| Abstract|| |
Introduction: The aim of this study was to assess whether epidermal growth factor receptor (EGFR) overexpression was a significant prognostic factor in clear cell renal cell carcinoma (CRCC) and whether its prognostic significance was affected by immunohistochemical expression patterns. Materials and Methods: Immunohistochemistry was performed on 100 cases of CRCC using an antibody against EGFR. Tumors were grouped by nuclear grade (NG) as low-NG (NG1, 2) or high NG (NG3, 4), and by pathological stage as localized (pT1, 2), or locally invasive (pT3, 4). Clinical disease was grouped by clinical stage as early stage (stage I, II), or late stage (stage III, IV). Evaluation of the EGFR overexpression was based on cytoplasmic (EGFR Cyt ), and membranous (EGFR Mem ) staining. Results: EGFR Cyt correlated with high NG (P = 0.001), lymphovascular invasion (P = 0.028), regional lymph node involvement (P = 0.027), metastasis (P = 0.001), late stage (P = 0.003), cancer-specific death (P = 0.036), and was a predictor for disease-specific survival (P = 0.012) whereas EGFR Mem correlated with only local invasion (P = 0.021) and perirenal invasion (P = 0.009) and did not show any correlation with cancer-specific death or disease specific survival. Conclusion: Our findings suggest that EGFR overexpression is an important prognostic factor in CRCC, and its prognostic value differs significantly with respect to the location of EGFR immunostaining. This prognostic difference may give direction on the management and treatment of CRCC patients.
Keywords: Clear cell renal cell carcinoma, epidermal growth factor receptor, prognosis, survival
|How to cite this article:|
Kankaya D, Kiremitci S, Tulunay O, Baltaci S. Prognostic impact of epidermal growth factor receptor on clear cell renal cell carcinoma: Does it change with different expression patterns?. Indian J Pathol Microbiol 2016;59:35-40
|How to cite this URL:|
Kankaya D, Kiremitci S, Tulunay O, Baltaci S. Prognostic impact of epidermal growth factor receptor on clear cell renal cell carcinoma: Does it change with different expression patterns?. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Nov 19];59:35-40. Available from: http://www.ijpmonline.org/text.asp?2016/59/1/35/178219
| Introduction|| |
The epidermal growth factor receptor (EGFR), a transmembrane glycoprotein, is the first receptor identified of a family of receptors known as the type I receptor tyrosine kinases, or ErbB receptors. Ligand binding to the EGFR and receptor dimerization activate downstream signaling pathways, which culminate in cell cycle progression, inhibition of apoptosis and several tumor promoting activities.  The role of EGFR in the pathogenesis and progression of various malignant tumors has long been known, and therapy choices targeting the EGFR pathway have been approved for several cancers in an advanced stage. ,,
It has been shown that renal cell carcinomas (RCC), especially the clear cell renal cell carcinoma (CRCC) type, frequently display EGFR overexpression.  This has been explained by the mechanism that the vast majority of CRCCs harbor abnormalities of von Hippel-Lindau (VHL) gene, a tumor suppressor gene, and the loss of VHL gene function results in hypoxia-inducible factor (HIF)-mediated EGFR autocrine signaling culminating on EGFR overexpression.  However, data in the literature with respect to the prognostic role of EGFR on CRCC are conflicting, and there are only a few of studies investigating its prognostic significance by considering different immunohistochemical expression patterns. ,,,,,
The histological heterogeneity of RCCs and assessment of EGFR expression with different methods seem to be substantial causes of these controversial reports in the literature.
The prognostic importance of EGFR expression in CRCC should be clarified by determining standardized criteria for the evaluation of EGFR expression. This may provide progression on the development of targeted therapy choices against EGFR, especially for advanced CRCC cases of which treatment options are very limited.
The aim of this study was to assess whether EGFR overexpression was a significant prognostic factor in CRCC and whether its prognostic significance was affected by immunohistochemical expression patterns.
| Materials and methods|| |
The study consisted of 100 consecutive CRCC cases who underwent radical nephrectomy between 1995 and 2007 at our University. Hematoxylin and eosin stained histological sections were reviewed and histological grade, pathological stage were determined according to the Fuhrman grading system and TNM staging system, respectively. , Clinical stage was determined according to "College of American Pathologists Cancer Protocol 2005" which was based on TNM, 6 th edition. Lymphovascular invasion, an invasion of renal vein, renal sinus, perirenal tissues, regional lymph node involvement, and presence of metastasis were noted. Clinicopathological parameters including patient age, gender, multifocality, clinical stage, and clinical follow-up findings were retrieved from the records of urology department. Tumors were grouped by nuclear grade (NG) as low-NG (NG1, NG2) or high NG (NG3, NG4), by pathological stage (pT) as localized (pT1, pT2), or locally invasive (pT3, pT4), by clinical stage as early stage (stage I, II) or late stage (stage III, IV).
EGFR expression (clone SP9, 1/100, Neomarkers) was investigated by immunohistochemistry (IHC). 4 μm sections of representative blocks of each tumor were mounted on poly-lysine coated slides. All steps of the immunohistochemical analysis, including deparaffinization, antigen retrieval, and counterstaining were performed on an automatic immunostainer (Benchmark XT Staining Module, Ventana Medical Systems) by using Streptavidin-biotin complex immunodetection system. Protease solution (Ventana) was used for antigen retrieval. Diaminobenzidine was used as chromogen and followed by hematoxylin counterstaining. Positive control tissue was used as recommended by the suppliers.
The evaluation was based on cytoplasmic (EGFR Cyt ) [Figure 1]a and membranous (EGFR Mem ) [Figure 1]b expression. According to the EGFR expression patterns, tumors were grouped as EGFR Cyt (presence/absence of cytoplasmic expression), EGFR Mem (presence/absence of membranous expression), and EGFR Group ("dominantly membranous, dominantly cytoplasmic, and no expression). EGFR Group is subclassified as "dominantly membranous"/"dominantly cytoplasmic" according to the prominent staining pattern which is more extensive than the other and as "no expression" in case of no staining.
|Figure 1: Epidermal growth factor receptor expression patterns in CRCC. (a) Membranous dominant (b) cytoplasmic dominant overexpression with epidermal growth factor receptor (only color on the web)|
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The intensity of the immunostaining (scale 0-3) was multiplied by the percentage of cells with positive staining to give a score of 0-300. The mean value of the staining scores was determined as a cut-off value, and expressions with staining scores higher than the cut-off value were assigned as positive. The cut-off values were 98 and 62 for EGFR Mem and EGFR Cyt respectively.
The relationship between immunohistochemical expressions and clinicopathologic parameters were compared using the Chi-square test or Fisher's exact test. Their effects on disease-free survival were investigated by using the Kaplan-Meier method and compared by the log-rank test. P ≤0.05 was considered statistically significant.
| Results|| |
The median age of the patients was 58 (33-77), and the male/female ratio was approximately 2:1. The median size (long diameter) of the tumors was 7 cm (1.5-20 cm), and 9% of the tumors were multifocal. Median follow-up was 24 months (1-144 months), and 30 patients had cancer death. Thirty-eight patients developed metastasis, of which 13 were at the presentation. Thirteen patients had regional lymph node metastasis at presentation, and 87 patients were N0. Tumor characteristics were summarized in [Table 1].
CRCCs with EGFR Cyt overexpression showed an increased incidence of high NG (78% vs. 23.7%, P = 0.001), lymphovascular invasion (24.4% vs. 8.5%, P = 0.028), regional lymph node involvement (22% vs. 6.8%, P = 0.027), metastasis (58.5% vs. 23.7%, P = 0.001), late stage disease (70.7% vs. 40.7%, P = 0.003), and cancer specific death (43.6% vs. 23.2%, P = 0.036) [Figure 2]a, whereas EGFR Mem overexpression showed such correlation only with local invasion (59.5% vs. 36.2%, P = 0.021) and perirenal invasion (57.1% vs. 31%, P = 0.009) [Figure 2]b. Tumors with cytoplasmic dominant overexpression showed more multifocality (22.2% vs. 4.5%, P = 0.018), high NG (88.9% vs. 31.3%, P = 0.001), local invasion (55.6% vs. 46.3%, P = 0.047), lymphovascular invasion (29.6% vs. 10.4%, P = 0.035), regional lymph node involvement (29.6% vs. 7.5%, P = 0.009), metastasis (59.3% vs. 31.3%, P = 0.022), and more cancer specific death (57.7% vs. 23.8%, P = 0.002) in the EGFR Group [Figure 2]c. Moreover, the cytoplasmic overexpression of EGFR was found to be a significant prognostic parameter (P = 0.009) according to the Kaplan-Meier curves and the log-rank test survival analysis [Figure 3]a], whereas no such correlation was found with EGFR Mem overexpression [Figure 3]b]. In keeping with that, dominantly cytoplasmic overexpression in the EGFR group also showed correlation with disease specific survival [Figure 3]c.
|Figure 2: Correlation of epidermal growth factor receptor expression with clinicopathological parameters due to staining patterns. (a) EGFRCytop (b) EGFRMemb and (c) EGFRGroup. EFGR: Epidermal growth factor receptor|
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|Figure 3: Kaplan-Meier curves for (a) EGFRCytop (b) EGFRMemb (c) EGFRGroup|
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| Discussion|| |
Clear cell renal cell carcinoma (CRCC) constitutes the majority of RCC, which is the most common renal tumor in adults.  Current prognostic factors as histological grade or stage are helpful, but insufficient in predicting patient outcome and the biologic aggressiveness of CRCC since tumors with the same stage and differentiation may behave very differently. Therapy choices are also limited especially for advanced stage tumors as they are usually resistant to conventional oncologic therapies and even to immunotherapies. Thus, identification of molecular markers which contribute to the progression of RCCs is essential to define outcomes of patients and give direction to new therapy modalities.
EGFR is a transmembrane growth factor receptor which belongs to the HER tyrosine kinase receptor family. Along with the determination of its pathogenetic and prognostic relevance with several cancers, it has come up as an important candidate for targeted therapy trials. ,,
It has been reported in the literature that RCCs frequently display EGFR overexpression, and the CRCC subtype show the highest frequency of EGFR expression when compared with other RCC subtypes.  This has been explained by the mechanism that the vast majority of CRCCs harbor abnormalities of VHL gene, a tumor suppressor gene, and the loss of VHL gene function results in HIF-mediated EGFR autocrine signaling culminating on EGFR overexpression.  However, studies dealing with the significance of EGFR as a prognostic parameter in RCC ,,,,, are conflicting.
We detected that EGFR expression is an important prognostic parameter for patients with CRCC, and its prognostic value shows variation depending on the pattern of expression. Cytoplasmic staining of EGFR showed a prominent correlation with unfavorable clinicopathological parameters, tumor grade and stage, and was found to be a poor prognostic parameter, whereas such relevance was not found for membranous expression. Several IHC studies in the literature have shown that EGFR staining in RCC is associated with cell proliferation,  higher tumor grade, the development of metastasis,  and poor prognosis, , in contrast to other studies, demonstrating no relationship to tumor characteristics; tumor grade or stage,  and prognosis  in RCC.
The main cause of this discrepancy in the literature may be the usage of different methods to show EGFR expression in the studies. To the extent we can see, in the majority of the published studies, only membranous EGFR expression were taken into account by overlooking cytoplasmic expression and those considering both of cytoplasmic and membranous expression were limited. , In keeping with our results, some studies reported that cytoplasmic EGFR staining correlated with higher tumor grade and stage, , but there is only one study which sustains the prognostic significance of cytoplasmic EGFR staining on CRCC.  There are some other studies, in contrary with our results, proposing the prognostic relevance of membranous EGFR staining, discounting cytoplasmic staining. ,, Usage of the tissue microarray technique may partly contribute to the disagreement among studies, as it may give discrepant results from the whole-mount sampling that we have used in our study, especially when tumors have focal antigen content.
Corresponding to our results, the prognostic importance of the cytoplasmic location of EGFR immunostaining has previously been emphasized in squamous cell carcinoma of the lung by cell culture experiments.  It has been speculated that the association of cytoplasmic EGFR expression and poor prognosis, may be based on the changes in ligand-EGFR complex internalization and activation of associated signaling pathways, which may have not been triggered in the course of membranous distribution of EGFR.
The underlying mechanism of EGFR overexpression in RCCs remained elusive due to the limited studies on this subject. Hirsch et al., indicated the underlying mechanism of EGFR overexpression as gene amplification in nonsmall cell lung carcinomas,  whereas Dacic et al. reported that protein expression does not necessarily require gene amplification, suggesting that other mechanisms such as gene mutation and transcriptional or posttranscriptional factors might have a role on it, as they have not found gene amplification in any of their EGFR positive nonsmall cell lung carcinoma cases.  There are a few studies in the literature, investigating the underlying mechanism of EGFR protein overexpression in the gene level in RCC. ,, These studies demonstrated gene amplification in a very small proportion of RCCs (<1%), but polysomy were detected more frequently, reaching to 41% in one study.  In these studies, the increase of EGFR gene copy number in RCC cases were found to be correlated with membranous overexpression of EGFR. No exon 18 through 21 mutations, which was found to determine the responsiveness to EGFR targeted therapy in other cancers, was identified in a study of 63 RCC cases, which is the only study investigating EGFR gene mutation in RCCs in the literature. , Almost all of the molecular studies on RCCs focus on the cases with membranous EGFR expression, and there is no molecular study on the RCC cases with cytoplasmic overexpression.
As it is well-known, Her-2/neu, which is another member of EGFR family, is overexpressed in several cancers, especially breast and stomach. Only, membranous expression of Her-2/neu has been known as an indicator of poor prognosis for these cancers and patients who will take Her-2/neu targeted therapy have been selected by Her-2 immunohistochemical scoring which is based solely on membranous staining (neglecting cytoplasmic staining). The importance of Her-2/neu membranous expression in these cancers has been explained by the strong correlation between membranous expression and Her-2/neu gene amplification, which is not detected for cytoplasmic staining. When we come to EGFR and its relevance to RCC, there is no such strong correlation between EGFR membranous expression and gene amplification or any other underlying genetic changes which will raise the importance of membranous expression. Moreover, the prognostic role of the cytoplasmic EGFR expression in RCCs which came up with a few previous studies and the present study should be elucidated by further molecular studies to underlie the molecular mechanism of this expression detected on protein level.
There are a few studies in the literature that have analyzed anti-EGFR therapy in RCC. ,,,,, They did not strongly promote the efficiency of anti-EGFR therapies in RCC, and this has been explained by the failure of appropriate patient selection.
| Conclusion|| |
Our findings suggest that EGFR overexpression is an important prognostic factor in CRCC, and its prognostic value differs significantly with respect to the location of EGFR immunostaining. The cytoplasmic overexpression of EGFR indicates short survival, and it may be a predictive marker of responsiveness to EGFR targeted therapies in CRCC. We suggest focusing on CRCC cases with cytoplasmic EGFR overexpression by planning future molecular studies to elucidate the underlying mechanism of this expression in the gene level. This may offer insight on evaluating the usage of EGFR targeted therapy for patients with CRCC.
Financial support and sponsorship
This research was supported by the grant from the scientific and technological research council of Turkey (TUBITAK) (Project No: 108S120).
Conflicts of interest
There are no conflicts of interest.
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Department of Pathology, School of Medicine, Ankara University, Sihhiye 06100, Ankara
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]