Effect of EDTA decalcification on estrogen receptor and progesterone receptor immunohistochemistry and HER2/neu fluorescence in situ hybridization in breast carcinoma

Abstract

Bone is the most common site of metastasis in breast carcinoma (BC). Treatments for metastatic BC depend on various factors, including the tumor's estrogen receptor (ER), progesterone receptor (PR), and HER2 status. Bone biopsies require decalcification which may affect the accuracy of ER and PR immunohistochemistry (IHC) and HER2 situ hybridization (FISH) studies. Ethylenediaminetetraacetic acid (EDTA) decalcifying solutions have been theorized to have no significant impact on ER and PR IHC or HER2 FISH analyses. We completed a prospective study of the effect of EDTA decalcification on ER and PR IHC and HER2 FISH in 29 cases of BC. Samples from 29 BC resections were collected and formalin fixed between 12 and 24h. Control samples were routinely processed, whereas test samples were placed in EDTA for 48h. ER and PR slides were blinded, randomized, and evaluated. Blinded samples underwent HER2 FISH assays where an average HER2 copy number and HER2/CEP17 ratio were calculated. Paired differences between EDTA and control samples were compared for ER and PR positivity, average HER2 copy number, and HER2/CEP17 ratios using paired-samples t-tests (PST) and Wilcoxon signed-rank test (WSR). PST and WSR tests yielded no significant difference between EDTA and control tissue for ER% (PST: P=1; WSR: P=0.916), PR% (PST: P=0.973; WSR: P=0.984), HER2 copy number (PST: P=0.124; WSR: P=0.103), and HER2/CEP17 ratio (PST: P=0.25; WSR: P=0.105). The use of EDTA in bony tissue is therefore a valid decalcification method to ensure accurate assessment of ER and PR IHC and HER2 FISH in metastatic BC.