Limits...
Activating K-Ras mutations outwith 'hotspot' codons in sporadic colorectal tumours - implications for personalised cancer medicine.

Smith G, Bounds R, Wolf H, Steele RJ, Carey FA, Wolf CR - Br. J. Cancer (2010)

Bottom Line: Colorectal tumours (n=106) were screened for additional K-Ras mutations, phenotypes compared in transformation and Ras GTPase activating assays and gene and pathway changes induced by individual K-Ras mutants identified by microarray analysis.The identification of mutations outwith previously described hotspot codons increases the K-Ras mutation burden in colorectal tumours by one-third.Future mutation screening to facilitate optimal patient selection for treatment with EGFR-targeted therapies should therefore be extended to codon 146, and in addition should consider the unique molecular signatures associated with individual K-Ras mutations.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Institute, Ninewells Hospital and Medical School, Dundee, UK.

ABSTRACT

Background: Response to EGFR-targeted therapies in colorectal cancer patients has been convincingly associated with Kirsten-Ras (K-Ras) mutation status. Current mandatory mutation testing for patient selection is limited to the K-Ras 'hotspot' codons 12 and 13.

Methods: Colorectal tumours (n=106) were screened for additional K-Ras mutations, phenotypes compared in transformation and Ras GTPase activating assays and gene and pathway changes induced by individual K-Ras mutants identified by microarray analysis. Taqman-based gene copy number and FISH analyses were used to investigate K-Ras gene amplification.

Results: Four additional K-Ras mutations (Leu(19)Phe (1 out of 106 tumours), Lys(117)Asn (1 out of 106), Ala(146)Thr (7 out of 106) and Arg(164)Gln (1 out of 106)) were identified. Lys(117)Asn and Ala(146)Thr had phenotypes similar to the hotspot mutations, whereas Leu(19)Phe had an attenuated phenotype and the Arg(164)Gln mutation was phenotypically equivalent to wt K-Ras. We additionally identified a new K-Ras gene amplification event, present in approximately 2% of tumours.

Conclusions: The identification of mutations outwith previously described hotspot codons increases the K-Ras mutation burden in colorectal tumours by one-third. Future mutation screening to facilitate optimal patient selection for treatment with EGFR-targeted therapies should therefore be extended to codon 146, and in addition should consider the unique molecular signatures associated with individual K-Ras mutations.

Show MeSH

Related in: MedlinePlus

Ras GTPase activating assays. Ras GTPase activating assays were carried out for each of the novel Kirsten-Ras (K-Ras) mutations and western blotting used to assess the expression of (A) active GTP-bound K-Ras (B) total K-Ras and (C) β-actin (loading control). The K-Ras G12V construct was included as a positive control for K-Ras protein in the active GTP-bound conformation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2837563&req=5

fig4: Ras GTPase activating assays. Ras GTPase activating assays were carried out for each of the novel Kirsten-Ras (K-Ras) mutations and western blotting used to assess the expression of (A) active GTP-bound K-Ras (B) total K-Ras and (C) β-actin (loading control). The K-Ras G12V construct was included as a positive control for K-Ras protein in the active GTP-bound conformation.

Mentions: To assess which K-Ras mutations were in the active GTP-bound conformation, a Raf-1 binding assay was carried out as described in Materials and Methods. Raf-1 selectively binds GTP-bound Ras (rather than the inactive GDP-bound form) which, following immunoprecipitation, can be visualised by western blotting. Figure 4 illustrates the results of our analysis where, consistent with our focus formation experiments, the K-Ras G12V and L19F, K117N and A146T mutations are clearly in the active GTP-bound conformation. In contrast, the R164Q mutation, like wt K-Ras, was not GTP-bound.


Activating K-Ras mutations outwith 'hotspot' codons in sporadic colorectal tumours - implications for personalised cancer medicine.

Smith G, Bounds R, Wolf H, Steele RJ, Carey FA, Wolf CR - Br. J. Cancer (2010)

Ras GTPase activating assays. Ras GTPase activating assays were carried out for each of the novel Kirsten-Ras (K-Ras) mutations and western blotting used to assess the expression of (A) active GTP-bound K-Ras (B) total K-Ras and (C) β-actin (loading control). The K-Ras G12V construct was included as a positive control for K-Ras protein in the active GTP-bound conformation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2837563&req=5

fig4: Ras GTPase activating assays. Ras GTPase activating assays were carried out for each of the novel Kirsten-Ras (K-Ras) mutations and western blotting used to assess the expression of (A) active GTP-bound K-Ras (B) total K-Ras and (C) β-actin (loading control). The K-Ras G12V construct was included as a positive control for K-Ras protein in the active GTP-bound conformation.
Mentions: To assess which K-Ras mutations were in the active GTP-bound conformation, a Raf-1 binding assay was carried out as described in Materials and Methods. Raf-1 selectively binds GTP-bound Ras (rather than the inactive GDP-bound form) which, following immunoprecipitation, can be visualised by western blotting. Figure 4 illustrates the results of our analysis where, consistent with our focus formation experiments, the K-Ras G12V and L19F, K117N and A146T mutations are clearly in the active GTP-bound conformation. In contrast, the R164Q mutation, like wt K-Ras, was not GTP-bound.

Bottom Line: Colorectal tumours (n=106) were screened for additional K-Ras mutations, phenotypes compared in transformation and Ras GTPase activating assays and gene and pathway changes induced by individual K-Ras mutants identified by microarray analysis.The identification of mutations outwith previously described hotspot codons increases the K-Ras mutation burden in colorectal tumours by one-third.Future mutation screening to facilitate optimal patient selection for treatment with EGFR-targeted therapies should therefore be extended to codon 146, and in addition should consider the unique molecular signatures associated with individual K-Ras mutations.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Institute, Ninewells Hospital and Medical School, Dundee, UK.

ABSTRACT

Background: Response to EGFR-targeted therapies in colorectal cancer patients has been convincingly associated with Kirsten-Ras (K-Ras) mutation status. Current mandatory mutation testing for patient selection is limited to the K-Ras 'hotspot' codons 12 and 13.

Methods: Colorectal tumours (n=106) were screened for additional K-Ras mutations, phenotypes compared in transformation and Ras GTPase activating assays and gene and pathway changes induced by individual K-Ras mutants identified by microarray analysis. Taqman-based gene copy number and FISH analyses were used to investigate K-Ras gene amplification.

Results: Four additional K-Ras mutations (Leu(19)Phe (1 out of 106 tumours), Lys(117)Asn (1 out of 106), Ala(146)Thr (7 out of 106) and Arg(164)Gln (1 out of 106)) were identified. Lys(117)Asn and Ala(146)Thr had phenotypes similar to the hotspot mutations, whereas Leu(19)Phe had an attenuated phenotype and the Arg(164)Gln mutation was phenotypically equivalent to wt K-Ras. We additionally identified a new K-Ras gene amplification event, present in approximately 2% of tumours.

Conclusions: The identification of mutations outwith previously described hotspot codons increases the K-Ras mutation burden in colorectal tumours by one-third. Future mutation screening to facilitate optimal patient selection for treatment with EGFR-targeted therapies should therefore be extended to codon 146, and in addition should consider the unique molecular signatures associated with individual K-Ras mutations.

Show MeSH
Related in: MedlinePlus