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Journal of Clinical Oncology, Vol 23, No 31 (November 1), 2005: pp. 8119-8120 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.03.2318
In Reply:Yale Medical School, New Haven, CT
University of Oklahoma Health Sciences Center, Oklahoma City, OK Karlin et al present experiments that raise concern regarding the specificity of a widely used anti-EGFR polyclonal antibody preparation Ab 1005 (sc-03, Santa Cruz Biotechnology, Santa Cruz, CA) that we employed in our study showing relationships between EGFR, ErbB2, and phosphorylated ErbB-2 expression.1 Ab 1005 is a polyclonal antibody raised against a 12-mer peptide corresponding to EGFR amino acids 1005 to 1016. This peptide sequence has 75% homology to the corresponding ErbB-2 sequence, with the discordant amino acids spaced evenly in the pattern ABAAABAAABAA, where A is an identical residue shared between the two receptors, and B is a divergent residue. In Figure 1 of our article we characterized the specificity of the Ab 1005 preparation we used in experiments similar to those presented by Karlin et al; and further characterized the reagent for phosphorylation state specificity. This figure demonstrates reactivity of Ab 1005 with EGFR immunoprecipitated from A431 cells (using a different precipitating EGFR antibody, Ab528), with reactivity equal for receptor in the phosphorylated (EGF-stimulated) or unphosphorylated (mock-stimulated) state. Furthermore, we demonstrated lack of cross reactivity with ErbB-2 in either the phosphorylated or unphosphorylated state, using ErbB-2 that was immunoprecipitated from BaF3 hematopoietic cells that had been transfected with the ErbB2 gene. The reactivity distribution we observed by immunohistochemistry on the clinical specimens was also consistent with a lack of cross reactivity, as only 35% of ErbB-2-positive breast tumors showed immunohistochemical staining for EGFR. Our series included many cases with the highest ErbB-2 staining that had undetectable EGFR staining. Previously published work by some of us (S.M.E., D.M., A.D.T.) also included verification of EGFR staining patterns using a monoclonal antibody MU207 for comparison (clone E30; BioGenex, San Ramon, CA). In that study, we used 40 invasive breast cancers and compared reactivity with these anti-EGFR antibodies (pAb 1005 and mAb E30), side by side with the anti-ErbB-2 antibody CB11. We saw similar reactivity patterns with pAb 1005 and mAb E30, and a distinct subset of tumors reactive with CB11.2 Karlin et al present immunoblotting experiments using cell lysates without immunoprecipitation.1 Using anti-EGFR Ab 2232, a band is detected only in MCF10A cells and EGFR-transfected 293T cells, whereas there is no detectable band in ErbB2-transfected 293T cells, ZR75-1 or SKBR3 cells. It is curious that EGFR is not detected in SKBR3 cells, since they are known to express high levels of EGFR in addition to (higher levels of) ErbB-2.3 This raises the question of whether Ab 2232 might recognize EGFR only from certain cell lines, possibly as a consequence of post-translational modifications. Examples of modifications affecting antibody reactivity include phosphorylation state,4 glycosylation pattern (eg, EGFR antibody E2760, clone 29.1, Sigma, St Louis, MO), conformational state,5 oligomerization state, or altered protein structure (alternative splice variant or cleaved form). Alternatively, a threshold effect may explain inability of Ab 2232 to detect EGFR under some circumstances. In distinction to Ab 2232, the Ab 1005 did recognize a band in SKBR3 cells, although it had slower mobility than that from EGFR-transfected 293T cells, and it comigrated with a band from ErbB2-transfected 293T cells that was recognized by anti-ErbB-2 antibody and also by Ab 1005, suggesting that under these experimental conditions, the Ab 1005 preparation used was cross reacting with ErbB-2. We suggest that it is possible that low level cross reactivity with ErbB-2 could be obtained with the polyclonal anti-EGFR antibody preparation used by Karlin et al when exceptionally high amounts of ErbB-2 are deposited on the membrane, as would be the case with the high level expression expected in ErbB2 gene-transfected 293T cells. The weak reactivity with SKBR3 cells shown in the cell block staining in panel B does not help to clarify the reactivity because, as mentioned above, SKBR3 cells express significant levels of EGFR. In any polyclonal preparation, a small population of antibodies may crossreact, which may explain reactivity seen at only extremely high levels of homologous receptor. In addition, being a polyclonal antibody, it is likely that there is batch-to-batch variation in the composition of the antibody preparation, and it is possible that the batch that Karlin et al used for their experiments may have cross reactivity that was absent in the batch we used for our experiments that were performed several years ago (circa 1999). In conclusion, Karlin et al raise important issues that serve as a reminder that all antibody-based work must be interpreted with some degree of caution. This is especially true for immunohistochemical experiments where, unlike immunoblotting, molecular weight confirmation of appropriate reactivity is lacking. All investigators should maintain vigilance regarding these issues. Karlin et al demonstrate that with high levels of ErbB-2, the lot of Ab 1005 that they used reacts with ErbB-2 in immunoblots. The lack of ErbB-2 immunoblot cross reactivity observed with the batch of antibody we used, our finding of a large percentage of breast tumors staining strongly for ErbB-2 with no detectable EGFR staining, our parallel supporting studies with other reagents,2 and the supporting conclusions from other investigators who have found HER2/EGFR coexpression associated with poor outcomes6 lead us to believe that our data and conclusions are sound. Authors' Disclosures of Potential Conflicts of Interest The authors indicated no potential conflicts of interest. REFERENCES
1. DiGiovanna MP, Stern DF, Edgerton S, et al: ErbB-2 activation, epidermal growth factor receptor expression and prognosis in breast cancer patients. J Clin Oncol 23:1152-1160, 2005
2. Thor AD, Edgerton SM, Liu S, et al: Gelsolin as a negative prognostic factor and effector of mobility in erbB-2-positive epidermal growth factor receptor-positive breast cancers. Clin Cancer Res 7:2415-2424, 2001
3. Bangalore L, Tanner AJ, Laudano AP, et al: Antiserum raised against a synthetic phosphotyrosine-containing peptide selectively recognizes p185neu/erbB-2 and the epidermal growth factor receptor. Proc Natl Acad Sci U S A 89:11637-11641, 1992 4. DiGiovanna MP: Phosphorylation sensitivity of the commonly used anti-p185 neu/erbB2 monoclonal antibody clone 3B5 suggests selective usage of autophosphorylation sites. Analytical Biochem 247:167-170, 1997[CrossRef][Medline]
5. Johns TG, Adamas TE, Cochran JR, et al: Identification of the epitope for the epidermal growth factor receptor-specific monoclonal antibody 806 reveals that it preferentially recognizes an untethered form of the receptor. J Biol Chem 279:30375-30384, 2004 6. Suo Z, Risberg B, Kalsson MG, et al: EGFR family expression in breast carcinomas. c-erbB-2 and c-erbB-4 receptors have different effects on survival. J Pathol 196:17-25, 2002[CrossRef][Medline]
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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