HER-2 NEU EXPRESSION IN NODE-NEGATIVE BREAST-CANCER - DIRECT TISSUE QUANTITATION BY COMPUTERIZED IMAGE-ANALYSIS AND ASSOCIATION OF OVEREXPRESSION WITH INCREASED RISK OF RECURRENT DISEASE/

The HER-2/neu proto-oncogene (also known as c-erb B-2) is homologous w ith, but distinct from, the epidermal growth factor receptor. Amplific ation of this gene in node-positive breast cancers has been shown to c orrelate with both earlier relapse and shorter overall survival. In no de-negative breast cancer patients, the subgroup for which accurate pr ognostic data could make a significant contribution to treatment decis ions, the prognostic utility of HER-2/neu amplification and/or overexp ression has been controversial. The purpose of this report is to addre ss the issues surrounding this controversy and to evaluate the prognos tic utility of overexpression in a carefully followed group of patient s using appropriately characterized reagents and methods. In this repo rt we present data from a study of HER-2/neu expression designed speci fically to test whether or not overexpression is associated with an in creased risk of recurrence in node-negative breast cancers. From a coh ort of 704 women with node-negative breast cancer who experienced recu rrent disease (relapsed cases) 105 were matched with 105 women with no recurrence (disease-free controls) after the equivalent follow-up per iod. Immunohistochemistry was used to assess HER-2/neu expression in a rchival tissue blocks from both relapsed cases and their matched disea se-free controls. Importantly, a series of molecularly characterized b reast cancer specimens were used to confirm that the antibody used was of sufficient sensitivity and specificity to identify those cancers o verexpressing the HER-2/neu protein in this formalin-fixed, paraffin-e mbedded tissue cohort. In addition, a quantitative approach was develo ped to more accurately assess the amount of HER-2/neu protein identifi ed by immunostaining tumor tissue. This was done using a purified HER- 2/neu protein synthesized in a bacterial expression vector and protein lysates derived from a series of cell lines, engineered to express a defined range of HER-2/neu oncoprotein levels. By using cells with def ined expression levels as calibration material, computerized image ana lysis of immunohistochemical staining could be used to determine the a mount of oncoprotein product in these cell lines as well as in human b reast cancer specimens. Quantitation of the amount of HER-2/neu protei n product determined by computerized image analysis of immunohistochem ical assays correlated very closely with quantitative analysis of a se ries of molecularly characterized breast cancer cell lines and breast cancer tissue specimens. Breast cancer cells with no overexpression of HER-2/neu had an average oncoprotein content of 0.18 +/- 0.02 (SE) pg /cell, whereas the content of those with overexpression was as follows : 2.01 +/- 0.73 pg/cell in cancers with overexpression but no amplific ation; 2.60 +/- 0.47 pg/cell in cancers with 2 to 5-fold amplification and overexpression; and 3.38 +/- 0.29 pg/cell in cancers with more th an 5-fold amplification and overexpression. HER-2/neu overexpression w as identified in 41 of 105 relapsed cases and in 21 of 105 matched dis ease-free controls. The risk of developing recurrent disease in node-n egative women with any level of HER-2/neu overexpression was 3.0 times that of women whose breast cancer lacked overexpression while the gro up of patients with high overexpression had a risk of recurrence 9.5 t imes greater than those whose breast cancers had normal expression (P = 0.0001). Analysis of various subgroups showed significantly increase d risks in pre- and postmenopausal women as well as in women with estr ogen receptor-negative and small (T1A) breast cancers indicating the i ncreased risk of recurrence extended across several subgroups of node- negative breast cancer patients. Our results demonstrate that HER-2/ne u overexpression is an independent indicator of increased risk of deve loping recurrent disease in women with node-negative breast cancer. Al though frozen tissue is optimal for analysis, formalin-fixed, paraffin -embedded tissue can yield meaningful results when reagents of suffici ent sensitivity and specificity are used. Finally, the quantitative am ount of oncoprotein in tumor cells can be determined using immunohisto chemistry in conjunction with computerized image analysis.