Clonal Evolution and Therapy Related Selection Of TP53 Mutations In Chronic Lymphocytic Leukemia Patients
Authors: Plevova Karla , Malcikova Jitka , Trbusek Martin , Pavlova Sarka , Sebejova Ludmila , Tichy Boris , Doubek Michael , Brychtova Yvona , Mayer Jiri , Pospisilova Sarka 
Published: October 2019

Abstract
Recent studies of clonal evolution in chronic lymphocytic leukemia (CLL) show that new copy number alterations (CNAs) emerge in disease relapse in about 30% of cases. Furthermore, selection of TP53 mutations (mut-TP53) was recognized as a therapy related event in approximately one fifth of treated CLL patients and was associated with higher disease aggressiveness and inferior clinical outcome. However genomic abnormalities accompanying mut-TP53 selection have not been described so far. Therefore, we analyzed clonal evolution of leukemic cells derived from patients who acquired a new TP53 mutation after therapy. TP53 analysis was performed in consecutive samples by FASAY and direct sequencing. Genomic DNA was isolated from separated CD19+ or mononuclear cells and analyzed using Affymetrix arrays (Cytogenetics 2.7M, or CytoScan High Density). Array results were compared with I-FISH and metaphase cytogenetics based on CpG/IL-2 stimulation. We identified 38 CLL patients with intact TP53 gene before therapy but with newly acquired mut-TP53 during disease relapse. Out of this specific cohort, 18 cases (47%; Cohort I) with available pre-treatment and relapse material were selected for the microarray analysis of clonal and subclonal CNAs and copy number neutral loss of heterozygosity (cnLOH). Median interval between collection of the pre-treatment and relapsed samples was 39 months (range 14-81 months), patients received 1-4 lines of therapy in the meantime (median 2 lines). In addition, a control group of 13 chemorefractory patients was analyzed in the same manner: 8 harbored mut-TP53 from diagnosis (Cohort II) and 5 manifested wild-type TP53 (wt-TP53) during the whole follow-up (Cohort III) (median interval 19 vs. 60 months; median number of therapies 2 vs. 3 in the meantime). Following recurrent aberrations were detected in initial samples from Cohort I: gains in 2p (2 pts), deletions in 11q22 (5 pts), 13q14 (9 pts), 14q24 (2 pts), and 17p13 (2 pts), and trisomy 12 (2 pts). Interestingly, no difference in initial genomic complexity (defined by ≥3 CNAs in the first sample) was observed between Cohorts I and III, i.e., the patients with selection of mut-TP53 and control wt-TP53 cases (12/18; 67% vs. 4/5; 80%). This observation may suggest that factors other than genomic complexity predispose to selection of new TP53 mutations. Concerning the relapsed samples, patients with mut-TP53 selection acquired on average the highest number of additional chromosomal defects per case [5.5 changes per patient vs. 3 changes in Cohort III (always wt-TP53) vs. 2.5 changes in Cohort II (always mut-TP53)]. In 10/18 (56%) patients from Cohort I, mut-TP53 selection was accompanied by inactivation of the other allele through either deletion (9 pts) or cnLOH (1 pt). Further, the heterozygous mutation accompanied by intact second allele was selected in 6/18 patients (33%), and in the remaining 2 patients (11%), deletion of the second allele was already present in initial sample. Regarding the other aberrations accompanying mut-TP53 appearance, we observed co-selection of monoallelic del(13q) in 4/9 initially 13q-intact cases and evolution from mono- to biallelic del(13q) in 1/9 initially 13q-monoallelic cases. This observation seems to be in contrast with the published data showing a relative stability of del13q14, or even displacement of the corresponding clones with this deletion in samples having high genomic complexity. In our cohort, we observed del(13q) elimination in disease relapse in one case only. In both control cohorts, no evolution of 13q14 locus was detected. Furthermore, complex deletions evolved on chromosome 6 in 3/18 (17%) patients in Cohort I. The region was also affected in Cohort II (always mut-TP53) but not in Cohort III (always wt-TP53), which suggests that emergence of CNAs on chromosome 6 might be associated with TP53 inactivation in general. Other recurrent CNAs emerging in patients with mut-TP53 selection were amp(2p), del(4p), del(9q21) and del(18p); each detected in 3/18 cases. To summarize, selection of TP53 defects during CLL relapse seems to be accompanied by accumulation of specific CNAs, mainly deletions. We assume that these CNAs may be a consequence of genomic instability generally associated with TP53 defects, but these CNAs may potentially also drive disease aggressiveness. Supported by CZ.1.05/1.1.00/02.0068, MSM0021622430, MUNI/A/0723/2012, NT13493-4/2012, NT13519-4/2012.

Disclosures:
No relevant conflicts of interest to declare.