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 Institute's 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.