Abstract
In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1mut) 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 NOTCH1mut 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 NOTCH1mut to verify the FA variation over time. Our data revealed that with the ddPCR approach the incidence of NOTCH1mut in CLL was much higher (53.4%) than expected. However, longitudinal ddPCR analysis of CLL cases showed a statistically significant reduction of the NOTCH1mut FA detected at diagnosis after treatment (median FA 11.67 % vs 0.09 %, respectively, p = 0.01); the same difference, in terms of NOTCH1mut FA, was observed in the relapsed cases compared to the NOTCH1mut 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 NOTCH1mut in CLL than has previously been reported, and showed that the NOTCH1mut 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.
Highlights
Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with highly variable clinical manifestations, ranging from asymptomatic at the time of diagnosis to a progressive symptomatic disease that is poorly responsive to the common immunochemotherapeutic regimens [1,2]
NOTCH1mut were detected by NGS in 11% of monoclonal B cell lymphocytosis (MBL) and 13.4% of chronic lymphocytic leukemia (CLL) Binet stage A patients [27]; this mutation was frequently observed at a low clonal level, in MBL patients, and sequential analyses demonstrated that the www.impactjournals.com/oncotarget
Our data confirmed the association of NOTCH1mut with IGHVunmutated CLL [6, 15, 23,24]; our finding that among all the CLL patients with NOTCH1mut those with IGHV-unmutated had the higher mutated allelic burden seems worthy of note
Summary
Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with highly variable clinical manifestations, ranging from asymptomatic at the time of diagnosis to a progressive symptomatic disease that is poorly responsive to the common immunochemotherapeutic regimens [1,2]. Apart from generation (NGS) and Sanger sequencing analysis, the presence of c.7541-7542delCT NOTCH1 mutations (NOTCH1mut) can be investigated by amplification www.impactjournals.com/oncotarget refractory mutation system PCR, as previously reported [6, 9, 13, 15]. All of these methods of investigation, with the exception of NGS, provide only qualitative and not quantitative information. We validated a c.7541-7542delCT NOTCH1 specific mutation assay based on digital droplet PCR (ddPCR) technology. The aims of our study were to analyze: a) a c.7541-7542delCT NOTCH1 mutation assay based on ddPCR; b) the NOTCH1mut allelic burden in CLL patients at diagnosis to assess its prognostic role; c) longitudinal ddPCR in a few cases harboring NOTCH1mut (including patients who required therapy and those who were managed with a “watch and wait” approach)
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