Phosphorylated AKT and MAPK expression in primary tumours and in corresponding metastases and clinical outcome in colorectal cancer patients receiving irinotecan-cetuximab

Monoclonal antibodies against the ligand-binding site of the epidermal growth factor receptor (EGFR) have been shown to improve global outcome of metastatic colorectal cancer patients [1‐5]. The introduction of K-RAS mutational status for patients selection in this setting appeared to possess the necessary potential for a full translation into clinical practice of the concept of targeted therapy [5‐7].

In fact although we are now able to exclude from anti-EGFR treatment patients with putative refractory colorectal tumours (i.e. those harboring a K-RAS mutant status), we are still unable to select responding patients among those without K-RAS mutations. Clinical observations suggested that a non negligible proportion of patients, ranging from 40% to 70%, does not seem to benefit from the use of anti-EGFR targeted antibodies even in the absence of a mutation of the K- RAS gene (i.e. K-RAS wild-type) [2, 4‐7].

Preclinical observations demonstrated that binding of specific ligands, such as the epidermal growth factor (EGF) and transforming growth factor α (TGF-α) to the EGFR, results in the dimerisation of the receptor with the subsequent initiation of the intracellular signalling pathways cascade. A major downstream signalling route is via the Ras-Raf-MAP-kinase. Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs, ERK1, and ERK2, which ultimately regulate transcription of molecules involved in cell proliferation [8, 9]. Another important target in EGFR signalling is phosphatidylinositol 3-kinase (P13K) and the downstream protein-serine/threonine kinase AKT. This latter protein kinase transduces molecular signals triggering crucial steps for cell growth and survival [8‐10].

It has been also hypothesized that the activation of the downstream signalling pathway (AKT and MAPK) could be responsible for EGFR aberrant activity even in the absence of a detectable EGFR expression [11‐14]. In this case targeting the receptor via monoclonal antibodies would probably be clinically irrelevant. In fact in non small cell lung cancer patients it has been suggested that TKIs responsiveness might be predicted by EGFR downstream proteins such as activated (phosphorylated) AKT [15, 16]. However, data regarding the in vivo EGFR-driven molecular profile in colorectal cancer are conflicting and consequently at the present no speculations are possible about its role in determining resistance and/or sensitivity to EGFR-targeted drugs.

In a series of 28 metastatic colorectal patients treated with gefitinib monotherapy, biologic evaluation of total and activated EGR, activated AKT, MAP-kinase and Ki 67 on paired pre- and 1 week post- treatment tumour samples could not confirm a gefitinib-induced decreased expression of these molecular markers [17].

Moreover no significant correlation has been found between pAKT expression and clinical outcome in metastatic colorectal cancer patients treated with cetuximab [18]. However patients were not stratified for K-RAS status and therefore firm conclusions were not possible.

A further potential confounding factor in this setting is the evidence that AKT and MAPK expression in primary colorectal tumours may not correlate with the expression in corresponding metastases and therefore AKT and MAPK [19].

We tested the interaction between phosphorylated AKT and MAPK in primary colorectal tumours and corresponding metastases and clinical outcome in terms of response rate (RR), progression free survival (PFS) and overall survival (OS) in order to identify a group of K-RAS wild type patients more likely to benefit from EGFR-targeted treatment.

Seventy-two patients were included in our study, 51 males (70%) and 21 females (30%). Median age at diagnosis was 65 years (range 36–80). Most of the patients (61%) were treated with the irinotecan-cetuximab combination and had more than 1 metastatic sites. Main clinical characteristics are summarised in Table 1. In 37 cases the corresponding metastatic sites (all liver metastases) were available for AKT and MAPK analysis (Table 2).

The introduction of K-RAS testing for colorectal cancer patients offered the long-awaited clinical opportunity to exclude resistant tumours from the treatment with anti-EGFR antibodies. However a not negligible proportion of colorectal tumours, ranging from 40 to 70% in different series, is still refractory to this therapeutic approach even in presence of a K-RAS wild type status [2‐5]. Unfortunately to date it is not possible to further characterize, either biologically or clinically, this particular group of patients, who, in the end, receive an ineffective therapy at a cost of unwanted side-effects.

Many biological factors have been investigated with the aim to better identify responding patients in this setting, however initial observations were often conflicting and limited to a small proportion of patients thus precluding their effective use into the clinical practice [23‐28]. The example of b-RAF is clearly emblematic. In fact preliminary data about the role of b-RAF mutational status seemed initially promising for a straightforward application in the clinical practice, but a large subsequent analysis from the CRYSTAL trial demonstrated that b-RAF although possessing a possible prognostic role had no predictive value [29, 30].

In our analysis we suggested that activated AKT and MAPK expression in liver metastases could represent possible molecular determinants for the prediction of clinical outcome in K-RAS wild type colorectal cancer patients receiving irinotecan-cetuximab. These observations are in accordance with the biological assumption that the activation of the downstream signalling pathway (pAKT and pMAPK) could be responsible for EGFR aberrant activity independently from the receptor itself ultimately making treatment strategies targeting the EGFR ineffective. A prior study in colorectal cancer patients could not confirm any correlation between pAKT expression in primary tumours or metastases and either response or survival [18]. However in this analysis the role of phosphorylated AKT was assessed irrespectively of K-RAS status and this may represent a possible confounding factor for data interpretation. An analysis presented by Baba et al. showed that pAKT expression was correlated to good prognosis in colorectal cancer patients. In this latter study the analysis was conducted on primary tumours and no information was available about the treatment administered to the patients. Taken together these and our observations seem then to suggest that more than a prognostic factor pAKT is a predictive factor in presence of anti-EGFR inhibitors.

A further relevant finding from our analysis is that only AKT and MAPK status in metastases is relevant for the prediction of patients outcome. The variation (or conservation) of the primary colorectal tumours molecular profile in distant metastases is a current, but unresolved issue in molecular and clinical oncology. Published analyses in colorectal cancer patients indicated that the mutational status of some molecular determinants such as K-RAS and BRAF is almost completely unaltered from primary to corresponding metastases [31]. On the contrary other biological markers such as EGFR or PTEN, for example, demonstrated a significant variation [18, 32].

A discordant pAKT and pMAPK expression in primary colorectal tumours and metastases has been previously suggested. A shift in pAKT and pMAPK expression from primary to metastases has been in fact reported in as much as 30% of all cases [18, 19]. In the present study a non concordant status between primary tumours and metastases for AKT and MAPK was noticed respectively in 27% and 29% of examined cases thus confirming previous reports. More importantly our data demonstrated that this different expression pattern had a biological effect in modulating the activity of EGFR targeted antibodies, thus reinforcing the hypothesis that when treating metastatic disease with molecularly targeted agents attention should be paid to the biological markers expression profile in metastases.

We could speculate that in colorectal tumours with pAKT and pMAPK expression a therapeutic strategy targeting these molecular factors may be more appropriate than anti-EGFR therapies. However these assumptions should be demonstrated before a possible application into the clinic.

We believe that our findings along with others already reported by other groups may help further composing the molecular mosaic of K-RAS wild type colorectal cancer patients especially in view of a prospective validation.