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Introduction/Background* The treatment of cervical high-grade squamous intraepithelial lesion (cHSIL) by topical imiquimod (Aldara®) is investigated as an alternative for surgical large loop excision of the transformation zone (LLETZ), because of the latter‘s risk of causing cervical insufficiency and subsequent premature birth in following pregnancies. Imiquimod is effective in ~60% of cHSIL patients, at present we are not able to select women likely to succesfully respond. Therefore, studies on predictive biomarkers are needed to enable personalised therapy and to prevent unnecessary potential side effects. Here, we performed an in-depth analysis of the role of the pre-existing immune microenvironment in cHSIL in response to topical imiquimod. Methodology Histologically confirmed cHSIL of 35 patients biopsied before and 10 weeks after treatment with topical imiquimod were analyzed by two multispectral seven-color immunofluorescence panels to investigate the T cell (CD3, CD8, FOXP3, PD1, TBET, TIM3, DAPI) and Myeloid cell (CD68, CD163, CD11c, CD14, CD33, PDL1, DAPI) composition in relation to treatment response. All 70 samples were scanned with the Vectra multispectral imaging system. Cells were automatically identified using a deep learning multispectral image analysis approach (inForm software). Result(s)* Our data show that the immune microenvironment of complete responders (CR) prior to imiquimod therapy is characterized by a coordinated infiltration with T helper cells (activated PD1+/type 1 Tbet+) and pro-inflammatory M1 macrophages (CD68+CD163-) and dendritic cells (CD11c+). The lesions of non-responders (NR) lacked such a pro-inflammatory response and displayed an impaired influx of these pro-inflammatory lymphoid and myeloid cells. In contrast, the NR showed an increased infiltration by immunosuppressive regulatory T cells (CD3+FOXP3+). After 10 weeks of topical imiquimod application, the influx of pro-inflammatory CD4+ and CD8+ T cells was further increased in the CR but not in the NR patients, and the infiltration by macrophages was decreased. Conclusion* Response of cHSIL to topical imiquimod is associated with the presence of a pre-existing pro-inflammatory process, resulting in the coordinated influx of several types of immune cells, which is then further amplified. Our findings indicate major potential of the immune microenvironment as predictive biomarker for the selection of cHSIL patients responding to topical imiquimod immunotherapy.

Introduction/Background Vulvar squamous cell carcinoma (VSCC) consists of three subtypes; HPV-related, HPV-negative TP53 wildtype and HPV-negative mutated TP53 (HPVposVSCC, HPVnegVSCC/p53wt, and HPVnegVSCC/p53abn, respectively) which are all treated by mutilating radical surgery and/or (chemo)radiotherapy. Despite the fact that the immune system plays a key role in cancer, the knowledge on its effect in VSCC is limited at best. A study, elucidating the clinical impact of tumor-immunity in VSCC was, therefore, performed with the aim to foster the development of immunotherapeutic approaches. Methodology Sixty-five patients with early-stage VSCC were categorized based on HPV and p53 status. Archived tissues were analyzed for expression of CD3, CD8, FoxP3, PD-1, and pan-keratin in randomly selected areas using immunofluorescence. Additional phenotyping of T cells was performed ex-vivo on VSCC and blood samples by flow cytometry. Healthy vulvar tissue and blood served as controls. Results T-cell infiltration of VSCC was highly variable between patients, ranging from completely absent to very high numbers, and differed per VSCC subtype. Approximately 80% of the HPVposVSCC showed high T-cell infiltration, followed by 60% of the HPVnegVSCC/p53wt, and 40% of the HPVnegVSCC/p53abn. Importantly, high T-cell infiltration was associated with longer recurrence-free period and overall survival, irrespective of the HPV and p53 status. In-depth analysis of tumor-infiltrating T cells with flow cytometry confirmed the tumor-specific presence of activated effector memory T cells in VSCC and revealed that most of the CD4+ and CD8+ T cells expressed PD-1. Conclusion This study is the first to show a strong correlation between T-cell infiltration and clinical outcome. Our data suggest the application of two immunotherapeutic strategies depending on immune phenotype. The high expression of PD-1 in T-cell infiltrated tumors alludes to anti-PD1 blockade, whereas VSCC tumors with low numbers of intratumoral T cells should be stimulated with inflammatory reagents to stimulate local immune responses. Disclosure Nothing to disclose.