To the Editor: Jimenez et al. reported the levels of concordance between FISH and three antibodies for Her-2/Neu status in 34 cases of invasive breast carcinoma. The antibodies employed (TAB 250 from Zymed Laboratories, the CB11 from Ventana Laboratories, and the DAKO polyclonal) are all well-described in the literature. The utility of the article stems from a head-to-head comparison between the antibodies using an automated (Ventana) stainer with well-defined protocols.

Their results showed that of the three antibodies, CB11 had the worse sensitivity, detecting only 8 of the 10 cases scored as 3+ by the other two antibodies and determined to be amplified using FISH.

We have been using CB11 (Ventana Laboratories) on the Ventana automated immunostainer for 2 years and have found that antigen retrieval with heat (microwave for 14.0 minutes in a pressure cooker with 0.1 mm EDTA PH 8.0) is essential for optimum performance with this antibody. This also is recognized by the manufacturer, as they include a recommendation for heat retrieval with their antibody.

We have since characterized the performance of the three antibodies with or without antigen retrieval using a panel of nine breast and ovarian cell lines. All of the staining was performed on the Ventana automated stainer. The most sensitive technique was the DAKO polyclonal used as specified in the article by Jimenez et al. With retrieval, the CB11 and TAB 250 showed equivalent sensitivities. Without antigen retrieval, both CB11 and the DAKO antibody showed poor reactivity with decreased sensitivity (CB11) or decreased specificity (DAKO).

The need for standardized protocols for Her-2/Neu testing is paramount. In formalin fixed paraffin-embedded tissue, we are now recognizing that epitope retrieval is essential for optimizing many of the antibodies (1) and that comparisons between antibodies without such retrieval are meaningless.

  • Judith Hugh, M.D.

  • Randy Barley, M.Sc.

  • Laith Dabbagh, M.Sc.

  • Cross Cancer Institute

  • Edmonton, Alberta, Canada

REFERENCE

1. Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. HER-2/neu protein expression in breast cancer evaluated by immunohistochemistry: a study of interlaboratory agreement. Am J Clin Pathol 2000; 113: 251–8.

In reply: As noted in the Materials and Methods section, we employed staining methods as recommended by each vendor, including Ventana. This included antigen retrieval on the stains that employed the CB11 antibody. In addition, the two amplified cases that did not demonstrate 3+ staining were repeated, showing similar results. The reason(s) for lack of correlation with in situ hybridization in these two cases is unclear.

We disagree somewhat with the statement by Dr. Hugh et al. that the point of our article was a comparison of antibody reagents. Rather, the study was designed as a comparison between immunohistochemistry and in situ hybridization for analyzing the status of Her2/Neu.

We also disagree with their implication that antigen retrieval is essential for all immunohistochemical studies. Our experience has shown that antigen retrieval should be investigated as a component of the overall evaluation of immunohistochemical reagents. It is not necessarily essential, or even desirable, for all staining protocols.

  • Daniel W. Visscher, M.D.

  • Pamela Tabaczka, B.S., M.T. (ASCP)