E cadherin

IHC was performed on 5 μm sections using an automated immunostainer (Ventana BenchMark ULTRA, Ventana Medical Systems, Oro Valley, AZ, USA) and protein expression was quantified using the histoscore method as previously described^{51 (link)}. The following antibodies were used: AXL (Cell Signaling Technology, Leiden, The Netherlands, #8661, dilution 1:100), pAkt (Cell Signaling Technology, #4060, dilution 1:50), phospho-PRAS40 (Cell Signaling Technology, #2997, dilution 1:100), E-cadherin (Roche #5973872001, RTU), β-catenin (Roche #5269016001, RTU), vimentin (Roche #5278139001, RTU), and N-cadherin (Abcam, Cambridge, UK, #ab18203, dilution 1:100).

IHC staining was performed using the Ventana Bench Mark ultra autostainer and the Ventana Ultra View universal DAB detection kit (Ventana Medical System Inc., Roche Tucson, Arizona, USA). The primary antibodies including ER, PR, HER2, Ki-67, E-cadherin, p120, AE1/AE3, calponin and p63 were purchased from Roche Ventana. All IHC stains were carried out with appropriate positive and negative controls.
The status of ER and PR were determined following ASCO/CAP guideline [10 (link)]. The ratio of nuclei-positive tumor cells to all tumor cells represented Ki-67 expression level and a Ki-67 expression was high when the ratio was ≥20% [9 (link)]. E-cadherin and p120 were employed to discriminate lobular carcinomas from ductal carcinomas. Double immunostainings were performed with AE1/AE3/P63 or AE1/AE3/calponin to confirm the presence of an invasive component.
A dual-probe FISH test using the PathVysion HER2 DNA probe Kit (Vysis Inc., Downers Grove, IL) was performed on the same specimen as IHC test following the manufacturer’s instructions. FISH results were interpreted by two experienced pathologists independently.

For analyses, paraffin blocks were obtained, cut, and stained by hematoxylin and eosin (H&E) in order to select the best histological area. Subsequently, selected tissue areas were placed into the TMA by circling the identified area in the corresponding block. Cylindrical core biopsies were then extracted from each paraffin block using a 20-μm² stylet and placed into a new recipient block. Selected adequate cases had tumors that occupied at least 10% of the core area. Each case was processed in triplicate to prevent tissue loss during cutting. Sections from each tissue array block were cut, de-paraffinized, and dehydrated for H&E and immunohistochemical procedures. Our IHC analyses included the following primary antibodies: PD-L1: Cat # SK00521-k (Dako, Carpinteria, CA, USA); all other antibodies: E-cadherin: Cat # 7904497 MLH1: Cat # 06472966001; PMS2: Cat # 06419216001; MSH2: Cat # 05269270001; MSH6: Cat # 5929911001); HER2: Cat # 05278368001; p16: Cat # 06695221001; p53: Cat # 5278074001 were from Roche Diagnostics (Basel, Switzerland). Protein expression levels were determined in a manually prepared TMA [38 (link),39 (link)] from deparaffinized sections obtained from archival tumor samples.