In the validation phase, rectal cancer patients with stage III/IV showed a 4.79-fold change (P=0.006) in ALDH1 expression, 1.70-fold change in E cadherin, 4.84-fold change in vimentin (P=0.02), and 11.76-fold change in Twist expression (P=0.003) with respect to stage II rectal tumor when normalized using GAPDH and PUM1 reference genes. This evidence concerns the gene TWIST1 and rectal neoplasm.