Original article
Positional gene expression analysis identifies 12q overexpression and amplification in a subset of neuroblastomas

https://doi.org/10.1016/j.cancergencyto.2004.02.009Get rights and content

Abstract

Neuroblastoma is a heterogeneous disease with variable clinical behaviors. Unique molecular features are associated with clinically relevant subgroups. We performed a comprehensive microarray gene expression analysis of 95 neuroblastomas in an effort to define clinically important molecular subtypes. A subset of tumors overexpressed several contiguous genes located at 12q13∼q15 and were studied further. By microarray, 5 of 95 neuroblastomas had overexpression of genes mapped to 12q13.1∼q15, suggesting an amplification event in this region. Positional expression mapping identified the narrowest region of overlap containing 21 genes, with 11 genes overexpressed in all five cases. Fluorescence in situ hybridization demonstrated 3 neuroblastomas with more than a 10-fold increase in 12q gene copies and 9 with 3- to 5-fold increases. Amplification and overexpression of genes at 12q13∼q15 were observed in a small subset of neuroblastomas. Although amplification of 12q has been previously reported in neuroblastoma cell lines, this is the first demonstration in tumor samples, and it defines a distinct subset that has not been described previously. The expressed genes mapped closely to the complex amplicon reported in sarcomas, and they identify critical genes and pathways affected by 12q gene amplification.

Introduction

Neuroblastoma is a biologically and clinically heterogenous disease. The clinical spectrum can range from spontaneous regression and maturation to aggressive dissemination despite multimodal therapy. Molecular genetics have been used to characterize and define clinically relevant subgroups of neuroblastoma [1], [2], [3], and several genetic alterations correlate with outcome and response to therapy (e.g., 1p loss, 17q gain, MYCN amplification, etc.) [4], [5], [6], [7]. Recent genome-wide analysis has identified novel regions of frequent allelic imbalance in neuroblastoma that may also be associated with distinct clinical subgroups [8]. In an effort to further define molecular subsets of neuroblastoma and to identify critically important genes, we performed genome-wide expression studies of human neuroblastic tumor samples. A subset of neuroblastomas was found to have high-level overexpression of genes at 12q13∼q15. The 12q region is altered in some tumors and is believed to harbor genes that may contribute to tumorigenesis. Amplification of 12q has been detected in sarcomas and gliomas, and several genes in this region are candidate oncogenes. In addition, allelic imbalance of 12q has been detected in human neuroblastoma and amplification has been reported in neuroblastoma cell lines [9], [10], [11], [12], [13], [14], [15], [16]. We further characterized the frequency of 12q gene overexpression and amplification in neuroblastomas and identified candidate oncogenes.

Section snippets

Patient tumor samples

A total of 129 samples were subjected to expression analysis, including 95 neuroblastomas, 12 ganglioneuromas, 12 cell lines, and 10 normal bone marrows.

Samples were snap-frozen immediately after surgical removal. Of the 95 neuroblastomas, 8 were stage 4S, 5 were stage 1, 13 were stage 2, 16 were stage 3, and 53 were stage 4 (International Neuroblastoma Staging System [INSS]). Histologic sections of the frozen tissue samples were reviewed and manually dissected to provide consistency and avoid

Contiguous genes at 12q are overexpressed in neuroblastoma

We performed a genome-wide expression analysis of 95 neuroblastomas using oligonucleotide arrays. Of the 95 neuroblastoma tumor samples, 5 demonstrated strong overexpression of contiguous genes at 12q13∼q15 (at least 2 standard deviations above the mean), and a focused analysis of gene expression in the region was carried out. All predicted genes at 12q13∼q15 currently listed in the human genome databases were identified (http://bioinformatics.weizmann.ac.il/cards/, //www.ncbi.nlm.nih.gov/LocusLink/

Discussion

The diverse clinical and molecular features of neuroblastomas have been widely investigated. At lease three disease patterns are recognized: (1) infants with widespread disease that can spontaneously regress without medical intervention (stage 4S); (2) local-regional disease that may recur but does not metastasize to the bone or bone marrow (stages 1–3); and (3) systemic disease with widespread metastasis that responds to cytotoxic therapy but frequently becomes resistant to medical treatment

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