Epidermal growth factor receptor and cyclin D1 are independently amplified and overexpressed in esophageal squamous cell carcinoma

Abstract

Purpose: To assess the status of EGFR, HER-2, and CCND1 at the gene and protein levels in esophageal squamous cell carcinoma.

Methods: Dual-color FISH assays were performed using DNA probes for EGFR/CEP 7, HER-2/CEP 17, and CCND1/CEP 11. The respective proteins, furthermore, was assessed in IHC assays and correlated with patient and tumor characteristics.

Results: From 55 ESCCs, 8 (15%) tumors showed gene amplification and 20 (36%) had gene overrepresentation (balanced gene and chromosome 7 polysomy) for EGFR. High-level protein expression was frequent (49%), positively correlated with gene copy numbers (kappa=0.4), and associated with well-differentiated histology (p=0.02). For HER-2, gene amplification was detected in a single tumor (2%) and protein overexpression was rare (9%). CCND1 gene was amplified in 23 (42%) tumors; likewise, CCND1 protein overexpression was common (58%) and prevailed in gene overrepresentation or amplification. Only 1 patient showed gene amplification for both EGFR and CCND1. Survival was not associated with EGFR or CCND1 gene/protein status, whereas negative patients for HER-2 protein had a better survival than positive patients (p=0.04).

Conclusions: Frequent overexpression and gene amplification of EGFR and CCND1 make these molecules and their pathways potential therapeutic targets for ESCC. In addition, EGFR and CCND1 appeared to be independently altered suggesting alternative mechanisms for pathway activation. Therapeutic agents targeting these molecules are urged to be tested in clinical trials and comprehensive biological analyses should be included to properly interpret the outcome.

Fig. 1

Gene (red signal) and chromosome (green signal) detected by fluorescence in situ hybridization (FISH) in esophageal squamous cell carcinoma (ESCC; probes as described in the text). A Balanced trisomy and B gene amplification for EGFR. C Balanced disomy and D gene amplification for HER-2. E balanced polysomy and F gene amplification for CCND1

Fig. 2

Protein expression detected by IHC in ESCC: for EGFR in A negative/low (×40) and B high (×100); for HER-2 in C negative (×40) and D 2+ overexpressed (×100); and for CCND1 in E negative (×40) and F high (×100) levels of expression

Fig. 3

Comparison of dichotomized gene patterns and protein expression levels for EGFR, HER-2, and CCND1 in ESCC. The gene copy numbers were dichotomized as low (FISH patterns BD+BT) and high (FISH patterns BP+GA) copy numbers per cell. The protein expression was dichotomized in low level (IHC negative for HER-2 and CCND1; score ≤300 for EGFR) and high level of expression (IHC positive for HER-2 and CCND1; score >300 for EGFR)