Tumors were categorized into molecular subgroups according to a modified TransPORTEC classifier that recapitulates the four subgroups of the TCGA as follows: (1) mismatch repair deficient (MMR-D, surrogate to microsatellite instability hypermutated in the TCGA classification system); (2) p53 abnormal (p53 abn, surrogate to copy-number high); (3) polymerase-ϵ (POLE) ultramutated; and (4) “no specific molecular profile” (NSMP, surrogate to copy-number low) [29,30]. A tissue microarray was constructed on primary tumor samples as previously described [26]. The following monoclonal antibodies were used for chromogenic immunohistochemistry: MLH1 (ES05, Dako, Santa Clara, CA, USA); MSH2 (G219-1129, BD Biosciences, San Jose, CA, USA); MSH6 (EPR3945, Abcam, Cambridge, UK); PMS2 (EPR3947, Epitomics, Burlingame, CA, USA); p53 (DO-7, Dako); and L1CAM antibody clone 14.10 (SIG-3911, Covance, Princeton, NJ, USA). Tissue microarray slides were scanned with a three-dimensional Histech Pannoramic 250 Flash II scanner by Fimmic Oy (Helsinki, Finland). Slide images were managed and analyzed with WebMicroscope Software (Fimmic Oy). Virtual slides were scored by a pathologist blinded to clinical data. Equivocal cases were examined by a second investigator and a consensus was reached. MMR status was considered deficient when a complete loss of nuclear expression in carcinoma cells of one or more MMR proteins (MLH1, MSH2, MSH6, PMS2) was detected by immunohistochemistry. Aberrant p53 staining was defined as strong and diffuse nuclear staining or completely negative (“null”) staining in carcinoma cells. Weak and heterogeneous staining was classified as wild-type expression. Stromal and inflammatory cells served as internal controls for MMR and p53 staining. A membranous staining of ≥10% was considered positive for L1CAM expression [26]. POLE exonuclease domain mutation (EDM) screening of hot spots in exons 9, 13, and 14 was performed by direct sequencing [31]. Only samples with high-quality sequence for all four examined POLE hot spots were included in the study.

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