Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement

Circulating miR-1246 and miR-485-3p as Promising Biomarkers of Clinical Response and Outcome in Melanoma Patients Treated with Targeted Therapy

Despite the significant improvements in advanced melanoma therapy, there is still a pressing need for biomarkers that can predict patient response and prognosis, and therefore support rational treatment decisions. Here, we investigated whether circulating miRNAs could be biomarkers of clinical outcomes in patients treated with targeted therapy. Using next-generation sequencing, we profiled plasma miRNAs at baseline and at progression in patients treated with BRAF inhibitors (BRAFi) or BRAFi + MEKi. Selected miRNAs associated with response to therapy were subjected to validation by real-time quantitative RT-PCR. Receiver Operating Characteristics (ROC), Kaplan–Meier and univariate and multivariate Cox regression analyses were performed on the validated miR-1246 and miR-485-3p baseline levels. The median baseline levels of miR-1246 and miR-485-3p were significantly higher and lower, respectively, in the group of patients not responding to therapy (NRs) as compared with the group of responding patients (Rs). In Rs, a trend toward an increase in miR-1246 and a decrease in miR-485-3p was observed at progression. Baseline miR-1246 level and the miR-1246/miR-485-3p ratio showed a good ability to discriminate between Rs and NRs. Poorer PFS and OS were observed in patients with unfavorable levels of at least one miRNA. In multivariate analysis, a low level of miR-485-3p and a high miR-1246/miR-485-3p ratio remained independent negative prognostic factors for PFS, while a high miR-1246/miR-485-3p ratio was associated with an increased risk of mortality, although statistical significance was not reached. Evaluation of miR-1246 and miR-485-3p baseline plasma levels might help clinicians to identify melanoma patients most likely to be unresponsive to targeted therapy or at higher risk for short-term PFS and mortality, thus improving their management

Heart failure in Eastern Veneto: prevalence, hospitalization rate, adherence to guidelines and social costs

Heart failure is a preminent problem of public health, requiring innovating methods of health services organization. Nevertheless, data are still not available on prevalence, hospitalization rate, adherence to Guidelines and social costs in the general Italian population. The necessity to identifying patients with heart failure derives from the efficacy of new therapeutic interventions in reducing morbidity and mortality. In this study we aimed to identify, in a subset of the Eastern Veneto population, patients with heart failure through a pharmacologic-epidemiologic survey. The study was divided in 5 phases: 1) identification of patients on furosemide in the year 2000 in the ASL 10 of Eastern Veneto general population, through an analysis of a specific pharmaceutic service database; 2) definition of the actual prevalence of heart failure in a casual sample of these patients, through data base belonging to general practitioners, cardiologists, or others. Diagnosis was based on the following criteria: a) previous diagnosis of heart failure; b) previous hospitalization for heart failure; c) clinical evidence, with echocardiographic control in unclear cases; 3) survey of hospitalizations; 4) evaluation of adhesion to guidelines, through both databases and questionnaires; 5) analysis of the social costs of the disease, with a retrospective “bottom up” approach. From a total population of 198.000 subjects, we identyfied 4502 patients on furosemide. In a casual sample of 10.661 subjects we defined a prevalence of heart failure in Eastern Veneto of 1.1%, that rised to 7.1% in octuagenarians. The prescription of life saving drugs was satisfactory, while rather poor was the indication to echocardiography and to cardiologic consultation. Hospitalization rate for DRG 127 was low: 2.1/1000 inhabitants/year in the general polulation and 12.5 /1000 inhabitants/year in patients >70 years of age. Yearly mortality was 10.3%. Social costs were elevated (15.394 €/patient/year), due to a relevant sanitary component (hospital 53%, drugs 28%) and particularly a to an indirect cost component. In conclusion, the assumption of furosemide lends itself as a good marker for identifying patients with heart failure. Patient identification is simple, cheap and cost-efficient, and can be easily reproduced in other regional areas