MMR proteins expression was analyzed by IHC assay, using monoclonal antibodies directed against MutL homolog 1 (MLH1: VENTANA MLH1, M1), PMS homologue 2 (PSM2: VENTANA PMS2, EPR3947), mutS homologue 2 (MSH2: VENTANA MSH2, G219-1129) and mutS homologue 6 (MSH6: VENTANA MSH6, 44). The staining was regarded positive when the tumor nuclei stained positively with the same intensity of the internal positive control including infiltrating lymphocytes, stromal cells and adjacent non-neoplastic epithelium. All cases with loss (absence) of nuclear immunostaining or reduced protein expression, when compared with internal positive control, were considered negative. An abnormal MMR expression was represented by a patchy and/or weak expression consisting of a nuclear loss associated with a gain in cytoplasmic staining or a heterogeneous expression within adjacent tumor areas.15 (link)
Microsatellite instability status (MSI) molecular test will be performed by comparison of the allelic profiles of the mononucleotide repeat markers BAT-25, BAT-26, NR-21, NR-24, and NR-27 in tumor and corresponding normal tissue. EBER using VENTANA EBER Probe was performed to assess EBV status.