Tissue micro-arrays of 250 metastatic breast cancer patients who received first-line chemotherapy together with Herceptin were obtained from Clinomics Biosciences (Pittsfield, MA, USA). All of the tissues were obtained under institutional IRB approval. The histology of the tumors varied with infiltrating ductal carcinoma being most common. All patients received radiotherapy postsurgery. The tissue samples in the array were taken before treatment and were all taken from the primary tumor unless otherwise noted. ErbB2 expression had been determined by the Herceptest on the original biopsies for all patients. Patient response was based upon the case histories at last follow-up as decided by the independent investigators for the clinical trials from which the samples were obtained. Patients who were free of disease at the time of examination after therapy were classified as disease free. Patients whose tumors had not progressed at the time of examination were classified as having stable disease. Patients who were disease free or had stable disease were grouped together as nonprogressors. Patients who redeveloped the disease after therapy or whose tumors progressed were classified as progressors.
EGFR and erbB2 immunostaining was performed by using the prediluted EGFR and erbB2 antibodies from Ventana Medical Instruments, Inc. (VMSI, Tucson, AZ, USA). erbB3 (1
10) and Heregulin (1
25) antibodies were obtained from NeoMarkers (Fremont, CA, USA). TGF-α
and IGF-IR antibodies were obtained from Oncogene Sciences (San Diego, CA, USA) and NeoMarkers (Fremont, CA, USA), respectively. EGFR, ErbB2, erbB3, IGF-IR, Heregulin and TGF-α
were immunostained using the ‘BenchMark’ (VMSI) with I-VIEW (VMSI) detection chemistry. Phospho-specific HER2 (p-HER2, Y1248), phospho-specific ERK (p-ERK), phospho-AKT (p-AKT) and phospho-S6 ribosomal protein (p-S6) antibodies were obtained from Cell Signalling Technology (Beverly, MA, USA), and immunostained using a labelled streptavidin peroxidase technique. Slides for p-S6 ribosomal protein, p-ERK and p-AKT were processed with antigen retrieval using 0.1
M citrate buffer, pH 6.0 in the ‘decloaker’ (Biocare Corp.) and the sections incubated overnight with the primary antibodies at 4°C. The next day, the slides were placed onto the Autostainer (Dako Corp.) and the ‘LSAB2 kit (Dako) was used as the detection chemistry. DAB (Dako) was used as the chromagen. Slides for p-HER2 were processed with antigen retrieval using 1
EDTA, pH 8.0 solution and processed manually using the Vector Elite detection system. After immunostaining, all slides were counterstained manually with 4% ethyl green (Sigma). ErbB2, EGFR, erbB3, IGF-IR, TGF-α
, Heregulin, p-ERK, p-AKT and p-S6 ribosomal protein or phosphorylation levels were quantified using alkaline phosphatase or peroxidase techniques and microscope-based image analysis of immunohistochemical stained slides (Bacus et al, 1997
). Quantification was by means of a CAS 200 image analyser, as previously described (Bacus and Ruby, 1993
). Slides for p-HER2 were scored manually following the criteria of the Herceptest. For the purpose of the analysis, tumors were classified as negative or positive for all antibodies based upon the level of staining. Statistical analysis was performed to quantify frequencies and calculate Pearson χ2
tests of significance for interactions between variables. Comparisons were performed only on samples for which all relevant data was available.