*2.2.3 Amphiregulin (AREG) and epiregulin (EREG) expression*

The EGFR ligands AREG and EREG are overexpressed in colorectal cancer at both the mRNA and protein levels [70, 71], and suppression of *AREG* or *EREG* gene expression reduces the therapeutic efficacy of cetuximab in tumor cell lines [72]. Accordingly, multiple studies have found evidence that the extent of expression of these ligands is related to efficacy of anti-EGFR therapy [71, 73–79].

For example, in the randomized phase III PICCOLO study of panitumumab and irinotecan *vs* irinotecan alone in fluorouracil-resistant mCRC, high messenger RNA (mRNA) expression of *EREG* or *AREG* (defined as either *EREG* or *AREG* in the top tertile for mRNA level) was a predictive marker for anti-EGFR therapy benefit in patients with wild-type *RAS* tumors. In contrast, patients with mutant *RAS* tumors gained no panitumumab therapy benefit regardless of ligand status [80]. Similarly, in the CO.17 study of cetuximab in chemotherapy-refractory mCRC, wild-type *KRAS* patients with high *EREG* gene expression obtained benefit from cetuximab therapy, while no benefit was observed in patients with low *EREG* expression; patients with mutant *KRAS* tumors showed no improvement on anti-EGFR therapy irrespective of *EREG* expression levels [76]. A retrospective analysis of the single-arm phase II NCT 00508404 study of first-line panitumumab plus FOLFIRI similarly also found a higher overall response rate for patients with wild-type *RAS* tumors and high *vs* low *AREG* expression [81]. A meta-analysis including eight studies that used anti-EGFR therapy alone or in combination with chemotherapy reported that *AREG*/*EREG* mRNA overexpression was associated with longer overall survival in patients with wild-type *RAS* tumors who received cetuximab or panitumumab treatment; *AREG* overexpression was further associated with longer PFS. In contrast, *AREG* and *EREG* was found not to have predictive value in patients with mutant *KRAS* tumors [82]. Given these encouraging data, further examination of these ligands in prospective controlled trials appears warranted.

### *2.2.4 BRAF mutation*

The *BRAF* gene encodes a serine-threonine protein kinase that is an integral member of the RAS/MAPK signaling pathway. Approximately 10% of colorectal cancers harbor activating mutations in *BRAF*, with a valine (V) to a glutamic acid (E) substitution at codon 600 (V600E) accounting for more than 95% of alterations [16]. Mutations in *BRAF* are mutually exclusive with *KRAS* mutations in CRC [83]. Patients with mCRC who possess a *BRAF* mutation have significantly poorer prognosis as measured by PFS and OS, and mutational analysis is recommended

for prognostic stratification in guidelines from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology [84]. The relatively low mutation prevalence and strong association with prognosis in the metastatic setting have hampered conclusive evaluation of *BRAF* status as a predictive biomarker for anti-EGFR therapy in individual trials.

Two meta-analyses of randomized studies of anti-EGFR antibodies have been conducted with inconsistent findings. The first meta-analysis of eight randomized controlled trials published in seven studies concluded that there was insufficient evidence to demonstrate that mCRC patients with wild-type *RAS*/mutant *BRAF* tumors attain a different treatment benefit from anti-EGFR therapy as compared to patients with wild-type *RAS*/wild-type *BRAF* tumors [85]. However, the second meta-analysis of 10 randomized controlled trials from nine reports focusing on wild-type *RAS*/mutant *BRAF* tumors reported that anti-EGFR therapy provided no benefit in these patients, indicating presence of mutation as a marker of drug resistance. Based on these uncertain data, current guidelines for the treatment of mCRC do not recommend *BRAF* mutations as a biomarker for response to anti-EGFR therapy [84].

### *2.2.5 PIK3CA mutation*

Phosphoinositide 3-kinases (PI3K) are a family of heterodimeric lipid kinases which consist of regulatory (p85) and catalytic (p110) subunits. PI3K is a key signaling mediator downstream of EGFR involved in the regulation of cell metabolism, growth, proliferation and survival. The *PIK3CA* gene encodes the catalytic subunit, p110α, which, when mutated in cancer, results in constitutively active PI3K signaling. *PIK3CA* mutations are present in approximately 10–20% of colorectal cancers, with missense mutations in exon 9 (helical domain) and exon 20 (kinase domain) being the most common alterations [86, 87]. Notably, biochemical studies comparing mutant p110α proteins have established that exon 9 and exon 20 substitutions have different mechanisms of action. Exon 9-mutant p110α protein induces cell transformation independently of binding to p85 but requires interaction with RAS-GTP, whereas exon 20-mutant p110α protein is active in the absence of RAS-GTP binding but is dependent on the interaction with p85 [88].

*PIK3CA* mutations have been investigated as a potential predictor of anti-EGFR therapy efficacy, with studies considering mutation status overall or for exons 9 and 20 separately. Again, conclusive analyses from individual studies have been hampered by the relatively low mutation prevalence, with *PIK3CA* mutations tending to co-occur with *KRAS* mutations [87]. A series of meta-analyses have been conducted to consolidate findings, indicating that *PIK3CA* mutations as a whole are associated with a lack of anti-EGFR therapy response in patients with wild-type *RAS* tumors [89–93]. Some studies further suggest that the predictive power may be confined to exon 20 mutations, although sample size remains limited [90, 91, 94]. However, these meta-analyses have included many of the same studies, as well as observed and acknowledged between-study heterogeneity. Further investigations are needed before definitive conclusions regarding the predictive value of *PIK3CA* mutations for clinical decision making can be drawn, and *PIK3CA* mutational analysis of colorectal carcinoma tissue for therapy selection outside of a clinical trial is currently not recommended [84].

#### *2.2.6 PTEN loss*

PTEN is a negative regulator of the PI3K/AKT pathway downstream of EGFR through its lipid phosphatase activity. PTEN is a tumor suppressor gene

**117**

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

in colorectal cancer, with inactivating mutations or loss of protein expression observed in approximately 5% and 30% of sporadic colorectal cancers [87, 95, 96]. With respect to response to anti-EGFR therapy, a number of studies have indicated an association with PTEN loss and lack of response to cetuximab and panitumumab [97–100], although other reports have not identified this relationship [101–103]. There are also data to suggest that some discordance in PTEN protein expression may exist between primary tumors and metastases [104]. Several meta-analyses have considered published findings, supporting the notion that loss of PTEN protein expression and/or mutation are predictive of worse outcomes in patients with wild-type *KRAS* tumors treated with anti-EGFR therapy [91, 92, 105]. However, given a high level of variability in methods for assessment of PTEN expression between studies, including IHC scoring algorithms, and the potential inconsistency in expression between primary and metastatic tumor samples, loss of PTEN expression cannot yet be regarded as a reliable predictive biomarker. Further investigation and prospective large randomized clinical trials are still required to fully confirm the role of PTEN in anti-EGFR therapy

The introduction of multiple chemotherapy and biological therapy options for the treatment of CRC over the past few decades have driven an increased need for predictive biomarkers to select the most appropriate therapy for each patient. Biomarker guided treatment selection is critical to improving patient outcomes, reducing exposure to ineffective lines of treatment that are associated with significant toxicities and costs. For the use of the anti-EGFR antibodies cetuximab and panitumumab, current best clinical practice mandates that assessment of all common mutations in *KRAS* and *NRAS* be undertaken at the time of diagnosis of mCRC. Sidedness is also an important factor and it is recommended to limit anti-EGFR therapy to cases with left-sided primary tumor

Mutations and amplifications of several genes other than *RAS* have been investigated as potential predictive biomarkers of response to anti-EGFR therapy. These include *EGFR* gene copy number, *BRAF* and *PIK3CA* mutation as well as PTEN loss (mutation and loss of protein expression). The individual frequencies of all of these mutations and amplifications are low and methodologies to determine DNA copy number or protein expression have been highly variable across studies, thus whether these alterations are true biomarkers for anti-EGFR therapy resistance remains uncertain. Expression of the EGFR ligands AREG and EREG are an interesting avenue to explore, but current evidence is insufficient to recommend routine testing in clinical practice. Skin toxicity is a potential predictive marker in wild-type *RAS* patients receiving anti-EGFR therapy, but prospective randomized data are required to demonstrate clinical utility and determine how this information is best used to inform patient management (dose escalation *vs* treatment

While significant progress has been made in identifying predictive biomarkers for anti-EGFR therapy, with *RAS* mutation status and tumor sidedness endorsed as clinical diagnostics, many patients treated with cetuximab and panitumumab as selected by these parameters still do not experience treatment benefit. Further basic biology and clinical studies are clearly warranted to improve our understanding of EGFR signaling to identify novel biomarkers predictive of anti-EGFR therapy

response and to develop more refined companion diagnostics.

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

resistance.

**3. Conclusion**

location [53].

discontinuation).

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

in colorectal cancer, with inactivating mutations or loss of protein expression observed in approximately 5% and 30% of sporadic colorectal cancers [87, 95, 96].

With respect to response to anti-EGFR therapy, a number of studies have indicated an association with PTEN loss and lack of response to cetuximab and panitumumab [97–100], although other reports have not identified this relationship [101–103]. There are also data to suggest that some discordance in PTEN protein expression may exist between primary tumors and metastases [104]. Several meta-analyses have considered published findings, supporting the notion that loss of PTEN protein expression and/or mutation are predictive of worse outcomes in patients with wild-type *KRAS* tumors treated with anti-EGFR therapy [91, 92, 105]. However, given a high level of variability in methods for assessment of PTEN expression between studies, including IHC scoring algorithms, and the potential inconsistency in expression between primary and metastatic tumor samples, loss of PTEN expression cannot yet be regarded as a reliable predictive biomarker. Further investigation and prospective large randomized clinical trials are still required to fully confirm the role of PTEN in anti-EGFR therapy resistance.
