Characterization of p53 Abnormalities in CLL Patients in Relation to IGVH Mutation Status and Previous Treatment.

Background: Although the defects in the p53 gene predispose CLL patients for inferior outcome, little is known about the reasons leading to inactivation of this tumor-suppressor. The p53 abnormalities were reported to be associated with unmutated IgVH subtype and may thus arise as a consequence of its more aggressive behaviour, but some reports point also to potentially damaging chemotherapy including alkylating agents. Aims: The aims of the study were to determine the spectrum of p53 mutations in CLL patients treated or monitored at our center and to correlate the data to mutation status of IgVH and to the previous treatment. Methods: We analyzed the status of the p53 gene in 144 patients diagnosed with CLL of all stages using functional analysis in yeasts (FASAY) supplemented by Western-blotting detection of p53 protein expression and I-FISH detection of p53 deletions. We used PCR and direct sequencing to analyze the IgVH rearrangements and mutation status. Results: Our comprehensive approach for monitoring of p53 abnormalities provided us the overall frequency of inactivation within the expected range (14%; usually reported between 10–15%). We noticed a very good overlap between the mutation of one and deletion of the second allele (93%) and between the mutation and corresponding p53 protein over-expression (92%). All the identified mutations in the p53 gene were unambigously determined by direct sequencing from yeast colonies harboring mutated phenotype. We did not find any p53 mutation (n=15) more than once and thus we do not see in our population neither the hot-spots published for CLL in p53 mutation database IARC (codons 248 and 273) nor the hot-spots reported in literature (codons 209 and 143). Inactivation of p53 ocurred markedly more frequently in subtype with unmutated IgVH compared to mutated one (16.7% vs. 5.3%). Moreover, two of the three IgVH mutated cases harbored just p53 deletion without accompanying mutation, slightly over background level. We noticed a very low frequency of the immunoglobulin gene VH3-21 (4.3% in our study vs. 11.2% reported by Tobin et al., Blood2002; 99: 2262–2264), which was recently reported to be mostly mutated and to be associated with p53 inactivation. Four of the six our cases manifested unmutated IgVH, with two of them showing deletions of p53 or ATM (p53-regulatory kinase), respectively. The overall treatment, which included alkylating agents in 43/48 cases, was markedly more frequent within the subgroup with germ-line IgVH (in 49% of patients) compared to group harboring mutated IgVH (in 7% of patients - none of them with p53 inactivation). In the former subgroup the p53 inactivation in the untreated and treated patients was very similar (17% vs. 20%). Conclusions: The spectrum of p53 mutations in our population is different from those reported in other studies. The inactivation of the p53 was in our study associated with unmutated IgVH locus and does not seem to be primarily the consequence of previous chemotherapy including alkylating agents. Supported by grants NR8445-3/2005, NR8443-3/2005 and NR8448-3/2005 provided by IGA of Ministry of Health of the Czech Republic.