Enrichment of CD56dimKIR+CD57+ highly cytotoxic NK cells in tumor infiltrated lymph nodes of melanoma patients

An important checkpoint in the progression of melanoma is the metastasis to lymph nodes. Here, to investigate the role of lymph node NK cells in disease progression, we analyze frequency, phenotype and functions of NK cells from tumor-infiltrated (TILN) and tumor-free ipsilateral lymph nodes (TFLN) of the same patients. We show an expansion of CD56dimCD57dimCD69+CCR7+KIR+ NK cells in TILN. TILN NK cells display robust cytotoxic activity against autologous melanoma cells. In the blood of metastatic melanoma patients the frequency of NK cells expressing the receptors for CXCL8 receptor is increased compared to healthy subjects, and blood NK cells also express the receptors for CCL2 and IL6. These factors are produced in high amount in TILN and in vitro switch the phenotype of blood NK cells from healthy donors to the phenotype associated with TILN. Our data suggest that the microenvironment of TILN generates and/or recruits a particularly effective NK cell subset

miR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators

BACKGROUND: Targeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance.METHODS: The miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy.RESULTS: miR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor.CONCLUSIONS: Deregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment. Video Abstract

Restricted Mean Survival Time and Cure-Rate Modeling in Estimating Relapse-Free Survival Benefit With Adjuvant Dabrafenib + Trametinib Treatment in Melanoma

Abstract not available

High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients

BACKGROUND: Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. METHODOLOGY/PRINCIPAL FINDINGS: We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504+/-315) or caveolin-1 (619+/-310) were significantly increased in melanoma patients as compared to healthy donors (223+/-125 and 228+/-102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. CONCLUSIONS/SIGNIFICANCE: We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients

Adjuvant Dabrafenib plus Trametinib in Stage III BRAF-Mutated Melanoma

BACKGROUND Combination therapy with the BRAF inhibitor dabrafenib plus the MEK inhibitor trametinib improved survival in patients with advanced melanoma with BRAF V600 mutations. We sought to determine whether adjuvant dabrafenib plus trametinib would improve outcomes in patients with resected, stage III melanoma with BRAF V600 mutations. METHODS In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned 870 patients with completely resected, stage III melanoma with BRAF V600E or V600K mutations to receive oral dabrafenib at a dose of 150 mg twice daily plus trametinib at a dose of 2 mg once daily (combination therapy, 438 patients) or two matched placebo tablets (432 patients) for 12 months. The primary end point was relapse-free survival. Secondary end points included overall survival, distant metastasis-free survival, freedom from relapse, and safety. RESULTS At a median follow-up of 2.8 years, the estimated 3-year rate of relapse-free survival was 58% in the combination-Therapy group and 39% in the placebo group (hazard ratio for relapse or death, 0.47; 95% confidence interval [CI], 0.39 to 0.58;

Sex-specific effect of RNASEL rs486907 and miR-146a rs2910164 polymorphisms' interaction as a susceptibility factor for melanoma skin cancer

The genetics of melanoma is complex and, in addition to environmental influences, numerous genes are involved or contribute toward melanoma predisposition. In this study, we evaluated the possible interaction between miR-146a and one of its putative targets ribonuclease L (RNASEL) in the risk of sporadic melanoma. Polymorphisms rs2910164 in miR-146a and rs486907 in the RNASEL gene have both independently been associated with the risk of different cancers, and an interaction between them has been observed in nonmelanoma skin cancer. Polymorphisms rs2910164 G/C and rs486907 A/G were genotyped by restriction fragment length polymorphism analysis in 304 sporadic melanoma patients and 314 control individuals. Genotype distribution between cases and controls for each of the two polymorphisms was compared using Fisher's exact test. Epistasis between the two polymorphisms was tested by a logistic regression model. In the present study, we observed a sex-specific effect of the miR-146a rs2910164 C allele restricted to individuals carrying the RNASEL rs486907 A allele as well. Men carrying this allelic combination have the highest risk of melanoma, whereas it seems to have no effect or even an opposite relationship to melanoma risk in the female population. The results reported in the present study suggest a sex-specific interaction between miR-146a and RNASEL genes in melanoma skin cancer susceptibility, and could account for possible discordant results in association studies when stratification according to sex is not performed

Enrichment of KIR+CD57+ highly cytotoxic NK cells in sentinel lymph nodes of melanoma patients

Background\ud NK cells contribute to melanoma cell recognition and anti-tumor immunity, which is traditionally analyzed using human peripheral blood NK cells. An important checkpoint in the progression of malignant melanoma is the metastasis to lymph nodes.\ud \ud Materials and methods\ud To investigate the role of lymph node NK cells in disease progression, we analyzed frequency, phenotype and functions of NK cells purified from either tumor infiltrated lymph nodes or tumor-free ipsilateral lymph nodes of the same patients. Lymph node NK cells were compared to peripheral blood NK cells from either melanoma patients or healthy donors.\ud \ud Results\ud The data showed an expansion of CD56dimCD57+CD69+CCR7+KIR+ NK cells in tumor infiltrated lymph nodes. This phenotype corresponds to a recently described fully mature and highly cytotoxic NK cell population, and indeed we found that these lymph node NK cells displayed robust anti-tumor activity against autologous melanoma cells. The NK cells trafficking from periphery to the tumor draining lymph nodes have been investigated and the chemokines pattern identified. Moreover, the presence of a high proportion of KIR+CD57+CD56dim in the infiltrated lymph nodes was associated with an improved patients’ survival.\ud \ud Conclusions\ud Our data suggest that NK cells from tumor infiltrated lymph nodes are attractive candidates to improve current NK cell-based immunotherapy of melanoma

Enrichment of CD56(dim)KIR + CD57 + highly cytotoxic NK cells in tumour-infiltrated lymph nodes of melanoma patients

An important checkpoint in the progression of melanoma is the metastasis to lymph nodes. Here, to investigate the role of lymph node NK cells in disease progression, we analyze frequency, phenotype and functions of NK cells from tumour-infiltrated (TILN) and tumour-free ipsilateral lymph nodes (TFLN) of the same patients. We show an expansion of CD56(dim)CD57(dim)CD69 + CCR7 +KIR+ NK cells in TILN. TILN NK cells display robust cytotoxic activity against autologous melanoma cells. In the blood of metastatic melanoma patients, the frequency of NK cells expressing the receptors for CXCL8 receptor is increased compared with healthy subjects, and blood NK cells also express the receptors for CCL2 and IL-6. These factors are produced in high amount in TILN and in vitro switch the phenotype of blood NK cells from healthy donors to the phenotype associated with TILN. Our data suggest that the microenvironment of TILN generates and/or recruits a particularly effective NK cell subset