Droplet digital PCR analysis of NOTCH1 gene mutations in chronic lymphocytic leukemia

Abstract

In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1^mut) have been associated with adverse prognostic features but the independence of these as a prognostic factor is still controversial. In our study we validated a c.7541-7542delCT NOTCH1 mutation assay based on droplet digital PCR (ddPCR); we also analyzed the NOTCH1^mut allelic burden, expressed as fractional abundance (FA), in 88 CLL patients at diagnosis to assess its prognostic role and made a longitudinal ddPCR analysis in 10 cases harboring NOTCH1^mut to verify the FA variation over time. Our data revealed that with the ddPCR approach the incidence of NOTCH1^mut in CLL was much higher (53.4%) than expected. However, longitudinal ddPCR analysis of CLL cases showed a statistically significant reduction of the NOTCH1^mut FA detected at diagnosis after treatment (median FA 11.67 % vs 0.09 %, respectively, p = 0.01); the same difference, in terms of NOTCH1^mut FA, was observed in the relapsed cases compared to the NOTCH1^mut allelic fraction observed in patients in complete or partial remission (median FA 4.75% vs 0.43%, respectively, p = 0.007). Our study demonstrated a much higher incidence of NOTCH1^mut in CLL than has previously been reported, and showed that the NOTCH1^mut allelic burden evaluation by ddPCR might identify patients in need of a closer clinical follow-up during the “watch and wait” interval and after standard chemotherapy.