The number of lymph nodes evaluated ranged between 1 and 61 with mean and median of 12 and 11, respectively. MMR status was evaluated by IHC according to MLH1, MSH2 and MSH6 expression . Based on this Lenalidomide order analysis, the TMA included 1031 MMR-proficient tumors and 194 MMR-deficient tumors. Table 1 Characteristics of CRC patient cohort (n=1420)*. Overall survival was defined as primary endpoint. Follow-up data were available for 1379 patients with mean/median and IQR event-free follow-up time of 67.7/68 and 45�C97 months. Immunohistochemistry Standard indirect immunoperoxidase procedures were used for immunohistochemistry (IHC; ABC-Elite, Vector Laboratories, Burlingame, CA). Briefly, slides were dewaxed and rehydrated in distilled water. Endogenous peroxidase activity was blocked using 0.
5% H2O2. Sections were incubated with 10% normal goat serum (DakoCytomation, Carpinteria, CA) for 20 min and incubated with primary antibody at room temperature. Primary antibodies used were specific for MPO (clone 59A5 Novocastra, Newcastle, UK), CD15 (clone Carb-1, Leica Biosystems, Nussloch, Germany), CD16 (clone 2H7, Novocastra), CD68 (clone PG-M1, Dako, Glostrup, Denmark), FOXP3 (clone 236A/E7, Abcam, Cambridge, UK) and CD8 (clone C8/144B, DakoCytomation, Switzerland). Subsequently, sections were incubated with peroxidase-labelled secondary antibody (DakoCytomation) for 30 min at room temperature. For visualization of the antigen, sections were immersed in 3-amino-9-ethylcarbazole plus substrate-chromogen (DakoCytomation) for 30 min, and counterstained with Gill��s hematoxylin.
Evaluation of Immunohistochemistry MPO+ and CD15+ tumor infiltrating cells were counted for each punch (approximately one high power [20x] field) by a trained research fellow [R.D.]. Data were independently validated by two additional investigators [L.To. and C.H.] and a high Spearman correlation coefficient (=0.82) and a highly significant (p<0.0001) correlation between measurements was observed. Evaluation of MLH1, MSH2, MSH6, CD16, CD68, CD8 and FOXP3 specific stainings in the CRC TMA under investigation was published previously , , . Flow Cytometry Analyses Following Institutional Review Board approval (63/07), tissues from surgically removed CRC and adjacent normal mucosa were minced, centrifuged, and resuspended in RPMI 1640 medium supplemented with 5% foetal calf serum, 2 mg/ml collagenase IV, 0.
1 mg/ml hyaluronidase V, and 0.2 mg/ml DNAse I (Sigma Aldrich, Basel, Switzerland). Following a 1 hour digestion, cell suspensions Brefeldin_A were filtered and centrifuged. For phenotypic analysis of surface markers, cells were stained with mAbs for 15 minutes on ice in PBS, washed once with PBS 0.5% FCS, 0.5 M EDTA buffer and fixed in lysis buffer from BD Bioscience (110). Samples were then permeabilized in BD fixation/permeabilization buffer.