Colorectal cancer (CRC) is the second most common cause of cancer-related death in developed countries, as in the last few years the incidence has been increasing with decreasing age at diagnosis. The 5-year relative survival rate is approximately 45%, demonstrating an improvement from 30 years ago, when the survival rate was 30% 1 . The irinotecan and oxaliplatin have improved survival while the development of monoclonal antibodies against growth factor receptors, such as monoclonal antibodies against epidermal growth factor receptor (EGFR), has augmented their effects 2 3 .

EGFR (ErbB1) is a glycoprotein composed of an extracellular ligand-binding domain, a transmembrane region, and an intracellular tyrosine kinase domain. The receptor is a member of the ErbB family of receptor tyrosine kinases, including ErbB1, ErbB2 (HER-2), ErbB3, and ErbB4, and it is encoded by the c-erbB protooncogene. In normal and malignant cells, the activation of EGFR receptor cascades has multiple consequences, such as cell growth, differentiation, and proliferation 4 5 . Overexpression of membrane EGFR (mEGFR) has been found to correlate with poor prognosis in several cancers, including colorectal 6 7 . However, there are results that indicate that there is an independent relationship between EGFR expression and CRC prognosis 8 9 . Several studies have shown that EGFR levels are a poor predictor of response to anti-EGFR therapies. In clinical trials evaluating the efficacy of cetuximab, treatment response was not related to the levels of mEGFR expression 10 11 . On the other hand, current data suggest that nuclear EGFR (nEGFR) expression contributes to acquired resistance to cetuximab 12 . It has been shown that mEGFR can be transported from the plasma membrane to the nucleus. Thus, nEGFR has two functions, first, as transcription factor it interacts with STAT3 and E2F1 to mediate transcription of cyclin-D1, iNOS, B-Myb and Aurora Kinase A, and second in direct phosphorylation of the proliferating cell nuclear antigen 13 14 15 . Activated cyclin-D1 controls cell cycle, particularly in the transition from G1 to S-phase. Despite a well-established role of cyclin-D1 in cell cycle progression, previous data on cyclin-D1 and clinical outcome in colon cancer have been conflicting 16 17 . According to some studies, cyclin-D1 expression has been associated with poor and according to others with good prognosis, while most results revealed no independent prognostic value of cyclin-D1 18 19 .

At the same time, membrane and nuclear EGFR expression in CRC, according to our knowledge, has not been published so far, and the present study was conducted with the aim to explore the correlation between mEGFR, nEGFR and cyclin-D1 expression on tumor cells. We hypothesized that such investigation could have predictive value by identifying biologically and probably clinically different subsets of CRC that may have diverse response to anti-EGFR therapies. Furthermore, the methods of tissue processing and EGFR scoring systems were not homogeneous among studies, so that reproducibility of data remains a major issue for clinical application of the test. Therefore, our second aim was to observe how the scoring system for EGFR expression correlates with gene EGFR expression.

In previous immunohistochemical studies, there was considerable discrepancy in the frequency and distribution of EGFR overexpression in CRC. Many of these studies gave inconclusive information on the association of the protein expression and clinicopathologic features 8 9 10 11 . Furthermore, in clinical trials evaluating the efficacy of cetuximab, treatment response was not related to the levels of EGFR expression, since many patients with EGFR expressing CRC failed to respond 10 11 , or those with EGFR negative tumor responded to therapy 12 . Several technical reasons have supported the lack of this association, such as prolonged storage, tissue-fixation methods, the antibodies used, the detection techniques and criteria used on result evaluation. In addition, according to some authors, the possible reasons for EGFR levels being a poor predictor of response to anti-EGFR therapies include disparity between the form or epitope of EGFR detected by immunohistochemistry and the one targeted by anti-EGFR monoclonal antibodies 22 . Recent emerging data suggest the existence of a new mode of EGFR signaling pathway in which activated EGFR undergoes nuclear translocation, and based on in vitro study nEGFR may play a functional role in the response to molecular therapeutic agents 12 . These intriguing findings emphasize the relevance of evaluating both the membrane and nuclear EGFR expression in CRC in order to provide more independent information on the protein association with clinicopathologic features. The present study, to our knowledge, is the first with concurrently examined mEGFR and nEGFR expression in the same cohort of CRC patients. The results confirmed the heterogeneity in mEGFR and nEGFR expression without any correlation between these proteins. Clinical significance of the present findings needs further investigation.

In our study, strong mEGFR was demonstrated in less than 20% of CRC cases, while other studies report on EGFR overexpression in up to 82% of cases 23 . Along with different methodologies used, other studies reporting a higher percentage of mEGFR expression also used less strict criteria in defining membrane overexpression. In the present analysis, only tumors with moderate to strong complete membrane staining in more than 50% of tumor cells were interpreted as strong expression, which is probably the major cause of such a low prevalence of mEGFR overexpression. Strong mEGFR expression found in our study was associated with gene amplification, found in approximately 6% of CRC samples, while moderate mEGFR was observed in those tumors that showed chromosome 7 polysomy. The result obtained is in agreement with studies where EGFR amplification has been reported to correlate with expression, although the authors stress that amplification does not reliably predict EGFR overexpression, or that overexpression of EGFR caused by amplification comprised only a small portion of the cells in these tumors 22 . The relatively low prevalence of strong mEGFR expression (overexpression) associated with the prevalence of EGFR gene amplification in CRC samples supports the evaluation of immunohistochemical staining used in our study as probably being more objective. However, in our cohort of CRCs, the strong nEGFR expression did not correlate either with strong mEGFR or with EGFR gene amplification, suggesting that gene amplification is probably not a significant event that would lead to higher nuclear translocation of the EGFR membrane receptor, as found in breast cancer model 24 .

In comparison to clinicopathologic characteristics, the only significant association was found between mEGFR expression and histologic grade of CRC, or more precisely, low grade carcinomas were more characterized with negative or weak mEGFR expression. This observation was also demonstrated in other studies, where an association with more advanced stage, lymphovascular invasion, and poor prognosis was also found 6 7 .

Strong nEGFR expression was confirmed in more than 50% of cases and strong cyclin-D1 expression in CRC samples was obtained in nearly the same percentage. There are no studies on the clinical significance of nEGFR expression in CRC, except for those referring to the breast, oropharyngeal squamous and ovarian cancer 14 24 25 26 , with the expression associated with worse prognosis 12 24 25 , increased proliferation and cyclin-D1 expression 25 . In the present study, nEGFR was associated with cyclin-D1. The clinical significance of these findings should be further investigated. Yet, it is important to discuss the finding reported by Li et al. 12 that HER family ligands are up-regulated in the cells with acquired resistance to cetuximab. The authors conclude that nEGFR may prove a viable molecular target, and according to our findings we presume that these CRC could be immunohistochemically recognized.

In our study, strong cyclin-D1 expression was observed in 57% of CRC cases. Those CRC with strong cyclin-D1 expression showed positive correlation with nEGFR, as also found in breast cancer 25 . According to previous studies, increased cyclin-D1 expression occurs in one-third of colonic tumors as an early event during the multistage process of colon carcinogenesis 16 . Although some studies demonstrated cyclin-D1 as an independent indicator of poor prognosis 17 , a large cohort study suggests that cyclin-D1 expression is independently associated with good prognosis in colon cancers It is very common to assume that oncogene activation (or tumor suppressor inactivation) is associated with aggressive tumor behavior. However, as commented by the authors, this hypothesis does not always hold true, since it is well recognized that MSI, which is known to cause inactivation of a number of tumor suppressors (including TGFBR2, BAX, and many others), is associated with better patient outcome 27 .

Standardized chemotherapy regimens result not only in improved efficacy but also in an increased toxicity and treatment costs, therefore requiring identification of decision-making tools to select patients who are likely to benefit from them. Both KRAS mutation and EGFR disomy represent two negative-predictive factors able to impair clinical responsiveness to anti-EGFR therapy with monoclonal antibodies 28 29 30 ; however, our findings indicate the importance of further investigation of standardized protocols for immunohistochemical analysis of both mEGFR and nEGFR, which will be applicable in clinical practice.

The authors declare that they have no competing interests.

AD participated in the design of the study and drafted the article. AD and SP participated in collection of references and evaluation of the data. RDD and IB participated in collection of the data. MŽB performed the statistical analysis. NJ participated in the design, coordination and reviewing the manuscript. All authors read and approved the final manuscript.