M. Eva Alonso

Genetic and epigenetic alteration of the NF2 gene in sporadic meningiomas

The role of the NF2 gene in the development of meningiomas has recently been documented; inactivating mutations plus allelic loss at 22q, the site of this gene (at 22q12), have been identified in both sporadic and neurofibromatosis type 2–associated tumors. Although epigenetic inactivation through aberrant CpG island methylation of the NF2 5′ flanking region has been documented in schwannoma (another NF2‐associated neoplasm), data on participation of this epigenetic modification in meningiomas are not yet widely available. Using methylation‐specific PCR (MSP) plus sequencing, we assessed the presence of aberrant promoter NF2 methylation in a series of 88 meningiomas (61 grade I, 24 grade II, and 3 grade III), in which the allelic constitution at 22q and the NF2 mutational status also were determined by RFLP/microsatellite and PCR‐SSCP analyses. Chromosome 22 allelic loss, NF2 gene mutation, and aberrant NF2 promoter methylation were detected in 49%, 24%, and 26% of cases, respectively. Aberrant NF2 methylation with loss of heterozygosity (LOH) at 22q was found in five cases, and aberrant methylation with NF2 mutation in another; LOH 22q and the mutation were found in 16 samples. The aberrant methylation of the NF2 gene also was the sole alteration in 15 samples, most of which were from grade I tumors. These results indicate that aberrant NF2 hypermethylation may participate in the development of a significant proportion of sporadic meningiomas, primarily those of grade I. ©2005 Wiley‐Liss, Inc.

Analysis of p73 gene in meningiomas with deletion at 1p

The p73 gene has been mapped to 1p36.33, a chromosome region that is frequently deleted in a wide variety of neoplasms including meningiomas. The protein encoded by p73 shows structural and functional similarities to p53 and may thus represent a candidate tumor suppressor gene. To determine whether p73 is involved in the development of meningiomas, we examined 30 meningioma samples with proven 1p deletion for mutations of p73. Sequence analysis of the entire coding region of the p73 gene revealed previously reported polymorphisms in eight cases. A tumor-specific missense mutation as a result of an A-to-G transition with an Asn204Ser change was found in one meningioma that nevertheless retained the normal allele. These results suggest that if p73 plays a role in meningioma carcinogenesis, it must be in a manner different from the Knudson two-hit model.

DNA methylation of multiple promoter-associated CpG islands in meningiomas: relationship with the allelic status at 1p and 22q

The purpose of this research was to examine the DNA methylation profile of meningiomas. Accordingly, we examined the DNA methylation status of ten tumor-related genes (RB1, p16INK4a, p73, MGMT, ER, DAPK, TIMP-3, p14ARF, THBS1, and Caspase-8) in 98 meningiomas (68 grade I; 27 grade II; and 3 grade III samples) using methylation-specific PCR and sequencing. The most frequently methylated genes were THBS1 (30%), TIMP-3 (24%), p16INK4a (17%), MGMT (16%), p73 (15%), ER (15%), and p14ARF (13%), whereas methylation was relatively rare in the other genes (<10%). Methylation occurred in at least one gene in 77.5% of the cases and in three or more genes in 25.5%. Methylation was tumor specific since it was absent in the controls: two non-neoplastic meningeal samples and two non-neoplastic brain samples. The frequency of aberrant gene methylation in grade I versus grade II–III tumors showed some differences for TIMP-3, THBS1, MGMT, p16INK4a and p73; these differences reached statistical significance for TIMP-3: 18% in grade I versus 40% in grade II–III (P<0.02). Our previous loss of heterozygosity studies provided the allelic constitution at 1p and 22q for 60 of the 98 meningiomas included in this report. The level of aberrant promoter methylation increased in tumors (30 samples) displaying 1p loss (either isolated or as concurrent deletion at 1p/22q; P=0.014). These meningiomas primarily accumulated the epigenetic changes of THBS1 (14/30; 47%; P<0.005), TIMP-3 (12/30; 40%; P<0.05), p73 (10/30; 26%; P<0.02) and p14ARF/p16INK4a(7/30 each one; 23%; not significant). Our findings indicate that aberrant DNA methylation of promoter-associated CpG islands in meningiomas contributes to the development of these tumors.

Aberrant CpG island methylation of multiple genes in ependymal tumors.

Aberrant methylation of promoter CpG islands in human genes represents an alternative mechanism for genetic inactivation, and contributes to the development of human tumors. Nevertheless, thus far, few reports have analyzed methylation in ependymomas. We determined the frequency of aberrant CpG island methylation of several tumor-associated genes: p16INK4a, RB1, MGMT, DAPK, TIMP3, THBS1, TP73, NF2 and Caspase 8 in a group of 27 ependymomas, consisting of 22 WHO grade II samples and five anaplastic WHO grade III tumors. The respective methylation indices (number of genes methylated/total genes analyzed) for both tumor groups was 0.195 and 0.198. Overall methylation rates greater than 20% were detected in MGMT, TIMP3, THBS1 and TP73. NF2 and Caspase 8 each presented hypermethylation in less than 10% of cases, and the cell-cycle regulators RB1/p16INK4a were hypermethylated in 4% and 18% of the samples, respectively, mostly affecting the low-grade forms. Our findings suggest that methylation commonly contributes to the inactivation of cancer-related genes in ependymomas.

Analysis of the NF2 gene in oligodendrogliomas and ependymomas

Allelic losses of chromosome 22 are commonly found in ependymomas and oligodendrogliomas, suggesting that at least one tumor suppressor gene on chromosome 22 must be inactivated during the multistep process of tumorigenesis in these glial tumors. The neurofibromatosis 2 gene (NF2) located at 22q12, is a candidate tumor suppressor gene potentially involved in the pathogenesis of gliomas. Because there have been only a few studies of the NF2 gene in glial tumors other than astrocytoma, we screened the entire 17 NF2 exons for mutations in a series of 47 nonastrocytic tumors, including 40 oligodendrogliomas and 7 ependymomas. Only one mutation was detected, a 59-base pair insertion in exon 3 from a spinal anaplastic ependymoma. These results concur with previous findings proposing preferential inactivation of the NF2 gene in a subgroup of ependymomas, and suggest that the NF2 gene is not the target of chromosome 22 aberrations in oligodendrogliomas.

CpG island methylation of tumor-related genes in three primary central nervous system lymphomas in immunocompetent patients

We have determined the promoter CpG island methylation status of O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione-S-transferase P1 (GSTP1), death-associated protein kinase (DAPK), p14(ARF), thrombospondin-1 (THBS1), tissue inhibitor of metalloproteinase-3 gene (TIMP-3), p73, p16(INK4A), RB1, and TP53 genes in three primary central nervous system lymphomas (PCNSL). Five genes (GSTP1, DAPK, TIMP-3, p16(INK4A), and RB1) were hypermethylated in two samples, whereas MGMT, THBS1, and p73 were aberrantly methylated in only one sample. No case presented CpG island methylation for the p14(ARF) and TP53 genes. These findings concur with previous data suggesting a frequent inactivation of p16(INK4A) and very limited involvement of TP53 in PCNSL and also provide insights into the epigenetic molecular involvement of other tumor-related genes in this neoplasm.

Real-Time Quantitative PCR Analysis of Gene Dosages Reveals Gene Amplification in Low-Grade Oligodendrogliomas

Proto-oncogene amplification is an important alteration that is present in about 45% to 50% of high-grade human gliomas. We studied this mechanism in 8 genes (cyclin-dependent kinase-4 [CDK4], MDM2, MDM4, renin-angiotensin system-1, ELF3, GAC1, human epidermal growth factor receptor-2, and platelet-derived growth factor receptor-A gene) in a series of 40 oligodendrogliomas (World Health Organization (WHO) grade II, 21; WHO grade III, 13; and WHO grade II-III oligoastrocytomas, 6) using real-time quantitative polymerase chain reaction. Amplification of at least 1 of these genes was detected in 58% of samples (23/40). By histopathologic grade, 67% of grade II oligodendrogliomas (14/21), 46% of grade III anaplastic oligodendrogliomas (6/13), and 50% of mixed oligoastrocytomas (3/6) were positive for amplification of at least 1 gene. CDK4, MDM2, and GAC1 were the most frequently involved genes (12/40 [30%], 12/40 [30%], and 13/40 [33%], respectively). Our findings demonstrate gene amplification in low-grade samples indicating that it is an important alteration in the early steps of oligodendroglioma development and, therefore, might be considered a molecular mechanism leading to malignant progression toward anaplastic forms.

Real-time Quantitative Pcr Analysis of Regions Involved in Gene Amplification Reveals Gene Overdose in Low-grade Astrocytic Gliomas

We have studied gene amplification of genes located in 1q32 (GAC1, ELF3, MDM4, and ren1), 4q11 (PDGFR-α), and in 12q13-14 (MDM2 and CDK4) using quantitative real-time PCR in a group of 86 tumors consisting of 44 WHO grade IV glioblastomas (GBM) (34 primary and 10 secondary tumors), 21 WHO grade III anaplastic astrocytomas (AA), and 21 WHO grade II astrocytomas (AII). Gene amplification was present in 56 of the 86 samples (65%) in at least 1 gene in our series. GAC1 (51%) and MDM4 (27%) were the most frequently amplified genes within the 1q32 amplicon, and their higher amplification frequency was statistically significant (P < 0.05, χ2) in the low-grade astrocytomas. Concordant coamplification was determined for ELF3 and ren1 or ren1 and MDM4 in the grade III-IV tumors. MDM2 amplification was significantly more frequent in primary GBM (16%) than was in secondary GBM (0%). The present study shows that gene amplification in the studied regions is already present in low-grade astrocytic tumors and that amplification of some genes may represent another molecular marker to differentiate primary from secondary GBM.