Representative tumor areas were selected by experienced pathologists, evaluated in hematoxylin-and-eosin (H&E) sections, and employed for TMA inclusion selection, as previously described [46 (link)]. MMR status was determined using IHC with specific primary antibodies for MLH1 (clone ES05, #IR079), PMS2 (clone EP51, #IR087), MSH2 (clone FE11, #IR085), and MSH6 (clone EP49, #IR086), and with the Envision detection kit (all from Agilent Technologies, Santa Clara, CA, USA). Entire sections from the original tissue block were examined when the case was not assessable in the TMA.
The presence of dMMR was considered when the complete loss of nuclear expression of one or more markers in tumor cells was observed. Normal stromal tissue cells demonstrating nuclear staining served as an internal positive control [47 (link)]. Additionally, MSH3 expression was analyzed with a primary specific antibody (clone RM405, #275928, Abcam, Cambridge, UK).
The presence of dMMR was considered when the complete loss of nuclear expression of one or more markers in tumor cells was observed. Normal stromal tissue cells demonstrating nuclear staining served as an internal positive control [47 (link)]. Additionally, MSH3 expression was analyzed with a primary specific antibody (clone RM405, #275928, Abcam, Cambridge, UK).
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