Epr3947

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.

Four 3‐μm‐thick sections of FFPE samples were made for IHC analysis and stored at 37°C overnight. We used two auto‐staining systems. Protein expression was evaluated with mutL homolog 1 (MLH1) (M1; Ventana Medical Systems; Roche Group), mutS homolog 2 (MSH2) (G219‐1129; Ventana Medical Systems), mutS homolog 6 (MSH6) (44; Ventana Medical Systems), and postmeiotic segregation 1 homolog 2 (PMS2) (EPR3947; Ventana Medical Systems) antibodies using the Ventana BenchMark XT system (Roche). Protein expression was evaluated with MLH1 (ES05; Agilent), MSH2 (FE11; Agilent), MSH6 (EP49; Agilent), and PMS2 (EP51; Agilent) antibodies using the Autostainer Link48 (Agilent).²⁷, ²⁸ All sections were evaluated by a medical technologist (K.A.) and a pathologist (T.O). Tumors with completely absent nuclear staining of at least one of MLH1, MSH2, MSH6, or PMS2 were classified as dMMR.

A combination of multiple test platforms was used to determine the MSI or MMR status of the profiled samples, including fragment analysis (FA, Promega, Madison, WI), IHC (MLH1, M1 antibody; MSH2, G2191129 antibody; MSH6, 44 antibodies; and PMS2, EPR3947 antibody (Ventana Medical Systems, Inc., Tucson, AZ) and NGS (7000 target microsatellite loci were examined and compared to the reference genome hg19 from the University of California). The three platforms generated highly concordant results as previously reported^{41 (link)} and in the rare cases of discordant results, the MSI or MMR status of the tumor was determined in the order of IHC, FA, and NGS.