Primera generación

PCR-SSCP showed abnormal migration of the PCR product during electrophoresis in at least one exon of the p53 gene either in the first or second biopsy sample in 103 out of 132 cases (78.0%) (Figure 3.19). The results from DNA sequence analysis of these samples indicated that 44 out of 132 cases (33.3%) had p53 mutations in at least one of the biopsy

samples (Table 3.19, 3.20, Figure 3.20). These can be divided by grade at the first operation into 20 grade 2 cases (out of 45 cases, 44.4%), 12 grade 3 cases (out of 27 cases, 44.4%), and 12 grade 4 cases (out of 60 cases, 20.0%).

The incidence of p53 mutation in grade 4 tumours was significantly lower than in grade 2 tumours (p = 0.007, Pearson Chi-Square) or in grade 3 tumours (p = 0.018, Pearson Chi-Square). The incidence of p53 mutation can be divided further by grade at the first and second operations as shown in Table 3.20. There was no difference in the incidence of p53 mutation in the grade 2 and grade 3 tumours diagnosed at the first operation which subsequently progressed or recurred as grade 3 or 4 at the second operation. There was no difference in the incidence of p53 mutation in the grade 4 tumours which had one or two operations.

O f the 39 cases with two operations which had p53 mutations, irrespective of grade, 11 cases had the same mutations in both the first and second biopsy samples, 12 cases had different mutations between the first and second biopsy samples, 2 cases had mutations in the first biopsy sample only, and 10 cases had mutations in the second biopsy sample only. There were 4 cases in which information was available only in one of the biopsy samples because of a failure in amplification of PCR products (Table 3.22).

In the 11 cases which had the same mutation in the first and second biopsy samples, 7 were originally diagnosed as grade 2, 3 were grade 3 and 1 was a grade 4 tumour. In the 12 cases in which the mutations were located at a different sites or which had a second mutation during recurrence or progression, 5 cases were originally diagnosed as grade 2 ,4 cases as grade 3 and 3 cases as grade 4 (Table 3.23). One out of 5 cases of grade 2 tumour (ID 71) had an additional silent mutation in the second biopsy. The original mutation in this case was an A to C transversion at the first base of codon 239 in exon 7 with an amino acid change from asparagine (AAC) to histidine (CAC). In the second biopsy sample there was also a sequence change at exon 4 with a G to C transversion at the second base of codon 72 producing an amino acid change from arginine (CGC) to proline (CCC). The second base change at codon 72 in exon 4,

which changes an arginine to proline has been reported as a polymorphism which occurs in about 35% of the population (Buchman et al., 1988; Olschwang et al., 1991). However, in this case (ID 71) the amino acid at codon 72 in the first biopsy sample was arginine but changed to proline in the second biopsy sample and should be regarded as a mutation because there is a sequence change between the first and second biopsy samples.

There were 2 cases which had a p53 mutation present in the first biopsy sample only. Of 10 cases which had a mutation in the second biopsy sample only, 6 were originally diagnosed as grade 2, 3 as grade 3, and 1 as grade 4. Of the 6 cases in grade 2, 5 progressed to grade 3 and 1 case progressed to grade 4 (Table 3.24). Of the 4 cases in which information was available only in one of the biopsy samples, there were 2 cases in which the mutation was found in the first biopsy sample but DNA from the second biopsy sample failed to be amplified by PCR.

The highest incidence of mutations in this series was in exon 5 in which mutations occurred in 19 out of 32 cases (59.4%) in the first biopsy sample and 17 out of 35 cases (48.6%) in the second biopsy sample. The most common location for mutation in exon 5 was at codon 175 and this occurred in 8 out of 32 cases (25.0%) in the first biopsy sample and in 9 out of 35 cases (25.7%) in the second biopsy sample. The second most common location of p53 mutation in exon 5 was at codon 176 which was detected in 4 out of 32 cases (12.5%) in the first biopsy sample and in 3 out of 35 (8.6%) in the second biopsy sample.

Polymorphisms of the p53 gene was found in 37 out of 132 cases (28%), consisting of 17 cases from grade 2, 7 cases from grade 3 and 13 cases from grade 4 tumour (Table 3.25). There was no difference in frequency o f p53 polymorphism between grades of tumour. The most common location of p53 polymorphism in this series was at codon 76 of exon 4 which is characterised by a C to G transversion at the second base resulting in an amino acid change from alanine (GCA) to glycine (GGA). O f 33 cases in which p53 polymorphism was detected in the first biopsy sample, in 30 cases (91%) the polymorphism was located at codon 76.

Five of these cases also had a second polymorphism at codon 72. The polymorphism at codon 72 is characterised by a G to C transversion at the second base resulting in an arginine (CGC) to proline (CCC) amino acid change. Polymorphisms were detected in the second biopsy sample in 24 out of 132 cases. Of these, 22 cases (91.6%) had a polymorphism at codon 76 and 2 cases were additionally polymorphic at codon 72 (Table 3.26).

ID 27 exon 4 ID 94 exon 5 ID 131 exon 6 ID 61 exon m

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Figure 3.19 Representative samples which showed band shift in SSCP gel (C = control, T = tumour, T1 = tumour from T* biopsy sample, T2 = tumour from 2"‘* biopsy sample).

3.. Exon 4 riD 2 1 )