6 Study Tol et al. [49] Hecht et al. [50] Pathology Research International Setting First-line treatment First-line treatment Treatment CAPOX + bevacizumab + cetuximab vs. CAPOX + bevacizumab FOLFOX + bevacizumab + panitumumab vs. FOLFOX + bevacizumab Table 2: Continued. KRAS genotype No. of patients ORR (%) mPFS (mos) mOS (mos) WT Mut WT Mut WT Mut WT Mut 158 98 156 108 201 135 203 125 50.0 59.2 61.4∗ 45.9 50 47 56 44 10.5∗ 8.1 10.6 12.5 9.8 10.4 11.5 11.0 21.8 17.2 22.4 24.9 20.7 19.3 24.5 19.3 ∗ Statistically signiﬁcant improvement †Statistically signiﬁcant improvement for the combination of cetuximab and irinotecan only. ORR: overall response rate; mPFS: median progression-free survival; mOS: median overall survival; N.R.: not reported; BSC: best supportive care. The mitogen-activated protein kinase kinase (MEK, also known as MAP2K) is downstream of BRAF in the RAS-RAF- MEK signaling cascade of the EGFR and uses extracellular signal-regulated kinase (ERK) as a substrate (see Figure 1) [67]. A number of MEK inhibitors such as AS703026, AZD6244 and RO5068760 have been or currently are being investigated in phase 1 and 2 clinical trials [68, 69]. The development of several MEK inhibitors has been halted because of either very low response rates or due to ocular toxicity [70]. These agents have however shown substantial preclinical activity in tumor cell lines harboring the BRAF V600E gene mutation [71]. It has been established that KRAS has a number of downstream eﬀectors that are not blocked by MEK inhibition, and indeed BRAF mutant cell lines were found to be more sensitive to MEK inhibitors than KRAS mutant cells [71]. It is imperative to be able to identify which patients are likely to respond to MEK inhibitors, and it appears that those with BRAF mutations are a good start. Given that KRAS signaling operates through a number of downstream eﬀectors, those with KRAS mutations may require a combination of targeted agents. Preclinical evidence suggests that BRAF gene ampliﬁcation is a mechanism of resistance to both MEK and BRAF inhibitors and a combination of these inhibitors may be a strategy to overcome this [72]. An additional EGFR pathway is the PTEN/PI3K/AKT pathway [phosphatase and tensin homologue gene (PTEN)]. PTEN encodes a phosphatase which uses phosphatidylinositol-3,4,5-triphosphate (PIP-3) as a major substrate [73]. Loss of PTEN function leads to increased PIP- 3 concentration, with resultant AKT hyperphosphorylation protecting tumor cells from apoptosis [73]. Roughly 60% of primary CRCs have a hyperphosphorylated AKT [74]. PTEN loss, activating mutations of phosphatidylinositol 3- kinase catalytic alpha polypeptide (PIK3CA) and activating mutations in KRAS/BRAF/MAPK confer resistance to apoptosis induced by cetuximab [75]. In patients with KRAS WT tumors treated with a cetuximab-based regimen, PTEN loss was associated with a signiﬁcantly shorter OS [60]. Approximately one third of CRCs harbor activating somatic mutations in PIK3CA, and it has been reported that these mutations are predictive of lack of beneﬁt from antiEGFR therapy [76]. Additional genetic alterations which could confer resistance to antiEGFR monoclonal antibodies include an inhibitor of PI3K signaling; coampliﬁcation of PAK4 (p-21-activated protein kinase) and AKT, which are downstream mediators of PI3K signaling; and ampliﬁcation of IRS2 (insulin receptor substrate 2), which is an upstream activator of PI3K signaling [77, 78]. 4. Strategies to Overcome Resistance A number of approaches to the problem of resistance to antiEGFR monoclonal antibody therapy have been stud- ied and are ongoing. Combining antiEGFR monoclonal antibodies with cytotoxic chemotherapy has already been discussed. Erlotinib and geﬁtinib, two oral small molecule EGFR inhibitors, are inactive by themselves [79, 80]. The combination of erlotinib with capecitabine and oxaliplatin in previously treated patients and the combination of geﬁtinib with FOLFOX were investigated in small phase II studies with favorable results, however randomized trials with chemotherapy alone as a control are needed [81–83]. Dual antiEGFR therapy with antiEGFR monoclonal antibodies plus antiEGFR TK inhibitors may overcome resistance to either drug alone. A 41% response rate was reported for the combination of cetuximab and erlotinib in patients with refractory disease, however this was limited to patients with KRAS and BRAF WT tumors [84]. EGFR and vascular endothelial growth factor (VEGF) have several signal transduction pathways in common, with preclinical data revealing that antiEGFR and antiVEGF drug combinations have synergistic activity [85]. The BOND-2 study randomized patients with irinotecan- and oxaliplatin- refractory but bevacizumab na¨ıve disease to cetuximab and bevacizumab with or without irinotecan [21]. Response rates, TTP and OS favored the triple drug regimen, however, these results did not hold up in a subsequent study of this combination [21, 22]. Two subsequent randomized phase III trials have shown that combinations of antiEGFR