Pathway anti her 2 neu 4b5

Human epidermal growth factor receptor 2 (HER2) status was detected using immunohistochemistry (IHC) staining with Her2 antibody (cat. # PATHWAY anti-HER-2/neu (4B5), Ventana, Tucson, AZ, USA). Standard American Society of Clinical Oncology / College of American Pathologists ASCO/CAP IHC classification for HER2 positivity (0, 1+, 2+ and 3+) was used. In cases were IHC results were considered equivocal (HER2 overexpression 2+), fluorescent in situ hybridization (FISH) analysis was performed. Evaluation of mismatch repair (MMR) protein expression was performed using antibodies to MutL homolog 1 (MLH1) (cat. no.# 790–4535, Ventana, PMS1 homolog 2, mismatch repair system component (PMS2) (cat. no. #760–4531, Ventana, Tucson, AZ, USA), mutS homolog 6 (MSH6) (cat. no. #287R-26, Cell Marque, Rocklin, CA, USA) and MutS protein homolog 2 (MSH2) (cat. no. # G219–1129, Cell Marque, Rocklin, CA, USA). Loss of MMR protein expression defined the patient as microsatellite instability high (MSI-H). PD-L1 expression was determined using IHC staining procedure with Dako Autostainer Link 48 platform (Agilent, Santa Clara, CA, USA) and an automated staining protocol validated for the PD-L1 IHC 22C3 pharmDx assay (Agilent). PD-L1 scoring was done according to FDA approved scoring guideline for PD-L1 in GC.

Dulbecco’s modified Eagle medium (DMEM), F-12 nutrient mixture (Ham’s F-12) and penicillin-streptomycin solution (PS) were purchased from Invitrogen (Carlsbad, CA, USA). Antibiotic-antimycotic mixed stock solutions (AAMS, 100×) were purchased from Nacalai Tesque Inc., (Kyoto, Japan). Fetal calf serum (FCS) was purchased from Biowest (Riverside, MO, USA). Cryopreservation reagents, Cell Banker, were purchased from Nippon Zenyaku Kogyo (Fukushima, Japan). Matrigel basement membrane matrix was purchased from Corning (Corning, NY, USA).
PAXgene tissue fix containers were purchased from QIAGEN (Tokyo, Japan). PATHWAY anti-HER-2/neu (4B5), INFORM HER2 Dual ISH DNA Probe Cocktail Assay (780-4422), and EBER 1 DNP Probe (760-1209) were purchased from Roche Diagnostics (Tokyo, Japan)