In an attempt to standardize the immunohistochemical detection of ERBB2, we evaluated six normal breast samples and 58
breast carcinomas for (1) the effect of four different
fixatives (
Bouin's fluid, buffered
formalin, alcoholic
formalin, and
methacarn), and (2) the sensitivity and specificity of eight commercially available
antibodies. ERBB2 gene copy numbers and
RNA transcripts were measured by Southern and Northern blot analysis in all samples. The BT-474 and MCF7 cell lines were processed in the same way as the tissue samples and used a s references of activation or the normal state of the ERBB2 gene, respectively. Complete concordance between immunohistochemistry and molecular biology was observed in 43/58 cases (74 per cent). However, fixation and processing protocols significantly affected the reactivity of the
antigenic determinants. The most consistent results were obtained with NCLCB11 on frozen sections and with Tab250 on
paraffin-embedded sections. For prospective studies, the use of alcoholic
formalin in association with a highly specific antibody (Tab250, 9G6, NCLCB11, or OA-11-854) gave the best results.
Methacarn appears to be a reliable
fixative, but was studied in only 28 cases. For retrospective studies, the most important parameter to take into account is the sensitivity of the antibody. Thus, with Bouin's fixation, 3B5 was the only reliable antibody, whereas with buffered
formalin, Tab250 gave the most consistent results. This study will help inter-laboratory comparisons, and in determining the prognostic significance of ERBB2 expression in
breast carcinomas using a standardized methodology.