G219 1129

Immunohistochemistry was performed on 4-µm paraffin sections. The primary antibodies specific for dMMR proteins were as follows: MSH2 (Roche G219-1129), MLH1 (Roche M1), MSH6 (Abcam EPR3945), and PMS2 (Roche A16-4). All dMMR protein staining was performed using an autoimmunostainer (BenchMark Ultra, Ventana Medical Systems).

Fully automated immunostaining was performed on 4-μm-thick FFPE sections using a BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ). IHC for MMR proteins was performed according to standard antibody protocols using the following antibodies: mouse anti-MLH1 (Ventana/Roche M1, pre-dilute antibody), mouse anti-MSH2 (Roche G219-1129, pre-dilute antibody), rabbit anti-MSH6 (Roche SP93, pre-dilute antibody), and mouse anti-PMS2 (Roche A16-4, pre-dilute antibody). Tumors lacking MLH1, MSH2, PMS2, or MSH6 expression were considered dMMR, whereas tumors that expressed MLH1, MSH2, PMS2, or MSH6 were considered pMMR.
EBV status was determined by in situ hybridization with EBV-encoded small RNA (EBER) probes (INFORM EBER, Roche, ready-to-use) according to product protocols. Strong EBER signals were interpreted as EBV-positive.

Pedigree analyses were done for all the families within the study. For those fulfilling clinical criteria for potential presence of Lynch syndrome according to Bethesda Guidelines [24 (link)], we proceeded with immunohistochemistry to evaluate the expression of mismatch repair proteins. Immunohistochemistry (IHC) was performed on 3-μm-thick tissue sections from paraffin-embedded, formalin-fixed tumour. The OptiView DAB IHC Detection kit on the Benchmark ULTRA staining module (Ventana) was used according to the manufacturer’s instructions. Staining with four antibodies MLH1 (M1, Ventana), MSH2 (G219–1129, Ventana), MSH6 (SP93, Ventana) and PMS2 (EPR3947, Ventana) were evaluated. A case was reported as MMR-deficient (dMMR) when displaying total or partial nuclear loss of expression in tumour cells, with retained expression in adjacent normal tissue as a positive control. Expression was reported as MMR proficient (pMMR) when nuclear staining was retained both in tumour cells and positive internal controls. In case of loss of expression of one or more of these proteins, we did further a DNA sequencing analysis on the genes of interest. For those who fulfilled criteria for HDGC according to The International Gastric Cancer Linkage Consortium [13 (link)], CDH1genetic screening was performed using DNA sequencing.

We reviewed haematoxylin and eosin (H&E)-stained sections prepared for pathological diagnosis before constructing tissue microarrays (TMAs). A set of TMAs from 89 patients were constructed as follows. Two representative tumour cores (2 mm in diameter) were obtained from formalin-fixed, paraffin-embedded (FFPE) tissue blocks representative of the lesions. Then, the cores were embedded in paraffin and serial 4 µm-thick sections were prepared for H&E staining, IHC, and ISH.
The primary antibodies used for IHC were purchased from Ventana (Tucson, AZ, USA) as follows: an anti-PD-L1 rabbit monoclonal antibody (SP263), anti-HER2 rabbit monoclonal antibody (4B5), anti-EGFR mouse monoclonal antibody (5B7), anti-mutL homolog 1 (MLH1) mouse monoclonal antibody (M1), anti-mutS homolog 2 (MSH2) mouse monoclonal antibody (G219-1129), anti-mutS homolog 6 (MSH6) rabbit monoclonal antibody (44), and anti-postmeiotic segregation increased 2 (PMS2) rabbit monoclonal antibody (EPR3947). A BenchMark ULTRA System (Ventana) was used for IHC. Chromogenic ISH to detect EBV-encoded RNA (EBER) was performed using fluorescein-labelled oligonucleotide probes (INFORM EBER probe, Ventana) with enzymatic digestion (ISH protease 3, Ventana) and an iViewBlue detection kit (Ventana) with the BenchMark ULTRA staining system.

Formalin-fixed paraffin-embedded (FFPE) tumor specimens were prepared by the standard procedures in Department of Diagnostic Pathology, Kyoto University Hospital. A board-certificated pathologist selected multiple and separate cancer lesions from a single tumor. Specimens of FFPE cancer spheroids were prepared as previously reported [14 (link)]. These specimens were sectioned at 4-μm thickness, and stained with H&E or immunostained for MLH-1 (M1, Ventana, Tucson, AZ, USA), MSH2 (G219-1129, Ventana), MSH6 (EPR3945, Abcam, Cambridge, UK), or PMS2 (EPR 3947, Ventana) followed by hematoxylin counterstaining for nuclei.