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In neuroblastoma, tumor biopsies are used for prognostic evaluation and risk assessment by molecular genetic analyses such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array CGH). Analysis of primary tumors by array CGH can be hampered by the lack of sufficient tumor cells due to small biopsy size or availability of invaded bone marrow only. Given the importance of accurate assessment of genetic alterations in the diagnostic work-up of patients with neuroblastoma, we evaluated the possibility to analyze bone marrow metastases in patients with disseminated disease. Disseminated neuroblastoma cells were isolated from bone marrow aspirates by using either laser capture microdissection (LCM) or magnetic activated cell sorting (MACS). The array CGH profiles of these isolated metastases were compared to array CGH profiles and/or FISH data of the corresponding primary tumor. Here, we show that the major recurrent DNA copy number alterations detected in primary neuroblastoma tumors (i.e., 1p, 3p and 11q deletion, 17q gain and MYCN amplification) can be detected, with high sensitivity and specificity, in the disseminated neuroblastoma cells isolated from the bone marrow aspirates, using an array platform with high coverage for these regions. Moreover, we demonstrate that for archived material, for example, for retrospective studies, LCM is the method of choice, while for fresh bone marrow aspirates, acquired at the time of diagnosis, MACS is superior., (Copyright © 2011 UICC.)

Partial gain of chromosome arm 17q is the most frequent genetic change in neuroblastoma (NB) and constitutes the strongest independent genetic factor for adverse prognosis. It is assumed that 1 or more genes on 17q contribute to NB pathogenesis by a gene dosage effect. In the present study, we applied chromosome 17 tiling path BAC arrays on a panel of 69 primary tumors and 28 NB cell lines in order to reduce the current smallest region of gain and facilitate identification of candidate dosage sensitive genes. In all tumors and cell lines with 17q gain, large distal segments were consistently present in extra copies and no interstitial gains were observed. In addition to these large regions of distal gain with breakpoints proximal to coordinate 44.3 Mb (17q21.32), smaller regions of gain (distal to coordinate 60 Mb at 17q24.1) were found superimposed on the larger region in a minority of cases. Positional gene enrichment analysis for 17q genes overexpressed in NB showed that dosage sensitive NB oncogenes are most likely located in the gained region immediately distal to the most distal breakpoint of the 2 breakpoint regions. Interestingly, comparison of gene expression profiles between primary tumors and normal fetal adrenal neuroblasts revealed 2 gene clusters on chromosome 17q that are overexpressed in NB, i.e. a region on 17q21.32 immediately distal to the most distal breakpoint (in cases with single regions of gain) and 17q24.1, a region coinciding with breakpoints leading to superimposed gain., ((c) 2007 Wiley-Liss, Inc.)

The recurrent loss of 3p segments in neuroblastoma suggests the implication of 1 or more tumor suppressor genes but thus far few efforts have been made to pinpoint their detailed chromosomal position. To achieve this goal, array-based comparative genomic hybridization was performed on a panel of 23 neuroblastoma cell lines and 75 primary tumors using a tiling-path bacterial artificial chromosome array for chromosome 3p. A total of 45 chromosome 3 losses were detected, including whole chromosome losses, large terminal deletions and interstitial deletions. The latter, observed in cell lines as well as a number of distal deletions detected in primary tumors, allowed us to demarcate 3 minimal regions of loss of 3.6 Mb [3p21.31-p21.2, shortest regions of overlap (SRO)1], 1.4 Mb (3p22.3-3p22.2, SRO2) and 3.8 Mb (3p25.3-p25.1, SRO3) in size. The present data significantly extend previous findings and now firmly establish critical regions on 3p implicated in neuroblastoma. Interestingly, the 2 proximal regions coincide with previously defined SROs on 3p21.3 in more frequent tumors including lung and breast cancer. As such, similar tumor suppressor genes may play a critical role in development or progression of a variety of neoplasms, including neuroblastoma.

Deletions of the short arm of chromosome 3 are often observed in a specific subset of aggressive neuroblastomas (NBs) with loss of distal 11q and without MYCN amplification. The critical deleted region encompasses the locus of the von Hippel-Lindau gene (VHL, 3p25). Constitutional loss of function mutations in the VHL gene are responsible for the VHL syndrome, a dominantly inherited familial cancer syndrome predisposing to a variety of neoplasms, including pheochromocytoma. Pheochromocytomas are, like NB, derived from neural crest cells, but, unlike NB, consist of more mature chromaffin cells instead of immature neuroblasts. Further arguments for a putative role of VHL in NB are its function as oxygen sensitizer and the reported relation between hypoxia and dedifferentiation of NB cells, leading to a more aggressive phenotype. To test the possible involvement of VHL in NB, we did mRNA expression analysis and sought evidence for VHL gene inactivation. Although no evidence for a classic tumor suppressor role for VHL in NB could be obtained, a strong correlation was observed between reduced levels of VHL mRNA and low patient survival probability (p=0.013). Furthermore, VHL appears to have predictive power in NTRK1 (TRKA) positive tumor samples with presumed favorable prognosis, which makes it a potentially valuable marker for more accurate risk assessment in this subgroup of patients. The significance of the reduced VHL expression levels in relation to NB tumor biology remains unexplained, as functional analysis demonstrated no clear effect of the reduction in VHL mRNA expression on protein stability of its downstream target hypoxia-inducible factor alpha., (Copyright (c) 2006 Wiley-Liss, Inc.)

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine tumor of the skin. Cytogenetic studies have indicated that deletions and unbalanced translocations involving chromosome 1 short arm material occur in 40% of the investigated cases. Recurrent chromosomal imbalances detected by comparative genomic hybridization (CGH) analysis were loss of 3p, 10q, 13q and 17p and gains of 1q, 3q, 5p and 8q. In order to study genomic aberrations occurring in MCC in further detail, we combined karyotyping, CGH and multiplex-fluorescence in situ hybridization (M-FISH), a strategy that proved to be successful in the analysis of other malignancies. Analysis of 6 MCC cell lines and 1 MCC tumor revealed mostly near-diploid karyotypes with an average of 5 chromosomal rearrangements. The observed karyotypic changes were heterogeneous, with 3-27 breakpoints per case, leading to imbalance of the involved chromosomal regions that was confirmed by CGH. Chromosomal rearrangements involving the short arm of chromosome 1, the long arm of chromosome 3 and gain of 5p material were the most frequently observed abnormalities in our study. In keeping with previous observations, this series of MCCs showed no evidence for high-level amplification. We provid a detailed description of chromosomal translocations occurring in MCC that could be useful to direct future intensive investigation of these chromosomal regions., (Copyright 2002 Wiley-Liss, Inc.)

Merkel cell carcinoma (MCC) is a rare, highly metastatic skin tumor of neuroectodermal origin. The disease shares clinical and histopathological features with small cell lung carcinoma (SCLC). The genetic mechanisms underlying the development and tumor progression of MCC are poorly understood. We recently showed by comparative genomic hybridization (CGH) that the pattern of chromosomal abnormalities in MCC resembles that of SCLC. One of the most frequently observed losses involved the entire chromosome 10 or partial loss of the chromosome 10 long arm (33% of examined MCC cases). The PTEN tumor-suppressor gene has been mapped to 10q23.3 and was shown to be mutated in a variety of human cancers including SCLC. Germline PTEN mutations have been observed in familial predisposing cancer syndromes including Cowden disease. Interestingly, an association between Cowden syndrome and Merkel cell carcinoma has been reported. To study the possible role of PTEN in MCC oncogenesis, loss of heterozygosity (LOH) analysis for the 10q23 region was performed on 26 MCC tumor samples from 23 MCC patients. The PTEN locus was deleted in 9 of 21 (43%) informative MCC tumor samples [7 of 18 (39%) MCC patients]. Despite this high frequency of LOH at 10q23, mutation and homozygous deletion screening of the PTEN gene revealed only one tumor with a nonsense mutation and a second with a homozygous deletion of exon 9. These data suggest that either alternative mechanisms lead to inactivation of the PTEN gene or that other tumor-suppressor genes at chromosome 10 are implicated in the development of MCC., (Copyright 2001 Wiley-Liss, Inc.)