Immunohistochemical analysis of pathway HER2 4B5 performance in breast carcinoma expressing HER4.

Abstract

16 Background: The ability of pharmacodiagnostic antibodies to specifically detect their target antigen is essential to their performance. Non-clinical protein based models for specificity of two human epidermal growth factor 2 (HER2) pharmacodiagnostic antibodies, Oracle CB11 and PATHWAY HER2 4B5 (4B5) (Ventana Medical Systems Inc., Tucson, AZ, USA), have been reported to demonstrate affinity to both HER2 and HER4. To date there has been no evidence showing that affinity of 4B5 for HER4 impacts the IHC clinical HER2 assay. We therefore sought to investigate whether there exists any risk of the 4B5 antibody detecting HER4-positive but HER2-negative cases as falsely positive due to a hypothetical cross reactivity to HER4. Methods: A HER4 IHC assay was developed by screening ten HER4 antibodies for specificity by staining FFPE blocks of recombinant HEK-293 cells expressing HER1, HER2, HER3 and HER4. Specific HER4 antibodies were then evaluated in breast carcinoma to identify a clone that provided distinct membrane staining in tumour cells. This pattern was used as a reference in order to detect any potential interference of HER4 detection in determining the HER2 score. The HER4 antibody E200 (Epitomics) was selected and used to evaluate a cohort of breast carcinoma cases. The HER2 expression in these cases was then assessed using anti-HER2 clone PATHWAY HER2 4B5 and a HER2 reference antibody, clone SP3 (Spring Bioscience). Results: 117 out of 241 (48%) breast tissue cases evaluated were HER4 positive for membrane staining by IHC using E200. The scoring criteria for determining the HER4 score was based on the system used to score HER2, however a positive score indicated any membrane staining and therefore a 1+, 2+ or 3+ score was considered HER4-positive. 4B5 exhibited similar staining patterns as SP3 in co-expressing HER4 and HER2 tissues indicating that 4B5 was detecting only the HER2 antigen. Eighteen cases were HER4-positive by E200 and level 0 by 4B5 including a HER4 level 3+ case. Conclusions: PATHWAY HER2 4B5, a clinically relevant pharmacodiagnostic antibody for detecting HER2 expression in breast carcinoma, does not demonstrate cross reactivity to HER4 in IHC of FFPE clinical tissues.