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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.

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Focus formation assays. NIH3T3 cells were transfected with pEF.6 or plasmids containing wt or mutant K-Ras and foci visualised after crystal violet staining. (A) Cells transfected with empty vector or wt K-Ras were compared with previously described hotspot mutations. (B) The transforming potential of L19F, K117N, A146T and R164Q mutations were compared with the hotspot mutation G12V and wt K-Ras. All experiments were performed in duplicate and each set of transfections was repeated three times. (C) The combined results of all transfections, wherein total foci counts are presented ± SD are illustrated.
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fig3: Focus formation assays. NIH3T3 cells were transfected with pEF.6 or plasmids containing wt or mutant K-Ras and foci visualised after crystal violet staining. (A) Cells transfected with empty vector or wt K-Ras were compared with previously described hotspot mutations. (B) The transforming potential of L19F, K117N, A146T and R164Q mutations were compared with the hotspot mutation G12V and wt K-Ras. All experiments were performed in duplicate and each set of transfections was repeated three times. (C) The combined results of all transfections, wherein total foci counts are presented ± SD are illustrated.

Mentions: Phenotypes associated with the various K-Ras mutations have previously not been systematically evaluated. To compare the transformation potential of the K-Ras mutants, therefore, NIH3T3 cells were transiently transfected with plasmids expressing wt K-Ras and the K-Ras mutations G12V, G12D, G13D, Q61H, L19F, K117N, A146T and R164Q. Equivalence of plasmid loading was assessed spectrophotometrically and by western blotting for K-Ras (data not shown). Cells were stained with crystal violet and foci counted 21 days after transfection, as described in Materials and Methods (Figure 3). No foci were observed in untransfected cells or in cells transfected with wt K-Ras, whereas abundant foci were seen with each hotspot K-Ras mutation (Figure 3A). Codon 12 mutations had slightly greater transforming potential than codon 13 mutations and consistently greater transforming potential than the codon 61 Q61H mutation (Figure 3C). After transfection of additional K-Ras mutations, significant focus formation was observed for codon 117 and 146 mutations (Figure 3B). In contrast, the codon 164 mutation was phenotypically equivalent to wt K-ras with no evidence of foci formation and the codon 19 mutation generated low but consistent numbers of isolated foci (Figure 3C).


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)

Focus formation assays. NIH3T3 cells were transfected with pEF.6 or plasmids containing wt or mutant K-Ras and foci visualised after crystal violet staining. (A) Cells transfected with empty vector or wt K-Ras were compared with previously described hotspot mutations. (B) The transforming potential of L19F, K117N, A146T and R164Q mutations were compared with the hotspot mutation G12V and wt K-Ras. All experiments were performed in duplicate and each set of transfections was repeated three times. (C) The combined results of all transfections, wherein total foci counts are presented ± SD are illustrated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837563&req=5

fig3: Focus formation assays. NIH3T3 cells were transfected with pEF.6 or plasmids containing wt or mutant K-Ras and foci visualised after crystal violet staining. (A) Cells transfected with empty vector or wt K-Ras were compared with previously described hotspot mutations. (B) The transforming potential of L19F, K117N, A146T and R164Q mutations were compared with the hotspot mutation G12V and wt K-Ras. All experiments were performed in duplicate and each set of transfections was repeated three times. (C) The combined results of all transfections, wherein total foci counts are presented ± SD are illustrated.
Mentions: Phenotypes associated with the various K-Ras mutations have previously not been systematically evaluated. To compare the transformation potential of the K-Ras mutants, therefore, NIH3T3 cells were transiently transfected with plasmids expressing wt K-Ras and the K-Ras mutations G12V, G12D, G13D, Q61H, L19F, K117N, A146T and R164Q. Equivalence of plasmid loading was assessed spectrophotometrically and by western blotting for K-Ras (data not shown). Cells were stained with crystal violet and foci counted 21 days after transfection, as described in Materials and Methods (Figure 3). No foci were observed in untransfected cells or in cells transfected with wt K-Ras, whereas abundant foci were seen with each hotspot K-Ras mutation (Figure 3A). Codon 12 mutations had slightly greater transforming potential than codon 13 mutations and consistently greater transforming potential than the codon 61 Q61H mutation (Figure 3C). After transfection of additional K-Ras mutations, significant focus formation was observed for codon 117 and 146 mutations (Figure 3B). In contrast, the codon 164 mutation was phenotypically equivalent to wt K-ras with no evidence of foci formation and the codon 19 mutation generated low but consistent numbers of isolated foci (Figure 3C).

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