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

Janivette Alsina, David H. Gorski, F. Joseph Germino, Weichung Shih, Shou En Lu, Zhi Gang Zhang, Jin Ming Yang, William N. Hait, James S. Goydos

Research output: Contribution to journalArticlepeer-review

32 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)6419-6425
Number of pages7
JournalClinical Cancer Research
Issue number17
StatePublished - Dec 15 2003

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research


Dive into the research topics of 'Detection of Mutations in the Mitogen-Activated Protein Kinase Pathway in Human Melanoma'. Together they form a unique fingerprint.

Cite this