Alterations in epidermal growth factor receptors 1 and 2 in esophageal squamous cell carcinomas

Esophageal cancer (EC) is among the ten most incident tumors in the world, and esophageal squamous cell carcinoma (ESCC) is the most frequent type of EC. In addition to its high incidence, ESCC ranks fifth in cancer mortality in men and eighth in women. ESCC mortality and incidence rates are similar, with the 5 year overall survival rate being below 15% [1, 2]. The poor prognosis of ESCC patients results from late stage diagnosis and the poor efficacy of treatment, with systemic chemotherapy having mainly a palliative role [3]. Although a number of cytotoxic drugs have been used to treat ESCC patients, overall survival rates have not improved [4].

Therefore, the development of new therapy modalities, particularly targeted therapies based on the knowledge of the biology and genetics of the disease may offer a potential for improving treatment response and life quality for ESCC patients [5]. Drugs targeting the human epidermal growth factor receptors (HER) may act in two manners: as tyrosine kinase activity inhibitors (TKIs) or as receptor blocking monoclonal antibodies (mAbs) [6]. A number of these drugs, such as gefitinib used to treat non-small cell lung cancer, cetuximab used to treat patients diagnosed with advanced colorectal cancer, and particularly trastuzumab used to treat breast cancer patients, have shown substantial improvement in tumor response when compared with conventional chemotherapy [7‐9].

Among HER family members, EGFR (HER1) and HER2 are the most commonly altered receptors in human malignancies [10]. These receptors are mainly involved in cell proliferation and survival through activation of PI3K-Akt [11], STAT3 [12], and Ras-Raf-MAPK signaling pathway, with the latter described as the main pathway activated by EGFR [13]. The most common EGFR alterations found in tumors are mRNA and protein overexpression, often associated with gene amplification, followed by mutations in specific hotspots located in the region that encodes the tyrosine-kinase domain of the receptor [14]. The increased expression of EGFR is mainly found in head and neck cancers, in which 70-90% of the cases show this profile [15]. Complementary, EGFR mutations were firstly reported in lung cancer patients who had greater response to treatment with EGFR tyrosine kinase inhibitors. These mutations are generally found in the exons 18-21 of the gene and are more prevalent in Asian non-smoker women with lung adenocarcinoma [16]. The role of HER2 in tumorigenesis is a consequence of abnormally increased protein expression, as a result of gene amplification. This phenomenon is observed in more than 25% of breast cancer patients and more recently in about 15-25% of gastric cancer patients [17, 18].

In addition to the alterations in HER receptors, mutations in genes involved in the signaling pathways activated by these receptors are also correlated with the carcinogenesis process and failure of therapeutic response to HER inhibitors [14]. For instance, colorectal cancer patients who present mutations in KRAS or BRAF do not respond to panitumumab, a monoclonal antibody against EGFR, recently approved by FDA as a monotherapy against metastatic colorectal carcinoma [19].

Since EGFR and HER2 alterations may predict a successful response to HER target specific therapy, and ESCC has a very poor prognosis with currently available treatments, it is essential to analyze possible alterations of these receptors in ESCC, to evaluate the potential of use of anti-HER therapy to treat ESCC patients.

The present study revealed that ESCC of Brazilian patients, who largelly present typical western characteristics, do not present mutations in hot spots of EGFR (exons 18-21), K-RAS (codons 12 and 13) and BRAF (V600E), and only a minor proportion (4%) present overexpression of EGFR or HER2. These results indicate that common alterations in EGFR and HER2 receptors and in the Ras-Raf-MAPK signalling pathway, observed in many other epithelial tumors, are rare in ESCC from Brazilian patients.

EGFR alterations in cancer can be divided mostly in two categories: mutations in exons 18-21, which encode the tyrosine kinase portion of the receptor, and gene and protein overexpression. EGFR mutations are mostly observed in lung tumors, and curiously they are more prevalent in Asian women diagnosed with adenocarcinoma who never smoked [16]. The most frequent EGFR mutations are deletions in exon 19 and a point mutation in codon 858 of exon 21, known as L858R (T2573G; ID: rs121434568) [16]. Patients who carry these mutations in EGFR tend to have a better response to gefitinib, an EGFR-TKI, whereas patients with the wild-type genotype show a better response to conventional chemotherapy [7]. This could be explained by the fact that the mutated receptor possess a greater affinity to the drug in comparison with ATP, and therefore cannot initiate the phosphorylation cascade downstream through the signaling pathways that lead to proliferation and cell survival. However, about 50% of lung cancer patients treated with EGFR-TKI acquire a secondary mutation that confers drug resistance, the T790M (C2369G; ID: rs121434569), located in exon 21 of the gene, which reduces the affinity of the ATP-binding site for the drug [15]. In addition to lung cancer, other tumors present low frequencies of EGFR mutations, like head and neck cancers, with no more than 7% of the patients carrying these alterations [28]. Our results showed no mutations in exons 18 to 21 of EGFR in 135 ESCC patients. So far, few studies were published that analyzed mutations in EGFR in ESCCs [29‐31]. Among these, only one report found mutations in this gene (in 14% of tumors). However this study was carried out with Chinese patients, who usually present a different set of etiological factors when compared to western patients. Furthermore, the authors used the Scorpions Amplification Refractory Mutation System, a non-conventional methodology for the identification of mutations [31]. Our study also identified two synonymous polymorphisms: one at codon 787, in exon 20, with a G>A transition, found in more than 79% of the patients, without any significant difference to controls, and another at codon 836, in exon 21, with a C>T transition in only 2% of the patients.

It is estimated that 33-50% of epidermal tumors present overexpression of EGFR [14], being observed in more than 90% of head and neck tumors [15]. In addition to protein overexpression, around 10-17% of the head and neck tumors present EGFR gene amplification, as shown by FISH analysis [28]. In 2006, the FDA approved the use of cetuximab, a chimeric anti-EGFR mAb, for the treatment of patients with head and neck tumors presenting overexpression of this protein. The use of cetuximab was approved for the first time in 2004 for the treatment of colorectal cancer, which has high response rates to this drug (about 47% of the patients) [8], although there is no concordance in the literature about the role of EGFR expression as a biomarker for response to this targeted therapy [32‐34]. More recently, Panitumumab, a humanized anti-EGFR mAb, was also approved to colorectal cancer treatment, with good results in therapeutic efficacy [35]. However, several reports showed that mutations in genes involved in the Ras-Raf-MAPK pathway, like KRAS and BRAF, are important biomarkers for colorectal tumor patient response to anti-EGFR mAbs. These mutations turn these proteins constitutively activated, resulting in a receptor-independent activation of the pathway, what culminates in the resistance to treatment with anti-EGFR mAbs [36]. The most frequent mutations observed in colorectal cancer patients are found at codons 12 and 13 of KRAS, in approximately 35% of the patients, and the V600E mutation of BRAF, found in about 15% of the cases [19, 34]. Head and neck tumors present mutations in KRAS and BRAF, but in very low frequencies, with 6% of the patients carrying a mutation in KRAS and 3% in BRAF [37]. In our study, 11% of ESCC tumors presented elevated EGFR mRNA levels in comparison with the normal adjacent mucosa, while only 4% showed protein overexpression. Previous studies analyzing EGFR expression in ESCC showed protein overexpression in more than 40% of ESCC patients, with 15% of cases presenting gene amplification [30, 38]. This difference may be explained by the different methodologies used to score EGFR staining by IHC. In this study we evaluated EGFR staining score by the method reported by Pierker et al. [26], where a sample with weak staining is not considered positive for EGFR expression. In the other studies [30, 38], the scoring method adopted was less stringent. Nevertheless, differences among the populations that took part in our and in the other studies may also explain this difference.

We found no alterations in hotspots of KRAS and BRAF in ESCC patients. This data is in accordance with the study developed by Hollstein and colleagues, who previously described the absence of mutations in KRAS in ESCC of patients from Normandy (France) and Uruguay [39], while no study had investigated BRAF mutations in ESCC so far. Therefore, our results both on EGFR hot-spot mutations and expression suggest that the EGFR-Ras-Raf-MAPK pathway is not associated with esophageal carcinogenesis.

HER2 overexpression, as a consequence of gene amplification, was initially seen to be present in around 25% of breast cancer patients, and more recently in a similar percentage of stomach and esophagogastric junction tumors [40]. These findings became even more relevant with the possibility to use a HER2-specific antibody, trastuzumab, to treat these patients [41]. Breast cancer patients, who present HER2 overexpression and gene amplification, and are treated with trastuzumab present a response rate of 62%, that is substantially higher when compared with 32% achieved with conventional chemotherapy [9]. Our work demonstrated that 7% of the ESCC tumors show high HER2 mRNA levels compared to the adjacent tissue, whereas 22% showed protein overexpression. Gene amplification was confirmed in 4% of the cases by FISH, a frequency comparable to that of increased mRNA levels. Some studies focused on ESCC already described a 3-fold higher frequency of patients with score 2+ for HER2 in comparison with those with score 3+. Besides, those reports also showed that every sample classified as score 3+ presented HER2 amplification, similarly to our findings [42, 43]. Interestingly, the frequency of cases with high HER2 mRNA expression and gene amplification is much lower than those with protein overexpression, which could be explained by HER2 biology. It has been described previously that dimmers containing HER2 generally tend to remain longer in the plasma membrane and are not targeted for proteolytic degradation, returning to the membrane in a process called recycling [44]. This phenomenon could explain why cases scored as 2+, considered as protein overexpression, do not show gene amplification.

A limitation of this study was that although we initially had 241 tumor samples, these were divided into smaller groups according to the different assays performed, due to the heterogeneity in sample amount and quality. Although this solution may have generated results with a limited number of samples in some of the analyses, a sufficient statistical power was reached in all cases [27]. Therefore, we may suggest that HER-activated pathway does not play a predominant role in esophageal carcinogenesis in the vast majority of cases. Furthermore, the absence of any EGFR, KRAS and BRAF mutations as well as a frequency of HER overexpression of less than 10% may also suggest that these modifications could be lethal to esophageal cells during transformation. In accordance with this speculation, Kim and colleges showed that EGFR-induced human esophageal tumor presents a strong TUNEL staining [45], what suggests that EGFR overexpression tends to induce apoptosis pathways in esophageal cells. However, other in vitro studies are still necessary to confirm this hypothesis.

This study shows that most ESCC patients do not have the molecular profile for anti-HER targeted therapy. Thus, other markers should be investigated in the pursuit of new treatments that could increase survival and life quality of these patients.