TP53 Abnormalities in Chronic Lymphocytic Leukemia Exhibit a Disease Specific Profile: Meta-Analysis of 270 Mutations.

TP53 mutations have been shown to occur in 5–40% of CLL patients depending on the clinical background (early stage/refractory disease). They are associated with a poor response to standard chemotherapy with alkylators and purine analogues and dismal clinical course. Because previous studies have examined small cohorts without detailed molecular characterization, the exact TP53 mutation profile and a correlation of TP53 mutation pattern, allele status and associated molecular genetics has not been possible.

We performed a large scale mutation analysis of TP53 at four centres and pooled the data to characterize the pattern of TP53 mutations in CLL. The methods of mutational analysis included DHPLC (n=149 mutations), FASAY assay (n=63), direct sequencing (n=28) and a TP53 custom re-sequencing chip (n=30). We performed a comparison to a group of 463 patients without TP53 mutation.

We found 270 mutations in 256 patients with CLL (14 with 2 mutations) out of over 1000 screened cases. Transversions, small deletions and insertions were observed less commonly (86/270; 44/270; 9/270 resp.). Missense mutations appeared in 74% of cases, compared to frameshift (20%), nonsense (4%) and splice site (2%) mutations. As expected, the majority (246/270) of mutations were located in the DNA binding domain of p53, predominantly represented by missense mutations (80%). Outside this central core domain, frame-shift (52%) and nonsense mutations (26%) were more frequent. Transitions were found in 131 of 270 mutations, with only 41 occurring at methylated CpG sites (15%). Interestingly, the number of G-A mutations was markedly higher in comparison with C-T mutations at the CpGs (27 vs. 14). Since the former alteration may reflect a C-T transition on non-coding, transcribed DNA strand, we hypothesize that these mutations might be selected for during a prolonged G1-phase, which is characteristic for CLL cells.

Genomic aberrations detected by FISH were available for 261/270 cases. Most mutations (201/270) were accompanied by deletion of the other allele (17p-). Nonetheless, TP53 mutations were also found in cases with no aberrations (n=18) and 13q- as the sole abnormality (n=24). There appeared to be a higher frequency of frameshift mutations in the 17p- subgroup (22% vs. 13%) although the comparison did not reach statistical significance. Interestingly, trisomy 12 (without 17p- or 11q-) was only found in a single case with TP53 mutation compared to 54/463 in the cohort without mutation (P<0.0001). We quite frequently observed a deletion 11q- (36/270) accompanying the abnormalities of one (14/60) as well as of both (22/201) TP53 alleles (mutation/deletion). 11q deletion may not be a simple alternative to p53 inactivation, as it has been proposed previously.

Chemotherapy before mutation analysis was noted in 106 cases. The mutational profile was not different in the cohorts with and without prior therapy suggesting that the mechanism underlying the development of mutations may be similar independent of treatment.

When comparing the predicted functional activity of the mutated TP53 in different cytogenetic subgroups (http://p53.free.fr/Database/p53_recomendations.html) we observed a significantly lower residual activity (WAF1 promotor) of the missense mutations in the 17p- subgroup compared to the patients with 13q- (single) and a TP53 mutation (median 6,815 vs. 10,31 p<0,0001).

Seventy percent of missense mutations (140/201) were located in 30 different codons. The amino acids most frequently mutated were at positions 179, 209, 248 and 273. This indicates that the classical hot spots are also commonly mutated in CLL. Codons 175, 248, and 273 made up for 11% of the mutations, but we identified three other commonly mutated codons (179, 209, 220) that also made up for 10% of the mutations in CLL.

The mutation pattern of TP53 shows a comparatively small amount of transitions at CpG sites indicating a relatively negligible contribution of endogenous mutability at methylated cytosine. In addition, CLL is characterized by a high incidence of deleterious frame-shift mutations compared to other cancers. The high frequency of mutations at codon 209 in our cohort suggests this as a new hot spot of TP53 abnormalities associated with CLL.