Objective
The objective of the study was to quantify vessel type and density in lichen sclerosus (LS) to find a marker for its malignant potential.
Study Design
Quantitative analysis was performed on paraffin-embedded tissue samples of 28 patients with LS (7 adjacent to vulvar squamous cell carcinoma, 21 solitary) and immunohistochemical staining for CD34 (vascular and lymphangiogenic lymph endothelial cells), D2-40 (lymphatic-specific marker), and α-SMA (pericyte marker). Electron microscopy was performed on fresh tissue.
Results
No significant differences in vessel density or other vessel parameters could be demonstrated between the 2 groups. In hyalinized lesions, vessel diameter, and α-SMA positivity was reduced compared with nonhyalinized lesions. Electron microscopy revealed detachment of pericytes from vascular endothelial cells and increased thickening of basement membrane, whereas endothelial cell function did not appear strongly impaired.
Conclusion
Malignant potential of LS cannot be predicted by vessel characteristics. Hyalinization in LS is associated with pericyte detachment from the basal lamina of vascular endothelial cells.
Lichen sclerosus (LS) is a chronic inflammatory skin disease that may occur in any cutaneous surface but has a distinct preference for the anogenital area. It is more commonly found in women than men, and extragenital LS can be found in up to 20% of all women suffering from vulvar LS. The etiology of LS remains elusive.
Multiple associations with autoimmune disorders, sexual hormones, infection, or repeated trauma (itch and scratch hypothesis/Koebner phenomenon) have been reported, and genetic as well as immunologic factors are thought to play a role. The classic histological features of lichen sclerosus are epidermal thinning, decreased rete ridge length, band-like dermal inflammation of varying intensity, with or without edema and/or hyalinization.
LS is diagnosed in all age groups, including infancy, but is most prevalent in postmenopausal women. An incidence of 1:300-1000 in gynecological and dermatological female patients is estimated, but the exact numbers are unknown.
Women suffering from genital LS have a 4-6% risk of developing vulvar squamous cell carcinoma (SCC). Furthermore, in 20-60% of the cases of vulvar SCC, LS can be found in adjacent areas. Because of the risk of malignant progression, it is current practice that all patients with vulvar LS undergo regular check-ups, although there is no evidence that this follow-up prevents the development of vulvar SCC or results in earlier detection of a malignancy.
Until now, there is no biomarker available that can identify vulvar LS lesions in which SCCs are more likely to develop. Microvessel density (MVD) has been suggested to be of predictive value for tumor development and progression in skin tumors and multiple types of gynecological tumors. The induction of the angiogenic response is considered a key step in the transition from a premalignancy toward an invasive neoplasm, and high MVD is related to poor survival in vulvar cancer patients. Furthermore, Raspollini et al suggested that MVD could identify those cases of LS that have the potential to evolve to vulvar SCC.
In addition to vascular endothelial cells, pericyte number and function could have a prognostic value. Pericytes envelop microvascular endothelial blood vessels and are key regulators of vessel homeostasis. In bladder and colorectal tumors, loss of pericyte function is associated with poor prognosis. Pericytes have not been studied in vulvar lesions.
Edema and hyalinization may be considered surrogate markers for the (dys)functioning of microvasculature: edema and deposition of proteins (hyalinization) may be the results of leaky microvessels and/or inadequate drainage. Although edema and hyalinization are both manifestations of vascular malfunction, hyalinization is thought to occur after the edematous phase in LS.
The aim of this study was to analyze MVD and pericyte characteristics in vulvar LS associated with vulvar SCC and solitary vulvar LS to establish a possible diagnostic significance in the identification of malignant potential.
Materials and methods
Sample selection
Paraffin-embedded tissue of 28 LS patients were selected from the archives of the Department of Pathology, Radboud University Nijmegen Medical Centre (Nijmegen, The Netherlands). Two distinct groups of patients were distinguished: solitary LS (n = 21), and LS directly adjacent to vulvar squamous cell carcinoma (n = 7). LS was considered to be solitary when the patient had no history of differentiated vulvar intraepithelial neoplasia (VIN) and/or vulvar SCC prior to or after the biopsy. All hematoxylin and eosin (H&E)–stained slides were reexamined by an expert gynecopathologist (John Bulten). In each lesion, the presence of edema and hyalinization was scored.
Because the stored tissue samples were anonymously studied, this part of the study was exempt from institutional review board approval.
For electron microscopy, fresh 4 mm biopsies of LS lesions were obtained and split in 2. Half was processed for H&E staining and the other fixed in 2.5% glutaraldehyde dissolved in 0.1 M sodium cacodylate buffer (pH 7.4). Tissues were obtained according to local ethical guidelines and approved by the local regulatory committee.
Immunohistochemistry of CD34, D2-40, and α-SMA
Paraffin sections (4 μm) were mounted onto Superfrost slides (Menzel-Gläser, Braunschweig, Germany) and dried overnight at 37°C. Tissues were dewaxed in xylene, rehydrated through graded alcohol baths, and rinsed 3 times in phosphate-buffered saline (PBS; pH 7.4) for 10 minutes. Following an antigen retrieval step (sodium citrate [0.01 M; pH 6.0], 95°C, 10 minutes), tissue sections were preincubated with 20% normal goat serum in PBS and subsequently incubated with the primary antibodies for CD34 (Dako, Glostrup, Denmark, 1:750), D2-40 (Dako, 1:100), or α-SMA (1:15,000; NeoMarkers, Fremont, CA) for 60 minutes at room temperature. All antibodies were diluted in PBS containing 1% bovine serum albumin.
Slides were rinsed in PBS for 10 minutes and incubated with biotinylated antirabbit immunoglobulin G (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. A biotin-avidin alkaline phosphatase complex was generated according to standard procedures (Vector Laboratories). Alkaline phosphatase was visualized with Fast Blue (20 ml Tris HCl [pH 8.2], 4 mg Naphtol AS-MX, 4.8 mg levamisole, and 20 mg Fast Blue BB salt; Sigma-Aldrich, Steinheim, Germany) and counterstained with Nuclear Fast Red (Vector Laboratories). Slides were mounted with Imsol (Klinipath, Duiven, The Netherlands) and subsequently with Permount (Fisher Scientific, Fairlawn, NJ). Negative controls (buffer only) and positive controls (normal skin) were applied in each run.
Quantification of CD34, D2-40, and α-SMA staining
Image acquisition was performed using a 3CCD color video camera (Sony DXC-950P; Sony Corp, Tokyo, Japan) mounted on a conventional light microscope (Axioskop 2 plus; Carl Zeiss AG, Jena, Germany) and attached to a personal computer with frame grabber card (Matrox Meteor-II Multichannel; Matrox Imaging, Dorval, Canada). Images were acquired using a ×20 objective (Plan Neofluar, NA = 0.5, resulting in specimen level pixel size of 0.39 μm 2 ). Prior to analysis of the immunohistochemical staining, an image of an empty microscopic field was acquired, which was used for correction for unequal illumination. Image acquisition and analysis were performed using a custom written macro in KS400 image analysis software (Carl Zeiss).
Thresholds were determined from a set of training slides and were found adequate for almost all slides analyzed in this study. When the initial thresholds led to unrealistic patterns, adjustment was performed by the operator (data not shown). For each patient, MVD, mean vessel area, and mean vessel perimeter in the lichenoid area between the band-like inflammatory infiltrate and epidermis was calculated per surface area in multiple nonoverlapping images (range, 3–13; mean, 6.4).
Electron microscopy
Processing of tissue samples for electron microscopy was performed as described previously. After presence of LS was confirmed in H&E, the glutaraldehyde-fixed tissue fragments were postfixed in cacodylate-buffered 1% osmiumtetroxide containing 1% potassiumhexacyanoferrate(II) for 1 h, dehydrated, and embedded in Epon 812 (Merck, Darmstadt, Germany). Ultrathin sections were cut on an ultratome (Leica, Reichert Ultracuts, Wien, Austria), and contrasted with 4% uranyl acetate for 45 min and subsequently with lead citrate for 4 min at room temperature. Sections were examined in a Jeol 1200EX-II electron microscope (JEOL, Tokyo, Japan).
Statistical analysis
Calculation of vessel parameters per patient was performed using Statistical Package for the Social Sciences (version 15.0.1; SPSS, Chicago, IL). Because the vessel variables were not normally distributed, the nonparametric Mann-Whitney U test was used to analyze differences between the groups. The relationship between the presence of edema or hyalinization in the 2 groups of LS was calculated according to the Fisher’s exact test. P < .05 was considered to be statistically significant.
To test our hypothesis that no differences exists for MVD between SCC-associated and solitary lesions and also between nonhyalinized and hyalinized lesions, equivalence testing was used next to the commonly performed testing for differences between groups. The null hypothesis for equivalence testing is that the difference between the means of 2 groups exceeds a certain threshold ε, with alternative hypothesis that no such difference exists. The critical t value for rejecting the null hypothesis may be computed from the inverse noncentral F distribution, with noncentrality parameter λ = n 1 n 2 ε 2 /(n 1 +n 2 ). In the present study, the constant ε was fixed at a value of 20% of the average MVD for the respective experiment, which we consider an appropriate threshold for a difference of physiological significance.
Results
Clinicopathological features
The histopathological analysis of all cases of LS showed hyperkeratosis, a thin epidermis, and a variable chronic inflammatory cell infiltrate. The presence of edema and hyalinization was variable and recorded for each patient. We found no cases of edema without hyalinization and half of the cases showed hyalinization without edema, confirming that hyalinization occurs after the edema ( Table 1 ). The presence of hyalinization was not related to the presence of SCC adjacent to the LS ( Table 2 ; P = .673).
| Variable | Edema | No edema | Total |
|---|---|---|---|
| Hyalinized | 7 | 13 | 20 |
| Nonhyalinized | 0 | 8 | 8 |
| Total | 7 | 21 | 28 |
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