Anticipating High-Risk Chromosome Clonal Evolution in Chronic Lymphocytic Leukemia: A Next Generation Sequencing and Longitudinal FISH Combined Study

Background: Chronic lymphocytic leukemia (CLL), the most common leukemia of adults in Western countries, is clinically characterized by a variable clinical outcome, ranging from indolent to agressive cases. The acquisition of new 17p and/or 11q chromosomal lesions during the disease course (high-risk chromosomal clonal evolution), detected either by FISH or conventional cytogenetics has been shown to confer an adverse prognosis. In a high proportion of these patients, a concomitant p53 and ATM mutation can be found in the remaining allele. Next Generation Sequencing (NGS) of tumors is now an affordable, rapid and comprehensive technology for detecting somatic coding mutations and its depth-sensitivity enables reliable detection of subclonal mutations not detectable by classic methods. The aim of this study was to assesed whether the presence of a detectable p53 or ATM mutation by NGS might anticipate a high risk chromosomal clonal evolution during the follow up.

Methods: To this end, we performed targeted NGS sequencing of blood samples from 168 CLL patients at diagnosis who did not present a 17p and/or 11q deletion by FISH and did not meet criteria for active treatment. We designed a TruSeq Custom Amplicon panel (TSCA, Illumina) targeting 12 genes recurrently mutated in CLL, including p53 and ATM. In genes with well-defined mutational hotspots only these regions were targeted; otherwise the entire coding sequence of the gene was sequenced. The panel covers a total of 46605 base pairs with 305 amplicons. Libraries prepared from 250 ng DNA were subjected to 250 bp paired-end sequencing. A second FISH was perfomed in the course of the disease if progression data fullfilling criteria for starting therapy was observed or during of the third year of disease follow up otherwise.

Results: With a median age of 71 y.o. (range, 43-95) and a slight male predominance (56%), the median follow up time of our cohort was 43 months (24-104). Median absolute lymphocyte count at baseline was 17660/uL (interquartile range, 7300-25250), with a 49% and 33% of ZAP70 and CD38 positive cases, respectively. At baseline, 52% of patients presented a 13q deletion and 13% a trisomy 12.Twenty-eight percent of patients presented, at least, a panel mutation, being NOTCH1 the gene most frequenly mutated (n=14).Thirteen patients (9%) developed a high risk chromosomal clonal evolution during the follow up: 8 patients acquired a 17p deletion and 5 cases a 11q deletion. In eight out of these cases, a p53/ATM mutation could be found in the baseline simple, with a clonal size ranging from 4% to 50% (median=9%). The presence of a p53/ATM mutation was associated with de development od a high-risk chromosomal clonal evolution (p=0.02)

Conclusions: Our study shows, in a clinical setting, that the use of targeted next-generation sequencing technology can anticípate the high-risk chromosomal clonal evolution during the follow up in a subset of patients with chronic lymphocytic leukemia,