Research Article Long-term Survival and Clinical Benefit from Adoptive T-cell Transfer in Stage IV Melanoma Patients Is Determined by a Four-Parameter Tumor Immune Signature Sara M. Melief 1 ,Valeria V.Visconti 1 , Marten Visser 1 , Merel van Diepen 2 , Ellen H.W. Kapiteijn 1 , Joost H. van den Berg 3 , John B.A.G. Haanen 3 , Vincent T.H.B.M. Smit 4 , Jan Oosting 5 , Sjoerd H. van der Burg 1 , and Els M.E. Verdegaal 1 Abstract The presence of tumor-infiltrating immune cells is associated with longer survival and a better response to immunotherapy in early-stage melanoma, but a comprehensive study of the in situ immune microenvironment in stage IV melanoma has not been performed. We investigated the combined influence of a series of immune factors on survival and response to adoptive cell transfer (ACT) in stage IV melanoma patients. Metastases of 73 stage IV melanoma patients, 17 of which were treated with ACT, were studied with respect to the number and functional phenotype of lymphocytes and myeloid cells as well as for expression of galectins-1, -3, and -9. Single factors associated with better survival were identified using Kaplan–Meier curves and multivariate Cox regression analyses, and those factors were used for interac- tion analyses. The results were validated using The Cancer Genome Atlas database. We identified four parameters that were associated with a better survival: CD8 þ T cells, galectin- 9 þ dendritic cells (DC)/DC-like macrophages, a high M1/M2 macrophage ratio, and the expression of galectin-3 by tumor cells. The presence of at least three of these parameters formed an independent positive prognostic factor for long-term sur- vival. Patients displaying this four-parameter signature were found exclusively among patients responding to ACT and were the ones with sustained clinical benefit. Cancer Immunol Res; 5(2); 170–9. Ó2017 AACR. Introduction Melanoma is the most aggressive form of skin cancer and has long been recognized as a highly immunogenic tumor and a good target for immunotherapy (1). In different types of cancer, includ- ing melanoma, the presence of type I cytokine–oriented tumor- infiltrating lymphocytes (TIL) has been associated with improved survival (2). Indeed, a strong ongoing immune response was linked to spontaneous regression in about half of the primary melanomas (3) and longer survival of patients with stage I–III primary and regionally metastasized melanoma (4–6). More recently, a large study in patients with stage IV (distant metastases) melanoma revealed that even at this stage, intratumoral T-cell content was associated with improved survival (7). However, the predictive value for survival was not so strong, indicating that other immune-related factors previously studied in primary mel- anoma may also play a role (8–12); this involvement of other immune parameters was also suggested by studies at the gene expression level (13, 14). In parallel, studies showing that a strong intratumoral T-cell infiltrate fosters a better response to PD-1 checkpoint therapy (15) and autologous tumor cell vaccination (11), but also that intratumoral macrophages can hamper CTLA-4 checkpoint therapy (16), suggest that the tumor's immune con- texture may also influence the response to immunotherapy. In this study, we have expanded on earlier studies (4, 7) by assessing the influence of a series of immune factors in the metastatic tumor microenvironment in a large group of stage IV melanoma patients with up to 10 years of follow-up since metas- tasis. We identified four parameters, each of which was associated with better survival. These parameters comprised CD8 T cells, the presence of galectin-9 þ dendritic cells (DC)/DC-like macro- phages, a higher M1/M2 macrophage ratio, and galectin-3 expres- sion by tumor cells. The presence of at least three parameters was an independent prognostic factor for survival, which was validat- ed by analysis of these parameters in stage IV melanoma patients in The Cancer Genome Atlas (TCGA) database. Furthermore, with the introduction of targeted therapies and checkpoint inhibitors, adoptive cell transfer (ACT) has mostly become a salvage therapy (17) for treatment of stage IV melanoma patients. Analysis of the predictive value of this signature for the response to ACT revealed that the pretreatment tumors of patients without clinical benefit 1 Department of Clinical Oncology, Leiden University Medical Center, Leiden, the Netherlands. 2 Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, the Netherlands. 3 Division of Immunology, The Netherlands Cancer Institute, Amsterdam, the Netherlands. 4 Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands. 5 Bioinformatics Center of Expertise, Leiden University Medical Center, Leiden, the Netherlands. Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). Corresponding Author: Els M.E. Verdegaal, Department of Clinical Oncology, Leiden University Medical Center, P.O. Box 9600, Leiden 2300 RC, the Nether- lands. Phone: 317-1526-3464; Fax: 317-1526-6760; E-mail: e.verdegaal@lumc.nl doi: 10.1158/2326-6066.CIR-16-0288 Ó2017 American Association for Cancer Research. Cancer Immunology Research Cancer Immunol Res; 5(2) February 2017 170 on June 18, 2020. © 2017 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from Published OnlineFirst January 10, 2017; DOI: 10.1158/2326-6066.CIR-16-0288