ATM Gene Alterations in CLL Patients Induce Gene Profile Clusters and Predict Disease Progression.

Chronic lymphocytic leukemia (CLL) presents a highly variable clinical course, with some patients surviving for many years without requiring treatment, while others have rapidly progressing disease, require aggressive treatment and show a short life expectancy. Several biological features of the leukemic cells - the mutational status of the immunoglobulin (Ig) heavy (H) chain variable (V) genes, cytogenetic alterations, ZAP-70 and CD38 antigen expression, p53 dysfunctions – bear prognostic value and allow to stratify patients into risk categories. The deletion of the chromosome region 11q22–q23 that occurs in 10–20% of cases represents the second most common genetic abnormality in CLL and defines a subgroup of patients characterized by advanced progressive disease and poor survival. The ATM (ataxia telangiectasia-mutated) gene is located in this region and several studies suggest that the 11q deletion results in ATM gene inactivation. The ATM gene consists of 66 exons, 62 (5–66) of which are coding exons. The ATM protein is a nuclear serine/threonine kinase of 350 kDa induced by chromosomal double-strand breaks that arise endogenously or after exposure to DNA-damaging agents, including ionizing radiations and drugs. It protects the integrity of the genome in several ways: it regulates the arrest of the cell cycle at the G1/S and G2/M phases to prevent the processing of damaged DNA; it activates DNA repair pathways and induces apoptosis if the DNA damage cannot be repaired. In the present study we examined the status of the ATM gene by DHPLC (Denaturing High Performance Liquid Cromatography) in a series of untreated CLL patients studied at the Division of Hematology, “Sapienza” University of Rome. In addition, a multiplex gene dosage analysis of the ATM gene was performed by MLPA (Multiplex Ligation Probe Amplification) in all patients. The results obtained have been correlated with the molecular status of the IgV_H genes, the presence of cytogenetic abnormalities, ZAP-70 and CD38 expression, p53 mutations, and with the gene expression profile. Mutational screening of the ATM gene identified mutations in 8 of the 57 CLL patients studied (14%): 6 missense, 8095C>T (Pro2699Ser), 8071C>T (Arg2691Cys), 2476A>C (Ile826Leu) and, in 3 patients, 1435G>T (Asp479Tyr); 1 frameshift: 2502insA; 1 splicing mutation: IVS29+5G>A. In addition, 14 different variants and polymorphisms of unknown functional significance were found. The 8 ATM mutated cases showed the following features: 6 were IgVH unmutated, 4 were ZAP70+, 5 were CD38+, 4 harbored a 13q14 deletion, only 1 patient showed a 17p13 deletion and 2 a 14q32 deletion; furthermore, 2/8 patients showed a p53 gene mutation; none showed the 11q deletion. MLPA analysis identified 6 patients with 11q deletion and all of them showed at least 20% of cells positive for the 11q23 deletion evaluated by FISH; only 1 case showed a 17p13 deletion. In these cases, the IgVH gene was always unmutated and ZAP70 was positive; 4 cases were also CD38+. ATM gene mutated and deleted CLL cases showed a peculiar gene profile characterized by the up-regulation and down-regulation of genes involved in apoptosis and DNA repair function. On the contrary, no gene profile difference was found in patients carrying ATM gene polymorphisms. Patients with ATM gene mutated and deleted showed a short time to treatment, 23 and 19 months respectively. The results of this study indicate that ATM gene alterations - mutations or deletions - account for 24% of untreated CLL patients, are associated with a defined gene profile and are characterized by a poor prognosis. It is important to underline that these patients have been evaluated before any treatment and that ATM mutations have been found in the absence of 11q23 deletions. ATM alterations appear to represent the most frequent genetic abnormality with unfavorable prognostic implications in CLL and should be investigated prior to initiating therapy in order to plan an appropriate treatment algorithm.