TP53 mutation and human papilloma virus status as independent prognostic factors in a Norwegian cohort of vulva squamous cell carcinoma.

INTRODUCTION: Vulva squamous cell carcinoma (VSCC) develops through two separate molecular pathways-one involving high-risk human papilloma virus infection (HPV-associated), and the other without HPV infection (HPV-independent) often involving TP53 mutation. HPV-associated VSCC generally has a better progression-free survival than HPV-independent VSCC. The aim of this study was to determine TP53 mutation status using immunohistochemistry, compare different methods of HPV detection and correlate both with survival in a retrospective cohort of 123 patients with VSCC. MATERIAL AND METHODS: Immunohistochemistry for p53, Ki67 and p16INK4A (a surrogate marker for HPV infection) was performed on formalin-fixed paraffin-embedded tissues from a cohort of surgically treated VSCC patients to identify molecular subtypes of VSCC. Presence of HPV infection was detected by HPV DNA PCR and HPV mRNA in situ hybridization (ISH). The Pearson chi-square test and multivariable Cox regression model were used to investigate the association of different parameters with progression-free survival and disease-specific survival (DSS), and Kaplan-Meier curves were used to show the association of different parameters with survival. RESULTS: The results of p53 and p16INK4A immunohistochemistry confirmed three VSCC subtypes associated with different prognosis. The TP53 mutation status was identified as an independent prognostic factor of worse progression-free survival (p = 0.024) after adjustment for FIGO stage. p16INK4A immunohistochemistry, mRNA ISH, and DNA PCR had excellent concordance in terms of HPV detection. According to the multivariable Cox regression model, the presence of hrHPV mRNA correlated significantly with increased progression-free survival (p = 0.040) and DSS (p = 0.045), after adjustment for other confounders. CONCLUSIONS: p53 and p16INK4A immunohistochemistry stratify VSCC cohort into three subtypes with TP53mutated patients having the worst prognosis. The detection of hrHPV mRNA by ISH was an independent predictor of increased survival. Thus, the combined detection of p53 and HPV mRNA might improve risk stratification in VSCC.

Positive p16 Immunostaining Is an Independent Prognostic Variable for Disease-free Survival and Overall Survival in Patients With Squamous Cell Carcinoma of the Vulva Treated With Radical Surgery and Inguinofemoral Lymphadenectomy: An Italian Single Center Retrospective Study.

BACKGROUND/AIM: The expression of the cyclin-dependent kinase inhibitor p16 correlates with the presence of human papillomavirus. The purpose of this investigation was to assess the prognostic relevance of p16 expression in patients with vulvar squamous cell carcinoma (VSCC) treated with radical surgery followed by adjuvant (chemo) radiation in selected cases. PATIENTS AND METHODS: Seventy-eight patients were analyzed retrospectively. RESULTS: Positive p16 immunostaining was detected in 19 (24.4%) patients. Five-year disease-free survival (DFS) and 5-year overall survival (OS) were better in p16-positive compared to p16-negative patients (83.9% versus 37.3% p=0.002 and 91.7% versus 57.6%, p=0.003, respectively). p16 expression retained prognostic relevance at multivariate analysis for both DFS and OS. CONCLUSION: p16 expression was detected in 24.4% of patients with VSCC and was found to be an independent prognostic variable for both DFS and OS.

Evaluating automated infrared thermography and vulva exposure tracking as components of an estrus detection platform in a commercial dairy herd.

The primary objective of this study was to develop an automated infrared thermography platform (Estrus BenchMark) capable of measuring skin temperature and tail movements as a means of identifying cows in estrus. The secondary objective was to evaluate the accuracy of Estrus BenchMark to detect estrus compared to in-line milk progesterone (P4) analysis (Herd Navigator System) in a commercial dairy herd managed under a robotic milking system. Data were collected on forty-six cows from 45 to 120 d after calving. Cows were flagged in estrus when milk P4 fell below 5 ng/mL. The Estrus BenchMark true positive estrus alerts (Sensitivity; Se%) were compared to Herd Navigator System estrus alerts at different time-windows (±12 h, ±24 h, ±48 h, and ±72 h) relative to the Estrus BenchMark estrus alerts for all the estrus alerts (AE) and confidence-quality estrus (CQE; >80% quality) alerts identified by Herd Navigator System. The Estrus BenchMark captured skin temperature and tail movements resulting in vulva exposure (left tail movements, LTail; right tail movements, RTail; and pooled tail movements, PTail) for each milking event. Skin temperature tended to increase when the milk P4 concentration (Least-Squares Means ±â€¯SE) dropped for AE (estrus day [d 0]; P4; 3.51 ±â€¯0.05 ng/mL, Skin temperature; 33.31 ±â€¯2.38 °C) compared with d -7 (P4; 20.22 ±â€¯0.73 ng/mL; Skin temperature: 32.05 ±â€¯3.77 °C). The increase in skin temperature, however, was significant in cows with CQE > 80% at d 0 (32.75 ±â€¯0.29 °C) compared to d -7 (31.80 ±â€¯0.28 °C). The prevalence of tail movements to expose vulva was greater (P = 0.01) in AE at d 0 (LTail: 62.50%; PTail; 68.75%; and RTail: 56.25%) compared with d -7 (LTail: 18.75%; PTail: 9.37%: and RTail: 9.37%), and d +4 (LTail: 9.37%; PTail: 9.37%; and RTail: 12.5%). Moreover, the higher prevalence of tail movements at d 0 was observed in cows with CQE > 80% (LTail; 65%, PTail; 80%, and RTail; 70%) compared to those with CQE < 80%. The highest Estrus BenchMark Youden index (YJ; 0.45), diagnostic odds ratio (DOR; 9.04), and Efficiency (0.77) were achieved for AE in a ±48 h window and at ±72 h window for CQE (YJ; 0.66, DOR; 25.29, and Efficiency 0.76) relative to Herd Navigator System estrus alerts. The highest Estrus BenchMark resulted in 58% estrus detection rates for AE and 80% for cows with CQE compared to the Herd Navigator System.

Evaluation of a handheld device for the measurement of beta-hydroxybutyrate in capillary blood obtained by the puncture of the vulva as well as in venous whole blood in cattle.

A negative energy balance is a common condition in high yielding dairy cows causing the production of ketone bodies (KB), including beta-hydroxybutyrate (BHB), defined as subclinical ketosis (SCK) if clinical signs are missing. The aim of the present study was to evaluate a handheld electronic device for the detection of SCK (BHB-concentration > 1.2 mmol/l), in capillary blood and venous whole blood in cows (WellionVet BELUA, MED TRUST Handels GmbH, Marz, Austria) as well as the feasibility of the puncture of the external vulva with a single use lancet. For this purpose, the blood BHB-concentration was tested in 250 venous and capillary blood samples and compared to the results of a certified laboratory. The majority (76.3%) of the animals displayed no signs of discomfort related to the puncture and in 74.2% the procedure was successful on the first attempt. The BHB-concentrations detected in capillary blood showed good agreement with the reference method, both in capillary (correlation coefficient 0.94 (p⟨0.001), Kappa-value 0.89) and venous whole blood (correlation coefficient of 0.95 (p⟨0.001), Kappa-value 0.89). Altogether, 98% of all the samples were correctly classified as SCK or non-SCK by the handheld device in capillary blood (sensitivity 0.96, specificity 0.98) and 97.4% in venous whole blood (sensitivity 0.889, specificity 0.991), respectively. An increase in the correlation by the adaptation of the cut off level could not be achieved for both sampling sites.

Comparative study of vulva and abdominal skin microbiota of healthy females with high and average BMI.

BACKGROUND: Obesity is known to modulate human health in a number of ways including altering the microbiome of the gut. Very few studies have examined the how obesity may affect the microbiomes of sites distant to the gut. We hypothesized that vulva and abdominal skin may be especially susceptible to body mass index (BMI)-induced alterations in biophysical properties and the microbiome due increased maceration and skin folds at those sites. The aim of this study was to determine if high BMI (≥30) was associated with alterations in the biophysical properties and microbiomes of vulva and abdominal skin. RESULTS: The vulvar microbial communities of healthy reproductive-aged females were examined using 16S rRNA sequencing techniques. Our results show that vulvar pH of women with high body mass index (BMI) was statistically higher than that of women with average BMI. Phylogenetic analysis of the vulvar microbiota indicated that women with average BMI have a predominately Lactobacillus-dominated flora, whereas women with high BMI and higher pH were predominately colonized by Finegoldia and Corynebacterium. This BMI-associated shift in microbiota was not observed in samples collected from the exposed skin around the belly, indicating the effect is not global. CONCLUSION: These results indicate that physiological changes associated with changes in BMI may modulate the vulva microbiome.

Reduction in ERRα is associated with lichen sclerosus and vulvar squamous cell carcinoma.

OBJECTIVE: ERRs (estrogen-related receptors) regulate energy metabolism, the cell cycle and inflammatory processes in both normal and cancer cells. Chronic inflammation induced by lichen sclerosus (LS) or human papilloma virus (HPV) precedes vulvar squamous cell carcinoma (vulvar SCC). We investigated the expression of ERRα, ERRß and ERRγ in normal vulvar skin, LS as well as LS-dependent and LS-independent/HPV-related vulvar SCC. METHODS: A total of 203 samples were analyzed for ERRα, ERRß and ERRγ by using immunohistochemistry. These included 37 normal vulvar skin samples, 110 LS samples, 6 vulvar intraepithelial neoplasia (VIN) samples and 50 vulvar SCC samples. RESULTS: A substantial reduction in or disappearance of ERRα was detected in all vulvar SCC samples. A total of 79% of childhood-onset LS and 51% of adulthood-onset LS lesions showed decreases in ERRα staining. A gradual reduction in ERRα cytoplasmic staining was observed from healthy vulvar skin to precursor lesions and further to SCC. Nuclear ERRα staining was observed in 8/33 (24%) LS-dependent and 10/17 (59%) LS-independent SCC samples. CONCLUSIONS: ERRα, a key regulator of cell energy metabolism, may play a role in the pathogenesis of both LS and vulvar SCC.

F-actin accumulates in the vulva of female Strongyloides venezuelensis.

Little is known about the actin cytoskeleton architecture in female Strongyloides venezuelensis and thus to investigate the distribution and concentration of actin, female worms were labelled with phalloidin-rhodamine and visualized under confocal microscopy. Our results demonstrate that filamentous actin accumulates in the vulva and the concentration of F-actin at this site suggests its important role, especially during oviposition, in the life cycle of S. venezuelensis.

Clear cells of Toker in the developing anogenital region of male and female fetuses.

The clear cells of Toker are a mysterious population of intra-epidermal glandular cells. They were originally described in nipples, but were recently observed in the vulva as well. It was hypothesized that intra-epidermal embryonic remnants or underlying glands were a potential source. The embryological aspects were investigated by studying specimens of the anogenital region of 18 male and 15 female fetuses between 12 and 39 weeks gestation. The search for Toker cells was enhanced by cytokeratin (CK) 7 immunohistochemistry. The investigation showed that Toker cell elements are a normal, though highly variable constituent of the developing anogenital region. The study revealed the following: (1) single intra-epidermal glandular vesicles near follicular anlages in interlabial sulcuses of female fetuses of 15 and 16.5 weeks gestation; (2) CK7+ solitary cells, clusters, and vesicles which were related to developing intra-epidermal follicular canal tracks and tended to disperse inside the epidermis in fetuses of approximately 18 weeks gestation; (3) dispersed CK7+ cells in fetuses of 19-23 weeks gestation; (4) characteristic CK7+ Toker cell proliferations in fetuses more than 23 weeks gestation. These observations indicate that in the anogenital region, primordial follicular cells programmed to participate in the formation of apocrine and mammary-like glands, become displaced into the epidermis where they disperse, and proliferate into Toker cell populations. However, the proximity of Toker cells to CK7+ cells in excretory ducts of late fetal apocrine and mammary-like glands suggested a possible additional source. Consequences for Toker cells of the breast and primary Paget disease are discussed.

Reactive fibroblastic and myofibroblastic proliferation of the vulva (Cyclist's Nodule): A hitherto poorly described vulval lesion occurring in cyclists.

Perineal nodules occurring in male cyclists are reported in the literature, although the histologic features are not extensively documented. There has been little description of similar lesions in the female population. We describe 4 cases in which a vulval nodule or swelling developed in competitive female cyclists aged 15 to 45 years. The lesions were unilateral and occurred on the right or left labium majus (2 cases each). The histologic features were similar in all cases and consisted of a haphazard admixture of adipose tissue, variably cellular hyalinized tissue containing bland spindle-shaped fibroblasts, blood vessels, and nerve fibers. In some areas, thick cords of fibrous tissue imparted a keloid-like appearance. Other histologic features included plump mesenchymal cells with round or ovoid nuclei and abundant eosinophilic cytoplasm resulting in an epithelioid, plasmacytoid, or ganglion-like appearance (2 cases), a lymphocytic infiltrate around blood vessels (3 cases), foci of fat necrosis (1 case), and collections of elastic fibers (2 cases). One case recurred, the histologic features of the recurrent lesion being identical to the original. The overall morphologic appearances, especially in the cases with plump mesenchymal cells, bore some resemblance to proliferative fasciitis. Immunohistochemically, the cells were estrogen receptor positive and the plump mesenchymal cells were smooth muscle actin positive, in keeping with myofibroblasts. Desmin, S100, CD34, and HMGA2 were negative. Pathologists should be aware of this pseudoneoplastic lesion occurring on the vulva, which arises in a specific clinical setting and has the potential to be misdiagnosed as a variety of other mesenchymal lesions. We term this lesion as reactive fibroblastic and myofibroblastic proliferation of the vulva or "cyclist's nodule."

Recuperação completa da doença de Paget extramamária decorrente da vulva (DPEM r-V) após tratamento com Imiquimode / Full recovery of recurrent extramammary Paget's disease of the vulva(rEMPD-V) after imiquimod treatment

Apesar de rara, a doença de Paget extramamária recorrente da vulva (DPEMr-V) é uma condição grave porque, subjacente à malignidade interna, podem acompanhar lesões cutâneas superficiais. A doença de Paget extramamária é uma condição caracterizada por erupção cutânea crônica tipo eczema de pele ao redor da região anogenital em homens e mulheres. Sob o microscópio,é muito parecida com o tipo mais comum da doença de Paget mamária, que ocorrena mama. A doença de Paget extramamária ocorre mais comumente em mulheres com idades entre 50 a 60 anos. Contudo, a excisão cirúrgica é o padrão geralmente aceito para a DPEMr-V. As taxas de recorrência da DPEMr-V são altas, apesar da intervenção cirúrgica agressiva. O tratamento tópico com imiquimod creme a 5% pode ser eficaz na remoção de lesões. Relatamos o caso de uma mulher de 72 anos com DPEMr-V comprovada por biópsia, tratada com sucesso com imiquimod, com aplicações três vezes por semana, durante 6 semanas.

Although rare, extramammary Paget's disease (EMPD) is a serious condition because underlying internal malignancy may accompany superficial cutaneous lesions.Extramammary Paget disease is characterised by a chronic eczema-like rash of the skin around the anogenital regions of males and females. Underthe microscope it looks very similar to the more common type of mammary Paget´s disease that occurs on the breast. Extramammary Paget disease mostcommonly occurs in women aged between 50-60 years. Although surgical excision is the generally accepted standard of care for EMPD. The EMPD-Vrecurrence rates are high despite aggressive surgical intervention, treatment with topical imiquimod 5 percent cream has reportedly been efficacious inclearing lesions.We report the case of a 72-year-old woman with biopsy-proven EMPD-V of the thigh treated successfully with imiquimod application thrice weekly for 6 weeks.

In vivo imaging of Caenorhabditis elegans glycans.

The nematode Caenorhabditis elegans is an excellent model organism for studies of glycan dynamics, a goal that requires tools for imaging glycans in vivo. Here we applied the bioorthogonal chemical reporter technique for the molecular imaging of mucin-type O-glycans in live C. elegans. We treated worms with azidosugar variants of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylmannosamine (ManNAc), resulting in the metabolic labeling of their cell-surface glycans with azides. Subsequently, the worms were reacted via copper-free click reaction with fluorophore-conjugated difluorinated cyclooctyne (DIFO) reagents. We identified prominent localization of mucins in the pharynx of all four larval stages, in the adult hermaphrodite pharynx, vulva and anus, and in the tail of the adult male. Using a multicolor, time-resolved imaging strategy, we found that the distribution and dynamics of the glycans varied anatomically and with respect to developmental stage.

Evaluating lotion transfer to skin from feminine protection products.

BACKGROUND/PURPOSE: Adding lotions or emollients to the surface of a variety of paper products improves overall consumer comfort. Techniques to measure lotion transfer to skin are needed. METHODS: Two approaches were compared to assess lotion transfer from impregnated feminine pads: (1) pad application to the popliteal fosssa, an adaptation of the previously described 'behind-the-knee (BTK)' test model, and (2) a clinical in-use study in which pads were worn in the usual manner. In both approaches, dressings attached to the skin were used to absorb lotion transferred from the pad. Lotion transfer was first evaluated after 3 h in both protocols, and then after either 6 h (BTK method) or after and 24 h (clinical use test). RESULTS: In the BTK model, lotion transfer was a function of both the total amount of lotion contained on the topsheet and the type of absorbent material in the core of the pad. Pads containing absorbent gelling material transferred lotion more effectively than pads containing cellulose absorbent cores. In the in-use clinical study, the results were directionally similar, but statistical significance was not reached. CONCLUSIONS: An adaptation of the BTK test method provides an effective means of evaluating the transfer of lotion formulations from feminine protection pads.

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.

Tissue-specific differences in human transfer RNA expression.

Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms.

[Expression of cyclin D1 and p16 protein in vulvar white lesion].

OBJECTIVE: To investigate the relationship between cell cycle protein (cyclin D1 and p16) expression and vulvar white lesion. METHODS: Biopsies from 34 cases with vulvar white lesion, including 12 cases with lichen sclerosus (LS), 18 with squamous hyperplasia (SH) and 4 SH accompanied with LS, were examined for protein expression of cyclin D1 and p16 using immunohistochemical techniques. Normal vulvar tissues from 11 patients with other benign gynecologic diseases were used as control. RESULTS: Fifty-six percent of patients with vulvar white lesion were immunopositive for cyclin D1 protein, which was significantly higher than that of control group (9%, P < 0.05); but there was no significant difference between LS (58%) and SH patients (50%, P > 0.05) in expression of cyclin D1 protein. Immunopositive expression of p16 protein in patients was 6%, with no significant difference from the control group (0, P > 0.05). CONCLUSIONS: Cyclin D1 and p16 are important factors modulating cell cycle. The interrupt of balance between these two factors derived from abnormal expression of cyclin D1 may be one of the causes of vulvar white lesion.

Fetal microchimerism is not involved in the pathogenesis of lichen sclerosus of the vulva.

OBJECTIVES: The aim of this study was to investigate a possible relationship between fetal cell microchimerism and lichen sclerosus of the vulva. We searched for the presence of male cells and DNA in vulval tissue samples. METHODS: Paraffin-embedded skin biopsy samples from 15 women affected with vulval lichen sclerosus who gave birth to at least one son were analyzed for the presence of microchimeric male cells using fluorescence in situ hybridization (FISH) and fluorescent PCR. We included three lichen sclerosus samples originating from women without male offspring, six vulval specimens without pathological finding originating from autopsies and seven male gingival specimens as controls. RESULTS: Nucleated cells containing Y-chromosome specific sequences were neither detected at any site of the lesions nor in normal vulval specimens by using FISH. These results were confirmed by the use of PCR amplification demonstrating only DNA sequences specific for the X chromosome. No female microchimerism was detected in the male gingival samples. CONCLUSION: Despite the limited number and size of the samples, we conclude that persistent male fetal cells are not involved in the pathogenesis of lichen sclerosus of the vulva, since we consistently could not detect Y-chromosome specific sequences by using two molecular techniques.

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.

Human papillomavirus infection and p16(INK4a) protein expression in vulvar intraepithelial neoplasia and invasive squamous cell carcinoma.

OBJECTIVE: Vulvar intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. The purpose of this study was to investigate the functional properties of VIN related to expression of p16(INK4a) protein as well as to detection of human papillomavirus (HPV) type 16 by real-time polymerase chain reaction (RT-PCR) analysis. METHODS: A total of 49 vulvar biopsy samples were examined by hematoxylin-eosin staining from benign/reactive lesions, condyloma acuminatum, VIN, and invasive squamous cell carcinoma (SCC). JC8 mouse monoclonal antibodies were used that recognize p16(INK4a) epitope at a dilution of 1:25. The reaction pattern for p16(INK4a) was graded in each sample between 0 and 3+. RT-PCR analysis of formalin-fixed paraffin-embedded sections determined positivity for HPV type 16. RESULTS: p16(INK4a) immunoreactivity was different in VIN 1, VIN 2, VIN 3, and squamous cell carcinoma. Strong expression of p16(INK4a) protein was observed in 92% (22 of 24) of VIN 2 and VIN 3 lesions and 100% (4 of 4) of invasive SCCs. Two (67%) of 3 VIN 2 lesions, 17 (81%) of 21 VIN 3 lesions, and 4 (100%) of 4 SCCs were positive for HPV type 16 by PCR analysis. Two (20%) of 10 VIN 1 lesions were immunoreactive for p16(INK4a), with only 1 lesion positive for HPV type 16. No p16(INK4a) immunoreactivity was observed in any of the benign/reactive and condyloma acuminatum lesions. In addition, none of the benign/reactive or condyloma lesions were positive for HPV type 16 by RT-PCR analysis. CONCLUSIONS: Upregulation of INK4a gene occurs in vulvar carcinogenesis. p16(INK4a) is not a sensitive marker for differentiation of benign vulvar squamous epithelium from condyloma acuminatum or VIN 1 lesions because most VIN 1 lesions are p16(INK4a) negative. Expression of p16(INK4a) may aid in the diagnosis of HPV-related lesions and as such may be of value as a surrogate marker in the diagnosis of vulvar premalignant and malignant lesions.