Vulvar Epithelium Research Articles

(096) UNDERSTANDING THE RELATIONSHIP BETWEEN CHRONIC PAIN AND DEPRESSION IN LOCALIZED PROVOKED VULVODYNIA

Abstract Introduction Localized provoked vulvodynia (LPV) is a disease characterized by chronic pain in the vulva in response to touching the vulvar vestibular epithelium. As with other chronic pain disorders, vulvodynia is often found accompanied with depression, and depressive symptoms aggravate pain severity and persistence. While there is clear evidence of a connection between chronic vulvar pain and depression, the mechanism of this bidirectional relationship is largely unknown. Accumulating evidence suggests that inflammation plays a central role in depression. Since the pathophysiology of LPV also involves inflammation, we aim to better understand these relationships by pharmacologically inhibiting the enzyme that plays a key role in both inflammation and depression: soluble epoxide hydrolase (sEH). Objective To elucidate the role of sEH in localized provoked vulvodynia, which might be involved in the chronic pain and depression comorbidity prevalent in vulvodynia patients, and discuss the clinical significance of using sEH inhibitors, such as 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), for patients suffering from this condition. Methods Vulvar punch biopsies obtained from painful sites within the vulvar vestibule and non-painful sites within the external vulva of 6 LPV patients and 6 age/race-matched healthy controls were used to generate a library of primary fibroblast strains. Cells were treated with the inflammatory instigator interleukin-1β (IL-1β) and TPPU. Quantitative PCR, Western blotting, and enzyme-linked immunosorbent assays were used to assess the effects of sEH inhibition. Statistical analysis was performed using GraphPad Prism 10.0.2. Comparisons among groups were performed using the one-way analysis of variance (ANOVA) or two-way ANOVA, followed by post-hoc Tukey test. Results Vulvodynia patients produce increased levels of pro-inflammatory cytokines, namely interleukin-6 (IL-6) and prostaglandin E2 (PGE2), when compared with controls. Interestingly, mRNA (P = 0.0091) and protein (P = 0.0198) levels of sEH were significantly lower in the painful vestibule of these patients. When sEH was inhibited with TPPU, we observed a significant decrease in IL-6 levels (P = <0.0001), but not PGE2 (P = 0.6452). These preliminary findings are of fundamental importance as cytokines can directly contribute to the development of depressive symptoms. Conclusions Patients with vulvodynia are more likely to develop depressive symptoms than patients without vulvar pain. Likewise, patients with depression are more likely to develop vulvodynia than patients without depression. Our results suggest that sEH may play a role in this bidirectional mechanism, which could serve as a novel therapeutic target for chronic pain and depression comorbidity. Disclosure No.

Cell cycle perturbation uncouples mitotic progression and invasive behavior in a post-mitotic cell

The acquisition of the post-mitotic state is crucial for the execution of many terminally differentiated cell behaviors during organismal development. However, the mechanisms that maintain the post-mitotic state in this context remain poorly understood. To gain insight into these mechanisms, we used the genetically and visually accessible model of C. elegans anchor cell (AC) invasion into the vulval epithelium. The AC is a terminally differentiated uterine cell that normally exits the cell cycle and enters a post-mitotic state before initiating contact between the uterus and vulva through a cell invasion event. Here, we set out to identify the set of negative cell cycle regulators that maintain the AC in this post-mitotic, invasive state. Our findings revealed a critical role for CKI-1 (p21CIP1/p27KIP1) in redundantly maintaining the post-mitotic state of the AC, as loss of CKI-1 in combination with other negative cell cycle regulators—including CKI-2 (p21CIP1/p27KIP1), LIN-35 (pRb/p107/p130), FZR-1 (Cdh1/Hct1), and LIN-23 (β-TrCP)—resulted in proliferating ACs. Remarkably, time-lapse imaging revealed that these ACs retain their ability to invade. Upon examination of a node in the gene regulatory network controlling AC invasion, we determined that proliferating, invasive ACs do so by maintaining aspects of pro-invasive gene expression. We therefore report that the requirement for a post-mitotic state for invasive cell behavior can be bypassed following direct cell cycle perturbation.

Clinical and morphopathological assay in vulvovaginal candidiasis.

Candida vulvovaginitis is characterized by the appearance of inflammatory changes in the vaginal and vulvar epithelium secondary to infection with Candida species. The purpose of this study was to analyze and compare the clinical, microbiological, and histopathological aspects of pregnant and non-pregnant patients, symptomatic or asymptomatic in the case of candida vaginitis and to correlate the microscopic aspects with the symptoms before applying the local treatment with Nystatin. The study presents a retrospective analysis of the management of vaginitis in 166 pregnant or non-pregnant patients during 2021-2022. We observed the structure of the Malpighian squamous epithelium without keratinization present on the vaginal mucosa and the structure of the subepithelial connective tissue, which shows increased numerical values of inflammatory and vascular cellularity in the case of candida vaginitis symptomatic compared to asymptomatic ones. We noticed also in the microscopic study that in cases of asymptomatic patients before treatment, the number of inflammatory cells and blood vessels situated immediately under the epithelium was significantly lower compared to their number in symptomatic patients before treatment. Analyzing the results obtained after the administration of the treatment proposed by us, we can say that local Nystatin treatment is beneficial and safe for pregnant and non-pregnant patients and is a good alternative for patients with recurrent vulvovaginal candidiasis.

A cross-sectional study of contact allergens in feminine hygiene wipes: a possible cause of vulvar contact dermatitis.

The present study aims to investigate the presence and prevalence of potential allergens in the most used feminine hygiene wipes. An internet-based search was performed to identify best-selling name brand and generic feminine hygiene wipes. Each unique wipe was analyzed and compared to the North American Contact Dermatitis Group 80 allergens. We found contact allergens are frequently present in feminine hygiene wipes, most commonly fragrances, other scented botanicals in the form of essences, oils, and fruit juices, and vitamin E (tocopherol). All wipes analyzed in this study contained potential allergens. The inability to eliminate commercial names from analysis could have introduced bias. Vaginal and vulvar epithelia are highly susceptible to contact allergens, often found in products marketed for feminine hygiene and cleanliness. Providers should caution patients against trusting product labeling claims to avoid incidental contact allergy and encourage simply cleansing the vulva with water.

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.

Menstrual Products as a Source of Environmental Chemical Exposure: A Review from the Epidemiologic Perspective.

Menstrual bleeding is a regular, common occurrence in a substantial portion of the population. Menstruators may use more than 10,000 menstrual products over the lifetime. Given the potential for environmental chemicals in menstrual products to be absorbed by the vulvar and vaginal epithelium into systemic circulation, we reviewed the available data on menstrual products as a source of environmental chemical exposure. Nearly two dozen studies have been conducted measuring environmental contaminants in menstrual products; all have detected environmental chemicals but had discrepant conclusions on exposure risks. Only three human studies have investigated menstrual product use and environmental chemical concentrations and all observed associations. Detection of environmental chemicals in menstrual products, in combination with challenges of exposure assessment, scarcity of human studies, and the exceedingly common occurrence of menstrual bleeding, motivates the need for further research. We provide recommendations to move this field forward.

Reciprocal EGFR signaling in the anchor cell ensures precise inter-organ connection during Caenorhabditis elegans vulval morphogenesis.

ABSTRACTDuring Caenorhabditis elegans vulval development, the uterine anchor cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval cell fates and then invades the underlying vulval epithelium. By doing so, the AC establishes direct contact with the invaginating primary vulF cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood. Using a conditional let-23 EGF receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the primary vulval cells, delayed AC invasion and disorganized adherens junctions at the contact site forming between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the primary vulval cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. Thus, EGFR signaling in the AC ensures the precise alignment of the two developing organs.

Vulvar intraepithelial neoplasia. Literature review

Vulvar intraepithelial neoplasia (VIN) is the proliferation of atypical basal cells in the vulvar epithelium. The global VIN incidence has recently doubled; its incidence among white women under 35 years of age has almost tripled with a tendency for further growth. Such an increase in the number of usual-type VIN cases in young women is primarily attributed to infection with highly oncogenic human papillomavirus. The second type of dysplasia, namely differentiated VIN, is usually found in older women and is associated with chronic dystrophic diseases of the vulva, most frequently with lichen sclerosus of the vulva. VIN diagnosis is quite challenging; no screening programs for this disorder have been developed so far. Patients with VIN practice self-treatment for a long time, which aggravates their condition and might trigger the development of vulvar cancer. Several treatment options are currently available; however, their efficacy worldwide is not high.

Verruciform xanthoma of the vulva in the context of lichen sclerosus: A mimicker of HPV and p53-independent intraepithelial neoplasia

A 67-year-old woman presented with a 2 x 1.5 cm white, asymptomatic verrucous plaque on the left labia minora. She was known for vulvar lichen sclerosus et atrophicus (LSA) for 1.5 years, treated with high-potency topical corticosteroids. A biopsy of the verrucous lesion demonstrated a small fragment of parakeratotic vulvar epithelium with papillomatosis and moderate basal atypia, spongiosis and mitoses. Neutrophils were present, forming microabscesses in the stratum corneum. The granular layer was lost in those acanthotic areas. The differential diagnosis included vulvar verruciform lichen simplex chronicus, verrucous carcinoma, vulvar acanthosis with altered differentiation, differentiated exophytic vulvar intraepithelial neoplasia or verruciform xanthoma. We demonstrate how accurately diagnosing verruciform xanthoma in the context of inflammatory dermatoses of genital sites is crucial to avoid overtreating as a preneoplastic mimicker lesion.

Aberrant activation of the Hedgehog signalling pathway in squamous cell carcinoma of the vulva as a potential target for cancer therapy

In a previous study, we showed that the Hedgehog (Hh) signalling pathway is aberrantly activated in vulval squamous cell carcinoma (VSCC). In this study, we further validated our findings on a prospective cohort of primary VSCC cases, where immunohistochemical staining confirmed that key Hh pathway components were overexpressed in VSCC compared to normal vulval epithelium. We also undertook a series of in vitro studies to determine the extent of Hh pathway activation in VSCC-derived cell lines, and examine the consequences of pathway inhibition on the growth of these cells. We found that of six cell lines tested, four displayed elevated baseline Hh pathway activity that was dependent on SHH ligand, or in one case, a PTCH1 gene mutation. Hh signalling appeared necessary to sustain cell growth, as SHH ligand depletion with Robotikinin or SMO inhibition, either with chemical inhibitors (Itraconazole or LDE-225) or SMO-specific siRNA, attenuated GLI1 activity and cell proliferation in both monolayer and organotypic raft culture. Furthermore, treatment of Hh-dependent cell lines with SMO inhibitors sensitised cells to Cisplatin. Findings from our study offer us the opportunity to explore further the development of targeted chemotherapy for women with VSCC driven by aberrant Hh activation.

Glucocorticoid Receptor Beta and Its Prognostic Value on Treatment Response in Chronic Vulvar Dermatitis

Background: Chronic vulvar dermatitis (CVD) is the most prevalent disease in gynecologic dermatology. The treatment mainly depends on topical glucocorticoids (TGC) but is challenged by insufficient treatment response. On a histological level, the upregulation of the glucocorticoid receptor β (GRβ), an inhibitor of the active glucocorticoid receptor α (GRα), is discussed as mechanism of glucocorticoid insensitivity. Objectives: To analyze whether the expression of GRβ protein at baseline in keratinocytes may predict responsiveness to TGC in patients with CVD. Methods: In this retrospective cohort study, clinical and biological data of 25 women with a histological diagnosis of chronic vulvar eczema were analyzed. Randomization was done according to the responsiveness to TGC treatment (responsive vs. nonresponsive). Clinical data and the expression of GRβ in the immunohistochemical stained biopsies were examined. Results: Fifty-two percent of women with CVD were nonresponsive to TGC. GRβ was abundantly expressed in the cytoplasma of keratinocytes of the vulvar epithelium, but no difference in the level of expression was found among GC responsive and nonresponsive patients in the semiquantitative (p = 0.376) and quantitative analysis (p = 0.894). Conclusion: GRβ is highly expressed in keratinocytes of the vulvar epidermis affected by CVD, but GRβ expression was not increased in patients nonresponsive to TGC compared to responsive patients. Thus, the failure mechanism in nonresponders still remains to be elucidated.

Programmed Death Ligand-1 (PD-L1) expression is up-regulated and related to the pattern of invasion in FIGO Stage I vulvar squamous cell carcinomas

The immune checkpoint protein programmed death ligand-1 (PD-L1) is expressed in different types of cancer and is a potential prognostic factor as well as therapeutic target. This study evaluated PD-L1 expression in the neoplastic progression of vulvar epithelia with respect to the pattern of infiltration in FIGO stages I keratinizing squamous cell carcinomas (SCC). Normal squamous vulvar epithelia (n=20), usual type vulvar intraepithelial neoplasia (uVIN, n=23), differentiated VIN (dVIN, n=21) and FIGO stage I SCC (n=35) were immunostained for PD-L1. In SCC a cohesive growth with well-delineated borders was considered as pushing, dissociative growth in small groups or single cells was defined as diffuse pattern of infiltration. Immunostaining was done with a monoclonal anti PD-L1 antibody (clone SP263, Ventana) and scored to determine up-regulation and overexpression (score 0/1+, 0-5% immunoreactive cells; score 2+, >5 to 50% immunoreactive cells; score 3+, >50% immunoreactive cells). PD-L1 immunoexpression was comparable in normal epithelia and VINs (score 0/1+, n=59; score 2+, n=5, in VINs only; score 3+, n=0), was significantly increased (P<0.0001) in SCC (score 0/1+, n=13; score 2+, n=16; score 3+, n=6), and was related to a diffuse pattern of infiltration (P<0.0001). Staining was accentuated at the invasive margins of SCC frequently. PD-L1 expression is up-regulated in the neoplastic cells of vulvar low stage SCC, related to the development of an invasive phenotype reflecting the initiation of cancer immunoediting, and to an aggressive diffuse type of stromal invasion.

Fractional Microablative CO2 Laser-Related Histological Changes on Vulvar Tissue in Patients With Genitourinary Syndrome of Menopause.

Fractional CO2 laser has been proposed as an effective treatment for the genitourinary syndrome of menopause (GSM). However, the effects of laser treatment on vulvar tissue have never been assessed. We aimed to assess histological changes related to fractional CO2 laser in vulvar tissue from GSM patients. A single-center observational prospective cohort study was performed enrolling all GSM patients from July 2017 to October 2018. Patients underwent three outpatient vulvovaginal applications of fractional CO2 laser and vulvar biopsy before and after treatment. Rates of histological changes in vulvar tissue, the difference in means of Vulva Health Index (VuHI), Vaginal Health Index (VHI), Visual Analogue Scale scores for GSM symptoms, and procedure-related pain, and rate of patient'soverall satisfaction with treatment were assessed. Univariate comparisons between continuous variables were performed by using the paired t-test(α error = 0.05). Of 20 enrolled patients, 18 underwent all laser applications, and 15 underwent both vulvar biopsies. 93.3% of patients showed remodeling of vulvar connective tissue; 80% showed improvement in vulvar epithelium trophism and 86.7% showed neovascularization. Differences in means between before and after treatment were significant for VuHI, VHI, and all GSM symptoms. Means ± standard deviation of the degree of pain at each laser application were 4.4 ± 0.9, 3.7 ± 1.6, and 2.9 ± 1.9. The rate of overall satisfaction with the treatment was 72.2%. Fractional CO2 laser leads to a restoration of the normal architecture of vulvar tissue, with significant improvement in GSM-related signs and symptoms, and overall satisfaction with the treatment in most GSM patients. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Immunolocalization of c-Fos, ELOVL5 and oestradiol in the ewe vulva in relation to oestrus behaviour after treatment with lipopolysaccharide.

Sudden activation of the stress axis by a lipopolysaccharide endotoxin (LPS) significantly reduces ewes' sexual attractivity to rams by delaying all signs of oestrous behaviour. To understand mechanisms involved in attracting male interest, we examined c-Fos (nuclear activation), ELOVL5 (enzyme involved in pheromone synthesis) and oestradiol receptors (ER) using immunohistochemistry on ewe vulval tissue at 0, 31 and 40hr in the ovarian follicular phase with or without exposure to LPS at 28hr (5 groups of 4 ewes per group). While there was intense staining for immunoreactive (IR)-c-Fos and IR-ELOVL5 in the vulval epithelium and sebaceous glands, there were no differences in intensity between groups of ewes. The absence of IR-ER staining in vulval epithelium and sebaceous/sweat glands was unexpected. Differences in ram behaviour towards ewes in the ovarian follicular phase and after LPS treatment do not appear to involve quantitative changes in vulval c-Fos, ELOVL5 or ER, but subtle qualitative differences in individual-specific compounds (attraction pheromones) remain an option.

Structure of Epithelial and Stromal Compartments of Vulvar and Vaginal Tissue From Women With Vulvo-Vaginal Atrophy Taking Ospemifene

IntroductionVulvo-vaginal atrophy affects the daily lives of most post-menopausal women. We know that ospemifene intake can induce vaginal epithelial improvements within a few weeks; however, direct evidence of the effects of ospemifene on the human vulva and on connective tissue of both the vagina and vulva are lacking. AimTo evaluate the changes induced by ospemifene on epithelium thickness, glycogen content proliferation index, collagen content, and type I/III collagen ratio in vulvar and vaginal tissue of post-menopausal women. Methods20 women who attended our gynecologic clinic for planned surgery were recruited for the study. 11 subjects were taking ospemifene at the time of inclusion, and 9 subjects who were not taking ospemifene were selected as control group. Vaginal and vulvar biopsies were taken during surgery. Histological features and glycogen content were evaluated by standard hematoxylin-eosin and periodic acid–Schiff staining, total collagen and collagen type I/III ratio were evaluated by hydroxyproline assay and Sirius red staining, while the expression of Ki67 was evaluated by immunohistochemistry. Main Outcome MeasureWe analyzed histological features of the epithelial and stromal layer of the vaginal and vulvar vestibule mucosa. ResultsVaginal and vulvar biopsies from women taking ospemifene showed an increased epithelium thickness, glycogen content, and proliferation index compared with the control group. Collagen content was also higher in women taking ospemifene, while an increased ratio between type I and III collagen fibers was found only at vaginal level. Clinical ImplicationsOur study shows that the effectiveness of ospemifene on vaginal tissue also extends to the vulvar vestibule. Strength & LimitationsThis study provides direct evidence of the impact of ospemifene on vaginal and vulvar tissue. A specifically designed longitudinal study may further support our findings. ConclusionOspemifene intake is associated with a marked improvement of various morphological and physiological features of both vaginal and vulvar vestibule epithelium, including the collagen content of the tissues.Alvisi S, Baldassarre M, Gava G, et al. Structure of Epithelial and Stromal Compartments of Vulvar and Vaginal Tissue From Women With Vulvo-Vaginal Atrophy Taking Ospemifene. J Sex Med 2018;15:1776–1784.

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.

Cell Invasion In Vivo via Rapid Exocytosis of a Transient Lysosome-Derived Membrane Domain

Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in Caenorhabditis elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume by more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion, which enables protrusion growth. Together these studies define a netrin-dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

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.

Histological changes in the vulva and vagina from ovariectomised rats undergoing oestrogen treatment.

The purpose of this study was to assess the histological changes occurring in the vagina and vulva in ovariectomised female rats, as well as the response to the administration of injectable oestrogens. We used 30 female Wistar white rats, distributed as follows: group 1 - the control group, group 2 - the operated but untreated rats, and groups 3, 4 and 5 - operated rats, to which oestrogenic treatment was administered (Estradiol, Estradurin, Sintofolin) at a dosage of 0.2 mg/rat/day. After 14 days of treatment, all animals were sacrificed and vaginal and vulvar biopsies were taken from all groups. In group 2, we encountered structural changes of the vaginal mucosa, with severe atrophy and alterations in the thickness of the vagina and vulva. In groups 3, 4 and 5 we found marked hyperplasia of the vaginal and vulvar epithelium, eosinophilic and mast cell infiltration in the chorion. Our study proves that the histopathological changes during anoestrus after administration of oestrogens are cell hyperplasia, thickening of the superficial mucosal layer, eosinophilic and mast cells infiltrations, and chorionic congestion. Furthermore, we demonstrated that Estradiol therapy induces the most evident histological changes when compared to synthetic oestrogens such as Estradurin or Sintofolin.

Abstract A18: G1 cell cycle arrest is required for invasive behavior

Abstract The ability for a cell to breach the surrounding basement membrane and adopt an invasive phenotype is a critical, but poorly understood step in the progression of cancer. Like many aspects of cancer cell behavior, the genetic programs that regulate invasive behavior are likely shared with normal cell biological processes, as invasion occurs during embryonic development and immune surveillance. Due to difficulties of studying this dynamic behavior in vivo, elucidating the genetic and epigenetic controls of cell invasive activity has been difficult. Our laboratory uses a unique in vivo model to examine cell invasion that combines functional genomic and genetic tools with single-cell high resolution visual analyses. We examine anchor cell (AC) invasion into the vulval epithelium during the larval development of the model roundworm, C. elegans. Our data functionally links G1 cell cycle arrest to acquisition of invasive behavior. Previously we have identified that a single transcription factor, the conserved nuclear hormone receptor nhr-67 (NR2E1/TLX) is required in the AC to prevent the invasive AC from entering the cell cycle. NHR-67 maintains the AC in G1 cell cycle arrest, in part through upregulation of the cyclin-dependent kinase inhibitor cki-1 (p21CIP1/p27Kip1). Loss of nhr-67 results in non-invasive mitotic ACs that fail to express matrix metalloproteinases (MMPs) and actin regulators or form invadopodia, F-actin rich membrane-localized protrusions that are required for invasion. Strikingly, AC invasion can be rescued through induction of G1 arrest, preventing cell division and promoting differentiation. Downstream of G1 arrest, the AC requires the activity of the conserved histone deacetylase HDA-1, a key regulator of cell differentiation, to regulate the expression of pro-invasive genes and localize invadopodia. Through loss-of-function RNA interference (RNAi) screening, we have identified new cell cycle regulatory components (lin-35/RB, skr-2/SKP1, cdc-14/CDC14A, cul-1/ Cullin-1 and cul-4/ Cullin-4B) and epigenetic modifiers (let-418/Mi-2/CHD3 and Swi/snf-components) that function with cki-1 and hda-1, respectively, to maintain G1 cell cycle arrest and differentiate the invasive phenotype. Together our results suggest that the acquisition of the invasive phenotype is a post-mitotic differentiated state, which may help explain the paradoxical reports that the invasive fronts of many metastatic cancers are non-proliferative. Citation Format: Abraham Q. Kohrman, Mana Chandhok, Wan Zhang, David Q. Matus. G1 cell cycle arrest is required for invasive behavior. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A18.