Prognostic significance of intratumoral vascular endothelial growth factor as a marker of tumour angiogenesis in epithelial ovarian cancer.

OBJECTIVES: The aim of this study was to examine the prognostic significance of vascular endothelial growth factor (VEGF) in epithelial ovarian cancer (EOC). METHODS: Surgical specimens of 105 patients with primary EOC FIGO Stages 1 to 4, who underwent surgical staging, were investigated. Expression of VEGF was evaluated by immunohistochemical staining using related monoclonal antibodies. The correlation of this data with survival and established prognostic factors such as histological grade, FIGO stage and residual tumour status was evaluated. Multivariate analysis and correlation tests were performed. RESULTS: The results of VEGF expression were correlated with clinicopathological variables and overall survival. No correlation between the VEGF expression and clinicopathological factors was identified. However, VEGF expression was found to be significantly correlated to survival, and a prognostic factor independent of the stage of disease and residual tumour status (p < 0.0001). CONCLUSION: High intratumoral VEGF expression, a marker of angiogenesis, appeared to be an independent prognostic factor for overall survival in women with EOC. Angiogenic evaluation of patients with EOC may play a role in predicting a subgroup of patients with aggressive disease. These patients could be the target of front-line molecular targeted therapy with anti-angiogenic agents.

Cervical intraepithelial neoplasia treatment in Human Immunodeficiency Virus-positive women.

We set out to consider the level of agreement between referral and treatment pathology and to investigate the effectiveness of standard surgical treatment for cervical intraepithelial neoplasia (CIN) in Human Immunodeficiency Virus (HIV)-positive women. This was a case-note review of all women who underwent treatment for CIN between 1995 and 2004. Information on the referral and follow-up smear and biopsy results and the status of the excision margins at treatment were collected. A total of 71 women had at least one large loop excision of the transformation zone (LLETZ) for CIN. Agreement between the referral smear and biopsy was poor (kappa = 0.20) and between the referral and treatment pathology was only fair (kappa = 0.37). Ten treatment samples showed no histological evidence of CIN and were excluded from analysis of the presence of CIN at the resection margins. In only 32.8% of treatment samples were both margins clear of CIN. A high pre-LLETZ CD4 count was strongly associated with clear margins. A total of 55.6% patients had CIN at follow-up, despite both margins being clear. The follow-up smear/biopsy had decreased by >or=1 grade of CIN in only 50.8% patients. Our results show a high degree of discrepancy between cytology/biopsy and LLETZ histology in HIV-positive women. Additionally, there is often incomplete clearance of CIN at the resection margins emphasing the need for close follow-up after surgery.

The association between polycystic ovaries and endometrial cancer.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) are assumed to be at increased risk of endometrial cancer (EC), albeit of a more differentiated type with better prognosis than in normal women. This study was designed to test these assumptions, as evidence for them is lacking. METHODS: The prevalence of polycystic ovaries (PCO), as a marker of PCOS, was investigated in ovarian sections from 128 women with EC and 83 with benign gynaecological conditions. The expression of the prognostic markers p53, Ki67, Bcl2 and cyclin D1 was also investigated by immunohistochemistry in endometrial tumours from 11 women with PCO and 16 with normal ovaries. RESULTS: Overall, PCO were similarly prevalent in women with EC (8.6%) and benign controls (8.4%); however, in women aged <50 years, PCO were more prevalent in women with EC (62.5 versus 27.3%, P = 0.033). Cyclin D1-expressing endometrial tumours tended to be more prevalent in women with PCO compared to normal ovaries (36.4 versus 6.25%, respectively, P = 0.071). Bcl2-, p53- and Ki67-expressing tumours were similarly prevalent. CONCLUSIONS: The association between PCOS and EC appears confined to premenopausal women. The tendency for cyclin D1-expressing endometrial tumours to be more prevalent in women with PCO challenges the assumption that EC prognosis is improved in women with PCOS.

Microvessel quantification in benign and malignant ovarian tumors.

Microvessel density (MVD) in 92 paraffin sections of ovarian samples of different histologic subtypes was correlated with microvessel counts from 58 corresponding frozen sections. Anti-human von Willebrand factor antibody was used as an endothelial marker. MVD was performed in neovascular hotspots using a Quantimet 500+ Image Analyzer. The highest vessel density (HVD) and average vessel density (AVD) of three fields at the x 200 and x 400 magnification were recorded. There was a strong correlation between the HVD and AVD at the x 200 and x 400 magnifications when comparing fixed with frozen sections (correlation coefficients at x 200 for the HVD was 0.37, P = 0.005 and AVD was 0.30, P = 0.02; correlation coefficients at x 400 for the HVD was 0.38, P = 0.003 and AVD was 0.37, P = 0.004). In the fixed tissue, the HVD and AVD at both these magnifications were significantly greater in the group containing functional cysts; this was also the case for the frozen sections. These findings are consistent with the development of a microcirculation necessary for the growth and maturation of such cysts, and this appears to be greater than that in tumors. The good correlation between MVD in fixed and frozen sections suggests that such observations represent a true reflection of ovarian angiogenesis in both physiologic and pathologic states.

Cell cycle and apoptotic proteins in relation to ovarian epithelial morphology.

OBJECTIVE: To assess the relationship between the expression of cell cycle and apoptotic proteins and the morphological appearance of the surface epithelium in non-neoplastic ovaries. METHODS: The subjects for this study were 79 women who had undergone oophorectomy for benign conditions at the North Middlesex Hospital, London, and Royal Free Hospital, London, and whose ovaries had been reported on routine histology as entirely normal or containing physiological cysts or endometriosis. The epithelial morphology was reassessed on haematoxylin and eosin-stained paraffin wax sections using nine cytological and architectural parameters associated with premalignant intraepithelial changes. A 'score' was obtained for each ovary. Expression of p53, Ki67, cyclin D1 and Bcl-2 in the surface, cystic and endometriotic epithelium was assessed in corresponding sections using standard immunohistochemistry. RESULTS: The median score for the morphological changes was significantly higher in the sections, which expressed p53 compared to those which did not. This difference remained significant in a subanalysis of the sections, which did not contain endometriosis. No relationship was identified between the morphological score and the expression of Ki67, Bcl-2 and cyclin D1. CONCLUSION: Increased intraepithelial abnormality as assessed by an epithelial morphological score of ovarian sections is associated with expression of the p53 cell cycle protein. This lends credence to the hypothesis that the ovarian surface or cystic epithelium goes through an identifiable precursor or "premalignant" phase before the development of invasive disease. Further work is required to characterise the changes that take place before the development of malignancy in ovarian epithelium.

TP53 mutations in vulval lichen sclerosus adjacent to squamous cell carcinoma of the vulva.

Non-neoplastic epithelial lesions of the vulva (NNEDV) lichen sclerosus (LS) and squamous hyperplasia (SH) have been implicated in the pathogenesis of squamous cell carcinoma of the vulva (SCC). To date, there have been no recognisable precursor lesions for SCC associated with NNEDV. TP53 is the most frequent genetic change in human cancers and can indicate both aetiology and molecular pathogenesis of tumours. A total of 27 SCC patients underwent immunohistochemistry (IHC) and TP53 mutational analysis using microdissection and direct sequencing. There were 19 patients with areas of adjacent epidermis: 17 had NNEDV (four SCCs had more than one adjacent lesion) and two had normal epidermis. In all, 70.4% of the SCCs, 40% LS and 22.2% SH demonstrated overexpression of p53. In total, 77.8% of SCCs, 46.7% of LS and 22.2% SH demonstrated mutations in TP53, with the majority of lesions having a mutation in codon 136. Eight cases were identified where the same mutation was identified in the SCC and in the adjacent area. These data suggest that TP53 mutations develop in NNEDV and are intrinsic to the clonal evolution that leads to SCC. The type of mutation detected is more likely to occur due to endogenous cellular changes rather than exogenous carcinogen exposure.

The effect of topical corticosteroids on Ki67 and p53 expression in vulval lichen sclerosus.

BACKGROUND: Topical corticosteroids have become the treatment of choice for genital lichen sclerosus (LS) and are believed to be required for long-term relief of symptoms. OBJECTIVE: To compare vulval LS that had been treated with topical corticosteroids, vulval LS that had not received topical corticosteroids, and histologically normal vulval skin. METHODS: We used immunohistochemistry to look for Ki67 expression and abnormal p53 expression. RESULTS: We found a statistically significant difference for p53 overexpression, with increased levels seen when comparing corticosteroid-treated LS with normal genital skin (P = 0.011). Ki67 expression was also significantly higher in the corticosteroid-treated group compared with normal genital skin (P = 0.001), and increased levels were also found in the treated group compared with untreated LS (P = 0.05). CONCLUSIONS: Our data suggest that topical corticosteroids have an effect on cell cycle proteins in genital skin and, in particular, genital skin with LS changes.

The role of p53 and Ki67 in Paget's disease of the vulva and the breast.

OBJECTIVE: Paget's disease of the vulva (PDV) and Paget's disease of the breast (PDB) are uncommon diseases, accounting for approximately 1% of all vulval neoplasms and 0.5-4% of all breast cancers, respectively. In 10-30% of vulval cases an invasive adenocarcinoma is present. In such cases the disease is often aggressive and recurrence rate is high. This is in contrast to PDB where the general consensus is that almost all cases are associated with an in situ or invasive ductal carcinoma. Our aim was to examine the presence of the tumor suppressor protein p53 and the proliferation marker Ki67 in PDV and PDB and correlate any differences in the expression of these two proteins with the presence of an underlying carcinoma. METHODS: Immunohistochemistry was performed on 52 archival cases of PDV, which included 10 with associated invasive adenocarcinoma of the vulva, and on 37 archival cases of PDB, including 26 with available associated ductal carcinoma in situ (DCIS) or invasive carcinoma of the breast. All cases were formalin-fixed and paraffin wax-embedded. Monoclonal antibodies were used with microwave antigen retrieval. Streptavidin-biotin-horseradish peroxidase and 3,3'-diaminobenzidine detection methods were employed to visualize antibody binding and staining. A section was scored positive for p53 if more than 10% of cell nuclei were stained brown and Ki67 was expressed as a percentage of positive cells to the nearest 5% of cells showing nuclear positivity (Ki67 staining index). RESULTS: p53 was expressed in 15 of 52 (29%) PDV cases and 5 of 37 (13%) cases of PDB. Four of the ten cases (40%) of PDV associated with invasive disease expressed p53 compared with 11 of 42 (26%) cases without invasive disease. The mean Ki67 staining index for PDV associated with invasion was 19%, and for that without invasion, 16%. In the breast cases, the mean staining index was 11%. CONCLUSION: Our data suggest that p53 may have a role to play in PDV progression, and may be a late event in some cases, especially those associated with invasive disease. Ki67 has no apparent prognostic role in PDV as there was no significant difference between those cases associated with and those without invasive disease. Neither p53 nor Ki67 appears to have a prognostic role to play in PDB.

Cyclin D1 and retinoblastoma protein in vulvar cancer and adjacent lesions.

Abnormalities in the cell cycle are associated with tumorigenesis but have not yet been identified in squamous cell carcinoma (SCC) of the vulva or in adjacent vulvar lesions. The purpose of this study was to identify cell cycle protein expression (cyclin D1 and retinoblastoma protein [pRb]) in vulvar SCC and in adjacent potentially premalignant lesions: lichen sclerosis (LS), squamous cell hyperplasia (SH), and vulvar intraepithelial neoplasia (VIN). Using immunohistochemical techniques, 57 SCCs were analyzed with 19 adjacent areas showing LS, 13 showing SH, 11 VIN, and six normal epithelium. Fifty-one percent of SCCs showed abnormal cyclin D1 expression and 37% showed abnormal pRb. Abnormal cyclin D1 expression in the adjacent areas was as follows: 53% in LS, 31% in SH, 18% in VIN, and 0% in normal. Abnormal pRb expression was as follows: 42% in LS, 62% in SH, 46% in VIN, and 33% in normal. Only 10 lesions showed abnormal expression of both proteins. Abnormal expression of cyclin D1 in SCC was statistically significant compared with adjacent normal epithelium. In SCC lesions, abnormal cyclin D1 expression was associated with greater depth of invasion. Abnormal pRb in SCC was associated with poor tumor grade. Cyclin D1 and pRb are separately involved in the progression of vulvar cancer, and changes in the expression of these proteins may represent an early stage of malignant transformation in vulvar disease.

Quantification of vascular endothelial growth factor-A in leiomyomas and adjacent myometrium.

Although uterine leiomyomas constitute the commonest benign tumour in women, the regulation of their growth is poorly understood. It is believed that angiogenesis, the process by which new capillaries develop from pre-existing blood vessels, may be involved. We therefore investigated the expression of vascular endothelial growth factor-A (VEGF-A), a primary regulator of angiogenesis, in leiomyoma tissue and the adjacent myometrium in 36 pre-menopausal women undergoing hysterectomy for leiomyomas, with or without prior treatment with gonadotrophin-releasing hormone analogue (GnRHa). In 5 microm sections prepared from archival paraffin-wax blocks, VEGF-A was demonstrated by standard immunohistochemistry using a monoclonal antibody. VEGF-A was expressed in 14 of 18 (77.8%) leiomyoma sections from women without GnRHa pretreatment, and in 15 of 18 (83%) of those from women with prior treatment. VEGF-A expression in the adjacent myometrium was much lower, being noted in two of 18 (11.1%) sections from women without prior GnRHa treatment and in one of 18 (5.5%) sections from tissue that had been subject to prior down-regulation. Moreover, when VEGF-A expression was present, expression was strong in leiomyomas (> or =20 focal areas/cm(2)), but not in adjacent myometrium. The differential expression of VEGF-A antigen in leiomyomas compared with the adjacent myometrium indicates that local angiogenesis may be important in the development and growth of these tumours. GnRHa therapy does not appear to alter this pattern of VEGF-A expression.

Cell cycle proteins as molecular markers of malignant change in vulvar lichen sclerosus.

Lichen sclerosus (LS) has a known association with the development of squamous cell carcinoma of the vulva. The purpose of this study was to investigate molecular markers, which could indicate premalignant changes. Multiple sequential vulvar biopsies were taken over a period of 11 years from a patient with longstanding LS. Immunohistochemical staining was used to demonstrate a range of molecular markers. Increased expression of p53 and Ki67 was found in areas of squamous hyperplasia (SH) and differentiated vulvar intraepithelial neoplasia (dVIN) which correlated with the subsequent development of invasive squamous cell carcinoma (SCC). Molecular changes have been found to accompany histologic changes in the progression of vulvar LS to malignancy. Such markers may prove a useful addition in the clinical management of these conditions.

Role of angiogenesis in benign, premalignant and malignant vulvar lesions.

OBJECTIVE: To investigate the presence of angiogenic factors in benign, premalignant and malignant vulvar lesions. STUDY DESIGN: Immunohistochemical demonstration of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) in normal vulvar skin, lichen sclerosus, vulvar intraepithelial neoplasia (VIN) and vulvar cancer. RESULTS: VEGF was found in the majority of vulvar cancers but only a minority of VIN lesions. PD-ECGF was found in the majority of lesions. CONCLUSION: Demonstration of angiogenesis may suggest which preinvasive lesions will progress to invasive cancer.

Axon regeneration and vascularisation of nerve grafts. An experimental study.

An experimental model has been designed to assess the effect of vascularisation on axon regeneration in nerve grafts. The vascular status of the grafts has been demonstrated by microangiography and histology. Rat sciatic nerve grafts in which the vascular pedicles were left intact retained a normal vascular pattern which was not adversely affected by wrapping the graft in a polythene sleeve. In devascularised grafts, revascularisation commenced at three days and was complete at nine days. If the devascularised grafts were wrapped in a polythene sleeve, revascularisation was impeded and at fifteen days the middle segment of the graft was avascular and infarcted. The rate of axon regeneration was measured electrophysiologically in the above four groups of nerve grafts. There was a linear relationship between the rate of axon regeneration with time post-graft, axon growth proceeding at a mean rate of 1.150mm/day (S.E. +/- 0.084) after a mean delay of 4.85 days. There was no significant difference in the rate of axon regeneration in the four groups.

Mesothelioma associated with the syndrome of inappropriate secretion of antidiuretic hormone.

A patient with a malignant mesothelioma developed the syndrome of inappropriate secretion of antidiuretic hormone. The electrolyte abnormalities were corrected by treatment with demethylchlortetracycline. Arginine vasopressin concentrations were increased in serum and urine. It is suggested that the syndrome might have been mediated by secretion of antidiuretic hormone from the posterior pituitary, because arginine vasopressin was not detected in the patient's tumor using a sensitive radioimmunoassay.