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In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3+ and CD8+ cell infiltrate, with few PD1+ cells, rare CD4+ cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B+ NK and T cells, including CD4+ T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4+ T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade. Trial registration: NCT02460224. Registered 02/06/2015.

Background: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Co