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

Abstract

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 imiquimodinduced clearance of HPV results in normalization of counts for certain immune cells and is strongly correlated with histologic regression of the disease.Vulvar intraepithelial neoplasia (VIN) is a premalignant disorder that is classified into differentiated type VIN, which is associated with lichen sclerosus, and usual type VIN (uVIN), 1 which is caused by a persistent infection with a high-risk or oncogenic HPV (hrHPV, usually HPV-type 16, 18 or 33). 2 Over the last decades, the incidence of uVIN has increased, most likely due to a rise in the incidence of HPV infections. 3 Lifetime risk to become infected with HPV in western societies is around 80% and $40% of all sexually active, female adolescents are at least once infected with hrHPV. [4][5][6][7] When hrHPV persists (in less than 10% of cases), premalignant disorders of the anogenital tract, such as uVIN, can develop. 8-10 uVIN has invasive potential (10% of untreated cases will progress in 1-4 years) and needs to be treated proactively. 11,12 The host immune response is of critical importance in determining clearance or persistence of an HPV-infection. In natural life, during the early stages of a viral infection, CpGrich regions in the viral DNA are recognized by toll-like receptor (TLR) 7 and 9 on the cell surface of immature dendritic cells (DCs). Upon binding, TLRs activate kinase cascades eventually activating NFjB, which will result in the production of cytokines, adhesions molecules and other effectors of the innate immune response. 13 Other receptor and adhesion molecules at the DC surface can bind viral antigen and will internalize and digest the antigen, which is followed by expression on the cell membrane in major histocompatibility complex...

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

“…16 Although there was no significant difference between responding and nonresponding patients, the nonresponding group on average had the highest number of intraepithelial CD141 cells suggesting that these nonresponding patients immunologically were more similar to the recurrent uVIN and vulvar cancer patients. It might be perceived that the intraepithelial CD141 or single CD141 cell population, thus is unfavorable.…”

“…This is in line with previous studies, the focus of which was mainly on T cell infiltrates and LCs/DCs in uVIN in relation to responses to immunotherapy. 5,7,8,16,17,21,43,44 From these studies it was concluded that the epithelium of uVIN lesions was suppressed as it lacked a strong CD81 infiltration and displayed lower numbers of Langerhans cells. 16,17,21 In contrast, the dermis of uVIN lesions was thought to be the most active region as there was abundant infiltration of CD41 and CD81 T cells as well as mature DCs.…”

“…15 Studies of the microenvironment in uVIN revealed that it is characterized by especially dermal immune activity as higher numbers of innate and adaptive immune cells are found in the stroma when compared to healthy control tissue. 16,17 In contrast, the epidermis shows reduced numbers of CD81 T cells, CD1a1 mature Langerhans cells (LCs) and immature CD2071 LCs, …”

“…15 Studies of the microenvironment in uVIN revealed that it is characterized by especially dermal immune activity as higher numbers of innate and adaptive immune cells are found in the stroma when compared to healthy control tissue. 16,17 In contrast, the epidermis shows reduced numbers of CD81 T cells, CD1a1 mature Langerhans cells (LCs) and immature CD2071 LCs, while the number of intraepithelial macrophages is increased in uVIN. 16,17 Intralesional macrophages are derived from tissue immigrating monocytes and are generally categorized into tumor suppressive type 1 macrophages (M1) which produce interleukin 12 and tumor necrosis factor a and tumor promoting type 2 macrophages (M2) which are known to produce anti-inflammatory cytokines.…”

“…16,17 In contrast, the epidermis shows reduced numbers of CD81 T cells, CD1a1 mature Langerhans cells (LCs) and immature CD2071 LCs, while the number of intraepithelial macrophages is increased in uVIN. 16,17 Intralesional macrophages are derived from tissue immigrating monocytes and are generally categorized into tumor suppressive type 1 macrophages (M1) which produce interleukin 12 and tumor necrosis factor a and tumor promoting type 2 macrophages (M2) which are known to produce anti-inflammatory cytokines. 13,18,19 High numbers of tumor-associated macrophages induce tumor growth, progression and poor survival rates (reviewed in Ref.…”