320 Tumor cell-intrinsic signaling mediates T cell exclusion and promotes resistance to checkpoint blockade therapy in non-small cell lung cancer

BackgroundLung cancer is the leading cause of cancer-related death worldwide.1 Immunotherapies such as checkpoint blockade therapy (CBT) can be an effective approach to treat patients with metastatic tumors. However, only a fraction of patients is responsive to CBT treatments.2Patients with a non-T cell-infiltrated tumor microenvironment correlate with a poor response to CBT and T cell infiltration can be influenced by tumor cell-intrinsic signaling pathways.3 4 Therefore, understanding the tumor cell-intrinsic mechanisms affecting anti-tumor immune response will aid us to design better treatments for lung cancer patients. Preliminary work in our group showed that patients with non-small cell lung cancer (NSCLC) can be segregated according to the expression of a T cell gene signature into T cell-infiltrated and non-T cell-infiltrated cohorts. Using an unbiased pathway analysis, we identified that upregulation of specific gene modules significantly correlate with low expression of the T cell gene signature gene (non-T cell-infiltration). In this project, we aim to investigate how the overexpression of one specific pathway in NSCLC cells impacts the anti-tumor immune responses.MethodsWe used a lung cancer cell line derived from a KrasG12D/+ and Tp53-/- mouse (KP) to overexpressed our gene of interest (KP-A). Tumors were induced by subcutaneous or orthotopic implantation and were treated with anti-PD-L1 and anti-CTLA-4 blocking antibodies and analyzed for tumor burden. Infiltration of cytotoxic T cells and regulatory T cells were evaluated by fluorescence microscopy. Additionally, we engineered the KP-A and KP cell lines to express the model antigen SIY which allowed us to characterize tumor-specific T cell responses and utilize SIY-reactive TCR-transgenic T cells.ResultsWe identified that the overexpression of our gene of interest in KP cells impairs the response to CBT mediated by T cell exclusion from the tumor. Analyses of tumor-reactive T cells indicated that T cell activation and differentiation into effector T cells was not affected, however, effector T cells failed to infiltrate KP-A tumors. We are currently investigating the molecular mechanism whereby our gene of interest mediates T cell exclusion.ConclusionsOur results strongly suggest that tumor cell-intrinsic activation of specific pathways in NSCLC promote immune evasion and contribute to immunotherapy resistance. Understanding the molecular and immunological mechanisms mediating T cell exclusion from the lung tumor microenvironment will facilitate the development of novel combination treatment strategies for NSCLC patients.AcknowledgementsThis work was supported by a postdoctoral fellowship from the Ludwig Center at MIT’s Koch Institute for Integrative Cancer ResearchReferencesChen Z, Fillmore CM, Hammerman PS, Kim CF, Wong KK. Non-small-cell lung cancers: a heterogeneous set of diseases. Nat Rev Cancer 2014;14:535–546.Hellmann MD, Paz-Ares L, Bernabe Caro R, Zurawski B, Kim SW, Carcereny Costa E, Park K, Alexandru A, Lupinacci L, de la Mora Jimenez E, Sakai H, Albert I, Vergnenegre A, Peters S, Syrigos K, Barlesi F, Reck M, Borghaei H, Brahmer JR, O’Byrne KJ, Geese WJ, Bhagavatheeswaran P, Rabindran SK, Kasinathan RS, Nathan FE, Ramalingam SS. Nivolumab plus ipilimumab in advanced non-small-cell lung cancer. N Engl J Med 2019;381:2020–2031.Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature 2017;541:321–330.Nguyen KB, Spranger S. Modulation of the immune microenvironment by tumor-intrinsic oncogenic signaling. J Cell Biol 2020;219.Ethics ApprovalAll mouse experiments in this study were approved by MIT’s Committee on Animal Care (CAC) - DHHS Animal Welfare Assurance # D16-00078

Tumor Escape Phenotype in Bladder Cancer Is Associated with Loss of HLA Class I Expression, T-Cell Exclusion and Stromal Changes

Cancer eradication and clinical outcome of immunotherapy depend on tumor cell immunogenicity, including HLA class I (HLA-I) and PD-L1 expression on malignant cells, and on the characteristics of the tumor microenvironment, such as tumor immune infiltration and stromal reaction. Loss of tumor HLA-I is a common mechanism of immune escape from cytotoxic T lymphocytes and is linked to cancer progression and resistance to immunotherapy with the inhibitors of PD-L1/PD-1 signaling. Here we observed that HLA-I loss in bladder tumors is associated with T cell exclusion and tumor encapsulation with stromal elements rich in FAP-positive cells. In addition, PD-L1 upregulation in HLA-I negative tumors demonstrated a correlation with high tumor grade and worse overall- and cancer-specific survival of the patients. These changes define common immuno-morphological signatures compatible with cancer immune escape and acquired resistance to therapeutic interventions across different types of malignancy. They also may contribute to the search of new targets for cancer treatment, such as FAP-expressing cancer-associated fibroblasts, in refractory bladder tumors.

PTEN Loss Correlates with T cell Exclusion across Human Cancers

Background: Recent evidences had shown that loss in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was associated with immunotherapy resistance, which may be attributed to the non-T-cell-inflamed tumor microenvironment. The impact of PTEN loss on tumor microenvironment, especially regarding T cell infiltration across tumor types is not well understood.Methods: Utilizing The Cancer Genome Atlas (TCGA) and publicly available dataset of immunotherapy, we explored the correlation of PTEN expressing level or genomic loss with tumor immune microenvironment and response to immunotherapy. We further investigated the involvement of PI3K-AKT-mTOR pathway activation, which is known to be the subsequent effect of PTEN loss, in the immune microenvironment modulation.Results: We reveal that PTEN mRNA expression is significantly positively correlated with CD4/CD8A gene expression and T cells infiltration especially T helpers cells, central memory T cell and effector memory T cells in multiples tumor types. Genomic loss of PTEN is associated with reduced CD8+ T cells, type 1 T helper cells, and increased type 2 T helper cells, immunosuppressed genes (e.g. VEGFA) expression. Furthermore, T cell exclusive phenotype is also observed in tumor with PI3K pathway activation or genomic gain in PIK3CA or PIK3CB. PTEN loss and PI3K pathway activation correlate with immunosuppressive microenvironment, especially in terms of T cell exclusion. PTEN loss predict poor therapeutic response and worse survival outcome in patients receiving immunotherapy.Conclusion: These data brings insight into the role of PTEN loss in T cell exclusion and immunotherapy resistance, and inspires further research on immune modulating strategy to augment immunotherapy.

PTEN loss correlates with T cell exclusion across human cancers

Background Recent evidences had shown that loss in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was associated with immunotherapy resistance, which may be attributed to the non-T-cell-inflamed tumor microenvironment. The impact of PTEN loss on tumor microenvironment, especially regarding T cell infiltration across tumor types is not well understood. Methods Utilizing The Cancer Genome Atlas (TCGA) and publicly available dataset of immunotherapy, we explored the correlation of PTEN expressing level or genomic loss with tumor immune microenvironment and response to immunotherapy. We further investigated the involvement of PI3K-AKT-mTOR pathway activation, which is known to be the subsequent effect of PTEN loss, in the immune microenvironment modulation. Results We reveal that PTEN mRNA expression is significantly positively correlated with CD4/CD8A gene expression and T cells infiltration especially T helpers cells, central memory T cell and effector memory T cells in multiples tumor types. Genomic loss of PTEN is associated with reduced CD8+ T cells, type 1 T helper cells, and increased type 2 T helper cells, immunosuppressed genes (e.g. VEGFA) expression. Furthermore, T cell exclusive phenotype is also observed in tumor with PI3K pathway activation or genomic gain in PIK3CA or PIK3CB. PTEN loss and PI3K pathway activation correlate with immunosuppressive microenvironment, especially in terms of T cell exclusion. PTEN loss predict poor therapeutic response and worse survival outcome in patients receiving immunotherapy. Conclusion These data brings insight into the role of PTEN loss in T cell exclusion and immunotherapy resistance, and inspires further research on immune modulating strategy to augment immunotherapy.

Cytokine and Chemokine Signals of T-Cell Exclusion in Tumors

The success of cancer immunotherapy in solid tumors depends on a sufficient distribution of effector T cells into malignant lesions. However, immune-cold tumors utilize many T-cell exclusion mechanisms to resist immunotherapy. T cells have to go through three steps to fight against tumors: trafficking to the tumor core, surviving and expanding, and maintaining the memory phenotype for long-lasting responses. Cytokines and chemokines play critical roles in modulating the recruitment of T cells and the overall cellular compositions of the tumor microenvironment. Manipulating the cytokine or chemokine environment has brought success in preclinical models and early-stage clinical trials. However, depending on the immune context, the same cytokine or chemokine signals may exhibit either antitumor or protumor activities and induce unwanted side effects. Therefore, a comprehensive understanding of the cytokine and chemokine signals is the premise of overcoming T-cell exclusion for effective and innovative anti-cancer therapies.

Integrated digital pathology and transcriptome analysis identifies molecular mediators of T-cell exclusion in ovarian cancer

Close proximity between cytotoxic T lymphocytes and tumour cells is required for effective immunotherapy. However, what controls the spatial distribution of T cells in the tumour microenvironment is not well understood. Here we couple digital pathology and transcriptome analysis on a large ovarian tumour cohort and develop a machine learning approach to molecularly classify and characterize tumour-immune phenotypes. Our study identifies two important hallmarks characterizing T cell excluded tumours: 1) loss of antigen presentation on tumour cells and 2) upregulation of TGFβ and activated stroma. Furthermore, we identify TGFβ as an important mediator of T cell exclusion. TGFβ reduces MHC-I expression in ovarian cancer cells in vitro. TGFβ also activates fibroblasts and induces extracellular matrix production as a potential physical barrier to hinder T cell infiltration. Our findings indicate that targeting TGFβ might be a promising strategy to overcome T cell exclusion and improve clinical benefits of cancer immunotherapy.

495 Basal cell carcinoma demonstrates a T-cell exclusion immune phenotype in contrast to other anti-PD-1 therapy responsive cutaneous malignancies

BackgroundBasal cell carcinoma (BCC) is considered an immunogenic tumor based on the high tumor mutational burden (TMB), increased incidence in immunocompromised patients, and responsiveness to imiquimod, a toll-like receptor agonist therapy. However, anti-PD-1 immunotherapy response rates in patients with advanced BCC appear less than that seen with other advanced cutaneous malignancies. Molecular profiles of BCC tumors were analyzed to determine immune phenotypes and resistance mechanisms in comparison to other anti-PD-1 therapy responsive cutaneous malignancies.MethodsNext generation sequencing on DNA (NGS; NextSeq and Novaseq), PD-L1 immunohistochemistry (SP-142 and 28–8 antibody clones, cutoff >5% tumor staining) and mRNA gene expression level (Whole Transcriptome Sequencing, NovaSeq) data from BCC (N=69), melanoma (N=914), and cutaneous squamous cell carcinoma (SCC) tumors (N=165) at Caris Life Sciences (Phoenix, AZ) were analyzed. Tumor mutational burden (TMB) was calculated by counting all non-synonymous missense mutations that had not been previously described as germline alterations. Microenvironment cell population counter1 was used to estimate cell population abundance in the TME. Gene set enrichment analysis (GSEA) was performed on transcriptomes.2 Statistical significance was set at P value or false discovery rate (FDR) < 0.05.ResultsOf the 69 BCC tumors with NGS data, the most frequent mutations were in PTCH1 (82%), P53 (73%) and ARID1A (42%); additional relevant mutations included SMO (18%), JAK1 (9%), PI3KCA (6%), APC (4%), and CTNNB1 (3%). TMB was significantly greater in BCC compared to melanoma (median 30.5 vs 12 mut/Mb, P<0.0001) and similar to SCC (median 29.5 mut/Mb, P=0.9389). PD-L1 positivity was 1/23 (4%) in BCC, 215/831 (26%) in melanoma, and 81/147 (56%) in SCC. Interferon gamma and T-effector immune gene analyses showed significantly lower expression in BCC compared to melanoma and SCC (e.g., IFNg TPM=0.26 (BCC) vs 0.65 and 0.58 (melanoma, SCC, both P<0.01). BCC demonstrated the lowest CD-8 T-cell fractions and the highest neutrophil and cancer associated fibroblast (CAF) fractions compared to melanoma and SCC. Angiogenesis and TGF-beta gene sets were enriched in BCC compared to melanoma (NES=1.5, FDR=0.046 and NES=1.35, FDR=0.055, respectively), but not compared to SCC (NES=0.90, FDR=0.57 and NES=0.94, FDR=0.60, respectively).ConclusionsWhile BCC tumors demonstrated a high TMB, a markedly lower level of adaptive anti-tumor immunity in comparison to other cutaneous malignancies was observed. T-cell exclusion mechanisms mediated through CAFs and desmoplasia, with upregulation of TGF-beta and angiogenic signaling, may play a role. Further investigation into abrogation of these mechanisms is warranted to develop improved anti-PD-1 based therapies for BCC.ReferencesBecht E, Giraldo NA, Lacroix L, Buttard B, Elarouci N, Petitprez F, Selves J, Laurent-Puig P, Sautès-Fridman C, Fridman WH, de Reyniès A. Estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression. Genome Biol 2016; 17(1):218.Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005;102(43):15545–50.

733 Integrative molecular profiling of high-grade primary prostate cancer identifies patients with a biomarker profile that favors the combination of standard of care (SOC) therapy with immunotherapy

BackgroundProstate cancer (PCa) is primarily driven by androgen receptor (AR) signaling and has a highly immunosuppressive microenvironment. Although genomic and histopathological differences between low- and high-grade primary PCa (lgPCa and hgPCa) have been reported, an integrative assessment of multiple molecular features in the context of disease grade and metastatic outcome is lacking. We propose that a subset of hgPCa patients who relapse under SOC may benefit from adjuvant immune-checkpoint blockade (ICB) added to SOC to overcome immune suppression.MethodsWe analyzed treatment naive prostatectomy tissue from a cohort of 124 primary PCa patients (n= 58, Gleason score ≤6; n= 66, Gleason score ≥ 8). We performed RNAseq expression profiling, whole-exome sequencing (WES) and immunohistochemistry. We employed digital spatial analysis in tumor vs. stromal regions to characterize differences in CD8+ T-cell topology between lgPCa and hgPCaResults1.Comparisons in lg vs. hgPCA: Digital spatial analysis assessing the proximity of CD8+ T-cells to tumor cells revealed a T-cell exclusion phenotype that is more prominent in hgPCa, whereas evaluation of overall CD8+ T-cell density in tumor and stromal regions did not differentiate disease grades. HgPCa had a higher frequency of at least one functional mutation in either TP53, RHPN2, or KMT2D genes compared to lgPCa. Assessment of MHC-I deficiency by IHC and mRNA revealed that hgPCa has significantly lower MHC-I protein expression compared to lgPCa. Interestingly, MHC-I loss in hgPCa associated with a T-cell exclusion phenotype. Moreover, RNAseq gene expression signatures revealed higher expression of tumor-associated macrophage (TAMs), T-regs, Cancer-Associated Fibroblasts (CAFs), DNA damage repair (DDR) genes and lower Interferon-γ (IFN- γ) expression in hgPCa compared to lgPCa. Overall, hgPCa is characterized by a combined phenotype of ‘MHCIloss/IFN- γ low/CAFhigh/TAMhigh/T-reghigh/DDRhigh’. 2.Comparisons within hgPCA that develop metastasis: Unsupervised analysis of molecular features in hgPCa patients that developed metastases identified a subset of patients that exhibit a less immunosuppressive phenotype with lower tumor AR expression, retained tumor MHC-I expression, moderate CD8+ T-cell infiltration and a high IFN-γ RNA signature (figure 1), suggesting potential benefit from ICB therapyAbstract 733 Figure 1Unsupervised analysis of primary hgPCa that develop metastasis. Features used for unsupervised classification into Cluster 1 and Cluster 2 are: IHC CD8+ cell proximity to tumor cells (IHC_Infiltrated/Desert/Excluded), CD8+ overall cell density in tumor area, MHCI IHC H-score in tumor (% of tumor cells that are positive X stain intensity), RNAseq signatures for AR pathway in tumor, T-cell exhaustion, Interferon-y, Macrophage M1, Neuroendocrine phenotypes and DNA repair pathway. Patient cohort annotated for at least one mutations in driver genes (TP53, RHPN2, KMT2D), percent tumor expression of MHCI IHC (>25% high, <25% low), CD8 infiltration type in relation to tumor and cancer subtypes as defined by Mortensen mRNA profiling (Mortensen at al, Science Reports 2015).ConclusionsOur analysis suggests that hgPCa is characterized by low antigenicity as assessed by loss of MHC-I protein expression and an immunosuppressive microenvironment rich in CAFs, macrophages, T-regs and T-cell exclusion phenotypes. Unlike lgPCa, hgPCA can have a poor prognosis (within 5 years relapse). However, a subset of hgPCa patients that metastasized while on SOC exhibited a biomarker profile that might benefit from combination of SOC with ICBEthics ApprovalThis study was approved by BMS Cambridge Massachusetts Institutional Biosafety Committee, approval number CAM_2020_12050_6Consent‘Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.’

Overcoming primary and acquired resistance to anti-PD-L1 therapy by induction and activation of tumor-residing cDC1s

The ability of cancer cells to ensure T-cell exclusion from the tumor microenvironment is a significant mechanism of resistance to anti-PD-1/PD-L1 therapy. Evidence indicates crucial roles of Batf3-dependent conventional type-1 dendritic cells (cDC1s) for inducing antitumor T-cell immunity; however, strategies to maximize cDC1 engagement remain elusive. Here, using multiple orthotopic tumor mouse models resistant to anti-PD-L1-therapy, we are testing the hypothesis that in situ induction and activation of tumor-residing cDC1s overcomes poor T-cell infiltration. In situ immunomodulation with Flt3L, radiotherapy, and TLR3/CD40 stimulation induces an influx of stem-like Tcf1+ Slamf6+ CD8+ T cells, triggers regression not only of primary, but also untreated distant tumors, and renders tumors responsive to anti-PD-L1 therapy. Furthermore, serial in situ immunomodulation (ISIM) reshapes repertoires of intratumoral T cells, overcomes acquired resistance to anti-PD-L1 therapy, and establishes tumor-specific immunological memory. These findings provide new insights into cDC1 biology as a critical determinant to overcome mechanisms of intratumoral T-cell exclusion.