Finally the contribution of each variables to the predictive capability of the final model was investigated by comparing the AUC value in the model with that of the same model without the variable itself. The analysis of cfDNA may have the potential to complement or replace the existing cancer tissue and blood biomarkers in the future. In order to reach this goal, specific and sensitive analytical procedures must be developed and optimized to compute proper circulating target molecules showing differences between patients and healthy subjects. It is now widely accepted that a single biomarker cannot fully distinguish between controls and patients and consequently an approach based on different markers would be preferable in order to achieve a stronger predictive ability. It has been demonstrated that in prenatal screening, a combination of multiple markers, each with limited sensitivity and/or specificity, can lead to a more powerful screening test. Similarly, Schneider and Mizejewski suggest to develop a multi-marker screening approach for cancer diagnosis. Unfortunately this strategy has been proven unsuccessful, notwithstanding the high number of new biomarkers reported in the literature, even if some examples on prostate ovarian and colorectal cancer clearly showed that multi-marker screening can have its place in early cancer detection. The study presented here tests the diagnostic potential of four markers associated to cfDNA in identifying melanoma patients. Particular efforts were dedicated to the technical aspects of the methods adopted for each single parameter allowing to reach accurate and reproducible measurements. We evaluated total cfDNA concentration by a qPCR assay for the single copy gene APP, as well as DNA fragmentation represented by the integrity index 180 bp/67 bp. On the other hand, tumour contribution to cfDNA was assessed by quantifying BRAFV600E PLX-4720 918505-84-7 mutated alleles and RASSF1A promoter methylation. These markers have been used in a panel in all patients, thus representing a simple model potentially adoptable by any laboratory. Following the standard approach for the clinical validation of biomarkers for early detection the next step will be focused on the assessment of the impact of these biomarkers on clinical practice including the identification of the most suitable thresholds to use for the early detection of melanoma by clinicians. Our preliminary results show that by jointly considering the panel of biomarkers here investigated the highest predictive capability is given by total cfDNA followed by integrity index 180/ 67 and methylated RASSF1A. According to these results, an approach based on the simultaneous determination of the three biomarkers could be suggested to improve the diagnostic performance in melanoma. Alternatively, as reported in Figure 5, a more parsimonious sequential approach could be adopted using preselection by cfDNA, followed by further selection using integrity index 180/67 and/or methylated RASSF1A. We plan to evaluate the prognostic role of both these approaches as soon as the follow-up time of our case study will be adequate. However preliminary data, obtained in a subgroup of patients submitted to an additional blood draw 2 weeks after surgery, show a decrease of the four biomarkers, suggesting the potential role of these test as useful tools for monitoring patients after initial diagnosis/surgery. Even though each biomarker investigated in the present work is not exclusively associated with melanoma, their combination reveals a high specificity for melanoma detection. Wilson’s disease is an autosomal recessively inherited disorder that leads to copper accumulation and, consequently, to hepatic damage and neuropsychological symptoms. The causative mutations affect the copper-transporting P-type ATPase ATP7B.