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Our understanding of the pathogenesis of Paget's disease of the vulva and the breast remains limited. Current evidence supports the fact that angiogenesis plays an important role in the pathogenesis of several diseases. Therefore, we sought to define its role, as correlated with microvessel density, in Paget's disease of the vulva and the breast. Microvessels were analysed using anti-von Willebrand factor antibody in 105 cases of Paget's disease of the vulva and the breast comprising 71 cases of Paget's disease of the vulva, including 8 cases with invasive disease, and 34 cases of Paget's disease of the breast. The latter included 12 cases with DCIS, 5 cases with both DCIS and invasive carcinoma, and 6 with carcinoma alone. Eleven cases had no underlying tumour identified. Increased microvessel density was demonstrated in Paget's disease of the breast with DCIS and with carcinoma alone compared to Paget's disease of the breast alone, P < 0.08 and P < 0.013, respectively. There were no significant differences in microvessel density in the vulval cases. Neovascularisation is an important process in the development of Paget's disease of the breast. Other biological and molecular processes are more involved in the pathogenesis of Paget's disease of the vulva.

Aims: Loss of retinoblastoma protein expression and overexpression of cyclin D1 have been implicated in the development and progression of some cancers. Paget's disease of the vulva (PDV) and Paget's disease of the breast (PDB) are uncommon conditions and the pathogenesis of these diseases is still unclear. The aim was to examine the expression of the retinoblastoma and cyclin D1 proteins in PDV and PDB and to correlate any differences between PDV and PDB, and in the presence or absence of an underlying carcinoma., Methods and Results: Seventy-two archival cases of PDV including 10 with invasive disease and 36 cases of PDB were evaluated immunohistochemically for the expression of cyclin D1 and retinoblastoma protein. Forty-four percent (32/72) of cases of PDV showed loss of expression of the retinoblastoma protein, compared with 67% (24/36) of PDB cases. Fifty-nine percent (41/69) of PDV overexpressed cyclin D1. In PDB, 8% (3/34) overexpressed cyclin D1. There were no significant differences in the expression of retinoblastoma and cyclin D1 in PDV cases with or without underlying invasive disease. There were significant differences between the expression of retinoblastoma (P = 0.03) and cyclin D1 (P < 0.001) in PDV compared with PDB., Conclusions: The differences in the expression of cyclin D1 and retinoblastoma may indicate the differences in the pathogenesis of PDV and PDB.

The growth and metastasis of many cancers is due in part to loss of cell-cell adhesion. E-cadherin, plakoglobin and beta-catenin are important in cell adhesion. Our aim was to examine the presence of these molecules in Paget's disease of the vulva and Paget's disease of the breast, and to correlate any differences in their expression with the presence of invasive disease or an underlying carcinoma. Sixty-three archival cases of Paget's disease of the vulva, including eight associated with invasive disease, and 23 archival cases of Paget's disease of breast, which included 10 cases with ductal carcinoma in situ alone, four cases with both ductal carcinoma in situ and invasive carcinoma, and five cases with underlying invasive carcinoma alone, were analysed immunohistochemically for expression of E-cadherin, plakoglobin and beta-catenin proteins. The respective mRNAs were also detected by in situ hybridisation using digoxigenin-labelled cRNA probes. Seventy-six percent (41/54) of Paget's disease of vulva cases had >50% of Paget cells expressing the E-cadherin protein, compared with 28 % (2/7) of Paget's disease vulva with invasive disease. This result was significant, with a P-value of 0.039. Twenty-five percent (14/55) of the intraepidermal Paget's disease of the vulva cases had >50% of Paget cells expressing the plakoglobin protein, compared with 12% (1/8) of cases of Paget's disease of vulva with invasive disease, and for beta-catenin, 9% (5/55) of the non-invasive Paget's disease of the vulva had >50% of Paget cells expressing beta-catenin, compared with 12% (1/8) of Paget's disease of the vulva cases with invasive disease. Sixty-five percent (15/23) of the Paget's disease of the breast had >50% of Paget cells expressing E-cadherin, and for plakoglobin and beta-catenin it was 17% (4/23) and 28% (6/21), respectively. The results were not significant. The results suggest that reduced expression of E-cadherin may have a role to play in the pathogenesis of invasive Paget's disease of the vulva. Abnormal plakoglobin expression may be involved in the formation of some cases of Paget's of the vulva and the breast.

Objective: To perform a pilot study to investigate the relationship between localized, provoked vulvodynia of the vestibule and inflammatory cytokine expression., Study Design: Women with a diagnosis of localized, provoked vulvodynia had tissue samples taken for vulvar expression of Interleukin 1alpha and 1beta and tumor necrosis factor alpha and compared to those of a control group., Results: The study group did not show a significant increase in expression of inflammatory markers., Conclusion: There was no evidence in this study that localized, provoked vulvodynia is an inflammatory condition, as previously thought. This may be helpful in explaining why some women are resistant to medical or antiinflammatory treatment and may allow treatment to be prescribed more effectively.

Purpose of Investigation: Epithelial ovarian cancer (EOC) is the leading cause of death from gynaecological malignancy in the UK. The pathogenesis of this disease is poorly understood. Our hypothesis was that chlamydial infection might play a role in the pathogenesis of EOC., Methods: 122 serum samples of patients undergoing surgery for benign or malignant gynaecological conditions were analysed. There was a total of 41 patients with EOC (33.6%), 27 with benign cystadenomas (22.1%) and 54 with normal ovaries (44.3%)., Results: There was a higher incidence of IgA seropositivity and lower incidence of IgG seropositivity in the EOC group compared with the other groups; however, this was not statistically significant. There was no statistical difference in the serum IgM antibodies to chlamydia in the three different groups., Conclusion: Although chronic infection and persistent inflammation may contribute to the pathogenesis of EOC, and chlamydia is a common genital tract pathogen, our study did not find an association between chlamydia and EOC.

Background: Although uterine fibroids are very common, their pathogenesis and clinical behaviour are poorly understood. Since they may be prevalent in some families, we investigated whether such a prevalence was associated with distinctive clinical and molecular features., Methods: A case-control questionnaire study of 300 multi-ethnic women with uterine fibroids at a London university hospital was undertaken, with review of case notes and immunohistochemical determination of vascular endothelial growth factor (VEGF-A) in fibroids., Results: When compared with families with sporadic fibroids, familial prevalence of fibroids was associated with a higher incidence of abdominal swelling (59.1% versus 41.6%; P=0.037), menorrhagia (84.4% versus 51.9%; P=0.042), dysmenorrhoea (64.4% versus 46.3%; P=0.004), dyspareunia (43.2% versus 27.9%; P=0.012) and family history of cancers (52.3% versus 32.4%; P<0.01). The fibroids were also more multiple (mean +/- SEM: 7 +/- 0.86 versus 3 +/- 0.42; P<0.011) and strong VEGF-A expression in fibroids was more common in the familial group (64% versus 28%). Racial distribution was the same in both groups (blacks 49%, whites 33.4%, others 18.6%)., Conclusions: Familial prevalence of uterine fibroids is associated with distinct clinical and molecular features that differ from those found when fibroids occur sporadically in families.

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.

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.

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.

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.

Objective: A pilot study was performed to investigate the relationship between vulvar vestibulitis syndrome and estrogen receptor expression., Study Design: Women with a diagnosis of vulvar vestibulitis syndrome had tissue samples taken for vulvar estrogen receptor-alpha expression and this was compared with a control group., Results: The study group showed a significant decrease in estrogen receptor expression, and 50% of the samples did not exhibit any receptor expression., Conclusion: There appears to be a subgroup of women with vulvar vestibulitis syndrome who exhibit abnormal estrogen receptor-alpha expression. This may be helpful in explaining why some women are resistant to medical treatment and may allow treatment to be prescribed more effectively.

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.

Angiogenesis, the development of new blood vessels from the 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 will discuss various angiogenic stimulators and inhibitors in epithelian ovarian cancer (EOC), including vascular endothelial growth factor and platelet derived endothelial cell growth factor/thymidine phosphorylase. The analysis of tumour vascularisation by microvessel density will also be discussed and the relevance of these markers of angiogenesis in the prognosis of EOC will be assessed.

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.

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.

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.

Risk factors for squamous cell vulval cancer (SCC) remain unclear though there have been associations with lichen sclerosis, smoking, and vulval intraepithelial neoplasia (VIN). We studied 191 patients who had been referred to the vulval clinic at the Royal Free Hospital and who had both blood group and histopathology results available. Seventy-two percent of patients with SCC and non-neoplastic epithelial disorders of the vulva (NNEDV) were found to be in blood group A with only 17% in blood group O. Those with SCC associated with VIN had only 30% in blood group A with 50% in blood group O. The control population showed that 38% of the population were in blood group A and 43% were in blood group O. Our results suggest that blood group A is prevalent in patients with SCC associated with NNEDV but not in those women with squamous vulval cancer and associated VIN.

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.

This pilot study investigated the use of the non-invasive cytospin monolayer technique in the diagnosis and screening of neoplastic and non-neoplastic vulval disease. Twenty-three patients (age range 34-86 years) attending a vulval disease clinic had brush cytology performed. The samples were prepared with a cytospin monolayer technique and the slides Papanicolaou-stained. Subsequent cytological interpretation and diagnosis were performed without knowledge of the clinical history and correlated with follow-up biopsy histopathology from each patient. Twenty-eight cytospin samples were analysed in total, of which 11 (39%) contained dyskaryotic cells which were assessed and a predicted VIN/AIN grade given. Ten of 11 samples (91%) reported as dyskaryotic had VIN/AIN on biopsy histology. One of 11 samples (9%) was reported as containing occasional squamous cells with borderline nuclear features and, although the corresponding biopsy did not show VIN, basal atypia was reported. One patient had features suggesting invasive carcinoma on cytology which was verified on subsequent biopsy. The 15 cases in which no dyskaryotic cells were identified did not show VIN or AIN on subsequent histology. Two cases were acellular and considered inadequate for cytological interpretation. The cytospin monolayer technique allows the diagnosis of neoplastic from non-neoplastic vulval disease. It is a quick, inexpensive and non-invasive method that may have a role in diagnosis, screening and surveillance of patients.

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.

Objective: The goal of this study was to assess the relationship between ovulation induction, nulliparity, and ovarian epithelial dysplasia., Methods: This retrospective cohort study was performed in one teaching and one district general hospital in London. The subjects, 83 women who had undergone hysterectomy and bilateral oophorectomy and whose ovaries were reported as "normal," were divided into three groups: ovulation induction (13), nulliparity (20), and fertile controls (50). These ovaries were independently reviewed by two pathologists who assigned a score of 0, 1, or 2 to nine epithelial cytological and architectural features. The main outcome measure was the total dysplasia score, which was used to quantify the degree of ovarian epithelial abnormality in the three groups., Results: The mean dysplasia score was significantly higher in the women who had undergone ovulation induction than in the fertile controls (7.92 vs 5.70, P = 0.012). The magnitude of the difference between the ovulation induction group and controls remained similar after adjusting for age, parity, and duration of oral contraceptive use (2.17, 95% CI: -0.11-4.44). However, the statistical significance of this difference was reduced (P = 0.062). We did not find any evidence of a difference in dysplasia score between nulliparous women and controls, neither before (P = 0.85) nor after adjusting for age and duration of oral contraceptive use (P = 0.87)., Conclusions: These results suggest a possible association between ovarian epithelial dysplasia and ovulation induction therapy, in accord with previous reports of increased risk of ovarian cancer in women with a history of fertility treatment. The higher dysplasia score could be attributable to the drugs used to induce ovulation or to a genetic susceptibility to ovarian cancer., (Copyright 2001 Academic Press.)

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.

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.

Tumors of the pituitary gland are usually benign adenomas and account for 10% of all intracranial neoplasms. Five pituitary tumors have previously been reported to harbor multiple allelic deletions. Of these, three displayed particularly aggressive biological behavior, whereas there were no clinical details provided for the others. This study was designed to test the hypothesis that genetic deletions are a marker of invasive behavior and to identify the loci most commonly involved. Accordingly, we studied two cohorts of pituitary tumors, classified radiologically as invasive or noninvasive, for loss of heterozygosity (LOH). There is a significantly higher frequency of LOH in invasive tumors (10.8% of all loci examined) compared to noninvasive tumors (2.4%; P < 0.001). Of the 11 loci investigated, 75% of the allelic deletions identified in invasive tumors were found at 4 loci: 11q13, 13q12-14, 10q, and 1p. Twenty of 47 invasive tumors had evidence of at least 1 allelic deletion, whereas 14 of 20 had more than 1. Of the 6 tumors with only 1 deletion, 5 involved the 11q13 locus, suggesting that this is an early change in the transition from noninvasive to invasive adenoma. Comparison of invasive and noninvasive tumors demonstrates a significantly higher frequency of deletions affecting 11q13 (P < 0.001), 13q12-14 (P < 0.05), and 10q26 (P < 0.05) in invasive tumors. In addition, allelic deletion correlates with increasingly invasive behavior (modified Hardy classification), as 73% of grade 4 tumors compared to 33% of grade 3 and 9.5% of grade 1 and 2 tumors demonstrated LOH at any locus. Furthermore, in some tumors we identified a breakpoint between markers intragenic and extragenic to the retinoblastoma gene (Rb1) on chromosome 13q, suggesting that tumor suppressor genes other than or in addition to Rb1 may be involved in pituitary tumorigenesis. This was further supported by the presence of Rb protein in two of four tumors where the genetic loss extended to include the intragenic marker D13S153. Early identification of tumors with likely invasive potential by means of genetic analysis (LOH) may provide useful information on potential tumor behavior and aid tumor management in a manner that is not possible using routine histological methods. A large prospective study is required in patients without radiological evidence of invasion to assess the value of LOH in predicting outcome and for planning treatment.

In the endocrine pancreas, alpha 2-adrenoceptor stimulation reduces glucose-induced insulin secretion from islet B-cells. There is, however, controversy with regard to the effects of alpha 2-agonists on islet A-cell function. In this paper, we have used RNA samples prepared from whole rat islets and from FACS-purified rat A- and B-cells to study alpha 2-adrenoceptor subtype expression. RNase protection assays detected transcripts encoding alpha 2a and alpha 2b subtypes in the RNA pool of rat islets. Reverse transcription-PCR revealed that transcripts for all three alpha 2-adrenoceptor subtypes are present in rat islet cells in purified A-cell RNA. In contrast, RT-PCR of islet B-cell RNA yielded products corresponding to alpha 2a and alpha 2c, with no evidence for the presence of alpha 2b. Thus, the results reveal that both islet cell types express more than one receptor subtype and suggest that the distribution of the subtypes may differ between rat islet A- and B-cells.

Sequences from cDNA molecules encoding alpha 2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human alpha 2C2-, alpha 2C4- and alpha 2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of alpha 2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic beta-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both alpha 2 C2 and alpha 2 C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding alpha 2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each alpha 2-adrenoceptor subtype in sections of human pancreas. All three subtypes of alpha 2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffin-embedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three alpha 2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple alpha 2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of alpha 2-adrenoceptor subtype mRNA species in pancreatic beta-cells was confirmed by Northern blotting of RNA extracted from the clonal beta-cell line, HIT-T15. Transcripts encoding each of the three cloned alpha 2-adrenoceptor subtypes were detected in HIT-T15 cells. Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with beta 2-adrenoceptor mRNA revealed expression of this species in islet beta-cells but not in the exocrine tissue of the pancreas.

We describe studies to determine if susceptibility to pituitary tumours is associated with the putatively high risk GSTM1 null and CYP2D6 EM genotypes. Frequency distributions of these genotypes were similar in cases and controls though the frequency of CYP2D6 PM and GSTM1 B tended to be lower (P = 0.072 and P = 0.095 respectively) in the tumour group. Immunopositivity for p53 was found in 18/97 tumours. In these samples GSTM1 null (39%) and CYP2D6 EM (56%) frequencies were not different to those in controls. The frequencies of CYP2D6 PM and GSTM1 B in the p53 immunonegative tumours tended to be lower (P = 0.055 and P = 0.1185 respectively) than in controls. Mutations in gsp and ras were studied using the polymerase chain reaction and allele specific oligonucleotide analysis. Eight of 19 somatotrophinomas demonstrated mutations in gsp; frequencies of GSTM1 null and CYP02D6 EM were similar to controls. No ras mutations were identified in 55-tumour studies. The data indicate the GSTM1 null and CYP2D6 EM genotypes are not associated with altered expression of p53 or, mutation of gsp and ras in these adenomas and, suggest the CYP2D6 PM genotype is associated with a reduced risk of pituitary adenomas and, that GSTM1*B confers greater protection than GSTM1*A.

A 30-year-old presented in 1984 with a clinically nonfunctional tumour which subsequently developed into a metastatic corticotrophinoma from which he died despite surgery in 1984, 1986 and 1991 and external radiotherapy in 1986. Molecular genetic analysis of tumour and metastatic tissue revealed loss of heterozygosity at loci on the autosomes 1p, 3p, 10q26, 11q13 and 22q12. Tissue taken at surgery in 1986 also revealed positive cytoplasmic immunostaining for p53 protein. No such staining was evident in tissue taken at first surgery in 1984. Further analysis of invasive pituitary adenomas may reveal loci associated with such behaviour, enabling better prediction of subsequent clinical outcome than is possible using standard histological techniques, and delivery of early, aggressive treatment to those tumours which show molecular markers associated with a poor prognosis.

The p53 protein, a negative regulator of cell growth, plays an important role in the pathogenesis of many human tumours following gene mutation and/or deletion. We screened a large number of sporadic pituitary tumours for p53 protein accumulation suggestive of gene mutation. Samples were divided into benign adenomas (n = 95) and invasive tumours with local or distant invasion (n = 26). All main tumour classes were represented. Putative p53 mutations were detected by immunohistochemistry on paraffin-embedded sections using polyclonal CM-1 and monoclonal DO-7 and PAb1801 antibodies. Results were compared to normal post-mortem pituitary tissue controls (n = 17). p53 protein accumulation was detected in invasive tumours (16%), but only in corticotrophinomas (2/4) and non-functional tumours (4/15). In non-invasive adenomas, protein accumulation was observed only in ACTH-secreting tumours where 50% were positive (16/32). No protein accumulation was identified in any control tissue. These results indicate that p53 protein accumulation may play a role in the development of Cushings adenomas and in the progression of non-functional tumours to the invasive state.

The p53 protein, a negative regulator of cell growth, plays an important role in the pathogenesis of many human tumours following gene mutation and/or deletion. We screened a large number of sporadic pituitary tumours for p53 protein accumulation suggestive of gene mutation. Samples were divided into benign adenomas (n = 95) and invasive tumours with local or distant invasion (n = 26). All main tumour classes were represented. Putative p53 mutations were detected by immunohistochemistry on paraffin-embedded sections using polyclonal CM-1 and monoclonal DO-7 and PAb1801 antibodies. Results were compared to normal post-mortem pituitary tissue controls (n = 17). p53 protein accumulation was detected in invasive tumours (16%), but only in corticotrophinomas (2/4) and non-functional tumours (4/15). In non-invasive adenomas, protein accumulation was observed only in ACTH-secreting tumours where 50% were positive (16/32). No protein accumulation was identified in any control tissue. These results indicate that p53 protein accumulation may play a role in the development of Cushings adenomas and in the progression of non-functional tumours to the invasive state.

Tumor formation may result from the activation of dominant oncogenes or by inactivation of recessive, tumor suppressor genes. The role of such mutations in the development of pituitary tumors has been studied. Tumors from 88 patients, representing the 4 major classes of adenoma, were investigated. In DNA extracted from matched leukocyte and tumor samples, allelic deletions were sought with 15 probes identifying restriction fragment length polymorphisms on chromosomes 1, 5, 10, 11, 13, 17, 20, and 22. Evidence of amplification or rearrangement of 10 recognized cellular oncogenes (N-ras, mycL1, mycN, myc, H-ras, bcl1, H-stf1, sea, kraS2, and fos) was sought in tumor DNA. Activating dominant mutations of Gs alpha were detected using the polymerase chain reaction to amplify exons 7-10 and hybridizing the product to normal and mutant allele-specific oligonucleotides. Allelic deletions on chromosome 11 were identified in 16 tumors (18%) representing all 4 major subtypes. Deletions on other autosomes were observed in less than 6% of tumors. Three adenomas had deletions on multiple autosomes, 2 of these were aggressive and recurrent. Mutations of Gs alpha were confirmed to be specific to somatotrophinomas, being identified in 36% of such tumors in this series. No evidence of amplification or rearrangement of other recognized cellular oncogenes was found. Inactivation of a recessive oncogene on chromosome 11 is an important and possibly early event in the development of the four major types of pituitary adenoma, whereas activating mutations of Gs alpha are confirmed to be specific to somatotropinomas. Two aggressive tumors were found to have multiple autosomal losses, suggesting a multistep progression in the development of tumors of this phenotype.

The frequency of the GSTM1 0 polymorphism at the glutathione S-transferase M1 locus has been determined in controls and patients with pituitary adenomas by using the polymerase chain reaction to amplify genomic DNA in the exon 4-5 region of the gene. The frequency of the genotype in patients with prolactinomas, non-functional adenomas, corticotrophinomas and somatotrophinomas varied between 52-67% compared with 44% in the controls. In the patients with prolactinomas the frequency of the genotype (67%) was significantly greater than in controls with odds ratio analysis indicating that GSTM1 0 individuals have a 2.56-fold greater risk of developing this adenoma.

mRNA and protein populations were studied in the brains of Maudsley reactive (MR) and Maudsley nonreactive (MNR) rat strains, which exhibit differing levels of emotionality. Translational analysis of forebrain mRNA indicated that the relative levels of two translation products (42 kDA, pI 5.0; 30 kDa, pI 5.8) were increased in the MR compared to the MNR strain. In addition, a charge-shift variant of a 36 kDa protein was present in the MR strain. Analysis of brain protein patterns indicated that a protein of 39 kDa, pI 5.0, was found to be more abundant in MR compared with MNR strains in both frontal cortex and hippocampus and the relative level of one protein (40 kDa, pI 5.8) was decreased in the frontal cortex.

Poly(A+) mRNA was extracted from the post-mortem brain of schizophrenics (9 subjects), unipolar depressives (5 subjects) and controls (10 subjects) and used to direct the in vitro translation of radiolabelled protein in a cell-free reticulocyte-lysate system. Protein species were analysed on two-dimensional gels. Over 200 products were detected and, from these, 74 well-resolved species were chosen for further analysis. The optical density of each product was quantified by image analysis and normalised with respect to overall gel intensity. It was found that 7 novel, uncharacterised protein species, ranging from molecular weights (Mr) 17 kDa to 38 kDa and apparent isoelectric points (pI) 5.7-7.1, changed significantly in intensity in the psychotic groups compared to controls. One species changed only in the schizophrenia group (Mr = 26 kDa, pI = 5.8, 18% of control intensity) and 3 changed only in the depressive group (Mr = 38 kDa, pI = 6.2, 540% of control; Mr = 34 kDa, pI = 6.2, 6% of control; Mr = 17 kDa, pI = 5.7, 238% of control). Three further protein species were common to both psychotic groups (one species decreased in both schizophrenia and depression, Mr = 33 kDa, pI = 5.8; two species showed opposing intensity changes, decreasing in schizophrenia and increasing in depression, Mr = 35 kDa, pI = 7.1; Mr = 23 kDa, pI = 6.1). None of these changes was a function of post-mortem delay or mode of death. It is quite likely that such protein species reflect the abundance of specific mRNAs and target gene systems associated with the disease state.(ABSTRACT TRUNCATED AT 250 WORDS)

Total cellular polyadenylated RNA (poly(A)+ RNA, mRNA) was prepared after guanidinium thiocyanate extraction of frozen brain tissue from age-matched controls and patients suffering from schizophrenia and unipolar depression. These mRNA populations were analysed by in vitro translation followed by two-dimensional gel analysis. Data were obtained from fluorograms derived from 10 different schizophrenic patients, 10 different controls and 5 different depressive patients. The relative concentrations of mRNA species coding for 4 translation products (33 kDa, pI 5.8; 26 kDa, pI 5.8; 35 kDa, pI 7.1; 23 kDa, pI 6.1) were significantly reduced in schizophrenia compared to controls when determined by computerised image analysis of the fluorograms. In the case of depression, the relative concentrations of mRNA species coding for 6 translation products were significantly altered, 4 being increased (38 kDa, pI 6.2, 17 kDa, pI 5.7, 35 kDa, pI 7.1; 23 kDa, pI 6.1) and two decreased (34 kDa, pI 6.2; 33 kDa, pI 5.8). Three translation products were altered in both schizophrenia and depression, one (33 kDa, pI 5.8) being altered according to the same trend, a decrease relative to controls, but two (35 kDa, pI 7.1; 23 kDa, pI 6.1) being altered differently in schizophrenia (reduced) and depression (increased). The effects of post mortem delay, mode of death and drug treatment on mRNA composition were also examined and found not to affect the levels of these translation products significantly. The significance of these changes will be discussed in relation to their relevance of biological mechanisms in the psychoses.

Poly (A+ mRNA species, isolated from 100-day-old rat brain, were analysed by in vitro translation and two-dimensional gel electrophoresis. The synthesis of selected protein species was compared to actin on the basis of [35S]methionine incorporation. The estimated molar abundance of translation products varied from abundant species at 0.78% of the total to several are species, detectable below the 0.02% level. If these synthesis rates reflect the abundance of particular mRNAs in the mixture, this sensitivity limit compares well with accepted values using differential cDNA screening techniques. This analysis provides evidence that in vitro translation methodology is able to detect rarer mRNA species than is usually expected--these include similar abundance classes to library screening procedures.

Messenger RNA, obtained from post-mortem brain of 10 schizophrenics, five depressed patients and 10 control subjects, was characterised with respect to a number of parameters. It was found that post-mortem delay was not the major factor in determining RNA yield, size (as determined by cDNA synthesis) and biological activity. Biological activity, as determined by in vitro translation in a reticulocyte-lysate system, could be observed using messenger RNA from periods of 0 to 84 hours post-mortem. Two-dimensional gel analysis of the newly-synthesised radiolabelled products obtained from this material revealed several hundred individual species but no consistent degradation of any particular species with post-mortem delay. It is suggested, therefore, that premortem changes are as important as post-mortem changes in determining RNA yield, size and biological activity. Although no consistent difference could be found between patients and controls using any of these parameters, this study confirms that, by isolating messenger RNA from post-mortem human brain, valuable information can be gained on gene expression in psychiatric disorders.