Anti-Mitotic Activity and Downstream Immune Response of Tumor Treating Fields (TTFields) Therapy

Effect of X-irradiation on Epidermal Immune Function: Decreased Density and Alloantigen-Presenting Capacity of Ia+ Langerhans Cells and Impaired Production of Epidermal Cell-Derived Thymocyte Activating Factor (ETAF)

Helichrysum arenarium (L.) (Asterales:Asteraceae) Moench is a therapeutic plant which contains etheric oil, flavones and flavon glycosides, sterins, bitter substances and tannins having various coumarins. This plant is thought to have important characteristics such as diuretic effect, dropping stones and sand from the kidney, regulating digestive disorders, strengthening the immune system, and having antibiotic and antioxidant effects. Additionally, this plant is traditionally used in liver and biliary tract diseases and also shows anti-inflammatory and detoxifying properties. Model organism Galleria mellonella L. (Lepidoptera:Pyralidae) is an invertebrate species that is frequently used to study the effects of human pathogens and various pesticides, hormones, etc. on immune system. In our study, we examined the effect of various doses of H. arenarium on the hemocyte count and behavior of G. mellonella larvae. According to the findings obtained at the end of our study, H. arenarium caused an increase in hemocyte count with the injection of 0.25% and 0.5% doses compared to the untreated and DMSO groups. At the same time, the 0.25% and 0.5% doses showed a strong encapsulation-melanization response and an increase in phenoloxidase enzyme activity over 24 h compared to the other injected groups. Based on these results, H. arenarium extract has an anti-mitotic activity at high doses (above 0.5%). This effect may be due to the fact that the plant extract supports mitosis at a certain dose, while being toxic when exceeding it.

Preclinical animal models provide a key tool for non-invasive detection and quantitative measurement of long-term tumor burden. Here, Ramasamy and colleagues applied real-time non-invasive micro-CT imaging to demonstrate the therapeutic efficacy of silibinin in preventing lung tumor development through modulation of inducible nitric oxide synthase (iNOS). Micro-CT imaging revealed a significant and time-dependent decrease in the number and size of lesions in silibinin-treated wild-type animals, but not in iNOS‐/‐ mice. Further, CT results correlated with ex vivo tumor data measured at the end of the experiment. These results show the utility of micro-CT imaging in evaluating lung cancer chemopreventive agents.B-cell chronic lymphocytic leukemia (B-CLL) is a common leukemia of adults. In this study, Zauli and colleagues evaluated the combination of Dasatinib (a multi-kinase inhibitor) plus Nutlin-3 (a nongenotoxic activator of the p pathway) in primary B-CLL patient cells and in B leukemic cell lines. They found that the drug combination induced synergistic cytotoxicity in both p53wild-type and p53mutated/deleted leukemic cells. Further, Akt down-regulation was found to be important in mediating the antileukemic activity of Dasatinib+Nutlin-3. These findings suggest that the combination of Dasatinib and Nutlin-3 represents an innovative therapeutic strategy for B-CLL.TDiamond and colleagues performed a phase I clinical study of the Aurora and angiogenic kinase inhibitor ENMD-2076 in patients with advanced solid tumors. Antitumor activity was seen in several tumor types, including hepatocellular carcinoma, triple-negative breast cancer, and platinum-resistant ovarian cancer. ENMD-2076 exhibited both antimitotic and broad anti-angiogenic activity, and was tolerable up to 160 mg/m2 orally once daily with continuous dosing. Consistent with inhibition of the target kinases, doselimiting hypertension occurred. These findings support future clinical trials evaluating this agent, including an ongoing phase II study in platinumresistant ovarian cancer.Tumor antigen NY-ESO-1 is a major target in human cancer vaccine studies, due to its tumor-restricted expression and strong immunogenicity. Here, Karbach and colleagues investigated the efficacy of NY-ESO-1 recombinant protein combined with the adjuvant CpG 7909 to prime antigen-specific naive B- and T-cells in prostate cancer patients. They observed induction of NY‐ESO‐1‐specific immune responses in a high proportion of NY‐ESO‐ 1‐naive patients (regardless of the level of NY-ESO-1 expression in the autologous tumor). These results demonstrate the strong immunogenicity of this vaccine formulation in vivo, and suggest that it may be effective in preventing the outgrowth of NY‐ESO‐1‐expressing cancers.

Advanced gastrointestinal stromal tumors (GIST) are typically treated with tyrosine kinase inhibitors, and imatinib is the most commonly used standard of care in first line treatments. The use of this and other tyrosine kinase inhibitors is associated with objective tumor responses and prolongation of progression-free and overall survival, but the treatment of metastatic disease is non-curative due to the selection or acquisition of secondary mutations and the activation of alternative kinase signaling pathways, leading to resistance and disease progression after an initial response. The present preclinical study evaluated the potential use of the fibroblast growth factor receptor inhibitors infigratinib and dovitinib alone or in combination with the mitogen-activated protein kinase inhibitor binimetinib in mouse models of GIST with different sensitivity or resistance to imatinib. Patient- and cell-line-derived GIST xenografts were established by bilateral, subcutaneous transplantation of human GIST tissue in female adult nu/nu NMRI mice. The mice were treated with dovitinib, infigratinib, or binimetinib, either alone or in combination with imatinib. The safety of treated animals was assessed by well-being inspection and body weight measurement. Antitumor effects were assessed by caliper-based tumor measurement. H&E staining and immunohistochemistry were used for assessing anti-mitotic and pro-apoptotic activity of the experimental treatments. Western blotting was used for assessing effects of the agents on kinase signaling pathways. Anti-angiogenic activity was assessed by measuring tumor vessel density. Dovitinib was found to have antitumor efficacy in GIST xenografts characterized by different imatinib resistance patterns. Dovitinib had better efficacy than imatinib (both at standard and increased dose) and was found to be well tolerated. Dovitinib had better efficacy in a KIT exon 9 mutant model, highlighting a role of patient selection in clinical GIST trials with the agent. In a model with KIT exon 11 and 17 mutations, dovitinib induced tumor necrosis, most likely due to anti-angiogenic effects. Additive effects combining dovitinib with binimetinib were limited.

Background Tumor-infiltrating lymphocytes (TILs) can be used to predict immune response in multiple tumors. Currently, the evaluation of TILs relies on pathological and molecular examine on resected tumor samples. Enlarged regional lymph nodes could be attributed to tumor involvement or local inflammation with active immune response. We therefore aimed to investigate the role of inflamed lymph nodes in distinguishing the hot tumor from cold tumor. Methods Fresh-frozen tumor samples from pathologically-confirmed node-negative colorectal cancer patients with inflamed nodes (n= 22) and non-inflamed nodes (n= 23) were used to generate the genome-wide DNA methylation profile using Infinium Methylation EPIC BeadChip. The inflamed and non-inflamed nodes were defined as the pathologically-confirmed negative regional lymph nodes with enlarged size (≥ 5mm) and normal size (< 5mm) in radiological imaging, respectively. A MeTIL algorithm that integrates CD3+ T cell-specific differentially methylated positions was constructed and validated by immunohistochemical assay to evaluate TIL-based tumor immune response in bulk tumor samples. Results The baseline characteristics were balanced between patients with inflamed and non-inflamed nodes. The high abundance of TILs was significantly associated with inflamed nodes (P= 0.025). In addition, we identified a subgroup of superhot tumors characterized by inflamed nodes, enriched TILs and favorable overall survival outcomes (HR 0.53, 95% CI: 0.36-0.97; P = 0.032). Conclusions Inflamed regional lymph nodes in radiological imaging indicate potential active immune response in tumor. Our findings provide an easily accessible biomarker for cancer immunotherapy. Citation Format: Dingcheng Shen, Xiaolin Wang, Meijin Huang, Yanxin Luo, Huichuan Yu. Inflamed lymph nodes mark active tumor immune response in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5546.

Although immunotherapy yields striking results in various malignancies, results in pancreatic cancer have been disappointing. Pancreatic cancer has been characterized as a non-cytotoxic T-cell infiltrated tumor, and this may explain the low response rate of immune checkpoint antibodies. A highly immunosuppressive tumor microenvironment and dense desmoplastic stroma in established pancreatic tumors are considered to be the main reasons. Dendritic cells (DCs) are the most potent activators of the immune system and DC vaccination have been shown to successfully induce immune responses in various non-immunogenic malignancies. DCs loaded with an allogeneic mesothelioma tumor cell lysate has proven to be safe, feasible and clinically active in mesothelioma patients. Mesothelioma and pancreatic cancer share tumor characteristics and tumor antigens. We therefore argue that this off-the-shelf vaccine may be beneficial for the treatment of pancreatic cancer. In a murine model we assessed the effectiveness of mesothelioma-lysate loaded DCs against pancreatic adenocarcinoma. C57BL/6 mice were vaccinated with bone-marrow derived DCs both prior to and subsequent to inoculation with subcutaneous syngeneic pancreatic tumor cells (KPC3). DCs were generated with a GM-CSF bone marrow culture and stimulated overnight with CpG along with either pancreatic (KPC3) or mesothelioma tumor (AE17) lysate. Lysates were generated by freeze-thawing and sonification. Mice were challenged with KPC3 tumors and tumor sizes were monitored over time. Immune responses were determined by flow-cytometry of cells in peripheral blood, spleen and tumor. Tumor-specific T-cell responses were investigated by co-culturing purified splenic CD8+ T-cells with IFNg-treated pancreatic cancer cells. DC vaccination preceding tumor challenge led to a significant increase of systemic CD4+ and CD8+ T-cells frequencies in treated mice compared to untreated mice and were persistent over time (p<0.001). This also holds true for effector memory, activated and proliferating CD4+ and CD8+ T-cells. In addition, treated mice displayed significantly delayed tumor growth accompanied by increased frequencies of intratumoral T-cells compared to untreated mice (p<0.05). No difference in immune activation was observed between mice receiving DCs stimulated with pancreatic cancer or mesothelioma lysate. In both groups, a negative correlation between tumor size and tumor infiltrating lymphocyte frequency was found. No differences in intratumoral regulatory T-cells were measured between groups. Interestingly, vaccination with DCs stimulated with CpG in the absence of tumor lysate did not delay tumor growth. To further denote the induction of a tumor-specific T-cell response of both pancreatic and mesothelioma lysate-DC vaccination, the frequencies of CD69+, IFNg+, CD107a+ and granzyme B+ expressing CD8+ T-cells were increased upon stimulation with pancreatic cancer cells compared to untreated mice (P<0.001). Vaccination in a therapeutic setting also demonstrated identical robust systemic immune responses; however, no retardation in tumor growth or survival benefit was observed. In line with this observation, tumor analysis of treated mice revealed a lack of infiltrating lymphocytes in the tumor microenvironment, explaining the lack of therapeutic efficacy.The results demonstrate the potency of mesothelioma lysate-DC therapy generating a robust tumor-specific response with delay in tumor growth in a pancreatic cancer murine model. These findings warrant further testing of mesothelioma lysate-DC vaccination in a clinical setting, and therefore we are currently setting up a trial starting with resected pancreatic cancer patients having no clinical signs of established tumor. Citation Format: Sai Ping S. Lau, Priscilla P. Kinderman, Melanie M. Lukkes, Floris F. Dammeijer, Heleen H. Vroman, Menno M. van Nimwegen, Thorbald T. van Hall, Sjoerd S.H. van der Burg, Joachim J.G.J.V. Aerts, Nadine A.G. Pronk-van Montfoort, Casper C.H.J. van Eijck. Allogeneic tumor-lysate loaded dendritic cells induce anti-tumor immunity and tumor responses in pre-clinical models of pancreatic adenocarcinoma: Towards clinical trials [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B117.

Interactions between various components in the tumor microenvironment and dysregulated immune responses are thought to play important roles in cancer development. To better understand the role of immune cells in tumor pathogenesis and destruction, we computationally model the microenvironment of an aggressive brain tumor glioblastoma multiforme (GBM). We downloaded GBM patient RNA-seq data from the Cancer Genome Atlas (TCGA). Using cluster analysis, we identified 16 clusters, each containing 10 to 933 genes that show a statistical enrichment of immune response related gene ontology terms. Utilizing a panel of RNA-seq data from normal cell types, we constructed global and cluster specific regression models to characterize the expression profiles of GBM samples in the clusters of interest as linear combinations of normal cell and reference GBM expression profiles. Simulated data was used to validate that the regression model coefficients accurately reflect the contributions of normal cell types to the expression profiles of tumor samples. Based on the regression analysis, we were able to uncover high variability in the composition of microenvironment across the TCGA cohort, suggesting diverse immune responses in tumors. The results from global regression analysis were then associated with common genetic alterations in GBM and with GBM subclassification. Especially the estimated macrophage, granulocyte, and CD8+ T lymphocyte proportions show significant differences between different GBM subgroups. Accumulation of immune cells was increased in the mesenchymal and neural subtype compared to other subtypes. Furthermore, estimated immune cell proportions were associated with alterations in EGFR and NF1. Taken together, our analysis provided a characterization of the immunomicroenvironment in GBM and linked immune cell responses to typical GBM alterations and GBM subtypes. More detailed characterization of diverse immune responses will facilitate patient stratification and might provide tools for personalized immunotherapy in the future. Citation Format: Suvi Luoto, Juha Kesseli, Matti Nykter, Kirsi J. Granberg. Different immune cell responses are associated with glioblastoma subclassification and typical genetic alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4148.

2630 Background: Human papillomavirus 16 (HPV-16) infection is associated with several cancer types with limited treatment options in the locally R/M settings. Furthermore, immune checkpoint inhibitors have limited activity against advanced HPV-cancers. TG4001 is a viral vector vaccine targeting HPV E6 and E7 antigens. We have previously shown in a single-arm, dose finding phase 1 study that TG4001 combined with PD-L1 blockade using avelumab was safe and associated with a response rate of 22%. Herein we report preliminary data on immunogenicity and clinical activity of TG4001 in a randomized phase 2 study comparing TG4001 plus avelumab versus avelumab alone. Methods: Eligibility criteria: R/M HPV16+ anogenital cancer including cervical, vulvar, vaginal, penile, and anal cancers; immunotherapy naïve and with no more than one prior line of chemotherapy. HPV-16 positivity was required and centrally determined using a PCR based assay. Patients were randomly assigned 1:1 to receive either TG4001 plus avelumab (Vaccine arm) or avelumab alone. Randomization was stratified by tumor type (cervical, anal, genital). TG4001 was administered s.c at 5·107 pfu, Q1w for 5 weeks, then Q2w until month 6 followed by Q12w until progressive disease and avelumab i.v at 800 mg Q2w until progressive disease. PBMCwere collected at baseline, day 43 and day 85 to assess T-cell responses against E6 and E7 antigens using ex-vivo IFNg ELISPOT and immunophenotyping of circulating T cells. Vaccine immune response (IR) was defined as onset of a new T-cell response against either antigen or amplification of a pre-existing response under treatment. Tumor response was assessed using RECIST 1.1. Results: 59 pts had been randomized by the time of data cut-off. IR was assessed in 24 pts in the vaccine arm and 16 pts in the avelumab arm. T cells against E6 or E7 antigens at baseline were rare with only 4 pts having a low intensity ELISPOT readout against either target prior to initiation of treatment. 11/24 pts in the vaccine arm had an IR, while none of the patients in the avelumab arm had an IR. Occurrence of IR was detected at day 43 and tended to gain in intensity at D85. In the vaccine arm, IR was associated with tumor response with 3 clinical responders among 11 pts with positive IR versus 1 response observed among the 13 patients without IR. Conversely, only 2 progressive disease pts among 11 positive IR compared favorably to the 6/13 vaccine arm pts with no IR. Remarkably, a complete clinical response was observed in a vaccine treated pt exhibiting the strongest E6 and E7 IR. Conclusions: TG4001 can induce a de novo immune response against HPV-16 antigens E6 and E7 in advanced HPV-16 cancers. The data suggest that these IRs are associated with anti-tumor response and that TG4001 vaccination in combination with ICI has the potential to drive clinical benefit in R/M anogenital cancers. Clinical trial information: NCT03260023 .

We have shown that ObRb, the leptin receptor, is overexpressed in colorectal cancer cells, and that this may influence the patients' outcome. We investigated colonocytes as leptin targets and characterized their pivotal role in antitumor immune response. Cytokine and chemokine mRNAs in HT29 cells were measured by targeted arrays. In vitro, normal colonocytes and human colon cancer cells (HT29, Caco-2, SW480, and HCT116) were used to investigate ObRb transduction system and cytokine releases. Animal colonocytes and CD8 splenocytes and human HT29, HCT116, and CD8(+) cells from blood donors were used to investigate the lymphocyte response to the colonocytes when stimulated by leptin. Leptin-induced cytokine releases in the normal colonic mucosa and tumor growth and cytokine releases within tumors in vivo were measured in male rats and nude mice, respectively. Statistical analysis was done by Fisher's exact and Mann-Whitney U tests. Various cytokines and their receptors were produced in normal and tumoral colonocytes in response to leptin by increasing nuclear factor-kappaB activation. Interleukin-8 (IL-8) was the main cytokine produced in vitro. The levels of IL-8 and its receptor, CXCR1, were higher in tumors than in homologous normal mucosa. Systemic leptin enhanced the proinflammatory cytokines in normal colonocytes and in HT29 xenografted tumor colonocytes. Colonocyte-derived products after leptin treatment stimulated perforin and granzyme B expressions in normal CD8(+) T cells in vitro. Leptin triggers an inflammatory response in tumor tissue by directly stimulating colonocytes, which can recruit T cytotoxic cells in the tumor microenvironment.

<div>Abstract<p>We have shown that ObRb, the leptin receptor, is overexpressed in colorectal cancer cells, and that this may influence the patients' outcome. We investigated colonocytes as leptin targets and characterized their pivotal role in antitumor immune response. Cytokine and chemokine mRNAs in HT29 cells were measured by targeted arrays. <i>In vitro</i>, normal colonocytes and human colon cancer cells (HT29, Caco-2, SW480, and HCT116) were used to investigate ObRb transduction system and cytokine releases. Animal colonocytes and CD8 splenocytes and human HT29, HCT116, and CD8<sup>+</sup> cells from blood donors were used to investigate the lymphocyte response to the colonocytes when stimulated by leptin. Leptin-induced cytokine releases in the normal colonic mucosa and tumor growth and cytokine releases within tumors <i>in vivo</i> were measured in male rats and nude mice, respectively. Statistical analysis was done by Fisher's exact and Mann-Whitney <i>U</i> tests. Various cytokines and their receptors were produced in normal and tumoral colonocytes in response to leptin by increasing nuclear factor-κB activation. Interleukin-8 (IL-8) was the main cytokine produced <i>in vitro</i>. The levels of IL-8 and its receptor, CXCR1, were higher in tumors than in homologous normal mucosa. Systemic leptin enhanced the proinflammatory cytokines in normal colonocytes and in HT29 xenografted tumor colonocytes. Colonocyte-derived products after leptin treatment stimulated perforin and granzyme B expressions in normal CD8<sup>+</sup> T cells <i>in vitro</i>. Leptin triggers an inflammatory response in tumor tissue by directly stimulating colonocytes, which can recruit T cytotoxic cells in the tumor microenvironment. [Cancer Res 2008;68(22):9423–32]</p></div>

<div>Abstract<p>We have shown that ObRb, the leptin receptor, is overexpressed in colorectal cancer cells, and that this may influence the patients' outcome. We investigated colonocytes as leptin targets and characterized their pivotal role in antitumor immune response. Cytokine and chemokine mRNAs in HT29 cells were measured by targeted arrays. <i>In vitro</i>, normal colonocytes and human colon cancer cells (HT29, Caco-2, SW480, and HCT116) were used to investigate ObRb transduction system and cytokine releases. Animal colonocytes and CD8 splenocytes and human HT29, HCT116, and CD8<sup>+</sup> cells from blood donors were used to investigate the lymphocyte response to the colonocytes when stimulated by leptin. Leptin-induced cytokine releases in the normal colonic mucosa and tumor growth and cytokine releases within tumors <i>in vivo</i> were measured in male rats and nude mice, respectively. Statistical analysis was done by Fisher's exact and Mann-Whitney <i>U</i> tests. Various cytokines and their receptors were produced in normal and tumoral colonocytes in response to leptin by increasing nuclear factor-κB activation. Interleukin-8 (IL-8) was the main cytokine produced <i>in vitro</i>. The levels of IL-8 and its receptor, CXCR1, were higher in tumors than in homologous normal mucosa. Systemic leptin enhanced the proinflammatory cytokines in normal colonocytes and in HT29 xenografted tumor colonocytes. Colonocyte-derived products after leptin treatment stimulated perforin and granzyme B expressions in normal CD8<sup>+</sup> T cells <i>in vitro</i>. Leptin triggers an inflammatory response in tumor tissue by directly stimulating colonocytes, which can recruit T cytotoxic cells in the tumor microenvironment. [Cancer Res 2008;68(22):9423–32]</p></div>

Tumors with high mutation rate such as those of melanoma and non-small cell lung cancers (NSCLC) have shown greatest response to the emerging immune therapies. These tumors harbor a large number of somatic mutations, some of which could be translated into neoantigens, potentially evoking host immune response. To what degree self antigens, many of which are cancer testis (CT) antigens, contribute to enhanced immune response in these tumors has never been systematically studied. Here we show that cancers with higher mutation rate frequently express a larger number of CT antigens than cancers with fewer mutations. In NSCLC, tumors with more than 300 non-synonymous mutations (75th percentile, n = 158) express more than 3 times as many CT antigens as those tumors with fewer than 100 mutations (25th percentile, n = 162), with a P-value of 1.6e-13. In head and neck cancers, tumors with more than 140 non-synonymous mutations (75th percentile, n = 79) express nearly twice as many CT antigens as those tumors with fewer than 60 mutations (25th percentile, n = 80), with a P-value of 0.004. Additionally, in NSCLC, tumors with high mutation rate are associated with higher expression of CD8B and granzyme B, markers representing cytotoxic lymphocytes. Tumors with more expressed CT antigens are also associated with higher expression of these markers. These data suggest that CT antigens, along with neoantigens, may contribute to a stronger host immune response in these hypermutative tumors. Citation Format: Junping Jing, Patrick Mayes, Yan Degenhardt, James R. Brown, Philippe Sanseau. Higher mutation rate is associated with more frequent expression of cancer/testis antigens in human tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 525.

Macroautophagy (hereafter autophagy) degrades and recycles intracellular components to sustain metabolism and survival during starvation. Host autophagy promotes tumor growth by providing essential tumor nutrients. Autophagy also regulates immune cell homeostasis and function and suppresses inflammation. Although host autophagy does not promote a T-cell anti-tumor immune response in tumors with low tumor mutational burden (TMB), whether this was the case in tumors with high TMB was not known. Here we show that autophagy, especially in the liver, promotes tumor immune tolerance by enabling regulatory T-cell function and limiting stimulator of interferon genes, T-cell response and interferon-γ, which enables growth of high-TMB tumors. We have designated this as hepatic autophagy immune tolerance. Autophagy thereby promotes tumor growth through both metabolic and immune mechanisms depending on mutational load and autophagy inhibition is an effective means to promote an antitumor T-cell response in high-TMB tumors.

NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer

15 Figure 1 Preclinical Study on 4T1 Murine Mouse Model -Ablation Modalities Comparison. Schematic representation of the experimental design. PEF (Pulsed Electric Field) and RFA (Radiofrequency) ablation were applied at...