Ultrasensitive detection of NOTCH1 delCT mutations in chronic lymphocytic leukemia by ddPCR reveals high frequency of subclonal mutations and predicts clinical outcome in cases with trisomy 12

Abstract NOTCH1 is recurrently mutated in chronic lymphocytic leukemia (CLL), most commonly as a two base-pair frameshift deletion (c.7541_7542delCT). This mutated allele encodes a truncated form of the receptor (P2514fs*4) lacking the C-terminal PEST degradation domain, and results in increased NOTCH1 signaling duration. NOTCH1 mutation has been associated with poor clinical outcomes in CLL. We sought to validate a highly sensitive and quantitative droplet digital PCR (ddPCR) assay for the NOTCH1 delCT mutation which we anticipated would perform well as compared to Sanger sequencing and allele-specific PCR. Performance characteristics of this assay were tested on 126 samples from an unselected CLL cohort, and a separate cohort of 85 samples from patients with trisomy 12 CLL. The delCT mutation was detected at allele frequencies as low as 0.024% and found 25% of unselected cases and 55% of trisomy 12 cases were positive at the 0.024% detection threshold. Mutational burdens ≥1% were found to be significantly associated with shorter overall survival (OS) in patients with trisomy 12+ disease in multivariate analysis (median OS 9.1 vs. 13 years with hazard ratio of 2.34; p=0.031). Mutational burdens