Page 195 - Strategies for non-invasive managementof high-grade cervical intraepithelial neoplasia - prognostic biomarkers and immunotherapy Margot Maria Koeneman

damage-associated molecular patterns (DAMPs). TLRs also play a role in cervical HPV infection and oncogenesis. The viral oncogenes E6 and E7 have been shown to inhibit the activity of NF-kB in response to TLR stimulation, thereby reducing antigen presentation. This enables HPV to evade local immune response and enables persistent infection.[48, 49, 51] Additionally, two TLRs seem to play an active role in cervical carcinogenesis.[49] TLR4 and TLR9 were found to be gradually upregulated in progression from cervical dysplasia to cervical cancer and their stimulation may contribute to cervical carcinogenesis through mechanisms described above.
Imiquimod in treatment of high-grade CIN
Imiquimod is an agonist of TLR7 and TLR8. The general antitumoral effect of imiquimod is mediated through at least three mechanisms.[52] The most important effect is agonistic activity towards TLR7 and -8, which activates NF-kB, inducing cell-mediated immunity. Imiquimod leads to a profound T-helper (Th)-1-weighted cellular immune response. TLR-independent effects are interference with adenosine receptor signaling pathways, which augments pro- inflammatory activity, and induction of apoptosis at higher concentrations through activation of a mitochondrial pathway. Imiquimod was shown to induce regression of high-grade CIN lesions in a clinical study, but the specific biological mechanisms of imiquimod in CIN lesions have not been completely elucidated.[15] Both TLR7 and TLR8 are found in the epithelium and stroma of cervical epithelium, but expression is limited.[53] One study found a mildly increased expression of TLR7 in CIN and cervical carcinoma as compared to normal epithelium, but this finding was not confirmed in a later study.[53, 54] This does not argue for a significant upregulation of TLR during cervical carcinogenesis, indicating that TLR7/8 are most likely not pro- carcinogenic in the human cervix. A clinical study indicated that a higher expression of cervical TLR7 in HPV16-positive women was a significant predictor of HPV clearance during follow-up. [55] As such, stimulation of TLR7/8 may contribute to HPV clearance and possibly regression of CIN. More specific evidence regarding the biological mechanisms of imiquimod in HPV induced precancerous lesions is available from studies in VIN. Two immunological studies – in a limited number of women – showed that imiquimod treatment did not lead to a general induction or enhancement of HPV16-specific CD4+ and CD8+ T-cell responses.[56, 57] This indicates an effect of imiquimod other than induction of a specific immune response through TLR 7/8. Interestingly, a stronger preexisting HPV-specific type-1 T-cell response was associated with a more favorable clinical outcome upon topical imiquimod treatment. The authors propose that the efficacy of imiquimod in VIN may be explained by the induction of additional pro-inflammatory signals, through Langerhans cells and macrophages and by stimulation of natural killer cells and Th1 cells via indirect mechanisms. This may restore the aberrant immunological environment that characterizes VIN (and CIN) lesions and allow for lesion regression. Indeed, a later study by the authors showed a normalization of immune cell counts in patients that cleared HPV and showed histologic regression after imiquimod treatment.[58] Additional evidence for a TLR independent effect of imiquimod was found in a cell line study on primary human skin derived keratinocytes. [51] In this study, it was shown that the effects of imiquimod treatment on NF-κB activation and cytokine secretion was independent of TLR 7/8. Furthermore, it was found that undifferentiated primary keratinocytes do not express TLR 7/8. The antitumoral effects of imiquimod in high-grade CIN may therefore be the result of two simultaneous mechanisms: first, stimulation of TLR7/8 in
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