Nivel de desempeño obtenido en cada criterio de evaluación de la

Over expression o f p53 is a common finding in human malignant tumours and has been investigated in relation to vulval cancer in a number o f studies (Table XII). 69% o f SCCs showed over expression o f p53 compared to 39-87% in other studies

1999; Lerma et al, 1999; Ngan et al, 1999; Scheistroen et al, 1999). However, several o f these studies used different scoring systems and also different antibodies (Table XII).

p53 over expression can be the result o f several mechanisms. The p53 protein is normally found in minute amounts in m any cells, but when these cells are exposed to genotoxic stimuli, wt p53 levels increase rapidly and initiate a programme o f

apoptosis or cell cycle arrest. In addition to increased wt p53 levels, p53 over

expression can be the result o f a mutation or by the binding o f proteins that neutralise wt p53, e.g. Mdm-2. In this work it was found that there was no over expression o f Mdm-2 and therefore it was not likely that the p53 protein was being stabilised by Mdm-2.

In this study HPV-related SCC (e.g. SCCs associated with VIN) showed over

expression o f p53, which was unexpected as E6 from HPV binds to p53 and leads to

p53 degradation. The over expression o f p53 in these SCCs could be due to several mechanisms e.g. that the SCC contains low levels HPV-DNA. Alternatively, there may be a defect in the ubiquitin degradative pathway and hence accumulation o f the p53 protein occurs.

Ki67 expression in SCC

Ki67 was expressed in the majority o f SCCs with a mean o f 24.1± 21.0. Others have also shown the presence o f Ki67 in SCC. Hendricks et al (1994) also described two patterns o f staining: diffuse and localised. In this study 70% showed the localised pattern and 23% showed the diffuse pattern but in 7% SCCs were found with both staining patterns. The biological significance o f these staining patterns is still uncertain since the localised pattern was predominantly seen in well-differentiated SCCs and patients with vascular invasion compared to those without vascular invasion, which tended to show a diffuse pattern. Others have demonstrated a worse survival in those with a diffuse pattern (Hendricks et al, 1994; Hantschmann et al, 2000).

A surprising statistical difference was demonstrated between well-differentiated SCCs showing an increased proliferation rate compared to the poorly differentiated SCCs,

although this was thought to be through the small numbers o f the poorly differentiated tumours (n=3) compared to the well-differentiated SCCs (n=47). A further reason is demonstrated when looking at prognosis; others have shown that it is not the

proliferation rate that appears important but the staining pattern, but in this study numbers are too small to compare staining patterns for the poorly differentiated SCC and survival rates.

This study has demonstrated a significant correlation between Ki67 staining and p53 over expression in SCC, which could indicate a loss o f wt p53 resulting in a loss o f cell cycle control and therefore increased proliferation. However, it is also known that p53 has other mediators which can also play a part in cell cycle regulation.

Correlation with clinicopathological variables

There was no correlation between p53 over expression and stage, lymphatic/vascular spread, size or depth as has been reported in other studies (Emanuels et al, 1999; Scheistroen et al, 1999). McConnell et al ^1997) showed that well and moderately differentiated tumours were more likely to over express p53, this was also

demonstrated in this work.

p53 and Ki67 expression in adjacent lesions:

All epidermal changes except for normal skin adjacent to vulval cancer showed a substantial percentage o f specimens with p53 over expression. To date, there have been few studies (Kagie et al, 1997a; McConnell et al, 1997) examining p53 over expression in the lesions adjacent to SCCs. In this work 57% o f the VINs adjacent to SCC showed p53 staining which is a variance w ith other reports which have shown lower percentages (Kagie et al, 1997a; McConnell et al, 1997). This may reflect the different methodology used, or sampling o f areas away from the SCCs in other studies, since it was frequently unclear if the lesion was adjacent to SCC (Pilotti et al,

1995; Ngan et al, 1999). The role that HPV and p53 play in VIN lesions needs further study to understand the process that results in the over expression o f the p53 protein. Other groups have also shown lower p53 expression in NNEDV lesions (e.g. LS or SH), though again this could be the result o f different methodology (Kohlberger et al, 1995).

p53 has been found to be an early marker o f high-risk lesions; often preceding invasion in some cancers for example in bronchial lesions 25% o f the earliest neoplastic lesions will show p53 mutations (Sundaresan et al, 1992). Others have demonstrated p53 alterations (either increased expression or mutation) at or before the stage o f severe dysplasia and in some normal mucosa in the oropharynx (Nees et al, 1993). This work has also demonstrated an increase in both p53 and Ki67 expression several years prior to the development o f SCC (Rolfe et al, 2001; Chapter 8) when examining the malignant progression from LS lesions.

The mean Ki67 staining index was higher in all lesions adjacent to SCC compared to normal vulval skin; even normal vulval skin adjacent to SCC had an increased proliferation rate compared with normal vulval tissue, though this did not reach statistical significance. The pattern o f staining varied between different types o f lesion, with both VIN and SH showing a diffuse pattern throughout the epidermis while LS and normal vulval tissue showed staining in the basal/parabasal layer. Changes in Ki67 staining pattern has also been described in dysplasias e.g. colon (Andersen et al, 1998).

In conclusion these data suggest that the over expression o f p53 is associated with the progression o f vulval cancer and changes in its expression represent an early stage o f malignant transformation in vulval disease. Such p53 changes are generally associated with increased cell proliferation rates.

4.3. ii

21

p21 (Figures 27) was scored as described in Chapter 2 (Table VI) however,

the majority o f SCCs demonstrated abnormal p21 expression. Therefore

p2 1 was scored as a percentage and then divided into three groups as

described by Stein et al (1998), 0-<10% was counted as negative, 11-< 50% as + and

>50% as ++. Stein et al (1998) showed that p21 is prognostic in bladder

Figure 27a: Demonstrating p21 in VIN adjacent to SCC. Magnification x 100.

WSÊM

Figure 27b: expression in SCC, with VIN seen overlying the SCC also

expressing Magnification x 200.

Figure 27c: in LS adjacent to SCC.

Magnification x 200.

Figure 27d: in normal vulva adjacent to SCC. Magnification x 200. A 4. f.: r * ' t / s.,, \ y

Figure 27e: SH adjacent to SCC demonstrating p21Wafl^^'^^ expression. Magnification x 200.

Due to lack o f clinical tissue forty-eight SCCs were stained for p21 83.3% o f SCCs demonstrated positive staining (i.e. >10%) (Figure 27b), this was demonstrated to be statistically significantly different when compared to normal epidermis not adjacent to SCC (p=0.000009). There was also a statistical significant difference demonstrated when comparing SCC associated with NNEDV and SCCs associated with VESf especially if one compares <10% as normal and >10% as abnormal (p=0.03) with the majority o f SCCs associated with NNEDV demonstrating abnormal

p2 1 expression.

When splitting the p21 into the three groups described by Stein et al (1998)

the only statistical significant difference was demonstrated in the 0-<1 0% group (p=0.034) with the majority o f SCCs associated w ith VIN demonstrating this group.

16.6% were counted as negative (8/48), 54.2% (26/48) were + and 29.2% (14/48) showed ++. In adjacent normal epidermis 33.3% were negative (n=2/6) while 6 6.6% showed + staining with none identified with ++ staining. Normal vulval skin not

adjacent to SCC showed no p21 expression.

p53 and p21 p21 and p53 are believed to be linked through the cell

cycle pathway. When combining over expression o f p53 with p21 expression

(> 10%) 54.2% o f the SCCs showed expression o f both proteins this compared with

29.2% showing no over expression o f p53 but positive p21 and both proteins

being negative (6.2%). A statistical significant difference was demonstrated between SCCs with both proteins showing expression compared to adjacent normal epidermis (p=0.02) and normal vulval skin not adjacent to SCC (p=0.005) with SCCs

demonstrating over expression o f both these proteins.