The Paget Trial: A Multicenter, Observational Cohort Intervention Study for the Clinical Efficacy, Safety, and Immunological Response of Topical 5% Imiquimod Cream for Vulvar Paget Disease.

BACKGROUND: Vulvar Paget disease is a rare skin disorder, which is most common in postmenopausal Caucasian women. They usually present with an erythematous plaque that may show fine or typical "cake icing" scaling or ulceration that may cause itching, pain, irritation, or a burning sensation. Although most cases are noninvasive, vulvar Paget disease may be invasive or associated with an underlying vulvar or distant adenocarcinoma. The histological evidence of so-called "Paget cells" with abundant pale cytoplasm in the epithelium confirms the diagnosis. The origin of these Paget cells is still unclear. Treatment of choice is wide local excision with negative margins. Obtaining clear surgical margins is challenging and may lead to extensive and mutilating surgery. Even then, recurrence rates are high, ranging from 15% to 70%, which emphasizes the need for new treatment options. A number of case reports, retrospective case series, and one observational study have shown promising results using the topical immune response modifier imiquimod. OBJECTIVE: This study aims to investigate the efficacy, safety, and immunological response in patients with noninvasive vulvar Paget disease using a standardized treatment schedule with 5% imiquimod cream. METHODS: Topical 5% imiquimod cream might be an effective and safe treatment alternative for vulvar Paget disease. The Paget Trial is a multicenter observational cohort study including eight tertiary referral hospitals in the Netherlands. It is ethically approved by the Medical-Ethical Committee of Arnhem-Nijmegen and registered in the Central Committee on Research Involving Human Subjects (CCMO) Register by as NL51648.091.14. Twenty patients with (recurrent) noninvasive vulvar Paget disease will be treated with topical 5% imiquimod cream three times a week for 16 weeks. The primary efficacy outcome is the reduction in lesion size at 12 weeks after end of treatment. Secondary outcomes are safety, immunological response, and quality of life. Safety will be assessed by evaluation of adverse events and tolerability of treatment. To evaluate the immunological response, various immunological markers will be tested on biopsy specimens taken before, during, and after treatment. Quality of life will be assessed with three questionnaires taken before, during, and after treatment. RESULTS: First results are expected in the summer of 2018. TRIAL REGISTRATION: ClinicalTrials.gov NCT02385188; https://clinicaltrials.gov/ct2/show/NCT02385188 (Archived by WebCite at http://www.webcitation.org/6sXygHuhP).

Treatment of vulvar intraepithelial neoplasia with topical imiquimod: seven years median follow-up of a randomized clinical trial.

OBJECTIVE: Recently we reported on the efficacy of imiquimod for treating vulvar intraepithelial neoplasia (VIN) in a placebo-controlled, double-blinded randomized clinical trial (RCT). Four weeks after treatment, a complete response was observed in 35% of patients and a partial response in 46%. All complete responders remained disease-free at 12 months follow-up. In the current investigations, we assessed long-term follow-up at least 5 years after the initial RCT. METHODS: Twenty-four of 26 imiquimod-treated patients who had participated in the initial RCT were seen for follow-up. Primary endpoint was durability of clinical response to imiquimod assessed by naked eye vulvar examination and histology. Long-term clinical response was correlated to lesion size before start of the initial RCT. Secondary endpoints were mental health, global quality of life, body image and sexual function in relation with long-term clinical response. RESULTS: Median follow-up period was 7.2 years (range 5.6-8.3 years). VIN recurred in one of nine complete responders. Of the initial partial responders, two became disease-free after additional imiquimod treatment. In the other partial responders, VIN recurred at least once after the initial RCT. In long-term complete responders, lesion size at study entry was smaller and these patients had a significantly better global quality of life at follow-up than patients with residual disease and/or recurrence after imiquimod treatment. CONCLUSIONS: In case of a complete response, imiquimod is effective in the long-term. Furthermore, patients with a long-term complete response had a significantly better global quality of life than patients who recurred after imiquimod treatment.

Nonsteroidal anti-inflammatory drugs do not interfere with imiquimod treatment for usual type vulvar intraepithelial neoplasia.

Imiquimod has been shown to be an effective treatment for usual type vulvar intraepithelial neoplasia (uVIN). Since local inflammation and burning are common side effects, patients often use nonsteroidal anti-inflammatory drugs (NSAIDs). Our study investigated whether NSAID-use, which has been documented to inhibit the cell-mediated immune response, interferes with the outcome of imiquimod treatment. Monocyte-derived dendritic cells (moDCs) and Langerhans cells (moLCs) were cultured in the presence of NSAIDs. The expression of relevant surface markers (CD80, CD86, CD40, HLA-DR, CCR6 and CCR7), stimulatory function, and cytokine production were evaluated. Furthermore, we analyzed in uVIN patients whether frequent NSAID-use had an effect on the clinical response and on immunocompetent cell counts before and after imiquimod treatment. Although an effect was observed on the expression of moDC and moLC maturation markers, NSAIDs did not affect the ability of moDCs and moLCs to stimulate allogeneic T-cell proliferation, or the production of cytokines in an allogeneic T-cell stimulation assay. In agreement with this, in uVIN patients treated with imiquimod, no interference of frequent NSAID-use with clinical outcome was observed. However, we did notice that high CD1a(+) and CD207(+) cell counts in frequent NSAID-users before treatment seemed to predict a favourable response to imiquimod treatment. Our data indicate that NSAID-use does not seem to interfere with moDC and moLC function and does not interfere with immunomodulatory properties of imiquimod in uVIN patients. Therefore, NSAIDs can safely be used to reduce imiquimod side effects in uVIN patients during treatment.

Update on intraepithelial neoplasia of the vulva: proceedings of a Workshop at the 2009 World Congress of the International Society for the Study of Vulvovaginal Diseases, Edinburgh, Scotland, September 2009.

A workshop on updates on intraepithelial neoplasia of the vulva was held at the 2009 World Congress of the International Society for the Study of Vulvovaginal Diseases in Edinburgh, Scotland, September 2009. This is a review of the information presented.

Imiquimod-induced clearance of HPV is associated with normalization of immune cell counts in usual type vulvar intraepithelial neoplasia.

Recently, we reported on the efficacy of imiquimod for treatment of usual type vulvar intraepithelial neoplasia (uVIN). A histologic regression of uVIN to normal tissue was observed in 58% of patients. As success of treatment is related to clearance of high-risk human papilloma virus (HPV), the aim of our study was to assess differences in immune cell counts and in the expression of p16(INK4a) in VIN tissue before and after imiquimod treatment, in relation to HPV clearance and clinical response. Vulvar tissue samples taken prior to imiquimod treatment and 4 weeks after treatment were tested for the presence of HPV. Previously determined immune cell counts (CD1a, CD207, CD208, CD123/CD11c, CD94, CD4, CD8 and CD25/HLA-DR) in epidermis and dermis of 25 VIN patients and 19 healthy controls were completed with the counts for CD14 and CD68. The expression of p16(INK4a) was investigated by immunohistochemistry in 15 patients. Before imiquimod treatment, both HPV cleared and HPV noncleared patients showed mainly in the dermis significantly upregulated immune cell counts compared to healthy controls. However, in patients that cleared HPV and showed histologic regression already 4 weeks after imiquimod treatment, immune cell counts and p16(INK4a) expression were normalized. In conclusion, our data indicate that imiquimod-induced clearance of HPV results in normalization of counts for certain immune cells and is strongly correlated with histologic regression of the disease.

Disturbed patterns of immunocompetent cells in usual-type vulvar intraepithelial neoplasia.

Genital infection with human papillomavirus (HPV) is usually transient, as the immune system is capable of eliminating the virus. When immunity "fails" and the infection persists, vulvar intraepithelial neoplasia (VIN) may develop. In this study, we examined the distribution of inflammatory cells in 51 patients with HPV-associated usual-type VIN and in 19 healthy controls. Frozen vulvar tissue samples were tested for the presence of HPV-DNA, and immunohistochemical staining for the markers CD1a, CD207, CD208, CD123/CD11c, CD94, CD4, CD8, and CD25/HLA-DR was performed. Cells were counted in both the epidermis and dermis over at least 2 mm of basal membrane length. In the epidermis of VIN patients, CD1a(+) and CD207(+) (Langerin) dendritic cells (DC) and CD8(+) T cells were significantly lower than in controls, whereas the number of CD123(+)/CD11c(-) plasmacytoid DCs (pDC) was significantly increased. No significant changes were observed for CD208(+) DCs, CD94(+) natural killer (NK) cells, CD4(+) T cells, and CD25(+)/HLA-DR(+) regulatory T cells. In the dermis of VIN patients, elevated numbers of CD208(+), CD123(+)/CD11c(-), CD94(+), CD4(+), CD8(+), and CD25(+)/HLA-DR(+) cells were observed when compared with healthy controls. The numbers of CD1a(+) and CD207(+) DCs were not different between groups. In summary, high-risk HPV-related usual-type VIN lesions are characterized by an immunosuppressive state in the epidermis, showing a reduction of immature myeloid DCs (mDC) and CD8(+) T cells. In the dermis, inflammatory activation is reflected by the influx of mature mDCs and pDCs, NK cells, and T cells, suggesting that the cellular immune response on viral HPV infection occurs in the dermis of VIN patients.

Reduced local immunity in HPV-related VIN: expression of chemokines and involvement of immunocompetent cells.

Usual type VIN is a premalignant disorder caused by persistent HPV infection. High prevalence of VIN in immuno-suppressed women suggests that a good innate and adaptive immune response is important for defense against HPV. Here, we explored expression levels of chemokines and related these to the presence or absence of immuno-competent cells (dendritic and T-cells) in affected (HPV-positive VIN) and non-affected (HPV-negative) vulvar tissues from the same patients. Combining microarray data with quantitative real-time RT-PCR, it was observed that several important chemokines were differentially expressed between VIN and control samples (up-regulation of IL8, CXCL10, CCL20 and CCL22 and down-regulation of CXCL12, CCL21 and CCL14). Furthermore, an increased number of mature dendritic cells (CD208+) seemed to be bottled up in the dermis, and although a T-cell response (increased CD4+ and CD8+ cells) was observed in VIN, a much larger response is required to clear the infection. In summary, it seems that most mature dendritic cells do not receive the proper chemokine signal for migration and will stay in the dermis, not able to present viral antigen to naive T-cells in the lymph node. Consequently the adaptive immune response diminishes, resulting in a persistent HPV infection with increased risk for neoplasia.

Treatment of vulvar intraepithelial neoplasia with topical imiquimod.

BACKGROUND: Alternatives to surgery are needed for the treatment of vulvar intraepithelial neoplasia. We investigated the effectiveness of imiquimod 5% cream, a topical immune-response modulator, for the treatment of this condition. METHODS: Fifty-two patients with grade 2 or 3 vulvar intraepithelial neoplasia were randomly assigned to receive either imiquimod or placebo, applied twice weekly for 16 weeks. The primary outcome was a reduction of more than 25% in lesion size at 20 weeks. Secondary outcomes were histologic regression, clearance of human papillomavirus (HPV) from the lesion, changes in immune cells in the epidermis and dermis of the vulva, relief of symptoms, improvement of quality of life, and durability of response. Reduction in lesion size was classified as complete response (elimination), strong partial response (76 to 99% reduction), weak partial response (26 to 75% reduction), or no response (< or =25% reduction). The follow-up period was 12 months. RESULTS: Lesion size was reduced by more than 25% at 20 weeks in 21 of the 26 patients (81%) treated with imiquimod and in none of those treated with placebo (P<0.001). Histologic regression was significantly greater in the imiquimod group than in the placebo group (P<0.001). At baseline, 50 patients (96%) tested positive for HPV DNA. HPV cleared from the lesion in 15 patients in the imiquimod group (58%), as compared with 2 in the placebo group (8%) (P<0.001). The number of immune epidermal cells increased significantly and the number of immune dermal cells decreased significantly with imiquimod as compared with placebo. Imiquimod reduced pruritus and pain at 20 weeks (P=0.008 and P=0.004, respectively) and at 12 months (P=0.04 and P=0.02, respectively). The lesion progressed to invasion (to a depth of <1 mm) in 3 of 49 patients (6%) followed for 12 months (2 in the placebo group and 1 in the imiquimod group). Nine patients, all treated with imiquimod, had a complete response at 20 weeks and remained free from disease at 12 months. CONCLUSIONS: Imiquimod is effective in the treatment of vulvar intraepithelial neoplasia. (Current Controlled Trials number, ISRCTN11290871 [controlled-trials.com].).

Detection of human papillomavirus (HPV) 16-specific CD4+ T-cell immunity in patients with persistent HPV16-induced vulvar intraepithelial neoplasia in relation to clinical impact of imiquimod treatment.

PURPOSE: Topical application of the immune response modifier imiquimod is an alternative approach for the treatment of human papillomavirus (HPV)-positive vulvar intraepithelial neoplasia (VIN) and aims at the immunologic eradication of HPV-infected cells. We have charted HPV16-specific immunity in 29 patients with high-grade VIN and examined its role in the clinical effect of imiquimod treatment. EXPERIMENTAL DESIGN: The magnitude and cytokine polarization of the HPV16 E2-, E6-, and E7-specific CD4+ T-cell response was charted in 20 of 29 patients by proliferation and cytokine bead array. The relation between HPV16-specific type 1 T-cell immunity and imiquimod treatment was examined in a group of 17 of 29 patients. RESULTS: HPV16-specific proliferative responses were found in 11 of the 20 patients. In eight of these patients, T-cell reactivity was associated with IFNgamma production. Fifteen of the women treated with imiquimod were HPV16+, of whom eight displayed HPV16 E2- and E6-specific T-cell immunity before treatment. Imiquimod neither enhanced nor induced such immunity in any of the subjects. Objective clinical responses (complete remission or >75% regression) were observed in 11 of the 15 patients. Of these 11 responders, eight patients displayed HPV16-specific type 1 CD4+ T-cell immunity, whereas three lacked reactivity. Notably, the four patients without an objective clinical response also lacked HPV16-specific type 1 T-cell immunity. CONCLUSIONS: HPV16-specific IFNgamma-associated CD4+ T-cell immunity, although not essential for imiquimod-induced regression of VIN lesions, may increase the likelihood of a strong clinical response (P = 0.03).

Is the assumed natural history of vulvar intraepithelial neoplasia III based on enough evidence? A systematic review of 3322 published patients.

OBJECTIVE: To establish the true natural history of VIN III from literature data. METHODS: In a systematic review, data of women with VIN III indexed in several computer databases were pooled. The effect of treatment was correlated with recurrences and progression of VIN III. RESULTS: Ninety-seven articles met the inclusion criteria. Data of 3322 patients were available. The mean age at diagnosis of VIN III was 46. This decreased over time, although not significantly (P = 0.08). Recurrences were seen as often after local excision as after vulvectomy. The percentage of recurrences was lower, but not absent, after free surgical margins than after involved surgical margins (P < 0.001). 6.5% of the 3322 patients progressed to an invasive vulvar carcinoma. Occult carcinomas were diagnosed in 3.2% of patients and 3.3% carcinomas were diagnosed during follow-up. The mean age at diagnosis of invasive vulvar carcinoma was 52 years. Nine percent of 88 untreated patients progressed in 12 to 96 months to invasive vulvar carcinoma. Only 1.2% of the 3322 patients showed complete regression, mostly during the first 10 months after diagnosis, 41% of which was related to pregnancy. CONCLUSION: Evidence exists that VIN III may progress to invasive vulvar carcinoma. However, the available literature suggests that the progression rate to invasive vulvar carcinoma is low. The incidence of invasion as found in this systematic review is probably even too high, because overreporting of (micro)invasive cases cannot be excluded. Only a prospective registration using a standardized pathology examination will provide information about the real natural history of VIN III.

Imiquimod in the treatment of multifocal vulvar intraepithelial neoplasia 2/3. Results of a pilot study.

OBJECTIVE: To investigate the efficacy of topical treatment with imiquimod 5% cream, an immune response modifier, in patients with vulvar intraepithelial neoplasia (VIN) 2/3. STUDY DESIGN: Fifteen women (aged 35-51) with histologically proven multifocal VIN 2/3 without invasion, were entered into a prospective, observational, pilot study. Imiquimod 5% cream was applied by the patient to the vulvar lesions one to three times a week at night. Clinical response was analyzed by macroscopic examination and categorized as complete (CR) or partial (PR). RESULTS: Four patients achieved CR (27%) and nine patients, PR (60%) after 6-34 weeks of treatment. Two patients discontinued medication. CR was reached after 6, 7, 11 and 30 weeks of treatment. CONCLUSION: This pilot study showed the potential beneficial effect of imiquimod 5% cream in multifocal VIN 2/3. In contrast to current surgical treatment, imiquimod focuses on the cause of VIN and preserves the anatomy and function of the vulva. Therefore, imiquimod may prove to be the treatment of choice in multifocal, high grade VIN.