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 journalArticle

30 Citations (Scopus)

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

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Mitogen-Activated Protein Kinases
Melanoma
Mutation
Point Mutation
Neoplasm Metastasis
Site-Directed Mutagenesis
Up-Regulation
Western Blotting
Mitogen-Activated Protein Kinase 3
Mitogen-Activated Protein Kinase 1
Research Design
Complementary DNA
Antibodies
Incidence
Genes

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

Cite this

Alsina, J., Gorski, D. H., Germino, F. J., Shih, W., Lu, S. E., Zhang, Z. G., ... Goydos, J. S. (2003). Detection of Mutations in the Mitogen-Activated Protein Kinase Pathway in Human Melanoma. Clinical Cancer Research, 9(17), 6419-6425.
Alsina, Janivette ; Gorski, David H. ; Germino, F. Joseph ; Shih, Weichung ; Lu, Shou En ; Zhang, Zhi Gang ; Yang, Jin-Ming ; Hait, William N. ; Goydos, James S. / Detection of Mutations in the Mitogen-Activated Protein Kinase Pathway in Human Melanoma. In: Clinical Cancer Research. 2003 ; Vol. 9, No. 17. pp. 6419-6425.
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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.",
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Alsina, J, Gorski, DH, Germino, FJ, Shih, W, Lu, SE, Zhang, ZG, Yang, J-M, Hait, WN & Goydos, JS 2003, 'Detection of Mutations in the Mitogen-Activated Protein Kinase Pathway in Human Melanoma', Clinical Cancer Research, vol. 9, no. 17, pp. 6419-6425.

Detection of Mutations in the Mitogen-Activated Protein Kinase Pathway in Human Melanoma. / Alsina, Janivette; Gorski, David H.; Germino, F. Joseph; Shih, Weichung; Lu, Shou En; Zhang, Zhi Gang; Yang, Jin-Ming; Hait, William N.; Goydos, James S.

In: Clinical Cancer Research, Vol. 9, No. 17, 15.12.2003, p. 6419-6425.

Research output: Contribution to journalArticle

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T1 - Detection of Mutations in the Mitogen-Activated Protein Kinase Pathway in Human Melanoma

AU - Alsina, Janivette

AU - Gorski, David H.

AU - Germino, F. Joseph

AU - Shih, Weichung

AU - Lu, Shou En

AU - Zhang, Zhi Gang

AU - Yang, Jin-Ming

AU - Hait, William N.

AU - Goydos, James S.

PY - 2003/12/15

Y1 - 2003/12/15

N2 - 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.

AB - 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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Alsina J, Gorski DH, Germino FJ, Shih W, Lu SE, Zhang ZG et al. Detection of Mutations in the Mitogen-Activated Protein Kinase Pathway in Human Melanoma. Clinical Cancer Research. 2003 Dec 15;9(17):6419-6425.