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Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention. Electronic supplementary material The online version of this article (10.1007/s00262-020-02748-9) contains supplementary material, which is available to authorized users.

Patients with HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis than patients with non-HPV-induced OPSCC. The role of the immune response in this phenomenon is yet unclear. We studied the number of T cells, regulatory T cells (Tregs), T helper 17 (Th17) cells and IL-17+ non-T cells (mainly granulocytes) in matched HPV-positive and HPV-negative OPSCC cases (n = 162). Furthermore, the production of IFN-γ and IL-17 by tumor-infiltrating T cells was analyzed. The number of tumor-infiltrating T cells and Tregs was higher in HPV-positive than HPV-negative OPSCC (p

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3(+)), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3(+)), and T helper 17 (Th17) cells (i.e., IL-17(+)CD3(+)) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17(+) non-T cells. This suggests that IL-17(+) non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Harnessing a patient’s immune system by attenuating endogenous immune checkpoints on T-cells has led to dramatic, durable tumor rejection for multiple tumor types. Nonetheless, many cancers, including pancreatic ductal adenocarcinoma (PDAC), still do not respond to immunotherapy. PDAC remains largely resistant to all therapies, leading to its notoriety as one of the most lethal malignancies, with a 6% 5-year survival rate. We aimed to find novel, targetable mechanisms by which tumors escape the adaptive immune system and resist immunotherapy. We conducted a high throughput screen of 1280 compounds producing a synergistic effect with T-cell killing to assess differences and similarities among distinct pathways inclusive between PDAC and melanoma. These screens identified several aurora kinase inhibitors as synergistic with autologous T-cell killing of tumor target. Here we present results underlying the mechanism by which aurora kinase inhibition enhances autologous T-cell mediated killing and may improve T-cell-mediated cancer immunotherapy. These findings may contribute to the design of new therapeutic combination strategies for enhancing immunotherapy treatment of melanoma, PDAC, and other “immunologically cold” tumors. Note: This abstract was not presented at the meeting. Citation Format: Emily L. Ashkin, Deborah Silverman, Simone Punt, Soraya Zorro Manrique, Leila Williams, Yunfei Wang, Patrick Hwu. A novel compound screen for enhancing T-cell based immunotherapy identifies aurora kinases as a targetable mechanism for tumor immune escape in pancreatic ductal adenocarcinoma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3956.

Harnessing the immune system through the attenuation of endogenous immune checkpoints on T-cells has led to dramatic, durable tumor rejection in multiple solid tumors; however, most cancer types remain resistant to immunotherapy. It is imperative to understand the unique mechanisms by which these lethal malignancies evade the immune system in order to design efficacious therapies. Research by several groups is elucidating how overall mutational burden, tumor stroma, and patient microbiome predict response to immunotherapies in immune-resistant cancers. However, the frequency and distribution of driver mutations in the tumors themselves differ between immunogenic and non-immunogenic cancer types and may play a role in immune escape. Whereas p53, the most commonly mutated gene in cancer, is mutated in 70-90% of non-immunogenic tumors like pancreatic adenocarcinoma (PDAC), it is mutated in only 10-15% of immunogenic skin cutaneous melanoma cases. Loss of p53 through truncating mutations mediates tumor escape from apoptosis and senescence. Furthermore, many p53 missense mutations (mtp53) not only lose wild-type activity (LOF), but acquire novel gain-of-function (GOF) activities which promote oncogenesis and resistance to therapy. Pre-clinical data suggest that mtp53 differentially mediates tumor escape from immune surveillance by altering the innate immune response, including NK cell function and macrophage phenotype, thereby allowing tumorigenesis through chronic local immunosuppression. Few studies have been completed, however, to demonstrate the role of mtp53 in regulating the adaptive immune response. Understanding the role of p53 and its mutants in the regulation of T-cell function in cancer would provide a novel framework by which to understand and overcome resistance to cancer immunotherapy in many deadly cancer types. We hypothesize that mtp53 mediates evasion of T-cell anti-tumor activity, and that gain-of-function pathways downstream of mtp53 drive this process. Here we elaborate on previously presented work, elucidating how GOF vs. LOF mtp53 influences T-cell infiltration and killing using both novel model systems. Ultimately, these results may define a new role for mtp53 in influencing the immune system, and provide a rationale for developing effective combination strategies to improve response to immunotherapy. Citation Format: Deborah A. Silverman, Emily Ashkin, Benjamin Whitfield, Simone Punt, Soraya Zorro Manrique, Yunfei Wang, Anil Korkut, Leila Williams, Minying Zhang, Eran Kotler, Moshe Oren, Anirban Maitra, Patrick Hwu. Tumoral p53 mutations differentially mediate poor T-cell infiltration and autologous T-cell killing in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2372.

Programmed death-ligand 1 (PD-L1) is expressed in various immune cells and tumor cells, and is able to bind to PD-1 on T lymphocytes, thereby inhibiting their function. At present, the PD-1/PD-L1 axis is a major immunotherapeutic target for checkpoint inhibition in various cancer types, but information on the clinical significance of PD-L1 expression in cervical cancer is largely lacking. Here, we studied PD-L1 expression in paraffin-embedded samples from two cohorts of patients with cervical cancer: primary tumor samples from cohort I (squamous cell carcinoma, n=156 and adenocarcinoma, n=49) and primary and paired metastatic tumor samples from cohort II (squamous cell carcinoma, n=96 and adenocarcinoma, n=31). Squamous cell carcinomas were more frequently positive for PD-L1 and also contained more PD-L1-positive tumor-associated macrophages as compared with adenocarcinomas (both P

Cervical cancer is a leading cause of morbidity and mortality among women worldwide, especially in the developing countries (Munoz, 2000; Rock et al, 2000). Infection with oncogenic types of human papillomavirus (HPV) is an important factor in the development of cervical cancer (Schwartz et al, 2001; Waggoner, 2003). The persistent HPV infection induces an inflammatory response. Inflammation is an important component in the majority of tumour types. The outcome of this inflammatory response surrounding the cancer cells is dependent on the composition of the inflammatory infiltrate and locally produced signalling molecules (Coussens and Werb, 2002). Although inflammatory cells within the neoplastic lesion are capable of generating an anti-tumour response, this does not efficiently occur (Elgert et al, 1998; Manna and Mohanakumar, 2002). Inflammatory cells are attracted to the tumour site by locally produced cytokines and chemokines (Balkwill, 2003; Vicari et al, 2004). Cervical cancer cells are known to produce an extensive range of cytokines and chemokines, such as CCL2, GM-CSF, TNFα and interleukin (IL)-12 (Clerici et al, 1997; Hazelbag et al, 2001; Zijlmans et al, 2006). In addition to attracting inflammatory cells, these cytokines and chemokines influence the activation status and function of infiltrating antigen-presenting cells and stromal cells, thus influencing the course of the disease (Kusmartsev and Gabrilovich, 2002; Balkwill, 2003). In a previous study, we have shown that high expression levels or undetectable levels of IL-12p40 mRNA in cervical carcinoma are associated with an improved overall survival compared with low amounts of IL-12p40 that were associated with poor survival (Zijlmans et al, 2007). As IL-12 is known to stimulate effector cell populations, such as cytotoxic T cells and natural killer cells (Strobl, 2003; Trinchieri, 2003), our results suggest a dual role for IL-12p40. The IL-12 cytokine family includes IL-12, IL-23, IL-27 and IL-35 (Xu et al, 2010). From this family, IL-12 and IL-23 share the IL-12p40 subchain. Interleukin-12 is composed of IL-12p40 and IL-12p35, whereas IL-23 is composed of IL-12p40 and IL-23p19. Interleukin-23 has, amongst others, an important role in the IL-17/IL-23 pathway, resulting in the maintenance and expansion of Th17 cells (Korn et al, 2009). In addition to IL-23, IL-1 and IL-6 are thought to have an important role in the induction of Th17 cells in humans (Acosta-Rodriguez et al, 2007). The effect of IL-23 on cancer progression or cancer eradication is still not clear (Langowski et al, 2006; Shan et al, 2006). To further delineate the role of IL-12p40 in cervical carcinoma, we have quantified the mRNA expression levels of IL-23p19, and compared its expression level with IL-12p35 and IL-12p40 to investigate the relative importance of IL-12 and IL-23 in the tumour microenvironment. In addition, we have investigated the role of IL-1 and IL-6 in the tumour microenvironment by determining both the number of IL-1-positive cells and the number of IL-6-positive cells. Finally, we have assessed the association between IL-23p19, IL-12p35, IL-12p40, high number of IL-6-positive cells and clinicopathological parameters.

Adoptive T cell therapy (ACT) produces durable responses in some cancer patients; however, most tumors are refractory to ACT and the molecular mechanisms underlying resistance are unclear. Using two independent approaches, we identified tumor glycolysis as a pathway associated with immune resistance in melanoma. Glycolysis-related genes were upregulated in melanoma and lung cancer patient samples poorly infiltrated by T cells. Overexpression of glycolysis-related molecules impaired T cell killing of tumor cells, whereas inhibition of glycolysis enhanced T cell-mediated antitumor immunity in vitro and in vivo. Moreover, glycolysis-related gene expression was higher in melanoma tissues from ACT-refractory patients, and tumor cells derived from these patients exhibited higher glycolytic activity. We identified reduced levels of IRF1 and CXCL10 immunostimulatory molecules in highly glycolytic melanoma cells. Our findings demonstrate that tumor glycolysis is associated with the efficacy of ACT and identify the glycolysis pathway as a candidate target for combinatorial therapeutic intervention.

Background Deep invasion of the normal surrounding tissue by primary cervical cancers is a prognostic parameter for postoperative radiotherapy and relatively worse survival. However, patients with tumor-specific immunity in the blood at the time of surgery displayed a much better disease free survival. Here we analyzed if this was due to a more tumor-rejecting immune population in the tumor. Methods Tumor sections from a group of 58 patients with deep normal tissue-invading cervical tumors were stained for the presence of immune cells (CD45), IFNγ-producing cells (Tbet) and regulatory T cells (Foxp3) by immunohistochemistry. The slides were scanned and both the tumor area and the infiltration of the differently stained immune cells were objectively quantified using computer software. Results We found that an increased percentage of tumor occupied by CD45+ cells was strongly associated with an enhanced tumor-infiltration by Tbet+ cells and Foxp3+ cells. Furthermore, the area occupied by CD45+ immune cells, Tbet+ cells but not Foxp3+ cells within the tumor were, in addition to the lymph node status of patients, associated with a longer disease free survival and disease specific survival. Moreover, interaction analyses between these immune parameters and lymph node status indicated an independent prognostic effect of tumor infiltrating Tbet+ cells. This was confirmed in a multivariate Cox analysis. Conclusions The area occupied by a preferentially type I oriented CD45+ cell infiltrate forms an independent prognostic factor for recurrence-free and disease-specific survival on top of the patient’s lymph node status. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0664-0) contains supplementary material, which is available to authorized users.

Galectins are proteins that bind β-galactoside sugars and provide a new type of potential biomarkers and therapeutic targets in cancer. Galectin-1, -3 and -9 have become the focus of different research groups, but their expression and function in cervical cancer is still unclear. The aim of this study was to determine the phenotype of galectin-1, -3 and -9 expressing cells and the association with clinico-pathological parameters in cervical cancer. Galectin expression was scored in tumor cells, tumor epithelium infiltrating immune cells and stromal cells in squamous cervical cancer (n = 160). Correlations with clinico-pathological parameters and survival were studied according to the REMARK recommendations. We additionally investigated whether the galectins were expressed by tumor cells, fibroblasts, macrophages and T cells. Galectin-1 and -9 were both expressed by tumor cells in 11% of samples, while 84% expressed galectin-3. Strong galectin-1 expression by tumor cells was an independent predictor for poor survival (hazard ratio: 8.02, p = 0.001) and correlated with increased tumor invasion (p = 0.032) and receiving post-operative radiotherapy (p = 0.020). Weak and positive tumor cell galectin-3 expression were correlated with increased and decreased tumor invasion, respectively (p = 0.012). Tumor cell expression of galectin-9 showed a trend toward improved survival (p = 0.087). The predominant immune cell type expressing galectin-1, -3 and -9 were CD163+ macrophages. Galectin-1 and -3 were expressed by a minor population of T cells. Galectin-1 was mainly expressed by fibroblasts in the tumor stroma. To conclude, while tumor cell expression of galectin-9 seemed to represent a beneficial response, galectin-1 expression might be used as a marker for a more aggressive anti-cancer treatment.

The role of interleukin (IL)-17 in cancer remains controversial. In view of the growing interest in the targeting of IL-17, knowing its cellular sources and clinical implications is crucial. In the present study, we unraveled the phenotype of IL-17 expressing cells in cervical cancer using immunohistochemical double and immunofluorescent triple stainings. In the tumor stroma, IL-17 was found to be predominantly expressed by neutrophils (66%), mast cells (23%), and innate lymphoid cells (8%). Remarkably, T-helper 17 (Th17) cells were a minor IL-17 expressing population (4%). A similar distribution was observed in the tumor epithelium. The Th17 and granulocyte fractions were confirmed in head and neck, ovarian, endometrial, prostate, breast, lung, and colon carcinoma. An above median number of total IL-17 expressing cells was an independent prognostic factor for poor disease-specific survival in early stage disease (p = 0.016). While a high number of neutrophils showed at trend toward poor survival, the lowest quartile of mast cells correlated with poor survival (p = 0.011). IL-17 expressing cells and neutrophils were also correlated with the absence of vaso-invasion (p < 0.01). IL-17 was found to increase cell growth or tightness of cervical cancer cell lines, which may be a mechanism for tumorigenesis in early stage disease. These data suggest that IL-17, primarily expressed by neutrophils, predominantly promotes tumor growth, correlated with poor prognosis in early stage disease. Strikingly, a high number of Th17 cells was an independent prognostic factor for improved survival (p = 0.026), suggesting Th17 cells are part of a tumor suppressing immune response.

Cervical adenocarcinoma comprises approximately 15 % of cervical cancer cases. This histological subtype has different characteristics than cervical squamous cell carcinoma, which may influence disease progression. To study whether the infiltration of T cell subpopulations was correlated with cervical adenocarcinoma patient survival, similar to squamous cell carcinoma, the tumor-infiltrating T cells, Tregs, Th17 cells and IL-17+ cell frequencies were analyzed in a cohort of cervical adenocarcinoma patients (n = 67). Intraepithelial, stromal and total cell frequencies were scored using triple immunofluorescence. The majority of Tregs were present in the tumor stroma, while other T cells and IL-17+ cells infiltrated the tumor epithelium three times more frequently. A high total number of Tregs were significantly correlated with improved disease-specific and disease-free survival (p = 0.010, p = 0.007). Within the tumor epithelium, a high T cell frequency was significantly correlated with improved disease-free survival (p = 0.034). In particular, a low number of both Tregs and IL-17+ cells were correlated with poor disease-specific survival (p = 0.007). A low number of Tregs combined with Th17 cells present were also correlated with poor survival (p = 0.018). An increased number of IL-17+ cells were significantly correlated with the absence of vaso-invasion (p = 0.001), smaller tumor size (p = 0.030) and less infiltration depth (p = 0.021). These results suggest that Tregs and IL-17+ cells represent a beneficial immune response, whereas Th17 cells might represent a poor response in cervical adenocarcinoma. This contrasts with the correlations described in squamous cell carcinoma, suggesting that the local immune response in cervical adenocarcinoma contributes differently to tumor growth than in squamous cell carcinoma. Electronic supplementary material The online version of this article (doi:10.1007/s00262-015-1678-4) contains supplementary material, which is available to authorized users.

9572 Background: Checkpoint inhibitors (IT) have revolutionized treatment options for patients (pts) with advanced melanoma. Recently, we proposed a framework of 7 parameters (PM) describing requirements for a sufficient anti-tumor immune response (the “cancer immunogram”). In a first analysis we tested pts from our ipi cohort for outcome. Using this framework pts benefitting the most from treatment with ipi might be selected upfront, and in the future also from other checkpoint inhibitors. Methods: Using Kaplan-Meier- and Cox-regression-analysis correlations between 6 of these PM at baseline and overall survival (OS) were investigated in a single center cohort of pts treated with 3 mg/kg ipi for metastatic melanoma. Results of the 7thPM are currently being finalized. Results: PM assessed were: LDH (≤ULN vs > ULN), erythrocyte sedimentation rate (ESR,≤ULN vs > ULN), absolute lymphocyte count (ALC, < 1000/mm3 vs ≥1000/mm3) and IHC on tumor material for CD8+ T-cell infiltration ( < median vs ≥median), PD-L1 expression ( < 1% vs ≥1% positive cells) and MHC-class I expression (loss vs weak/positive). We analyzed 179 pts treated with ipi between 2010 and 2013. Median age was 55 years (19–88) and 61% of pts were male. Minimum follow-up of pts alive was 42 months(mo). Data were available as follows:177 cases LDH, 159 ALC/ESR, 118 PD-L1, 99 CD8 and 68 MHC-class I. Median OS was 7.1 mo. In univariable analysis LDH (HR 2.5, 95%CI 1.8-3.5), ALC (HR 1.6, 95%CI 1.1-2.4), ESR (HR 2.4, 95%CI 1.5-3.8) and CD8 (HR 1.8 95%CI 1.2-2.8) were significant for OS ( p< 0.01). Pts with all 4 favorable PM had a median OS of 25.1mo(95%NR), pts with 3 PM 15.8 mo(95%CI 7.0-24.7), 2 PM 8.1 mo(95%CI 1.0-15.3), 1 PM 4.8 mo(95%CI 3.3-6.3) and no favorable PM 1.7 mo(95%CI 0–4.9). In multivariable analysis LDH(HR 2.4,95%CI 1.5-4.0) and CD8(HR 1.7,95%CI 1.1-2.8) were the only independent PM. Pts with a normal LDH and high CD8 had a median OS of 15.8 mo(95%CI 9.5-22.2) vs 3.8 mo(95%CI 1.8-5.7) for pts that did not ( p< 0.01). Conclusions: In conclusion, low values of baseline LDH and high values of CD8 are the strongest PM associated with a favorable outcome in pts with advanced melanoma, treated with ipi. The other PM added low effect on the pts outcome upon ipi.

Meeting abstracts Cervical cancer is the second leading cause of cancer death in young women worldwide. The aim of the current study was to identify which combination of immunological and vascular factors in the tumor microenvironment of cervical carcinoma is most significant for survival. Using

Cervical cancer is typically well infiltrated by immune cells. Because of the intricate relationship between cancer cells and immune cells, we aimed to identify both cancer cell and immune cell expressed biomarkers. Using a novel approach, we isolated RNA from flow-sorted viable EpCAM+ tumor epithelial cells and CD45+ tumor-infiltrating immune cells obtained from squamous cell cervical cancer samples (n = 24). Total RNA was sequenced and differential gene expression analysis of the CD45+ immune cell fractions identified TCL1A as a novel marker for predicting improved survival (p = 0.007). This finding was validated using qRT-PCR (p = 0.005) and partially validated using immunohistochemistry (p = 0.083). Importantly, TCL1A was found to be expressed in a subpopulation of B cells (CD3-/CD19+/CD10+/CD34-) using multicolor immunofluorescence. A high TCL1A/CD20 (B cell) ratio, determined in total tumor samples from a separate patient cohort using qRT-PCR (n = 52), was also correlated with improved survival (p = 0.027). This is the first study demonstrating the prognostic value of separating tumor epithelial cells from tumor-infiltrating immune cells and determining their RNA expression profile for identifying putative cancer biomarkers. Our results suggest that intratumoral TCL1A+ B cells are important for controlling cervical cancer development.