EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma

Abstract

Background: The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.

Methods: The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.

Results: Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient's survival, while there was no connection with pathological stage.

Conclusion: In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.

Figure 1

EGFR protein and gene status in clear cell renal cell carcinoma. Immunohistochemical expression of EGFR protein, shown at lower (×10) and higher magnification (×20), was designated as discontinuous or continuous membrane staining of different intensity: (a) weak and moderate (+/++) discontinuous and continuous membrane immunostaining, and (b) strong continuous immunostaining (+++);. (c) chromosome 7 copy number was analyzed in tumor cells using fluorescence in situ hybridization (FISH) with an α-satellite DNA probe for chromosome 7 (centromere 7, green signal; EGFR gene, red signal); tumor nuclei showed disomy of chromosome 7 without EGFR gene amplification (d) tumor nuclei showed polysomy with a greater number of red and green signals than in normal cells; (c and d magnification × 100).

Figure 2

Chromosome 7 polysomy in different groups of clear cell renal cell carcinoma (CCRCC) regarding EGFR immunohistochemical staining intensity: (a) the number of tumors categorised as polysomic decreases with attenuation of staining intensity (p = 0.019, Pearson's χ²-test). Black bars represent percentage of tumors with chromosome 7 polysomy, white bars represent percentage of tumors without chromosome 7 polysomy; (b) the number of tumor cells showing chromosome 7 polysomy is declining with attenuation of EGFR immunohistochemical staining (p = 0.004, one-way ANOVA).

Figure 3

Chromosome 7 polysomy and membrane EGFR protein expression determined as percentage of EGFR immunohistochemicaly positive cells per tumor (%), EGFR total H-score (tH-score) and EGFR continuous H-score (cH-score) in clear cell renal cell carcinoma (CCRCC). Low and high categories of EGFR immunohistochemical staining are designated according to median cut off value. Student's t-test is showing higher percentage of tumor cells with chromosome 7 polysomy to be significantly associated with high expression of all EGFR immunohistochemical staining categories: (a) EGFR % (p = 0.001); (b) EGFR tH-score (p < 0.001); (c) EGFR cH-score (p < 0.001).

Figure 4

Kaplan-Meier survival analysis according to EGFR protein expression determined as H-score of continuous immunohistochemical staining (cH-score) and total immunohistochemical staining (tH-score) in clear cell renal cell carcinoma (CCRCC): (a) The log-rank test showed significantly shorter overall survival in patients with tumors showing high continuous EGFR protein expression that was above median H-score values (p = 0.046). The 5-year survival rate was 59% for patients whose tumors showed low EGFR cH-score vs. 40% for patients whose tumors showed high EGFR cH-score; (b) on the contrary, the log-rank test showed no significant association between EGFR tH-score and survival (p = 0.074). The 5-year survival rate was 57% for patients whose tumors showed low EGFR tH-score and 40% for patients whose tumors showed high EGFR tH-score.