroblastomas have allowed classification into low-, intermediate- and high-risk groups. This allows the most appropriate intensity of therapy to be selected - from observation alone to aggressive, multimodality therapy. Future therapies will focus increasingly on the genes and biological pathways that contribute to malignant transformation or progression.

Neuroblastoma: biological insights into a clinical enigma.

Neuroblastoma is a childhood cancer that can be inherited, but the genetic aetiology is largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at chromosome bands 2p23-24 using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate germline missense mutations in the tyrosine kinase domain of ALK that segregated with the disease in eight separate families. Resequencing in 194 high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. Nine of the ten mutations map to critical regions of the kinase domain and were predicted, with high probability, to be oncogenic drivers. Mutations resulted in constitutive phosphorylation, and targeted knockdown of ALK messenger RNA resulted in profound inhibition of growth in all cell lines harbouring mutant or amplified ALK, as well as in two out of six wild-type cell lines for ALK. Our results demonstrate that heritable mutations of ALK are the main cause of familial neuroblastoma, and that germline or acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy.

/pdf/identification-of-alk-as-a-major-familial-neuroblastoma-19e91b95ds.pdf

Identification of ALK as a major familial neuroblastoma predisposition gene

Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.

Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma

Publisher Summary This chapter focuses on the proteins of the Myc network that are essential regulators of cell growth and differentiation. The identification of the Myc partner, Max, in 1991 and the subsequent realization that this protein is the essential dimeric partner for all known c-Myc functions was a major boost to the field and led to a number of very interesting observations and findings. The chapter focuses on c-Myc's role as a transcription factor in the regulation of cell growth, apoptosis, and transformation. The most exciting recent findings suggest that the Myc network not only includes proto-oncoproteins (c-, N-, and L-Myc) but, with the Mad family proteins, also potential tumor suppressors. This together with the fact that Myc proteins as well as Max are essential, as deduced from homozygous disruption of the genes in mice, places the Myc network in a central position in the regulation of cell growth and homeostasis. Genes that have been generated by the duplication of and divergence from an ancestral gene(s) are grouped into families. The myc family of protooncogenes has most likely arisen through such duplications. It currently consists of three well-characterized members; c-myc, N-myc, and L-myc. Two additional genes, B-myc and S-myc, have been identified only in rodents. The c-, N-, and L-myc genes share similar genomic organization and the corresponding proteins contain several regions of high sequence homology. The identification of the Myc dimerization partner Max has significantly advanced our understanding of the molecular function of c-Myc.

Proteins of the Myc Network: Essential Regulators of Cell Growth and Differentiation

Cellular oncogenes comprise a class of genes whose aberrant expression or function may be involved in the development of tumours1. Indeed, several naturally occurring animal and human tumours are associated with consistent alterations in the structure or genomic position of particular cellular oncogenes2–4. Recently, we isolated a DNA segment having limited similarity to c-myc (termed N-myc) from a human neuroblastoma cell line5. Although N-myc was present as a single copy in normal cells, it was selectively amplified up to 140-fold in tumour cells from human neuroblastomas5. Now, we have used somatic cell hybrids to show that N-myc is normally localized on the distal short arm of chromosome 2, and in situ hybridization to localize N-myc to chromosome 2p23–24. Further, in situ hybridization localizes amplified N-myc in neuroblastoma cells to homogeneously staining regions (HSRs) on different chromosomes. Thus, our results suggest that amplification and translocation of N-myc may be interrelated processes associated with human neuroblastoma, and demonstrate that lhe site of N-myc amplification is quite variable and bears no apparent relationship to either the normal single-copy locus or recognized sites of non-random chromosome alteration in human neuroblastoma.

Chromosome localization in normal human cells and neuroblastomas of a gene related to c- myc

The chromosomal locations of the genes for the common alpha subunit of the glycoprotein hormones and the beta subunit of chorionic gonadotropin in humans and mice have been determined by restriction enzyme analysis of DNA isolated from somatic cell hybrids. The CG alpha gene (CGA), detected as a 15-kb BamHI fragment in human DNA by hybridization to CG alpha cDNA, segregated with the chromosome 6 enzyme markers ME1 (malic enzyme, soluble) and SOD2 (superoxide dismutase, mitchondrial) and an intact chromosome 6 in human-rodent hybrids. Cell hybrids containing portions of chromosome 6 allowed the localization of CGA to the q12 leads to q21 region. The greater than 30- and 6.5-kb BamHI CGB fragments hybridizing to human CG beta cDNA segregated concordantly with the human chromosome 19 marker enzymes PEPD (peptidase D) and GPI (glucose phosphate isomerase) and a normal chromosome 19 in karyotyped hybrids. A KpnI-HindIII digest of cell hybrid DNAs indicated that the multiple copies of the CG beta gene are all located on human chromosome 19. In the mouse, the alpha subunit gene, detected by a mouse thyrotropin (TSH) alpha subunit probe, and the CG beta-like sequences (CG beta-LH beta), detected by the human CG beta cDNA probe, are on chromosomes 4 and 7, respectively.

Chromosome assignment of genes encoding the alpha and beta subunits of glycoprotein hormones in man and mouse.

Using human-mouse somatic cell hybrids containing different parts of chromosome 6 and a DNA probe of the oncogene (v-myb) of avian myeloblastosis virus (AMV), we regionally mapped by Southern blot techniques the human cellular myb (c-myb) protooncogene to 6q21----qter.

Regional assignment of human protooncogene c-myb to 6q21----qter.

A panel of 22 monoclonal antibodies (MAbs) recognizing 21 distinct human cell surface antigens was tested by mixed hemadsorption assays for reactivity with a large number of rodent-human somatic cell hybrids containing different subsets of the human chromosome complement. The serological typing results permit the assignment of six gene loci determining cell surface antigens to human chromosomes 3, 6, 11, 19, 20, and 22. In addition, analysis of hybrids retaining deleted copies (but no normal homologs) of specific human chromosomes provides regional assignments for 18 gene loci, located on seven different chromosomes. These findings extend and refine the genetic map for human cell surface antigens and identify new selectable markers for defined chromosome segments.

Karl Heinz Grzeschik

Papers

Chromosome localization in normal human cells and neuroblastomas of a gene related to c- myc

Chromosome assignment of genes encoding the alpha and beta subunits of glycoprotein hormones in man and mouse.

Regional assignment of human protooncogene c-myb to 6q21----qter.

Definition of selectable cell surface markers for human chromosomes and chromosome segments in rodent-human hybrids.

Die pathologische Anatomie des menschlichens Genoms