In our screen, SMGs were enriched in four pathways known to be important in cancer, including the cell cycle, NOTCH signaling, PI3K signaling, and cell adhesion. In addition, analyses of the KEGG pathway revealed overrepresented mutations of hedgehog (Hh) signaling and MAPK signaling, and a high percentage of truncating mutations were observed in chromatin-remodeling genes (Figure 6A). Eleven recurrent mutated genes involving the cell-cycle regulatory pathway were identified, and TP53, CDKN2A, and RB1 (MIM 614041) accounted for 88%, 8%, and 2%, respectively (Table S3). Moreover, frequent truncating mutations were observed in KMT2D and KMT2C (14%), CREBBP and EP300 (13%), and KDM6A (3%). Genes involved in the PI3K-AKT-mTOR pathway were mutated in 29% of 104 tumors, and PIK3CA was the most significantly altered gene (17%). Immunohistochemical analysis confirmed the presence of PIK3CA, AKT1, and GLI1 in tumors (compared with matched normal tissue) in the 104-individual WGS and WES cohorts (Figure 6B). Hence, these data shed light on the essential role of dysregulation of these critical pathways in tumorigenesis of ESCC.