The TP53 mutation profile in chronic lymphocytic leukemia (CLL) and the correlation of TP53 mutations with allele status or associated molecular genetics are currently unknown. We performed a large mutation analysis of TP53 at four centers and characterized the pattern of TP53 mutations in CLL. We report on 268 mutations in 254 patients with CLL. Missense mutations appeared in 74% of cases compared with deletions and insertions (20%), nonsense (4%) and splice site (2%) mutations. The majority (243 of 268) of mutations were located in the DNA-binding domain. Transitions were found in 131 of 268 mutations, with only 41 occurring at methylated CpG sites (15%), suggesting that transitions at CpGs are uncommon. The codons most frequently mutated were at positions 175, 179, 248 and 273; in addition, we detected a common 2-nt deletion in the codon 209. Most mutations (199 of 259) were accompanied by deletion of the other allele (17p-). Interestingly, trisomy 12 (without 17p-) was only found in one of 60 cases with TP53 mutation (without 17p-) compared with 60 of 16 in the cohort without mutation (P ¼ 0.006). The mutational profile was not different in the cohorts with and without previous therapy, suggesting that the mechanism underlying the development of mutations may be similar, independent of treatment.

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.

The prognosis of CLL with 17p deletion is very poor. While it is generally accepted that inactivation of p53 (by mutation) underlies refractoriness of CLL with 17p deletion, no study has analyzed a large cohort of CLL patients with 17p deletion with respect to TP53 mutations and investigated other mechanisms of p53 inactivation. In order to assess the incidence of TP53 mutations in CLL with 17p deletion we identified a large cohort of CLL cases with 17p deletion (n=217) and studied TP53 mutations in 124 of these patients. We used DHPLC to screen for TP53 mutations (Exons 2–11). Aberrant DHPLC profiles lead to sequencing of the respective exons. A sub-group of cases were also studied by direct sequencing and with an array based TP53 mutation platform (AmpliChip Roche molecular systems) to confirm the absence of mutations. In addition, detailed genetic studies (VH-mutation status, ZAP70, FISH) were available for the patients. The median size of the 17p- clone was 73% (10–97%). In addition to the 17p-, 13q- was observed in 99 cases (46%), but 11q-, +12, 6q-, and +8q were rarely seen (8.8%, 11.5%, 5.1%, and 3.7%, resp.). Most cases with 17p deletion had an unmutated VH status (126/168 (75%)). We found mutations in the protein coding region of TP53 in 100 of 124 CLL patients (81%) with 17p deletion. Only few cases had more than one mutation (4/100). The majority of mutations were located in the DNA binding domain of p53. We found no mutations in exons 2, 3, and 11. Most mutations were missense mutations (72%), while splice site mutations (5%), nonsense mutations (5%) and frame shift mutations (18%) were less common. In the vast majority of the cases the estimated clone size of the TP53 mutation correlated very closely with the percentage of cells with a single signal (FISH results). The size of the 17p- clone was significantly higher in cases where a mutation was detected (65.5%) compared to cases where a mutation was not found (42.7%; p=0.0003). When we separated the cohort into quartiles based on the 17p- clone size we found increasing proportions of TP53 mutation, suggesting that sensitivity may cause the lower rate of TP53 mutation detection. As further evidence that mutations may also be observed in cases with 17p deletion in a sub-clone, we also observed mutations in cases with less than 20% of cells (5/11) carrying the 17p deletion. The analysis of follow-up samples in a number of these cases with low grade 17p-deletion showed definite evidence for the selection of the p53 deficient clone (mutation and deletion). In spite of this clear evidence for a classical TP53-related tumor suppressor mechanism underlying the resistance to chemotherapy in cases with 17p deletion, there remain cases where no mutation in the exons of TP53 can be detected by DHPLC, direct sequencing, and array-based p53 mutation analysis, suggesting that in these cases alternative mechanisms lead to inactivation of p53. These mechanisms were investigated by p21/p53 FACS and Western blotting in selected cases with a high 17p- clone size. When we assayed the p21/p53 levels after irradiation and in un-irradiated controls, we found different patterns; we observed cases with type A dysfunction (high basal p53 and failure to upregulate p21)(n=2), type B dysfunction (failure to upregulate p21 and p53; n=2), but also normal induction of p53 and p21 (n=1). The current study supports the role of p53 inactivation by TP53 mutation underlying the chemo-resistance of CLL with 17p deletion. The extent of mutations of the remaining allele and the demonstration of coexisting mutations even in cases with deletions in only the minority of cells suggest that p53 is the main biological target of 17p deletion and its clinical consequence.