Prognostic implications of EGFR protein expression in sporadic colorectal tumors: Correlation with copy number status, mRNA levels and miRNA regulation

Abstract

Sporadic colorectal cancer (sCRC) is the third most frequent cancer worldwide and the second most common cause of cancer-related deaths (mainly due metastatic dissemination). We investigated the immunohistochemical expression of frequently altered proteins in primary tumors from 51 patients (25 liver metastatic and 26 non-metastatic cases) with a median 103 months follow-up (103 months). We evaluated EGFR copy number (using SNP arrays and FISH) and its expression and regulation (by mRNA and miRNA arrays). We found differences between metastatic and non-metastatic sCRCs for MLH1 (p = 0.05), PMS2 (p = 0.02), CEA (p < 0.001) and EGFR (p < 0.001) expression. EGFR expression was associated with lymph node metastases (p = 0.001), liver metastases at diagnosis (p < 0.001), and advanced stage (p < 0.001). There were associations between EGFR expression-, EGFR gene copy number- and EGFR mRNA levels. We found potential interactions of two miRNAs targeting EGFR expression, (miR-134 and miR-4328, in non-metastatic and metastatic tumors, respectively). EGFR expression was associated with a worse outcome (p = 0.005). Multivariate analysis of prognostic factors for overall survival identified that, the expression of EGFR expression (p = 0.047) and pTNM stage (p < 0.001) predicted an adverse outcome. EGFR expression could be regulated by amplification or polysomies (in metastatic tumors), or miRNAs (miRNA-134, in non-metastatic tumors). EGFR expression in sCRC appears to be related to metastases and poor outcome.

Figure 1

Clinical, biological and immunohistochemical markers of sCRC patients showing the impact on overall survival in the univariate analysis: (A) Age, (B) gender, (C) site of primary tumor, (D) TNM stage at diagnosis, (E) tumor size, (F) carcinoembryonic antigen (CEA) serum levels, (G) microsatellite instability, (H) EGFR expression, (I) β-catenin expression, (J) CEA expression, (K) c-Myc expression, (M) Her2 expression, (N) Ki-67 expression, and (O) p53 expression.

Figure 2

Copy number alterations (CNAs) and EGFR gene expression levels detected in primary tumors from patients with metastatic and non-metastatic sCRC. CNA status assessed by Affymetrix 500‐K single nucleotide polymorphism (SNP)‐array platform (panel A) and FISH techniques [probes for identifying chromosome 7 centromere (7p11; green spots) and EGFR gene (7p11.2; red spots)] (panel B). EGFR gene expression profile analyzed by oligonucleotide arrays (Affymetrix PrimeView Human Gene Expression microarray) (panel C) and immunostaining techniques (20x) (panel D). Panel (B,D) correspond to a sample of the same patient.

Figure 3

Correlation between EGFR gene expression using oligonucleotide arrays and CNA by SNP arrays in patients with sporadic colorectal cancer at diagnosis. The graphs show the regression lines for Y as a function of X (solid line) and for X as a function of Y (dashed line). If these regression lines are approximately perpendicular, it indicates that X and Y are not linearly correlated. The closer the lines, the greater the correlation.

Figure 4

Hierarchical cluster analysis based on EGFR expression regulated by 98 miRNAs present in our array (out of 134 miRNAs total candidates) of the non-metastatic (n = 25) and metastatic cases (n = 26) included in the study.

Figure 5

Validation of the impact of EGFR expression on overall survival in an independent series of sCRC patients from the GEO database (n  =  32): panel (A), EGFR expression showing the impact on overall survival; panel (B), EGFR gene expression levels detected in primary tumors from patients with metastatic and non-metastatic sCRC; and panel (C), correlation between EGFR gene expression using oligonucleotide arrays and CNA by SNP arrays in patients with sporadic colorectal cancer at diagnosis.

Figure 6

TMA: (A) TMA paraffin block; (B) TMA slide, hematoxylin-eosin; (C) hematoxylin-eosin panoramic view, 3-µm section.