Despite the general depletion of somatic mutations at TFBSs in gastrointestinal tumors, several studies have reported an increased mutation rate specifically at CBSs in gastrointestinal tumors15,18,40. Indeed, when we examined all CBS across the genome, we found a 3-fold increased mutation rate at CBSs (11 mutations/Mb) compared to their 1 Kb flanking regions (3.6 mutations/Mb). In addition, the mutation frequencies at CBSs were very different among tumors of different molecular subtypes. The somatic mutation rate was 7.1 and 4.7-fold higher at CBSs compared to flanking regions in CIN and GS tumors, respectively (Fig. 4a–e). There was no enrichment of somatic mutations at CBSs in MSI tumors, likely due to impaired DNA MMR. Surprisingly, EBV tumors, which are not MMR-deficient, only had a modest 1.7-fold increase in mutation load at CBSs. The enrichment of somatic mutations at CBSs is therefore unlikely the result of differential DNA repair alone.Fig. 4Analysis of CBS mutations in different gastric cancer subtypes. a–d Mutation count per tumor around CBSs in the four gastric cancer subtypes. e Elevated mutation rates at CBSs compared to flanking regions. f Somatic substitution patterns within CTCF motifs for hotspot mutations and all mutations, respectively. g The negative log P-values of mutation recurrence of all CBSs evaluated with a CBS-specific background model. CBS hotspots identified in Fig. 3a are highlighted in magenta. The horizontal line marks the Bonferroni adjusted P-value of 0.01