bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A30: Combined IDO inhibitor and OX40L-armed-oncolytic-adenovirus therapy improves survival in murine glioblastoma
Authors: Nguyen Teresa T. , Shin Dong Ho , Jiang Hong , Lang Frederick , Gomez-Manzano Candelaria , Fueyo Juan
Published: March 2020

Glioblastoma diagnoses associate with upregulation of the immunosuppressive indoleamine-2,3-dioxygenase (IDO) enzyme, which correlates with poor prognoses. IDO inhibitors are immunometabolic adjuvants that can reverse protumor immunosuppression through increasing effector T-cell metabolism. To further stimulate antitumor T-cell activation, the OX40L-armed-oncolytic-adenovirus (Delta-24-RGDOX) can be used, which has shown to induce immune mechanisms that activate antitumor cytotoxic properties of T cells. Thus, we hypothesized that combining IDO inhibitors and Delta-24-RGDOX synergizes to produce a therapeutic effect in glioblastoma. In this study, we used a syngeneic glioblastoma orthotopic immunocompetent model to test the efficacy of combining Delta-24-RGDOX and an IDO inhibitor (1MT or indoximod). Delta-24-RGDOX treatment resulted in a significant survival benefit compared to the control groups (mean survival, 46.5 vs. 38.5 days, P=0.02). Interestingly, the combination of Delta-24-RGDOX and 1MT produced a higher percentage of long-term survivors compared to single therapy with Delta-24-RGDOX (50% vs. 20%, P=0.03). Moreover, when these long-term glioblastoma survivors were rechallenged with the same glioblastoma cells, we observed 100% survival, indicating the establishment of immune memory by the combination therapy. Furthermore, functional studies showed a significant increase in antitumor activity of splenocytes from glioblastoma-bearing combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Additionally, immunophenotyping by flow cytometry revealed that glioblastoma-bearing combination-treated mice yielded the highest levels of PD-1/TIM-3/CD8+ T cells compared to all other treatment groups and decreased levels of immune-tolerant populations, including regulatory T cells and myeloid-derived suppressor cells, compared to Delta-24-RGDOX-only treated mice, and correlated with complete tumor elimination. Most importantly, these data support the use of immunometabolic adjuvants combined with virotherapy as a treatment for glioblastoma to improve its dismal outcomes.

Citation Format: Teresa T. Nguyen, Dong Ho Shin, Hong Jiang, Frederick Lang, Candelaria Gomez-Manzano, Juan Fueyo. Combined IDO inhibitor and OX40L-armed-oncolytic-adenovirus therapy improves survival in murine glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A30.
Abstract A33: High-throughput prediction of MHC Class I and Class II neoantigens with MHCnuggets
Authors: Shao Xiaoshan M. , Bhattacharya Rohit , Huang Justin , Sivakumar Ashok , Tokheim Collin , Zheng Lily , Hirsch Dylan , Kaminow Ben , Omdahl Ashton , Bonsack Maria , Riemer Angelika B. , Velculescu Victor E. , Anagnostou Valsamo , Pagel Kymberleigh , Karchin Rachel
Published: March 2020

Computational prediction of binding between neoantigen peptides and major histocompatibility complex (MHC) proteins is an emerging biomarker for predicting patient response to cancer immunotherapy. Current neoantigen predictors focus on in silico estimation of MHC binding affinity and are limited by low positive predictive value for actual peptide presentation, inadequate support for rare MHC alleles, and poor scalability to high-throughput data sets. To address these limitations, we developed MHCnuggets, a deep neural network method to predict peptide-MHC binding. MHCnuggets is the only method to handle binding prediction for common or rare alleles of MHC Class I or II, with a single neural network architecture. Using a long short-term memory network (LSTM), MHCnuggets accepts peptides of variable length and is capable of faster performance than other methods. When compared to methods that integrate binding affinity and HLAp data from mass spectrometry, MHCnuggets yields a fourfold increase in positive predictive value on independent MHC-bound peptide (HLAp) data. We applied MHCnuggets to 26 cancer types in TCGA, processing 52.6 million allele-peptide comparisons in under 2.3 hours, yielding 103,587 candidate immunogenic missense mutations (IMMs). IMM hotspots occurred in 36 genes, including 22 driver genes. Predicted IMM load was significantly associated with increased immune cell infiltration (p<2e-16), including CD8+ T cells. Notably, only 0.15% of predicted immunogenic missense mutations were observed in >2 patients, with 65% of these derived from driver mutations. Our results provide a new method for neoantigen prediction with high performance characteristics and demonstrate its utility in large data sets across human cancers.

Citation Format: Xiaoshan M. Shao, Rohit Bhattacharya, Justin Huang, Ashok Sivakumar, Collin Tokheim, Lily Zheng, Dylan Hirsch, Ben Kaminow, Ashton Omdahl, Maria Bonsack, Angelika B. Riemer, Victor E. Velculescu, Valsamo Anagnostou, Kymberleigh Pagel, Rachel Karchin. High-throughput prediction of MHC Class I and Class II neoantigens with MHCnuggets [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A33.
Abstract A34: Cancer immunotherapy clinical trials in K-MASTER project
Authors: Kim Ju Won , Sung Jae Sook , Jung Hee Jun , Choi Jung Yoon , Lee Soo Hyeon , Chang Won Jin , Choi Yoon Ji , Park Kyong Hwa , Kim Yeul Hong
Published: March 2020

The K-MASTER cancer precision medicine diagnosis and treatment enterprise (K-MASTER project) is a precision medicine cancer treatment clinical trial platform, which uses next-generation sequencing (NGS) panel to screen the actionable mutations in 10,000 patients with refractory solid tumors and assigns the patients to matched clinical trials. K-MASTER project was initiated in Korea in June 2017. From October 2017, we have analyzed NGS -based cancer panel to detect genomic variations in Korean cancer patients. Tumor DNA was analyzed by K-MASTER panel, which allows to detect variations of 375 genes. As of September 3, 2019, total 4,240 cancer patients were registered via KM-00 screening protocol. The mean depth of K-MASTER panel was 701x. The patient whose cancer has been confirmed of certain genomic variants can participate in matched clinical studies. As of September 2019, 11 clinical trials are successfully in process and another 6 are awaiting kickoff under the name of K-MASTER project. There are 3 ongoing immunotherapy clinical trials: avelumab for metastatic colon cancer with microsatellite instability-high or POLE mutation (KM-01), nivolumab for advanced solid tumor with DNA repair pathway aberration (KM-06), and spartalizumab combined with lacnotuzumab for esophageal squamous cell carcinoma (KM-12). Among them, the KM-01 study closed its registration and is waiting for data analysis. Other studies are actively enrolling patients along with KM-00 screening protocol. In addition, various new immunotherapy studies are preparing for initiation: basket trial of nivolumab plus ipilimumab for high tumor mutational burden solid tumors (KM-09), phase II study of CAST cell therapy (KM-15), alternative scheduling trial for pembrolizumab (KM-19), and phase II/III trial of novel PD-1 inhibitor INCMGA00012 (KM-20) are on their way to start. In conclusion, K-MASTER project is efficient to detect actionable variations and function as a reliable path between patient and new immunotherapeutic agent. Nationwide NGS-based screening is not only feasible but also effective in recruiting participants in molecular basket trials as well as immunotherapy trials. This novel knowledge provides an intriguing background to investigate new target approaches and to progress precision medicine. (This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute [KHIDI], funded by the Ministry of Health &Welfare, Republic of Korea [Grant number: HI17C2206].)

Citation Format: Ju Won Kim, Jae Sook Sung, Hee Jun Jung, Jung Yoon Choi, Soo Hyeon Lee, Won Jin Chang, Yoon Ji Choi, Kyong Hwa Park, Yeul Hong Kim. Cancer immunotherapy clinical trials in K-MASTER project [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A34.
Abstract A43: Exploiting off-target effects of estrogen receptor-targeting drugs to sensitize breast cancer to immune killing
Authors: Wolfson Benjamin , Schlom Jeffrey , Hodge James
Published: March 2020

1 in 8 women will be diagnosed with breast cancer within their lifetime. The majority of women are diagnosed with estrogen receptor-positive (ER+), progesterone receptor-positive (PR+), or HER2+ breast cancers. The standard of care for ER+ breast cancer is treatment with selective estrogen receptor modulator (SERM) and selective estrogen receptor degrader (SERD) drugs. While these are highly effective for ER+ breast cancers and additional targeted drugs exist for PR+ and HER2+ cancers, there are currently no targeted therapeutic strategies for the 10-15% of women who are diagnosed with triple-negative breast cancer (TNBC). We hypothesize that estrogen receptor-targeting drugs are capable of inducing phenotypic changes to sensitize breast tumor cells to immune-mediated killing regardless of their ER status. In this study, we used in vitro killing assays and molecular characterization of ER+ and ER- cell lines to identify that SERM and SERD drugs sensitize breast cancer to killing by healthy-donor natural killer (NK) cells regardless of their ER status. While tamoxifen (SERM) and fulvestrant (SERD) had no cytotoxic or antiproliferative effect on ER- cells, pretreatment with either resulted in increased NK-mediated cell lysis. Through RNAseq analysis of tamoxifen-treated ER+ and ER- cells, we identified increased activation of both apoptotic and death receptor signaling pathways. These pathways play a key role in immunogenic modulation, indicating a potential mechanism for tamoxifen-induced tumor sensitization to immune cell lysis. It has previously been reported that in addition to binding to ER, both tamoxifen and fulvestrant bind to G protein-coupled estrogen receptor 1 (GPR30). We found that GPR30 is expressed in both ER+ and ER- breast cancer cells. Using the specific GPR30 agonist G-1, we demonstrated that targeted activation of GPR30 signaling resulted in increased NK cell killing. These data indicate that the observed ER-independent immunogenic modulation of breast cancer cells may be occurring through GPR30. Together, these findings demonstrate a novel effect of SERM and SERD drugs on the interaction between both ER+ and ER- tumor cells with the immune system. This study is the first to demonstrate the potential use of ER-targeting drugs in immunotherapy combinations in an ER-agnostic manner and may inform novel immunotherapy strategies in breast cancer.

Citation Format: Benjamin Wolfson, Jeffrey Schlom, James Hodge. Exploiting off-target effects of estrogen receptor-targeting drugs to sensitize breast cancer to immune killing [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A43.
Abstract A48: Inactivation of Fbxw7 impairs dsRNA sensing and confers resistance to PD-1 blockade
Authors: Gstalder Cécile , Liu David , Miao Diana , Devine Alexander , Lutterbach Bart , Pancholi Priya , Shettigar Megha , Buchbinder Elizabeth , Carter Scott , Manos Michael , Rojas-Rudilla Vanesa , Brennick Ryan , Gjini Evisa , Lako Ana , Rodig Scott , Yoon Charles , Freeman Gordon , Hodi F. Stephen , Van Allen Eliezer M. , Haq Rizwan
Published: March 2020

Immunotherapies such as anti-PD-1 antibodies can induce durable responses in a subset of cancer patients, but intrinsic or acquired resistance occurs in most cases. Understanding the molecular mechanisms that drive resistance and response to immunotherapies could therefore lead to alternative treatment strategies exhibiting greater efficacy and precision. To identify new tumor cell-intrinsic mechanisms of resistance to PD-1 blockade, we evaluated metastatic melanoma patients who exhibited complete regression of all but one metastatic site following anti-PD-1 therapy. In one patient, we observed a loss-of-function mutation in the tumor suppressor gene F-Box and WD repeat domain containing 7 (FBXW7)—specifically in the resistant tumor. Using an immunocompetent, anti-PD-1 sensitive melanoma mouse model, we demonstrated that Fbxw7 loss of function in tumor cells causes resistance to PD-1 blockade. We showed that the genetic deletion of Fbxw7 in tumor cells alters the tumor immune microenvironment—including decreased effector cells and increased suppressive cells—in addition to diminished responses to viral sensing and interferon signaling pathways in vivo. Mechanistically, we found that Fbxw7 is necessary for the expression of the dsRNA sensors Mda5 and Rig-I. As a consequence, Fbxw7 is required for dsRNA-induced type I interferon production and interferon signaling in tumor cells. Conversely, restoration of the dsRNA signaling pathway in Fbxw7-deficient cells is sufficient to increase MHC-I expression and to suppress growth of Fbxw7-deficient tumors, thereby promoting antitumor immunity. Our findings establish a hitherto unrecognized role of the tumor suppressor gene FBXW7 in tumor immunity and sensitivity to immunotherapy. Collectively, these findings establish a novel tumor-intrinsic pathway of resistance and suggest that therapeutic reactivation of viral sensing pathways could improve clinical responses to checkpoint inhibitors in genomically defined populations.

Citation Format: Cécile Gstalder, David Liu, Diana Miao, Alexander Devine, Bart Lutterbach, Priya Pancholi, Megha Shettigar, Elizabeth Buchbinder, Scott Carter, Michael Manos, Vanesa Rojas-Rudilla, Ryan Brennick, Evisa Gjini, Ana Lako, Scott Rodig, Charles Yoon, Gordon Freeman, F. Stephen Hodi, Eliezer M. Van Allen, Rizwan Haq. Inactivation of Fbxw7 impairs dsRNA sensing and confers resistance to PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A48.
Abstract A59: Inhibition of Glycogen Synthase Kinase 3 (GSK-3) synergizes with anti-PD-1 by potentiating tumor-infiltrated CD8+ T-cells
Authors: Krueger Janna , Taylor Alison , Kazanova Alexandra , Jeidane Saloua , Motta Vinicius , Watson Ian , Rudd Christopher E.
Published: March 2020

Anti-programmed cell death-1 (anti-PD-1) blockade in tumor therapy results in an expansion of CD8+ T cells and can restore the function of exhausted CD8+ T cells. We identified GSK-3 as a key upstream kinase of regulating PD-1 expression. Inhibition of GSK-3 in CD8+ T cells increased Tbx21 (Tbet), which suppressed Pdcd1 (PD-1) transcription (Taylor et al., Immunity 2016). In controlling spontaneous pulmonary metastasis of B16 melanoma and EL-4 lymphoma cells, GSK-3 blockade is as effective as anti-PD-1 blocking antibody (Taylor et al., Cancer Research 2018). Given the clinical importance in devising strategies to overcome tumor resistance to immune checkpoint blockade, we assessed whether the combined use of GSK-3 small-molecule inhibitor (SMI) and anti-PD-1 can overcome resistance to anti-PD-1 monotherapy. Indeed, GSK-3 SMI and anti-PD-1 synergized in therapy against solid B16 tumors that are resistant to monotherapy, leading to delayed tumor growth. Further, the synergy of GSK-3 SMI and anti-PD-1 therapy has been successfully confirmed by using adoptive transfer of OT1 cells into B16-OVA tumor-bearing mice. High-dimensional profiling of tumor-infiltrated lymphocytes (TILs) using mass cytometry (CyTOF) showed that the combination therapy gave an increase in cellularity of CD8+ effector memory T cells, which correlated to better tumor control. Transcriptome analysis of these CD8+ TILs highlighted the increased cytotoxicity as shown by significant changes of granzymes. Our findings outline a next-generation approach of potentiating PD-1 antibody therapy with SMIs of GSK-3 that synergizes as therapy by boosting the cellularity and cytotoxicity of tumor infiltrated CD8+ T cells.

Citation Format: Janna Krueger, Alison Taylor, Alexandra Kazanova, Saloua Jeidane, Vinicius Motta, Ian Watson, Christopher E. Rudd. Inhibition of Glycogen Synthase Kinase 3 (GSK-3) synergizes with anti-PD-1 by potentiating tumor-infiltrated CD8+ T-cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A59.
Abstract A62: Inhibition of tumor growth by novel CART redirected against cells expressing high levels of fibroblast activation protein
Authors: Noguera-Ortega Estela , Todd Leslie , Monslow James , Sun Jing , Albelda Steven M. , Pure Ellen
Published: March 2020

Cancer-associated fibroblasts (CAFs) support tumor growth and metastasis of virtually all solid tumors, making them a potential “universal therapeutic target.” However, there are challenges to designing CAF targeted therapies. First, CAFs are heterogeneous, consisting of tumor-promoting and tumor-restraining populations. Second, molecular targets expressed strictly by tumor-promoting CAFs have yet to be defined. Nonetheless, Fibroblast Activation Protein (FAP) is selectively overexpressed on tumor-promoting CAFs. Moreover, deleting FAP+ cells inhibits tumor growth in preclinical models due to reductions in stroma-dependent signals that support tumor cell survival, growth, and progression and in stroma-derived immunosuppressive factors and can enhance efficacy of other therapies when given in combination. Prior studies in preclinical models suggest, however, that selective elimination of FAPhigh cells while sparing normal FAP+ cells that typically express lower levels of FAP may be necessary to avoid toxicity due to on-target, off-tumor effects. Thus, the aim of this study was to generate a FAP-chimeric antigen receptor (CAR) that selectively targets FAPhigh cells but that exhibits minimal activity against FAPlow cells for use in future clinical trials. We generated a FAP-CAR containing a scFv based on a monoclonal antibody (mAb) we generated that reacts with human and mouse FAP, the CD8 transmembrane region, and the CD3z and 41BB cytoplasmic domains. We show that primary human T cells transduced with this novel FAP-CAR exhibit strong reactivity against cells expressing high levels of human or mouse FAP but not FAPlow or FAP-negative cells. Moreover, these FAP-CART cells inhibited the growth of human FAP-negative A549 tumor xenografts infiltrated by FAP+ murine CAFs in NSG mice. Based on these characteristics, this novel FAP-CAR will be developed for use in clinical trials for human cancer patients.

Citation Format: Estela Noguera-Ortega, Leslie Todd, James Monslow, Jing Sun, Steven M. Albelda, Ellen Pure. Inhibition of tumor growth by novel CART redirected against cells expressing high levels of fibroblast activation protein [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A62.
Abstract A66: Intracavity infusions of autologous cellular immunotherapy in the treatment of refractory malignant pleural and ascites: A Chinese population-based cohort study
Authors: Qiao Guoliang
Published: March 2020

Objectives: we have explored the potential efficacy and safety of autologous adoptive T cells represented by dendritic cell (DC)-cytokine-induced killer (CIK) infused within cavity in advanced cancer patients with malignant pleural effusion and ascites.

Methods: The apheresis for collection of peripheral blood mononuclear cells was performed in 37 recruited patients. DC-CIKs were expanded ex vivo until qualified for intracavity perfusions for three consecutive times. The quantitative volume of malignant effusions was recorded by radiologic imaging assay. The clinical adverse reactions were analyzed.

Results: For the quantitative assay for those effusions, 1 patient has achieved complete response, 13 patients have achieved PR, 12 patients have stable diseases, and 11 patients have progressive diseases, respectively, yielding a total clinical control rate of 70.3%. After adjusting for competing risk factors, number of concurrent metastasis distribution of organs (p=0.007) and infused number of CIKs (p=0.013) remained independent factors associated with malignant effusion remission. Further exploration demonstrated that the infused CD3+/CD8+ and CD8+/CD28- T-cell populations were able to determine the clinical responses (P<0.05). Moreover, elevated peripheral blood CD3+/CD8+ and declined CD4+/CD25+ T-cell population were noticed in malignant effusion remission group compared with non-remission group (P <0.05). Fewer adverse reactions were recorded.

Conclusions: We have provided the novel therapeutic approach by autologous cellular immunotherapy with confirmed clinical efficacy and safety in the stably controllable malignant effusions. The intracavity infusions were featured by the immunologic T-cell recovery and clinical remission of those effusions.

Note:This abstract was not presented at the conference.

Citation Format: Guoliang Qiao. Intracavity infusions of autologous cellular immunotherapy in the treatment of refractory malignant pleural and ascites: A Chinese population-based cohort study [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A66.
Abstract A8: Immuno-phenotyping of tumor-specific CD8 T cells using high-dimensional mass cytometry
Authors: Roumanes David , Fehlings Michael , Yadav Mahesh , Nardin Alessandra
Published: March 2020

There is strong evidence that tumor-specific T cells play a central role in tumor rejection. However, few tools can simultaneously detect rare antigen-specific T cells and deeply characterize their function and phenotype. immunoSCAPE leverages the high-dimensional immune profiling capabilities of mass cytometry combined with a unique technology for the identification of antigen-specific T cells to support the development of immunotherapy strategies. By applying cytometry by time of flight in conjunction with combinatorial peptide-MHC tetramer staining and high-performance dimensional analysis tools, we broadly and sensitively map T-cell reactivity against MHC-class I epitopes while concurrently performing in-depth characterization of these rare antigen-specific T cells. Here, we have applied our unique capabilities to detect and characterize circulating neoantigen-specific T cells in lung carcinoma patients undergoing atezolizumab treatment. Out of 782 neoantigen candidates, T-cell reactivity against 13 unique neoantigens was detected within patients who responded to atezolizumab treatment, while T cells within nonresponder patients recognized 7 neoantigens. Additionally, neoantigen-specific CD8 T cells from treatment responders exhibit a differentiated effector phenotype similar to CMV or EBV-specific T cells, whereas neoantigen-specific CD8 T cells from nonresponders display a memory-like phenotype, suggesting that the properties of tumor-reactive T cells may be associated with clinical response to anti-PD-L1 treatment. By providing insight into the nature and function of tumor-specific T cells, immunoSCAPE’s unique high-dimensional immune profiling platform is a valuable tool to guide the development of novel immunotherapy strategies through assessment of drug biologic activity, definition of mechanism of action, and identification of biomarkers of patient response.

Citation Format: David Roumanes, Michael Fehlings, Mahesh Yadav, Alessandra Nardin. Immuno-phenotyping of tumor-specific CD8 T cells using high-dimensional mass cytometry [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A8.
Abstract A80: Mapping immune landscape in clear cell renal carcinoma by single-cell genomics
Authors: Vishwakarma Ajaykumar , Bocherding Nick , Chimenti Michael , Vishwakarma Purshottam , Nepple Kenneth , Salem Aliasger , Jenkins Russell W. , Zhang Weizhou , Zakharia Yousef
Published: March 2020

Human clear cell renal cell carcinoma (ccRCC) is one of the most immunologically distinct tumor types due to high levels of tumor-infiltrating immune cells including T cells, yet not every patient responds to immunotherapy. Interestingly, in contrast to other cancers, infiltration with cytotoxic CD8+ T cells is associated with poorer overall survival in ccRCC, suggesting that subpopulations of CD8+ and other immune cells may underlie this observation. To characterize the tumor immune microenvironment of ccRCC, we applied single-cell RNA sequencing along with T-cell receptor sequencing to map the transcriptomic heterogeneity of 25,688 individual CD45+ lymphoid and myeloid cells in matched tumor and blood from patients with ccRCC. We identified multiple and distinct immune cell phenotypes for B and T (CD4 and CD8) lymphocytes, natural kill (NK) cells, and myeloid cells. Evaluation of T-cell receptor (TCR) sequences revealed limited shared clonotypes between patients, whereas tumor-infiltrating T-cell clonotypes were frequently found in peripheral blood, albeit in lower abundance. Evaluation of myeloid subsets revealed unique gene programs defining monocytes, dendritic cells, and tumor-associated macrophages. In summary, here we have leveraged scRNA-seq to refine our understanding of the relative abundance, diversity, and complexity of the immune landscape of ccRCC. This report represents the first such characterization of the ccRCC immune landscape using scRNA-seq. With further characterization and functional validation, these findings may identify novel subpopulations of immune cells amenable to therapeutic intervention.

Citation Format: Ajaykumar Vishwakarma, Nick Bocherding, Michael Chimenti, Purshottam Vishwakarma, Kenneth Nepple, Aliasger Salem, Russell W. Jenkins, Weizhou Zhang, Yousef Zakharia. Mapping immune landscape in clear cell renal carcinoma by single-cell genomics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A80.
Abstract A81: Identifying cellular immune components that correlate with response to immunotherapy in breast cancer using murine models
Authors: Mehta Anita K. , Castrillion Jessica A. , Sotayo Alaba , Mittendorf Elizabeth A. , Letai Anthony G. , Guerriero Jennifer L. , Cheney Emily
Published: March 2020

The heterogeneity of the tumor microenvironment may contribute to the lack of durable responses of immunotherapy in breast cancer. To understand factors that contribute to tumor immune cell heterogeneity, we report a detailed analysis and comparison of the immune tumor microenvironment of the autochthonous MMTV-PyMT murine breast cancer model resembling luminal B breast cancer and corresponding syngeneic models. We obtained tumors from MMTV-PyMT mice and used them to generate syngeneic models using either 1E6, 1E5, or 1E4 cells injected into the mammary fat pad of FVB/NJ wild-type mice. When tumors reached 100 mm3, tumors were harvested and quantitative flow-cytometry and NanoString analysis was performed. We have identified that the number of cells inoculated to generate syngeneic tumors significantly influences tumor latency, the tumor immune microenvironment, and the response to immune checkpoint blockade (ICB). Compared to the autochthonous model, the 1E6 and 1E5 model had significantly more tumor-infiltrating lymphocytes (TILs; CD3+, CD4+, and CD8+) and the highest proportion of PD-L1-positive myeloid cells. We found that increased TILs and expression of PD-L1 on myeloid cells were the best predictors of response to PD-L1 or CTLA-4 therapy but that tumor cell expression of PD-L1 and T-cell expression of PD-1 did not correspond to beneficial outcome of treatment. Both the 1E6 and 1E5 models responded to PD-L1 and/or CTLA-4 ICB therapy, whereas the 1E4 and autochthonous models were resistant. These matched sensitive and resistant tumor models represent a unique opportunity to further interrogate the TME in breast cancer.

Citation Format: Anita K. Mehta, Jessica A. Castrillion, Alaba Sotayo, Elizabeth A. Mittendorf, Anthony G. Letai, Jennifer L. Guerriero, Emily Cheney. Identifying cellular immune components that correlate with response to immunotherapy in breast cancer using murine models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A81.
Abstract A82: Direct and indirect regulators of epithelial-to-mesenchymal transition mediated immunosuppression in breast carcinomas
Authors: Dongre Anushka , Rashidian Mohammad , Eaton Elinor Ng , Reinhardt Ferenc , Nepal Sunita , Ploegh Hidde , Weinberg Robert
Published: March 2020

The epithelial-to-mesenchymal transition (EMT) is a cell-biologic program that confers mesenchymal traits on carcinoma cells and drives their metastatic dissemination. We recently demonstrated that epithelial and mesenchymal carcinomas recruit distinct immune cells to their tumor microenvironments (TME) and differ in their susceptibility to checkpoint blockade. Specifically, mesenchymal tumors recruit immunosuppressive cells to their TME and are refractory to checkpoint blockade, while epithelial tumors recruit CD8+ T cells instead and are sensitive to checkpoint blockade. The mechanism(s) underlying such EMT-mediated immunosuppression remains elusive. Furthermore, the biologic relevance of various immune cells infiltrating epithelial versus mesenchymal tumors requires further understanding. Using a combination of novel epithelial and mesenchymal tumor models arising in syngeneic immunocompetent hosts combined with transcriptomic approaches, we demonstrate that mesenchymal but not epithelial tumors are specifically associated with an immunosuppressive gene signature. Furthermore, while CD8+ T cells play a critical role in regulating tumor progression and responses to checkpoint blockade in epithelial breast tumors, they do not do so in mesenchymal breast tumors. On the contrary, mesenchymal tumors recruit and rely on M2-macrophages to generate an immunosuppressive TME and mount refractory responses to checkpoint blockade. Finally, we demonstrate that mesenchymal tumors utilize a combination of carcinoma cell-intrinsic direct and indirect mechanism(s) to assemble an immunosuppressive TME. This work suggests that epithelial and mesenchymal breast carcinomas have dramatically different immunologic profiles. Thus, the residence of carcinoma cells in the epithelial or mesenchymal states can dictate their corresponding responses to checkpoint blockade immunotherapies. This in turn can therefore have a direct translational impact on breast cancer patients.

Citation Format: Anushka Dongre, Mohammad Rashidian, Elinor Ng Eaton, Ferenc Reinhardt, Sunita Nepal, Hidde Ploegh, Robert Weinberg. Direct and indirect regulators of epithelial-to-mesenchymal transition mediated immunosuppression in breast carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A82.
Abstract A84: Clinical correlates and rationale for PEGylated adenosine deaminase 2 as a novel cancer immunotherapy
Authors: Saatci Ozge , Londoño Luz Marina , Clift Renee , Thompson Curt , LaBarre Mike , Sahin Ozgur , Cekic Caglar
Published: March 2020

Extracellular adenosine accumulates in solid tumors due to direct release or due to dephosphorylation of ATP, ADP, and AMP by extracellular enzymes such as CD39 and CD73. Adenosine signaling promotes metastasis and suppress antitumor immune responses. CD73 expression is associated with poor survival and chemotherapy resistance in triple-negative breast tumors. Adenosine deaminase 2 is a serum protein that catalyzes the deamination of adenosine to inosine. Expression of Adenosine deaminase 2 increases in pathologic conditions such as inflammation and cancer. Here we show that expression of the gene coding for ADA2 (CECR1) in lung and breast tumors is associated with increased expression of gene signatures associated with immune checkpoint blockade responsiveness. Our analyses have also indicated that increased ADA2 expression is associated with favorable outcomes among Her2+ and triple-negative breast cancer patients. Further stratification based on the expression of adenosine-generating enzymes, CD39/CD73, revealed that increased ADA2 expression is particularly associated with increased survival among patients with elevated CD39 and CD73 expression. ADA2 expression is found to be higher in stroma areas of the tumor as compared with tumor cells. Stromal expression but not tumor expression of ADA2 is associated with increased T-cell costimulation and infiltration scores. To support clinical evidence in preclinical models and to show the potential of PEGylated Adenosine Deaminase 2 (PEGADA2) as a novel therapy, we have shown that an engineered high-catalytic activity PEGADA2 variant (PEGADA2HCA) can slow growth of EMT6 syngeneic breast tumors. PEGADA2HCA also completely reversed adenosine inhibition of human T-cell activation. PEGADA2HCA treatment in humanized mice caused a linear dose-exposure relationship after repeat dosing. These results provide a strong rationale for testing PEGylated Adenosine Deaminase 2 alone or in combination in a clinical setting.

Citation Format: Ozge Saatci, Luz Marina Londoño, Renee Clift, Curt Thompson, Mike LaBarre, Ozgur Sahin, Caglar Cekic. Clinical correlates and rationale for PEGylated adenosine deaminase 2 as a novel cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A84.
Abstract A95: Neoantigen sequence similarity to pathogens and commensals determines immune phenotype of cancer samples and patient survival
Authors: Balogh Gergo Mihaly , Koncz Balazs , Asztalos Leo , Kemeny Lajos , Papp Balazs , Pal Csaba , Manczinger Mate
Published: March 2020

The immune system has to destroy pathogens and cancer, while tolerating commensal species and self-proteins. Neoantigens mediate the immune response against tumors. While tumors often carry many different neoantigens, the immune response is ineffective in a number of cases. Accordingly, only a small subset of neoantigens are immunogenic and are able to induce destructive responses. Why is it so? Numerous studies have shown that neoantigens similar to pathogens are more immunogenic. Recently, the role of the commensal flora in antitumor immunity has also come into research focus. We propose that molecular mimicry of cancer neoantigens to certain pathogenic as well as commensal species determines whether immune-mediated destruction or tolerance is formed. Consequently, the amino acid sequences of neoantigens in a given sample determine its immune phenotype and, thus, the survival of cancer patients. We analyzed cancer samples of The Cancer Genome Atlas. We identified neoantigens using established methods, and defined T cell-exposed motifs on neoantigen sequences. We determined the prevalence of these motifs in 1,303 commensal and 183 pathogen species. We report significant and relevant differences in the motif signature of cancer samples belonging to different immune subtypes: i) Inflammatory type samples carried numerous motifs of pathogens, while ii) TGF-beta dominant samples carried motifs of commensal bacteria and pathogens associated with immune suppression. At the same time, iii) neoantigens of immunologically quiet samples contained no or only a few motifs associated with pathogens or the microbiome. We clustered patients based on the motif signature of their cancer samples. Patients in different clusters showed significantly different survival. The effect of motif signature remained significant after controlling for patient demographics and missense mutation count in a Cox model. We conclude that neoantigen sequence signature determines the type of immune response against cancer cells and, thus, patient survival. These modified sequences are able to induce not only immune-mediated destruction, but also immune tolerance, which depends on its similarity to pathogen and commensal species.

Citation Format: Gergo Mihaly Balogh, Balazs Koncz, Leo Asztalos, Lajos Kemeny, Balazs Papp, Csaba Pal, Mate Manczinger. Neoantigen sequence similarity to pathogens and commensals determines immune phenotype of cancer samples and patient survival [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A95.
Abstract A96: Correlation of estrogen receptor beta with immune cells in colon cancer patients
Authors: Topi Geriolda , Lydrup Marie Louise , Sjölander Anita
Published: March 2020

Background: The estrogen receptor β (ERβ) is the predominant ER in the colon mucosa. We have previously reported that high expression of ERβ is independently associated with a better prognosis in patients with colorectal cancer (CRC), and the induction of this receptor in colon cancer cell lines resulted in upregulation of antitumorigenic proteins and downregulation of protumorigenic proteins. In addition, we have also shown that high mast cell density (MCD) was associated with longer survival of CRC patients.

Aim: To investigate the correlations between ERβ, mast cells (MCs), and prostaglandin D2 (PGD2) synthase.

Materials and Methods: A tissue microarray (TMA) of primary CRCs from 314 patients was stained for the expression of ERβ, MCs, and PGD2 synthase. Immunohistochemistry was used to evaluate the staining intensity by using the immunoreactive score (IRS).

Results: Patients with high ERβ expression had higher number of MCs, and there was a significant positive correlation between ERβ and MCs (r = 0.12, P = 0.03). In addition, patients with high ERβ expression had significantly higher activity of PGD synthase, which is produced mainly from MCs and is known to have antitumor effects. Furthermore, a strong, positive, and significant correlation was observed between ERβ expression and PGD synthase (r = 0.42, P < 0.0001).

Future Plans: We aim to correlate the expression of ERβ with other immune cells and factors important for the tumor microenvironment, such are CD3+, CD8+, T-bet, and PDL-1.

Conclusion: High ERβ expression correlates with an antitumor immune prolife in CRC patients. This strengthens our hypothesis that the induction of ERβ expression in CRC patients will potentially improve the disease outcome.

Citation Format: Geriolda Topi, Marie Louise Lydrup, Anita Sjölander. Correlation of estrogen receptor beta with immune cells in colon cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A96.
Abstract A98: Cancer-associated fibroblasts promote immunosuppression by inducing NOX2-expressing monocytic MDSCs in lung squamous cell carcinoma
Authors: Xiang Handan , Ramil Carlo , Hai Josephine , Zhang Chunsheng , Wang Huijun , Watkins Amanda A. , Afshar Roshi , Georgiev Peter , Song Xuelei , Sun Dongyu , Loboda Andrey , Jia Yanlin , Moy Lily Y , Chi An , Brandish Philip E.
Published: March 2020

Cancer-associated fibroblasts (CAFs) are activated fibroblasts that constitute the stromal component in the tumor microenvironment (TME). Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, their roles and potential mechanisms by which they may contribute to immune suppression in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining the TCGA dataset. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. To elucidate additional immunosuppressive mechanisms of CAF-educated MDSCs, we performed proteomic profiling and identified the NADPH oxidase 2 (NOX2) complex to be highly enriched in MDSCs. Pharmacologic inhibition of NOX2 activity in CAF-induced MDSCs restored CD8+ T-cell proliferation. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrates that NOX2 inhibition abrogates the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

Citation Format: Handan Xiang, Carlo Ramil, Josephine Hai, Chunsheng Zhang, Huijun Wang, Amanda A. Watkins, Roshi Afshar, Peter Georgiev, Xuelei Song, Dongyu Sun, Andrey Loboda, Yanlin Jia, Lily Y Moy, An Chi, Philip E. Brandish. Cancer-associated fibroblasts promote immunosuppression by inducing NOX2-expressing monocytic MDSCs in lung squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A98.
Abstract B104: Immunomodulatory effect of 15-hydroxy prostaglandin dehydrogenase in colon cancer
Authors: Satapathy Shakti R. , Sjolander Anita
Published: March 2020

Cyclooxygenase-2, the key player in the biosynthesis of prostaglandin-E2 (PGE2) is often upregulated in colon cancer. In addition, TNF-α, the prime regulator of COX-2 and mPGES-1, also contributes to increased synthesis of PGE2. However, 15-hydroxy prostaglandin dehydrogenase, a tumor suppressor playing major role in PGE2 catabolism, is often downregulated in colon cancer. Interestingly, colon cancer patients expressing high levels of cysteinyl leukotriene receptor-2 (CysLT2R) show good prognosis and therefore, we investigated a potential link between CysLT2R-signaling and 15-PGDH in metastatic colon cancer. Elevation of 15-PGDH expression by LTC4, a CysLT2R ligand, exhibited antitumor activity in colon cancer cells with significant phosphorylation of β-catenin with concurrent activation of CASPASE-3 and dramatic downregulation of TNF-α as well as NFκβ1 mRNA expression in HT-29 and Caco-2 colon cancer cells. The interaction between programmed death ligand 1 (PD-L1) and programmed death receptor 1 (PD-1) of cancer cells and T cells, respectively, leads to T-cell inactivation and tumor escape. We observed that recombinant TNF-α stimulated colon cancer cells expressed high PD-L1 compared to the unstimulated counterparts, which was downregulated by LTC4 treatment. Protumorigenic role and association of tumor-associated macrophage (TAMs) with PD-L1 was also evident with increase in CD-47 mRNA expression. Human (SW480, SW620) and mouse (MC-38) colon cancer cells exposed to conditioned media of chemically differentiated THP-1 and RAW 264.7 cells, respectively, showed abundant expression of PD-L1 in both mRNA and protein level along with CD-47 in mRNA level, which was inhibited by LTC4 stimulation via activation of CysLT2R in a 15-PGDH dependent manner. Hence restoration of 15-PGDH expression through CysLT2R-signaling promotes immunomodulatory effect, indicating a promising therapeutic alternative for metastatic colon cancer with high TAM infiltration.

Citation Format: Shakti R. Satapathy, Anita Sjolander. Immunomodulatory effect of 15-hydroxy prostaglandin dehydrogenase in colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B104.
Abstract B13: Immunogenic chemotherapy alters microbiome-induced T-cell activation
Authors: Smith Peter L. , Dalgleish A.G.
Published: March 2020

Introduction: The efficacy of cancer immunotherapy is influenced by the patient’s intestinal microbiome. The local and systemic effects of the microbiome on immune function are thought to depend upon a combination of microbial metabolites produced collectively from the diverse bacterial species present in the intestine and the activation of intestine-resident immunocytes such as antigen-presenting cells and T lymphocytes. The interactions between the microbiome and the immune response are regulated by Intestinal epithelial cells, which together maintain intestinal immune homeostasis. The oral chemotherapeutics lenalidomide (Len) and cyclophosphamide (CP) are currently licensed for the treatment of numerous cancers. These agents are also potent immune modulators and have been studied alongside checkpoint inhibition (CPI) with promising results. Few studies have sought to investigate the effect of these immune-modulatory agents on immune responses activated by the intestinal microbiome. In this study the immunomodulatory properties of low-dose Len and CP on IEC function and T-cell activation were investigated in combination with microbial TLR ligands and short-chain fatty acids (SFCA), immunogenic metabolites produced by intestinal bacteria.

Results: Both Len and CP altered cytokine expression from IEC in response to stimulation by TLR ligands including lipopolysaccharide, peptidoglycan, and flagellin. Combinations of SCFA modulated the viability and proinflammatory effects of CP on IEC. SCFA and LEN in combination enhanced the expression of antimicrobial peptides from IEC. LEN enhanced IFN-γ and IL-17A expression by CD8+ and CD4+ T-cells. Combinations of LEN and the SCFA acetate further enhanced IFN-γ and IL-17A expression. Cytokines expressed by IEC predispose towards the development of Th2 immune responses; however, in the presence of IEC expressed Th2 skewing cytokines including IL-33 and TSLP, LEN promoted IFN-γ expressing Th1 responses. CP inhibited the ability of the SCFA butyrate to induce T-regulatory cells while Len mitigated the Th1 inhibitory effect of butyrate.

Conclusions: LEN and SCFA synergize to drive Th1 responses while CP mitigate their immune-regulatory properties. Both LEN and CP prime IEC function. Collectively these data provide evidence supporting the ability of low-dose LEN and CP to alter components of intestinal immune network consisting of IEC, Immunocytes, and the microbiome. These findings have implications for the homeostasis of intestinal immunity and the maintenance of the intestinal microbiome during chemotherapeutic treatment. The influence of these agents on the microbiome and CPI immunotherapy is under investigation.

Citation Format: Peter L. Smith, A.G. Dalgleish. Immunogenic chemotherapy alters microbiome-induced T-cell activation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B13.