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


CONCLUSIONS
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.
Sato-Kuwabara
Yukie
Y
Department of Anatomic Pathology, Hospital AC Camargo, São Paulo, SP, Brazil. yukiesato@gmail.com
Neves
José I
JI
Fregnani
José H T G
JH
Sallum
Rubens A
RA
Soares
Fernando A
FA
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism / Q000401:mortality; D004938:Esophageal Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism / Q000401:mortality; D005260:Female; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008875:Middle Aged; D011379:Prognosis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D015996:Survival Rate
D018719:Erb-b2 Receptor Tyrosine Kinases