Pr1294

Between 2006 and 2018, 146 hormone receptor–positive (HR+) BC patients were treated with NET in both routine and clinical trial settings in the Multidisciplinary Breast Clinic of the Antwerp University Hospital. All patients (n = 56) who had HR (+), HER‐2 negative BC and had undergone surgery after NET were analysed in our study. Only 44 of these patients could be included in this study due to the fact that one of them had a pathological complete response and the tissue blocks of 11 patients had been exhausted. All patients had HR+ and HER2‐ BC and ER+/HER2‐ was divided into Luminal A like tumours and Luminal B like tumours and a Ki67 cut‐off (≥20%) was used to split these two subtypes.²³ Oestrogen receptor (ER) and progesterone receptor (PR) expression was assessed using monoclonal antibodies EP1 (Ready‐to‐use (RTU) kit; Dako) and PR1294 (1:50; Dako) clones, respectively, and scored according to the Allred method. Allred scores of 3–8 were considered positive. Ki‐67 was stained using MIB‐1 (RTU kit Dako). Clinicopathological and follow‐up data of all patients were collected from hospital medical records. The absence of residual invasive carcinoma in the resected breast specimen and in all sampled regional lymph nodes after NET was defined as pCR.

IHC was performed as previously described.^{28 (link)} Briefly, 10 μm paraffin sections were subjected to antigen retrieval in citrate buffer. Primary antibodies used were as follows: ERα (SP1, ThermoFisher, 1:100), PR (1294, DAKO, 1:500), CK5 (NCL-L-CK5, Leica Biosystems, 1:500), CK8/18 (NCL-L-5D3, Leica Biosystems, 1:500), and CA2 (SAB2700301, Sigma, 1:1600). Secondary antibodies were anti-Rabbit Ig MP-7401 (Vector, 1:2000; Burlingame, CA, USA) or anti-Mouse Ig MP-7402 (Vector, 1:2000). Development used the ImPACT DAB Peroxidase Substrate Kit (Vector). Images were captured using the Aperio Digital Pathology system (Leica Biosystems) and assembled in Adobe Photoshop CC with minimal linear changes to contrast. Quantification of the percentage of positive cells was performed using the Imagescope software (Leica) that used algorithms tuned for nuclear or cytoplasmic staining.