**6.2 EGFR inhibitors**

The epidermal growth factor receptor (EGFR) is involved in signalling upstream of the RAS-RAF-MEK-ERK pathway. Monoclonal antibodies directed against EGFR, cetuximab and panitumumab have shown to be effective in metastatic CRC; however, KRAS mutation is a negative predictor of EGFR treatment response and upfront testing is recommended before starting treatment [36, 37].

As previously discussed, KRAS and BRAF mutations are mutually exclusive. Given the common signalling pathway, BRAF mutation has also been proposed to be a negative predictive marker of EGRF antibody treatment response. In the first line setting, the PRIME study evaluated the addition of panitumumab to FOLFOX. In BRAF-MT tumours, panitumumab added no benefit to survival (HR 0.9, P = 0.76) [42]. Similarly, the phase III MRC COIN trial showed no benefit in the addition of cetuximab to first-line oxaliplatin based chemotherapy, irrespective of KRAS or BRAF mutation status [43]. In the second line setting, the PICCOLO study reported no effect of panitumumab in combination with irinotecan on PFS, but a significant negative effect on OS (HR 1.84, P = 0.029). Cetuximab was also evaluated against best supportive care in the phase III CO.17 trial [44]. For BRAF MT tumours, there were no responses and no change to survival in the sample size of 13 (HR 0.84, P = 0.81).

Given the small numbers of BRAF-MT patients in these trials, there have been a number of meta-analyses evaluating the BRAF mutation as a predictive marker of EGFR therapy. Therkildsen et al. reviewed KRAS, NRAS, BRAF, PIK3CA and PTEN mutations in patients with KRAS exon 2 wild-type patients. Of the 1267 patients in 17 studies treated with either cetuximab or panitumumab in both first line and subsequent like therapies, 128 patients had BRAF V600E mutations [45]. There was a significant decrease in overall response rate (17 vs. 45%). BRAF mutation was also linked to shorter PFS (HR 2.95) and OS (HR 2.52) compared to BRAF wild-type tumours.

Pietrantonio et al. examined the impact of cetuximab and panitumumab on PFS, OS and overall response rate (ORR) [46]. This meta-analysis included 9 phase III trials and 1 phase II trial (across first-line, second-line and chemotherapy refractory settings). 463 RAS wild-type/BRAF MT CRC patients were identified. The addition of EGFR antibody therapy did not significantly improve PFS (HR 0.88, P = 0.33), OS (HR 0.91, P = 0.63) and ORR (relative risk 1.31, P = 0.25).

A further meta-analysis was published in 2015 but Rowland et al. [47]. It included 8 randomised control trials that had also been included in the analysis by Pietrantonio et al., but differed by excluding 2 trials; 1 by Tveit et al. [48] due to lack of OS and PFS data and Stintzing et al. [49] as the control arm included

**41**

*BRAF Mutation and Its Importance in Colorectal Cancer*

trend to improved ORR (71.4 vs. 22.2%, P = 0.1262).

cal agents such as BRAF inhibitors as discussed below.

bevacizumab. In addition, the statistical analysis differed as Rowland et al. compared BRAF MT patients with BRAF wild-type. 351 patients were identified with BRAF mutation, of which 330 with the V600E mutation. The HR for PFS was 0.86 for RAS wild-type/BRAF MT compared with 0.62 for RAS wild-type/BRAF wild-type tumours with a test of interaction that nears but does not reach statistical significance (P = 0.07). There was no difference for OS either, the HR for RAS wild-type/BRAF MT tumours was 0.97 compared with 0.81 for RAS wild-type/ BRAF wild-type (test of interaction, P = 0.43). It concluded that there was insufficient evidence to definitively state that RAS wild-type/BRAF MT individuals derive a different treatment benefit from EGFR antibodies compared with RAS wild-type/

More recently, the triplet chemotherapy regime, FOLFOXIRI, has been studied in combination with panitumumab in the VOLFI trial [50]. This was a randomised phase II trial of patients with RAS WT, unresectable metastatic CRC. 96 patients were included, of which, 16 patients with BRAF MT disease. The primary endpoint was ORR. The addition of panitumumab significantly improved ORR in the overall population (85.7 vs. 54.5%, P = 0.0013), and in the BRAF MT population, there was

Thus, while there exists a significant body of evidence that suggests minimal clinical benefit of EGFR antibody treatment in BRAF MT metastatic CRC, it is not definitive and therefore remains an option for therapy in discussion with the patient. This primarily relates to anti-EGFR as the sole biological agent however anti-EGFR therapy may have a definite role when combined with additional biologi-

BRAF represents a therapeutic target in cancer as, unlike KRAS, it is a relatively unidirectional MEK-ERK effector. Inhibition of BRAF with vemurafenib (PLX4032) has been demonstrated to significantly benefit patients with unresectable or metastatic BRAF V600E MT melanoma, improving progression free survival and OS, with a response rate of 48% [51]. In sharp contrast, BRAF inhibition in mCRC is disappointing. An expansion phase II study examined vemurafenib in patients with BRAF MT mCRC who have had at least one line of prior therapy [52]. Of the 21 patients treated, 1 patient had a partial response and 7 other patients had stable disease by RESIST criteria. The median PFS was only 2.1 months and ORR of 5%. Although there were signs of efficacy, the authors concluded that single-agent vemurafenib did not show any meaningful clinical activity in patients with BRAF

These results were similar to a histology-independent phase II "basket" trial of vemurafenib. 122 patients with BRAF V600 MT malignancies were enrolled into 7 prespecified cohorts, including 37 with mCRC [53]. Vemurafenib, as a single agent, was given to 10 patients with mCRC. Response was poor, with 50% having stable disease and the rest progressing on therapy. The remaining 27 patients with mCRC received combination of vemurafenib and cetuximab, and the results will be

There are several mechanisms of resistance identified that reduce the efficacy of BRAF inhibition in mCRC. For example Prahallad et al. identified that BRAF inhibition with vemurafenib in mCRC cells causes a rapid activation of EGFR through an ERK-dependent negative feedback loop [54]. Unlike in melanoma, CRC cell lines express high levels of activated EGFR. Blockade of EGFR with either EGFR monoclonal antibodies or small-molecule kinase inhibitors (gefitinib and erlotinib)

was showed to work synergistically with BRAF inhibition.

*DOI: http://dx.doi.org/10.5772/intechopen.82571*

BRAF wild-type patients.

**6.3 BRAF inhibition in mCRC**

V600E MT mCRC.

discussed later in the chapter.

*BRAF Mutation and Its Importance in Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.82571*

*Advances in the Molecular Understanding of Colorectal Cancer*

performance status given the overall survival data.

**6.2 EGFR inhibitors**

showed a statistically significant benefit to progression free survival and trend towards improved overall survival at the expense of greater incidence of grade three toxicities [39]. An exploratory analysis of the BRAF-MT cohort (25 patients in a pooled population) reported a median PFS of 11.8 months, median OS of 24.1 months and an impressive response rate of 72%, including one patient with complete response [40]. This was followed up by the open label phase III TRIBE study comparing FOLFIRI plus bevacizumab with FOLFOXIRI with bevacizumab [41]. In the molecular subgroup analysis, 28 out of 391 cases were BRAF mutant. There was a trend towards benefit in overall survival (19.0 months in the FOLFOXIRI plus bevacizumab arm vs. 10.7 months in the FOLFIRI plus bevacizumab arm, HR 0.54); however, this was not statistically significant. This was also seen in median PFS (7.5 vs. 9.5 months, HR 0.57) and best overall response (56 vs. 42%). While not statistically significant, this regime has been proposed in the first line setting for BRAF-MT mCRC patients with good

The epidermal growth factor receptor (EGFR) is involved in signalling upstream

As previously discussed, KRAS and BRAF mutations are mutually exclusive. Given the common signalling pathway, BRAF mutation has also been proposed to be a negative predictive marker of EGRF antibody treatment response. In the first line setting, the PRIME study evaluated the addition of panitumumab to FOLFOX. In BRAF-MT tumours, panitumumab added no benefit to survival (HR 0.9, P = 0.76) [42]. Similarly, the phase III MRC COIN trial showed no benefit in the addition of cetuximab to first-line oxaliplatin based chemotherapy, irrespective of KRAS or BRAF mutation status [43]. In the second line setting, the PICCOLO study reported no effect of panitumumab in combination with irinotecan on PFS, but a significant negative effect on OS (HR 1.84, P = 0.029). Cetuximab was also evaluated against best supportive care in the phase III CO.17 trial [44]. For BRAF MT tumours, there were no responses and no change to survival in the sample size of 13 (HR 0.84,

Given the small numbers of BRAF-MT patients in these trials, there have been a number of meta-analyses evaluating the BRAF mutation as a predictive marker of EGFR therapy. Therkildsen et al. reviewed KRAS, NRAS, BRAF, PIK3CA and PTEN mutations in patients with KRAS exon 2 wild-type patients. Of the 1267 patients in 17 studies treated with either cetuximab or panitumumab in both first line and subsequent like therapies, 128 patients had BRAF V600E mutations [45]. There was a significant decrease in overall response rate (17 vs. 45%). BRAF mutation was also linked to shorter PFS (HR 2.95) and OS (HR 2.52) compared to BRAF wild-type tumours.

Pietrantonio et al. examined the impact of cetuximab and panitumumab on PFS, OS and overall response rate (ORR) [46]. This meta-analysis included 9 phase III trials and 1 phase II trial (across first-line, second-line and chemotherapy refractory settings). 463 RAS wild-type/BRAF MT CRC patients were identified. The addition of EGFR antibody therapy did not significantly improve PFS (HR 0.88, P = 0.33),

A further meta-analysis was published in 2015 but Rowland et al. [47]. It included 8 randomised control trials that had also been included in the analysis by Pietrantonio et al., but differed by excluding 2 trials; 1 by Tveit et al. [48] due to lack of OS and PFS data and Stintzing et al. [49] as the control arm included

OS (HR 0.91, P = 0.63) and ORR (relative risk 1.31, P = 0.25).

of the RAS-RAF-MEK-ERK pathway. Monoclonal antibodies directed against EGFR, cetuximab and panitumumab have shown to be effective in metastatic CRC; however, KRAS mutation is a negative predictor of EGFR treatment response and

upfront testing is recommended before starting treatment [36, 37].

**40**

P = 0.81).

bevacizumab. In addition, the statistical analysis differed as Rowland et al. compared BRAF MT patients with BRAF wild-type. 351 patients were identified with BRAF mutation, of which 330 with the V600E mutation. The HR for PFS was 0.86 for RAS wild-type/BRAF MT compared with 0.62 for RAS wild-type/BRAF wild-type tumours with a test of interaction that nears but does not reach statistical significance (P = 0.07). There was no difference for OS either, the HR for RAS wild-type/BRAF MT tumours was 0.97 compared with 0.81 for RAS wild-type/ BRAF wild-type (test of interaction, P = 0.43). It concluded that there was insufficient evidence to definitively state that RAS wild-type/BRAF MT individuals derive a different treatment benefit from EGFR antibodies compared with RAS wild-type/ BRAF wild-type patients.

More recently, the triplet chemotherapy regime, FOLFOXIRI, has been studied in combination with panitumumab in the VOLFI trial [50]. This was a randomised phase II trial of patients with RAS WT, unresectable metastatic CRC. 96 patients were included, of which, 16 patients with BRAF MT disease. The primary endpoint was ORR. The addition of panitumumab significantly improved ORR in the overall population (85.7 vs. 54.5%, P = 0.0013), and in the BRAF MT population, there was trend to improved ORR (71.4 vs. 22.2%, P = 0.1262).

Thus, while there exists a significant body of evidence that suggests minimal clinical benefit of EGFR antibody treatment in BRAF MT metastatic CRC, it is not definitive and therefore remains an option for therapy in discussion with the patient. This primarily relates to anti-EGFR as the sole biological agent however anti-EGFR therapy may have a definite role when combined with additional biological agents such as BRAF inhibitors as discussed below.

## **6.3 BRAF inhibition in mCRC**

BRAF represents a therapeutic target in cancer as, unlike KRAS, it is a relatively unidirectional MEK-ERK effector. Inhibition of BRAF with vemurafenib (PLX4032) has been demonstrated to significantly benefit patients with unresectable or metastatic BRAF V600E MT melanoma, improving progression free survival and OS, with a response rate of 48% [51]. In sharp contrast, BRAF inhibition in mCRC is disappointing. An expansion phase II study examined vemurafenib in patients with BRAF MT mCRC who have had at least one line of prior therapy [52]. Of the 21 patients treated, 1 patient had a partial response and 7 other patients had stable disease by RESIST criteria. The median PFS was only 2.1 months and ORR of 5%. Although there were signs of efficacy, the authors concluded that single-agent vemurafenib did not show any meaningful clinical activity in patients with BRAF V600E MT mCRC.

These results were similar to a histology-independent phase II "basket" trial of vemurafenib. 122 patients with BRAF V600 MT malignancies were enrolled into 7 prespecified cohorts, including 37 with mCRC [53]. Vemurafenib, as a single agent, was given to 10 patients with mCRC. Response was poor, with 50% having stable disease and the rest progressing on therapy. The remaining 27 patients with mCRC received combination of vemurafenib and cetuximab, and the results will be discussed later in the chapter.

There are several mechanisms of resistance identified that reduce the efficacy of BRAF inhibition in mCRC. For example Prahallad et al. identified that BRAF inhibition with vemurafenib in mCRC cells causes a rapid activation of EGFR through an ERK-dependent negative feedback loop [54]. Unlike in melanoma, CRC cell lines express high levels of activated EGFR. Blockade of EGFR with either EGFR monoclonal antibodies or small-molecule kinase inhibitors (gefitinib and erlotinib) was showed to work synergistically with BRAF inhibition.

More recently, it has been showed that BRAF inhibition can also lead to up regulation of other receptor tyrosine kinases including human epidermal growth factor receptor (HER) 2 and HER3 [55].

Activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway has also been implicated in BRAF inhibition resistance [56]. PI3K signalling is activated by direct mutational activation or amplification of PIK3CA and AKT1 or loss of PTEN [57]. Approximately 40% of CRC have been shown to have alterations in 1 of 8 PI3K pathway genes, which are almost always mutually exclusive to each other [58]. Genotyping of BRAF MT CRC has showed concomitant PI3KCA and PTEN mutations [59].

The Wnt/β-catenin pathway is also involved in cell proliferation, differentiation and survival and interacts with the RAS-RAF-MEK-ERK pathway at multiple points. It has been identified as an important step in tumourigenesis and alterations in the Wnt pathway have been identified more frequently in BRAF V600E MT CRC patient samples, potentially representing an alternative pathway of tumour development when BRAF is inhibited [60].

Based on these findings, BRAF inhibition has been combined with a number of different agents in order to attempt to overcome resistance and improve response.
