Determination of Her-2/neu status in breast carcinoma: Comparative analysis of immunohistochemistry and fluorescent in situ hybridization

Her-2/neu (H2N) status in breast carcinoma has been considered a prognostic factor that may have therapeutic implications; however, the correlation be tween H2N overexpression and gene amplification has not been completely def ined. A consecutive series of ductal carcinomas (34 invasive and 7 in situ) were analyzed by fluorescent in situ hybridization for H2N gene and chromo some 17 copy number using touch preps of intact cells and by immunohistoche mistry, using three different commercial antibodies to H2N protein (Zymed, clone 31G7; Ventana, clone CB11; and Dako, polyclonal) in corresponding for malin-fixed, paraffin-embedded tissue sections. Gene amplification was clas sified as unequivocal if more than five signals were present in more than 8 0% of the counted nuclei and absent if more than 80% of the nuclei counted contained two or fewer gene copies. Cases that did not fulfill the above cr iteria were considered equivocal for amplification. Immunostaining was clas sified as follows: 0 = no staining; 1+ = faint, incomplete membranous patte rn; 2+ = moderate, complete membranous pattern; 3+ = strong membranous patt ern. Of the 34 invasive tumors, 10 (29%) had unequivocal gene amplification . Furthermore, all had more than 10 copies of the gene in more than 60% of the counted nuclei. An additional nine cases (26%) had equivocal amplificat ion, which was usually the result of chromosome 17 aneuploidy (seven of nin e) or heterogeneity. With the Zymed and Dako antibodies, all tumors with 3 staining had unequivocal gene amplification and all cases with 2+, If, or 6 staining were negative or equivocal for gene amplification. With the Vent ana antibody, all cases with 3+ staining had unequivocal gene amplification , but two cases with unequivocal amplification by fluorescent in situ hybri dization exhibited 1+ staining. Moderate (2+) H2N staining was observed in one case, three cases, and five cases with the Ventana, Dako, and Zymed rea gents, respectively, and did not correlate with H2N gene copy number. Disco rdance between H2N and chromosome 17 copy number was not a useful means of defining amplification. Two cases of ductal carcinoma in situ with the Zyme d antibody and two with the Dako antibody showed 3+ staining despite lack o f unequivocal gene amplification. We conclude that (1) strong H2N immunosta ining is highly associated with gene amplification, although there is minor variation in sensitivity between different antibodies; (2) a subset of bre ast carcinomas (3 to 15%) demonstrate moderate H2N staining without evidenc e of amplification, and it is unclear whether they represent highly sensiti ve staining or are a subset of cases that show over-expression without ampl ification; (3) gene amplification, as detected by fluorescent in situ hybri dization, is associated with at least 10 gene copies per nucleus, and lower gene copy duplication (3 to 4/nucleus) is frequent, usually the result of chromosome 17 polysomy, and not associated with high-level overexpression; (5) overexpression of H2N without amplification may be more frequent in duc tal carcinoma in situ, implying a different role in the biology of preinvas ive versus invasive neoplasm.