Immunohistochemical Evaluation of Tumors

Tumor grading, tumor typing and immunohistochemistry (ER, PR, Ki-67) was performed on the pretreatment core biopsies on all patients. Pathological complete response (pCR) was determined on tumor resection specimens after completion of neoadjuvant chemotherapy, and was defined as no evidence of residual invasive and ductal disease in the breast and lymph nodes (ypT0,ypN0).
Immunohistochemistry was performed according to previously standardized protocols on an automated IHC platform (Dako Techmate 500) with citrate buffer for antigen retrieval [23 (link)] and observing the ASCO/CAP guidelines for immunohistochemistry [7 (link)]. The following primary antibodies and corresponding dilutions were used (DakoCytomation, Glostrup, Denmark): ER (clone 1D5, 1:100), PR (clone PgR636, 1:100) and Ki-67 (MIB-1, 1:200). Slides were assessed by quantitative image analysis (qIHC) using the Aperio Image Analysis toolbox (Leica Biosystems, Nussloch, Germany). Staining intensity and percentage of positive nuclei were recorded after manually segmenting tumor from adjacent stroma. Tumors with ER/PR Remmele scores greater than 3 or positive nuclei greater than 1% were considered hormone receptor positive.