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The purpose of this study was to investigate the effect of image preprocessing on radiomic features estimation from computed tomography (CT) imaging of locally advanced rectal cancer (LARC). CT images of 20 patients with LARC were used to estimate 105 radiomic features of 7 classes (shape, first-order, GLCM, GLDM, GLRLM, GLSZM, and NGTDM). Radiomic features were estimated for 6 different isotropic resampling voxel sizes, using 10 interpolation algorithms (at fixed bin width) and 6 different bin widths (at fixed interpolation algorithm). The intraclass correlation coefficient (ICC) and the coefficient of variation (CV) were calculated to assess the variability in radiomic features estimation due to preprocessing. A repeated measures correlation analysis was performed to assess any linear correlation between radiomic feature estimate and resampling voxel size or bin width. Reproducibility of radiomic feature estimate, when assessed through ICC analysis, was nominally excellent (ICC > 0.9) for shape features, good (0.75 < ICC ≤ 0.9) or moderate (0.5 < ICC ≤ 0.75) for first-order features, and moderate or poor (0 ≤ ICC ≤ 0.5) for textural features. A number of radiomic features characterized by good or excellent reproducibility in terms of ICC showed however median CV values greater than 15%. For most textural features, a significant (p < 0.05) correlation between their estimate and resampling voxel size or bin width was found. In CT imaging of patients with LARC, the estimate of textural features, as well as of first-order features to a lesser extent, is appreciably biased by preprocessing. Accordingly, this should be taken into account when planning clinical or research studies, as well as when comparing results from different studies and performing multicenter studies. [ABSTRACT FROM AUTHOR]