Topical imiquimod treatment of residual or recurrent cervical intraepithelial neoplasia lesions (TOPIC-2): A randomised controlled trial.

OBJECTIVE: To investigate the efficacy of imiquimod in women with residual or recurrent cervical intraepithelial neoplasia (rrCIN), compared with large loop excision of the transformation zone (LLETZ). DESIGN: Randomised controlled non-inferiority trial. SETTING: One academic and one regional hospital in the Netherlands. POPULATION: Thirty-five women with rrCIN were included in the study between May 2016 and May 2021. METHODS: Women were randomised to receive treatment with 5% imiquimod cream (12.5 mg) intravaginally (three times a week for a duration of 16 weeks) or a LLETZ procedure (standard treatment). MAIN OUTCOME MEASURES: The primary outcome was reduction to normal cytology at 6 months after starting treatment. Secondary outcomes were clearance of high-risk human papilloma virus (hr-HPV) in both groups and reduction to ≤CIN1 in the imiquimod group. Side effects were monitored. RESULTS: Treatment success was 33% (6/18) in the imiquimod group versus 100% (16/16) in the LLETZ group (P < 0.001), whereas HPV clearance was 22% (4/18) in the imiquimod group versus 88% (14/16) in the LLETZ group (P < 0.001). After the randomisation of 35 women, the futility of treatment with imiquimod was proven and the trial was prematurely finished. In the follow-up period, three patients remained without additional treatment, whereas all other patients underwent LLETZ, conisation or hysterectomy. In the LLETZ group none of the patients received additional treatment during 2 years of follow-up. CONCLUSIONS: This is the first randomised controlled trial to show that topical imiquimod has a significantly lower success rate in terms of reduction to normal cytology and hr-HPV clearance, compared with LLETZ, in women with rrCIN. Additionally, imiquimod has numerous side effects and after using imiquimod most women with rrCIN still required additional surgical treatment.

Immune-based biomarker accurately predicts response to imiquimod immunotherapy in cervical high-grade squamous intraepithelial lesions.

BACKGROUND: The complete response rate of cervical high-grade squamous intraepithelial lesion (cHSIL) patients to imiquimod immunotherapy is approximately 60%. Consequently, many patients are exposed to unnecessary adverse effects of imiquimod. On the other hand, conventional surgical large loop excision therapy is associated with increased risk of premature births in subsequent pregnancies. An in-depth analysis of the cHSIL immune microenvironment was performed in order to identify and develop a predictive biomarker for response to imiquimod, to maximize therapy efficacy and to avoid adverse effects in patients unlikely to respond. METHODS: Biopsies of 35 cHSIL patients, before and 10 weeks on imiquimod treatment, were analyzed by two multispectral seven-color immunofluorescence panels for T cell and myeloid cell composition in relation to treatment response. Based on these results a simplified immunohistochemical detection protocol was developed. Samples were scanned with the Vectra multispectral imaging system and cells were automatically identified using machine learning. RESULTS: The immune microenvironment of complete responders (CR) is characterized by a strong and coordinated infiltration by T helper cells (activated PD1+/type 1 Tbet+), M1-like macrophages (CD68+CD163-) and dendritic cells (CD11c+) prior to imiquimod. The lesions of non-responders (NRs) displayed a high infiltration by CD3+FOXP3+ regulatory T cells. At 10 weeks on imiquimod, a strong influx of intraepithelial and stromal CD4+ T cells was observed in CR but not NR patients. A steep decrease in macrophages occurred both in CR and NR patients, leveling the pre-existing differences in myeloid cell composition between the two groups. Based on the pre-existing immune composition differences, the sum of intraepithelial CD4 T cell, macrophage and dendritic cell counts was used to develop a quantitative simplified one color immunohistochemical biomarker, the CHSIL immune biomarker for imiquimod (CIBI), which can be automatically and unbiasedly quantified and has an excellent predictive capacity (receiver operating characteristic area under the curve 0.95, p<0.0001). CONCLUSION: The capacity of cHSIL patients to respond to imiquimod is associated with a pre-existing coordinated local immune process, fostering an imiquimod-mediated increase in local T cell infiltration. The CIBI immunohistochemical biomarker has strong potential to select cHSIL patients with a high likelihood to experience a complete response to imiquimod immunotherapy.

Importance of the Immune Microenvironment in the Spontaneous Regression of Cervical Squamous Intraepithelial Lesions (cSIL) and Implications for Immunotherapy.

Cervical high-grade squamous intraepithelial lesions (cHSILs) develop as a result of a persistent high-risk human papilloma virus (hrHPV) infection. The natural course of cHSIL is hard to predict, depending on a multitude of viral, clinical, and immunological factors. Local immunity is pivotal in the pathogenesis, spontaneous regression, and progression of cervical dysplasia; however, the underlying mechanisms are unknown. The aim of this review is to outline the changes in the immune microenvironment in spontaneous regression, persistence, and responses to (immuno)therapy. In lesion persistence and progression, the immune microenvironment of cHSIL is characterized by a lack of intraepithelial CD3+, CD4+, and CD8+ T cell infiltrates and Langerhans cells compared to the normal epithelium and by an increased number of CD25+FoxP3+ regulatory T cells (Tregs) and CD163+ M2 macrophages. Spontaneous regression is characterized by low numbers of Tregs, more intraepithelial CD8+ T cells, and a high CD4+/CD25+ T cell ratio. A 'hot' immune microenvironment appears to be essential for spontaneous regression of cHSIL. Moreover, immunotherapy, such as imiquimod and therapeutic HPV vaccination, may enhance a preexisting pro-inflammatory immune environment contributing to lesion regression. The preexisting immune composition may reflect the potential for lesion regression, leading to a possible immune biomarker for immunotherapy in cHSILs.

A pre-existing coordinated inflammatory microenvironment is associated with complete response of vulvar high-grade squamous intraepithelial lesions to different forms of immunotherapy.

Immunotherapy of vulvar high-grade squamous intraepithelial lesion (vHSIL) is investigated as an alternative for surgery, because of high comorbidity and risk of recurrence. Limited evidence exists on the role and composition of the immune microenvironment in current immunotherapeutic approaches for vHSIL. The vHSIL of 29 patients biopsied before treatment with imiquimod were analyzed by two multiplex seven-color immunofluorescence panels to investigate the pre-existing T-cell and myeloid cell composition in relation to treatment response. The samples were scanned with the Vectra multispectral imaging system. Cells were automatically phenotyped and counted with inForm advanced image analysis software. Cell counts and composition were compared to that of vHSIL patients before therapeutic vaccination (n = 29) and to healthy vulva (n = 27). Our data show that the immune microenvironment of complete responders (CR) to imiquimod resembled the coordinated infiltration with type 1 CD4+ and CD8+ T cells and CD14+ inflammatory myeloid cells also found in healthy vulva. However, more CD8+ T cells and FoxP3+ regulatory T cells were present in CR. The lesions of partial responders (PR) lacked such a coordinated response and displayed an impaired influx of CD14+ inflammatory myeloid cells. Importantly, complete responses after imiquimod or therapeutic vaccination showed the same dependency on a pre-existing coordinated type 1 T-cell and CD14+ myeloid cell infiltration. In conclusion, a good clinical outcome after two different forms of immunotherapy for vHSIL is associated with the presence of a primary inflammatory process resulting in the coordinated influx of several types of immune cells which is then amplified.

Pre-existing inflammatory immune microenvironment predicts the clinical response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 vaccination.

BACKGROUND: Vulvar high-grade squamous intraepithelial lesion (vHSIL) is predominantly induced by high-risk human papilloma virus type 16 (HPV16). In two independent trials, therapeutic vaccination against the HPV16 E6 and E7 oncoproteins resulted in objective partial and complete responses (PRs/CRs) in half of the patients with HPV16+ vHSIL at 12-month follow-up. Here, the prevaccination and postvaccination vHSIL immune microenvironment in relation to the vaccine-induced clinical response was investigated. METHODS: Two novel seven-color multiplex immunofluorescence panels to identify T cells (CD3, CD8, Foxp3, Tim3, Tbet, PD-1, DAPI) and myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI) were designed and fully optimized for formalin-fixed paraffin-embedded tissue. 29 prevaccination and 24 postvaccination biopsies of patients with vHSIL, and 27 healthy vulva excisions, were stained, scanned with the Vectra multispectral imaging system, and automatically phenotyped and counted using inForm advanced image analysis software. RESULTS: Healthy vulvar tissue is strongly infiltrated by CD4 and CD8 T cells expressing Tbet and/or PD-1 and CD14+HLA-DR+ inflammatory myeloid cells. The presence of such a coordinated pre-existing proinflammatory microenvironment in HPV16+ vHSIL is associated with CR after vaccination. In partial responders, a disconnection between T cell and CD14+ myeloid cell infiltration was observed, whereas clinical non-responders displayed overall lower immune cell infiltration. Vaccination improved the coordination of local immunity, reflected by increased numbers of CD4+Tbet+ T cells and HLA-DR+CD14+ expressing myeloid cells in patients with a PR or CR, but not in patients with no response. CD8+ T cell infiltration was not increased after vaccination. CONCLUSION: A prevaccination inflamed type 1 immune contexture is required for stronger vaccine-induced immune infiltration and is associated with better clinical response. Therapeutic vaccination did not overtly increase immune infiltration of cold lesions.

Human Papillomavirus Downregulates the Expression of IFITM1 and RIPK3 to Escape from IFNγ- and TNFα-Mediated Antiproliferative Effects and Necroptosis.

The clearance of a high-risk human papillomavirus (hrHPV) infection takes time and requires the local presence of a strong type 1 cytokine T cell response, suggesting that hrHPV has evolved mechanisms to resist this immune attack. Using an unique system for non, newly, and persistent hrHPV infection, we show that hrHPV infection renders keratinocytes (KCs) resistant to the antiproliferative- and necroptosis-inducing effects of IFNγ and TNFα. HrHPV-impaired necroptosis was associated with the upregulation of several methyltransferases, including EZH2, and the downregulation of RIPK3 expression. Restoration of RIPK3 expression using the global histone methyltransferase inhibitor 3-deazaneplanocin increased necroptosis in hrHPV-positive KCs. Simultaneously, hrHPV effectively inhibited IFNγ/TNFα-mediated arrest of cell growth at the S-phase by downregulating IFITM1 already at 48 h after hrHPV infection, followed by an impaired increase in the expression of the antiproliferative gene RARRES1 and a decrease of the proliferative gene PCNA. Knockdown of IFITM1 in uninfected KCs confirmed its role on RARRES1 and its antiproliferative effects. Thus, our study reveals how hrHPV deregulates two pathways involved in cell death and growth regulation to withstand immune-mediated control of hrHPV-infected cells.

Vaccination against Oncoproteins of HPV16 for Noninvasive Vulvar/Vaginal Lesions: Lesion Clearance Is Related to the Strength of the T-Cell Response.

PURPOSE: Therapeutic vaccination with human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides (SLP) is effective against HPV16-induced high-grade vulvar and vaginal intraepithelial neoplasia (VIN/VaIN). However, clinical nonresponders displayed weak CD8(+) T-cell reactivity. Here, we studied if imiquimod applied at the vaccine site could improve CD8(+) T-cell reactivity, clinical efficacy, and safety of HPV16-SLP (ISA101). EXPERIMENTAL DESIGN: A multicenter open-label, randomized controlled trial was conducted in patients with HPV16(+) high-grade VIN/VaIN. Patients received ISA101 vaccination with or without application of 5% imiquimod at the vaccine site. The primary objective was the induction of a directly ex vivo detectable HPV16-specific CD8(+) T-cell response. The secondary objectives were clinical responses (lesion size, histology, and virology) and their relation with the strength of vaccination-induced immune responses. RESULTS: Forty-three patients were assigned to either ISA101 with imiquimod (n = 21) or ISA101 only (n = 22). Imiquimod did not improve the outcomes of vaccination. However, vaccine-induced clinical responses were observed in 18 of 34 (53%; 95% CI, 35.1-70.2) patients at 3 months and in 15 of 29 (52%; 95% CI, 32.5-70.6) patients, 8 of whom displayed a complete histologic response, at 12 months after the last vaccination. All patients displayed vaccine-induced T-cell responses, which were significantly stronger in patients with complete responses. Importantly, viral clearance occurred in all but one of the patients with complete histologic clearance. CONCLUSIONS: This new study confirms that clinical efficacy of ISA101 vaccination is related to the strength of vaccine-induced HPV16-specific T-cell immunity and is an effective therapy for HPV16-induced high-grade VIN/VaIN. Clin Cancer Res; 22(10); 2342-50. ©2016 AACRSee related commentary by Karaki et al., p. 2317.

The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation.

High-risk human papillomaviruses (hrHPVs) infect keratinocytes and successfully evade host immunity despite the fact that keratinocytes are well equipped to respond to innate and adaptive immune signals. Using non-infected and freshly established or persistent hrHPV-infected keratinocytes we show that hrHPV impairs the acetylation of NFκB/RelA K310 in keratinocytes. As a consequence, keratinocytes display a decreased pro-inflammatory cytokine production and immune cell attraction in response to stimuli of the innate or adaptive immune pathways. HPV accomplishes this by augmenting the expression of interferon-related developmental regulator 1 (IFRD1) in an EGFR-dependent manner. Restoration of NFκB/RelA acetylation by IFRD1 shRNA, cetuximab treatment or the HDAC1/3 inhibitor entinostat increases basal and induced cytokine expression. Similar observations are made in IFRD1-overexpressing HPV-induced cancer cells. Thus, our study reveals an EGFR-IFRD1-mediated viral immune evasion mechanism, which can also be exploited by cancer cells.

Intraepithelial macrophage infiltration is related to a high number of regulatory T cells and promotes a progressive course of HPV-induced vulvar neoplasia.

Human papilloma virus (HPV)-induced usual-type vulvar intraepithelial neoplasia (uVIN) is infiltrated by myeloid cells but the type and role of these cells is unclear. We used triple immunofluorescent confocal microscopy to locate, identify and quantify myeloid cells based on their staining pattern for CD14, CD33 and CD163 in a cohort of 43 primary and 20 recurrent uVIN lesions, 21 carcinomas and 26 normal vulvar tissues. The progressive course of uVIN is characterized by an increase in both intraepithelial and stromal mature M1 and M2 macrophages. While the M2 macrophages outnumber M1 macrophages in healthy controls and uVIN, they are matched in number by M1 macrophages in cancer. Importantly, uVIN patients with a dense intraepithelial infiltration with mature CD14+ macrophages (irrespective of M1 or M2 type) displayed approximately a six times higher risk to develop a recurrence and a high number of these cells constituted an independent prognostic factor for recurrence. In addition, a dense intraepithelial CD14+ cell infiltration was associated with high numbers of intraepithelial CD4+ Tregs and low numbers of stromal CD8+TIM3+ T cells. Patients with low numbers of intraepithelial CD14+ cells and high numbers of stromal CD8+TIM3+ cells showed the best recurrence-free survival. These data clearly show the importance of the local immune response in HPV-induced vulvar neoplasia and may be of help in predicting the prognosis of patients or their response to immunotherapy.

Expression of coinhibitory receptors on T cells in the microenvironment of usual vulvar intraepithelial neoplasia is related to proinflammatory effector T cells and an increased recurrence-free survival.

Human papillomavirus-induced usual-type vulvar intraepithelial neoplasia (uVIN) are infiltrated by immune cells but apparently not cleared. A potential explanation for this is an impaired T cell effector function by an immunesuppressive milieu, coinfiltrating regulatory T cells or the expression of coinhibitory molecules. Here, the role of these potential inhibitory mechanisms was evaluated by a detailed immunohistochemical analysis of T cell infiltration in the context of FoxP3, Tbet, indoleamine 2,3-dioxygenase, programmed cell death 1, T cell immunoglobulin mucin 3 (TIM3), natural killer cell lectin-like receptor A (NKG2A) and galectins-1, -3 and -9. Paraffin-embedded tissues of primary uVIN lesions (n=43), recurrent uVIN lesions (n=20), vulvar carcinoma (n=21) and healthy vulvar tissue (n=26) were studied. We show that the vulva constitutes an area intensely surveyed by CD8+, CD4+, Tbet+ and regulatory T cell populations, parts of which express the examined coinhibitory molecules. In uVIN especially, the number of regulatory T cells and TIM3+ T cells increased. The expression of the coinhibitory markers TIM3 and NKG2A probably reflected a higher degree of T cell activation as a dense infiltration with stromal CD8+TIM3+ T cells and CD3+NKG2A+ T cells was related to the absence of recurrences and/or a prolonged recurrence-free survival. A dense coinfiltrate with regulatory T cells was negatively associated with the time to recurrence, most dominantly when the stromal CD8+TIM3+ infiltration was limited. This notion was sustained in vulvar carcinoma's where the numbers of regulatory T cells progressively increased to outnumber coinfiltrating CD8+TIM3+ T cells and CD3+NKG2A+ T cells.

Clinical characteristics associated with development of recurrence and progression in usual-type vulvar intraepithelial neoplasia.

OBJECTIVE: To identify clinical characteristics associated with recurrence and progression in patients with usual vulvar intraepithelial neoplasia (uVIN), which may function as prognostic factors and aid in the treatment of patients with human papillomavirus (HPV)-related disease of the genital tract. METHODS: A retrospective chart review was performed in 73 patients with uVIN treated at the Leiden University Medical Center between 1990 and 2012. All medical records were reviewed for demographics, treatment type, pathology reports, and recurrence and progression rates. RESULTS: The mean age of diagnosis was 43 years, and uVIN was symptomatic in 60.1% of the patients. The median follow-up time was 49 months. High-risk HPV was found in 86.3% of the patients. Smoking was reported in 76.8% of the patients. Eleven of 73 patients were immune compromised. Multicentric HPV-related disease of the cervix or vagina was reported in 75.3% of the patients. Recurrences were diagnosed in 50.7% of the patients after first treatment type that consisted of excision (45.2%), laser (34.2%), imiquimod (8.2%), and combination of excision and laser (12.3%). Higher recurrence rates were only correlated with multifocality of uVIN lesions. Excision, imiquimod therapy, and unifocal lesions showed an increased recurrence-free survival. Human papillomavirus type, smoking, multicentric disease, use of topical steroids, and positive surgical borders were not related to a shorter recurrence-free survival. Progression into vulvar carcinoma occurred in 11 (15.1%) of the patients, 4 of whom were immune compromised. These patients showed a shorter progression-free survival of 54 versus 71.5 months. CONCLUSION: There are no clinical characteristics that form prognostic factors in uVIN, except for multifocality of lesions, which is correlated with a higher recurrence rate. Furthermore, progression of uVIN to carcinoma was accelerated and increased in immune-compromised patients, suggesting that studies of local immunity in uVIN may reveal potentialprognostic factors and aid in the development of new treatment modalities.

HPV16 synthetic long peptide (HPV16-SLP) vaccination therapy of patients with advanced or recurrent HPV16-induced gynecological carcinoma, a phase II trial.

BACKGROUND: Human papilloma virus type 16 (HPV16)-induced gynecological cancers, in particular cervical cancers, are found in many women worldwide. The HPV16 encoded oncoproteins E6 and E7 are tumor-specific targets for the adaptive immune system permitting the development of an HPV16-synthetic long peptide (SLP) vaccine with an excellent treatment profile in animal models. Here, we determined the toxicity, safety, immunogenicity and efficacy of the HPV16 SLP vaccine in patients with advanced or recurrent HPV16-induced gynecological carcinoma. METHODS: Patients with HPV16-positive advanced or recurrent gynecological carcinoma (n = 20) were subcutaneously vaccinated with an HPV16-SLP vaccine consisting of a mix of 13 HPV16 E6 and HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant. The primary endpoints were safety, toxicity and tumor regression as determined by RECIST. In addition, the vaccine-induced T-cell response was assessed by proliferation and associated cytokine production as well as IFNγ-ELISPOT. RESULTS: No systemic toxicity beyond CTCAE grade II was observed. In a few patients transient flu-like symptoms were observed. In 9 out of 16 tested patients vaccine-induced HPV16-specific proliferative responses were detected which were associated with the production of IFNγ, TNFα, IL-5 and/or IL-10. ELISPOT analysis revealed a vaccine-induced immune response in 11 of the 13 tested patients. The capacity to respond to the vaccine was positively correlated to the patient's immune status as reflected by their response to common recall antigens at the start of the trial. Median survival was 12.6 ± 9.1 months. No regression of tumors was observed among the 12 evaluable patients. Nineteen patients died of progressive disease. CONCLUSIONS: The HPV16-SLP vaccine was well tolerated and induced a broad IFNγ-associated T-cell response in patients with advanced or recurrent HPV16-induced gynecological carcinoma but neither induced tumor regression nor prevented progressive disease. We, therefore, plan to use this vaccine in combination with chemotherapy and immunomodulation.

Treatment failure in patients with HPV 16-induced vulvar intraepithelial neoplasia: understanding different clinical responses to immunotherapy.

Failure of the immune system to launch a strong and effective immune response to high-risk HPV is related to viral persistence and the development of anogenital (pre)malignant lesions such as vulvar intraepithelial neoplasia (VIN). Different forms of immunotherapy, aimed at overcoming the inertia of the immune system, have been developed and met with clinical success. Unfortunately these, in principal successful, therapeutic approaches also fail to induce clinical responses in a substantial number of cases. In this review, the authors summarize the traits of the immune response to HPV in healthy individuals and in patients with HPV-induced neoplasia. The potential mechanisms involved in the escape of HPV-induced lesions from the immune system indicate gaps in our knowledge. Finally, the interaction between the immune system and VIN is discussed with a special focus on the different forms of immunotherapy applied to treat VIN and the potential causes of therapy failure. The authors conclude that there are a number of pre-existing conditions that determine the patients' responsiveness to immunotherapy. An immunotherapeutic strategy in which different aspects of immune failure are attacked by complementary approaches, will improve the clinical response rate.

Treatment with methotrexate of a cornual pregnancy following endometrial resection.

OBJECTIVES: Endometrial resection is a procedure often performed for treatment of menorrhagia. Despite the fact that amenorrhoea frequently ensues, some normal endometrium can remain present and become the implantation site of a pregnancy. Such an event is uncommon (0.7%); however, the likelihood of an ectopic pregnancy is increased. This case report calls the reader's attention to the risk of intra- and extrauterine pregnancies and the necessity for contraception after endometrial resection, even in cases where amenorrhoea supervenes. CASE: A 46-year-old woman with prior endometrial resection and subsequent amenorrhoea, was diagnosed with a cornual pregnancy. She was successfully treated with systemic methotrexate, which was given in an outpatient clinic. CONCLUSION: Clinicians should be aware of the increased likelihood of an ectopic pregnancy after endometrial resection. All women submitting to this procedure should be counselled about the need for contraception, even in cases where amenorrhoea develops.