Analysis of circulating tumor cells (CTC) in the peripheral blood of cutaneous melanoma patients provides information on the metastatic process and potentially improves patient management. The isolation by size of epithelial tumor cells (ISET) is a direct method for CTC identification in which tumor cells are collected by filtration as a result of their large size. So far, ISET has been applied only to CTC detection from epithelial cancer patients, and the technique has never been applied to cutaneous melanoma patients. We herein investigated the presence of CTC by ISET in the peripheral blood of 140 subjects (87 with cutaneous melanomas, 10 subjects undergoing surgery for melanocytic nevi, 5 patients with non-melanoma skin tumors, and 38 healthy volunteers). The identification of the cells trapped in filters as CTC was supported by positivity for immunohistochemical markers and for tyrosinase mRNA by real-time RT-PCR. CTC were neither detected in the controls nor in the in situ melanoma group. In contrast, CTC were shown in 29% of patients with primary invasive melanoma and in 62.5% of metastatic melanoma patients (P<0.01). CTC detection correlated with the presence of mRNA tyrosinase in blood samples, assayed by real-time RT-PCR (P=0.001). CTC detection corroborated by suitable molecular characterization may assist in the identification and monitoring of more appropriate therapies in melanoma patients.

For LMS involving the skin, it is advisable to recognize and indicate in the histopathology report the depth of dermal and/or subcutaneous extension, since even minimal subcutaneous involvement may be associated with late local recurrences and/or distant metastases, and therefore appropriate and long-term follow-up is needed.

Expression of the Wilms' tumor suppressor WT1 has been demonstrated in a variety of tumors and tumor cell lines, e.g., in breast cancer and melanoma cell lines. Its role is controversial, with evidence for both tumor-promoting and tumor-suppressing activities. In this paper, we show that WT1 is expressed in malignant melanoma in >80% of the tumor cells, but not in normal skin or benign melanocytic nevi in vivo. We detected an unusual shift of WT1 isoforms towards WT1(+17AA/+KTS) in melanoma. WT1 shared an overlapping expression with proliferating nuclear cell antigen and with Nestin and Zyxin, which are involved in melanoma cell proliferation. To investigate whether WT1 is directly involved in melanoma cell proliferation, we made use of an RNAi approach in vitro. WT1 silencing significantly reduced the expression of Nestin and Zyxin and resulted in inhibition of melanoma cell proliferation as determined by a reduced BrdU incorporation. These findings suggest a direct role of WT1 in melanoma proliferation, which might be mediated via Nestin and Zyxin. Furthermore, expression of WT1 in vivo clearly discriminates between benign acquired nevi and malignant melanomas and appears to be correlated with melanocytic atypia and malignancy.

Signaling mediated by the Notch receptor governs tissue development during embryonal organogenesis, while in adult tissues it contributes to maintenance of cellular differentiation, proliferation and apoptosis. In addition, control by the Notch pathway of stem cell self-renewal and multi-potency points to an expanding role of Notch signaling in the progression of solid tumors. Notch and its ligands are abundantly expressed in the epidermis, where Notch signaling functions as a molecular switch that intervenes in cell transition between different skin layers during the epidermal differentiation process. More recent findings obtained in melanoma and non-melanoma skin cancers show that Notch signaling has a dual action (either as an oncogene or as a tumor suppressor), depending on the tumor cell type and the synchronous activation of other intracellular signaling mechanisms. In this Review, we highlight the pleiotropic roles of the Notch signaling pathway in normal skin homeostasis and differentiation and focus on its altered regulation in the tumorigenesis of melanoma and non-melanoma skin cancer. Further understanding of the roles of Notch signaling in specific skin cancer types may provide a rationale for novel Notch-based therapeutic strategies.

Cutaneous melanoma preferentially metastasizes via the lymphatic route. However, the mechanisms of lymphatic invasion and metastasis to regional lymph nodes are poorly understood. Nitric oxide is a free radical molecule synthesized from L-arginine by nitric oxide synthases that plays a critical role in various physiological and pathological processes, including tumor growth and angiogenesis. We have tested whether inducible nitric oxide synthase expression correlates with lymphatic vessel density identified with D2-40 antibody and/or blood microvessel density identified with CD105/endoglin in a series of melanocytic nevi (n ¼ 28) and cutaneous melanomas (n ¼ 38), representative of various pT. Inducible nitric oxide synthase expression was significantly lower in melanocytic nevi in comparison with primary and metastatic melanomas (Po0.001). Mean microvessel density was significantly higher in primary and metastatic melanomas in comparison with melanocytic nevi (Po0.001 for intratumoral and P ¼ 0.001 for peritumoral vessels). Vertical growth phase melanomas showed a higher intratumoral microvessel density in comparison with radial growth phase melanomas (P ¼ 0.02). The number of peritumoral lymphatics was significantly lower in nevi as compared with primary and metastatic melanomas (P ¼ 0.01). No correlation between microvessel or lymphatic vessel and clinical outcome was found in melanomas. A significant direct correlation was observed between inducible nitric oxide synthase immunostaining in melanocytic tumor cells and the density of lymphatic vessels (peritumoral: P ¼ 0.001; intratumoral: P ¼ 0.08), and the density of peritumoral blood microvessel (P ¼ 0.02). Our findings support the hypothesis that inducible nitric oxide synthase is implicated not only in blood, but also in lymphatic vascular neoformation in melanoma. Mechanistic studies are needed to address the possibility that inducible nitric oxide synthase controls lymphangiogenesis, dissemination and lymphatic borne metastases. Keywords: iNOS; CD105; D2-40; microvessel density; melanoma; nevi Cutaneous melanoma metastasizes at least initially through the lymphatic route and lymph node metastasis is currently still a major determinant for patients staging and clinical management. The mechanisms of lymphatic invasion and metastasis to regional lymph nodes are mostly unknown and whether human tumors promote de novo lymphangiogenesis is still unclear. Although the pathways that regulate tumorrelated angiogenesis have been partially investigated, poor information is available on the processes that result in lymphangiogenesis in melanoma.Nitric oxide (NO) is a diatomic free radical molecule synthesized from L-arginine by NO synthases (NOS) that plays a critical role in various physiological and pathological processes, including tumor growth. Although the Ca 2 þ -dependent isoforms NOS1 and NOS3 are constitutively expressed by neural and endothelial cells, the Ca 2 þ -independent form (NOS2 or iNOS) can be induced by many cell types upon stimulation by inflamma...