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 [8–11]. 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 . 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 . 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 . 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 . 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 . 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 .
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, 24–26], with the expression associated with worse prognosis [12, 24, 25], increased proliferation and cyclin-D1 expression . 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.  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 . 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 . Although some studies demonstrated cyclin-D1 as an independent indicator of poor prognosis , 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 .