In brief, deparaffinised and dehydrated sections were incubated in 20% sodium bisulphate/2 �� standard saline citrate (SSC) at 43��C for 20min. After being washed with SSC, sections were treated with proteinase K (Boehringer-Mannheim, Mannheim, Germany) at 37��C for 25min. Subsequently, denaturation, hybridisation, and post-hybridisation washing were carried out according to the manufacturer’s novel protocol, and the sections were counterstained with 4��,6-diamidine-2��-phenylindole dihydrochloride (Oncor, Gaithersburg, MD, USA). Fluorescence in situ hybridisation analysis was performed using a fluorescence microscope (Olympus, Tokyo, Japan) equipped with Triple Bandpass Filter sets (Vysis). Signals were countered for at least 40 cancer nuclei per tumour.

In accordance with our previous studies with FISH, a cell was considered to show amplification when a definite cluster or more than 10 orange signals of HER-2 were observed (Takehana et al, 2002). Antibody-dependent cell-mediated cytotoxicity assay Peripheral blood mononuclear cells (PBMC) were separated from peripheral blood obtained from healthy donors and oesophageal SCC patients before treatment by centrifugation with Ficoll�CPaque (Pharmacia). After the target cells were labelled with 50��Ci of 51Cr for 60min, target cells (5 �� 103well?1) and PBMC from healthy donors or oesophageal cancer patients as effector cells were co-incubated at various effector/target ratios in 200��l of X-VIVO medium in a 96-well U-bottomed plate in triplicate with indicated doses of cetuximab or/and trastuzumab or a control antibody, rituxan.

After 6h of incubation, the radioactivity of the supernatant (100��l) was measured with a ��-counter. The percentage of specific lysis=100 �� (experimental c.p.m.?spontaneous c.p.m.)/(maximum c.p.m.?spontaneous c.p.m.). Apoptosis Each cell line (2 �� 105 cells) was incubated in 2ml of X-VIVO with control mAb alone, cetuximab (0.5��gml?1) alone, trastuzumab (10��gml?1) alone, and cetuximab in combination with trastuzumab at 37��C in a six-well plate. After incubation for 24h, apoptosis in each cell line was measured by staining with FITC-conjugated annexin-V and propidium iodide (PI) using a MEBCYTO Apoptosis kit (MBL, Nagoya, Japan) following the manufacturer’s recommendations.

MTT cell proliferation Brefeldin_A assay Each cell line (2500 cells) was incubated in 200��l of X-VIVO with indicated doses of cetuximab or/and trastuzumab, or a control antibody in a 96-well flat-bottomed plate in triplicate. After incubation for 96h at 37��C, 50��l of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 2mgml?1; Sigma, St Louis, MO, USA) was added to each well and incubation was carried out for 4h. Then, the supernatant was discarded and the crystal products were eluted with DMSO (50��lwell?1; Sigma).