Imiquimod is a good non-invasive treatment option for women with cervical high-grade squamous intraepithelial neoplasia (cHSIL), especially those with a possible (future) pregnancy wish. Complete response to imiquimod occurs in 55-73% of patients, however side-effects of imiquimod are common and can be extensive. Therefore, biomarkers which can predict response to imiquimod therapy are warranted, to increase therapy efficacy and to avoid side effects in patients who will not respond.

This prospective, multi-center cohort study aims to validate the potential of immune related biomarkers to predict the clinical response of patients with primary cHSIL to imiquimod, aims to explore the value of these immune biomarkers in recurrent/residual cHSIL to predict treatment responses for imiquimod and aims to explore their potential in spontaneous regression of cHSIL (CIN2).

RATIONALE: A persistent high risk Human Papilloma Virus (hrHPV) infection can cause (pre)malignant anogenital lesions of the cervix, vulva or vagina. Cervical high grade squamous intraepithelial lesions (cHSIL) have a malignant potential and require adequate therapy. The natural history of cHSIL is unpredictable: ~25% of cHSIL will regress, while 18% will progress to invasive cervical cancer. The standard treatment of histologically confirmed cHSIL is surgical excision by large loop excision of the transformation zone (LLETZ), with potential complications, such as hemorrhage, infection and an increased risk of preterm birth in subsequent pregnancies. Imiquimod cream has been studied as a non-invasive treatment alternative and the recent TOPIC-3 trial for cHSIL a complete response rate of 55% upon imiquimod therapy was reported. Imiquimod is now considered as a standard non-surgical therapy for patients with cHSIL in the Netherlands, especially in those patients with a future pregnancy wish. Side-effects of imiquimod therapy however are common and can be extensive, consisting mostly of local inflammation and burning, but also systemic adverse events such as headache and flu-like symptoms. Therapy adherence is challenging with up to 20% discontinuation of treatment due to the side effects and the 16 week treatment duration. As such, biomarkers which can predict response to imiquimod therapy are warranted, to increase therapy efficacy and to avoid side effects in patients who will not respond. Our previous work shows that clinical response to imiquimod in cHSIL is associated with a coordinated pre-existing type 1 T cell- and inflammatory myeloid cell infiltration and provided the first set of parameters that potentially can function together as a predictive biomarker CIBI (CHSIL Immune Biomarker for Imiquimod).

OBJECTIVE: This study aims to validate the potential of immune related biomarkers to predict the clinical response of patients with primary cHSIL to imiquimod and aims to explore the value of these immune biomarkers in recurrent/residual cHSIL to predict treatment responses for imiquimod and aims to explore their potential in spontaneous regression of cHSIL (CIN2).

STUDY DESIGN: Multicenter, real-life prospective cohort validation study.

STUDY POPULATION: We aim to include 316 women with a primary histological diagnosis of cHSIL, 50 patients with residual/recurrent histological diagnosis of cHSIL treated with imiquimod and 50 patients with cHSIL (e.g. CIN 2) not treated to await potential spontaneous regression according to the real life setting.

INTERVENTION: Patients are included in the study if they prefer imiquimod treatment, as standard care, for their cHSIL lesion or choose for expectative management of cHSIL (e.g. CIN2), according to the real-life clinical setting in the Netherlands. Patients are treated by a 16-week regime of imiquimod 5% cream, applied three times a week. Treatment efficacy will be evaluated after 20 weeks, by colposcopy with diagnostic biopsies, and at 6 months after completion of imiquimod therapy with cytology or if indicated histology. At inclusion, at 20 weeks and after 6 months a vaginal swab will be taken to evaluate the vaginal microbiome. Therapy adherence and side effects will be registered.

PRIMARY STUDY OBJECTIVES:

• Confirm the relationship between a complete clinical response to imiquimod and the increased epithelial and stromal infiltration of CD4+ T cells, CD11c+ cells and/or M1-like macrophages as well as the decreased infiltration with FoxP3+ Tregs in primary cHSIL.
• Validate the association of CIBI to a complete response to imiquimod treatment in primary cHSIL.
• Determine the sensitivity and specificity of CIBI in patients with primary cHSIL lesions to estimate the predictive value for therapy efficacy upon imiquimod treatment.

SECONDARY STUDY OBJECTIVES:

• Explore the CIBI as a predictive biomarker for therapy efficacy to imiquimod treatment in patients with residual/recurrent cHSIL (rrcHSIL).
• Explore the CIBI as a predictive biomarker for spontaneous regression of cHSIL (e.g. CIN2).
• Determine the vaginal microbiome in cHSIL patients treated with imiquimod or not treated to explore the potential interaction of the vaginal microbiome and composition of immune infiltrates in relation to imiquimod treatment and the relation to spontaneous regression.

NATURE AND EXTENT OF THE BURDEN AND RISKS ASSOCIATED WITH PARTICIPATION, BENEFIT AND GROUP RELATEDNESS: The burden associated with participation to this study is minimal since patients are included in accordance to real-life selection. If patients prefer imiquimod treatment for the therapy for their cHSIL lesion after consultation with the gynecologist they will be treated following a standard protocol, following the national guideline for cHSIL (CIN, AIS en VAIN.pdf). The burden for patients to participate in the study lies in an extra biopsy taken at colposcopy at 20 weeks and taking vaginal cultures for microbiome analysis. The benefit for the patients lies in extra support via telephonic consultation and close monitoring. For the patients in the observational arm with no treatment, no extra examinations will be performed according to the national guideline for cHSIL, only data and tissue will be used and two vaginal swabs taken. For the study cohort the benefit is limited but if we are able to identify a clinical predictive biomarker for spontaneous regression or imiquimod in cHSIL, this may increase therapy efficacy for future cHSIL patients by patient selection preventing unnecessary imiquimod therapy.

Inclusion Criteria:

• Primary cHSIL lesions (e.g. CIN3 or CIN 2), histologically confirmed by diagnostic biopsy Nota bene: In case of CIN 2, expectative management must be discussed according to the Dutch national guideline with the patient, if the patient prefers imiquimod therapy the patient can be treated with imiquimod and enrolled in the study, if the patient prefers expectative management they can be enrolled in the observational CIN 2 group.
• Recurrent or residual cHSIL lesions after initial LLETZ treatment (e.g. CIN2 or CIN3), histologically confirmed by diagnostic biopsy
• Age of 18 years or older

Exclusion Criteria:

• Concomitant diagnoses of VAIN (vaginal intraepithelial neoplasia e.g. vaginal HSIL)
• PAP (Papanicolaou) 4 cytology as indication for the baseline colposcopy at study entrance
• Adenocarcinoma in situ (AIS) diagnosis
• Previous imiquimod therapy for cHSIL
• Previous cervical malignancy
• Current malignant disease
• Immunodeficiency (including HIV/AIDS and immunosuppressive medication)
• Pregnancy
• Legal incapability
• Insufficient knowledge of the Dutch language

Only patients who have a cervix uteri are eligible.

Patients will be included in real-life outpatient clinic setting and treated according to standard Dutch therapy guidelines.

We aim to enroll 310 patients with primary cHSIL and 50 patients with rrcHSIL treated with imiquimod and 50 cHSIL (CIN 2) patients not treated in the observational arm.