Research article

Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using Fluorescence in situ Hybridization (FISH) and immunohistochemistry

Yukie Sato-Kuwabara^1* , José I Neves^1* , José HTG Fregnani^2* , Rubens A Sallum^3* and Fernando A Soares^1*

¹  Department of Anatomic Pathology, Hospital AC Camargo, Rua Antônio Prudente, 211, 1o. andar, São Paulo, SP, CEP: 01509-010, Brazil

²  Department of Morphology, School of Medical Sciences of Santa Casa of São Paulo, Rua Dr. Cesário Motta Jr., 61, São Paulo, SP, CEP: 01221-020, Brazil

³  Department of Abdominal Surgery, Hospital AC Camargo, Rua Antônio Prudente, 211, 1o. andar, São Paulo, SP, CEP: 01509-010, Brazil

author email corresponding author email* Contributed equally

BMC Cancer 2009, 9:6doi:10.1186/1471-2407-9-6

Published:	7 January 2009

Abstract

Background

Esophageal squamous cell carcinoma (ESCC) is the sixth most frequent neoplasia in Brazil. It is usually associated with a poor prognosis because it is often at an advanced stage when diagnosed and there is a high frequency of lymph node metastases. It is important to know what prognostic factors can facilitate diagnosis, optimize therapeutic decisions, and improve the survival of these patients. A member of the epidermal growth factor receptor (EGFR) family, c-erbB-2, has received much attention because of its therapeutic implications; however, few studies involving fluorescence in situ hybridization (FISH) analysis of HER-2/neu gene amplification and protein expression in ESCC have been conducted. The aim of this study was to verify the presence of HER-2/neu gene amplification using FISH, and to correlate the results with immunohistochemical expression and clinical-pathological findings.

Methods

One hundred and ninety-nine ESCC cases were evaluated using the Tissue Microarray (TMA) technique. A polyclonal antibody against c-erbB-2 was used for immunohistochemistry. Analyses were based on the membrane staining pattern. The results were classified according to the Herceptest criteria (DAKO): negative (0/1+), potential positive (2+) and positive (3+). The FISH reactions were performed according to the FISH HER2 PharmDx (DAKO) protocol. In each case, 100 tumor nuclei were evaluated. Cases showing a gene/CEN17 fluorescence ratio ≥ 2 were considered positive for gene amplification.

Results

The c-erbB-2 expression was negative in 117/185 cases (63.2%) and positive in 68 (36.8%), of which 56 (30.3%) were 2+ and 12 (6.5%) were 3+. No significant associations were found among protein expression, clinicopathological data and overall survival. Among the 47 cases analyzed, 38 (80.9%) showed no gene amplification while 9 (19.1%) showed amplification, as demonstrated by FISH. Cases that were negative (0/1+) and potential positive (2+) for c-erbB-2 expression by immunohistochemistry showed no gene amplification. However, all cases with gene amplification were positive (3+) by immunohistochemistry. According to univariate analysis, there was a significant difference (p = 0.003) in survival rates when cases with and without HER-2/neu amplification were compared.

Conclusion

Our data demonstrate the correspondence between gene amplification and protein expression of HER-2/neu. Gene amplification is an indicator of poor prognosis in ESCC.