The vulvar microbiome in lichen sclerosus and high-grade intraepithelial lesions.

Background: The role of the vulvar microbiome in the development of (pre)malignant vulvar disease is scarcely investigated. The aim of this exploratory study was to analyze vulvar microbiome composition in lichen sclerosus (LS) and vulvar high-grade squamous intraepithelial lesions (HSIL) compared to healthy controls. Methods: Women with vulvar lichen sclerosus (n = 10), HSIL (n = 5) and healthy controls (n = 10) were included. Swabs were collected from the vulva, vagina and anal region for microbiome characterization by metagenomic shotgun sequencing. Both lesional and non-lesional sites were examined. Biophysical assessments included trans-epidermal water loss for evaluation of the vulvar skin barrier function and vulvar and vaginal pH measurements. Results: Healthy vulvar skin resembled vaginal, anal and skin-like microbiome composition, including the genera Prevotella, Lactobacillus, Gardnerella, Staphylococcus, Cutibacterium, and Corynebacterium. Significant differences were observed in diversity between vulvar skin of healthy controls and LS patients. Compared to the healthy vulvar skin, vulvar microbiome composition of both LS and vulvar HSIL patients was characterized by significantly higher proportions of, respectively, Papillomaviridae (p = 0.045) and Alphapapillomavirus (p = 0.002). In contrast, the Prevotella genus (p = 0.031) and Bacteroidales orders (p = 0.038) were significantly less abundant in LS, as was the Actinobacteria class (p = 0.040) in vulvar HSIL. While bacteria and viruses were most abundant, fungal and archaeal taxa were scarcely observed. Trans-epidermal water loss was higher in vulvar HSIL compared to healthy vulvar skin (p = 0.043). Conclusion: This study is the first to examine the vulvar microbiome through metagenomic shotgun sequencing in LS and HSIL patients. Diseased vulvar skin presents a distinct signature compared to healthy vulvar skin with respect to bacterial and viral fractions of the microbiome. Key findings include the presence of papillomaviruses in LS as well as in vulvar HSIL, although LS is generally considered an HPV-independent risk factor for vulvar dysplasia. This exploratory study provides clues to the etiology of vulvar premalignancies and may act as a steppingstone for expanding the knowledge on potential drivers of disease progression.

The development of in vitro organotypic 3D vulvar models to study tumor-stroma interaction and drug efficacy.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) is a rare disease with a poor prognosis. To date, there's no proper in vitro modeling system for VSCC to study its pathogenesis or for drug evaluation. METHODS: We established healthy vulvar (HV)- and VSCC-like 3D full thickness models (FTMs) to observe the tumor-stroma interaction and their applicability for chemotherapeutic efficacy examination. VSCC-FTMs were developed by seeding VSCC tumor cell lines (A431 and HTB117) onto dermal matrices harboring two NF subtypes namely papillary fibroblasts (PFs) and reticular fibroblasts (RFs), or cancer-associated fibroblasts (CAFs) while HV-FTMs were constructed with primary keratinocytes and fibroblasts isolated from HV tissues. RESULTS: HV-FTMs highly resembled HV tissues in terms of epidermal morphogenesis, basement membrane formation and collagen deposition. When the dermal compartment shifted from PFs to RFs or CAFs in VSCC-FTMs, tumor cells demonstrated more proliferation, EMT induction and stemness. In contrast to PFs, RFs started to lose their phenotype and express robust CAF-markers α-SMA and COL11A1 under tumor cell signaling induction, indicating a favored 'RF-to-CAF' transition in VSCC tumor microenvironment (TME). Additionally, chemotherapeutic treatment with carboplatin and paclitaxel resulted in a significant reduction in tumor-load and invasion in VSCC-FTMs. CONCLUSION: We successfully developed in vitro 3D vulvar models mimicking both healthy and tumorous conditions which serve as a promising tool for vulvar drug screening programs. Moreover, healthy fibroblasts demonstrate heterogeneity in terms of CAF-activation in VSCC TME which brings insights in the future development of novel CAF-based therapeutic strategies in VSCC.

Reflectance confocal microscopy as a non-invasive imaging tool in vulvar high-grade squamous intraepithelial lesions and lichen sclerosus: A descriptive morphological study in patients and healthy volunteers.

Incorrect and delayed diagnosis of vulvar high-grade squamous intraepithelial neoplasia (vHSIL) and lichen sclerosus (LS) increases malignant progression risks and negatively impacts prognosis and quality of life. There is a need to improve diagnosis and monitoring. Reflectance confocal microscopy is a non-invasive imaging tool that visualizes skin structures at cellular resolution. The objectives were to explore feasibility and patient acceptability of vulvar RCM imaging and to identify RCM characteristics that are discriminative for vulvar HSIL and LS. This was a prospective, cross-sectional, observational clinical trial in patients with vHSIL and LS compared to healthy volunteers. RCM images and vulvar tissue samples were obtained. Five (5) patients with vHSIL, 10 patients with LS and 10 healthy volunteers were enrolled. In total, 100 image series of vulvar skin were obtained, including lesional and nonlesional sites. The RCM technique was considered acceptable for application by patients and healthy controls. Healthy vulvar skin was characterized by a homogenous, normal honeycomb patterned epidermis and a clear epidermal-dermal junctions. Vulvar HSIL and LS displayed an atypical honeycomb pattern of the epidermis and lymphocytic influx with presence of melanophages. Distinct features specifically observed in LS included the presence of hyalinised vessels and sclerotic areas in the dermis. RCM is a non-invasive imaging technique that is feasible and clinically acceptable to apply on vulvar skin, both in patients with premalignant lesions and healthy controls. Recognition and validation of disease-specific characteristics could make reflectance confocal microscopy a clinical tool to non-invasively aid identification of vulvar premalignancies.

Dermatoscopy and Optical Coherence Tomography in Vulvar High-Grade Squamous Intraepithelial Lesions and Lichen Sclerosus: A Prospective Observational Trial.

OBJECTIVE: This study aimed to examine potential discriminatory characteristics of dermatoscopy and dynamic optical coherence tomography (D-OCT) on vulvar high-grade squamous intraepithelial lesions (vHSIL) and lichen sclerosus (LS) compared with healthy vulvar skin. METHODS: A prospective observational clinical trial was performed in 10 healthy volunteers, 5 vHSIL and 10 LS patients. Noninvasive imaging measurements using dermatoscopy and D-OCT were obtained at several time points, including lesional and nonlesional vulvar skin. Morphologic features of vHSIL and LS were compared with healthy controls. Epidermal thickness and blood flow were determined using D-OCT. Patients reported tolerability of each study procedure, including reference vulvar biopsies. The main outcome measures were feasibility and tolerability of imaging modalities, dermatoscopy and OCT characteristics, OCT epidermal thickness and D-OCT dermal blood flow. RESULTS: The application of dermatoscopy and D-OCT is feasible and tolerable. In vHSIL, dermatoscopic warty structures were present. In LS, sclerotic areas and arborizing vessels were observed. Structural OCT in the vulvar area aligned with histology for hyperkeratosis and dermal-epidermal junction visualization. Currently, the OCT algorithm is unable to calculate the epidermal thickness of the uneven vulvar area. Dynamic optical coherence tomography showed statistically significant increased blood flow in LS patients (mean ± SD, 0.053 ± 0.029) to healthy controls (0.040 ± 0.012; p = .0024). CONCLUSIONS: The application of dermatoscopy and D-OCT is feasible and tolerable in vHSIL and LS patients. Using dermatoscopy and D-OCT, the authors describe potential characteristics to aid differentiation of diseased from healthy vulvar skin, which could complement clinical assessments.

Monitoring of Ex Vivo Cyclosporin a Activity in Healthy Volunteers Using T Cell Function Assays in Relation to Whole Blood and Cellular Pharmacokinetics.

Therapeutic drug monitoring (TDM) of calcineurin inhibitors (i.e., tacrolimus and cyclosporin A) is standard of care after solid organ transplantation. Although the incidence of acute rejection has strongly decreased, there are still many patients who experience severe side effects or rejection after long-term treatment. In this healthy volunteer study we therefore aimed to identify biomarkers to move from a pharmacokinetic-based towards a pharmacodynamic-based monitoring approach for calcineurin inhibitor treatment. Healthy volunteers received a single dose of cyclosporine A (CsA) or placebo, after which whole blood samples were stimulated to measure ex vivo T cell functionality, including proliferation, cytokine production, and activation marker expression. The highest whole blood concentration of CsA was found at 2 h post-dose, which resulted in a strong inhibition of interferon gamma (IFNy) and interleukin-2 (IL-2) production and expression of CD154 and CD71 on T cells. Moreover, the in vitro effect of CsA was studied by incubation of pre-dose whole blood samples with a concentration range of CsA. The average in vitro and ex vivo CsA activity overlapped, making the in vitro dose-effect relationship an interesting method for prediction of post-dose drug effect. The clinical relevance of the results is to be explored in transplantation patients on calcineurin inhibitor treatment.

The Human Vulvar Microbiome: A Systematic Review.

The link between cancer and the microbiome is a fast-moving field in research. There is little knowledge on the microbiome in ((pre)malignant) conditions of the vulvar skin. This systematic review aims to provide an overview of the literature regarding the microbiome composition of the healthy vulvar skin and in (pre)malignant vulvar disease. This study was performed according to the PRISMA guidelines. A comprehensive, electronic search strategy was used to identify original research articles (updated September 2021). The inclusion criteria were articles using culture-independent methods for microbiome profiling of the vulvar region. Ten articles were included. The bacterial composition of the vulva consists of several genera including Lactobacillus, Corynebacterium, Staphylococcus and Prevotella, suggesting that the vulvar microbiome composition shows similarities with the corresponding vaginal milieu. However, the vulvar microbiome generally displayed higher diversity with commensals of cutaneous and fecal origin. This is the first systematic review that investigates the relationship between microbiome and vulvar (pre)malignant disease. There are limited data and the level of evidence is low with limitations in study size, population diversity and methodology. Nevertheless, the vulvar microbiome represents a promising field for exploring potential links for disease etiology and targets for therapy.

Integrin αvß6 as a Target for Tumor-Specific Imaging of Vulvar Squamous Cell Carcinoma and Adjacent Premalignant Lesions.

Surgical removal of vulvar squamous cell carcinoma (VSCC) is associated with significant morbidity and high recurrence rates. This is at least partially related to the limited visual ability to distinguish (pre)malignant from normal vulvar tissue. Illumination of neoplastic tissue based on fluorescent tracers, known as fluorescence-guided surgery (FGS), could help resect involved tissue and decrease ancillary mutilation. To evaluate potential targets for FGS in VSCC, immunohistochemistry was performed on paraffin-embedded premalignant (high grade squamous intraepithelial lesion and differentiated vulvar intraepithelial neoplasia) and VSCC (human papillomavirus (HPV)-dependent and -independent) tissue sections with healthy vulvar skin as controls. Sections were stained for integrin αvß6, CAIX, CD44v6, EGFR, EpCAM, FRα, MRP1, MUC1 and uPAR. The expression of each marker was quantified using digital image analysis. H-scores were calculated and percentages positive cells, expression pattern, and biomarker localization were assessed. In addition, tumor-to-background ratios were established, which were highest for (pre)malignant vulvar tissues stained for integrin αvß6. In conclusion, integrin αvß6 allowed for the most robust discrimination of VSCCs and adjacent premalignant lesions compared to surrounding healthy tissue in immunohistochemically stained tissue sections. The use of an αvß6 targeted near-infrared fluorescent probe for FGS of vulvar (pre)malignancies should be evaluated in future studies.

Morphological and phenotypical features of ovarian metastases in breast cancer patients.

BACKGROUND: Autotransplantation of frozen-thawed ovarian tissue is a method to preserve ovarian function and fertility in patients undergoing gonadotoxic therapy. In oncology patients, the safety cannot yet be guaranteed, since current tumor detection methods can only exclude the presence of malignant cells in ovarian fragments that are not transplanted. We determined the need for a novel detection method by studying the distribution of tumor cells in ovaries from patients with breast cancer. Furthermore, we examined which cell-surface proteins are suitable as a target for non-invasive tumor-specific imaging of ovarian metastases from invasive breast cancer. METHODS: Using the nationwide database of the Dutch Pathology Registry (PALGA), we identified a cohort of 46 women with primary invasive breast cancer and ovarian metastases. The localization and morphology of ovarian metastases were determined on hematoxylin-and-eosin-stained sections. The following cell-surface markers were immunohistochemically analyzed: E-cadherin, epithelial membrane antigen (EMA), human epidermal growth receptor type 2 (Her2/neu), carcinoembryonic antigen (CEA), αvß6 integrin and epithelial cell adhesion molecule (EpCAM). RESULTS: The majority of ovarian metastases (71%) consisted of a solitary metastasis or multiple distinct nodules separated by uninvolved ovarian tissue, suggesting that ovarian metastases might be overlooked by the current detection approach. Combining the targets E-cadherin, EMA and Her2/neu resulted in nearly 100% detection of ductal ovarian metastases, whereas the combination of EMA, Her2/neu and EpCAM was most suitable to detect lobular ovarian metastases. CONCLUSIONS: Examination of the actual ovarian transplants is recommended. A combination of targets is most appropriate to detect ovarian metastases by tumor-specific imaging.