**2. Anti-EGFR therapy**

EGFR is a tyrosine kinase transmembrane receptor that belongs to the ErbB protein family. EGFR-mediated signaling has important roles in cell proliferation, survival and differentiation, and dysregulation is a central driver in multiple malignancies including colorectal cancer [4–6]. EGFR interacts with multiple ligands including epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), amphiregulin (AREG), epiregulin (EPR), betacellulin (BTC), heparinbinding EGF (HB-EGF), epigen (EPN), and neuregulin 1-4 (NRG1-4). Activation of EGFR following ligand binding triggers a variety of signaling cascades, including the RAS/MAPK, PI3K/AKT, PLCγ/PKC, SRC tyrosine kinase and STAT pathways. In addition, ligand binding can induce EGFR translocation to the nucleus where EGFR behaves as a co-transcriptional activator regulating key genes such as Aurora Kinase A (*AURKA*), Cyclin D1 (*CCND1*), Prostaglandin-Endoperoxide Synthase 2 (*PTGS2*) and MYB Proto-Oncogene Like 2 (*MYBL2*).

EGFR is overexpressed in colorectal tumors, with most estimates between 40% and 80% depending on the methods and cut-offs used, highlighting the receptor as a prime drug target in this malignancy [7, 8]. Two monoclonal antibodies targeting EGFR have been clinically approved for the treatment of mCRC including cetuximab (Erbitux®), a chimeric mouse-human IgG1 antibody, and panitumumab (Vectibix®), a humanized IgG2 antibody. Both antibodies bind the extracellular domain of EGFR, inhibiting ligand-induced tyrosine kinase activation and leading to EGFR cellular internalization and degradation, thereby preventing the activation of downstream signaling (**Figure 1**). Panitumumab has a higher binding affinity for EGFR than cetuximab [9], and cetuximab is thought to additionally lead to activation of the immune response through antibody-dependent cell-mediated cytotoxicity (ADCC) due to the IgG1 chimeric antibody structure [10, 11]. With respect to

**109**

**Figure 1.**

*Predictive Biomarkers for Monoclonal Antibody Therapies Targeting EGFR (Cetuximab…*

toxicity, panitumumab treatment is associated with significantly lower occurrence of grade 3–4 infusion reactions (allergic reactions) than cetuximab due to its fully humanized nature [12]. Despite these differences, cetuximab and panitumumab showed clinical equivalence in efficacy in refractory patients [12], and both are approved for use in combination with chemotherapy in the first and second line

*Targeting of the EGFR signaling pathway with anti-EGFR monoclonal antibodies. EGFR activation is triggered by ligand binding which results in the formation of receptor homo- or hetero-dimers. Receptor autophosphorylation at tyrosine residues within the cytoplasmic tail acts as a docking site for proteins with Src homology2 (SH2) and phosphotyrosine-binding domains (PTB), initiating cellular signaling via the RAS/MAPK, PI3K/AKT, STAT and PLCγ/PKC pathways. Ligand binding can further stimulate EGFR translocation into the nucleus, with nuclear EGFR interacting with transcription factors to drive expression of target genes including NOS2, PTGS2, AURKA, MYBL2 and CCND1. EGFR signaling in tumor cells promotes cell proliferation and survival, and this can be* 

In unselected patient populations, the response rate to anti-EGFR therapy is typically less than 30% [13], and for patients who initially respond to treatment most tumors become refractory within 3–12 months [14]. The need to identify biomarkers predictive of EGFR response is therefore vital, and numerous studies have explored resistance mechanisms to EGFR blockade. Findings have unraveled a variety of biomarkers and pathways that are associated with resistance to anti-EGFR therapy. As discussed below, this work has led to the endorsement of predictive testing for tumor *RAS* (*KRAS* and *NRAS*) mutation status and consideration of primary tumor location to guide the use of anti-EGFR therapy. Efforts to discover and validate additional biomarkers is ongoing to further refine treatment delivery are ongoing.

Genes of the RAS type GTPase family, comprising *KRAS*, *NRAS* and *HRAS*, are principal downstream mediators of activated EGFR signaling [15]. In colorectal cancer, *KRAS* and *NRAS* are major oncogenes, with activating mutations found in approximately 40% and 3–5% of cases, respectively [16]. Constitutive downstream signaling through oncogenic RAS proteins activates processes contributing to tumor progression and metastasis, independent of EGFR and other cell surface receptor

setting or as monotherapy for refractory disease.

*blocked with antibodies against the receptor (cetuximab and panitumumab).*

**2.1 Current predictive biomarkers for anti-EGFR therapy**

*2.1.1 KRAS and NRAS mutations*

*DOI: http://dx.doi.org/10.5772/intechopen.80690*

*Predictive Biomarkers for Monoclonal Antibody Therapies Targeting EGFR (Cetuximab… DOI: http://dx.doi.org/10.5772/intechopen.80690*

#### **Figure 1.**

*Advances in the Molecular Understanding of Colorectal Cancer*

United States Food and Drug Administration (FDA) for the treatment of mCRC, including 5-fluorouracil, irinotecan, capecitabine, oxaliplatin, bevacizumab, cetuximab, panitumumab, ziv-aflibercept, regorafenib, ramucirumab, and trifluridinetipiracil. The expansion of treatment options has resulted in an increased clinical need for predictive biomarkers to guide the effective use of therapy. Only a small proportion of patients will respond to any given therapy, and treatments are associ-

Predictive biomarkers for anti-cancer agents are best developed prospectively as companion diagnostics during the drug development process. However, these can also be developed retrospectively through analysis of samples and data from previously conducted randomized clinical trials. Another avenue for marker discovery are longitudinal studies of patients analyzing the emergence of drug resistant tumor clones, although mechanisms of intrinsic (primary) and acquired (secondary) drug resistance may differ. Predictive markers can provide either drug sensitivity (positive prediction of response) or resistance (negative prediction of response)

There are many challenges in the biomarker development process, such as the choice of analyte (e.g. urine, blood, tissue), cancer sampling procedures (e.g. circulating tumor cells, primary cancer, metastatic lesions), technology for marker evaluation (e.g. DNA, RNA or protein) and determination of clinically relevant cut-offs. In this chapter, we review development efforts for predictive biomarkers for patients with mCRC focusing on anti-EGFR antibody therapies. Our discussion will concentrate on markers of primary drug resistance; markers of acquired drug

EGFR is a tyrosine kinase transmembrane receptor that belongs to the ErbB protein family. EGFR-mediated signaling has important roles in cell proliferation, survival and differentiation, and dysregulation is a central driver in multiple malignancies including colorectal cancer [4–6]. EGFR interacts with multiple ligands including epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), amphiregulin (AREG), epiregulin (EPR), betacellulin (BTC), heparinbinding EGF (HB-EGF), epigen (EPN), and neuregulin 1-4 (NRG1-4). Activation of EGFR following ligand binding triggers a variety of signaling cascades, including the RAS/MAPK, PI3K/AKT, PLCγ/PKC, SRC tyrosine kinase and STAT pathways. In addition, ligand binding can induce EGFR translocation to the nucleus where EGFR behaves as a co-transcriptional activator regulating key genes such as Aurora Kinase A (*AURKA*), Cyclin D1 (*CCND1*), Prostaglandin-Endoperoxide Synthase 2

EGFR is overexpressed in colorectal tumors, with most estimates between 40% and 80% depending on the methods and cut-offs used, highlighting the receptor as a prime drug target in this malignancy [7, 8]. Two monoclonal antibodies targeting EGFR have been clinically approved for the treatment of mCRC including cetuximab (Erbitux®), a chimeric mouse-human IgG1 antibody, and panitumumab (Vectibix®), a humanized IgG2 antibody. Both antibodies bind the extracellular domain of EGFR, inhibiting ligand-induced tyrosine kinase activation and leading to EGFR cellular internalization and degradation, thereby preventing the activation of downstream signaling (**Figure 1**). Panitumumab has a higher binding affinity for EGFR than cetuximab [9], and cetuximab is thought to additionally lead to activation of the immune response through antibody-dependent cell-mediated cytotoxicity (ADCC) due to the IgG1 chimeric antibody structure [10, 11]. With respect to

ated with significant toxicities and often with high financial costs.

information depending on the biomarker-drug relationship.

resistance have been summarized in recent reviews [3, 4].

(*PTGS2*) and MYB Proto-Oncogene Like 2 (*MYBL2*).

**2. Anti-EGFR therapy**

**108**

*Targeting of the EGFR signaling pathway with anti-EGFR monoclonal antibodies. EGFR activation is triggered by ligand binding which results in the formation of receptor homo- or hetero-dimers. Receptor autophosphorylation at tyrosine residues within the cytoplasmic tail acts as a docking site for proteins with Src homology2 (SH2) and phosphotyrosine-binding domains (PTB), initiating cellular signaling via the RAS/MAPK, PI3K/AKT, STAT and PLCγ/PKC pathways. Ligand binding can further stimulate EGFR translocation into the nucleus, with nuclear EGFR interacting with transcription factors to drive expression of target genes including NOS2, PTGS2, AURKA, MYBL2 and CCND1. EGFR signaling in tumor cells promotes cell proliferation and survival, and this can be blocked with antibodies against the receptor (cetuximab and panitumumab).*

toxicity, panitumumab treatment is associated with significantly lower occurrence of grade 3–4 infusion reactions (allergic reactions) than cetuximab due to its fully humanized nature [12]. Despite these differences, cetuximab and panitumumab showed clinical equivalence in efficacy in refractory patients [12], and both are approved for use in combination with chemotherapy in the first and second line setting or as monotherapy for refractory disease.

In unselected patient populations, the response rate to anti-EGFR therapy is typically less than 30% [13], and for patients who initially respond to treatment most tumors become refractory within 3–12 months [14]. The need to identify biomarkers predictive of EGFR response is therefore vital, and numerous studies have explored resistance mechanisms to EGFR blockade. Findings have unraveled a variety of biomarkers and pathways that are associated with resistance to anti-EGFR therapy. As discussed below, this work has led to the endorsement of predictive testing for tumor *RAS* (*KRAS* and *NRAS*) mutation status and consideration of primary tumor location to guide the use of anti-EGFR therapy. Efforts to discover and validate additional biomarkers is ongoing to further refine treatment delivery are ongoing.

## **2.1 Current predictive biomarkers for anti-EGFR therapy**

## *2.1.1 KRAS and NRAS mutations*

Genes of the RAS type GTPase family, comprising *KRAS*, *NRAS* and *HRAS*, are principal downstream mediators of activated EGFR signaling [15]. In colorectal cancer, *KRAS* and *NRAS* are major oncogenes, with activating mutations found in approximately 40% and 3–5% of cases, respectively [16]. Constitutive downstream signaling through oncogenic RAS proteins activates processes contributing to tumor progression and metastasis, independent of EGFR and other cell surface receptor

kinases [15]. As anticipated from the biological mechanism, mutations in *KRAS* and *NRAS* genes have been found to render tumors insensitive to anti-EGFR therapy.

The majority of *KRAS* mutations (85–90%) in colorectal cancer occur in exon 2 at codons 12 and 13 [16]. Analyses of clinical trials of cetuximab or panitumumab over the last decade have provided conclusive evidence that patients with *KRAS* mutations in exon 2 do not benefit from anti-EGFR therapy when given as a single agent or combined with chemotherapy (**Table 1**) [17–24]. Retrospective analyses of the randomized phase III CO.17 and 20020408 studies which evaluated cetuximab or panitumumab plus best supportive care (BSC) *vs* BSC alone in patients with chemotherapy-refractory mCRC, respectively, found a significant improvement in outcomes for patients with wild-type *KRAS* exon 2 tumors, but no benefit of anti-EGFR therapy in patients who had mutant *KRAS* exon 2 tumors [19, 22]. Similar results for the first-line setting were subsequently reported for both retrospective and prospective analyses of several randomized clinical trials, including the phase II OPUS and phase III PRIME studies which examined cetuximab or panitumumab plus oxaliplatin, fluorouracil, and leucovorin (FOLFOX4) *vs* FOLFOX4 alone, respectively [23, 25], and the phase III CRYSTAL study which assessed the addition of cetuximab to irinotecan, infusional fluorouracil, and leucovorin (FOLFIRI) [18]. Prospective analysis of the randomized phase III 20050181 study which evaluated panitumumab plus FOLFIRI in the second-line setting further confirmed the predictive value of *KRAS* exon 2 mutation status [24].

There is evidence that *KRAS* codon 12 and 13 mutations may exhibit differential biological effects, including variable ratios of these codon mutations between tumor types [16] and weaker *in vitro* transforming activity for *KRAS* codon 13 as compared to codon 12 mutant proteins [26]. Accordingly, some studies have suggested that patients with KRAS glycine (G)-to-aspartate (D) transitions at codon 13 (G13D), the most common codon 13 variant in colorectal cancer, might derive some benefit from anti-EGFR therapy [27, 28]. A retrospective consortium analysis assessing patients with chemotherapy-refractory mCRC treated with cetuximab who participated in multiple clinical trials (CO.17, BOND, MABEL, EMR202600, EVEREST, BABEL and SALVAGE) or who received off-study treatment reported longer overall and progression-free survival among individuals with KRAS G13D-mutated tumors than with other *KRAS*-mutated tumors [27]. An analysis of the updated pooled data sets from the CRYSTAL and OPUS studies also reported that addition of cetuximab to first-line chemotherapy appeared to benefit patients with KRAS G13D-mutant tumors [28]. In contrast, a retrospective analysis of 110 patients treated with cetuximab, found that patients with KRAS G13D mutations were unlikely to respond to therapy [29], and similar findings were reported for a retrospective pooled analysis of three randomized phase III trials evaluating panitumumab therapy (20050203, first line; 20050181, second line; and 20020408, monotherapy) [30]. To resolve this controversy, the randomized phase II ICECREAM study prospectively assessed cetuximab monotherapy and cetuximab plus irinotecan in patients with KRAS G13D-mutated chemotherapyrefractory mCRC. In this study, no statistically significant improvement in disease control was observed for patients with this rare molecular subtype [31].

More recently, several retrospective analyses have indicated that not only *KRAS* exon 2 mutations but also *KRAS* exons 3 and 4 and *NRAS* exons 2, 3, and 4 mutations are negative predictive markers for anti-EGFR therapy [23, 32–36]. These additional mutations are observed in approximately 15–20% of wild-type *KRAS* exon 2 tumors [23, 32]. Reassessment of the randomized OPUS and PRIME studies of cetuximab or panitumumab plus FOLFOX4 *vs* FOLFOX4 alone in the first-line setting found that additional *RAS* mutations predicted a lack of response [23, 34], and corresponding observations were reported for the CRYSTAL study of cetuximab plus FOLFIRI [32]. Accordingly, analyses of single arms of the phase III FIRE-3 study

**111**

**Table 1.**

*Predictive Biomarkers for Monoclonal Antibody Therapies Targeting EGFR (Cetuximab…*

**Number of patients**

CRYSTAL FOLFIRI + C 178 11.4 0.56 0.41–

FOLFIRI 189 8.4 FIRE-3 FOLFIRI + C 199 10.3 0.97 0.88–

FOLFIRI +B 201 10.2 OPUS FOLFOX + C 38 12 0.53 0.27–

FOLFOX 49 5.8 PEAK FOLFOX + P 50 13 0.65 0.44–

FOLFOX + B 60 9.5 PRIME FOLFOX + P 259 10.1 0.72 0.58–

FOLFOX 253 7.9 20050181 FOLFIRI + P 208 6.4 0.7 0.54–

FOLFIRI 213 4.6 20020408 P + BCS 107 12.3 wks 0.45 0.34–

BSC 110 7.3 wks

BSC 113 1.9

CRYSTAL FOLFIRI + C 246 7.4 1.1 0.85–

FOLFIRI 214 7.5

FOLFIRI +B n.a n.a OPUS FOLFOX + C 92 5.6 1.54 1.04–

FOLFOX 75 7.8

FOLFOX + B n.a n.a PRIME FOLFOX + P 272 7.3 1.3 1.07–

FOLFOX 276 8.7 20050181 FOLFIRI + P 299 4.8 0.86 0.71–

FOLFIRI 294 4 20020408 P + BCS 76 7.4 wks 0.99 0.73–

BSC 95 7.3 wks CO.17 C + BSC 81 1.8 0.99 0.73–

BSC 83 1.8

CO.17 C + BSC 117 3.7 0.4 0.3–0.54 p < 0.001

FIRE-3 FOLFIRI + C n.a n.a n.a n.a n.a

PEAK FOLFOX + P n.a n.a n.a n.a n.a

**PFS (months)** **HR PFS** **95% CI p-Value**

p < 0.001

0.77

0.0615

0.029

0.004

0.007

p < 0.001

0.47

0.0309

p < 0.001

0.14

n.r.

0.96

0.76

1.99

1.04

0.96

0.90

0.90

0.59

1.42

2.29

1.60

1.05

1.36

1.35

evaluating cetuximab plus FOLFIRI and the phase II PEAK study evaluating panitumumab plus modified fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) in the first-line setting reported a more pronounced survival advantage for the wild-type *RAS* population as compared to the wild-type *KRAS* exon 2 population [36, 37]. Retrospective analysis of the randomized 20050181 study of panitumumab plus

*Summary of clinical trials and treatment effects within subgroups defined by RAS status in patients with* 

*Abbreviations: PFS, progression-free survival; HR, hazard ratio; CI, confidence interval; FOL, folinic acid; F, fluorouracil; IRI, irinotecan; OX, oxaliplatin; B, bevacizumab; C, cetuximab; P, panitumumab; n.r., not reported;* 

*DOI: http://dx.doi.org/10.5772/intechopen.80690*

**arms**

**Study Treatment** 

**RAS wild-type**

**RAS mutant**

*BSC, best supportive care.*

*metastatic colorectal cancer.*


*Predictive Biomarkers for Monoclonal Antibody Therapies Targeting EGFR (Cetuximab… DOI: http://dx.doi.org/10.5772/intechopen.80690*

*Abbreviations: PFS, progression-free survival; HR, hazard ratio; CI, confidence interval; FOL, folinic acid; F, fluorouracil; IRI, irinotecan; OX, oxaliplatin; B, bevacizumab; C, cetuximab; P, panitumumab; n.r., not reported; BSC, best supportive care.*

#### **Table 1.**

*Advances in the Molecular Understanding of Colorectal Cancer*

predictive value of *KRAS* exon 2 mutation status [24].

kinases [15]. As anticipated from the biological mechanism, mutations in *KRAS* and *NRAS* genes have been found to render tumors insensitive to anti-EGFR therapy. The majority of *KRAS* mutations (85–90%) in colorectal cancer occur in exon 2 at codons 12 and 13 [16]. Analyses of clinical trials of cetuximab or panitumumab over the last decade have provided conclusive evidence that patients with *KRAS* mutations in exon 2 do not benefit from anti-EGFR therapy when given as a single agent or combined with chemotherapy (**Table 1**) [17–24]. Retrospective analyses of the randomized phase III CO.17 and 20020408 studies which evaluated cetuximab or panitumumab plus best supportive care (BSC) *vs* BSC alone in patients with chemotherapy-refractory mCRC, respectively, found a significant improvement in outcomes for patients with wild-type *KRAS* exon 2 tumors, but no benefit of anti-EGFR therapy in patients who had mutant *KRAS* exon 2 tumors [19, 22]. Similar results for the first-line setting were subsequently reported for both retrospective and prospective analyses of several randomized clinical trials, including the phase II OPUS and phase III PRIME studies which examined cetuximab or panitumumab plus oxaliplatin, fluorouracil, and leucovorin (FOLFOX4) *vs* FOLFOX4 alone, respectively [23, 25], and the phase III CRYSTAL study which assessed the addition of cetuximab to irinotecan, infusional fluorouracil, and leucovorin (FOLFIRI) [18]. Prospective analysis of the randomized phase III 20050181 study which evaluated panitumumab plus FOLFIRI in the second-line setting further confirmed the

There is evidence that *KRAS* codon 12 and 13 mutations may exhibit differential biological effects, including variable ratios of these codon mutations between tumor types [16] and weaker *in vitro* transforming activity for *KRAS* codon 13 as compared to codon 12 mutant proteins [26]. Accordingly, some studies have suggested that patients with KRAS glycine (G)-to-aspartate (D) transitions at codon 13 (G13D), the most common codon 13 variant in colorectal cancer, might derive some benefit from anti-EGFR therapy [27, 28]. A retrospective consortium analysis assessing patients with chemotherapy-refractory mCRC treated with cetuximab who participated in multiple clinical trials (CO.17, BOND, MABEL, EMR202600, EVEREST, BABEL and SALVAGE) or who received off-study treatment reported longer overall and progression-free survival among individuals with KRAS G13D-mutated tumors than with other *KRAS*-mutated tumors [27]. An analysis of the updated pooled data sets from the CRYSTAL and OPUS studies also reported that addition of cetuximab to first-line chemotherapy appeared to benefit patients with KRAS G13D-mutant tumors [28]. In contrast, a retrospective analysis of 110 patients treated with cetuximab, found that patients with KRAS G13D mutations were unlikely to respond to therapy [29], and similar findings were reported for a retrospective pooled analysis of three randomized phase III trials evaluating panitumumab therapy (20050203, first line; 20050181, second line; and 20020408, monotherapy) [30]. To resolve this controversy, the randomized phase II ICECREAM study prospectively assessed cetuximab monotherapy and cetuximab plus irinotecan in patients with KRAS G13D-mutated chemotherapyrefractory mCRC. In this study, no statistically significant improvement in disease

control was observed for patients with this rare molecular subtype [31].

More recently, several retrospective analyses have indicated that not only *KRAS* exon 2 mutations but also *KRAS* exons 3 and 4 and *NRAS* exons 2, 3, and 4 mutations are negative predictive markers for anti-EGFR therapy [23, 32–36]. These additional mutations are observed in approximately 15–20% of wild-type *KRAS* exon 2 tumors [23, 32]. Reassessment of the randomized OPUS and PRIME studies of cetuximab or panitumumab plus FOLFOX4 *vs* FOLFOX4 alone in the first-line setting found that additional *RAS* mutations predicted a lack of response [23, 34], and corresponding observations were reported for the CRYSTAL study of cetuximab plus FOLFIRI [32]. Accordingly, analyses of single arms of the phase III FIRE-3 study

**110**

*Summary of clinical trials and treatment effects within subgroups defined by RAS status in patients with metastatic colorectal cancer.*

evaluating cetuximab plus FOLFIRI and the phase II PEAK study evaluating panitumumab plus modified fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) in the first-line setting reported a more pronounced survival advantage for the wild-type *RAS* population as compared to the wild-type *KRAS* exon 2 population [36, 37]. Retrospective analysis of the randomized 20050181 study of panitumumab plus

FOLFIRI in the second-line setting further found no benefit of panitumumab addition in patients with *RAS* mutations beyond *KRAS* exon 2 [35]. Low response rates for additional *RAS* mutations were also reported by a European consortium analyzing tumor samples from a large cohort of patients with chemotherapy-refractory mCRC treated with cetuximab and chemotherapy [38].

A systematic review and meta-analysis of nine randomized controlled trials for anti-EGFR therapy comprising a total of 5948 participants evaluated for *RAS* mutations has confirmed tumors without any *RAS* mutations to have significantly superior progression-free (PFS) and overall survival (OS) as compared to tumors with *RAS* mutations. No difference in PFS or OS benefit was evident between tumors with *KRAS* exon 2 mutations and tumors with other *RAS* mutations [33]. Treatment guidelines for mCRC now recommend *RAS* testing prior to start of anti-EGFR antibody therapy to exclude patients with mutated *RAS* [2, 21]. However, *RAS* mutations only account for approximately 35–50% of nonresponsive patients, and the search for additional biomarkers that predict resistance continues to be an active area of research as surveyed below.

### *2.1.2 Primary tumor location*

Colorectal cancers can be broadly grouped by their primary tumor location within the colon [39]. The left-sided colon, comprising the distal third of the transverse colon, splenic flexure, descending colon, sigmoid colon and rectum, are derived from the embryonic hindgut. The right-sided colon, comprising the proximal two-thirds of the transverse colon, ascending colon and caecum, is derived from the embryonic midgut. Baseline differences exist along the colorectal tract such as cell composition and function of the epithelium, the microbiome and gene expression. Strong evidence for the prognostic effect of primary tumor location is available from clinical studies in patients with mCRC, with right-sided tumors exhibiting a worse prognosis [40, 41]. Right- and left-sided cancers differ in their clinical and molecular characteristics: right-sided colon cancers are more likely to be diploid and have high-grade or mucinous histology, DNA mismatch-repair deficiency and microsatellite instability, CpG island methylation, *BRAF*, *TGFBR2* and *PIK3CA* mutations [41, 42], while left-sided cancers often show chromosome instability, *APC*, *KRAS*, *SMAD4* and *TP53* mutations [43]. Right-sided tumors have also been associated with more frequent overexpression of the EGFR ligands, EREG and AREG, and amplification of EGFR and human epidermal growth factor receptor 2 (HER2) [44, 45]. In cohort studies, the classification of tumor sidedness is variable, with right-sided tumors commonly defined as comprising the region from the ceacum to the splenic flexure.

Clinically, primary tumor location was not considered of particular interest in metastatic patients treated with anti-EGFR therapy, until the importance of sidedness as a biomarker was recognized. Retrospective surveys of clinical trials have indicated that while anti-EGFR therapy provides clinical benefit to patients with *RAS* wild-type mCRC, this benefit is specific to patients with left-sided tumors (**Table 2**). In the CRYSTAL and FIRE-3 studies of cetuximab in the first-line setting, patients with *RAS* wild-type left-sided tumors had better outcomes compared to the respective comparators (FOLFIRI alone and FOLFIRI plus bevacizumab), while limited efficacy was observed in patients with RAS wild-type right-sided tumors [46]. Benefit from cetuximab treatment specific to patients with *KRAS* wild-type left-sided tumors was further observed for the randomized phase III CALGB/SWOG 80405 study of cetuximab or bevacizumab with either irinotecan/5- FU/leucovorin (FOLFIRI) or oxaliplatin/5-FU/leucovorin (mFOLFOX6) [47]. Similar results for patients with *RAS* wild-type left-sided as compared to right-sided

**113**

cancers only [53].

*Predictive Biomarkers for Monoclonal Antibody Therapies Targeting EGFR (Cetuximab…*

**of patients**

CRYSTAL FOLFIRI 138 8.9 0.5 0.34–

FOLFIRI + C 142 12 PRIME FOLFOX 159 9.2 0.72 0.57–

FOLFOX + P 169 12.9

FIRE-3 FOLFIRI + B 149 10.7 0.9 0.71–

FOLFIRI + C 157 10.7 PEAK FOLFOX + P 53 14.6 0.65 0.21–

FOLFOX + B 54 11.5

CRYSTAL FOLFIRI 51 7.1 0.87 0.47–

FOLFIRI + C 33 8.1 PRIME FOLFOX 49 7 0.8 0.50–

FOLFOX + P 39 7.5

FIRE-3 FOLFIRI + B 50 9 1.44 0.92–

FOLFIRI + C 38 7.6 PEAK FOLFOX + P 22 8.7 0.84 0.18–

FOLFOX + B 14 12.6

*Abbreviations: PFS, progression-free survival; HR, hazard ratio; CI, confidence interval; FOL, folinic acid; F, fluorouracil; IRI, irinotecan; OX, oxaliplatin; B, bevacizumab; C, cetuximab; P, panitumumab; n.r., not reported;* 

FOLFOX/FOLFIRI + C 71 7.5

FOLFOX/FOLFIRI + C 173 12.7

FOLFOX/FOLFIRI + B 152 11.2 0.84 0.66–

FOLFOX/FOLFIRI + B 78 10.2 1.64 1.15–

**PFS (months)** **HR PFS** **95% CI**

0.72

0.90

1.06

1.14

2.0

1.62

1.26

2.36

2.26

3.79

**p-Value**

<0.001

n.r.

0.15

0.38

n.r.

0.66

n.r.

0.006

0.11

n.r.

tumors were reported for panitumumab for analyses of the PRIME (comparator: FOLFOX alone) and PEAK studies (comparator: FOLFOX plus bevacizumab) [48]. A meta-analysis integrating these data for the first-line setting is available [49]. For the second-line setting, a retrospective analysis of FIRE-3 study also found evidence of better outcomes for cetuximab treatment in patients with *KRAS* wild-type leftsided tumors as compared to right-sided tumors (comparator: bevacizumab) [50]. Similar results for panitumumab were reported in a preliminary efficacy analysis of the 20050181 study for *RAS/BRAF* wild-type patients (comparator FOLFIRI) [51]. A retrospective analysis of the CO.17 study in the treatment-refractory setting further observed that only individuals with *KRAS* wild-type left-sided tumors

*Summary of clinical trials and treatment effects within subgroups defined by primary tumor location in* 

Given the above evidence, NCCN guidelines now recommend the use of anti-EGFR antibody therapies for the treatment of *RAS* wild-type left-sided colon

appeared to benefit from cetuximab as compared to BSC [52].

*DOI: http://dx.doi.org/10.5772/intechopen.80690*

**Left-sided colorectal cancer**

**Right-sided colorectal cancer**

CALGB/ SWOG 80405

CALGB/ SWOG 80405

*BSC, best supportive care.*

*patients with metastatic colorectal cancer.*

**Table 2.**

**Study Treatment arms Number** 


*Predictive Biomarkers for Monoclonal Antibody Therapies Targeting EGFR (Cetuximab… DOI: http://dx.doi.org/10.5772/intechopen.80690*

*Abbreviations: PFS, progression-free survival; HR, hazard ratio; CI, confidence interval; FOL, folinic acid; F, fluorouracil; IRI, irinotecan; OX, oxaliplatin; B, bevacizumab; C, cetuximab; P, panitumumab; n.r., not reported; BSC, best supportive care.*

#### **Table 2.**

*Advances in the Molecular Understanding of Colorectal Cancer*

mCRC treated with cetuximab and chemotherapy [38].

active area of research as surveyed below.

*2.1.2 Primary tumor location*

FOLFIRI in the second-line setting further found no benefit of panitumumab addition in patients with *RAS* mutations beyond *KRAS* exon 2 [35]. Low response rates for additional *RAS* mutations were also reported by a European consortium analyzing tumor samples from a large cohort of patients with chemotherapy-refractory

A systematic review and meta-analysis of nine randomized controlled trials for anti-EGFR therapy comprising a total of 5948 participants evaluated for *RAS* mutations has confirmed tumors without any *RAS* mutations to have significantly superior progression-free (PFS) and overall survival (OS) as compared to tumors with *RAS* mutations. No difference in PFS or OS benefit was evident between tumors with *KRAS* exon 2 mutations and tumors with other *RAS* mutations [33]. Treatment guidelines for mCRC now recommend *RAS* testing prior to start of anti-EGFR antibody therapy to exclude patients with mutated *RAS* [2, 21]. However, *RAS* mutations only account for approximately 35–50% of nonresponsive patients, and the search for additional biomarkers that predict resistance continues to be an

Colorectal cancers can be broadly grouped by their primary tumor location within

the colon [39]. The left-sided colon, comprising the distal third of the transverse colon, splenic flexure, descending colon, sigmoid colon and rectum, are derived from the embryonic hindgut. The right-sided colon, comprising the proximal two-thirds of the transverse colon, ascending colon and caecum, is derived from the embryonic midgut. Baseline differences exist along the colorectal tract such as cell composition and function of the epithelium, the microbiome and gene expression. Strong evidence for the prognostic effect of primary tumor location is available from clinical studies in patients with mCRC, with right-sided tumors exhibiting a worse prognosis [40, 41]. Right- and left-sided cancers differ in their clinical and molecular characteristics: right-sided colon cancers are more likely to be diploid and have high-grade or mucinous histology, DNA mismatch-repair deficiency and microsatellite instability, CpG island methylation, *BRAF*, *TGFBR2* and *PIK3CA* mutations [41, 42], while left-sided cancers often show chromosome instability, *APC*, *KRAS*, *SMAD4* and *TP53* mutations [43]. Right-sided tumors have also been associated with more frequent overexpression of the EGFR ligands, EREG and AREG, and amplification of EGFR and human epidermal growth factor receptor 2 (HER2) [44, 45]. In cohort studies, the classification of tumor sidedness is variable, with right-sided tumors commonly

defined as comprising the region from the ceacum to the splenic flexure.

Clinically, primary tumor location was not considered of particular interest in metastatic patients treated with anti-EGFR therapy, until the importance of sidedness as a biomarker was recognized. Retrospective surveys of clinical trials have indicated that while anti-EGFR therapy provides clinical benefit to patients with *RAS* wild-type mCRC, this benefit is specific to patients with left-sided tumors (**Table 2**). In the CRYSTAL and FIRE-3 studies of cetuximab in the first-line setting, patients with *RAS* wild-type left-sided tumors had better outcomes compared to the respective comparators (FOLFIRI alone and FOLFIRI plus bevacizumab), while limited efficacy was observed in patients with RAS wild-type right-sided tumors [46]. Benefit from cetuximab treatment specific to patients with *KRAS* wild-type left-sided tumors was further observed for the randomized phase III CALGB/SWOG 80405 study of cetuximab or bevacizumab with either irinotecan/5- FU/leucovorin (FOLFIRI) or oxaliplatin/5-FU/leucovorin (mFOLFOX6) [47]. Similar results for patients with *RAS* wild-type left-sided as compared to right-sided

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*Summary of clinical trials and treatment effects within subgroups defined by primary tumor location in patients with metastatic colorectal cancer.*

tumors were reported for panitumumab for analyses of the PRIME (comparator: FOLFOX alone) and PEAK studies (comparator: FOLFOX plus bevacizumab) [48]. A meta-analysis integrating these data for the first-line setting is available [49]. For the second-line setting, a retrospective analysis of FIRE-3 study also found evidence of better outcomes for cetuximab treatment in patients with *KRAS* wild-type leftsided tumors as compared to right-sided tumors (comparator: bevacizumab) [50]. Similar results for panitumumab were reported in a preliminary efficacy analysis of the 20050181 study for *RAS/BRAF* wild-type patients (comparator FOLFIRI) [51]. A retrospective analysis of the CO.17 study in the treatment-refractory setting further observed that only individuals with *KRAS* wild-type left-sided tumors appeared to benefit from cetuximab as compared to BSC [52].

Given the above evidence, NCCN guidelines now recommend the use of anti-EGFR antibody therapies for the treatment of *RAS* wild-type left-sided colon cancers only [53].
