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Background: While macrophages (CD68+) have been associated with angiogenesis in some inflammatory and neoplastic processes by increasing the release of vascular endothelial growth factor (VEGF), their role in anal intraepithelial neoplasia (AIN) and anal squamous cell carcinoma has not been established. This study records macrophage infiltration in anal pre-invasive and invasive lesions in HIV+ and HIV- populations, and determines their relationship with angiogenesis., Materials and Methods: Sixty patients (31 HIV+) with AIN and anal SCC were studied. Paraffin sections were stained for CD68, VEGF and von Willebrand factor. The density of CD68 cells, the expression of VEGF and angiogenesis were quantified, and compared amongst groups and between HIV+ and HIV- populations., Results: All three parameters increased linearly as the lesions became more dysplastic, in HIV+ and HIV- groups. The CD68 count was statistically lower in HIV+ (p<0.005) compared with HIV- groups, while the differences in VEGF expression and in angiogenesis were not significant between HIV+ and HIV- populations., Conclusion: There was a significant decrease of macrophage infiltrate in the HIV+ group. The relative increase in VEGF expression and angiogenesis in the face of lower macrophage infiltration in HIV+ patients may be explained either by a greater release of angiogenic factors by macrophages, or by VEGF expression not being solely dependent on macrophage activation.

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.

Introduction: Most cases of anal carcinoma seem to develop from high grade anal intraepithelial neoplasia (AIN) caused by persistent anal warts. Similar pre-invasive epithelial genital lesions (e.g. those of the cervix and vulva) have been shown to be associated with increased angiogenesis. In this study we examined biopsies of anal lesions ranging from warts to invasive anal carcinoma, with the aim of assessing the degree of angiogenesis in pre-invasive anal lesions., Method: Samples from 70 patients (51 male) who had undergone excision biopsy or resection of anal wart lesions (20), low grade AIN (12), high grade AIN (27) and anal squamous cell carcinoma (SCC) (11) were studied. Samples (6) from normal HIV-anal skin were used as controls. The samples were stained for von Willebrand factor, a specific marker of endothelial cells. Angiogenesis was measured by microvessel density (MVD) analysis, quantifying the microvessels in the stroma adjacent to the epithelial lesion., Results: There was a statistically significant (P < 0.001) progressive increase in MVD between low grade AIN, high grade AIN and anal SCC. The difference in MVD between normal skin, warts and low grade AIN was not statistically significant., Conclusion: There are progressive abnormal patterns of angiogenesis in highly dysplastic lesions, similar to those found in cervical and vulvar pathology. These findings may have biological, prognostic and therapeutic implications.

Objectives: We compared microvessel density (MVD) in normal, benign, preneoplastic, and neoplastic (squamous cell carcinoma (SCC)) vulvar disease to ascertain if this parameter could identify cases with lichen sclerosus (LS) and high-grade vulvar intraepithelial neoplasia (VIN3) at risk of developing malignancy., Methods: Microvessels were immunohistochemically stained in paraffin wax-embedded vulvar tissue sections with anti-von Willebrand factor (vWF) antibody using the streptavidin-biotin-horseradish peroxidase complex technique. Three "hot spots" with the greatest MVD were identified within 200 microm of the subepithelial dermis under low magnification (x 40 and x 100). The highest (HVD) and average (AVD) MVDs were quantified for each sample under high magnification (x 200) using an image analysis system., Results: HVD and AVD showed similar significant differences. SCC had significantly the highest MVD followed by VIN3, normal vulva, and LS. LS had significantly the lowest MVD, even lower than that of normal vulva. Two cases of VIN3 had much higher HVD (9.16 and 9.61) and AVD (6.89 and 7.71) compared with the main cluster of cases., Conclusion: In vulvar LS, MVD, as assessed by HVD/AVD, is not a useful parameter in determining potential malignant progression, while in VIN3 this parameter could be valuable in identifying cases at greatest risk of progression to invasive disease.

Background: Anal squamous cell carcinoma (SCC) develops from dysplastic anal warts. This study quantifies the expression of p53 and Ki67 in pre-invasive and invasive anal lesions., Materials and Methods: Samples of 70 patients with anal warts (n = 20), low grade anal intraepithelial neoplasia (LG AIN) (n = 12), high grade anal intraepithelial neoplasia (HG AIN) (n = 27) and anal SCC (n = 11) were stained using immunohistochemical techniques. Eight patients with normal anal skin were used as controls., Results: Both the expression of p53 and Ki67 increased significantly (p < 0.001) and gradually as the lesions became dysplastic and invasive. The main increase in p53 expression was as the lesions progressed from anal warts (7.38 +/- 11.93-mean +/- SD) to low grade AIN (20.778 +/- 13.14)., Conclusion: p53 is involved in the progression of anal cancer and its expression increases from early in the development of pre-invasive anal lesions. p53 and Ki67 may be useful markers of early dysplasia and should be considered in the screening of high risk patients.

Squamous cell carcinoma of the vulva (SCC) accounts for about 4% of all gynaecological malignancies. It has an incidence of about 1.8 per 100,000. However, after the age of 75 the incidence of vulvar carcinoma peaks at approximately 20 per 100,000, making it as common as cervical carcinoma. Benign and pre-malignant vulvar lesions can be broadly divided into non-neoplastic epithelial disorders of the vulva (NNEDV), vulvar intraepithelial neoplasia (VIN) and Paget's disease of the vulva (PDV). NNEDV lesions include lichen sclerosus (LS) and squamous hyperplasia (SH). To date no solid prognostic tools are available to predict which pre-malignant vulvar lesions will progress to SCC. About 4.5% of patients develop SCC following a history of LS and ca. 4% of treated VIN lesions go on to become vulvar SCC. In PDV, 28% of patients have an underlying cancer. Angiogenesis, the development of new blood vessels from existing vasculature, is an essential component of solid tumour growth and metastasis. Several angiogenic factors are expressed by many tumours, suggesting that tumours promote their own vascularisation by activating the host endothelium. This review follows the progress of research in angiogenesis in vulvar disease as assessed by a variety of methods, such as in situ hybridisation (ISH), microvessel density measurements (MVD), immunohistochemically-detected angiogenic growth factor expression, including vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), and serum concentrations of VEGF. Thus, the potential of angiogenesis as a prognostic and predictive tool for identifying which pre-malignant lesions could progress to SCC is discussed. A relatively high MVD and strong VEGF expression may serve as prognostic indicators of survival in invasive SCC. MVD and VEGF expression are also predictive factors in identifying which VIN lesions are more likely to become invasive. However VEGF is not a predictive marker in cases of LS likely to progress to carcinoma. The expression of PD-ECGF/TP in all types of lesions tested prevents its use as a prognostic tool, unlike VEGF. However, PD-ECGF/TP is involved in PDV pathogenesis, but is not a marker of the malignant progression of PDV. In PDV, VEGF was not expressed even in those cases associated with invasive disease. Serum concentrations of VEGF play a functional role in vulvar carcinogenesis. Although associated with impaired disease-free and overall survival, pre-treatment serum concentrations of VEGF are not an independent predictor of outcome in patients with vulvar cancer. These studies suggest that further angiogenic research will be important in both the therapy and prognosis of malignant and pre-malignant vulvar disease.

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.

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.