Spontaneous and pronase-induced HER2 truncation increases the trastuzumab binding capacity of breast cancer tissues and cell lines

A subgroup of HER2-overexpressing breast tumours co-expresses p95 ^HER2, a truncated HER2 receptor that retains a functional HER2 kinase domain but lacks the extracellular domain, thus impairing trastuzumab binding. We evaluated p95^HER2 expression in 99 frozen breast carcinoma samples by western blot analysis. The HER2-positive cell line BT474 treated with pervanadate or pronase was used as a positive control for p95^HER2 expression. Immunohistochemistry was performed on parallel formalin-fixed, paraffin-embedded sections of the same case series using antibodies directed against either the intra- or extra-cellular binding domain of HER2. In particular, biotinylated trastuzumab (BiotHER) was used to evaluate the binding capacity of the humanized antibody. To avoid a subjective evaluation of the score values and the percentage of immunostained cells, the slides were scanned and automatically analysed. The number of cases with HER2 overexpression (score 3+) and HER2 gene amplification was higher in the p185^HER2-positive/ p95^HER2-positive samples than in the p185^HER2-positive/ p95^HER2-negative group. Automated analysis confirmed a significantly higher percentage of 3+ scored cells in p95^HER2-positive cases. Conversely, the percentage of 2+ scored cells was higher in p95 ^HER2-negative cases. The status of the HER2 extracellular domain was then studied using flow cytometry on BT474 cells after pronase enzymatic digestion using trastuzumab and pertuzumab, while the presence of HER2-HER3 dimers was studied using a proximity-ligation assay. In vitro experiments showed that short-term pronase digestion of BT474 cells produced two HER2 fragments (of 95 and 150 kDa, detectable in tissue specimens as well), increased the binding affinity of trastuzumab, reduced the rate of HER2-HER3 dimers, and did not interfere with pertuzumab-binding capacity. In conclusion, the presence of p95^HER2 as detected by western blot analysis does not compromise the immunohistochemical detection of HER2. Our data suggest that a reduction of the receptor steric hindrance as induced by enzymatic shedding may facilitate the binding capacity of trastuzumab.