396 reflect these issues. It is generally accepted that the detec- tion of p53 protein by IHC is a poor indicator of TP53 gene mutation status, because alternative molecular mechanisms can lead to protein stabilization in tumors, and some muta- tions lead to loss of protein stability [71]. Studies in which TP53 mutations were detected by gene sequencing report associations with poor prognosis in colon cancer patients [62, 67, 72]. It has been suggested that, to analyze the gene properly in clinical studies, TP53 mutation status should be assessed by DNA sequencing and data must be combined with TP53 protein expression information as determined by IHC. KRAS The KRAS proto-oncogene encodes a 21-kDa guanosine triphosphate/guanosine diphosphate binding protein in- volved in facilitating cellular response to extracellular stim- uli. Mutations within the KRAS gene (primarily at codons 12 and 13) abrogating GTPase activity and leading to downstream activation of RAS/RAF signaling are common (35%– 42%) and early events in colon tumorigenesis [73]. However, the role of KRAS mutation status as a prog- nostic and predictive biomarker in the adjuvant setting is controversial (Table 1). In a large meta-analysis, codon 12 glycine-to-valine mutations were found to be prognostic in patients with stage III disease [74, 75]. Smaller studies have shown KRAS mutation status to be associated with poor prognosis in patients with stage II [76] and stage III disease [69, 74, 75]. However, recent analyses from the CALGB 89803 (stage III colon cancer) and PETACC-3 (stage II and III) trials demonstrated KRAS mutation to not be a prognos- tic marker for patients treated with adjuvant 5-FU– based chemotherapy [77, 78]. In addition, the National Cancer In- stitute of Canada CO.17 trial recently demonstrated that tu- mor KRAS mutation status had no prognostic effect for OS in pretreated stage IV patients receiving best supportive care [79]. As a predictive marker in the adjuvant setting, most studies report no association between KRAS mutations and response to standard chemotherapy [62, 80 – 83]. In a Southwest Oncology Group trial, patients with stage III tu- mors with KRAS mutations gained no additional benefit from receiving 5-FU/LV compared with observation or LV alone. In contrast, patients with KRAS wild-type tumors sig- nificantly benefited from 5-FU/LV therapy [69]. Data from the CALGB 89803 study suggest that KRAS tumor muta- tion status is not prognostic or predictive for treatment with irinotecan plus 5-FU and LV in stage III colon tumors [84]. In contrast, because of the central role of KRAS down- stream in the EGFR signaling pathway, there is currently intense interest in KRAS mutation status as a predictive bio- Biomarkers in Colon Cancer Adjuvant Treatment marker in patients with advanced CRC treated with thera- pies targeted to EGFR. KRAS gene mutations activate the EGFR signaling pathway independently of ligand stimula- tion of the receptor, and thus bypass the efficacy of EGFR- targeting drugs. Single-arm studies [85– 87] and large randomized studies in first-line [88, 89] and in previ- ously treated [79, 90] mCRC patients have demonstrated KRAS tumor mutations to be predictive of a lack of re- sponse to the EGFR-targeted antibodies cetuximab and panitumumab. It is now common practice to reserve treatment with EGFR-targeting agents to wild-type KRAS CRC patients. BRAF The BRAF gene encodes a serine–threonine protein kinase that acts as a downstream effector of KRAS signaling and belongs to the RAS–RAF–mitogen-activated protein ki- nase/extracellular signal–related kinase kinase (MEK)– extracellular signal–related kinase (ERK) kinase pathway [91]. BRAF gene mutations are important in colorectal tu- morigenesis [91, 92]. The most frequently reported BRAF tumor mutation is a valine-to-glutamic acid amino acid (V600E) substitution that leads to the aberrant activation of the MEK–ERK pathway [93]. BRAF and KRAS mutations tend to be mutually exclusive events in tumors [94], with BRAF mutations occurring more frequently in MSI than in MSS tumors [44, 95]. In patients with stage IV CRC, BRAF mutations have been reported to be associated with poor prognosis [96], and in chemotherapy-refractory mCRC patients BRAF mu- tations have been reported to be predictive of a lack of re- sponse to EGFR-targeted agents [96]. In stage II and stage III colon cancer patients in the PETACC-3 study, BRAF mutations occurred in 7.9% of tumors and were found to not be prognostic of relapse-free survival, but they were prog- nostic for OS, particularly in patients with MSI-L and MSS tumors (HR, 2.2; p .0003) [77]. Other retrospective stud- ies have also demonstrated an association between BRAF mutation and poor prognosis in stage II–III [44] and stage I–IV [97] CRC patients. Interestingly, in those studies the good prognosis associated with patients with MSI-H tu- mors was abrogated in the presence of coincident BRAF mutations [44, 97]. In the adjuvant setting, BRAF mutation status appears to be a valid prognostic marker; however, associations of BRAF tumor mutations with different molecular subgroups may have to be considered in order to assess the impact of BRAF mutation status as a predictive marker for treatment in future studies in this setting.