Comparative Assays for the HER-2/neu Oncogene Status in Breast Cancer

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Abstract
Context.—Tumor marker assays, especially those used to indicate the right therapy, should be standardized. Objective.—To analyze the current methods for the HER-2/neu (h2n) oncogene status by immunohistochemical (IHC) analysis, fluorescence in situ hybridization (FISH), and chromogenic in situ hybridization (CISH) and compare those results with the chromosome 17 copy number and the status of the topoisomerase II alpha (TPIIα) gene. Design.—We tested 50 infiltrating ductal breast carcinomas (pTNM status varied from pT1 N0 to pT4 N1) using the Food and Drug Administration (FDA)–approved methods HercepTest and Pathway for overexpression of h2n. We also used FISH and CISH to test for h2n amplification and CISH to test for chromosome 17 (c17) and TPIIα. The p53 and Ki-67 factors were also evaluated by IHC analysis. Results.—h2n overexpression (3+) and amplification were observed in only 6 (12%) of 50 cases by IHC analysis, FISH, and CISH. Three cases that initially scored 3+ and 2+ had 4 to 5.95 signals (equivocal) by FISH but when corrected by the h2n/c17 ratio were nonamplified. TPIIα isomerase was amplified in only 2 (4%) of the 50 cases. Nineteen (38%) of the 50 cases were aneuploidic. All h2n amplified cases had high proliferative activity, but only 2 of 6 had p53 protein alterations. Conclusions.—The HercepTest and Pathway IHC assay h2n were fully concordant for the 3+ cases. The 3+ cases had to be confirmed in 75% of the tumor area examined. These 2 IHC assays were fully concordant with FISH and CISH. The 2 in situ hybridization (ISH) assays were 94% concordant for the 50 cases. The cutoff signal points for both ISH assays should be 6 or more. Thus, there is no need for the c17 ratio correction. Tumor heterogeneity appears not be a major problem, but our percentage of amplified cases is lower than previously reported. The FDA-approved IHC and ISH assays should give relatively uniform results when used following our recommendations.