Analysis of HER2 External Domain Epitopes in Breast and Gastric Cancers Expressing p95HER2
Lorenzo Daniele, Daniele Recupero, Giuseppe Viale, Mauro Risio, Angelo P Dei Tos, Caterina Marchio, Isabella Castellano, Anna Sapino. University of Turin, Turin, Italy; European Institute of Oncology, Milan, Italy; Institute for Cancer Research and Treatment (IRCC), Candiolo, Turin, Italy; General Hospital of Treviso, Treviso, Italy
Background: A subgroup of HER2 overexpressing breast tumors coexpresses p95HER2, a truncated HER2 receptor that retains a highly functional kinase domain but lacks the extracellular domain (ECD) and results in intrinsic trastuzumab resistance. In formalin-fixed paraffin-embedded tissues the expression of p95HER2 is supposed to reduce the expression of the ECD as detected by immunohistochemistry (IHC). Here we want to compare p95 HER2 expression by western blot (WB) with HER2 expression by IHC (both intracellular (ICD) and trastuzumab-binding ECD).
Design: p95HER2 expression was evaluated by WB in a series of 99 breast carcinomas and 23 gastric cancers. The HER2 positive cell line BT474 was treated with pervanadate (a compound known to induce shedding of the HER2 ECD) and used as positive control for p95HER2 expression. p185HER2 was considered overexpressed (++) if the WB band was greater than or equal to p185HER2 level in BT474 cells. Lower levels of p185HER2 were classified as p185+. Specimens were scored as positive for p95HER2 (p95HER2+) if a clear band was present at the same molecular weight of the p95 pervanadate-induced band in BT474 cell extracts.
IHC was performed using antibodies against both the ICD (polyclonal antibody A0485 by Dako and CB11 by Novocastra) and the ECD (TAB 250 by Zymed and BiotHER, biotinylated trastuzumab, in-house created) of HER2. IHC was analyzed using an automated scanning system (Aperio ScanScope XT).
Results: The number of cases showing HER2 overexpression (score 3+) was higher in the p185++/ p95HER2+ cohort than in the p185++/ p95HER2- series, using both the anti-ICD and the anti-ECD antibodies. Automated analysis of IHC stained slides confirmed a significantly higher percentage of 3+ scored cells in p95HER2+ cases as compared to the p95HER2- cases. Conversely, the percentage of 2+ scored cells was higher in p95HER2- cases. The percentage of cells scored 1+ did not significantly change between the two groups.
Conclusions: The presence of p95HER2 seems not to compromise trastuzumab binding, suggesting p95HER2 expression per se defines an aggressive subtype of HER2-positive cancers with distinct biological and clinical features resistant to trastuzumab. This is in contradiction with the assumption that the expression of p95HER2 or of other HER2 truncated forms could reduce the expression of the ECD which contains the trastuzumab-binding site.
Wednesday, March 21, 2012 1:00 PM
Poster Session VI # 12, Wednesday Afternoon