Ines Esteves Domingues Pires Da Silva

Reversal of NK-Cell Exhaustion in Advanced Melanoma by Tim-3 Blockade

Silva and colleagues show that Tim-3 expression in NK cells from melanoma patients correlates with poor prognosis; blockade of Tim-3 induces NK-cell proliferative and cytolytic capacity and responsiveness to growth factors, indicating that Tim-3 is a therapeutic target for patients with advanced melanoma. The immunoregulatory protein T-cell immunoglobulin- and mucin-domain–containing molecule-3 (Tim-3) mediates T-cell exhaustion and contributes to the suppression of immune responses in both viral infections and tumors. Tim-3 blockade reverses the exhausted phenotype of CD4+ and CD8+ T cells in several chronic diseases, including melanoma. Interestingly, natural killer (NK) cells constitutively express Tim-3; however, the role of Tim-3 in modulating the function of these innate effector cells remains unclear, particularly in human diseases. In this study, we compared the function of Tim-3 in NK cells from healthy donors and patients with metastatic melanoma. NK cells from the latter were functionally impaired/exhausted, and Tim-3 blockade reversed this exhausted phenotype. Moreover, Tim-3 expression levels were correlated with the stage of the disease and poor prognostic factors. These data indicate that Tim-3 can function as an NK-cell exhaustion marker in advanced melanoma and support the development of Tim-3–targeted therapies to restore antitumor immunity. Cancer Immunol Res; 2(5); 410–22. ©2014 AACR.

Serum‐based miRNAs in the prediction and detection of recurrence in melanoma patients

Identification of primary melanoma patients at the highest risk of recurrence remains a critical challenge, and monitoring for recurrent disease is limited to costly imaging studies. We recently reported our array‐based discovery of prognostic serum miRNAs in melanoma. In the current study, we examined the clinical utility of these serum‐based miRNAs for prognosis as well as detection of melanoma recurrence.

Reversal of natural killer cell exhaustion by TIM-3 blockade.

Natural killer (NK) cells are innate immune cells that become progressively exhausted in advanced stage cancer, crippling their ability to execute antitumor functions. We previously characterized the nature of NK cell exhaustion in metastatic melanoma patients, reporting a correlation with high expression of TIM-3. Blockade of this immune checkpoint molecule reversed the exhausted phenotype and improved NK cell function.

RSK1 activation promotes invasion in nodular melanoma

Materials and methods Expression of 141 signaling proteins was evaluated by protein pathway array in 3 Radial Growth Phase (RGP)/ SSM and 3 Vertical Growth Phase (VGP)/NM cell lines. The impact of p90-ribosomal-S6-kinase (RSK1) and its inhibition on proliferation, migration and invasion was assessed in SSM and NM cell lines, and confirmed using NM cells treated with a RSK inhibitor (BI-D1870) in microarray profiling studies. The effect of constitutive RSK1 activation in vivo was further studied using a zebrafish model.

Genetic associations of the interleukin locus at 1q32.1 with clinical outcomes of cutaneous melanoma

Background Due to high melanoma immunogenicity, germline genetic variants in immune pathways have been studied for association with melanoma prognosis. However, limited candidate selection, inadequate power, or lack of independent validation have hampered the reproducibility of these prior findings, preventing personalised clinical applicability in melanoma prognostication. Our objective was to assess the prognostic utility of genetic variants in immunomodulatory pathways for prediction of melanoma clinical outcomes. Methods We genotyped 72 tag single nucleotide polymorphisms (SNPs) in 44 immunomodulatory genes in a population sample of 1022 melanoma patients and performed Cox regression analysis to test the association between SNPs and melanoma recurrence-free (RFS) and overall survival (OS). We have further investigated the most significant associations using a fine mapping strategy and followed with functional analyses in CD4+ T cells in a subset of 75 melanoma patients. Results The most significant associations were found with melanoma OS for rs3024493 in IL10 at chromosome 1q32.1 (heterozygous HR 0.58, 95% CI 0.39 to 0.86; p=0.0006), a variant previously shown to be linked with autoimmune conditions. Multiple additional SNPs at 1q32.1 were also nominally associated with OS confirming at least two independent association signals in this locus. In addition, we found rs3024493 associated with the downregulation of interleukin 10 (IL10) secretion in CD4+ T cells. Conclusions We discovered novel associations of IL10 with melanoma survival at 1q32.1, suggesting this locus should be considered as a novel melanoma prognostic biomarker with potential for aiding melanoma patient management. Our findings also provide further support for an alternative role of IL10 in stimulation of anti-tumour immune response.

Enhanced immunohistochemical detection of neural infiltration in primary melanoma: is there a clinical value?

Neural infiltration in primary melanoma is a histopathologic feature that has been associated with desmoplastic histopathologic subtype and local recurrence in the literature. We tested the hypothesis that improved detection and characterization of neural infiltration into peritumoral or intratumoral location and perineural or intraneural involvement could have a prognostic relevance. We studied 128 primary melanoma cases prospectively accrued and followed at New York University using immunohistochemical detection with antihuman neurofilament protein and routine histology with hematoxylin and eosin. Neural infiltration, defined as the presence of tumor cells involving or immediately surrounding nerve foci, was identified and characterized using both detection methods. Neural infiltration rate of detection was enhanced by immunohistochemistry for neurofilament in matched-pair design (47% by immunohistochemistry versus 25% by routine histology). Immunohistochemical detection of neural infiltration was significantly associated with ulceration (P = .021), desmoplastic and acral lentiginous histologic subtype (P = .008), and head/neck/hands/feet tumor location (P = .037). Routinely detected neural infiltration was significantly associated with local recurrence (P = .010). Immunohistochemistry detected more intratumoral neural infiltration cases compared with routine histology (30% versus 3%, respectively). Peritumoral and intratumoral nerve location had no impact on clinical outcomes. Using a multivariate model controlling for stage, neither routinely detected neural infiltration nor enhanced immunohistochemical characterization of neural infiltration was significantly associated with disease-free or overall survival. Our data demonstrate that routinely detected neural infiltration is associated with local recurrence in all histologic subtypes but that improved detection and characterization of neural infiltration with immunohistochemistry in primary melanoma does not add to prognostic relevance.

Melanoma expression of matrix metalloproteinase-23 is associated with blunted tumor immunity and poor responses to immunotherapy

BackgroundMatrix metalloproteinase-23 (MMP-23) can block the voltage-gated potassium channel Kv1.3, whose function is important for sustained Ca2+ signaling during T cell activation. MMP-23 may also alter T cell activity and phenotype through cleavage of proteins affecting cytokine and chemokine signaling. We therefore tested the hypothesis that MMP-23 can negatively regulate the anti-tumor T cell response in human melanoma.MethodsWe characterized MMP-23 expression in primary melanoma patients who received adjuvant immunotherapy. We examined the association of MMP-23 with the anti-tumor immune response - as assessed by the prevalence of tumor-infiltrating lymphocytes and Foxp3+ regulatory T cells. Further, we examined the association between MMP-23 expression and response to immunotherapy. Considering also an in trans mechanism, we examined the association of melanoma MMP-23 and melanoma Kv1.3 expression.ResultsOur data revealed an inverse association between primary melanoma MMP-23 expression and the anti-tumor T cell response, as demonstrated by decreased tumor-infiltrating lymphocytes (TIL) (P = 0.05), in particular brisk TILs (P = 0.04), and a trend towards an increased proportion of immunosuppressive Foxp3+ regulatory T cells (P = 0.07). High melanoma MMP-23 expression is also associated with recurrence in patients treated with immune biologics (P = 0.037) but not in those treated with vaccines (P = 0.64). Further, high melanoma MMP-23 expression is associated with shorter periods of progression-free survival for patients receiving immune biologics (P = 0.025). On the other hand, there is no relationship between melanoma MMP-23 and melanoma Kv1.3 expression (P = 0.27).ConclusionsOur data support a role for MMP-23 as a potential immunosuppressive target in melanoma, as well as a possible biomarker for informing melanoma immunotherapies.

Plasmacytoid dendritic cells lead the charge against tumors.

Imiquimod is a TLR agonist that is used as an antitumor agent, mainly against skin tumors. Its clinical benefits are well described in several studies; however, the mechanisms behind its antitumor effects are not completely understood. In this issue of the JCI, Drobits and colleagues demonstrate that topical application of imiquimod suppresses cutaneous melanoma by TLR7-dependent recruitment and transformation of plasmacytoid dendritic cells into killer cells; this occurs independently of conventional adaptive immune mechanisms.

Abstract 5410: Tim-3 expression and function in natural killer cells from metastatic melanoma patients

BACKGROUND: Tim-3 (T-cell immunoglobulin domain and mucin domain 3) is a type-I glycoprotein receptor with pivotal roles in immune regulation and tolerance induction. Tim-3 has opposing roles in innate and adaptive immunity. It synergizes with TLR signaling in DCs inducing the production of inflammatory cytokines, while it acts as a negative regulator of Th1/Tc1 cell function by triggering T cell apoptosis upon interaction with its ligand, galectin-9. Tim-3 is also responsible for the functional exhaustion of T cells observed in some chronic infectious diseases and tumors, such as metastatic melanoma (MM). Natural Killer (NK) cells, innate immune cells which eliminate tumors and infected cells through cytotoxicity and IFNγ production, constitutively express Tim-3. Moreover, it has been described that some tumor cells (including melanoma cells) express and secrete galectin-9. However, little is known about the role of Tim-3 on NK cells, particularly in the presence of galectin-9, in healthy or melanoma patients. METHODS: Purified NK cells from PBMCs of 20 melanoma donors (MD) and 40 healthy donors (HD) were characterized according to the expression of Tim-3 by flow cytometry. Lamp-1 expression on NK cell surface was used to measure NK cell cytotoxicity against the following target cells: i) K562 cells which don9t express Galectin-9 and ii) Gmel galectin-9+ and Gmel galectin-9- sorted melanoma cells. IFNγ production was assessed by intracellular staining after 4 hours culture in presence of IL-12. RESULTS: MD NK cells express higher levels of Tim-3 compared to HD NK cells (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5410. doi:1538-7445.AM2012-5410

bcGST—an interactive bias-correction method to identify over-represented gene-sets in boutique arrays

Motivation Gene annotation and pathway databases such as Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes are important tools in Gene‐Set Test (GST) that describe gene biological functions and associated pathways. GST aims to establish an association relationship between a gene‐set of interest and an annotation. Importantly, GST tests for over‐representation of genes in an annotation term. One implicit assumption of GST is that the gene expression platform captures the complete or a very large proportion of the genome. However, this assumption is neither satisfied for the increasingly popular boutique array nor the custom designed gene expression profiling platform. Specifically, conventional GST is no longer appropriate due to the gene‐set selection bias induced during the construction of these platforms. Results We propose bcGST, a bias‐corrected GST by introducing bias‐correction terms in the contingency table needed for calculating the Fishers Exact Test. The adjustment method works by estimating the proportion of genes captured on the array with respect to the genome in order to assist filtration of annotation terms that would otherwise be falsely included or excluded. We illustrate the practicality of bcGST and its stability through multiple differential gene expression analyses in melanoma and the Cancer Genome Atlas cancer studies. Availability and implementation The bcGST method is made available as a Shiny web application at http://shiny.maths.usyd.edu.au/bcGST/. Supplementary information Supplementary data are available at Bioinformatics online.