Recurrent ATM and BIRC3 Mutations in Patients with Chronic Lymphocytic Leukemia (CLL) and Deletion 11q22-q23

Deletion of the long arm of chromosome 11 (11q22-q23) is one of the most common chromosome aberrations (∼12%) in CLL and is associated with rapid disease progression and shorter overall survival (OS). The commonly deleted region contains the genes ATM and BIRC3. ATM (chromosome 11q22.3) plays a central role in activating TP53, DNA recombination processes, and cell cycle control. ATMmut were described to occur in ∼35% of CLL patients with 11q deletions. The apoptosis inhibitor BIRC3 is located on chromosome band 11q22.2, 6.0 Mb proximal of ATM. BIRC3mut have recently been described with a mutation frequency of 4% in CLL (Rossi D, Blood 2012).

1. Determine the frequency of deletions and mutations of ATM and BIRC3 in CLL with 11q deletion. 2. Analyze the association of ATM and BIRC3 mutations with ATM and BIRC3 deletions. 3. Characterization of the landscape and prognostic impact of ATM and BIRC3 mutations.

The investigated cohort comprised 60 de novo CLL cases harboring del(11q) identified by chromosome banding analysis. The cohort comprised 47 males and 13 females, median age was 68.9 years. Survival data was available in 38 cases. For sensitive mutation detection of the complete coding region of ATM (3056 amino acids) and BIRC3 (604 amino acids) an amplicon deep-sequencing approach (454 Roche Life Sciences, Branford, CT) was developed. Patients were further characterized for mutation status of TP53 (n=60), SF3B1 (n=59), and IGHV (n=60). Additionally, patients were investigated by interphase FISH for ATM (n=60; MetaSystems, Altlussheim, Germany) and BIRC3 (n=39; BAC RP11–177O8, BlueGnome, Cambridge, UK) deletions.

Based on FISH results, all 60 cases showed an entire gene deletion of ATM. Of those cases investigated for BIRC3del, 34/39 (87.2%) cases showed a deletion, thus 5/39 (12.8%) carried an ATMdel only. Mutational screening of the ATM and BIRC3 genes identified 24 ATMmut in 19/60 (31.7%) patients and 3 BIRC3mut in 3/60 (5.0%) cases. Additional mutations were detected in TP53 (3/58; 5.2%), and the spliceosome machinery gene SF3B1 (10/59, 16.9%). 49/60 (81.7%) cases were IGHV unmutated, whereas 11/60 (18.3%) cases were IGHV mutated.

The majority of ATMmut were found to be missense mutations (n=15) followed by nonsense (n=4), frame-shift (n=4), and in-frame (n=1) variants. In more detail, only one mutation occurred in two distinct patients (p.Ile2888Thr). Most cases showed only one mutation (n=15, 78.9%), whereas 3 cases (15.8%) showed two and one case (5.3%) three mutations. Mutations were spread across the entire coding region (exon 3–59). The median mutational burden as assessed by deep-sequencing read counts was 38.5%, ranging from 4.0–89.5%.

For BIRC3 3 deletions (1–4 bp) were observed resulting in 2 frame-shift and 1 splice-site mutation. The median mutation load was 32.5% (range: 20.0%-92.0%). In addition, two cases with BIRC3mut showed also a BIRC3del (n=1 data not available). Interestingly, all of these 3 cases with BIRC3mut were wild-type in the ATM gene.

To further address the role of ATMmut association analyses with respect to age, gender, white blood cell count, haemoglobin level, and platelet count were performed, however, no correlations were observed. Further, ATMmut were not correlated with IGHV (3/11 vs 16/49, P=1.00), TP53 (0/3 vs 19/55, P=0.544) or SF3B1 (3/10 vs 15/49, P=1.00) mutation status. However, the presence of ATMmut in CLL with del(11q) was associated with shorter OS (76.2% vs 95.0% at 3 years), although this difference was not significant. However, when comparing OS of ATMmut cases to cases without del(11q) (n=1,245) we detected a trend towards a shorter OS (76.2% vs 94.2% at 3 years, P=0.149) in contrast to cases with del(11q), but without ATMmut in comparison to cases with no del(11q) (95.0% vs 94.2%, P=0.731).

1. BIRC3 was mutated in only 5.0%, whereas ATM was mutated in 31.7% of patients with del(11q). 2. No mutation hotspot regions were observed, thus analyses of the whole genes are mandatory. 3. ATMmut were associated with a trend to shorter OS in CLL with del(11q). In comparison to cases without del(11q), cases with ATMmut showed shorter OS, indicating a more relevant role of ATMmut on prognosis versus cases with a del(11q) only. 4. In 38 cases (63.3%) with del(11q) neither ATM nor BIRC3 mutations were detected suggesting that further genomic alterations which are not yet identified play a role in CLL with del(11q).

Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Kienast:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.