c-erb-2 amplification and overexpression are currently attracting a great deal of attention because a new adjuvant therapy using an antibody against the c-erbB-2 gene product, trastuzumab (Herceptin; Genentech, Inc., South San Francisco, CA), has proved effective in treating breast cancer with amplification and/or overexpression of c-erbB-2. Aberrations of c-erbB-2 have also been detected in ovarian, endometrial and gastric carcinomas at varied frequencies. Amplification of the c-erbB-2 locus (17q12-q21.32), overexpression of c-erbB-2 protein (p185) and serum levels of soluble c-erbB-2 protein fragments (p105) were examined in gastric cancer patients using fluorescence in situ hybridization (FISH), immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), respectively. Overexpression of c-erbB-2 protein was found in 29 (8.2%) of the 352 gastric carcinomas analyzed. In FISH analysis, all tumors with 3؉ immunostaining and 1 of 5 tumors with 2؉ staining showed high-level amplification of c-erbB-2. Pre-operative serum p105 was quantified in serum specimens from 129 patients with gastric cancer and 28 patients with benign diseases. There were no significant differences in the serum p105 levels among 11 patients with c-erbB-2-overexpressing carcinomas, 118 patients with c-erbB-2 non-overexpressing carcinomas and 28 controls, although a single case of gastric carcinoma overexpressing c-erbB-2 with extensive liver metastasis had a higher level than the cut-off value. The mechanisms of overexpression of p185 and highlevel amplification of c-erbB-2 in gastric adenocarcinomas seem similar to those well-established in breast cancers. Patients having gastric adenocarcinoma with c-erbB-2 amplification are potential candidates for a new adjuvant therapy using humanized monoclonal antibody.

Overexpression of HER-2 and the epidermal growth factor receptor (EGFR) has been observed in many cancers, sometimes accompanied by gene amplification. To assess whether novel chemotherapies targeting these overexpressed proteins may be effective for the treatment of colorectal cancers, we examined the exact frequency of HER-2 and EGFR overexpression, the relationship between gene amplification and protein expression, and the heterogeneity of gene amplification within and between primary and metastatic tumors. We evaluated 244 colorectal cancers immunohistochemically. All tumors found to overexpress HER-2 or EGFR were further analyzed for gene amplification by fluorescent in situ DNA hybridization. Overexpression of HER-2 and EGFR was found in 8 (3%) and 19 (8%) of the 244 colorectal carcinomas, respectively. Gene amplification was observed in 100 and 58% of the tumors exhibiting HER-2 and EGFR overexpression, respectively. HER-2 amplification in cancer cells was characterized by clusters of hybridization signals, suggesting amplicons in homogeneously staining regions that were predominant in most primary and metastatic tumors. EGFR amplification, observed as scattered signals reminiscent of amplicons in double minute chromosomes, or coamplification of EGFR with the centromeric regions was observed as a minor population within primary tumors, and found in variety of populations in metastatic tumors. Overexpression of HER-2 and EGFR were observed in only a small fraction of colorectal carcinomas, but were frequently accompanied by gene amplification. The HER-2 gene is located on chromosomal region 17q11.2-q12 and the EGFR gene is located on 7p12. These encode 185 kDa (p185) and 170 kDa plasma membrane glycoproteins, respectively. They share approximately 50% overall homology and are composed of an N-terminus extracellular ligand-binding domain, a transmembrane lipophilic segment, and a C-terminus intracellular region containing a tyrosine kinase domain.

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Abnormalities in c-erbB-2 have attracted a great deal of attention. Treatment using an antibody against the c-erbB-2 gene product is effective against breast cancers with amplification and/or overexpression of c-erbB-2. There is an urgent need to establish methodology for selecting patients who would benefit from this therapy. A total of 235 breast carcinomas were examined for c-erbB-2 protein overexpression by immunohistochemistry. Tissue sections with discernible immunostaining from 52 tumors, 70 negative tumors and smear imprints from 35 patients were examined by dual-color fluorescence in situ hybridization (FISH) using probes specific for c-erbB-2 and the centromeric region of chromosome 17. The concordance between gene amplification and protein overexpression was 95.7%. When the findings of the two FISH preparation techniques were compared, no discrepancies were found in 24 of the tumors. However, differences were seen in eight cases. In six of these cases the differences did not affect the presence or absence of amplification, but in the other two cases, considered to show low-level amplification on paraffin sections, polysomy 17 was detected instead. It was concluded that FISH is an excellent tool to detect gene amplification in particular, and FISH on touch imprints is a useful adjunct to differentiate between low-level amplification and polysomy 17.