Consistent with findings by Katainen et al. in colorectal cancer15, we found that somatic mutations at CTCF motifs, including the CBS hotspot mutations, were predominately A.T>C.G and A.T>G.C substitutions (Fig. 4f), suggesting that hotspot mutations are generated by the same mutational process as other CBS mutations. The mutation pattern at CBS hotspots was overall similar to that of all CBSs. However, while a conserved base at position 9 of the 19 bp CTCF binding motif was the most commonly mutated position at CBSs in general, the CBS hotspot mutations had the highest enrichment in the 4 bp sequence flanking the 5′ end of the CTCF motif. Furthermore, C>T changes, which are relatively common among all CBS mutations are much rarer among the CBS hotspot mutations (Fisher’s exact test P-value = 4.4 × 10−7). These differences could indicate a functional difference between CBS hotspot and non-hotspot mutations.