A cell line at passage 5th was lost at passage 10th. B) Control cells lines (i.e. non- melanoma cell lines) showing no ESR signal. DPPH arrow indicates the position of the standard free radical signal (1, 1-diphenyl-2-picrylhydrazyl). doi:10.1371/journal.pone.0048849.gNevus and melanoma samples of the “All set” were divided in subgroups according to sex and lesion body location (“Trunk”, “Limbs” and “Head and Neck”). Mann-Whitney Test revealed that in all subgroups (except “Limbs” location) a significantly different signal was found between nevi and melanomas (p#0.05). The superimposition of the selected peak of 8 nevi and 8 melanomas is reported in Figure S1. Additional statistical analyses were carried out within melanomas subgroups. Each subgroup was classified according to tumour thickness, (“High” or “Low” Breslow’s depth) (Table 1), i.e. a parameter AN 3199 chemical information strongly related to the prognosis, being “High Breslow” associated to a worse prognosis. The ESR signal was significantly higher in samples with “High Breslow” in all melanomas subgroups (p,0.05) except “Limbs” (Fig. 4A).An additional ANOVA analysis confirmed the highly significant difference of the melanomas ESR signal with “High Breslow’s depth” vs nevi and melanomas “Low Breslow” (Fig. 4B). All calculations reported in Fig. 3 and Fig. 4 were carried out on amplitudes values; each calculation has also been performed on double-integral values reaching almost superimposable results as compared to amplitudes (Fig. 5). A correlation analysis by Spearman Test carried out in the 52 melanoma samples indicated a strongly significant correlation (R = 0.57; p,0.0001) between ESR signal amplitude and the corresponding Breslow’s depth value expressed in millimetres. Similar results were observed using integral values (R 
= 0.42; p = 0.002). The variation of the eumelanin/pheomelanin ratio (a/b) (see methods) was also investigated indicating a significant difference ofMelanoma Diagnosis via Electron Spin ResonanceFigure 2. ESR spectra of murine- and human- melanoma and healthy tissues. A) Murine B16F10 melanoma cells were injected in 5 mice in order to produce primary melanomas. Mice were sacrificed 14 days after the cell injection and tumours were collected for ESR analysis. The spectra show the presence of a strong signal located at the same position as observed in human melanoma cells. Signal was stable over time (recorded after 2 hours and after 14 days upon frozen storage). B) Murine tissues from liver, kidney and heart do not show ESR signal in the same magnetic field range. C) ESR spectra of formalin-fixed paraffin-embedded sections of human melanoma, human nevus tissue and fresh mouse melanoma tissue. DPPH arrow indicates the position of the standard free radical signal (1, 1-diphenyl-2-picrylhydrazyl). doi:10.1371/journal.pone.0048849.gmelanomas “Low Breslow” vs “High Breslow” melanomas (p,0.004) and nevi vs “High Breslow” melanomas. (p,0.009) ANOVA analysis carried out on a/b ratio confirmed a significant difference (Fig. 4C). ROC analysis was then carried out to test the ability of ESR signal to discriminate nevi from melanomas in paraffin-embedded sections. The computed area under the ROC curve quantifies the ability to discriminate controls from melanoma Rubusoside site patients taking into account both sensitivity and specificity. A value of 1 indicates the ability to discriminate 100 of patients from controls and corresponds to a curve mostly left-shifted in the graph.A cell line at passage 5th was lost at passage 10th. B) Control cells lines (i.e. non- melanoma cell lines) showing no ESR signal. DPPH arrow indicates the position of the standard free radical signal (1, 1-diphenyl-2-picrylhydrazyl). doi:10.1371/journal.pone.0048849.gNevus and melanoma samples of the “All set” were divided in subgroups according to sex and lesion body location (“Trunk”, “Limbs” and “Head and Neck”). Mann-Whitney Test revealed that in all subgroups (except “Limbs” location) a significantly different signal was found between nevi and melanomas (p#0.05). The superimposition of the selected peak of 8 nevi and 8 melanomas is reported in Figure S1. Additional statistical analyses were carried out within melanomas subgroups. Each subgroup was classified according to tumour thickness, (“High” or “Low” Breslow’s depth) (Table 1), i.e. a parameter strongly related to the prognosis, being “High Breslow” associated to a worse prognosis. The ESR signal was significantly higher in samples with “High Breslow” in all melanomas subgroups (p,0.05) except “Limbs” (Fig. 4A).An additional ANOVA analysis confirmed the highly significant difference of the melanomas ESR signal 
with “High Breslow’s depth” vs nevi and melanomas “Low Breslow” (Fig. 4B). All calculations reported in Fig. 3 and Fig. 4 were carried out on amplitudes values; each calculation has also been performed on double-integral values reaching almost superimposable results as compared to amplitudes (Fig. 5). A correlation analysis by Spearman Test carried out in the 52 melanoma samples indicated a strongly significant correlation (R = 0.57; p,0.0001) between ESR signal amplitude and the corresponding Breslow’s depth value expressed in millimetres. Similar results were observed using integral values (R = 0.42; p = 0.002). The variation of the eumelanin/pheomelanin ratio (a/b) (see methods) was also investigated indicating a significant difference ofMelanoma Diagnosis via Electron Spin ResonanceFigure 2. ESR spectra of murine- and human- melanoma and healthy tissues. A) Murine B16F10 melanoma cells were injected in 5 mice in order to produce primary melanomas. Mice were sacrificed 14 days after the cell injection and tumours were collected for ESR analysis. The spectra show the presence of a strong signal located at the same position as observed in human melanoma cells. Signal was stable over time (recorded after 2 hours and after 14 days upon frozen storage). B) Murine tissues from liver, kidney and heart do not show ESR signal in the same magnetic field range. C) ESR spectra of formalin-fixed paraffin-embedded sections of human melanoma, human nevus tissue and fresh mouse melanoma tissue. DPPH arrow indicates the position of the standard free radical signal (1, 1-diphenyl-2-picrylhydrazyl). doi:10.1371/journal.pone.0048849.gmelanomas “Low Breslow” vs “High Breslow” melanomas (p,0.004) and nevi vs “High Breslow” melanomas. (p,0.009) ANOVA analysis carried out on a/b ratio confirmed a significant difference (Fig. 4C). ROC analysis was then carried out to test the ability of ESR signal to discriminate nevi from melanomas in paraffin-embedded sections. The computed area under the ROC curve quantifies the ability to discriminate controls from melanoma patients taking into account both sensitivity and specificity. A value of 1 indicates the ability to discriminate 100 of patients from controls and corresponds to a curve mostly left-shifted in the graph.