Ionship between the log odds and their values on the original (methylated RASSF1A) or logarithm (total cfDNA, integrity index 180/67 and BRAFV600E) scale was appropriate. As reported in Table 3, disease status was significantly associated with all the biomarkers in the JSI124 site logistic univariate analysis. Consequently the initial model of the logistic multivariate regression analysis was built by including all four biomarkers. As reported in Table 4, total cfDNA, integrity index 180/67 and methylated RASSF1A retained a statistically significant (p-value ,0.05) association with disease status in the multivariate final logistic model. The AUC values computed for each biomarker (univariate logistic model) indicated a weak/satisfactory level of predictive capability by ranging between 0.64 (BRAFV600E) to 0.85 (total cfDNA) (Table 3 and Figure 2). Of note for all the considered biomarkers the 95 Confidence Interval (95 CI) of the AUC fails to include the 0.5 value (i.e. absence of predictive capability). Overall, a good predictive capability was observed for the final logistic model with an AUC of 0.95 (95 CI: 0.91?.98) (TableFigure 5. Sequential approach. Diagnostic performance increment (in terms of AUC) achieved by moving 
from cfDNA alone (AUC = 0.85; 95 CI = 0.79?.92) to cfDNA and integrity index 180/67 (AUC = 0.89; 95 CI = 0.84?.95) and to cfDNA, integrity index 180/67 and methylated RASSF1A (AUC = 0.95; 95 CI = 0.91?.98). doi:10.1371/journal.pone.0049843.gCell-Free DNA Biomarkers in MelanomaParticular 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 (see Materials Methods). On the other hand, tumour contribution to cfDNA was assessed by quantifying BRAFV600E 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 14636-12-5 custom synthesis standard approach for the clinical validation of biomarkers for early detection [40] 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 (total cfDNA, integrity index 180/67 and methylated RASSF1A) 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 18204824 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 (5 years). However preliminary data (not shown),obtained in a subgroup of patients submitted to an additional blood draw 2 weeks after surgery, show a 23115181 decrease of the four biomarkers, suggesting the potential role of these test as useful tools for mon.Ionship between the log odds and their values on the original (methylated RASSF1A) or logarithm (total cfDNA, integrity index 180/67 and BRAFV600E) scale was appropriate. As reported in Table 3, disease status was significantly associated with all the biomarkers in the logistic univariate analysis. Consequently the initial model of the logistic multivariate regression analysis was built by including all four biomarkers. As reported in Table 4, total cfDNA, integrity index 180/67 and methylated RASSF1A retained a statistically significant (p-value ,0.05) association with disease status in the multivariate final logistic model. The AUC values computed for each biomarker (univariate logistic model) indicated a weak/satisfactory level of predictive capability by ranging between 0.64 (BRAFV600E) to 0.85 (total cfDNA) (Table 3 and Figure 2). Of note for all the considered biomarkers the 95 Confidence Interval (95 CI) of the AUC fails to include the 0.5 value (i.e. absence 
of predictive capability). Overall, a good predictive capability was observed for the final logistic model with an AUC of 0.95 (95 CI: 0.91?.98) (TableFigure 5. Sequential approach. Diagnostic performance increment (in terms of AUC) achieved by moving from cfDNA alone (AUC = 0.85; 95 CI = 0.79?.92) to cfDNA and integrity index 180/67 (AUC = 0.89; 95 CI = 0.84?.95) and to cfDNA, integrity index 180/67 and methylated RASSF1A (AUC = 0.95; 95 CI = 0.91?.98). doi:10.1371/journal.pone.0049843.gCell-Free DNA Biomarkers in MelanomaParticular 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 (see Materials Methods). On the other hand, tumour contribution to cfDNA was assessed by quantifying BRAFV600E 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 [40] 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 (total cfDNA, integrity index 180/67 and methylated RASSF1A) 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 18204824 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 (5 years). However preliminary data (not shown),obtained in a subgroup of patients submitted to an additional blood draw 2 weeks after surgery, show a 23115181 decrease of the four biomarkers, suggesting the potential role of these test as useful tools for mon.
