Impact of tumour RAS/BRAF status in a first-line study of panitumumab + FOLFIRI in patients with metastatic colorectal cancer

Background: To investigate tumour biomarker status and efficacy of first-line panitumumab+FOLFIRI for metastatic colorectal carcinoma (mCRC). Methods: 154 patients received first-line panitumumab + FOLFIRI every 14 days. Primary end point was objective response rate (ORR). Data were analysed by tumour RAS (KRAS/NRAS) and BRAF status, and baseline amphiregulin (AREG) expression. Results: Objective responses occurred more frequently in RAS wild type (WT) (59%) vs RAS mutant (MT) (41%) mCRC and in RAS WT/BRAF WT (68%) vs RAS or BRAF MT (37%) disease. Median response duration was longer in RAS WT (13.0 months) vs RAS MT (5.8 months) (hazard ratio (HR): 0.16). Median progression-free survival was longer in RAS WT vs MT (11.2 vs 7.3 months; HR, 0.37) and was also longer in RAS WT/BRAF WT vs RAS or BRAF MT (13.2 vs 6.9 months; HR, 0.25). Incidence of adverse events was similar regardless of RAS/BRAF status, and no new safety signals were noted. Among patients with RAS WT tumours, ORR was 67% with high AREG expression and 38% with low AREG expression. Conclusions: First-line panitumumab+FOLFIRI was associated with favourable efficacy in patients with RAS WT and RAS WT/BRAF WT vs MT mCRC tumours and was well tolerated.

either FOLFOX (panitumumab or cetuximab) or FOLFIRI (cetuximab). Subsequently, mutations were identified in KRAS and NRAS in 17% of patients with non-mutated KRAS exon 2 in the phase III PRIME trial of panitumumab þ FOLFOX vs FOLFOX alone (Douillard et al, 2013). As a result, EGFR inhibitor use was refined to include only those patients with RAS WT disease. At the same time, studies also identified BRAF as an important negative prognostic -though not predictive -marker for survival in patients with mCRC, regardless of treatment (Phipps et al, 2012;Douillard et al, 2013;Peeters et al, 2013Peeters et al, , 2014a. Tumour expression of the biomarker amphiregulin (AREG) has also been correlated with survival during anti-EGFR therapy (Jacobs et al, 2009;Loupakis et al, 2014).
The improved risk-benefit profile of EGFR inhibitor treatment in patients selected by RAS status was later verified in trials of firstand second-line FOLFOX and FOLFIRI in combination with panitumumab or cetuximab (Douillard et al, 2013;Heinemann et al, 2014a, b;Venook et al, 2014;Bokemeyer et al, 2015;Van Cutsem et al, 2015). Importantly, patients with RAS mutant (MT) tumours showed no improvement in efficacy with the addition of EGFR inhibitor compared with chemotherapy alone. Indeed, there is some evidence that EGFR inhibitors combined with FOLFOX in such patients are detrimental compared with FOLFOX alone (Douillard et al, 2013;Bokemeyer et al, 2015), and EGFR inhibitors should therefore not be given to patients with RAS MT tumours.
Panitumumab plus FOLFIRI demonstrated superiority over FOLFIRI alone in a second-line phase II trial that demonstrated improved progression-free survival (PFS) in patients with RAS WT mCRC, although overall survival (OS) was not significantly different in this study (Peeters et al, 2014b). The only published study of first-line panitumumab plus FOLFIRI in mCRC is a single-arm trial that indicated the efficacy and predictable safety profile of the combination in KRAS WT patients , although preliminary data have been presented from a phase II study in which panitumumab plus FOLFOX4 or FOLFIRI was evaluated in patients with KRAS WT colorectal cancer and liverlimited disease (Abad et al, 2014). Here, we present a retrospective analysis of this first-line trial of panitumumab plus FOLFIRI, reporting efficacy and safety data for first-line FOLFIRI plus panitumumab according to tumour RAS/BRAF status and AREG levels in patients with mCRC.

PATIENTS AND METHODS
Study design. This is a retrospective analysis of data from a phase II, single-arm study (NCT 00508404); full details of the study design have been presented previously . In brief, first-line panitumumab (6 mg kg À 1 ) þ FOLFIRI were administered every 14 days until progression, unacceptable toxicity or withdrawal of consent. If FOLFIRI was withdrawn or suspended as a result of toxicity, panitumumab could be continued, and vice versa.
Patients. Patients were adults with histologically or cytologically confirmed, radiologically measurable mCRC and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. Patients could be enrolled only if all disease sites were evaluated within 28 days before enrolment, and tissue from the primary or metastatic site was available. Those who had received prior systemic therapy (including anti-EGFR therapy) for mCRC (except adjuvant fluoropyrimidine-based chemotherapy given X 6 months before enrolment) were excluded. Full inclusion and exclusion criteria have been reported previously .
The study protocol was approved by the relevant independent ethics committees. The study was conducted in accordance with the regulations and guidelines of the International Conference on Harmonisation of Good Clinical Practice. All patients provided signed, informed consent before any study-related procedures were performed.
End points and analyses. The primary end point was the objective response rate (ORR) assessed using modified Response Evaluation Criteria in Solid Tumors (mRECIST v1.0) (Therasse et al, 2000). Secondary end points included disease control rate (DCR), duration of response (DoR), depth of response (DpR; defined as the percentage of tumour shrinkage at nadir or progression), PFS, and time to progression (TTP). Resection rates were also evaluated, as was early tumour shrinkage (ETS), defined as the percentage of patients with X30% or X20% tumour shrinkage at week 8 (exploratory analysis). Safety was evaluated in terms of the incidence and severity of adverse events (AEs), using the National Cancer Institute Common Toxicity Criteria version 3.0.
Data were analysed descriptively by tumour RAS/BRAF status. Tumour specimens were assayed for mutations in KRAS exons 3-4, NRAS exons 2-4 and BRAF exon 15 by bidirectional Sanger sequencing. Mutations in KRAS exon 2 were analysed by CEmarked DxS kit.
Baseline tumour AREG levels were analysed in the RAS WT and MT populations. Total RNA was extracted from formalin-fixed paraffin-embedded tissue samples and AREG expression levels were analysed by qualified reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays (see Supplementary Material for details). A cutoff point for AREG status was prespecified based on analysis of data from an earlier clinical trial (STEPP) (Lacouture et al, 2010). Cox proportional hazards (PH) models were used to evaluate AREG expression levels as a continuous covariate. Decision curves were used to estimate the PFS hazard ratio (HR) with increasing levels of baseline AREG expression. A Gaussian Process (GP) model was used to fit the PH model (Joensuu et al, 2012) using the GPstuff toolkit in MATLAB (Vanhatalo et al, 2013) -details are included in the Supplementary Material.

RESULTS
Ascertainment of tumour KRAS/BRAF mutation status. In total, 154 patients were enrolled in the study . One patient withdrew consent and three patients had no DNA and/or tumour tissue available. The RAS mutational analysis therefore included 150 patients, of whom 143 received at least one dose of panitumumab.
Overall, 38% of patients had KRAS exon 2 mutations, and 10% had RAS mutations beyond KRAS exon 2 (KRAS exon 3 and 4 mutations each in 3% of tumours; NRAS exon 2 and 3 mutations each in 2% of tumours; no tumour was found to carry an NRAS exon 4 mutation). Complete RAS data were available for 143 of 150 patients, of whom 69 (45%) had RAS WT tumours (i.e., WT for exons 2, 3 and 4 of both KRAS and NRAS). BRAF mutations were present in nine patients (6%), all of whom had tumours that were WT for RAS.
Patients. Baseline characteristics were generally well balanced between patients with RAS WT vs RAS MT mCRC, and between patients with RAS WT/BRAF WT and RAS or BRAF MT status (Table 1). More patients with RAS WT and RAS WT/BRAF WT mCRC had liver-limited metastases, whereas more RAS MT and RAS or BRAF MT patients had metastases at other sites only. The sum of the longest diameters of measurable target lesions was also slightly larger in patients with RAS WT and RAS WT/BRAF WT mCRC compared with the corresponding MT populations. The median follow-up time in the study was 34.0 weeks (range, 5-223 weeks).

DISCUSSION
This analysis represents the first reported RAS data beyond KRAS exon 2 for panitumumab þ FOLFIRI in the first-line treatment of mCRC, an indication that has recently been approved by the EMA. The results show consistently favourable efficacy for first-line panitumumab þ FOLFIRI treatment in patients with RAS WT/ BRAF WT tumours compared with MT mCRC tumours. This is consistent with the primary data for the KRAS analysis of this study , in which response rates for patients with KRAS WT (n ¼ 86) and KRAS MT (n ¼ 68) tumours were 56 and 38% and median PFS was 8.9 and 7.2 months, respectively. In the current analysis, extended RAS testing identified 69 patients with RAS WT tumours, and small increases in response rate (59%) and median PFS (11.2 months) were seen in these patients compared with the primary analysis population. The results are also consistent with previous RAS analysis data for panitumumab plus FOLFIRI in the treatment of mCRC (Cohn et al, 2011;Mitchell et al, 2011;Peeters et al, 2014b;Abad et al, 2014). Thus, patients whose tumours harbour RAS mutations beyond KRAS exon 2 are unlikely to benefit from addition of panitumumab to FOLFIRI. Consistent results have also been reported in studies with another anti-EFGR monoclonal antibody, cetuximab (Van Cutsem et al, 2015;Heinemann et al, 2014a, b), highlighting the importance of up-front tumour RAS testing in patients being considered for EGFR inhibitor therapy. As OS was neither an end point nor followed in this trial, efficacy of panitumumab þ FOLFIRI was consistent with that reported for RAS WT populations in first-line  1 1 1 1 1 1 1 1 1 1 1 1    patients should therefore be considered to be at high risk of rapid progression and should be managed accordingly.
No new safety signals were seen with the combination of panitumumab þ FOLFIRI in RAS WT/BRAF WT population; AEs were similar to those seen in the KRAS exon 2 WT population of this study  and in previous studies using this combination in patients with mCRC (Cohn et al, 2011;Mitchell et al, 2011;Peeters et al, 2014b). Overall 28% of patients withdrew from study treatment because of AEs. Consistent with existing data on EGFR inhibitors, there was a high incidence of skin toxicity. While there was no protocol-mandated proactive management of skin toxicity in the present study, it is now recommended for patients receiving EGFR inhibitors (Boone et al, 2007;Melosky et al, 2009). An earlier analysis of data from the present study showed a higher incidence of skin toxicity in patients with KRAS WT tumours than in those with KRAS MT tumours , which may reflect the higher mean cumulative panitumumab dose and longer duration of treatment (i.e., panitumumab cycles) received by the KRAS WT group. However, exposure-adjusted AE rates showed integument-related toxicity overall to be higher in the KRAS MT vs WT population. Thus there is no evidence of an association between tumour KRAS/RAS status and toxicity. Furthermore, despite the high incidence of skin toxicity, generic quality of life (QoL) instruments have shown no impact of EGFR inhibitors plus FOLFIRI on overall QoL (Melosky et al, 2009;Bennett et al, 2011;Thaler et al, 2012). Although proactive management of skin toxicity may have reduced the impact for patients, it may also be that the QoL tools used in the study provided too general an assessment to determine the true impact of this AE. Future trials of EGFR inhibitors should include skin-toxicity specific QoL assessment tools to support better understanding of the true impact of this AE on patient wellbeing.
Among patients with RAS WT mCRC, high AREG expression was associated with response to panitumumab þ FOLFIRI. Consistent with other studies (Jacobs et al, 2009;Baker et al, 2011;Pentheroudakis et al, 2013;Loupakis et al¸2014;Jonker et al, 2014;Stahler et al,2016), there was an interaction between RAS and AREG levels. A higher percentage of patients with RAS WT mCRC had high AREG levels compared with those with RAS MT mCRC, suggesting that AREG levels are associated with EGFR signalling. Further studies are needed to determine whether there is an AREG expression level below which there is little or no response to panitumumab þ FOLFIRI treatment. Biomarker studies remain critical to our understanding of targeted agents in mCRC, and warrant further investigation.
One of the strengths of this comprehensive study was the high percentage of patients from the original cohort who were available for RAS/BRAF testing -which was conducted centrally for all specimens -allowing the identification of additional risks for lack of response to treatment. It should be noted that the analyses were retrospective and exploratory in nature, although, as noted above, the results were consistent with previous analyses of efficacy by RAS mutation status in patients with mCRC.
In conclusion, first-line panitumumab þ FOLFIRI was associated with consistently favourable efficacy in patients with RAS WT/BRAF WT vs MT mCRC tumours and was well tolerated, despite the expected high incidence of skin toxicity. The combination of first-line panitumumab þ FOLFIRI also gave efficacy similar to that seen in the RAS WT populations in other first-line studies of EGFR-targeted agents plus FOLFIRI or FOLFOX. As per the licensed indication for panitumumab, therefore, patients with RAS mutations should not receive panitumumab treatment. Across all lines of therapy, determination of tumour RAS status improves identification of patients unlikely to respond to treatment with panitumumab compared with evaluation of KRAS exon 2 alone. The combination of panitumumab with FOLFIRI can be considered as an important treatment option for first-line patients with RAS WT/BRAF WT mCRC.