17p Deletion in CLL: Detailed Analysis of TP53 Mutations, Alternative Mechanisms of p53 Inactivation, Clone Size and Clonal Evolution

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.