p53 Inactivation in CLL: Pattern of 110 TP53 Mutations.
Authors: Zenz Thorsten , Trbusek Martin , Smardova Jana , Häbe Sonja , Denzel Tina , Malcikova Jitka , Doubek Michael , Schwarz Jiri , Dohner Hartmut , Stilgenbauer Stephan 
Published: April 2020

Abstract
TP53 mutations have been shown to occur in 5–15% of CLL patients and are associated with a poor response to treatment. Because previous studies have examined small cohorts without detailed molecular characterization, the exact correlation of TP53 mutation pattern, allele status and associated molecular genetics has not been possible. We undertook a large scale mutation analysis of TP53 in two centres and pooled the data to characterize the mutation pattern of TP53 in CLL. Two methods of mutational analysis (DHPLC exons 2–11 / FASAY assay) were used. We found 110 mutations in 106 patients with CLL. As expected, most mutations were located in the DNA binding domain of p53. Transitions were found in 54 of 110 mutations (9/110 at CpG sites), while transversions, small deletions and especially insertions were less common (33/110; 22/110; 1/110 resp.). Cytogenetics by FISH were available for 100/106 patients. Most mutations were accompanied by deletion of the other allele (17p-)(n=67). Nonetheless, TP53 mutations were also found in the good prognostic subgroups with normal karyotype (n=6) and 13q- as the sole abnormality (n=15). 17 patients with TP53 mutations had a mutated VH status vs. 79 with an unmutated VH status. We quite frequently observed a deletion 11q- (16/100), accompanying evenly the abnormalities of one as well as of both TP53 alleles (deletion/mutation). It indicates that this deletion may not be a simple alternative to p53 inactivation, as it has been proposed previously. Chemotherapy before mutation analysis was noted in 43/92 patients and was more frequent in those with aberration of one TP53 allele without reaching statistical significance (64% vs. 51% of previously treated patients bearing both inactivated alleles). The transitions, transversions and small deletions were evenly distributed among both untreated and treated patients. When comparing the predicted functional activity of the mutated TP53 in different cytogenetic subgroups (http://p53.free.fr/Database/p53_recomendations.html) we observed significantly lower residual activity of the missense mutations in the 17p- subgroup compared to the patients with 13q- (single) and a TP53 mutation (median 3,1 vs. 10,4 p=0,037). The codons most frequently mutated were at positions 209, 220, 234, suggesting that the classical hot spots (codons 175, 248, 273) may not be commonly mutated in CLL. The very low number of mutations which were identified at CpG dinucleotides implies a relatively negligible contribution of endogenous mutability at methylated cytosine. This is manifested also by a finding of only two mutations at the very prominent, heavily methylated hot-spot at position 248Arg. We assume that some other mechanisms of mutagenesis may play a relevant role in B-CLL. The current study characterizes the mutational profile of TP53 mutations in CLL. While the classical hot spot mutants were not observed at high frequencies we could identify 3 codons that made up for 12,7% of the mutations. The majority of mutations are accompanied by the inactivation of the other allele (deletion 17p). For the first time, the analysis of a large cohort of TP53 mutations will allow to investigate potential differences in the clinical course of monoallelic vs. biallelic aberrations of TP53.