The Role of p53 Expression in Patients with RAS/BRAF Wild-Type Metastatic Colorectal Cancer Receiving Irinotecan and Cetuximab as Later Line Treatment

The transmembrane tyrosine kinase epidermal growth factor receptor (EGFR or HER1, ErB1) belongs to the ErbB family along with ErbB-2, ErbB-3 and ErbB-4a and is a key driver of cell proliferation, survival, adhesion, migration and differentiation [1‐3]. Deregulation of the EGFR pathway was observed in several malignancies such as colorectal, non-small cell lung, breast, ovary, renal, head and neck, pancreatic, prostate, cervical and bladder cancer [1, 4, 5]. In this setting, EGFR overexpression/upregulation and hyperactivation might be responsible for the promotion of tumour cell growth, resistance to apoptosis, synthesis of angiogenic and growth factors and metastatic spread. As a consequence, EGFR blockade plays an established role as an anti-cancer strategy, especially in colorectal cancer (CRC), non-small cell lung cancer, and head and neck tumours [6‐14]. Presently, the anti-EGFR monoclonal antibodies cetuximab and panitumumab represent the cornerstone of treatment for RAS wild-type (WT) metastatic CRC (mCRC) [15, 16].

Unfortunately, not all patients with RAS WT mCRC are sensitive to EGFR-targeting agents and resistance to these drugs is still an open issue [17‐21]. Several potential mechanisms for the lack of efficacy of anti-EGFR have been investigated beyond N-RAS and K-RAS mutational status, such as BRAF V600E mutation [22‐26], PI3KCA exon 20 mutation, PTEN loss [22, 27‐29], Stat3 and Akt phosphorylation [22, 30‐32], HER2 amplification [22, 33‐36], HER3 expression [37], IGF1R activation [22, 38, 39], MET amplification [22, 40], altered vascular endothelial growth factor/vascular endothelial growth factor receptor expression [22, 41], EGFR gene copy number alteration and EGFR methylation [42, 43], but for most of these no definitive data are available.

Among the potential mechanisms of resistance, data suggest that TP53 mutations might play a role in tumour cell sensitivity to anti-EGFR antibodies. In fact, recent studies have suggested that activation of the EGFR pathway leads to malignant transformation only if the p53 protein is inactivated [44].

The p53 protein, which is encoded by the tumour suppressor gene p53 (TP53) located in chromosome 17p, is one of the most important tumour suppressors. Acting as a zinc-containing transcription factor [45‐49], it regulates downstream genes involved in DNA repair, cell-cycle arrest and apoptosis [50‐54]. More specifically, in the event of cellular stress signals such as genotoxic damage, oncogene activation, hypoxia or loss of intercellular adhesion, p53 takes on an active tetrameric form and stops cell-cycle progression and induces DNA repair, senescence and cell death through apoptosis to preserve the genomic integrity. Consequently, p53 has a crucial role in the regulation of cell proliferation and cancer development inhibition; for its pivotal role in protection from tumour development, it has been defined as “the guardian of the genome” [54‐61].

Mutations or deletions of the TP53 gene can be found in nearly 50% of human cancers and they lead to an impaired tumour suppressor function [62]. Tumours harbouring a TP53 mutation are usually characterised by genomic instability and poor prognosis [54, 63, 64]. Moreover, it was shown that in p53 mutants, the loss of WT p53 function is also associated with the gain of new oncogenic roles promoting cancer, metastasis, inhibition of apoptosis and drug resistance [64‐67].

Mutations in TP53 usually occur in 40–60% of patients with CRC. There are well-known logistical difficulties and resource limitations associated with direct sequencing of the TP53 gene. Therefore, most studies have used immunohistochemistry to detect mutant p53, with the assumption that a mutation is often associated with its overexpression. However, the lack of expression is generally indicative of WT TP53. Only in limited cases, a complete loss of p53 expression is associated with truncating mutations and loss of heterozygosity (LOH). Several trials documented conflicting results regarding the association between p53 over-expression and clinical outcome [68‐71].

The predictive function of TP53 mutations in patients with mCRC treated with targeted therapies has not been established so far. However, some evidence suggests that TP53 status may affect the response to anti-EGFR therapies as a consequence of the activity of p53 on the promoter of the EGFR [72‐77]. However, findings from previous analyses investigating the role of p53 in this setting were contradictory. Based on these considerations, we designed our study with the aim to assess the predictive/prognostic role of p53 abnormal expression in patients with mCRC treated with anti-EGFR therapy by using a validation cohort.

The emergence of drug resistance represents the major limitation to the development and use of molecularly targeted cancer therapies. In this scenario, only a few studies tried to identify the prognostic and the predictive role of p53 in patients treated with anti-EGFR therapies.

Preclinical data suggest that both WT and mutant TP53 can modulate EGFR expression in various tumours. Ludes-Meyers et al. showed that WT and mutant TP53 use different mechanisms to activate EGFR transcription. They identified the binding site for WT TP53 in an EGFR promoter gene-specific sequence and observed that the p53 DNA-binding capacity seemed to be related to this gene transcription activation; furthermore, this sequence appeared to be the same target site of positive and negative regulators of EGFR promoter activity. Conversely, mutant TP53 did not require the WT p53-binding site for EGFR promoter transactivation but the oligomerisation domain after recruitment by other transcription factors. As mutant p53 is often overexpressed in tumour cells, this could lead to strong and persistent activation of growth factor genes, such as high EGFR levels and consequent aberrant cell proliferation [72‐76]. A further study demonstrated that the loss of p53 in normal human keratinocytes is responsible for increased EGFR expression by a mechanism involving YY1 and Sp1 and does not require p53 binding to the EGFR promoter [77]. In summary, preclinical findings show that p53 modulates EGFR promoter activity both directly by DNA binding and indirectly through other transcription factors.

Other clinical studies evaluated the co-expression of mutant p53 and EGFR mutations/overexpression in different tumours, suggesting a correlation with poor prognosis and a lack of response to tyrosine kinase inhibitors, chemotherapy and radiotherapy in non-small cell lung cancer, head and neck, and hepatocellular malignancies [81‐84].

However, the clinical role of TP53 mutational status in CRC is still controversial. Several studies showed that patients with a tumour harbouring a TP53 mutation have a significantly poorer outcome than patients with WT TP53 tumours. Moreover, p53 overexpression has a negative prognostic impact. Conversely, other findings failed to demonstrate a correlation between p53 overexpression and clinical outcome in patients with CRC [81‐83]. Theodoropoulos et al. retrospectively evaluated the relationship between p53 and EGFR expression assessing the correlation with clinical/histological prognostic factors and the impact on prognosis or survival in 164 patients with CRC with at least a 5-year follow-up. Overexpression of EGFR and p53 were significantly associated with an advanced T stage, suggesting that both proteins cause a growth advantage in deep invasion and they are a late event in CRC carcinogenesis [85].

In contrast with previous findings, Shyhmin et al. revealed that WT TP53 may enhance sensitivity to EGFR inhibitors and radiation through cell-cycle arrest, apoptosis and DNA damage repair, in CRC and non-small cell lung cancer [86]. The other two studies showed that TP53 mutations may be predictive of cetuximab sensitivity and TP53 genotyping could be useful to optimise the selection of patients with mCRC who should benefit from cetuximab-based chemotherapy [87, 88].

Of note, most pathologists do not have access to TP53 sequencing, and therefore, they use p53 immunohistochemistry as a surrogate for TP53 mutational analysis. Immunohistochemistry is quick, easy to perform and less expensive than sequencing. Hence, p53 immunohistochemistry represents a feasible biomarker already in use in the clinical setting. On this basis, our study aimed to evaluate whether the p53 abnormal expression could influence the clinical outcome of a patient with RAS/BRAF WT mCRC, treated with anti-EGFR.

Globally, our analysis suggests that p53 overexpression may predict resistance to anti-EGFR treatment in patients with mCRC. In the exploratory cohort, a significant benefit in terms of all clinical outcomes (OS, PFS and RR) was observed in p53 WT patients, compared with those showing p53 over-expression. Furthermore, the p53 low expression status was more frequent in left-sided tumours. These results were confirmed in the validation cohort.

Furthermore, we analysed survival data from the first palliative therapy, which did not contain anti-EGFR antibodies. This analysis showed a significant benefit in terms of OS in p53 low expression patients compared with those showing p53 over-expression, in both cohorts. Conversely, no statistically difference in terms of PFS was observed in patients non-overexpressing p53 vs patients overexpressing p53 in the exploratory and validation cohorts. These data confirmed the prognostic role of p53 expression and suggested the absence of a predictive impact to response to therapies that do not contain anti-EGFR.

In the era of precision medicine, p53 analysis could improve the selection of patients who could benefit from anti-EGFR therapy. The identification of the optimal treatment strategy is successfully provided by a better knowledge of the mCRC heterogeneous nature. Therefore, the finding of p53 overexpression as a resistance mechanism to anti-EGFR therapies represents a further step toward personalised treatment.

Although retrospective, we believe that our analysis could provide a relevant addition to the biological picture underlying the mechanism of resistance to anti-EGFR monoclonal antibodies in patients with mCRC. Further prospective studies, with a control arm without anti-EGFR therapy, will be needed to validate the prognostic and predictive effect of p53 expression in this setting, to better refine the molecular profile of patients more likely to benefit from this crucial treatment strategy.