VENTANA anti-MLH1 (M1) Mouse Monoclonal Primary Antibody

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The review of hematoxylin and eosin slides were performed by two experienced pathologists (J.S.K. and S.W.H.), and one representative block was selected for each case. Immunohistochemistry was performed on the representative paraffin-embedded 4 μm-thick whole tissue sections using a BenchMark XT automated stainer (Ventana Medical Systems, Tucson, AZ, USA) for CD8 (clone CD/144B, 1:100; Cell Marque, Rocklin, CA, USA), p53 (clone DO-7, 1:3000; Novocastra, Buffalo Grove, IL, USA) and MMR proteins, and a Leica automated stainer (Bond Max) for PD-1 (clone NAT105, 1:100; Cell marque) and PD-L1 (clone E1L3N, 1:150; Cell Signaling, Danvers, MA, USA). Antibodies used for MMR proteins were anti-human MLH1 (clone M1, ready to use; Ventana, Oro Valley, AZ, USA), MSH2 (clone G219-1129, ready to use; Ventana), MSH6 (clone 44, ready to use; Ventana), and PMS2 (clone ERP3947, ready to use; Ventana) monoclonal antibodies. Immunohistochemical evaluation was performed by two experienced pathologists (J.S.K. and S.W.H.) blinded to clinical data. Loss of MMR proteins (MMR deficiency) was defined as a complete loss of nuclear staining in the tumor cells with positive internal controls (stromal cells, lymphocytes, or normal endometrium). CD8 expression was evaluated as the number of CD8+ T lymphocytes in 10 randomly selected high-power fields (HPFs). P53 staining was scored as 0 (completely negative), 1 (focal, weak to moderate positive), and 2 (diffuse > 80%, strong positive); scores of 0 and 2 were regarded as abnormal, and a score of 1 was regarded as normal [39]. PD-1 and PD-L1 expression in immune cells were evaluated as the percentage of PD-1+ or PD-L1+ immune cells relative to the total number of immune cells in tumor stroma. PD-L1 expression in tumor cells was evaluated as the percentage of PD-L1+ tumor cells showing membranous expression relative to total number of tumor cells.

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