An Endogenous Electron Spin Resonance (ESR) Signal Discriminates Nevi from Melanomas in Human Specimens: A Step Forward in Its Diagnostic Application

et al. (2012) An Endogenous Electron Spin Resonance (ESR) Signal Discriminates Nevi from Melanomas in Human Specimens: A Step Forward in Its Diagnostic Application. PLoS ONE 7(11): e48849. doi:10.1371/journal.pone.0048849 An Endogenous Electron Spin Resonance (ESR) Signal Discriminates Nevi from Melanomas in Human Specimens: A Step Forward in Its Diagnostic Application Eleonora Cesareo 0 Liudmila Korkina 0 Gerardino D'Errico 0 Giuseppe Vitiello 0 Maria Simona Aguzzi 0 Francesca Passarelli 0 Jens Z. Pedersen 0 Antonio Facchiano 0 Soheil S. Dadras, University of Connecticut Health Center, United States of America 0 1 Laboratory of Tissue Engineering & Skin Pathophysiology, Istituto Dermopatico dell'Immacolata IDI-IRCCS , Rome , Italy , 2 Department of Chemical Sciences, University of Naples ''Federico II'' , Naples, Italy, 3 Consorzio per lo Sviluppo dei Sistemi a Grande Interfase (CSGI), Florence , Italy , 4 Laboratory of Vascular Pathology, Istituto Dermopatico dell'Immacolata (IDI-IRCCS) , Rome , Italy , 5 Histopathology Service, Istituto Dermopatico dell'Immacolata (IDI-IRCCS) , Rome , Italy , 6 Department of Biology, University of Tor Vergata , Rome , Italy Given the specific melanin-associated paramagnetic features, the Electron Spin Resonance (ESR, called also Electron Paramagnetic Resonance, EPR) analysis has been proposed as a potential tool for non-invasive melanoma diagnosis. However, studies comparing human melanoma tissues to the most appropriate physiological counterpart (nevi) have not been performed, and ESR direct correlation with melanoma clinical features has never been investigated. ESR spectrum was obtained from melanoma and non-melanoma cell-cultures as well as mouse melanoma and non-melanoma tissues and an endogenous ESR signal (g = 2.005) was found in human melanoma cells and in primary melanoma tissues explanted from mice, while it was always absent in non-melanoma samples. These characteristics of the measured ESR signal strongly suggested its connection with melanin. Quantitative analyses were then performed on paraffin-embedded human melanoma and nevus sections, and validated on an independent larger validation set, for a total of 112 sections (52 melanomas, 60 nevi). The ESR signal was significantly higher in melanomas (p = 0.0002) and was significantly different between ''Low Breslow's and ''High Breslow's'' depth melanomas (p,0.0001). A direct correlation between ESR signal and Breslow's depth, expressed in millimetres, was found (R = 0.57; p,0.0001). The eu/pheomelanin ratio was found to be significantly different in melanomas ''Low Breslow's'' vs melanomas ''High Breslow's'' depth and in nevi vs melanomas ''High Breslow's depth''. Finally, ROC analysis using ESR data discriminated melanomas sections from nevi sections with up to 90% accuracy and p,0.0002. In the present study we report for the first time that ESR signal in human paraffin-embedded nevi is significantly lower than signal in human melanomas suggesting that spectrum variations may be related to qualitative melanin differences specifically occurring in melanoma cells. We therefore conclude that this ESR signal may represent a reliable marker for melanoma diagnosis in human histological sections. - Funding: This study was supported in part by a grant from Italian Ministry of Health Contract (RF07 Onc-25/3) and by Progetto Oncoproteomica ItaliaUSA 527B/ 2A/5 and PON01_02433/2 from MIUR, to AF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Co-author Antonio Facchiano is a PLOS ONE Editorial Board member. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Skin melanoma is one of the most aggressive tumours in humans, showing high mortality at the metastatic stage, and increasing incidence worldwide. [1] Melanoma accounts for about 4% of skin cancers, causing however about 80% of skin cancerrelated deaths in western countries. Despite promising recent improvement at the therapeutic level, [26] surgical excision remains at this moment the most effective treatment at early stages, while therapeutic interventions have weak efficacy at advanced phases due to high metastatic potential and resistance to currently available therapies. [7]. Improving early diagnosis may therefore strongly affect melanoma-related mortality. Melanoma diagnosis routinely starts from non-invasive dermatoscopy- and epiluminescence-based skin inspection, to identify phenotypic features of the pigmented lesion. [8] Trained dermatologists still experience significant error-rate giving misdiagnosis and delay in treatment, and formal diagnosis still requires to be confirmed by histological analysis. [9] Hence, alternative non-invasive procedures are needed to improve the early non-invasive diagnostic accuracy. Several reports indicate melanogenesis as a key process in the melanoma biology. [10,11] Melanin synthesis involves an oxidation/reduction reactions chain leading to the synthesis of final organic polymers. The intermediate free radicals formed within such process [12] give melanin paramagnetic properties. [13] Besides free radicals, melanin may also contain or interact with metal ions and paramagnetic gases (dioxygen, nitric oxide) which also contribute to its paramagnetic properties. [14] ESR spectroscopy is the technique of choice to detect and to investigate free radicals. As such, it has long been used in melanin basic research since melanin ESR signal is stable, [1517] resistant to chemical degradation, [18] and different in eumelanin from pheomelanin. [19,20]. Previous studies investigated ESR spectra of melanoma tissues under different conditions, [2129] including formaline fixed-, or frozen-, or paraffin-embedded specimens. However, a large study investigating ESR spectra in human melanoma specimens compared to human nevus specimens is still lacking at this moment. The main goal of the present study was to provide strong support to the use of ESR spectroscopy as a reliable diagnostic help in melanoma management. To this aim we identified an endogenous ESR signal (g = 2.005) in melanoma and nonmelanoma human cell lines, then investigated this signal in mouse melanoma tissues. Finally, we investigated ESR signal in human melanoma specimens compared to human nevus specimens. A specific ESR signal was found in melanoma human tissues, significantly different from the one recorded in nevus paraffinembedded specimens; ROC analysis showed that ESR signal is able to discriminate human melanoma sections from nevi, with very high accuracy. Cell Cultures Five human melanoma cell lines from both primary and metastatic melanomas were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to the manufacturers instructions. SKMEL-28 (ATCC number HTB-72), SKMEL-2 (ATCC number HTB-68) and amelanotic C32 (ATCC number (...truncated)