Normal Vulvar Epithelium Research Articles

ORF1p Is a Potential Novel Diagnostic Marker for Differentiated Vulvar Intraepithelial Neoplasia.

Accurate diagnosis of differentiated vulvar intraepithelial neoplasia (dVIN) can be challenging as histomorphologic features may be subtle and overlap with nondysplastic lesions. In practice, aberrant p53 expression supports the diagnosis, but a substantial percentage retains wild-type p53. Recently, the retrotransposon long interspersed nuclear element 1 has been detected in distinct cancer types. We have now investigated the expression of the long interspersed nuclear element 1 encoded protein ORF1p in dysplastic and nondysplastic vulvar samples to assess its diagnostic value. Specimens of dVIN (n=29), high-grade squamous intraepithelial lesions (n=26), inflammatory vulvar lesions (n=20), lichen sclerosus (n=22), and normal vulvar epithelia (n=29) were included. ORF1p and p53 expression was determined using immunohistochemistry. The majority of dVIN [27/29 (93%)] and high-grade squamous intraepithelial lesions [20/26 (77%)] showed distinct (i.e. moderate or strong) ORF1p expression in the basal and suprabasal or all epithelial layers, respectively. Of note, ORF1p was present in all 4 cases of dVIN with wild-type p53 staining pattern. In contrast, ORF1p was negative or weakly expressed in most inflammatory lesions [14/20 (70%)] and lichen sclerosus [18/22 (82%), P <0.001]. Normal control epithelium exhibited negative staining in all cases. In conclusion, ORF1p might be a useful diagnostic marker for dVIN, especially in cases with retained wild-type p53.

Evaluation of CHK1 activation in vulvar squamous cell carcinoma and its potential as a therapeutic target in vitro.

CHK1 is an important regulator of the cell cycle and DNA damage response, and its altered expression has been identified in various tumors. Chk1 inhibitors are currently being evaluated as monotherapy and as potentiators of chemotherapy in clinical settings. However, to our knowledge, no previous study has investigated either the activation status or the therapeutic potential of CHK1 targeting in vulvar cancer. Therefore, we examined the expression status of activated CHK1 forms pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 in 294 vulvar squamous cell carcinomas (VSCC) using immunohistochemistry and analyzed their relationships with various clinicopathological variables and clinical outcome. To aid translation of preclinical studies, we also assessed cell sensitivity to the Chk1 inhibition in two vulvar cancer cell lines. Compared to the levels of pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 in normal vulvar squamous epithelium, high nuclear pCHK1Ser345 expression was found in 57% of vulvar carcinomas, whereas low nuclear pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 expressions were observed in 58%, 64%, and 40% of the cases, respectively. Low levels of pCHK1Ser317 and pCHK1Ser280 in the nucleus correlated significantly with advanced tumor behaviors and aggressive features. None of pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 forms were identified as prognostic factors. In vitro inhibition of CHK1 by small molecular inhibitors or siRNA reduced viability by inducing DNA damage and apoptosis of vulvar cancer cell lines. In summary, we conclude that cellular functions regulated by CHK1 are phosphorylation/localization‐dependent and deregulation of CHK1 function occurs in VSCC and might contribute to tumorigenesis. Targeting CHK1 might represent as a useful antitumor strategy for the subgroup of VSCC harboring p53 mutations.

HPV-related vulvar diseases and perspectives of p16INK4a immunochemistry: a review of the literature.

Two different types of vulvar intraepithelial neoplasia (VIN), HPV-related and HPV-unrelated, should be considered as two separate entities with different management options. The incidence of HPV-related VIN is increasing worldwide and is implicated in carcinogenesis. Our objective is to investigate the use of p16INK4a immunostaining or p16INK4a/p53 double staining for the detection of HPV-related disease to overcome the problem that histological criteria often have significant overlap. A systematic literature search was carried out in the online databases PubMed, EMBASE, Cochrane Library, Clincaltrials.gov and Scopus. The key search terms were HPV, VIN, p16INK4a immunochemistry and p53. We found that nuclear and cytoplasmic immunostaining for p16INK4a was intense and diffuse in HPV-associated lesions and weak and focal in normal vulvar epithelium, nondysplastic lesions, lichen sclerosus and keratinizing vulvar squamous cell carcinoma. p53 nuclear immunostaining was always negative in HPV-related disease. Our findings indicated that p16INK4a or p16INK4a/p53 immunoreactivity, along with histological diagnosis, could be a convenient means to adequately classify VIN and its connection to HPV infection. Therefore, the clear recognition of HPV-associated VIN would lead to an appropriate strategy of treatment and follow-up.

MALDI Mass Spectrometry Imaging Reveals Decreased CK5 Levels in Vulvar Squamous Cell Carcinomas Compared to the Precursor Lesion Differentiated Vulvar Intraepithelial Neoplasia.

Vulvar cancer is the fourth most common gynecological cancer worldwide. However, limited studies have been completed on the molecular characterization of vulvar squamous cell carcinoma resulting in a poor understanding of the disease initiation and progression. Analysis and early detection of the precursor lesion of HPV-independent vulvar squamous cell carcinoma (VSCC), differentiated vulvar intraepithelial neoplasia (dVIN), is of great importance given dVIN lesions have a high level of malignant potential. Here we present an examination of adjacent normal vulvar epithelium, dVIN, and VSCC from six patients by peptide Matrix-assisted laser desorption/ionization Mass Spectrometry Imaging (MALDI-MSI). The results reveal the differential expression of multiple peptides from the protein cytokeratin 5 (CK5) across the three vulvar tissue types. The difference observed in the relative abundance of CK5 by MALDI-MSI between the healthy epithelium, dVIN, and VSCC was further analyzed by immunohistochemistry (IHC) in tissue from eight VSCC patients. A decrease in CK5 immunostaining was observed in the VSCC compared to the healthy epithelium and dVIN. These results provide an insight into the molecular fingerprint of the vulvar intraepithelial neoplasia that appears to be more closely related to the healthy epithelium than the VSCC.

ROCK1 as a novel prognostic marker in vulvar cancer

BackgroundVulvar carcinoma is an infrequent tumour, accounting for fewer than 3% of all malignant tumours that affect women, but its incidence is rising in the past few decades. In young women, the manifestation of the vulvar carcinoma is often linked to risk factors such as smoking and HPV infection, but most cases develop in women aged over 50 years through poorly understood genetic mechanisms. Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) has been implicated in many cellular processes, but its function in vulvar cancer has never been examined. In this study, we aimed to determine the prognostic value of ROCK1 gene and protein analysis in vulvar squamous cell carcinoma (VSCC).MethodsROCK1 expression levels were measured in 16 vulvar tumour samples and adjacent normal tissue by qRT-PCR. Further, 96 VSCC samples were examined by immunohistochemistry (IHC) to confirm the involvement of ROCK1 in the disease. The molecular and pathological results were correlated with the clinical data of the patients. Sixteen fresh VSCC samples were analyzed by array-based comparative genomic hybridization (aCGH).ResultsIn each pair of samples, ROCK1 levels were higher by qRT-PCR in normal tissue compared with the tumour samples (p = 0.016). By IHC, 100% of invasive front areas of the tumour and 95.8% of central tumour areas were positive for ROCK1. Greater expression of ROCK1 was associated with the absence of lymph node metastasis (p = 0.022) and a lower depth of invasion (p = 0.002). In addition, higher ROCK1 levels correlated with greater recurrence-free survival (p = 0.001). Loss of ROCK1 was independently linked to worse cancer-specific survival (p = 0.0054) by multivariate analysis. This finding was validated by IHC, which demonstrated enhanced protein expression in normal versus tumour tissue (p < 0.001). By aCGH, 42.9% of samples showed a gain in copy number of the ROCK1 gene.ConclusionsROCK1 is lower expressed in tumour tissue when compared with adjacent normal vulvar epithelia. In an independent sample set of VSCCs, lower expression levels of ROCK1 correlated with worse survival rates and a poor prognosis. These findings provide important information for the clinical management of vulvar cancer.

Phosphorylated S6 as an immunohistochemical biomarker of vulvar intraepithelial neoplasia

As life expectancy lengthens, cases of non-viral-associated vulvar squamous cell carcinoma and its precursor lesion, so-called differentiated vulvar intraepithelial neoplasia (VIN), continue to increase in frequency. Differentiated VIN often is difficult to recognize and failure to detect it before invasion results in morbidity and mortality. Thus, identification of a reliable biomarker for this type of lesion would be of great clinical benefit. Our recent studies have identified activation (ser235/236 phosphorylation) of ribosomal protein S6 (p-S6) in basal epithelial cells as an event that precedes and accompanies laminin γ2 overexpression in most preinvasive oral dysplasias. To test this as a potential biomarker of vulvar dysplasia, we immunostained seven differentiated VINs and nine papillomavirus-related ‘classic’ VINs, most of which were associated with carcinoma, for p-S6. All carcinomas, all differentiated VINs, and most classic VINs contained regions of p-S6 staining in the basal layer, whereas basal and parabasal cells of normal vulvar epithelium and hyperplastic and inflamed lesions lacking cellular atypia were p-S6 negative. Laminin γ2 was expressed in a subset of VINs, always occurring within basal p-S6 positive regions, as we had found previously for oral dysplasias. Lichen sclerosus is considered a potential precursor of vulvar carcinoma. Two lichen sclerosus lesions of patients with a concurrent carcinoma and one of six lichen sclerosus lesions without atypia or known concurrent carcinoma were basal p-S6 positive. In summary, there is a distinct difference in p-S6 basal cell layer staining between benign and neoplastic vulvar squamous epithelium, with consistent staining of differentiated VIN and of some lichen sclerosus lesions. These results support further studies to assess the potential of p-S6 as a biomarker to identify vulvar lesions at risk of progressing to invasive cancer.

Immunohistochemical Expression of SOX2 in Vulvar Intraepithelial Neoplasia and Squamous Cell Carcinoma

SOX2 is a transcription factor controlling pluripotency in both embryonic stem cells and adult tissue-specific stem cells. SOX2 has been reported as an important factor in squamous cell carcinomas (SCC) of different locations and is involved in tumorigenesis. We evaluated the expression of SOX2 in vulvar non-neoplastic and neoplastic epithelia to test whether it is related to neoplastic progression. SOX2 immunoexpression was evaluated in 101 formalin-fixed, paraffin-embedded archival vulvar epithelia consisting of normal squamous vulvar epithelia (n=25), lichen sclerosus (n=9), high-grade classic vulvar intraepithelial neoplasia (HG-VIN, n=16), differentiated vulvar intraepithelial neoplasia (d-VIN, n=18), and vulvar invasive keratinizing SCC (n=33). Immunoexpression of SOX2 was nuclear and increased stepwise from normal vulvar epithelia/lichen sclerosus to HG-VIN and d-VIN (P<0.0001), from HG-VIN and d-VIN to invasive SCC (P=0.0029), and followed the morphologic distribution of atypical squamous epithelial cells. Scores for normal vulvar epithelia versus lichen sclerosus and HG-VIN versus d-VIN, respectively, did not differ significantly. SOX2 expression increased from tumor Grade 1 to 3 (P=0.0124); there was no relation to recurrence and survival. This is the first study presenting SOX2 as overexpressed in vulvar intraepithelial and invasive squamous lesions. This overexpression apparently reflects an early event in the neoplastic transformation of vulvar squamous epithelia. However, SOX2 seems to play a role in histologic dedifferentiation to Grade 3 invasive SCC too, and may be relevant to vulvar carcinogenesis.

Expression of p15INK4b and p57KIP2 and Relationship with Clinicopathological Features and Prognosis in Patients with Vulvar Squamous Cell Carcinoma

BackgroundThe cyclin-dependent kinase inhibitors p15INK4b and p57KIP2 are important regulators of the cell cycle, and their abnormal expression has been detected in various tumors. However, little is known about the role of p15INK4b and p57KIP2 in the pathogenesis of vulvar carcinoma, and the prognostic impact is still unknown. In our current study, we examined the expression of p15INK4b and p57KIP2 in a large series of vulvar squamous cell carcinomas to elucidate the prognostic impact.MethodsExpression of p15INK4b and p57KIP2 were examined in 297 vulvar squamous cell carcinomas using immunohistochemistry. Both uni- and multivariate analysis of prognostic factors were performed, and correlations with clinicopathologic parameters were examined.ResultsCompared to the high levels of p15INK4b and p57KIP2 in normal vulvar squamous epithelium, low levels of p15INK4b and p57KIP2 were found in 82% and 44% of vulvar carcinomas, respectively. Low levels of p15INK4b and p57KIP2 correlated significantly with malignant features, including large tumor diameter (p = 0.03 and p = 0.001, respectively) and increased invasiveness (p = 0.003 and p = 0.04, respectively). Although p15INK4b and p57KIP2 levels could not be identified as prognostic markers, combined analysis of p14ARF/p15INK4b/p16INK4a showed that patients whose tumors expressed low levels of two or three of these INK4 proteins had a worse prognosis than those with only low levels of one or no protein (univariate analysis p = 0.02). The independent prognostic significance of these INK4 proteins was confirmed by multivariate analysis (p = 0.008).ConclusionsWe show for the first time that p15INK4b and p57KIP2 may be involved in the progression of vulvar carcinomas and the combined p14ARF/p15INK4b/p16INK4a status was a statistically independent prognostic factor.

Expression of Human Telomerase Reverse Transcriptase in Vulvar Intraepithelial Neoplasia and Squamous Cell Carcinoma: An Immunohistochemical Study With Survivin and p53

Human telomerase reverse transcriptase (hTERT), an enzyme that enables cells to overcome replicative senescence and to divide indefinitely, is overexpressed in many cancers and their precursor lesions. To test whether hTERT expression is related to neoplastic progression and resistance to apoptosis in vulvar epithelia. Immunoexpression of hTERT was evaluated in 101 formalin-fixed, paraffin-embedded archival vulvar epithelia consisting of normal squamous vulvar epithelia (n = 25), lichen sclerosus (n = 10), high-grade classic vulvar intraepithelial neoplasia (n = 16), differentiated vulvar intraepithelial neoplasia (n = 18), and vulvar invasive keratinizing squamous cell carcinoma (n = 32) and related to survivin and p53 expression. Immunostaining for all factors was scored for moderate and strong intensities with regard to quantity to determine upregulation and overexpression (score 0, 0% immunoreactive cells; score 1+, <5% immunoreactive cells; score 2+, 5% to 50% immunoreactive cells; score 3+, >50% immunoreactive cells). Score 3+ was considered as overexpression. Nuclear hTERT immunoexpression was closely related to survivin reactivity, increased from normal vulvar squamous epithelia to lichen sclerosus and to high-grade classic vulvar intraepithelial neoplasia, differentiated vulvar intraepithelial neoplasia, and invasive keratinizing squamous cell carcinoma (P < .001), and followed the morphologic distribution of atypical squamous epithelial cells. Overexpression of hTERT was comparable to that seen for p53 in invasive keratinizing squamous cell carcinoma (P = .62); significant differences were calculated for differentiated vulvar intraepithelial neoplasia (P = .003) and high-grade classic vulvar intraepithelial neoplasia (P = .001). Human telomerase reverse transcriptase is upregulated in vulvar intraepithelial neoplasia and invasive keratinizing squamous cell carcinoma compared with nonneoplastic squamous epithelia of the vulva as an apparently early and preinvasive event in the neoplastic transformation, with development of cellular longevity and resistance to apoptosis by survivin activation as associated features, independent of the etiology of vulvar intraepithelial neoplasia.

High levels of p53 expression correlate with DNA aneuploidy in (pre)malignancies of the vulva

The molecular pathogenesis of human papilloma virus-unrelated vulvar squamous cell carcinoma is not well known. Whether malignant progression of lichen sclerosus and differentiated vulvar intraepithelial neoplasia to vulvar squamous cell carcinoma could be accompanied by altered DNA content has not been studied extensively. DNA content in isolated nuclei of microdissected normal vulvar epithelium (n = 2), lichen sclerosus (n = 9), differentiated vulvar intraepithelial neoplasia (n = 13), and squamous cell carcinoma (n = 17) from 22 patients was measured via DNA image cytometry. For additional analysis, 6 differentiated vulvar intraepithelial neoplasia lesions were selected, bringing the number of patients to 28. p53 expression was determined by immunohistochemistry on consecutive tissue sections. Thirty-eight percent (5/13) of differentiated vulvar intraepithelial neoplasia lesions and 65% (11/17) of squamous cell carcinomas were DNA aneuploid or tetraploid. In lesions that contained differentiated vulvar intraepithelial neoplasia and adjacent squamous cell carcinoma, the ploidy status of differentiated vulvar intraepithelial neoplasia did not exceed that of squamous cell carcinoma. We observed a strong correlation between high p53 expression and DNA aneuploidy. This relation was also present at the level of a single nucleus, measured by sequential image cytometry of p53 immunohistochemistry followed by DNA image cytometry on formalin-fixed tissue sections. Similarly, we found p53-positive nonproliferating cells with increased DNA content in the superficial compartment of 6 additional solitary differentiated vulvar intraepithelial neoplasia lesions that were not associated with squamous cell carcinoma, indicating ascending aneuploid cells from the basal compartment. DNA ploidy measurements suggest that differentiated vulvar intraepithelial neoplasia has a higher malignant potential than lichen sclerosus and thus is a more likely precursor of squamous cell carcinoma. Furthermore, high p53 expression correlates with increased DNA content and aneuploidy; but it requires further research to unveil a possible causal relation.

MIB1 expression in basal cell layer: a diagnostic tool to identify premalignancies of the vulva

Lichen sclerosus, high-grade usual vulvar intraepithelial neoplasia (VIN) and differentiated VIN have a different malignant potential. The objective of this study was to quantify the proliferative activity in the basal region of the epithelium of vulvar premalignancies. Furthermore, we investigated whether MIB1 expression in the basal region of vulvar epithelium can be helpful in diagnosing differentiated VIN, which may be hard to discern from normal epithelium. MIB1 was used to immunohistochemically visualise proliferating cells within formalin-fixed, paraffin-embedded, archival tissue sections of different vulvar premalignancies (N=48) and normal vulvar epithelium (N=16). Automatic digital image analysis software was developed to quantify the proliferating fraction in different parts of the epithelium (MIB1 positivity index). MIB1 expression differed among the various vulvar premalignancies; a MIB1-negative basal cell layer was a distinct feature of normal vulvar epithelium. No MIB1-negative basal cell layer was noted in differentiated VIN or other vulvar premalignancies. Owing to this negative cell layer, the MIB1 proliferation index in normal vulvar epithelium was significantly lower than in vulvar premalignancies. In conclusion, MIB1 expression can be a helpful tool in diagnosing a premalignancy and has additional value especially to distinguish differentiated VIN neoplasia from normal vulvar epithelium, but cannot explain the differences in malignant potential.

Aberrant expression of α-dystroglycan in cervical and vulvar cancer

Objectives. Cervical and vulvar cancers develop through well-defined precursor lesions but their exact pathogenesis is still unknown. The dystroglycan complex is a transmembrane glycoprotein that forms a continuous link from the extracellular matrix to the actin cytoskeleton. Deregulated expression of dystroglycan has been reported in human malignancies and related to tumor differentiation and aggressiveness. In this study, expression of dystroglycan was evaluated in the multistep cervical and vulvar tumorigenesis. Methods. Expression of the dystroglycan complex was evaluated by immunostaining in lesions representing different stages of vulvar and cervical tumorigenesis using a monoclonal antibody which recognizes carbohydratic epitopes on the α-dystroglycan subunit. Results. α-dystroglycan was constantly detected in normal cervical epithelium with a mean percentage of positive cells higher than 80%. A progressive significant reduction in the mean percentage of positive cells was observed in low (67%) and high grade SIL (14%) and in invasive carcinomas (2.6%) of the cervix. In cancers, no differences were observed in terms of percentage of positive cells when cases were stratified according with either tumor grade or stage. A progressive significant reduction in the mean percentage of positive cells was also observed from normal vulvar epithelium (90%) to VIN1 (66%), VIN2 (28%) and invasive vulvar carcinomas (22%). No significant decrease in the α-dystroglycan staining was observed in squamous cell hyperplasia lesions (85%) while lichen sclerosus displayed a percentage of positive cells (47%) significantly lower than normal epithelium. Conclusions. Detection of α-dystroglycan is frequently lost in human cervical and vulvar tumorigenesis and further studies are warranted to verify whether evaluation of this molecule might serve as marker of risk progression of preneoplastic lesions and to better understand its significance in terms of cancer development.

Expression of the CDK inhibitor p27kip1 and oxidative DNA damage in non-neoplastic and neoplastic vulvar epithelial lesions

Vulvar cancer represents an important medical problem worldwide whose incidence is increasing at an alarming rate in young females. Several factors have been linked to vulvar cancer development, but its exact pathogenesis remains to be determined. Vulvar tumorigenesis proceeds through intermediate dysplastic lesions, known as vulvar intraepithelial neoplasias, frequently associated with non-neoplastic epithelial disorders of the vulva, such as lichen sclerosus and squamous cell hyperplasia. In this study, the expression of the CDK inhibitor p27Kip1 and the extent of endogenous oxidative DNA damage were evaluated in vulvar specimens, including normal tissues, lichen sclerosus, squamous cell hyperplasia, vulvar intraepithelial neoplasias and invasive squamous cell carcinomas. We found that p27Kip1 was constantly expressed in normal vulvar epithelium cells while a progressive significant reduction in the percentage of p27Kip1-positive cells was observed in vulvar intraepithelial neoplasias (77%) and in invasive carcinomas (64%). Mean percentage of positive cells in invasive carcinomas, but not in vulvar intraepithelial neoplasias, was also significantly lower than squamous cell hyperplasia lesions (78%) while lichen sclerosus displayed a percentage of positive cells (45%) significantly lower than both vulvar intraepithelial neoplasias and invasive carcinomas. 8-hydroxydeoxyguanosine (8-OHdG) is considered a sensitive biomarker for oxidative stress. We observed a progressive significant increase in the levels of 8-OHdG and in the percentage of positive cells from normal vulvar epithelium to vulvar intraepithelial neoplasias (25%) and to invasive carcinomas (64%). Squamous cell hyperplasia displayed an intermediate percentage of positive cells comparable to vulvar intraepithelial neoplasias 2 but significantly higher than vulvar intraepithelial neoplasias 1 and lower than invasive carcinomas. Lichen sclerosus staining was significantly lower than carcinomas but higher than vulvar intraepithelial neoplasias and squamous cell hyperplasia. These results demonstrate that expression of p27Kip1 is downregulated while oxidative DNA damage increases from early non-neoplastic epithelial alterations through vulvar intraepithelial neoplasias to invasive vulvar carcinomas. Thus, both parameters might play an important role in the development of this cancer and their study might contribute to our understanding of human vulvar carcinogenesis.

Overexpression of Wild-type p53 in Lichen Sclerosus adjacent to Human Papillomavirus-negative Vulvar Cancer

Human papillomavirus is a risk factor for vulvar cancer, whereas human papillomavirus-negative late onset vulvar carcinoma is associated with the dermatologic condition, lichen sclerosus. Human papillomavirus E6 protein targets TP53 for degradation and by inference it has been assumed that human papillomavirus-negative vulvar cancer is dependent upon the acquisition of p53 somatic mutations and subsequent allelic loss. To investigate this, TP53 expression, loss of heterozygosity, and p53 genomic sequence were examined in 29 cases of human papillomavirus-negative vulvar carcinoma with adjacent lichen sclerosus. We examined 37 cases of lichen sclerosus without vulvar carcinoma, 10 cases of nongenital lichen sclerosus, and 12 cases of normal vulvar epithelium served as controls. TP53 was evident in 72% of vulvar carcinoma, 48% in epithelium adjacent to vulvar carcinoma, but was minimal in normal samples. When lichen sclerosus cases were selected to exclude samples with absolutely no TP53 expression through probable failed antigen retrieval or homozygous p53 loss the number of epithelial cells expressing TP53 increased progressively from nongenital lichen sclerosus to lichen sclerosus without vulvar carcinoma, then to lichen sclerosus with vulvar carcinoma (p<0.0001). These data suggest elevated TP53 is a feature of vulvar lichen sclerosus. Seventy-four percent of vulvar carcinoma had chromosome 17p-linked loss of heterozygosity, whereas 47% of adjacent lichen sclerosus featured loss of heterozygosity, but only 31% of vulvar carcinoma had p53 mutations, a frequency less than reported previously. Seven percent of adjacent lichen sclerosus had mutations, showing for the first time the presence of an identical mutation to the matched vulvar carcinoma. These data, however, implicate p53 mutations as a later event in vulvar carcinoma and in marked contrast to the original expectation, our loss of heterozygosity data are consistent with loss of another locus (not p53) on 17p operating as a tumor suppressor in lichen sclerosus destined to develop vulvar carcinoma.

Expression of topoisomerase IIalpha, Ki-67, proliferating cell nuclear antigen, p53, and argyrophilic nucleolar organizer regions in vulvar squamous lesions.

It was the aim of this study to investigate the expression of topoisomerase IIalpha (topo IIalpha), Ki-67, proliferating cell nuclear antigen (PCNA), p53, and argyrophilic nucleolar organizer region (AgNOR) staining in normal vulvar epithelia (NE, N = 10), vulvar condylomas (VC, N = 24), vulvar intraepithelial neoplasia (VIN, N = 26), as well as squamous cell carcinomas (SCC, N = 22) of the vulva. Formalin-fixed, paraffin-embedded archival tissue sections were immunostained with monoclonal antibodies against topo IIalpha, p53, and PCNA, as well as an affinity-isolated prediluted ready-to-use Ki-67 antibody using a standard immunohistochemical method, and stained with a colloid silver solution for AgNORs. Immunostaining was quantitated by determining the percentage of positively staining nuclei in each sample to express the labeling indices (LIs) by counting the immunoreactive nuclei in 1000 epithelial cells per case for each antibody. In each specimen 200 nuclei were examined using a x100 oil emersion lens, and the mean number of AgNORs per nucleus (AC) was calculated. The LIs for topo IIalpha, Ki-67, and PCNA as well as ACs increased stepwise from NE to VCs, VIN lesions, and SCCs. In contrast to PCNA LIs and ACs, a consistent correlation in all four groups was found for Ki-67 and topo IIalpha, suggesting that the latter is a proliferation-associated marker in these tissues. p53 expression was seen 8.3% of VCs, 30.8% of VIN lesions, and 54.45% of SCCs. p53 LIs were not correlated with LIs for topo IIalpha or Ki-67 in SCCs. The LIs for topo IIalpha, Ki-67, PCNA, p53, and ACs were not related to tumor progression, FIGO stage, or tumor grade in SCCs. This study presents topo IIalpha and Ki-67 as useful proliferation-associated markers of vulvar epithelia.

Expression of Mucin 1 (MUC1) in Benign, Premalignant and Malignant Vulvar Tumors.

We investigated mucin 1(MUC1) expression in 10 vulvar condyloma acuminata(VCA), 15 vulvar intraepithelial neoplasia(VIN) and 30 vulvar squamous cell carcinomas (VSCC) using three different monoclonal antibodies(Ma695, CA15−3, and DF3), and compared the level of MUC1 expression with the clinicopathologic features of VSCC. Of the three monoclonal antibodies, Ma695 showed the strongest immunoreactivity. MUC1 expression levels were high in VSCC, low in VIN III, and undetectable in VIN I-II, VCA and normal vulvar epithelium. Patients with human papilloma virus(HPV)−negative VSCC showed higher MUC1 expression than HPV−positive patients. The MUC1 expression level gradually increased from well through moderately to poorly differentiated VSCC, and there was a statistically significant difference between moderately/poorly differentiated VSCC and well differentiated VSCC(p<0.05). These results indicate that MUC1 expression may be an effective tumor marker for VSCC, and suggest that MUC1 gene expression may be a late event in the growth of VSCC. Moreover, it appears likely that MUC1 expression correlates negatively with HPV infection. Since the appearance of MUC1 is linked to cell dedifferentiation, it may be related to the malignant transformation and progression of VSCC.

Lectins in the vulva. I. Normal vulvar epithelium and epithelium adjacent to vulvar intraepithelial neoplasia and squamous cell carcinoma.

Lectins were used to investigate the cell surface oligosaccharide expression in normal vulvar epithelium from premenopausal and postmenopausal volunteer women. In addition, histologically normal epithelium adjacent to high-grade vulvar intraepithelial neoplasia (VIN III) and adjacent to vulvar tumors was examined with lectins for evidence of a possible "field change" surrounding these vulvar lesions. Seventeen vulvar biopsies were obtained prospectively from volunteer women, and 20 and 40 cases, respectively, of VIN III and vulvar squamous cell carcinoma were randomly chosen from pathology archives. Thirteen of the 20 VIN cases and all 40 vulvar carcinomas contained at least 2 cm of histologically normal-appearing epithelium adjacent to the vulvar lesion suitable for analysis. No alterations to lectin binding in normal vulvar epithelium with respect to patient age, menopausal status, phase of menstrual cycle, estrogen therapy, or history of cervical intraepithelial neoplasia were shown. ABO blood group antigen status affected epithelial binding for lectins HPA and UEAI (p < 0.005 and p < 0.001, respectively). In addition, lectins SNA, MPA, and LCA identified markers of cellular differentiation and maturation. T-antigen expression (as shown by the lectin PNA) was almost universally present in histologically normal epithelium adjacent to VIN and vulvar tumors, contrasting with the lack of PNA binding in normal vulvar epithelium from volunteer women (p < 0.001 and p < 0.001), a finding suggestive of a local "field change" surrounding preinvasive and invasive vulvar lesions.

Histologic and biomolecular aspects of papillomatosis of the vulvar vestibule in relation to human papillomavirus

To determine whether human papillomavirus (HPV) plays a role in the genesis of papillomatosis of the vulvar vestibule. We conducted a study based on molecular hybridization and histology of biopsy material obtained from the inner surface of the labia minora of 25 women with papillomatosis of the vulvar vestibule who presented no abnormal clinical, cytologic, or colposcopic changes in the cervix or vagina. These women were compared with 24 women with condyloma acuminatum of the vulvar vestibule and with ten women with normal vulvar epithelium and no cervicovaginal changes. All patients included in the study were 35 years or younger, and none was pregnant. Papillomatosis of the vulvar vestibule was rarely found to be HPV positive by molecular hybridization (one of 25, 4%, by dot blot hybridization and one of 15, 6.67%, by polymerase chain reaction [PCR]). This result did not differ significantly from that obtained for the group with normal vulvar epithelium (none of 10 by dot blot and none of six by PCR), but did differ (P = .001) from the result obtained for the group with condyloma acuminatum of the vestibule (12 of 24, 50%, by dot blot and six of six, 100%, by PCR). The biomolecular study of vestibular papillomatosis showed that focal koilocytosis was not correlated with HPV infection. Papillomatosis of the vulvar vestibule is not associated with HPV and should be considered a paraphysiologic formation of the vulvar epithelium. The diagnosis of vulvar HPV infection should be avoided in the absence of more explicit clinical-histologic evidence, with no need for biopsies or unnecessary treatments.

Cytokeratin subtypes and involucrin in squamous cell carcinoma of the vulva. An immunohistochemical study of 41 cases

Histologic grade seems to be of limited prognostic significance in patients with vulvar carcinoma. However, the study of cytokeratin expression is of potential interest because it allows a more precise evaluation of the degree of squamous differentiation. This study was conducted to investigate whether differences in cytokeratin expression exist between normal vulvar epithelium and vulvar carcinoma and whether these differences are prognostically significant. The expression of several differentiation markers, i.e., cytokeratin (CK) 10, CK 13, and involucrin, was studied in samples of 41 vulvar carcinomas. The expression of CK 8, 10, 13, and 14 was compared with CK expression in normal vulvar epithelium and was correlated with tumor grade and tumor growth pattern. Tumor growth pattern was considered type A if infiltrating tumor cell nests showed a layer of small, basaloid cells bordering the surrounding mesenchymal tissue and was considered type B if this was not the case. Prognosis was based on whether disease recurred or not. Sixteen patients had disease recurrence. No prognostic significance of tumor grade was found. Tumor growth pattern was prognostically significant: in patients with a type A tumor, recurrence was observed less often than in patients with a type B tumor (P = 0.03). Cytokeratin 14, typical for basal cells of normal vulvar epithelium, was expressed in all tumors, whereas CK 8 was not expressed in any tumor. A relationship between tumor growth pattern and the concordant expression of differentiation markers was observed: in 55% of type A tumors and in none of type B tumors, concordant expression of CK 10, CK 13, and involucrin was found. The expression of differentiation markers in vulvar carcinoma is related strongly to the tumor growth pattern, and this pattern is prognostically significant.

Upregulation of integrin expression in benign vulvar warts

Benign anogenital warts demonstrate features both of hyperproliferation and of aberrant differentiation associated with human papillomavirus (HPV) infection. The expression of integrins has been examined in normal vulvar epithelium and benign vulvar warts to determine whether HPV infection is associated with changes in integrin expression, either during terminal differentiation or among the proliferating cell population. The expression of integrins in normal vulvar epithelium was similar to that seen in the epidermis, with little expression of alpha 1 and expression of alpha 2, alpha 3, and beta 1 at the periphery of basal cells. In vulvar wart epithelium, alpha 1 was not significantly expressed, but upregulation of alpha 2, alpha 3, and beta 1 was seen at the periphery of cells throughout the basal and spinous layers. In addition, alpha 2 staining was greater at foci along the basal layer of normal vulva, whilst such heterogeneity was not detected in wart sections. Expression of beta 4 and alpha V was confined to the basal and lateral aspects of normal basal cells, but expressed at the periphery of basal cells and to a lesser extent in epibasal and spinous layers of warts. The observed differences in integrin expression are consistent with those reported in other hyperproliferative epithelia.