Effects of Hydrochloric Acid and Formic Acid Decalcification on Breast Tumor Biomarkers and HER2 Fluorescence In Situ Hybridization.

ABSTRACT

Biomarker analysis of metastatic breast carcinoma (MBC) is routinely recommended by ASCO/CAP guidelines, and establishing a diagnosis of MBC often requires immunohistochemistry (IHC). The reliability of breast tumor biomarkers and breast-specific markers on decalcified tissues has not been extensively studied. We performed IHC studies on breast tumors exposed to hydrochloric acid (HCl) and formic acid (FA) decalcification solutions, and HER2 fluorescence in situ hybridization on a subset of these tumors to establish a protocol for handling bone specimens with suspicion for MBC. Fifteen fresh cases of primary breast carcinoma and 8 HER2+ paraffin-embedded core biopsy cases were studied. Fresh tissue was divided into 5 fragments to approximate a bone core biopsy. One fragment (control) was fixed in 10% neutral buffered formalin. The remaining fragments were also exposed to FA or HCl decalcification for 1 or 5 hours. All fragments were embedded in 1 block and tested with an IHC panel. The known HER2+ cases were exposed to either 1 or 5 hours of FA, and HER2 fluorescence in situ hybridization was also performed. Results were interpreted as follows H-scores for estrogen receptor, progesterone receptor, and GATA-3 were assigned from 0 to 300; HER2, cytokeratin 7, gross cystic disease fluid protein-15, Pax-8, TTF-1, cytokeratin 20, and mammaglobin were scored from 0 to 3+; and Ki67 from 0% to 100%. Mean scores were compared using the t test or Wilcoxon test for paired samples. No significant differences in mean score were seen between NF and 1 hour FA for any IHC immunoreactivity. After 5 hours of FA, only Ki67 average score was significantly less than NF. Mean scores for estrogen receptor, progesterone receptor, HER2, Ki67, and GATA-3 were significantly lower than NF in the tissue after either 1 or 5 hours of HCl. Mean scores for gross cystic disease fluid protein-15, mammaglobin, and cytokeratin 7 staining were not significantly lower than NF after 1 or 5 hours of HCl.