**3. Conclusion**

*Advances in the Molecular Understanding of Colorectal Cancer*

therapy in individual trials.

EGFR therapy [84].

*2.2.5 PIK3CA mutation*

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

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-

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-

*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].

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

GTP binding but is dependent on the interaction with p85 [88].

**116**

*2.2.6 PTEN loss*

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 location [53].

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 discontinuation).

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.
