v-ki-ras2 Kristen rat sarcoma viral oncogene homologue (KRAS), a proto oncogene, is a signal transducer modulated by the EGFR signaling pathway. It is the most frequently mutated gene in colorectal cancer (20%-40%)(Karapetis et al., 2008). KRAS activation induces activation of downstream components of the RAF-MAPK signaling pathway. KRAS is a cytoplasmic GTP-binding protein with low inherent GTPase activity. When the KRAS protein is bound to GTP, it relays signals of cellular proliferation and inhibition of apoptosis, acting as a typical oncogene as described in Figure 2.19 and 2.20. KRAS mutations were observed mainly in gene exon 2, resulting in abrogated GTPase activity and locking the KRAS protein in the active KRAS-GTP conformation. By activating the RAS/RAF/MAPK axis downstream of EGFR, these mutations render therapeutic modulation of EGFR irrelevant. The most frequent mutations are observed in codons 12 and 13 (Vaughn et al., 2011). Mutations in codons 61 and 146 are under investigation.
Figure 2.19: Activation of RAS pathway.
Figure Legend: The ligand EGF (Epidermal growth factor) binds to EGFR (Epidermal growth factor receptor) and leads to phosphorylation of tyrosine kinase domain of the receptor. Upon stimulation of EGF receptor, Grb2 an adaptor protein binds to the tyrosine kinase domain through its SH2 domain and simultaneously binds to another protein SOS. This process catalyzes removal of GDP from RAS .RAS then binds to GTP acquiring an active conformation leading to further downstream RAF and MEK activation through phosphorylation.
Binding of growth factors to receptor tyrosine kinases stimulates the autophosphorylation of specific tyrosines on the receptors. The phosphorylated receptor then binds to an adaptor protein called GRB2 which, in turn, recruits SOS to the plasma membrane. SOS is a guanine nucleotide exchange factor which displaces GDP from Ras, subsequently allowing the binding of GTP and consequently activating RAS.
Figure 2.20: Association of anti-EGFR therapy and KRAS mutations.
Figure Legend: Anti EGFR drugs block receptor signals thus preventing downstream events. In case of Wild type KRAS, when EGFR receptor is blocked, it stops signaling and tumor cells do not proliferate. Whereas in case of mutant KRAS, it is permanently turned on allowing the tumor to continue to proliferate.
Many retrospective studies and trials have shown the impact of KRAS mutational status on treatment efficacy with anti-EGFR monoclonal antibodies in metastatic colorectal cancer patients (Qiu et al., 2010). The studies have suggested that treatment with anti –EGFR produces better outcomes only in patients with wild type KRAS, whereas these drugs had no effect on mutant KRAS patients. Analysis of data from CRYSTAL and OPUS trials showed that addition of cetuximab with chemotherapy provided significant improvement in progression free survival and overall survival, in comparison to chemotherapy alone. Addition of cetuximab significantly reduced the risk of disease
progression by 34% in KRAS wild type patients and increased the likelihood of achieving a response by greater than 2 fold (Odds ratio:2.16, p<0.0001). In the re-analysis of CRYSTAL study done in 2015 additional RAS mutations were examined at KRAS exon 3 (codons 59 and 61), KRAS exon 4 (codons 117 and 146), RAS status was evaluable in 430 of 666 patients (64.6%) (Van Cutsem et al., 2015) (Allegra et al., 2015). Recently, reports have suggested that different KRAS mutations may have different biological characteristics with respect to treatment sensitivity. Tumors having mutations of KRAS codon 13, glycine to aspartate (G13D), have been suggested to retain cetuximab sensitivity and has improved outcomes in some patients during cetuximab therapy (Tejpar et al., 2012). In the reported studies from different population backgrounds the KRAS mutations frequency varies from 14%-40% (Ozen et al., 2013, Mao et al., 2012a, D. Lambrechts, 2009). These variations in patterns of KRAS mutations may be due to the racial differences and etiological factors. In view of the results of several clinical trials, KRAS mutation screening in codons 12 and 13 for metastatic colorectal cancer treatment has been recommended (Allegra et al., 2009). The use of cetuximab and bevacizumab has been approved only for patients with KRAS wild type tumors.
2.8.3.2 BRAF
V-raf murine sarcoma viral oncogene homologue B1 (BRAF) is a member of the RAF family acting downstream of KRAS in the MAPK cascade. The BRAF gene is another potential predictive factor. BRAF and KRAS mutations are mutually exclusive events in tumor (Fransen et al., 2004). The most frequently reported BRAF tumor mutation is a valine-to-glutamic acid amino acid (V600E) substitution that leads to the aberrant activation of the MEK–ERK pathway (Ikenoue et al., 2003). This mutation leads to a 500-fold increase in BRAF activity compared to the wild type form. BRAF mutations are used as exclusion criteria in the diagnosis of hereditary nonpolyposis colorectal cancer syndrome. Also, BRAF mutation is closely associated with MSI-H phenotype, MLH1 hypermethylation and CIMP high status (Zlobec et al., 2010). The predictive value of BRAF mutations in KRAS wild-type patients treated with anti-EGFR therapy has been demonstrated by several groups. In the study carried out by Di Nicanlotonio et al., it was seen that among 79 patients with wild type KRAS, 86% had wild type BRAF. No patient with a mutated BRAF had objective tumor response compared to 32% in patients with wild type BRAF(Di Nicolantonio et al., 2008). In the PETACC-3 study, BRAF mutations occurred in 7.9% of tumors in stage II and stage III colon cancer patients. These mutations were found to be prognostic for overall survival (Hazard ratio=2.2, p=0.003)(Roth et al., 2010). An analysis of 724 patients treated with irinotecan plus cetuximab showed mutated BRAF was present in 5% of patients and that it was associated with reduced responses compared with wild-type BRAF (6% versus 24%) (Tejpar and De Roock, 2009). In the CAIRO-2 study, the predictive and prognostic
value of BRAF was analyzed in 516 patients. 8% of patients had BRAF mutations and had decreased median free survival compared to those without mutation (5.9 versus 12.2 months, P = .003 without cetuximab; and 6.6 vs 10.4 months, P = .010 with cetuximab, respectively) (Tol et al., 2009). This finding suggests that BRAF can be a prognostic factor and not a predictive factor of cetuximab efficacy. Pooled analysis from CRYSTAL and OPUS data confirms that patients with mutated BRAF have worse prognosis than those with wild type BRAF. Based on these findings, BRAF genetic screening has been recommended in patients negative for KRAS mutations before treatment with anti- EGFR drugs.
2.8.3.3 NRAS
Neuroblastoma Ras viral oncogene homolog (NRAS) is a member of RAS family. Along with KRAS and BRAF, NRAS has also been evaluated recently as a potential predictive marker in metastatic colorectal cancer. The most frequently reported NRAS mutations are observed in codons 12, 13, 61, 117 and 146 (D. Lambrechts, 2009). Recently in a retrospective study the predictive value of NRAS was evaluated in KRAS wild type metastatic colorectal cancer patients. As opposed to KRAS mutations, the NRAS mutation frequency was low (3-5%). In the pooled retrospective analysis led by the European Consortium, the rates of response to cetuximab in a large cohort of patients were lower in patients with KRAS wild-type tumors bearing NRAS mutations (De Roock et al., 2010). The only randomized dataset available demonstrates a numeric lack of benefit from panitumumab, another EGFR-targeted monoclonal
significance from findings obtained in a 14-patient population is not feasible. In one recent study, it was observed that in metastatic colorectal cancer the Q61K NRAS mutation had a favorable response to bevacizumab (Janku et al., 2013). These results suggest that NRAS mutations merits further investigation as a potential biomarker predicting the efficacy of bevacizumab-based treatment.
2.8.3.4 PIK3CA
Phosphatidylinositide-3-kinases (PIK3) are lipid kinases that are divided in three classes, I, II and III. Only the a-type isoform of the catalytic subunit, PI3KCA, harbors oncogenic mutations that are present in 15–20% of all colorectal cancers(Jehan et al., 2009). PIK3CA mutations occurring in the “hotspots” located in exon 9 (E542K, E545K) and exon 20 (H1047R) (Samuels et al., 2005). It has been demonstrated that PIK3CA mutations confer resistance to apoptosis, whilst enhancing invasion capacity and metastatic potential. Several studies have demonstrated that PIK3CA mutations do not respond to anti- EGFR therapy and these mutant colorectal cancer patients have shorter progression free survival than wild type patients (Lièvre et al., 2010). The findings of a European consortium suggest that response to EGFR treatment can be predicted only if specific PIK3CA mutation status is co-evaluated with KRAS status (De Roock et al., 2010). These investigations suggest that combining mutational analysis for KRAS and PIK3CA could identify up to 70% patients with metastatic colorectal cancer who are unlikely to respond to treatment with an EGFR targeted monoclonal antibody (Sartore-Bianchi et al., 2009). In one of the recent studies a contradictory evidence was reported in which it was found that there was no strong rationale for using PIK3CA
mutations as a single marker for sensitivity to cetuximab in chemotherapy refractory metastatic colorectal cancer (Prenen et al., 2009). Since tumors with oncogenic PIK3CA are likely to be driven by PI3K as the primary source of growth, proliferation and survival, the use of selective PI3K inhibitors is being tested in ongoing trials. Several PI3K inhibitors are progressing from pre-clinical studies to phase I trials. These include XL147,GDC-0941,BGT226, XL765 and NVP-BEZ235 (Yuan and Cantley, 2008). This data from various trials needs to be validated in clinical applications in larger study groups due to occurrence of low frequency of PIK3CA mutations.
2.8.3.5 PTEN
Phosphatase and tensin homolog (PTEN) acts as a tumor suppressor gene through the action of its phosphatase protein product. This phosphatase is involved in the regulation of the cell cycle, preventing cells from growing and dividing too rapidly (Chu and Tarnawski, 2004). It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating Akt/PKB signaling pathway. PTEN loss or inactivation leads to hyperactivation of the PI3K signaling pathway. Loss of PTEN expression occurs in 30% of sporadic CRCs (Thomas and Grandis, 2004). There are only few studies which have demonstrated that loss of PTEN expression may be useful in predicting response to cetuximab. Frattini et al. reported that none of 11 patients with tumor PTEN loss responded to cetuximab-based treatment, whereas 10 (63%) of 16 patients with intact PTEN protein expression were partial responders (Frattini et al., 2007, Sartore-Bianchi et al., 2009). Further studies are required
Figure 2.21: Estimated Response Rate to EGFR inhibitors in Western
population with activating mutations in KRAS, BRAF, NRAS and PIK3CA Data according to -(Frattini et al., 2007).
2.8.3.6 TP53
The TP53 gene encodes a tumor suppressor protein p53 which is one of the most frequently mutated genes in human cancer. Activated p53 binds to the regulatory sequences of a number of target genes to initiate a program of cell cycle arrest, DNA repair, apoptosis, and angiogenesis (Vogelstein et al., 2000). Loss of function of TP53 is critical in tumorigenesis, and mutations which result in overexpression of the protein are frequent events in colorectal cancer. p53 alterations are more frequent in tumors that are aneuploid, non-mucinous, and do not show any MSI or CIMP molecular phenotypes (Westra et al., 2005).
Associations of TP53 tumor alterations with patient prognosis and response to adjuvant chemotherapy have been widely studied. The majority of translational studies carried out which aimed at determining whether TP53 mutation and overexpression of p53 have prognostic value in colorectal cancer (Popat et al., 2006). Few studies in colon cancer patients failed to demonstrate correlations between TP53 alterations and benefit from adjuvant therapy (Allegra et al., 2003). Similarly, a subset of functionally inactive mutations in TP53 predict poor survival in late stage colorectal cancer (Iacopetta et al., 2006). Oden- Gangloff
et al. suggests that TP53 mutations may be predictive of increased likelihood of
response to cetuximab treatment, particularly in patients with wild-type KRAS status (Slevin et al., 2008).