The studied material consisted of 14 meningiomas (10 frozen and four paraffin-embedded tissue samples) obtained from 14 unrelated Greek patients who had been operated on at the Evangelismos Hospital, Athens, Greece. The patients were mostly women (n = 8), with an age range of 37-72 years (median 52 years), and a family history free of other cases of meningioma or NF2. Three of the meningiomas were of grade I, eight of grade II and three of grade III (Table 1). The tumor samples were assigned a number and examined blindly.

Ten biopsies were frozen in isopentane, cooled in liquid nitrogen, and cryostat 6 mm sections were immunostained by a standard avidin-biotin-peroxidase technique (see below). In parallel, a sural nerve biopsy (obtained from a neuropathy patient without any histopathological findings) was analyzed as a control for immunostaining. The staining of each sample was densitometrically compared to that of the control, and considered to be – (i.e., at least 10X less intensity), + (i.e., about equal intensity), ++ or +++ (i.e., at least 10X r 100X more intensity, respectively). Briefly, the immunostaining method included:

1.   Incubation with normal rabbit serum diluted 1/5 with phosphate buffer solution (PBS) for 10 minutes.

2.   Incubation with primary antibody c-N-ras IgG1 F155-227 (OncogeneO; Research Products, Boston, MA, USA) in 1/50 dilution for 1 hour and washing with PBS.

3.   Incubation with a biotinylated F(ab)2 fragment of anti-mouse IgG in 1/20 dilution for 30 minutes and washing with PBS.

4.   Incubation with extravidin-conjugated peroxidase in 1/20 dilution for 30 minutes and washing with PBS.

5.   Incubation with chromogen 3-amino-9-ethylcarbazole.

6.   Examination and intensity analysis under a light microscope, and photographic documentation.

Sections from four fixed, wax-embedded tissue samples were cut at 6 mm and dried at room temperature. The sections were then de-waxed with three washes of xylene, washed three times with alcohol, and finally submerged in water. Then, the immunostaining method was applied.

Total DNA was isolated from a region of each menin­gioma sample immediately adjunct to the sections taken. The DNA extraction was performed using a Nucleon TissueO kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). A 200 bp fragment of exon 2 and a 76 bp fragment of exon 4 (including hot spot codons 57 and 198, respectively) were amplified under standard polymerase chain reaction (PCR) conditions. The sequences of the primers used [12] were as follows: for the codon 57 fragment (annealing temperature 60°C) S1 5’-TTGCTCACAGTG TCCTTCCC-3’ and S2 5’-TCAGCCCCACCAGTTTC ATC-3’; and for the codon 198 fragment (annealing temperature 62°C) C1 5’-GACTCCGGAAATGTGGGAG GA-3’ and C2 5’-GATTGGGCCTCACCT GGCTC-3’.

The PCR products were digested with the restriction enzyme DdeI and analyzed by agarose gel electrophoresis. Digested products of 109+91 bp, and 56+20 bp were detected only when the CGA codons was mutated to TGA (stop codon).

F = female; M = male; (p) paraffin-embedded tissue; (wt) homozygous wild-type allele; (m) mutant allele in the heterozygous state; [+] staining intensity comparable to control; [–] staining intensity less than control; [++]/[+++] staining intensity more than control.