Frequent promoter hypermethylation and transcriptional downregulation of the NDRG2 gene at 14q11.2 in primary glioblastoma.

The N-myc downstream-regulated gene 2 (NDRG2) at 14q11.2 has been reported to be downregulated in glioblastoma, and NDRG2 overexpression represses glioblastoma cell proliferation in vitro (Deng et al., Int J Cancer 2003;106;342-7). To further address the role of NDRG2 as a candidate tumor suppressor in human gliomas, we analyzed 67 astrocytic tumors (10 diffuse astrocytomas, 11 anaplastic astrocytomas, 34 primary glioblastomas and 12 secondary glioblastomas) for NDRG2 gene mutation, promoter methylation and expression at the mRNA and protein levels. Using real-time reverse transcription PCR analysis, we found decreased NDRG2 mRNA levels in primary glioblastomas as compared to diffuse and anaplastic astrocytomas. Similarly, immunohistochemistry revealed low or absent NDRG2 protein expression in primary glioblastomas. Mutational analysis of the entire NDRG2 coding sequence did not reveal any tumor-associated DNA sequence alterations. However, sequencing of sodium bisulfite-modified DNA identified hypermethylation of the NDRG2 promoter region in 21 of 34 primary glioblastomas (62%). Moreover, NDRG2 promoter hypermethylation was associated with decreased NDRG2 mRNA expression. In contrast to primary glioblastomas, NDRG2 promoter hypermethylation was detected in only 1 of 11 anaplastic astrocytomas (9%) and was absent in 10 diffuse astrocytomas and 12 secondary glioblastomas. Taken together, our data support NDRG2 as a candidate tumor suppressor gene that is epigenetically silenced in the majority of primary glioblastomas, but not in lower grade astrocytomas and secondary glioblastomas.

Genotype and protein expression after bone marrow transplantation for adrenoleukodystrophy.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is the most common inherited peroxisomal disorder. It is caused by impaired function of ALD protein that results in accumulation of very long-chain fatty acids in tissues and body fluids. So far, hematopoietic stem cell transplantation (HSCT) constitutes the only curative approach able to prevent the progression of cerebral X-ALD. However, biological mechanisms of this beneficial approach are still unknown. OBJECTIVE: To describe the effect of HSCT in a family with various X-ALD disease forms by ALD mutation and protein expression analysis. DESIGN, SETTING, AND PATIENTS: In a family with various X-ALD forms, an ALD mutation screening was performed. Two boys had cerebral X-ALD and underwent HSCT. One of them is alive and well without any further neurological deterioration, whereas his cousin died of transplantation-related complications at day 76. The postmortem specimens were analyzed by genotyping and immunohistochemical assays. RESULTS: All of the affected family members carry an as-yet-undescribed large ALD gene deletion (NC_000023:g.152512130-152520645del) resulting in a complete lack of ALD protein expression on immunofluorescence analysis. After engraftment, both patients who underwent HSCT showed complete chimerism in blood. Postmortem studies in 1 patient revealed both mutant and wild-type ALD sequences in each of 23 analyzed tissues, indicating mixed chimerism. Furthermore, immunohistochemical staining for ALD protein revealed no differences between patient and control tissues including blood cells, bone marrow, and glial cells as well as neurons. CONCLUSION: To our knowledge, our analysis provides the first evidence for the stable development of a wild-type X-ALD genotype and peroxisomal ALD protein expression in a great variety of human tissues following HSCT.

Integrative genomic analysis identifies NDRG2 as a candidate tumor suppressor gene frequently inactivated in clinically aggressive meningioma.

Although meningiomas are common central nervous system tumors, little is known about the genetic events responsible for malignant progression. In this study, we employed gene expression profiling to identify transcripts whose expression was lost in anaplastic (WHO grade III) versus benign (WHO grade I) meningioma. Approximately 40% of genes down-regulated in anaplastic meningioma were localized to chromosomes 1p and 14q. One specific gene located at 14q11.2, NDRG2, was consistently down-regulated in grade III meningioma, a finding which we validated at both the transcript and protein levels in independent sets of clinically and pathologically diverse meningiomas. Loss of NDRG2 expression was also seen in a subset of lower-grade meningiomas, including atypical meningiomas (WHO grade II) with clinically aggressive behavior. Furthermore, we found that the loss of NDRG2 expression was significantly associated with hypermethylation of the NDRG2 promoter. Collectively, these data identify NDRG2 as the first specific candidate tumor suppressor gene on chromosome 14q that is inactivated during meningioma progression. In addition, these findings highlight the utility of combining genomic, epigenetic, and expression data to identify clinically significant tumor biomarkers, and suggest that NDRG2 expression will be a useful and functionally relevant biomarker to predict aggressive behavior in patients with meningioma.

Molecular classification of human gliomas using matrix-based comparative genomic hybridization.

Gliomas are the most frequent primary brain tumors and comprise a group of morphologically, biologically and clinically heterogeneous neoplasms. The different glioma types are associated with distinct genetic aberrations, which may provide useful information for tumor classification as well as prediction of prognosis and response to therapy. To facilitate the molecular classification of gliomas, we established a genomic microarray that consists of bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones representing tumor suppressor genes, proto-oncogenes and chromosomal regions frequently gained or lost in gliomas. In addition, reference clones distributed evenly throughout the genome in approximately 15 Mbp intervals were spotted on the microarray. These customized microarrays were used for matrix-based comparative genomic hybridization (matrix CGH) analysis of 70 gliomas. Matrix CGH findings were validated by molecular genetic analyses of candidate genes, loss of heterozygosity studies and chromosomal CGH. Our results indicate that matrix CGH allows for the sensitive and specific detection of gene amplifications as well as low-level copy number gains and losses in clinical glioma samples. Furthermore, molecular classification based on matrix CGH data closely paralleled histological classification and was able to distinguish with few exceptions between diffuse astrocytomas and oligodendrogliomas, anaplastic astrocytomas and anaplastic oligodendrogliomas, anaplastic oligodendrogliomas and glioblastomas, as well as primary and secondary glioblastomas. Thus, matrix CGH is a powerful technique that allows for an automated genomic profiling of gliomas and represents a promising new tool for their molecular classification.

Candidate genes in breast cancer revealed by microarray-based comparative genomic hybridization of archived tissue.

Genomic imbalances in 31 formalin-fixed and paraffin-embedded primary tumors of advanced breast cancer were analyzed by microarray-based comparative genomic hybridization (matrix-CGH). A DNA chip was designed comprising 422 mapped genomic sequences including 47 proto-oncogenes, 15 tumor suppressor genes, as well as frequently imbalanced chromosomal regions. Analysis of the data was challenging due to the impaired quality of DNA prepared from paraffin-embedded samples. Nevertheless, using a method for the statistical evaluation of the balanced state for each individual experiment, we were able to reveal imbalances with high significance, which were in good concordance with previous data collected by chromosomal CGH from the same patients. Owing to the improved resolution of matrix-CGH, genomic imbalances could be narrowed down to the level of individual bacterial artificial chromosome and P1-derived artificial chromosome clones. On average 37 gains and 13 losses per tumor cell genome were scored. Gains in more than 30% of the cases were found on 1p, 1q, 6p, 7p, 8q, 9q, 11q, 12q, 17p, 17q, 20q, and 22q, and losses on 6q, 9p, 11q, and 17p. Of the 51 chromosomal regions found amplified by matrix-CGH, only 12 had been identified by chromosomal CGH. Within these 51 amplicons, genome database information defined 112 candidate genes, 44 of which were validated by either PCR amplification of sequence tag sites or DNA sequence analysis.

Microarray-based gene expression profiling of benign, atypical and anaplastic meningiomas identifies novel genes associated with meningioma progression.

To identify gene expression profiles associated with human meningiomas of different World Health Organization (WHO) malignancy grades, we analyzed 30 tumors (13 benign meningiomas, WHO grade I; 12 atypical meningiomas, WHO grade II; 5 anaplastic meningiomas, WHO grade III) for the expression of 2,600 genes using cDNA-microarray technology. Receiver operator curve (ROC) analysis with a cutoff value of 45% selection probability identified 37 genes with decreased and 27 genes with increased expression in atypical and anaplastic meningiomas, compared to benign meningiomas. Supervised classification of the tumors did not reveal specific expression patterns representative of each WHO grade. However, anaplastic meningiomas could be distinguished from benign meningiomas by differential expression of a distinct set of genes, including several ones associated with cell cycle regulation and proliferation. Investigation of potential correlations between microarray expression data and genomic aberrations, detected by comparative genomic hybridization (CGH), demonstrated that losses on chromosomes 10 and 14 were associated with distinct expression profiles, including increased expression of several genes related to the insulin-like growth factor (IGF) (IGF2, IGFBP3 and AKT3) or wingless (WNT) (CTNNB1, CDK5R1, ENC1 and CCND1) pathways. Taken together, our microarray-based expression profiling revealed interesting novel candidate genes and pathways that may contribute to meningioma progression.