DETAILED MUTATIONAL ANALYSIS OF TP53 GENE REVEALS HIGH INCIDENCE OF ADDITIONAL MINOR PROPORTION MUTATIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

Sborník

Background. Presence of aberrations in the tumor suppressor gene TP53 is one of the strongest prognostic markers of chronic lymphocytic leukemia (CLL). Besides 17p deletion the individual TP53 gene mutations were recently associated with unfavorable CLL prognosis. In addition to clonal TP53 mutations, minor mutations may be detected in some CLL patients. Nevertheless, frequency and importance of mutations present in minor cancer clones wasn’t investigated up to now. Aims. We employed sensitive TP53 mutational screening to determine the incidence of minor mutations in CLL patients. Methods. For precise mutational testing the combination of functional analysis (FASAY) and denaturing high-performance liquid chromatography (DHPLC; Varian) in connection with direct sequencing was used. Selected samples were investigated by ultra-deep next generation sequencing (NGS; GS Junior System, Roche). Results. Mutational status of the TP53 gene was assessed in 198 samples from 185 selected CLL patients (12 patients were analyzed repeatedly). We found 96 mutations in 63 patients. Notably, in 15 patients with major clonal mutation 20 minor gene mutations were clearly detected (1-3 mutations per patient). Among them, 10 missense mutations, 7 splicing mutations and 3 frameshift mutations were identified. The detected minor mutations were evaluated by ultra-deep NGS in 7 patients, and presence of all 11 tested mutations was confirmed (mutation proportion 3,24% - 6,62%). Interestingly, the patients with minor TP53 mutations were more frequently pre-treated by chemotherapy in comparison with patients who carried a sole mutation (P = 0.0031). Conclusions. Clonal TP53 mutations are often accompanied with additional minor mutations, particularly in patients after chemotherapy. Our results further support the role of treatment in selection of TP53 mutations in CLL patients. Evaluation of clinical impact of these minor clones requires systematic long-term analysis of large patients’ cohort.This study was supported by research proposal MSM0021622430 and projects CZ.1.05/1.1.00/02.0068, CZ.1.07/2.3.00/ 20.0045, CZ.1.07/2.4.00/17.0042, MUNI/A/0784/2011.

Haematologica, 2012; 97(s1):  55