Objective
Lichen sclerosus (LS) is a relatively common chronic inflammatory disorder of the skin and mucosal surfaces.
Study Design
A total of 29 women with histologically confirmed, active LS were recruited to this study with 2 aims. First, we evaluated the effectiveness of pimecrolimus treatment to LS not responding to conventional corticosteroid treatment. The second aim in this study was to provide information of in vivo effects of topical pimecrolimus in acute LS lesions, especially the inflammatory cell infiltration.
Results
In all, 25 of 29 women applied cream as recommended. After 2 months of treatment, 20 patients had reached partial or complete clinical remission. Histology showed decreased inflammatory lymphoid infiltrate with down-regulation of CD3 + T cells, CD8 + T cells, and CD57 + natural killer cells. Also macrophage marker CD68 staining showed down-regulation. There was no change in CD20 + B lymphocytes.
Conclusion
We conclude that calcineurin inhibitors are an effective treatment for patients not responding to corticosteroid treatment.
Lichen sclerosus (LS) is a relatively common chronic inflammatory disorder of the skin and mucosal surfaces affecting predominantly postmenopausal women. It has an anatomical predilection for genitalia, vulva representing the most common localization. Clinically, it results in pruritic, ill-defined patches. Microscopically, the classic features of LS include epidermal atrophy, hydropic degeneration of the basal keratinocytes, hyperkeratosis, and dermal sclerosis with a bandlike dermal infiltrate of chronic inflammatory cells. However, there is a substantial variation in the histology of LS. The band of inflammatory cells contains CD4 + and CD8 + T lymphocytes in an approximately equal ratio admixed with CD68 + macrophages. The etiology and specific disease mechanism of LS are unknown. However, genetic factors, autoimmunity, and autoantibodies have been implicated to play a role in the disease mechanism.
The topical immunomodulating calcineurin inhibitor pimecrolimus was specifically developed for the treatment of inflammatory skin diseases. As LS is a T lymphocyte–mediated disorder, calcineurin inhibitors have been introduced as an effective and well-tolerated therapeutic option for the treatment of vulvar LS.
The aim of this study was to investigate the effectiveness of pimecrolimus treatment in patients not responding to corticosteroid treatment and to investigate the effects of pimecrolimus on the inflammatory infiltrate in the dermis and epidermis of vulvar skin affected by LS. We performed immunohistochemical stainings for T-cell subsets (CD3, CD4, CD8, and CD57), B cells (CD20), and macrophage marker (CD68).
Materials and Methods
Patients and samples
A total of 29 patients affected by vulvar LS participated in this study. Eligible were those with active disease and in whom there was no response or an incomplete response to topical steroids. In all, 21 of 29 patients had been using corticosteroids regularly. Before commencing pimecrolimus therapy, the patients were advised to have a 2-week break from corticosteroid ointments.
The mean age was 66.5 years (range, 41–85 years). All women selected for inclusion had biopsy-proven LS. According to the medical records, they had LS for an average of 14.7 years (range 3–21 years). Patients were clinically assessed prior to commencing therapy and no precancerous lesions or evidence of human papillomavirus infection had ever been found. The ethical committee of our hospital and National Agency for Medicines reviewed the protocol and granted approval before the start of the study. Informed consent was signed by every patient. Pimecrolimus cream was commenced twice daily to the affected skin area. Approximately, patients used 30 g of 1% pimecrolimus cream per month. Skin biopsy specimens of approximately 4 mm were taken from affected area before treatment and >2 months of treatment. In both appointments, the gynecological examination was performed and the clinical stage of LS was defined.
Immunohistochemistry
Pretreatment and posttreatment specimens were formalin fixed, embedded in paraffin, and subsequently cut into 5-μm-thick sections and stained immunohistochemically. Immunohistochemical stainings were performed using automated slide stainer (BenchMark; Ventana Medical Systems, Tucson, AZ). Primary antibodies for CD3 (polyclonal, ready-to-use, Ventana Medical Systems), CD4 (clone IF6, diluted 1:20; Novocastra, Newcastle, United Kingdom), CD8 (clone C8/144B, ready-to-use; Ventana Medical Systems), CD20 (clone L26, ready-to-use; Ventana Medical Systems), CD57 (clone natural killer [NK]-1, ready-to-use; Ventana Medical Systems) and CD68 (clone KP-1, ready-to-use; Ventana Medical Systems) were applied according to manufacturers’ recommendations. Immunoreactivity was semiquantitatively analyzed by 2 pathologists independently (S.K. and P.O.V.). The samples with dissimilar scores were reevaluated and consensus was reached. A laboratory technician cut the slides, numbered them randomly, and applied staining. Pathologists were also unaware of clinical outcome at the time of analyzing the slides.
Intraepithelial lymphocytes (hematoxylin-eosin, CD3 + , CD4 + , CD8 + , CD57 + ) were graded as: –, no positive intraepithelial cells; +, a few positive scattered intraepithelial cells; ++, a moderate number of positive intraepithelial cells; and +++, a large number of positive intraepithelial cells. Stromal lymphocytes (hematoxylin-eosin, CD3 + , CD4 + , CD57 + ) were graded as: –, no positive or only a few cells present; +, some scattered stromal lymphocytes; ++, patched, moderate lymphocyte infiltrate; and +++, a bandlike strong lymphocyte infiltrate. CD20 + stromal B lymphocytes were graded as: –, no positive cells; +, a few positive cells; ++, small groups of positive cells; and +++, strong infiltrate forming frequently lymphoid follicle formation. Macrophage marker CD68 + stromal cells were graded as: –, no positive cells; +, a few positive cells; ++, moderate number of positive cells; and +++, a large number of positive cells.
Statistical methods
Statistical analysis was performed using McNemar bias test to analyze if there were histological and immunohistochemical changes between the pretreatment and posttreatment groups; P < .05 was considered statistically significant. First, the test was applied for all patients having used pimecrolimus as recommended and secondly, for those patients who had reached clinical remission.
Results
In total, 25 of 29 women (86%) applied pimecrolimus cream as recommended. Four of 29 women (14%) were excluded from the study; the main reason was infrequent use of medication. One patient used cream only once a day. Two patients experienced extensive burning and were not willing to continue after 3-7 days of pimecrolimus treatment. One patient did not response to pimecrolimus treatment and after 2 weeks decided to drop out.
Follow-up of the patients >2 months of treatment showed complete clinical remission in 19 of 25 patients (76%). In these patients all typical LS lesions (fissuring, white plaques, and inflammatory erythema excluding skin atrophy) disappeared. Partial remission was observed in 1 patient (4%). Patients with complete clinical remission experienced the loss of the subjective symptoms such as pruritus and pain. Despite regular use of pimecrolimus, 5 patients (20%) showed no response and they still had an active disease, although 3 patients experienced improvement of their symptoms.
We analyzed histologically and immunohistochemically pretreatment and posttreatment biopsy specimens of 22 patients. Two patients with complete remission and 1 patient with active disease were excluded from the histological and immunohistological studies due to the superficial posttreatment samples not showing dermis adequately.
After treatment, we could not see change in the thickness of epithelium and stromal sclerosis remained the same observed by hematoxylin-eosin–stained sections. However, lymphoid infiltrate identified histomorphologically ranged from scarce to dense lichenoid both in pretreatment and posttreatment samples. Pimecrolimus treatment decreased number of stromal and intraepithelial lymphocytes ( P = .0003 and P = .0045, respectively) ( Figure and Table ). Although achieving clinical remission and patients being symptom free, there was histologically no change in number of lymphocytes in 7 patients (41%) and we still could see active inflammation histologically (moderate or strong) in 4 patients (24%).
