Takahiro Tsujikawa

Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients.

Good bacteria help fight cancer Resident gut bacteria can affect patient responses to cancer immunotherapy (see the Perspective by Jobin). Routy et al. show that antibiotic consumption is associated with poor response to immunotherapeutic PD-1 blockade. They profiled samples from patients with lung and kidney cancers and found that nonresponding patients had low levels of the bacterium Akkermansia muciniphila. Oral supplementation of the bacteria to antibiotic-treated mice restored the response to immunotherapy. Matson et al. and Gopalakrishnan et al. studied melanoma patients receiving PD-1 blockade and found a greater abundance of “good” bacteria in the guts of responding patients. Nonresponders had an imbalance in gut flora composition, which correlated with impaired immune cell activity. Thus, maintaining healthy gut flora could help patients combat cancer. Science, this issue p. 91, p. 104, p. 97; see also p. 32 Gut bacteria influence patient response to cancer therapy. Preclinical mouse models suggest that the gut microbiome modulates tumor response to checkpoint blockade immunotherapy; however, this has not been well-characterized in human cancer patients. Here we examined the oral and gut microbiome of melanoma patients undergoing anti–programmed cell death 1 protein (PD-1) immunotherapy (n = 112). Significant differences were observed in the diversity and composition of the patient gut microbiome of responders versus nonresponders. Analysis of patient fecal microbiome samples (n = 43, 30 responders, 13 nonresponders) showed significantly higher alpha diversity (P < 0.01) and relative abundance of bacteria of the Ruminococcaceae family (P < 0.01) in responding patients. Metagenomic studies revealed functional differences in gut bacteria in responders, including enrichment of anabolic pathways. Immune profiling suggested enhanced systemic and antitumor immunity in responding patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplants from responding patients. Together, these data have important implications for the treatment of melanoma patients with immune checkpoint inhibitors.

Bruton Tyrosine Kinase–Dependent Immune Cell Cross-talk Drives Pancreas Cancer

UNLABELLED Pancreas ductal adenocarcinoma (PDAC) has one of the worst 5-year survival rates of all solid tumors, and thus new treatment strategies are urgently needed. Here, we report that targeting Bruton tyrosine kinase (BTK), a key B-cell and macrophage kinase, restores T cell-dependent antitumor immune responses, thereby inhibiting PDAC growth and improving responsiveness to standard-of-care chemotherapy. We report that PDAC tumor growth depends on cross-talk between B cells and FcRγ(+) tumor-associated macrophages, resulting in T(H)2-type macrophage programming via BTK activation in a PI3Kγ-dependent manner. Treatment of PDAC-bearing mice with the BTK inhibitor PCI32765 (ibrutinib) or by PI3Kγ inhibition reprogrammed macrophages toward a T(H)1 phenotype that fostered CD8(+) T-cell cytotoxicity, and suppressed PDAC growth, indicating that BTK signaling mediates PDAC immunosuppression. These data indicate that pharmacologic inhibition of BTK in PDAC can reactivate adaptive immune responses, presenting a new therapeutic modality for this devastating tumor type. SIGNIFICANCE We report that BTK regulates B-cell and macrophage-mediated T-cell suppression in pancreas adenocarcinomas. Inhibition of BTK with the FDA-approved inhibitor ibrutinib restores T cell-dependent antitumor immune responses to inhibit PDAC growth and improves responsiveness to chemotherapy, presenting a new therapeutic modality for pancreas cancer.

Trim32 Deficiency Enhances Th2 Immunity and Predisposes to Features of Atopic Dermatitis

Altered innate immunity is a feature of certain skin inflammatory diseases such as psoriasis and atopic dermatitis (AD). In this study, we provide evidence that deficiency in Trim32 (a tripartite motif [TRIM] protein with innate antiviral activity) contributes to a T helper type 2 biased response and predisposes to features of AD in mice. On treatment with the toll-like receptor 7 agonist imquimod (IMQ), Trim32 knockout mice displayed compromised psoriasiform phenotypes and defective T helper type 17 response. Instead, IMQ treatment of Trim32 knockout mice induced AD-like phenotypes with enhanced skin infiltration of eosinophils and mast cells, elevation of T helper type 2 cytokines/chemokines expression, and reduced expression of filaggrin protein expression. Furthermore, although the induction of phosphorylated Stat3 and RelA was compromised after IMQ treatment in the knockout mice, phosphorylated Stat6 was elevated. CC chemokine ligand 20 induction by tumor necrosis factor-α and IL-17A was reduced in Trim32-deficient keratinocytes, whereas CC chemokine ligand 5 induction by tumor necrosis factor-α and IL-4 was enhanced. In addition, Trim32 protein levels were elevated in mice treated with IMQ. Unlike Trim32 overexpression in psoriasis, TRIM32 levels were low in patients with AD. Based on Trim32 induction by IMQ, the lower levels of TRIM32 in AD skin compared with healthy control and psoriatic skin suggest a defective TRIM32 pathway in AD pathogenesis.

Multiplex immunohistochemistry for immune profiling of HPV-associated head and neck cancer

Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically distinct from HPV-negative HNSCC, and as such requires differential therapeutic approaches. Accumulating evidence indicates a significant linkage between the immune response within the tissue and pathogenesis of HPV-associated HNSCC. To further elucidate immune-related signatures in HPV-associated HNSCC, we performed multiplex histological analysis in de-identified tissue microarray sections including HPV-positive (n = 21), HPV-negative (n = 17), and normal oropharynx (n = 8). Following immunohistochemistry (IHC) for CD45, CD3, CD8, Foxp3, T-bet, GATA-3, RORgT, CD20, CD56, CD68, MHC class II, CSF1R, CD66b, tryptase, CD83, DC-SIGN, PD-1, and PD-L1, the cell intensity per mm2 ratio/composition, localization were quantitatively evaluated. The HPV-status was confirmed by HPV16/18 polymerase chain reaction and IHC for p16INK4a. IHC for p16 or EpCAM were utilized for defining tumor region. Infiltration of T cell populations including CD45+CD3+CD8+ T cells (P < 0.01), CD45+CD3+CD8−Foxp3+ regulatory T cells (P < 0.05) and CD45+CD3+CD8−Foxp3+T-bet+ Th1 cells (P < 0.01), CD45+CD20+CD3−B cells (P < 0.05), CD45+CD68+CD163− macrophages (P < 0.001), and CD45+Tryptase+ mast cells (P < 0.01) was significantly higher in the HPV-positive group than in the HPV-negative group. CD8/CD68 ratio of HPV-positive tumor was higher than that of HPV-negative tumor (P < 0.05), and the highest CD163−CD68+/CD163+CD68+ ratio was observed in the intra-tumor region of HPV-positive tumors. High PD-L1 expression on CD68+CD163+ macrophages and MHC class II+CD83+ dendritic cells was intensively observed in the intra-tumor region of the HPV-positive group while maturation of dendritic cells assessed by CD83/DC-SIGN ratio was significantly higher in the peritumoral stroma than intra-tumor regions (P < 0.05), indicating distinct immune regulatory mechanisms between intra and peritumoral regions. These signatures provide further evidence of anti-tumor immunity against HPV-positive head and neck cancer, and potentially lead to personalized treatment including immunomodulatory therapeutic targets specialized for the HPV/immune status.

Multiplexed immunohistochemistry image analysis using sparse coding

Multiplexed immunohistochemical (IHC) methods have been developed to evaluate multiple protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) tissue section. Since distinct populations of resident and recruited immune cells in tissues (and tumors) not only regulate progression of malignant disease, these also represent targets for novel immune-based therapies; thus, improved tissue biomarker assessment evaluating immune responses in situ are needed. To objectively identify distinct cell subsets in tissues and tumors, we adopted sparse coding approaches enabling modeling of data vectors as sparse linear combinations of basis elements, to audit cellular presence and phenotypes using image cytometry datasets with unbiased assessments. By doing comparative analyses between manual gating (ground truth) and sparse coding, we report that results are comparable as obtained by manual gating strategies, and demonstrate robustness and objectivity of this novel bioinformatics approach.

Abstract A19: Lymphatic vessels as a biomarker in human melanoma

The aim of our work is to assess the local lymphatic vasculature within the tumor microenvironment as a possible biomarker for response to immunotherapy. Targeting the immune checkpoint molecules CTLA-4 and PD-1 is showing unprecedented clinical impact in human melanoma patients. However, a significant subset of patients does not respond to therapy. Therefore efforts have been made to identify biomarkers predictive of survival and response to immunotherapy, and evaluation of the immune microenvironment was shown to have high prognostic power. Characterization of tumor-infiltrating T cells with respect to density, phenotype and location has been demonstrated to predict survival and metastasis in retrospective studies and might be superior to classical TNM staging. Despite these promising results current strategies are not 100% predictive and additional cell populations within the tumor microenvironment are likely to affect the overall anti-tumor immune response. Interestingly, the density of tumor-associated lymphatic vessels was found to be decreased in patients with metastatic colorectal cancer compared to non-metastatic patients. In addition, our recent work using metastatic melanoma samples from the Broad Institute9s TCGA database demonstrated that expression of lymphatic genes (LYVE-1 and podoplanin) correlates with expression levels of immune cell markers. In mice lacking dermal lymphatic vessels immune infiltration into experimental melanoma was significantly decreased. At the same time these tumors displayed decreased immune suppression and improved tumor control by transferred cytotoxic T cells. These results suggest that lymphatic vessels are essential for establishment of an efficient anti-tumor immune response, but have a role in negatively regulating anti-tumor immunity during tumor progression. We therefore hypothesize that local lymphatic vessel density within the tumor microenvironment predicts immune infiltrate and response to immunotherapy. To simultaneously evaluate immune and vascular components in human formalin-fixed samples of melanoma we are using a multiplex-immunohistochemistry-based approach, which allows for examination of up to twelve markers by sequential staining of a single marker at a time. Tissue regions that include tumor/stroma borders and show high CD8 + T cell infiltrates are selected for analysis. This is followed by tissue segmentation based on the presence of a tumor marker and automated detection of cell populations within intra-tumoral regions and the invasive margin at the tumor border. Analysis using image cytometry allows us to quantify and correlate presence and location of multiple cell types within the tumor and the surrounding stroma. Our work will contribute to improved stratification of cancer patients with respect to possible response to immunotherapy. Citation Format: Julia Femel, Takahiro Tsujikawa, Guillaume Thibault, Jamie Booth, Amanda W. Lund. Lymphatic vessels as a biomarker in human melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A19.

Abstract A013: Favorable changes in tumor microenvironment following intravenous dosing with live attenuated Listeria monocytogenes-based immunotherapy

Modification of the tumor microenvironment (TME) to promote immune-mediated tumor cell destruction is considered to be an essential step for effective immunotherapy. We are evaluating recombinant live-attenuated, double deleted Listeria monocytogenes (LADD) as an immunotherapy platform for the treatment of cancer in several clinical trials in diverse indications. One LADD strain, known as CRS-207, has been engineered to express the tumor-associated antigen mesothelin and is being tested in pancreatic, ovarian and mesothelioma malignancies. Using multi-dimensional immunohistochemistry of paired biopsies from three patients with mesothelioma, we demonstrate the recruitment and expansion of effector tumor-infiltrating lymphocytes, including CD8+ T cells, mature DCs, CD163− macrophages and NK cells, following two prime infusions of CRS-207. In several different syngeneic mouse tumor models, we demonstrate that treatment with LADD engineered to express endogenous tumor antigens also induced significant changes in the TME that were consistent with changes observed in cancer patients, including enhanced CD8+ T cell effector function, recruitment of critical antigen presenting cells and reduction of regulatory T cells, and these changes correlated with significant therapeutic benefit in the mouse. LADD-induced changes to the TME were required for synergistic therapeutic antitumor efficacy combined with immune checkpoint blockade, including targeting MC38 tumor-specific neoantigens. Together, these findings demonstrate that intravenous administration of recombinant LADD therapy induces favorable changes in the tumor microenvironment in mice and humans with promise for effective outcomes in human clinical trials. Citation Format: Weiwen Deng, Takahiro Tsujikawa, Nitya Nair, Thomas Hudson, Weiqun Liu, Chris S. Rae, Edward E. Lemmens, Anthony W. Desbien, William Hanson, Peter Lauer, Lisa M. Coussens, Dirk G. Brockstedt, Thomas W. Dubensky, Jr., Meredith L. Leong. Favorable changes in tumor microenvironment following intravenous dosing with live attenuated Listeria monocytogenes-based immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A013.