Cutaneous melanoma occurs in both familial and sporadic forms. We investigated a melanoma-prone family through linkage analysis and high-throughput sequencing and identified a disease-segregating germline mutation in the promoter of the telomerase reverse transcriptase (TERT) gene, which encodes the catalytic subunit of telomerase. The mutation creates a new binding motif for Ets transcription factors and ternary complex factors (TCFs) near the transcription start and, in reporter gene assays, caused up to twofold increase in transcription. We then screened the TERT promoter in sporadic melanoma and observed recurrent ultraviolet signature somatic mutations in 125 of 168 (74%) of human cell lines derived from metastatic melanomas, 45 of 53 corresponding metastatic tumor tissues (85%), and 25 of 77 (33%) primary melanomas. The majority of those mutations occurred at two positions in the TERT promoter and also generated binding motifs for Ets/TCF transcription factors.

Endothelial cells (ECs) provide angiocrine factors orchestrating tumor progression. Here, we show that activated Notch1 receptors (N1ICD) are frequently observed in ECs of human carcinomas and melanoma, and in ECs of the pre-metastatic niche in mice. EC N1ICD expression in melanoma correlated with shorter progression-free survival. Sustained N1ICD activity induced EC senescence, expression of chemokines and the adhesion molecule VCAM1. This promoted neutrophil infiltration, tumor cell (TC) adhesion to the endothelium, intravasation, lung colonization, and postsurgical metastasis. Thus, sustained vascular Notch signaling facilitates metastasis by generating a senescent, pro-inflammatory endothelium. Consequently, treatment with Notch1 or VCAM1-blocking antibodies prevented Notch-driven metastasis, and genetic ablation of EC Notch signaling inhibited peritoneal neutrophil infiltration in an ovarian carcinoma mouse model.

Purpose: Cytotoxic lymphocytes interact with human tumor cells via the activating immunoreceptor NKG2D, recognizing a variety of stress-associated MIC and ULBP surface molecules. However, tumors can escape from this immunosurveillance by shedding NKG2D ligands (NKG2DL), rendering the soluble products detectable in patients' sera. Experimental Design: To elucidate the clinical significance of NKG2DL diversity, we studied their expression on melanoma tissues and their presence as soluble molecules in sera from >200 melanoma patients and compared the latter with the well-established serum marker S100B. Results: Immunohistochemistry revealed a heterogeneous expression of MIC and ULBP2 molecules between and within melanoma metastases. Compared with MIC, ULBP2 was less frequently expressed. Accordingly, elevated levels of soluble ULBP2 (sULBP2) were detected in sera of melanoma patients less frequently than elevated levels of soluble MICA (sMICA), although both soluble NKG2DL (sNKG2DL) were significantly increased compared with sera of healthy controls (P < 0.0001). Strikingly, elevated concentrations of sULBP2, but not of sMICA, were strongly associated with disease progression (P < 0.0001) and tumor load (P = 0.0003). Elevated serum levels of either sNKG2DL correlated with reduced overall survival, albeit considerably stronger for sULBP2 (P < 0.0001) than for sMICA (P = 0.011). In early-stage (I-III) melanoma patients, only sULBP2 (P < 0.0001) but neither sMICA nor S100B revealed prognostic significance. Multivariate analysis identified sULBP2 (P = 0.0015) and S100B (P = 0.013) but not sMICA as independent predictors of prognosis. Conclusion: Our data reveal marked differences in the clinical significance of individual sNKG2DL. Only sULBP2 is an independent predictor of prognosis, the significance of which is superior to the well-established and widely used melanoma serum marker S100B. (Clin Cancer Res 2009;15(16):5208-15) Genetic and epigenetic alterations are hallmarks of cancer.These modifications, on the one hand, are a prerequisite for cancer development and progression but, on the other hand, expose tumor cells as altered self to the immune system. Genetic alterations can initiate an intrinsic DNA damage response that in turn induces the expression of surface ligands binding to the activating immunoreceptor NKG2D (1). NKG2D is expressed on innate, innate-like, and adaptive immune effectors such as natural killer (NK) cells, γδ cells, and αβ T cells, respectively. Whereas NKG2D engagement directly activates NK cells, in T cells it delivers costimulatory signals modulating T-cell receptor-mediated activation (2-4). Studies in different

This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function.

Purpose: CD8 þ T lymphocytes can kill autologous melanoma cells, but their activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here, we analyzed three consecutive melanoma lesions obtained within one year of developing stage IV disease for their recognition by autologous T cells. Experimental Design: One skin (Ma-Mel-48a) and two lymph node (Ma-Mel-48b, Ma-Mel-48c) metastases were analyzed for T-cell infiltration. Melanoma cell lines established from the respective lesions were characterized, determining the T-cell-stimulatory capacity, expression of surface molecules involved in T-cell activation, and specific genetic alterations affecting the tumor-T-cell interaction.Results: Metastases Ma-Mel-48a and Ma-Mel-48b, in contrast with Ma-Mel-48c, were infiltrated by T cells. The T-cell-stimulatory capacity was found to be strong for Ma-Mel-48a, lower for Ma-Mel-48b, and completely abrogated for Ma-Mel-48c cells. The latter proved to be HLA class I-negative due to an inactivating mutation in one allele of the beta-2-microglobulin (B2M) gene and concomitant loss of the other allele by a deletion on chromosome 15q. The same deletion was already present in Ma-Mel-48a and Ma-Mel-48b cells, pointing to an early acquired genetic event predisposing to development of b2m deficiency. Notably, the same chronology of genetic alterations was also observed in a second b2m-deficient melanoma model. Conclusion: Our study reveals a progressive loss in melanoma immunogenicity during the course of metastatic disease. The genetic evolvement of T-cell resistance suggests screening tumors for genetic alterations affecting immunogenicity could be clinically relevant in terms of predicting patient responses to T-cell-based immunotherapy. Clin Cancer Res; 20(24); 6593-604. Ó2014 AACR.

NKG2D operates as an activating receptor on natural killer (NK) cells and costimulates the effector function of ab CD81 T cells. Ligands of NKG2D, the MHC class I chain-related (MIC) and UL16 binding protein (ULBP) molecules, are expressed on a variety of human tumors, including melanoma. Recent studies in mice demonstrated that NKG2D mediates tumor immune surveillance, suggesting that antitumor immunity in humans could be enhanced by therapeutic manipulation of NKG2D ligand (NKG2DL) expression. However, signals and mechanisms regulating NKG2DL expression still need to be elucidated. Here, we asked whether the proinflammatory cytokine Interferon-c (IFN-c) affects NKG2DL expression in melanoma. Cell lines, established from MHC class I-negative and -positive melanoma metastases, predominantly expressed MICA and ULBP2 molecules on their surface. Upon IFN-c treatment, expression of MICA, in some cases, also of ULBP2 decreased. Besides melanoma, this observation was made also for glioma cells. Down-regulation of NKG2DL surface expression was dependent on the cytokine dose and the duration of treatment, but was neither due to an intracellular retention of the molecules nor to an increased shedding of ligands from the tumor cell surface. Instead, quantitative RT-PCR revealed a decrease of MICA-specific mRNA levels upon IFN-c treatment and siRNA experiments pointed to an involvement of STAT-1 in this process. Importantly, IFN-c-treated MHC class I-negative melanoma cells were less susceptible to NKG2D-mediated NK cell cytotoxicity. Our study suggests that IFN-c, by down-regulating ligand expression, might facilitate escape of MHC class I-negative melanoma cells from NKG2D-mediated killing by NK cells. ' 2008 Wiley-Liss, Inc.Key words: melanoma; MHC class I loss; natural killer cell; NKG2D; interferon-c Malignant melanoma is well characterized for its recognition by cytotoxic ab CD8 1 T lymphocytes (CTLs) that respond to tumorassociated peptide antigens presented in the complex with classical MHC class I surface molecules.1 Indeed, CTLs can eliminate metastatic tumors in melanoma patients, as demonstrated in different clinical trials of adoptive T cell transfer.2 The efficiency of tumor cell killing is determined by the strength of the antigen signal and co-stimulatory signals sensed by different activating receptors. Very recently, it was demonstrated that triggering of the co-stimulatory receptor NKG2D on CTLs strongly enhanced the T cells capacity to kill melanoma cells. 3Besides the importance of antigen-specific CTLs in immune surveillance of melanoma, one can assume that early detection and elimination of premalignant and malignant cutaneous melanocytes might be a major task of skin-resident innate NK cells and innate-like Vd1 gd T lymphocytes. 4 Both effectors are capable of preventing and inhibiting the growth of autologous human melanoma grafted into the skin of severe combined immunodeficient (SCID) mice.5 Interestingly, the receptor NKG2D is constitutively expressed on Vd1 gd T cells present in normal human sk...

Activating mutations in the TERT promoter were recently identified in up to 71% of cutaneous melanoma. Subsequent studies found TERT promoter mutations in a wide array of other major human cancers. TERT promoter mutations lead to increased expression of telomerase, which maintains telomere length and genomic stability, thereby allowing cancer cells to continuously divide, avoiding senescence or apoptosis. TERT promoter mutations in cutaneous melanoma often show UV-signatures. Non-melanoma skin cancer, including basal cell carcinoma and squamous cell carcinoma, are very frequent malignancies in individuals of European descent. We investigated the presence of TERT promoter mutations in 32 basal cell carcinomas and 34 cutaneous squamous cell carcinomas using conventional Sanger sequencing. TERT promoter mutations were identified in 18 (56%) basal cell carcinomas and in 17 (50%) cutaneous squamous cell carcinomas. The recurrent mutations identified in our cohort were identical to those previously described in cutaneous melanoma, and showed a UV-signature (C>T or CC>TT) in line with a causative role for UV exposure in these common cutaneous malignancies. Our study shows that TERT promoter mutations with UV-signatures are frequent in non-melanoma skin cancer, being present in around 50% of basal and squamous cell carcinomas and suggests that increased expression of telomerase plays an important role in the pathogenesis of these tumors.

Topical tacrolimus ointment 0.1% was safe and effective for the treatment of long-standing active lichen sclerosus.