In Reply:
Authors: Michael P. DiGiovanna, David F. Stern, Susan M. Edgerton, Ann D. Thor
Journal: Journal of Clinical Oncology (2005)

vector, EGFR, or increasing amounts of ErbB-2 plasmid (Fig 1A, lanes 4 to 8).As in the breast cancer cell lines, the anti-EGFR (1005) antibody used by the authors detected EGFR in cells transfected with EGFR plasmid (Fig 1A, panel 1, lane 5) as well as a protein that migrates at a slightly higher molecular weight than EGFR in the cells transfected with high amounts of ErbB-2 plasmid (Fig 1A, panel 1, lanes 6 to 8).Reprobing of this blot with the anti-EGFR (2232) antibody or with the ErbB-2 specific antibody confirmed that the transfected cells expressed the appropriate proteins (Fig 1A, panels 2 and 3, lanes 4 to 8).As suggested by the data with the breast cancer cell lines, the cross reactivity of the anti-EGFR (1005) antibody with ErbB-2 was only seen at high levels of transfected ErbB-2 expression comparable to the expression seen in the SKBr3 cell line (eg, Fig 1A, compare lanes 3 and 7 in panels 1 and 3).Our immunoblotting experiments with the anti-EGFR (1005) antibody were performed using 0.2 g/mL of antibody, the same as in the paper by DiGiovanna et al.Additional experiments, using a 10-fold lower dilution of the antibody showed similar cross reactivity (data not shown).The authors show an immunoblot that demonstrates no cross reactivity of the anti-EGFR (1005) with ErbB-2.Our data show that the cross reactivity is only apparent at high levels of ErbB-2.It is possible that the amount of ErbB-2 precipitated in the experiment shown in the paper is not sufficient to elicit cross reactivity.However, we observe cross reactivity in a physiologically relevant context since the SKBr3 cell line is derived from a patient with amplified and overexpressed ErbB-2.We next tested whether the anti-EGFR (1005) antibody cross reacted with ErbB-2 when used for immunohistochemistry.The anti-EGFR (1005) antibody stained both MCF10A and SKBr3 cells (Fig 1B, top panels).In contrast, a second anti-EGFR antibody (31G7 from Zymed, Carlsbad, CA) stained MCF10A cells but not SKBr3 cells (Fig 1B, middle panels).Similarly, an ErbB-2 specific antibody (CB11 from Biogenex, San Ramon, CA) stained SkBr3 cells, but not MCF10A cells (Fig 1B, lower panels).These data are consistent with the cross reactivity seen on the immunoblot for the anti-EGFR (1005) antibody.Together, our immunoblotting and immunohistochemistry data suggest that the anti-EGFR (1005) antibody cross reacts with high levels of ErbB-2, levels frequently seen in patients with breast cancer.In this light, the EGFR expression data from the paper by DiGiovanna et al could be interpreted as detecting ErbB-2 expression in those patients with the highest ErbB-2 expression rather than EGFR coexpression.Previous work has demonstrated that ErbB-2 activation and prognosis correlate with ErbB-2 expression levels and thus the prognostic information gained from EGFR expression in this article could be an indication of ErbB-2 levels rather than EGFR coexpression. 3 While our observations about the ErbB-2 cross reactivity of the anti-EGFR (1005) antibody do not rule out a role for EGFR in breast cancer, we feel that the data in the paper by DiGiovanna et al need to be interpreted with caution and that their observations should be confirmed with a more specific anti-EGFR antibody.