Micro-dissecting the Role of Melanoma-Associated Plasmacytoid Dendritic Cells/ Interferon Producing Cells

Cutaneous melanoma (CM) represents a heterogeneous group of melanocytic neoplasms. Het¬erogeneity of CM occurs at different level including epidemiology, clinical presentation, histopathology and molecular genetics. Primary cutaneous melanoma (PCM) has represented an extraordinary model to understand the role of the immune response to cancer. Melanoma patients mount a spontaneous T cell response against their tumor. Specifically, autologous T cells recognize tumor-specific antigens or differentiation antigens common to normal melanocytes. This anti-tumor T-cells response is only partially beneficial since it fails to reject the tumor in most of the instances. In fact, at some point during melanoma progres¬sion, responder T cells become ineffective, likely because of a local immunosuppressive process occur¬ring at the tumor sites. The local immune response to PCM has been largely investigated in the early 90’. Initially, occur¬rence of tumour-infiltrating lymphocytes (TILs) in PCM has been associated with a better prognosis. However, the clinical significance of this factor remained controversial and other subsequent reports suggested no relationship with prognosis. Among cells with potential implication in cancer immunity, plasmacytoid dendritic cells/ inter¬feron producing cells (from here on referred as PDCs) emerged as important players due to their capacity to produce high levels of I-IFN and differentiate into antigen-presenting dendritic cells in response to a variety of stimuli (e.g. viruses, toll-like receptor [TLR] ligands), thus bringing together innate and adaptive immunity. Circulating PDCs have been characterized in a wide variety of human neoplasms, including carcinomas from different sites, melanoma and hema¬topoietic malignancies. A significant fraction of these studies have been focused on circulating PDCs, which are frequently reduced in cancer patients compared to healthy donor. Tumor-associated-PDC (TA-PDC), the counterpart found at tumor sites, has been detected in primary carcinomas from different organs (breast, ovary, head and neck, lung, skin, cervix, prostate and liver), cutaneous melanoma and lymphomas by using various strategies. By using immunohistochemis¬try, many of these studies have been conducted on frozen sections and this has represented a significant limitation in the analysis of large patient cohorts, thus hindering the appropriate clinical significance of TA-PDC. In addition, the most frequently used PDC marker anti-CD123, also stains other cells including endothelial cells, macrophages and other myeloid cells. AIMS. The candidate hypothesizes that the study of TA-PDC can provide a unique perspective on the role and clini¬cal significance of PDCs in this neoplasm. Encouraged by recent upgrades in DigMic technology, the candidate proposes to analyze TA-PDC in a large and clinically-oriented cohort of PCM and benign nevi (NV) to establish their frequency of distribution and clinical significance (AIM1). PDCs produce high amounts of I-IFN, a pleiotropic cytokine with demonstrated clinical ben¬efit for a fraction of PCM patients and which has recently emerged as a coordinator in the interactions between tumor cells and the immune system. Some studies indicate that TA-PDCs are defective in type I IFN production, but instead secrete immunosuppressive soluble factors responsible for tumor progression. PDCs, including those purified from human cancer, might exert a tolerogenic function, pri¬marily by inducing IL-10 producing regulatory T-cells. In human PDCs, this polarization might depend on the expression of ICOS ligand or production of the immunosuppressive enzyme indoleamine-Pyrrole 2,3,-Dioxygenase (IDO). However, recent work has contradicted this view demonstrating a beneficial ef¬fector function of activated PDCs via production of Granzyme B and TRAIL. In this part of the study, the candidate will verify the capability of melanoma-associated PDC to produce I-IFN (AIM2). Results and conclusion. In this large scale clinically-oriented study, we have analyzed the content and function of plasmacytoid dendritic cells (PDCs) in primary cutaneous melanomas (PCM) by using a novel cell count strategy. The latter was based on automated analysis of the entire tumor area by using a digital microscopy approach. The DigMic platform results from the integration of Aperio Scanscope slide scanner with the digital pa¬thology image analysis solution Tissue Studio Definiens. Our findings indicate that PDC infiltration in PCM represents a common event across all stages. PCM-associated PDC infiltration occurs early during tumor evolution. In term of cell density (mm2 of PCM tumor area) and ratio with CD45RP+ LK, PDC content did not correlate with major prognostic endpoints. This is not surprising since major PDC function, such as type I-IFN production, appear to be hijacked by the PCM microenvironment as demonstrated by low MxA expression. Previously published works indicate that the inherent molecular profile of cancer cells can significantly modify cancer cell immunogenicity and the surrounding microenvironment. Emerging findings suggest that blockade of BrafV600E signaling cascade in PCM significantly affect the immunogenicity of melanoma cells in different ways. Remarkably, in our cohort PDC infiltration trended to¬ward an increase in PCM carrying the BrafV600E somatic mutation. This finding indicate that persistent signaling through Braf in melanoma cells might be relevant in the regulation of the chemokine milieu of the PCM. The candidate believe that these findings establish the groundwork for further mechanistic studies clarifying the PDC requirement during melanoma genesis.