Clone 44

Formalin-fixed paraffin-embedded tissue was collected from the archives of the pathology division of our hospital. Hematoxylin–eosin archival slides were revised by an expert pathologist and the diagnosis was confirmed in all cases. The most representative slide of each sample was selected for IHC in cases with available material. Specifically, three-micron-thick serial paraffin sections of each case were processed by IHC using an automated immunostainer (Ventana BenchMark Ultra AutoStainer, Ventana Medical Systems, Tucson, AZ, USA) with antibodies against p53 (clone DO-7, catalogue number 7800-2912,Ventana Medical Systems, Tucson, AZ, USA), ER (clone SP1, catalogue number 790-4324, Ventana Medical Systems, Tucson, AZ, USA), PgR (clone 1E2, catalogue number 790-2223, Ventana Medical Systems, Tucson, AZ, USA), and the MMR status, evaluating the protein expression of MLH1 (clone M1, catalogue number 760-5091, Ventana Medical Systems, Tucson, AZ, USA), PMS2 (clone EPR3947, catalogue number 760-5094, Ventana Medical Systems, Tucson, AZ, USA), MSH2 (clone G219-1129, catalogue number 760-5093, Ventana Medical Systems, Tucson, AZ, USA), MSH6 (clone 44, catalogue number 760-5092, Ventana Medical Systems, Tucson, AZ, USA). Appropriate positive controls were included for each IHC run.

Formalin-fixed, paraffin-embedded tumors were stained for MLH1, MSH2, MSH6, and PMS2 proteins. Mismatch repair protein loss is defined as the absence of nuclear staining in neoplastic cells but positive nuclear staining in lymphocytes and normal adjacent colonic epithelium (16 (link)). Primary monoclonal antibodies against MLH1 (clone M1, Ventana, prediluted), MSH2 (clone G219-1129, Ventana, prediluted), MSH6 (clone 44, Ventana, prediluted), and PMS2 (clone EPR3947, Ventana, prediluted) were applied.