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BackgroundThe 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.MethodsBiopsies 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.ResultsThe 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, pConclusionThe 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.

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., What's new? Premalignant vulvar high‐grade squamous intraepithelial lesions (vHSIL) are predominantly induced by human papilloma virus infection. The immune microenvironment in vHSIL and its role in immunotherapeutic approaches remain largely unknown. This study is the first to show that a complete clinical response in patients is associated with a pre‐existent coordinated influx of type 1 T cells and CD14+ myeloid cells, irrespective of the type of successful immunotherapy given (topical imiquimod therapy or therapeutic vaccination). This coordinated immune microenvironment closely resembles that of healthy vulvar tissue, suggesting that an impaired primary inflammatory process acts as an immune resistance mechanism in non‐complete responders.

BackgroundThe composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre-existing tumor-specific T cells. Here, the blueprint of the TIME associated with such an ongoing tumor-specific T-cell response was dissected in a unique prospective oropharyngeal squamous cell carcinoma (OPSCC) cohort, in which tumor-specific tumor-infiltrating T cells were detected (immune responsiveness (IR+)) or not (lack of immune responsiveness (IR−)).MethodsA comprehensive multimodal, high-dimensional strategy was applied to dissect the TIME of treatment-naive IR+ and IR− OPSCC tissue, including bulk RNA sequencing (NanoString), imaging mass cytometry (Hyperion) for phenotyping and spatial interaction analyses of immune cells, and combined single-cell gene expression profiling and T-cell receptor (TCR) sequencing (single-cell RNA sequencing (scRNAseq)) to characterize the transcriptional states of clonally expanded tumor-infiltrating T cells.ResultsIR+ patients had an excellent survival during >10 years follow-up. The tumors of IR+ patients expressed higher levels of genes strongly related to interferon gamma signaling, T-cell activation, TCR signaling, and mononuclear cell differentiation, as well as genes involved in several immune signaling pathways, than IR− patients. The top differently overexpressed genes included CXCL12 and LTB, involved in ectopic lymphoid structure development. Moreover, scRNAseq not only revealed that CD4+ T cells were the main producers of LTB but also identified a subset of clonally expanded CD8+ T cells, dominantly present in IR+ tumors, which secreted the T cell and dendritic cell (DC) attracting chemokine CCL4. Indeed, immune cell infiltration in IR+ tumors is stronger, highly coordinated, and has a distinct spatial phenotypical signature characterized by intratumoral microaggregates of CD8+CD103+ and CD4+ T cells with DCs. In contrast, the IR− TIME comprised spatial interactions between lymphocytes and various immunosuppressive myeloid cell populations. The impact of these chemokines on local immunity and clinical outcome was confirmed in an independent The Cancer Genome Atlas OPSCC cohort.ConclusionThe production of lymphoid cell attracting and organizing chemokines by tumor-specific T cells in IR+ tumors constitutes a positive feedback loop to sustain the formation of the DC–T-cell microaggregates and identifies patients with excellent survival after standard therapy.

BackgroundExpression of killer cell lectin-like receptor B1 (KLRB1), the gene encoding the cell surface molecule CD161, is associated with favorable prognosis in many cancers. CD161 is expressed by several lymphocyte populations, but its role and regulation on tumor-specific CD4+ T cells is unknown.MethodsWe examined the clinical impact of CD4+CD161+ T cells in human papillomavirus (HPV)16+ oropharyngeal squamous cell carcinoma (OPSCC), analyzed their contribution in a cohort of therapeutically vaccinated patients and used HPV16-specific CD4+CD161+ tumor-infiltrating lymphocytes and T cell clones for in-depth mechanistic studies.ResultsCentral and effector memory CD4+ T cells express CD161, but only CD4+CD161+ effector memory T cells (Tem) are associated with improved survival in OPSCC. Therapeutic vaccination activates and expands type 1 cytokine-producing CD4+CD161+ effector T cells. The expression of CD161 is dynamic and follows a pattern opposite of the checkpoint molecules PD1 and CD39. CD161 did not function as an immune checkpoint molecule as demonstrated using multiple experimental approaches using antibodies to block CD161 and gene editing to knockout CD161 expression. Single-cell transcriptomics revealedKLRB1expression in many T cell clusters suggesting differences in their activation. Indeed, CD4+CD161+ effector cells specifically expressed the transcriptional transactivatorSOX4,known to enhance T cell receptor (TCR) signaling via CD3ε. Consistent with this observation, CD4+CD161+ cells respond more vigorously to limiting amounts of cognate antigen in presence of interleukin (IL)-12 and IL-18 compared to their CD161- counterparts. The expression of CD161/KLRB1andSOX4was downregulated upon TCR stimulation and this effect was boosted by transforming growth factor (TGF)β1.ConclusionHigh levels of CD4+CD161+ Tem are associated with improved survival and our data show that CD161 is dynamically regulated by cell intrinsic and extrinsic factors. CD161 expressing CD4+ T cells rapidly respond to suboptimal antigen stimulation suggesting that CD161, similar to SOX4, is involved in the amplification of TCR signals in CD4+ T cells.

BackgroundOropharyngeal squamous cell carcinoma (OPSCC) is the most prevalent type of head and neck cancer. The survival of patients with OPSCC is tightly linked to the intratumoral presence of tumor-specific CD4+ and CD8+ T cells. Yet, immunotherapy is currently far from effective in OPSCC partly due to our limited understanding of its immune microenvironment.MethodsHere a multi-modal, high-dimensional approach was used to dissect the immune landscape in a unique cohort of pre-therapy OPSCC patient samples (n=20) in which intratumoral tumor-specific T cells were either detected (immune response positive, IR+) or not (IR-). This included imaging mass cytometry (Hyperion) for high-dimensional phenotyping, spatial localization and interaction analyses of the cells in the tumor mircoenvironment with our newly developed imaging processing pipeline employing machine learning, Nanostring PanCancer IO360 panel analysis of immune signaling pathways, and combined single-cell gene expression profiling and T cell receptor sequencing (scRNAseq) to characterize the transcriptional states of clonally expanded tumor-infiltrating T cells.ResultsImmune cell infiltration in IR+ tumors is stronger and highly coordinated, with a distinct spatial phenotypic signature characterized by microaggregates of tumor-resident (CD103+) CD8+ and CD4+ T cells and dendritic cells within the tumor cell beds, which retained after permutation based correction for differences in cell frequencies. Furthermore, the increased expression of CXCL12 and LTB produced by CD4+ T cells, both involved in the spatial organization of immune cell infiltration, and the clonal expansion of CD8+ T cells producing the DC-attracting chemokines CCL4 or XCL1 in IR+ OPSCC, indicate that tumor-reactive T cells act as a positive feedback loop in the formation of these aggregates. The impact of these chemokines on local immunity and clinical outcome was confirmed in an independent TCGA OPSCC cohort. In contrast, the IR- OPSCC signature comprised spatial interactions between lymphocytes and different subpopulations of immunosuppressive myeloid cells.ConclusionsOur study reveals that the chemokine-driven spatial immune signature of OPSCC has strong potential as a prognostic and predictive biomarker. While the immune signature of IR+ OPSCC suggests potential benefit from neoadjuvant immunotherapeutic approaches to limit the side effects of current radio(chemo)therapy, that of IR- OPSCC calls for strategies focused on stimulating T cells and counteracting immune suppressive mechanisms.

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.

BackgroundVulvar 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.MethodsTwo 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.ResultsHealthy 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.ConclusionA 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.

BackgroundImmunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the development of more effective treatments.MethodsWe exploited a mouse model where tumor-specific therapeutic vaccination results in tumor regression, followed by local recurrence and resistance. In depth studies on systemic, local and tumor intrinsic changes were performed with flow and mass cytometry, immunohistochemistry, transcriptomics and several perturbation studies with inhibitors or agonistic antibodies in mice. Main findings were recapitulated in vaccinated patients.ResultsFull tumor regression and cure of tumor-bearing mice is dependent on the magnitude of the vaccine-induced T-cell response. Recurrence of tumors did not involve classical immune escape mechanisms, such as antigen-presentation alterations, immune checkpoint expression, resistance to killing or local immune suppression. However, the recurrent tumors displayed a changed transcriptome with alterations in p53, tumor necrosis factor-α and transforming growth factor-β signaling pathways and they became immunologically cold. Remarkably, ex vivo cell-sorted recurrent tumors, directly reinjected in naïve hosts retained their resistance to vaccination despite a strong infiltration with tumor-specific CD8+ T cells, similar to that of vaccine-responsive tumors. The influx of inflammatory mature myeloid effector cells in the resistant tumors, however, was impaired and this turned out to be the underlying mechanisms as restoration of inflammatory myeloid cell infiltration reinstated the sensitivity of these refractory tumors to vaccination. Notably, impaired myeloid cell infiltration after vaccination was also associated with vaccine resistance in patients.ConclusionAn immunotherapy-induced disability of tumor cells to attract innate myeloid effector cells formed a major mechanism underlying immune escape and acquired resistance. These data not only stresses the importance of myeloid effector cells during immunotherapy but also demands for new studies to harness their tumoricidal activities.

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.

Background Vulvar squamous cell carcinoma (VSCC) has been suggested to consist of three subtypes; HPV-positive, HPV-negative mutated TP53 or HPV-negative TP53 wildtype, with different clinical courses. To analyze the immune infiltrate in these molecular subtypes and its impact on clinical outcome, an in-depth study of the tumor immune microenvironment was performed. Methods Sixty-five patients with invasive VSCC matched for age, FIGO stage and treatment modality, were grouped according to the presence of HPV and p53 protein expression status. Archived tissues were analyzed for intraepithelial and stromal 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 (n = 14) and blood samples by flow cytometry. Healthy vulvar samples and blood served as controls. Results Based on T-cell infiltration patterns about half of the VSCC were classified as inflamed or altered-excluded while one-third was immune-deserted. High intraepithelial helper T cell infiltration was observed in 78% of the HPV-induced VSCC, 60% of the HPVnegVSCC/p53wildtype and 40% of the HPVnegVSCC with abnormal p53 expression. A high intraepithelial infiltration with activated (CD3+PD-1+), specifically helper T cells (CD3+CD8−Foxp3−), was associated with a longer recurrence-free period and overall survival, irrespective of HPV and p53 status. Flow cytometry confirmed the tumor-specific presence of activated (CD4+PD-1++CD161−CD38+HLA-DR+ and CD8+CD103+CD161−NKG2A+/−PD1++CD38++HLA-DR+) effector memory T cells. Conclusion This is the first study demonstrating an association between intraepithelial T cells and clinical outcome in VSCC. Our data suggest that abnormal p53 expressing VSCCs mostly are cold tumors whereas HPV-driven VSCCs are strongly T-cell infiltrated. Electronic supplementary material The online version of this article (10.1186/s40425-019-0712-z) contains supplementary material, which is available to authorized users.

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 oncoproteins of HPV16 with synthetic long peptides (SLP) resulted in vHSIL regression in about half of the patients after 12 months. Several studies have shown that the immune microenvironment influences therapy outcome. Therefore, a thorough investigation of the vHSIL immune microenvironment before and after SLP vaccination was performed, and its impact on clinical response was studied. Methods: Two novel multiplex immunofluorescence panels were designed and fully optimized for formalin-fixed paraffin-embedded tissue, one for T cells (CD3, CD8, Foxp3, Tim3, Tbet, PD-1, DAPI) and one for myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI). Pre- and post-vaccination biopsies of 29 vHSIL patients, as well as 27 healthy vulva excisions, were stained, scanned with the Vectra multispectral imaging system, and automatically phenotyped and counted with inForm advanced image analysis software. Results: A pre-existing pro-inflammatory tumor microenvironment, marked by high numbers of CD4+ and CD8+ T cells expressing Tbet and/or PD-1 as well as CD14+ inflammatory macrophages, is a strong predictor for good clinical response to therapeutic HPV16 SLP vaccination. A clear stepwise increase in pre-vaccination infiltrating Tbet+, CD4+, CD8+ T cells and CD14+ macrophages, and decrease in Foxp3+ regulatory T cells was observed as response increased from non to partial to complete response. Moreover, the pre-vaccination immune microenvironment of complete responders resembled healthy vulva. Vaccination further increased infiltrating CD4+ and Tbet+ T cells and CD14+ macrophages, and decreased Foxp3+ regulatory T cells and CD68+CD163+ M2 macrophages in the complete and partial responders, but not in the non responders. Conclusion: Clinical responsiveness to therapeutic HPV16 SLP vaccination requires a pre-existing inflamed type 1 immune contexture in vHSIL. Hence, only patients with an inflamed microenvironment should be selected for monotherapy by therapeutic peptide vaccination, since this strategy is incapable of overcoming an immunologically cold tumor microenvironment. Citation Format: Ziena Abdulrahman, Noel F. de Miranda, Mariette I. van Poelgeest, Sjoerd H. van der Burg. A pre-existing inflammatory immune microenvironment predicts the clinical and immunological response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 peptide vaccination [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1582.

Vulvar squamous cell carcinoma (VSCC) develops via two different pathways: TP53 mutations in a background of lichen sclerosus or a persistent infection with high-risk human papilloma virus (HPV). The latter group of tumor responds better to treatment than the non-virally induced VSCC. This may be explained by a difference in the tumor immune microenvironment (TME).This review summarizes literature on TME of VSCC and its precursors, and extrapolates this to foster the development of new therapeutic strategies.Both types of VSCC and their precursors are infiltrated with variable numbers of M2 macrophages, regulatory T cells and CD8+ T cells, indicating that they express targetable tumor antigens. Type 1 T cell immunity in precursor lesions is associated with fewer recurrences and better clinical responses to immunotherapy. Escape of these lesions and progression toward VSCC is associated with the downregulation of HLA Class I, increased expression of co-inhibitory molecules, infiltration with immunosuppressive cells and the local production of immunosuppressive enzymes and cytokines. More in-depth studies of the VSCC TME are required to fully comprehend the impact of the immune system on VSCC, and subsequently to identify patients who will benefit from immunotherapeutic strategies.