MUTATION STATUS OF ATM GENE IN CLL PATIENTS HARBORING DELETION 11Q OR TP53 DEFECT

Sborník

Background. Abnormalities of ATM gene are frequent in chronic lymphocytic leukemia (CLL) patients and represent important predictive and prognostic factor. ATM defects are commonly assessed through monitoring of 11q deletion (11q-) using I-FISH. However, there are two aspects which may hamper setting aside affected patients properly: (i) 11q deletion does not mean ATM inactivation if the other allele remains intact (ii) there are patients who harbor ATM mutation without accompanying 11q-. Mutation analysis of ATM is complicated due to the extreme gene size and lack of preferential (hot-spot) mutations. Nevertheless, knowledge of ATM mutation status should significantly improve patient stratification. Aims. The aims were to establish an efficient and convenient system to detect ATM mutations and assess their frequency in high-risk CLL patients. Methods. We used the following complementary methodologies: (a) resequencing microarray (Affymetrix platform), which was designed to detect 1-nt substitutions (i.e. missense mutations, nonsense mutations, and substitutions in splicing sites) (b) western blotting (WB) to disclose patients with null ATM protein (c) functional testing based on induction of CDKN1A (p21) and BBC3 (PUMA) genes after treatment of CLL cells with fludarabine and doxorubicin in parallel; in case of ATM mutation, the former drug leads to the gene expression induction, while the latter does not. Results. The resequencing on microarray was performed in 107 CLL patients. We detected 16 ATM mutations (13 missense, 2 nonsense, and 1 splicing) in 15 patients (14%). In parallel analysis, 11 out of 107 patients (10%) showed null ATM protein on WB; among these patients, there were six with mutation detected on microarray, two patients with three mutations together identified by direct Sanger sequencing only (two short deletions, one missense), and in remaining three patients a suspect mutation is still under investigation by direct sequencing. In total, there were 20 patients (19%) with demonstrable ATM defect (presence of mutation and/or null protein level). Our third test consisting of the ATM functional assessment (see Methods) indicated mutation in 12 patients; in 9 cases the mutation has already been identified. Not all identified mutations in our study, however, resulted into “ATM dysfunctional status”. Proportions of patients with ATM defect in subgroups divided according to the presence of high-risk genetic features were following: 6 % (3/49) in patients having TP53 mutation and/or deletion 17p; 32 % (13/41) in patients with 11q-; 24% (4/17) in patients without any adverse genetic defect. This observation confirms the previous data showing an exclusivity of ATM and TP53 defects (although not strict) and suggests that the frequency of ATM mutations may not be dramatically different between the subgroups with or without 11q-. Summary and Conclusions. Our data confirms that ATM mutations do not automatically overlap with 11q- in CLL patients and are rare in TP53-defected subgroup. Several complementary methodologies should preferably be used to effectively assess ATM status. The work was supported by grants NS 9858-3, MUNI/A/0784/2011, and CZ.1.07/2.3.00/20.004

Haematologica, 2012; 97(s1):  49