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Detection of Mutations in the Mitogen-Activated Protein Kinase Pathway in Human Melanoma

¹ Divisions of Surgical Oncology,
² Medical Oncology,
³ Biometrics, and
⁴ Pharmacology, The University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey

Purpose: Recent studies suggest that activating point mutations in B-RAF may commonly occur in melanoma. We devised a method to detect point mutations in heterogeneous tissues containing both wild-type and mutant B-RAF and N-RAS genes by using site-directed mutagenesis to introduce new restrictions sites in the cDNA sequence when the specific point mutations are present. We used this technique to determine the incidence of mitogen-activated protein kinase (MAPK) mutations in human melanoma.

Experimental Design: We screened 85 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Western blotting was used to evaluate downstream up-regulation of the mitogen-activated protein kinase pathway in these tissues.

Results: Thirty-three samples (7 of 25 primaries, 15 of 25 regional metastases, 5 of 25 nodal metastases, and 6 of 10 distant metastases) harbored the V599E B-RAF mutation (39%), 12 contained a Q61R N-RAS mutation and 5 a Q61K N-RAS mutation. Western blotting with antiphosphorylated extracellular signal-regulated kinase 1/2 antibodies demonstrated up-regulation of the MAPK pathway in samples containing activating B-RAF or N-RAS mutations compared with wild-type samples. This method of detection was sensitive and specific with no false positives.

Conclusions: Activating mutations of the MAPK pathway were present in 60% of samples tested and caused activation of this cellular pathway that appears to be important in the pathogenesis of melanoma.

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