Alterations of the tumor suppressor genes CDKN2A (p16(INK4a)), p14(ARF), CDKN2B (p15(INK4b)), and CDKN2C (p18(INK4c)) in atypical and anaplastic meningiomas

Abstract

We investigated 67 meningothelial tumors (20 benign meningiomas, 34 atypical meningiomas, and 13 anaplastic meningiomas) for losses of genetic information from chromosome arms 1p and 9p, as well as for deletion, mutation, and expression of the tumor suppressor genes CDKN2A (p16(INKa)/MTS1), p14(ARF), CDKN2B (p15(INK4b)/MTS2) (all located at 9p21) and CDKN2C (1p32). Comparative genomic hybridization and microsatellite analysis showed losses on 1p in 11 anaplastic meningiomas (85%), 23 atypical meningiomas (68%), and 5 benign meningiomas (25%). One atypical meningioma with loss of heterozygosity on 1p carried a somatic CDKN2C mutation (c.202C>T: R68X). Losses on 9p were found in five anaplastic meningiomas (38%), six atypical meningiomas (18%), and one benign meningioma (5%). Six anaplastic meningiomas (46%) and one atypical meningioma (3%) showed homozygous deletions of the CDKN2A, p14(ARF), and CDKN2B genes. Two anaplastic meningiomas carried somatic point mutations in CDKN2A (c.262G>T: E88X and c.262G>A: E88K) and p14(ARF) (c.305G>T: G102V and c.305G>A: G102E). One anaplastic meningioma, three atypical meningiomas, and one benign meningioma without a demonstrated homozygous deletion or mutation of CDKN2A, p14(ARF), or CDKN2B lacked detectable transcripts from at least one of these genes. Hypermethylation of CDKN2A, p14(ARF), and CDKN2B could be demonstrated in one of these cases. Taken together, our results indicate that CDKN2C is rarely altered in meningiomas. However, the majority of anaplastic meningiomas either show homozygous deletions of CDKN2A, p14(ARF), and CDKN2B, mutations in CDKN2A and p14(ARF), or lack of expression of one or more of these genes. Thus, inactivation of the G(1)/S-phase cell-cycle checkpoint is an important aberration in anaplastic meningiomas.

Figure 1.

a and b: Demonstration of LOH at microsatellite markers on 1p (a) and mutation of the CDKN2C gene in the atypical meningioma MN56B (b). Microsatellite analysis of tumor DNA (lane T) from MN56B and corresponding blood DNA (lane B) showed LOH at markers D1S468 and D1S224 in the tumor DNA (a, arrowheads). MN56B carried a somatic nonsense CDKN2C mutation (c.202C>T: R68X). c and d:CDKN2A and p14^ARF point mutations in two anaplastic meningiomas (MN34, MN63B). SSCP/heteroduplex analysis was performed after restriction digestion of the PCR products with SmaI. Both tumors showed an aberrant band corresponding to an undigested double-stranded PCR product (c, arrowhead). On sequencing, point mutations altering the SmaI restriction site were found in both tumors but not in the corresponding blood DNAs (d). MN34 showed a nonsense mutation in CDKN2A (c.262G>T: E88X) and a missense mutation in p14^ARF (c.305G>T: G102V). The mutation identified in MN63B also affected both genes (c.262G>A: E88K in CDKN2A and c.305G>A: G102E in p14^ARF). The sequences shown are derived from reverse sequencing of the coding strand. Arrows point to the mutation sites.

Figure 2.

Demonstration of a complex LOH pattern on 9p in the anaplastic meningioma MN34. This particular tumor showed retention of heterozygosity at D9S157 and D9S171 (9p21), as well as LOH at markers located between D9S157 and D9S171 (shown is D9S974) and distal to D9S157 (D9S168 at 9p22-p23). Arrows point to lost alleles in tumor DNA.

Figure 3.

Demonstration of homozygous p14^ARF, CDKN2A and CDKN2B deletions by duplex-PCR (a) and Southern blot analysis (b). Shown are results for seven meningiomas. MN63B retained two copies of each gene whereas MN19 and MN65 carried hemizygous deletions. MN64, MN67, MN90, and MN91A all show homozygous deletions (arrows). INFG and 9qSTS were used as reference for the duplex-PCR analyses. D2S44 served as reference to adjust for differences in DNA loading of the individual lanes of the Southern blot. The sizes of the respective PCR fragments (a) and hybridized restriction fragments (b) are given on the right.

Figure 4.

Expression of transcripts from CDKN2A, p14^ARF, and CDKN2B in meningiomas. Shown are results obtained by duplex reverse transcription-PCR using β-2-microglobulin (B2M) mRNA as reference. The individual lanes correspond to: 1, MN10; 2, MN27; 3, MN37; 4, MN38; 5, MN41; 6, normal leptomeningeal tissue; 7, MN34; 8, MN63B; 9, MN85; 10, MN91A; 11, MN16; 12, MN40; 13, MN61; 14, MN89; 15, MN2; 16, MN45; 17 and 18, two different samples of normal leptomeningeal tissue; 19 and 20, two different samples of nonneoplastic brain tissue; 21, water control. Lanes 1 to 5 and 15 to 16 contain samples from benign meningiomas (World Health Organization grade I), lanes 11 to 14 from atypical meningiomas (World Health Organization grade II), and lanes 7 to 10 from anaplastic meningiomas (World Health Organization grade III). Transcripts from all three genes are present in the normal brain samples (lanes 19 to 20). The leptomeningeal tissue samples expressed p14^ARF mRNA and low levels of CDKN2B mRNA but lacked detectable CDKN2A transcripts. Most benign meningiomas expressed all three genes whereas transcripts from one or more of the genes were not detectable in tumors MN85, MN91A, MN61, MN89, and MN2 (arrows). Among these tumors, MN91A had a homozygous deletion involving CDKN2A, p14^ARF, and CDKN2B, whereas tumor MN61 showed hypermethylation of these genes (see Figure 5 ▶ ).

Figure 5.

Hypermethylation of the CpG islands in the first exons of CDKN2A, p14^ARF, and CDKN2B in the atypical meningioma MN61. Shown are results of sequencing of sodium bisulfite-modified tumor and corresponding blood DNA. All sequences are derived from reverse sequencing of the coding strand. Arrows point to methylated CpG residues in tumor DNA that were not modified by the bisulfite treatment.