A task force of the Japanese Breast Cancer Society has proposed a recommendation for adequate evaluation of hormone receptors in routine practice, in order to standardize handling of tissues, staining techniques and scoring systems. As a part of the study, several examinations were conducted to detect the effect of technical problems, including the influence of fixation time and other fixation and processing conditions, on the immunoreactivity for ERα.
There is little influence of prolonged fixation on the immunoreactivity for ERα, except for cases in which particularly over-fixed blocks are used. A delay in the onset of fixation could decrease the immunohistochemical findings of steroid receptors, compared with shorter or longer fixation, and the situation is similar to the fixation of a whole large surgical specimen in formalin in a big bucket. Incomplete fixation might be an important cause of heterogeneiety of immunoreactivity for ERα.
Manual and automated immunohistochemical (IHC) staining by DAKO (Glostrup, Denmark) and Biogenex (San Ramon, CA) and automated IHC staining by Ventana Medical Systems (Tucson, AZ) each employ different methods. Using a scoring system, in which the proportion of cells stained in each specimen was recorded as 0, less than 1%, 1% or more and less than 10%, and 10% or more, the intermethod variability of those IHC staining methods exhibited substantial multi-rater kappa values concerning the ER and PgR (kappa for ER according to the percentage of positive cells=0.67; PgR=0.72). Concerning intermethod consistency, the scoring system based on the percentage of positive cells was advantageous over other scoring systems, based on the intensity of nuclear staining.
Using double staining, patients with ER-positive and HER2-positive tumors can be classified as those with co-expressed tumors and those with differently expressed tumors. As such, the co-expressed tumor might be resistant to antiestrogen therapy in ERα-positive and HER2-positive breast cancer and double staining might lead to the development of new therapeutic strategies for hormone and HER2-positive breast cancer.

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