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

30 Scopus citations

Abstract

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
Volume9
Issue number17
StatePublished - Dec 15 2003

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

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