2 PTEN PIP3 Ligands EGF receptor PIP2 PI3K AKT mTOR Anti-EGFR MoABs RAS RAF Nucleus MEK ERK Figure 1: EGFR signaling pathway [4]. (Reprinted with permission from American Society of Clinical Oncology 2008. All rights reserved.) Anti-EGFR monoclonal antibodies have been evaluated in both untreated metastatic CRC and chemotherapy refrac- tory disease. Figure 2 summarizes the current treatment paradigm for metastatic colorectal cancer including the appropriate incorporation of antiEGFR monoclonal anti- body therapy which improves survival for appropriately selected patients [9]. Table 1 summarizes selected clinical trials of antiEGFR monoclonal antibodies in metastatic CRC. Response rates with single-agent antiEGFR monoclonal antibodies range from 9–12%, with much higher response rates observed when cetuximab is used in combination with chemotherapy [12–22]. When administered to unselected metastatic CRC patients, only a minority responded to EGFR inhibitor therapy. Therefore, a method to identify and predict sensitivity to these drugs was needed. 2. Prediction of Response to Anti-EGFR Monoclonal Antibodies The RAS family of proto-oncogenes include HRAS, KRAS, and NRAS [23]. KRAS (Kirsten ras sarcoma viral onco- gene) is a guanosine triphosphate-(GTP-) binding protein downstream of the EGFR and is a central component of the mitogen-activated protein kinase (MAPK) pathway, a component of the EGFR signaling cascade [23]. Roughly 40% of colorectal cancers are characterized by a mutation in the KRAS gene [24]. About 90% of these mutations occur in codons 12 and 13 in exon 2 of the KRAS gene, with the remaining mutations occurring in codons 61 and 146 (roughly 5% each) [25, 26]. Such KRAS mutations lead to EGFR-independent constitutive activation of the signaling pathway and predict for a lack of response and beneﬁt from antiEGFR monoclonal antibodies cetuximab and panitumumab [27–35]. De Roock et al. showed that codon 61 mutations predicted for lack of response to cetuximab similar to codon 12 and 13 mutations; however, codon 146 mutations did not aﬀect cetuximab eﬃcacy [26]. Failure to test for codon 61 mutations may miss Pathology Research International a signiﬁcant mutation which would confer resistance to antiEGFR monoclonal antibody therapy. There is a very high concordance of KRAS mutational status between the primary tumor and metastasis, ranging from 92–100% [36– 38]. However, KRAS mutation status heterogeneity between primary tumors, lymph nodes and distant metastases in 5– 10% of patients has been reported, with mixed responses to antiEGFR monoclonal antibody therapy in those with metastatic CRC [37, 39, 40]. Because of this, some clinicians have called for a reassessment of KRAS mutation status on metastatic foci in situations where only the primary tumor was assessed for KRAS status [41]. Table 2 summarizes clinical trials of antiEGFR mon- oclonal antibodies which included analysis of treatment eﬀect and KRAS mutation status. Amado et al. assessed the predictive role of KRAS mutational status in a randomized phase III trial comparing panitumumab monotherapy with best supportive care (BSC) in patients with chemotherapy refractory metastatic CRC [24]. This trial showed that the clinical beneﬁt associated with panitumumab was restricted to the KRAS WT population. KRAS mutations predicted for lack of clinical beneﬁt to panitumumab [24]. Similarly, Karapetis et al. showed that treatment with cetuximab signiﬁcantly improved OS and PFS in patients with KRAS WT tumors; however, in this chemotherapy-resistant patient population, those with mutated KRAS tumors did not beneﬁt [38]. Use of cetuximab as ﬁrst-line treatment for metastatic disease was investigated by Van Cutsem et al.; patients were randomly assigned to receive FOLFIRI with or without cetuximab [36]. A statistically signiﬁcant beneﬁt in PFS for patients with KRAS WT tumors receiving cetuximab and chemotherapy was conﬁrmed in a ﬁnal presentation of this trial [42]. Bokemeyer et al. investigated the use of cetuximab in combination with FOLFOX chemother- apy as initial treatment for metastatic disease [34]. A retrospective analysis of this data revealed that cetuximab and chemotherapy had a statistically signiﬁcant increased response rate and lower risk of disease progression compared with chemotherapy alone in patients with KRAS WT tumors [43]. Prospectively, panitumumab has been investigated with either FOLFOX or FOLFIRI chemotherapy in the ﬁrst-line metastatic setting [44, 45]. The addition of panitumumab to FOLFOX chemotherapy was associated with a statistically signiﬁcant improvement in PFS [44]. Taken together, the data in Table 2 supports that in metastatic CRC, KRAS WT and mutation status predict for potential sensitivity to, and deﬁnite resistance to, respectively, both antiEGFR monoclonal antibodies, regardless of prior treatment and irrespective of use as monotherapy or in combination. Notably, while KRAS status is an established predictor of response to antiEGFR monoclonal antibody therapy, it has been disproven as a prognostic marker. In contrast to KRAS mutational status, evaluation of EGFR expression of CRC cells has failed to demonstrate predictive value for antiEGFR monoclonal antibody therapy. Cunningham et al. reported that the intensity of EGFR staining by immunohistochemical analysis did not correlate with response rate to cetuximab [13]. Similar data has also been reported with panitumumab [46]. KRAS mutated CRC absent of antiEGFR monoclonal