Despite exclusion of 30-40% of patients with KRAS-mutant tumours, cetuximab-based regimens fail in more than half of patients bearing KRAS wild-type colorectal cancer
[5–10]. We sought to screen for additional predictive biomarkers in a retrospective clinical series of 226 cetuximab-treated advanced CRC patients and to look for hypothesis-generating hints of efficacy or resistance. Despite homogeneous management of all patients in standardised HeCOG recommended therapies, the retrospective nature of the translational research is an inherent limitation. Moreover, in the absence of a control arm with no treatment it is impossible to speculate on the prognostic (impact on outcome irrespective of therapy) or predictive (impact on benefit from cetuximab therapy) effect of the biomarkers studied. Finally, the administration of cetuximab across several lines of therapy combined with various chemotherapeutic agents constitutes an additional layer of heterogeneity, masking possible interactions and confounders. Moreover, survival analysis will not be robust and even tumor response may be influenced by the chemotherapy backbone to which cetuximab was added. Finally, it would be ideal for all of the tissue samples to have been collected prior to cetuximab therapy, as some of the biomolecules studied may have changed by the time patients received 1st, 2nd or 3rd line therapy.
The incidence of observed KRAS, BRAF, NRAS, PIK3CA mutations are in line with those in the literature, ranging from 30-43% for KRAS, 0.5-7.2% for BRAF, 2-4% for NRAS, 5-17% for PIK3CA
[8, 16–19]. In univariate analysis, BRAF mutations were associated with an 8-fold increased risk of death, while KRAS mutations incurred a weaker, less than 2-fold increase in the risk of death only when codon 12 mutations were analysed separately. Similar to our findings, Souglakos et al. reported a markedly increased risk of death with a multivariate HR of 5.1 for BRAF mutations in cetuximab-treated CRC patients
. Moreover, Modest et al. recently reported median survival times of 23.5 months in KRAS wild-type, 18.9 months in KRAS codon 12-mutated and only 13 months in BRAF-mutated CRC patients on cetuximab-based first-line therapy
. Seemingly, BRAF mutation seems to carry especially adverse prognosis in patients who receive anti-EGFR therapeutic modulation.
We observed no prognostic effect of NRAS, PIK3CA or KRAS mutations outwith codon 12. Despite initial reports of significant negative correlation between PIK3CA mutations and response to anti-EGFR moAbs, series by Prenen et al. and Saridaki et al. failed to find such an association
[10, 18]. De Roock et al. suggested that only exon 20 PIK3CA mutations were associated with resistance to cetuximab therapies
[8, 22]. In our series, only 5 tumours with exon 20 PIK3CA mutations were detected. Regarding KRAS, the same group reported in a pooled dataset of 579 cetuximab-treated CRC patients that those with codon 13 KRAS mutant tumours (G13D) had better outcomes (HR for death 0.50) compared to patients with other KRAS mutant tumours
. Those findings stood in our Greek population cohort, as the median survival of our cetuximab treated patients seemed to de-escalate from 29–33 months for KRAS wild type to 28 months for the KRAS codon 13 mutated group, dropping to 19 months for the KRAS codon 12 mutated group. Although Peeters et al. recently failed to find a prognostic difference between codon 12 and codon 13 KRAS mutations in CRC patients managed with panitumumab in three randomised trials, another group from Germany did so for cetuximab
[21, 23, 24]. KRAS codon 12 mutant CRC patients reached a median survival of only 18.9 months versus 23.5 months for KRAS wild type and 26.2 months for KRAS codon 13 mutant CRC patients on first line cetuximab+CAPIRI/CAPOX. Indeed in preclinical experiments, transforming potential, RAS/GTP activation, MAPK phosphorylation and tumour growth were markedly increased in the presence of KRAS codon 12 mutations, followed in decreasing order by codon 13, 61, 117 and 146 mutations
[25, 26]. Moreover, microarray profiling of multi-gene expression signatures associated with KRAS mutations revealed that codon 12 mutations were linked to a gene cluster distinct from all other KRAS mutations
. Further validation of these data is warranted in order to confirm or refute the hypothesis that specific molecular characteristics of KRAS mutations are determinants of cetuximab benefit.
We observed a strong correlation of increased tumoural mRNA of AREG, EREG with clinical benefit from cetuximab (>50% reduction in the risk of death), in keeping with data reported by four other groups
[11, 20, 27, 28]. Gene expession of the two ligands, which are collocalised on chromosome 4q13.3 and produced by tumour cells in autocrine fashion, was tightly correlated and occurred more often in KRAS and BRAF wild type tumours. The correlation of low EREG/AREG expression with KRAS or BRAF mutated status could be due to the constitutive activation of the RAS/RAF/MAPK pathway which makes activation of the EGFR pathway redundant biologically. Alternatively, it could be due to a negative feedback loop linking MAPK axis activation with suppression of the EGFR pathway.
Objective response to anti-EGFR therapy was correlated to continuous expression levels of AREG, EREG but only to categorical EREG mRNA distribution, intriguingly more strongly in KRAS mutated cases. The smooth nature of relationship between marker levels and outcome, resulting in lack of a universal cut-point applicable in all clinical situations has already been reported by Jacobs et al.
. In the presence of downstream activation of the EGFR/RAS/MAPK axis due to mutated KRAS effector, gene expression of AREG, EREG ligands would be biologically irrelevant for benefit from cetuximab. In our series this was the case for AREG, however tumour EREG mRNA retained predictive significance for survival both in KRAS wild-type as well as mutated cases, a counter-intuitive finding, further supported by the predictive significance of EREG for response, even in KRAS mutant patients. AREG and EREG are not biologically identical: AREG binds EGFR only, whereas EREG binds EGFR and HER4 and leads to a prolonged state of receptor activation
. Compared to AREG, tumour EREG mRNA expession was a stronger predictor of cetuximab benefit in KRAS wild type cases in three more series
[13, 20, 27]. Consequently, we may speculate that even in the presence of KRAS mutations, cetuximab binding to EGFR prevents high levels of EREG from activating HER1/HER4 heterodimers and thus abrogates signalling pathways distinct from RAS/MAPK. In fact, Baker et al. reported HER4 gene expression as one of the genes significantly associated with clinical benefit in 144 cetuximab-treated CRC patients
. However, our findings should be interpreted with caution as the number of EREG-high, KRAS-mutant cases analysed was low (n=10) and random effects cannot be excluded.
In our cohort, the survival of cetuximab-treated patients with KRAS wild type, AREG-low CRC (median 15 months) was as poor as the survival of patients with KRAS mutant tumours (17–22 months). If this finding is confirmed in independent series, AREG expression in KRAS wild type cases would emerge as a robust biomarker of cetuximab efficacy. Baker et al. suggested the use of a 4-gene score (AREG, EREG, DUSP6, SLC26A3) for the prediction of anti-EGFR treatment benefit in KRAS wild type CRC
. In the presence of low AREG and thus inactive EGFR pathway, cetuximab binding to HER dimers might elicit a paradoxical «pro-survival» cellular response via MAPK-dependent and MAPK-independent pathways, as suggested by Oliveras-Ferraros et al.
. Finally, the markedly increased risk of death (8-fold) of high AREG mRNA in KRAS mutant versus KRAS wild-type CRC patients in multivariate analysis could be explained if AREG is viewed as a protein regulated by KRAS-dependent transcription factors: the former group includes tumours bearing KRAS mutations with a markedly pro-survival, proliferative activating effect, whereas the latter group includes KRAS wild type tumours addicted to active EGFR signalling that is amenable to abrogation by cetuximab.