Tracing Dissemination of Melanoma Cells in Healthy Tissues

In the western world, melanoma incidence has constantly risen for the last 50 years, and it is currently reported as the most frequent tumor in 20 - 29 year old women. BRAF, NRAS and c-kit genes play an important role in cell proliferation and are mutated at very high frequency in melanoma. Despite the recent therapeutical breakthroughs obtained with the use of new drugs, metastatic melanoma remains still a life threatening disease. One of the main questions in melanoma concern the initial steps leading to metastatic spread, a better understanding being a key step to its prevention and the identification of new molecular mechanisms being implemental to the improvement of our therapeutical arsenal. The proposed work aims to study the hypothesis of early spread in human melanoma using the recently developed powerful techniques of e-ice cold PCR, as well as classical immunohistochemistry. To do so, investigators will take advantage on the fact that treatment of melanoma relies on a secondary excision of normal peritumoral skin and sentinel lymph node. This peritumoral tissue is large (measuring 2 to 6 cm diameter), contains lymphatics in the hypodermis, the tissue considered to host the metastatic route of melanocytes and remains partially available for analysis. All patients with stage Ib and II melanoma followed in the parisian cohort Melan-cohort, Cochin Hospital and Gustave Roussy Institute included between 2005 and 2009 will be found. A PCR analysis will be done on DNA extracted from paraffin embedded sections of primary tumors. Patients who display mutations in BRAF (BRAFV600E, BRAFV600K), NRAS (codon 61) or c-kit genes will be selected. The archival paraffin embedded tissues from healthy perilesional skin as well as from healthy sentinel lymph nodes will be obtained. Pyrosequencing and e-ice cold PCR targeting the mutations of the above genes at their usual positions will be done on DNA extracted from these specimens. Immunofluorescence anti-BRAFV600E or anti tumoral/initiating/stem cells will be done on same tissues. These simple techniques will test -using a sensitive molecular biology tool- whether in humans with melanomas, there is early dissemination of melanoma cells in histopathological healthy sentinel lymph node and peritumoral skin. The presence of these clonal cells in these healthy tissues will be correlated to the survival of the patients after 5 years and will allow the development of new therapeutic follow-up and strategies.