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
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.