Tumor Immune Contexture Research Articles

Molecular profiling and characterization of the tumor immune microenvironment (TME) in appendiceal carcinoma (AC).

3622 Background: The rarity of AC presents challenges in understanding disease pathogenesis. We previously showed that AC has higher rates of mutations in KRAS and GNAS and lower rates of TP53, APC, and PIK3CA than CRC. The appendix also has many lymphoid clusters and regulates IgA production in the large bowel, suggesting that AC may be subject to more lymphocytic regulation than CRC. We sought to characterize the molecular profile and TME across AC histopathological types. Methods: AC samples were analyzed by DNA sequencing (592 genes, NextSeq, or whole exome sequencing, NovaSeq), whole transcriptome sequencing (WTS, NovaSeq), and immunohistochemistry (IHC) for molecular profiling, including microsatellite instability (MSI), mismatch repair (MMR), PD-L1 (SP142), and tumor mutational burden (TMB). Microenvironment Cell Population-counter Quantiseq was used to quantify tumor immune contexture using WTS. AC histopathology was derived from pathology reports. Mann-Whitney U and ChiSquare tests were applied as appropriate, with P-values adjusted for multiple comparisons using Benjamini-Hochberg. Results: AC (N = 731) were grouped by histology: 5% goblet cell (GC), 6% high-grade adenocarcinoma (HGA), 14% low grade mucinous (LGM), 33% mucinous adenocarcinoma (MA), 1% pseudomyxoma peritonei (PMP), 6% signet ring cell carcinoma (SRC), and 35% adenocarcinoma not otherwise specified (NOS) (see Table). Age at collection and PD-L1+ (0-5.2%) did not differ by histopathology. Seven patients had dMMR (4 NOS and 3 MA). Median TMB was significantly higher in NOS vs. GC (4 mutations/megabase vs 2, q < 0.001), NOS vs. LGM (4 vs 3, q = 0.048), MA vs. GC (4 vs 2, q < 0.001), and MA vs. LGM (4 vs 3, q = 0.037). Distinct TME patterns were observed in NOS vs. MA (median cell fraction: dendritic cells 0.07 vs 0, q < 0.01; M2 macrophages 0.055 vs 0.042, q = 0.030; natural killer cells 0.034 vs 0.031, q = 0.011; CD4 T cells 0.006 vs 0, q = 0.044) and GC vs. MA (M1 macrophages 0.039 vs 0.057, q = 0.021; dendritic cells 0.012 vs 0, q < 0.01; B cells 0.061 vs 0.050, q = 0.013; M2 macrophages 0.074 vs 0.042, q < 0.01; natural killer cells 0.041 vs 0.031, q = 0.01; and neutrophils 0.017 vs 0.038 q = 0.045). T cell inflamed signature (TIS) and immune-oncology (IO) marker gene expression were higher in HGA and lower in LGM and MA. Somatic mutation rates differed by histopathology (see Table). Conclusions: There is significant heterogeneity in TMB, TME, and mutational profiles across AC histologies. MA has a particularly immune-cold TME shown by lower infiltration of lymphocytes, TIS, and IO gene expression. These findings are critical to identify novel biomarkers and develop new therapeutic strategies for AC. [Table: see text]

Abstract CT197: Profiling of tumor infiltrating T cells in malignant pleural/peritoneal mesothelioma (MPM) and ovarian cancer patients as part of a Phase 1 clinical trial of gavo-cel (TC-210)

Abstract Gavocabtagene autoleucel (gavo-cel; TC-210) is a T cell receptor fusion construct (TRuC) cell therapy directed against mesothelin. As part of the phase 1 dose-escalation clinical trial (NCT03907852), patients with advanced solid tumors expressing mesothelin were administered a single infusion of gavo-cel. Thirty-two patients with late-stage disease (23 MPM, 8 ovarian and 1 cholangiocarcinoma) received gavo-cel at doses ranging from 5 × 107 - 5 × 108 TRuC cells/m2. The disease control rate (defined as achieving a response by RECISTv1.1 criteria or having stable disease [SD] for at least 12 weeks post-infusion) was 77%. Dose level 3 (1 × 108 TRuC cells/m2 following lymphodepletion with cyclophosphamide and fludarabine) was selected as the Recommended Phase 2 Dose (RP2D) for the ongoing phase 2 portion of the trial. Spatial distribution of immune cells, particularly T cells, in the tumor and stromal regions of tumor biopsies has been shown to identify patients that may respond to immunotherapies. To understand the tumor immune contexture, we profiled the T cell compartment in biopsies from five patients collected prior to and eight weeks post treatment using multiplexed immunofluorescence. We observed that the MPM patient with a partial response (PR) had a greater number of T cells at baseline in tumor biopsies when compared with the other SD patients. Notably, this patient with a PR, had a 3.6-fold increase in CD3+CD8+ T cell infiltrates into the tumor bed. In contrast, 2 SD patients with MPM had a minimal increase of T cells in the tumor compartment at 8 weeks post infusion. The density of TILs 8 weeks post infusion was much lower in the SD patients compared to the PR subject, indicating a potentially insufficient number of infiltrating T cells. Interestingly, 2/2 SD patients with ovarian cancer had a decrease in CD3+CD8+ T cells infiltrating into the tumor, consistent with an immune exclusion phenotype. The PR patient with MPM had minimal increase in PD-L1 and CD155 (a ligand for the inhibitory receptor, TIGIT) from negligible levels at baseline, whereas both SD patients with MPM had marked increases in PD-L1 and CD155, indicating potential resistance mechanisms. In summary, this preliminary data suggests although CD8+ TILs are increased following treatment with gavo-cel, the tumor regions in patients with SD show evidence of immune surveillance escape via PD-L1 and CD155 upregulation, thus acting as putative mechanisms of resistance. However, as seen in the PR patient with MPM there appears to be a critical number of TILs associating with response. We are currently evaluating the tumor immune contexture in phase 2, focusing on differences across tumor types with regards to TIL content, T cell influx, and correlations with response. We will also explore additional markers such as PD-1 and TIGIT, to profile the exhaustion state of immune cells and their spatial relationship with infiltrating gavo-cel T cells. Citation Format: Munisha Smalley, Michael Ross, Samantha F. Fowler, Lauren McLaughlin, Erin Jeter, Kevin Zikaras, Philip D. Bardwell, Alfonso Quintás-Cardama, Robert Tighe. Profiling of tumor infiltrating T cells in malignant pleural/peritoneal mesothelioma (MPM) and ovarian cancer patients as part of a Phase 1 clinical trial of gavo-cel (TC-210) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT197.

Abstract 5476: Veracyte Biopharma Atlas for colorectal cancer: Combining multi-parameter approach and machine learning to capture the complexity of the tumor immune contexture

Abstract (a) Understanding the immune contribution has deeply modified the standard of care of patients, especially with the advent of immunotherapies (ITs). However, ITs are still efficient only on a minority of the patients, especially for colorectal cancer (CRC), a high-incidence cancer. One hypothesis to explain the heterogeneity of the response to IT is that the presence or absence of immune factors within the tumor micro-environment (TME) may critically impact the efficacy of treatments. In this context, a deeper molecular understanding of the TME could be key to stratify patients. Here, we describe a proof of concept of the Veracyte Biopharma Atlas for CRC based on a multi-omics approach which integrates a unique range of assays allowing to decipher the immune contexture of tumors and offer a personalized and dynamic “fingerprint” of the interactions between the tumor and the immune system. (b) Veracyte Biopharma Atlas characterizes the tumor microenvironment by analyzing information from 3 different modalities: (i) proteomics (Brightplex® multiplex immunohistochemistry panels), (ii) transcriptomics (RNA sequencing) and (iii) genomics (somatic mutations by exome sequencing). This deep characterization of the tumor and its micro-environment requires the integration of thousands of features. The innovative Veracyte Biopharma Atlas approach relies on linear (multimodal factor analysis) and non-linear (self-organizing map) approaches with graphical representation of the data to integrate this multi-omics information. (c) Gene expression profiling, genomics analysis and proteomics by multiplex spatial technology were performed on 47 CRC FFPE samples to characterize the immune contexture of this disease. Multimodal analysis was used to derive a unique atlas map highlighting clusters distinguished by complex features and their immune tumor contexture. Indeed, as examples, the atlas map allows the identification of a group of patients displaying high immune cell infiltration, including T cell and macrophages. They are also characterized by an immune genes signature. By contrast, another group of patients is poorly infiltrated by immune cells but exhibits an elevated M2/M1 ratio, favorable to tumor development. (d) This proof of concept lays the foundations of an atlas for CRC that will be extended to samples associated with clinical data to build a map of the disease. Later-on, by projecting data from new specimens onto this referential map, it will become possible to establish an Immunogram of the tumor microenvironment of individual patients to define the most appropriate treatment but also facilitate the development of new therapies. Citation Format: Laurent Vanhille, Alboukadel Kassambara, Chafik Hamdad, Margaux Mercadal, Alexia Papadopoulos, Pernelle Outters, Théo Vasse, Vanina Leca, Jérôme Galon, Jacques Fieschi. Veracyte Biopharma Atlas for colorectal cancer: Combining multi-parameter approach and machine learning to capture the complexity of the tumor immune contexture. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5476.

Tumor immune contexture predicts recurrence after prostatectomy and efficacy of androgen deprivation and immunotherapy in prostate cancer

BackgroundProstate cancer is one of the most common cancers in men with notable interpatient heterogeneity. Implications of the immune microenvironment in predicting the biochemical recurrence-free survival (BCRFS) after radical prostatectomy and the efficacy of systemic therapies in prostate cancer remain ambiguous.MethodsThe tumor immune contexture score (TICS) involving eight immune contexture-related signatures was developed using seven cohorts of 1120 patients treated with radical prostatectomy (training: GSE46602, GSE54460, GSE70769, and GSE94767; validation: GSE70768, DKFZ2018, and TCGA). The association between the TICS and treatment efficacy was investigated in GSE111177 (androgen deprivation therapy [ADT]) and EGAS00001004050 (ipilimumab).ResultsA high TICS was associated with prolonged BCRFS after radical prostatectomy in the training (HR = 0.32, 95% CI 0.24–0.45, P < 0.001) and the validation cohorts (HR = 0.45, 95% CI 0.32–0.62, P < 0.001). The TICS showed stable prognostic power independent of tumor stage, surgical margin, pre-treatment prostatic specific antigen (PSA), and Gleason score (multivariable HR = 0.50, 95% CI 0.39–0.63, P < 0.001). Adding the TICS into the prognostic model constructed using clinicopathological features significantly improved its 1/2/3/4/5-year area under curve (P < 0.05). A low TICS was associated with high homologous recombination deficiency scores, abnormally activated pathways concerning DNA replication, cell cycle, steroid hormone biosynthesis, and drug metabolism, and fewer tumor-infiltrating immune cells (P < 0.05). The patients with a high TICS had favorable BCRFS with ADT (HR = 0.25, 95% CI 0.06–0.99, P = 0.034) or ipilimumab monotherapy (HR = 0.23, 95% CI 0.06–0.81, P = 0.012).ConclusionsOur study delineates the associations of tumor immune contexture with molecular features, recurrence after radical prostatectomy, and the efficacy of ADT and immunotherapy. The TICS may improve the existing risk stratification systems and serve as a patient-selection tool for ADT and immunotherapy in prostate cancer.

Targeted Depletion of Hyaluronic Acid Mitigates Murine Breast Cancer Growth.

Simple SummaryBreast cancer often contains excessive stromal matrix components that serve as a barrier to therapy. One such component is hyaluronic acid (HA), which also plays important roles in tumor progression and spread. In this study, we engineered an attenuated Salmonella typhimurium secreting a bacterial hyaluronidase (YS-HAse) and show that it degrades HA within murine breast tumors, resulting in enhanced tumor permeability and growth control. YS-HAse represents a promising new therapeutic approach that could also be used alongside current treatments to improve their delivery and effectiveness.Hyaluronic acid (HA) is highly elevated in breast cancers compared to normal breast tissue and is associated with increased tumor aggressiveness and poor prognosis. HA interacts with cell-trafficking CD44 receptors to promote tumor cell migration and proliferation and regulates both pro- and anti-inflammatory cytokine production through tumor-associated macrophages. The highly negative charge of HA enables its uptake of vast amounts of water that greatly increases the tumor interstitial fluidic pressure, which, combined with the presence of other extracellular matrix components such as collagen, results in tumor stroma with abnormal vasculature, hypoxia, and increased drug resistance. Thus, the degradation of HA in breast cancer may attenuate growth and improve permeability to anticancer agents. Previous methods to deplete tumor HA have resulted in significant off-tumor effects due to the systemic use of mammalian hyaluronidases. To overcome this, we developed a hyaluronidase-secreting Salmonella typhimurium (YS-HAse) that specifically and preferentially colonizes tumors to deplete HA. We show that the systemic administration of YS-HAse in immunocompetent murine models of breast cancer enhances tumor perfusion, controls tumor growth, and restructures the tumor immune contexture. These studies highlight the utility of YS-HAse as a novel microbial-based therapeutic that may also be combined with existing therapeutic approaches.

The Immune Contexture of Liposarcoma and Its Clinical Implications.

Simple SummaryLiposarcomas (LPS) are malignancies arising from adipose tissue. Based on the histological appearance, five subtypes are distinguished: well-differentiated LPS, dedifferentiated LPS (DDLPS), myxoid LPS (MLPS), pleomorphic LPS, and myxoid pleomorphic LPS. Immune cells can infiltrate the tumor microenvironment (TME) of LPS and can either promote an efficient antitumor immune response or mediate immunosuppression paving the way for immune evasion of the tumor. The LPS subtypes display different TME characteristics and vary in regard to immune cell infiltration, ranging from the generally lowly infiltrated MLPS to the highly infiltrated DDLPS where immunological determinants predict response to novel antibody-based immunotherapy. Thus, immune cells in the TME can significantly affect response to therapy, disease progression, and patient survival. This review aims to decipher the immune contexture of LPS as well as its clinical association and highlights differences between the LPS subtypes that may have implications for the design of novel treatment strategies.Liposarcomas (LPS) are the most frequent malignancies in the soft tissue sarcoma family and consist of five distinctive histological subtypes, termed well-differentiated LPS, dedifferentiated LPS (DDLPS), myxoid LPS (MLPS), pleomorphic LPS, and myxoid pleomorphic LPS. They display variations in genetic alterations, clinical behavior, and prognostic course. While accumulating evidence implicates a crucial role of the tumor immune contexture in shaping the response to anticancer treatments, the immunological landscape of LPS is highly variable across different subtypes. Thus, DDLPS is characterized by a higher abundance of infiltrating T cells, yet the opposite was reported for MLPS. Interestingly, a recent study indicated that the frequency of pre-existing T cells in soft tissue sarcomas has a predictive value for immune checkpoint inhibitor (CPI) therapy. Additionally, B cells and tertiary lymphoid structures were identified as potential biomarkers for the clinical outcome of LPS patients and response to CPI therapy. Furthermore, it was demonstrated that macrophages, predominantly of M2 polarization, are frequently associated with poor prognosis. An improved understanding of the complex LPS immune contexture enables the design and refinement of novel immunotherapeutic approaches. Here, we summarize recent studies focusing on the clinicopathological, genetic, and immunological determinants of LPS.

OA09.06 Vascular Leakage Promotes Immunosuppression and is Associated with Reduced Patient Survival in Non-Small Cell Lung Cancer

Vascular hyperpermeability (leakage) facilitates tumor progression, metastasis, and limited delivery of therapeutics by fostering a hypoxic tumor microenvironment. Recently, pharmacological inhibition of vascular leakage enhanced tumor infiltration and activity of cytotoxic (CD8+) T-cells when used in combination with anti-PD1 blockade. However, the influence of vascular leakage on the tumor immune contexture and prognostic implications in human cancer patients remains to be investigated.

Tumor immune contexture is a determinant of anti-CD19 CAR T cell efficacy in large B cell lymphoma

Axicabtagene ciloleucel (axi-cel) is an anti-CD19 chimeric antigen receptor (CAR) T cell therapy approved for relapsed/refractory large B cell lymphoma (LBCL) and has treatment with similar efficacy across conventional LBCL subtypes. Toward patient stratification, we assessed whether tumor immune contexture influenced clinical outcomes after axi-cel. We evaluated the tumor microenvironment (TME) of 135 pre-treatment and post-treatment tumor biopsies taken from 51 patients in the ZUMA-1 phase 2 trial. We uncovered dynamic patterns that occurred within 2 weeks after axi-cel. The biological associations among Immunoscore (quantification of tumor-infiltrating T cell density), Immunosign 21 (expression of pre-defined immune gene panel) and cell subsets were validated in three independent LBCL datasets. In the ZUMA-1 trial samples, clinical response and overall survival were associated with pre-treatment immune contexture as characterized by Immunoscore and Immunosign 21. Circulating CAR T cell levels were associated with post-treatment TME T cell exhaustion. TME enriched for chemokines (CCL5 and CCL22), γ-chain receptor cytokines (IL-15, IL-7 and IL-21) and interferon-regulated molecules were associated with T cell infiltration and markers of activity. Finally, high density of regulatory T cells in pre-treatment TME associated with reduced axi-cel–related neurologic toxicity. These findings advance the understanding of LBCL TME characteristics associated with clinical responses to anti-CD19 CAR T cell therapy and could foster biomarker development and treatment optimization for patients with LBCL.

Abstract 253: Analyses of tumor-specific T cell dynamics and tumor immune contexture in microsatellite instability-high cancers in response to first-line therapy

Abstract Microsatellite instability-high (MSI-H) phenotype arises from defective DNA repair mechanisms and is found in many malignancies, including colorectal and endometrial cancers. Due to high neoantigen loads derived from tumor-specific mutations, evidence of tumor-infiltrating T cells and high response rates to checkpoint blockade (30-60%), MSI-H cancers can be leveraged to study the features of neoantigen-induced anti-tumor immunity in response to therapy and disease outcome. Previously we identified a set of recurrent frameshift (fs) mutations in MSI-H cancers that yielded immunogenic polyepitope neoantigens (neoAgs) eliciting CD8+ T cell responses. Here we investigate whether these shared fs-neoAgs engendered spontaneous T cell responses in MSI-H colorectal and endometrial cancer patients. Using peripheral blood mononuclear cells (PBMCs), we evaluated the frequency of the fs-neoAg-specific T cells by ex vivo IFN-γ ELISPOT upon fs-peptide stimulation. Additionally, we isolated naïve and memory T cells from MSI-H patient PBMCs and expanded fs-neoAg-specific T cells within each subset. Fs-neoAg-induced effector cytokine (IFN-γ, TNF-α) production was measured by flow cytometry and was detected only in the memory subset, supporting the hypothesis that shared fs-neoAgs prime CD8+ T cells in vivo to generate clonally-expanded memory T cells in MSI-H patients. To further evaluate the clonal expansion of fs-neoAg-specific T cells in MSI-H cancer patients, we identified fs-neoAg-specific T cell receptors (TCRs). T cell lines enriched for fs-neoAg-specific T cells were generated from patient PBMCs and sequenced for variable β chains of TCRs. These fs-neoAg-specific TCRs were monitored in the blood and tumor tissues, including those from primary and recurrent sites. Finally, to determine the spatial relationships between the infiltrating immune cells and the malignant cells at single cell resolution, we performed multiplexed immunohistochemistry using CODEX technology. A panel of 35 markers was used to evaluate the myeloid (HLA-DR, CD11c, CD14, CD68, CD206) and T (CD4, CD8, Foxp3) cell subsets, the activation (CD107a, CD69, Ki67) and exhaustion (PD-1, TIM3, LAG3) status of T cells, T cell states (TCF-1, TOX, PD-1) and the presence of tertiary lymphoid structures/antigen presenting cell niches (CD19, CD20, HLA-DR) in the tumor microenvironment of primary or recurrent MS-stable and MSI-H tumors from endometrial cancer patients. We also investigated potential immune escape mechanisms in MSI-H tumors, such as the loss of B2M and MHC-I expression. This study will provide insights into the quality and quantity of shared anti-tumor T cells within the periphery and tumor tissues and will help identify the immune correlates of response or resistance to therapy in MSI-H cancer patients. Citation Format: Cansu Cimen Bozkus, Mona Saleh, Rachel Brody, Stephanie V. Blank, Nina Bhardwaj. Analyses of tumor-specific T cell dynamics and tumor immune contexture in microsatellite instability-high cancers in response to first-line therapy [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 253.

Abstract 1708: Novel analysis method for in situ spatial phenotyping of cell populations in multimarker imagery

Abstract Spatial biology is the study of tissues within their own context. This has become increasingly important in immuno-oncology because of the need to understand complex tumor microenvironment architecture, especially regarding the immune contexture in and around solid tumors. The main method for understanding the spatial biology of tumors has been the use of multi-marker staining and multiplexed imaging methods in FFPE sections. This imaging data is used to segment the tissue into relevant compartments (tumor, stroma, etc.) and perform multiplex phenotyping of the cells. For highplex data (10+ markers), phenotyping has typically used an unsupervised clustering algorithm. This was a good starting point, but has many limitations, including a reliance on having a sufficient population of cells of each type, variability sample to sample, and no means of differentiating between biologically relevant or impossible phenotypes. Improving cellular phenotyping for highplex samples is a requirement for many of the new multiplexed imaging modalities that are being used. We have developed a novel method of phenotyping cells in situ in multiplexed imaging data that involves the biological concept of dividing markers into two user-defined categories: lineage markers and functional markers. Lineage markers are used to identify cellular phenotypes, while functional markers are measured afterwards for their expression levels in across each phenotype and in each individual cell. Lineage markers (e.g., CD45, α-SMA, CD8, cytokeratin, CD20, etc.) define the phenotype of the cell and are essentially binary; they are either present or not present with the correct biodistribution. Functional markers (PD-1, PD-L1, Ki67, etc.), on the other hand, are quantitatively measured in each cell. This new method first uses a supervised classification methodology using lineage markers to find cells with expected phenotypes. In a second step, the levels of functional markers are measured for each cell. Unexpected phenotypes in unclassified cells can then be explored. These data can then be used for downstream spatial/distance/hotspot analysis. During the setup of the analysis the user can explore and QC the data using an interactive data exploration tool which allows for a constant algorithm supervision and correction. Here we present data of the novel workflow for 7-plex (Vectra) and 20+-plex (Hyperion/CODEX) data and found that the new method increased the efficiency of setting up complex analysis workflows and improved analysis results. Fewer non-biological phenotypes were found, cell classifications were improved compared to auto-clustering, while still exploring unexpected phenotypes. Interactive exploration of the cell populations enabled quicker validation of results and deeper understanding of the tumor immune contexture. This new method will improve analyses of the next generation of highplex imaging datasets. Citation Format: Fabian T. Schneider, James R. Mansfield, Alessandro S. Massaro, Simon Haastrup, Rasmus A. Lyngby, Andreas Hussing, Johan Dore Hansen, Jeppe Thagaard. Novel analysis method for in situ spatial phenotyping of cell populations in multimarker imagery [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 1708.

Abstract 2082: Obesity dependent benefits of metformin for improved anti-tumor immunity and outcomes in non-small cell lung cancer

Abstract Metformin, the widely used and studied diabetes drug, has been reported to have anti-cancer effects. However, in retrospective clinical studies, the survival benefit associated with metformin use in cancer patients is often modest. Consequently, the design and conduct of sufficiently powered prospective clinical trials aimed at exploring this drug’s anti-tumor potential are hampered by the absence of predictive biomarkers. Testing the notion that specific patient characteristics such as obesity may impact the relative potency of metformin’s effects on lung cancer survival, we analyzed the patient outcomes of 756 non-small cell lung cancer (NSCLC) cases with early- (stage I/II, n = 490) or late-stage disease (stage III/IV, n = 266) that either did or did not use metformin (n = 121 and 635, respectively) while not receiving neoadjuvant therapy. We found that metformin use is associated with improved overall and recurrence free survival only in overweight and obese patients defined by a body mass index (BMI) greater than 25 kg/m2. Corroborative findings obtained from immunocompetent mouse lung cancer models aligned with these observations. In these studies, despite supporting accelerated growth of both primary and metastatic tumors compared to their normal weight counterparts, animals subjected to diet induced obesity (DIO) experienced significantly reduced tumor development upon metformin treatment. In contrast, the drug had little-to-no effect on tumor burden in normal weight controls. Furthermore, while flow cytometric evaluation of obese mouse-derived tumors identified several previously described mediators of immune dysfunction previously described in animals with DIO (e.g., widespread up-regulation of the immune checkpoint factors PD-1 and Lag3, elevated suppressor cell proportions and activity), metformin treatment was associated with their reversal specifically in the tumors of obese mice. Interestingly, the drug had little effect on the tumor immune contexture of normal weight mice, and gene expression analysis failed to reveal considerable alterations in canonical cancer pathways. Thus, our preclinical and clinical studies suggest that the anti-diabetic drug metformin has an anti-cancer effect in NSCLC that is restricted to overweight individuals and reflects its potential to direct favorable, context-specific immune reprogramming. This work also provides the rationale for using high BMI as a predictive biomarker of the anti-cancer effect of metformin in lung cancer. Citation Format: Joseph Barbi, Randall J. Smith, Robert Zollo, Stephanie N. Sass, Deschana Washington, Cara Petrucci, Aravind Srinivasan, Eric Kannisto, Santosh Patnaik, Sai Sai Yendamuri. Obesity dependent benefits of metformin for improved anti-tumor immunity and outcomes in non-small cell lung cancer [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 2082.

Abstract 1235: Presence of TLS and combined high densities of PD-L1+ macrophages &amp; CD8+ T cells predict long-term overall survival for patients with advanced NSCLC treated with durvalumab

Abstract Introduction: Predictive biomarkers of anti‒PD-(L)1 therapies have largely focused on the tumor - T cell axis where tumor cell PD-L1 expression has demonstrated its clinical utility in predicting overall survival (OS) in patients with advanced non-small cell lung cancer (NSCLC). Although, other immune cell subsets were shown to be associated with clinical efficacy, their relative impact and combined effect in predicting improved long-term survival warrant further investigation. Using computational image analysis of multiplex immunofluorescence (mIF) and immunohistochemistry (IHC) immune marker panels, we sought to identify single and combined biomarkers of the tumor immune contexture in association with long-term OS in advanced NSCLC patients treated with Durvalumab. Methods: Pre-treatment tumor samples from advanced NSCLC patients (n = 210) enrolled in durvalumab nonrandomized phase 1/2 trial (10 mg/kg Q2W, CP1108/NCT01693562), were stained using IHC and 6-marker mIF panels to detect markers of immune cells, cell functional state and tertiary lymphoid structure (TLS) (PD-L1, CD8, PD-1, Ki67, CD68, CD20, CD1c, NKp46, CD66b, ICOS, FOXP3). Cell marker density (cells/mm2), distribution and proximity were quantified and analyzed in association with overall survival. Results: Computational image analysis of the tumor immune contexture revealed a greater immune inflamed phenotype, both innate (macrophages, dendritic cells) and adaptive (T and B cells), in NSCLC patients with long OS &amp;gt;2 years compared to those with short OS &amp;lt; 1 year (fold change &amp;gt; 2, p &amp;lt; 0.0001). Patient subgroup with high density of individual immune subsets show a median OS (mOS) of 10-20 months (p &amp;lt; 0.01 high vs. low subgroups) while combined markers of innate and adaptive immune cells show an improved mOS &amp;gt; 2 years (p &amp;lt; 0.001). Specifically, among the key findings, combined biomarkers of CD68+ PD-L1+ macrophages and CD8+ T cells predicts for a significant increase in OS for patients with high vs low marker density (HR = 0.21, 95%CI 0.12 - 0.39, p &amp;lt;10-7; mOS 39.5 months [high], 6.5 months [low]). Whereas, in patients with high density of either single biomarkers CD68+ PD-L1+ macrophage or CD8+ T cells mOS is 20.2 or 18.4 months respectively. Moreover, high density of CD20+ B cells, reflective of presence of TLS, associates with improved OS (mOS NR [high], 11 months [low], p = 0.003). In addition, TLS enriched tumors show an increased level of macrophages expressing PD-L1 in synapsis with CD8+ T cells (activated PD1+ or proliferative Ki67+ T cells). Conclusion: These findings demonstrate the importance of both tertiary lymphoid structure and high pre-existing innate-adaptive immunity in driving long-term overall survival of durvalumab-treated patients with NSCLC and highlight the need for the development of multiparametric predictive biomarkers beyond tumor-T cell axis. Citation Format: Lina Meinecke, Jorge Blando, Thomas Herz, Michael Surace, Thomas Padel, Monica Azqueta Gavaldon, Harald Hessel, Farzad Sekhavati, Megha Saraiya, Anmarie Boutrin, Karma Da Costa, Jaime Rodriguez Canales, Ashok Gupta, Carl Barrett, Zachary Aaron Cooper, Ikbel Achour. Presence of TLS and combined high densities of PD-L1+ macrophages &amp; CD8+ T cells predict long-term overall survival for patients with advanced NSCLC treated with durvalumab [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 1235.

Immune Microenvironment of Muscular-Invasive Urothelial Carcinoma: The Link to Tumor Immune Cycle and Prognosis.

In this study, we investigated the relationship between the tumor immune microenvironment (TIME), histological differentiation and hypoxia in patients with muscular-invasive urothelial carcinomas (MIUC) after radical cystectomy. Forty-two cases of pT2-3N0M0 MIUCs underwent clinical, histological and immunohistochemical evaluation by counting CD8+, FOXP3+, CD68+, CD163+ cells and polymorphonuclear leukocytes (PMN) in intra-tumoral and peritumoral areas, assessing PD-L1 and GLUT1 expression for defining the impact of tumor immune contexture on patients’ outcomes. Five-year survival rates and overall survival were calculated. Most of the MIUCs demonstrated the immune-desert or immune-excluded TIME, reflecting altered mechanisms of T-cells’ activation or traffic into tumors. Tumor immune contexture was closely related to histological differentiation. CD8+ cells were scant in MIUCs with papillary and squamous differentiation, while basal-like or mesenchymal-like histological differentiation was associated with increased density of CD8+ cells. A high rate of PD-L1 expression (47.6%) was not related to immune cell infiltration. M2-macrophages predominated under CD8+ lymphocytes. The abundance of PMN and CD163+ macrophages in MIUCs was associated with high GLUT1 expression. CD8+, CD68+, FOXP3+ cells and PD-L1 status did not affect patients’ outcomes, while high CD163+ density and PMN infiltration were associated with the unfavorable outcome of patients with MIUC. These data drive the hypothesis that in MIUC, immune escape mechanisms are shifted towards the role of the innate immunity cells rather than CD8+ lymphocytes’ functioning.

128: DECIPHERING THE IMMUNOLOGICAL DETERMINANTS OF RESPONSE TO NEOADJUVANT CHEMO-RADIATION IN ESOPHAGEAL ADENOCARCINOMA

Abstract Background and aim The standard therapy for esophageal adenocarcinoma (EAC) is pre-operative neoadjuvant chemo-radiation (NACR) and surgery. Less that 30% of patients achieve a pathological complete response (pCR) and increased 5-year survival after NACR. Understanding the mechanisms of response to NACR is pivotal to better stratify patients and inform the design of more efficacious therapies. Cancer progression partly depends on defective immune surveillance, and the presence of tumor infiltrating T lymphocytes (TILs) is associated with better prognosis of several malignancies. We hypothesize that NACR induces immunogenic cell death that stimulates immune responses against tumor neoantigens (TNAs), derived from cancer somatic mutations, in the responders. We thus compared responders and non-responders for tumor immune contexture, immune molecular pathways, circulating immune markers and TNAs-specific T cells responses. Methods Pre-NACR tumor biopsies prospectively collected from consecutive EAC patients were subjected to DNA whole exome and RNA sequencing, high dimensional (HD) multiparametric flow cytometry and immune contexture analysis by histology and by proteome digital spatial profiling (n = 53 targets), while their peripheral blood mononuclear cells and plasma collected pre- and post- NACR were analyzed by HD flow cytometry and cytokine multiplexing assay respectively. Results Treatment-naïve EACs of NACR responders showed higher TNA load and enriched immune signatures compared to non-responders. Moreover, pre-treatment EACs of responders demonstrate augmented effector T cell infiltration and reduced Treg cells and M2-like pro-tumoral macrophages. High M2-like macrophages score associated with lower overall survival. Increased frequencies of circulating myeloid suppressor populations were detected both at baseline and after NACR in non-responder patients. These observations were associated with circulating levels of n = 30 cytokines/chemokines and with spatial expression of immune markers in tumor biopsies. Conclusion Collectively, these results support pre-existing immune-mediated mechanisms of anti-tumor response stimulated by NACR in the fraction of responder EAC patients, suggesting possible ways to stratify patients and direct them to more tailored neoadjuvant and/or adjuvant treatments, possibly in combination with personalized immunotherapy approaches.

B cells and tertiary lymphoid structures as determinants of tumour immune contexture and clinical outcome.

B cells are a major component of the tumour microenvironment, where they are predominantly associated with tertiary lymphoid structures (TLS). In germinal centres within mature TLS, B cell clones are selectively activated and amplified, and undergo antibody class switching and somatic hypermutation. Subsequently, these B cell clones differentiate into plasma cells that can produce IgG or IgA antibodies targeting tumour-associated antigens. In tumours without mature TLS, B cells are either scarce or differentiate into regulatory cells that produce immunosuppressive cytokines. Indeed, different tumours vary considerably in their TLS and B cell content. Notably, tumours with mature TLS, a high density of B cells and plasma cells, as well as the presence of antibodies to tumour-associated antigens are typically associated with favourable clinical outcomes and responses to immunotherapy compared with those lacking these characteristics. However, polyclonal B cell activation can also result in the formation of immune complexes that trigger the production of pro-inflammatory cytokines by macrophages and neutrophils. In complement-rich tumours, IgG antibodies can also activate the complement cascade, resulting in the production of anaphylatoxins that sustain tumour-promoting inflammation and angiogenesis. Herein, we review the phenotypic heterogeneity of intratumoural B cells and the importance of TLS in their generation as well as the potential of B cells and TLS as prognostic and predictive biomarkers. We also discuss novel therapeutic approaches that are being explored with the aim of increasing mature TLS formation, B cell differentiation and anti-tumour antibody production within tumours.

The Radiosensitivity Index Gene Signature Identifies Distinct Tumor Immune Microenvironment Characteristics Associated With Susceptibility to Radiation Therapy

Radiation therapy (RT) is a mainstay of cancer care, and accumulating evidence suggests the potential for synergism with components of the immune response. However, few data describe the tumor immune contexture in relation to RT sensitivity. To address this challenge, we used the radiation sensitivity index (RSI) gene signature to estimate the RT sensitivity of >10,000 primary tumors and characterized their immune microenvironments in relation to the RSI. We analyzed gene expression profiles of 10,469 primary tumors (31 types) within a prospective tissue collection protocol. The RT sensitivity of each tumor was estimated by the RSI and respective distributions were characterized. The tumor biology measured by the RSI was evaluated by differentially expressed genes combined with single sample gene set enrichment analysis. Differences in the expression of immune regulatory molecules were assessed and deconvolution algorithms were used to estimate immune cell infiltrates in relation to the RSI. A subset (n=2368) of tumors underwent DNA sequencing for mutational frequency characterization. We identified a wide range of RSI values within and across various tumor types, with several demonstrating nonunimodal distributions (eg, colon, renal, lung, prostate, esophagus, pancreas, and PAM50 breast subtypes; P < .05). Across all tumor types, stratifying RSI at a tumor type-specific median identified 7148 differentially expressed genes, of which 146 were coordinate in direction. Network topology analysis demonstrates RSI measures a coordinated STAT1, IRF1, and CCL4/MIP-1β transcriptional network. Tumors with an estimated high sensitivity to RT demonstrated distinct enrichment of interferon-associated signaling pathways and immune cell infiltrates (eg, CD8+ T cells, activated natural killer cells, M1-macrophages; q < 0.05), which was in the context of diverse expression patterns of various immunoregulatory molecules. This analysis describes the immune microenvironments of patient tumors in relation to the RSI gene expression signature.

The Effect of Hypoxia and Hypoxia-Associated Pathways in the Regulation of Antitumor Response: Friends or Foes?

Hypoxia is an environmental stressor that is instigated by low oxygen availability. It fuels the progression of solid tumors by driving tumor plasticity, heterogeneity, stemness and genomic instability. Hypoxia metabolically reprograms the tumor microenvironment (TME), adding insult to injury to the acidic, nutrient deprived and poorly vascularized conditions that act to dampen immune cell function. Through its impact on key cancer hallmarks and by creating a physical barrier conducive to tumor survival, hypoxia modulates tumor cell escape from the mounted immune response. The tumor cell-immune cell crosstalk in the context of a hypoxic TME tips the balance towards a cold and immunosuppressed microenvironment that is resistant to immune checkpoint inhibitors (ICI). Nonetheless, evidence is emerging that could make hypoxia an asset for improving response to ICI. Tackling the tumor immune contexture has taken on an in silico, digitalized approach with an increasing number of studies applying bioinformatics to deconvolute the cellular and non-cellular elements of the TME. Such approaches have additionally been combined with signature-based proxies of hypoxia to further dissect the turbulent hypoxia-immune relationship. In this review we will be highlighting the mechanisms by which hypoxia impacts immune cell functions and how that could translate to predicting response to immunotherapy in an era of machine learning and computational biology.

Tumor promoting roles of IL-10, TGF-β, IL-4, and IL-35: Its implications in cancer immunotherapy.

Cytokines play a critical role in regulating host immune response toward cancer and determining the overall fate of tumorigenesis. The tumor microenvironment is dominated mainly by immune-suppressive cytokines that control effector antitumor immunity and promote survival and the proliferation of cancer cells, which ultimately leads to enhanced tumor growth. In addition to tumor cells, the heterogeneous immune cells present within the tumor milieu are the significant source of immune-suppressive cytokines. These cytokines are classified into a broad range; however, in most tumor types, the interleukin-10, transforming growth factor-β, interleukin-4, and interleukin-35 are consistently reported as immune-suppressive cytokines that help tumor growth and metastasis. The most emerging concern in cancer treatment is hijacking and restraining the activity of antitumor immune cells in the tumor niche due to a highly immune-suppressive environment. This review summarizes the role and precise functions of interleukin-10, transforming growth factor-β, interleukin-4, and interleukin-35 in modulating tumor immune contexture and its implication in developing effective immune-therapeutic approaches.Concise conclusionRecent effort geared toward developing novel immune-therapeutic approaches faces significant challenges due to sustained mutations in tumor cells and a highly immune-suppressive microenvironment present within the tumor milieu. The cytokines play a crucial role in developing an immune-suppressive environment that ultimately dictates the fate of tumorigenesis. This review critically covers the novel aspects of predominant immune-suppressive cytokines such as interleukin-10, transforming growth factor-β, interleukin-4, and interleukin-35 in dictating the fate of tumorigenesis and how targeting these cytokines can help the development of better immune-therapeutic drug regimens for the treatment of cancer.

Enabling CAR-T cells for solid tumors: Rage against the suppressive tumor microenvironment.

Adoptive T cell therapies based on chimeric antigen receptors (CAR-T) are emerging as genuine therapeutic options for the treatment of hematological malignancies. The observed clinical success has not yet been extended into solid tumor indications as a result of multiple factors including immunosuppressive features of the tumor microenvironment (TME). In this context, an emerging strategy is to design CAR-T cells for the elimination of defined cellular components of the TME, with the objective of re-shaping the tumor immune contexture to control tumor growth. Relevant cell components that are currently under investigation as targets of CAR-T therapies include the tumor vasculature, cancer-associated fibroblasts (CAFs), and immunosuppressive tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs). In this review, we recapitulate the rapidly expanding field of CAR-T cell therapies that directly target cellular components within the TME with the ultimate objective of promoting immune function, either alone or in combination with other cancer therapies.

TAMI-65. FASN-MEDIATED LIPID SYNTHESIS HAMPERS ANTI-CANCER IMMUNITY OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most aggressive type of primary brain tumor in adults. Radiation therapy (RT) is an essential modality for GBM treatment and is recognized to stimulate anti-cancer immunity by at least generating type I interferon (IFN-I) responses. However, RT also exacerbates potent immune inhibitory mechanisms that facilitate immune evasion. Notably, increased lipid synthesis by the fatty acid synthase (FASN) is an emerging mechanism that can account for the deceiving treatment efficacy and immune escape of GBM. Therefore, we hypothesize that FASN-mediated lipid synthesis represents an innate immune evasion mechanism of irradiated GBM. Supporting this hypothesis, we observed that 10 gray (Gy) irradiation of murine GBM cell lines, GL261 and CT2A, upregulates FASN protein expression and increase cellular lipid content determined by BODIPY staining and electronic microscopy. Interestingly, this effect was abrogated when GBM cells were incubated with an inhibitor of FASN. Next, to ask whether FASN was impairing RT-induced IFN-I, GL261 and CT2A cells were engineered to express an inducible shRNA silencing FASN (GBMshFASN) or its non-silencing control (GBMshNS). Irradiation of GBMshNS cells enhanced the secretion of IFN-beta and CXCL10, but this effect was more pronounced when FASN was blocked. Finally, GBMshNS and GBMshFASN cells were orthotopically implanted in mice on day 0. On day 10, selective irradiation (10Gy) was performed to the tumor. Tumor growth and immune contexture were evaluated on day 17. Magnetic resonance imaging revealed that FASN knockdown reduces tumor growth independently from RT. However, in situ immunofluorescence of GBMshFASN tumors showed increased infiltration of CD8+ T cells and CD11c+ cells only in irradiated mice bearing GBMshFASN tumors. Altogether, our data suggest that RT rewires the energy supply of GBM by promoting FASN-mediated lipid synthesis to foster immune evasion. Targeting FASN is a promising strategy to promote anti-cancer immunity and sensitize irradiated GBM to immunotherapies.