Fig 1.

(A) NOTCH1 mutations in patients with adenoid cystic carcinomas (ACCs) occurred predominantly in the same negative regulatory region and Pro-Glu-Ser-Thr–rich domain (PEST) hotspots as those observed in T-cell acute lymphoblastic leukemia and are predicted to be activating. (B) Notch1 intracellular domain (NICD) immunostaining in ACC. (Bi) Positive uniform nuclear expression of NICD in solid form of ACC. (Bii) ACC negative for NICD expression. (C) In vitro reporter assay assessing Notch1 pathway activation induced by individual mutations and the combination of both mutations observed in an index patient. 293T cells were cotransfected with NOTCH1-wild-type (WT) or NOTCH1-mutant (mut) constructs and HES1AB-responsive luciferase reporter, HES1AB-Δ luciferase mutant form, or Renilla luciferase control. Firefly/Renilla luciferase activity was measured in cell lysates after 48 hours. The NOTCH1 S2467fs* and L1600Q comutations led to a statistically significant 2.2-fold increase in reporter activity compared with wild-type NOTCH1. ANK, ankyrin repeat domain; DM, double mutation; LNR, Lin12/NOTCH repeats; M1, NOTCH1 S2467fs* mutation; M2, NOTCH1 L1600Q mutation; TM, transmembrane domain. (†) P < .001.