Genomic and Biological Characterization of Exon 4 KRAS Mutations in Human Cancer

Abstract

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signalregulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3. Cancer Res; 70(14); 5901-11. ©2010 AACR.

Genomic dissection of the epidermal growth factor receptor (EGFR)/PI3K pathway reveals frequent deletion of the EGFR phosphatase PTPRS in head and neck cancers

“…Tumor samples with matched normal tissue were collected during routine clinical care with patient consent under an institutional review board-approved protocol and snap-frozen. Single-nucleotide polymorphism fingerprinting was used to ensure that all tumor samples were obtained from unique patients and correctly matched to normal tissue (28). To determine the copy number landscape of HNSCC, DNA from microdissected tumors was extracted and analyzed using the Agilent 1M Human Comparative Genomic Hybridization (CGH) microarray (aCGH).…”

Resistance to Anti-EGFR Therapy in Colorectal Cancer: From Heterogeneity to Convergent Evolution

“…(12)(13) Validation of extended RAS assessment for predicting lack of response to panitumumab (24)(25) First evidence on the role of NRAS mutations in predicting resistance to anti-EGFR moAbs (20) First retrospective evidence of KRAS mutations impairing response to anti-EGFR moAbs (10) and causal relation establishment (11) First comprehensive dissection of the EGFR pathway to investigate biomarkers of response to anti-EGFR moAbs (9) First multideterminant analysis of coexisting molecular alterations in individual CRC (44) identify additional biomarkers of resistance that could account for the heterogeneity in clinical response. Sequencing studies revealed that although more than 80% of KRAS variants occur in exon 2 at codons 12 and 13, oncogenic mutations also affect KRAS codons 59, 61, 117, and 146 (16)(17)(18). Additional mutations of the NRAS isoform occur at codons 12, 13, and 61 in approximately 3% to 5% of colorectal cancer samples ( 19 ).…”

Multiplex Mutation Screening by Mass Spectrometry

“…The level of sensitivity that we observed is in keeping with other studies using the MassARRAY platform, and exceeds the sensitivity of standard DNA sequencing (approximately 20% mutant allele). [33][34][35][36][37] Standard DNA input was 10 ng per multiplex well, but mutant alleles were readily detected with as little as 1 ng DNA per well ( Figure 1B). …”