INCORPORATION OF NEXT-GENERATION SEQUENCING INTO ATM GENE ANALYSIS IN PATIENTS WITH CLL AND MCL

ABSSUB-5145

Background: ATM kinase plays a key role in p53 activation after dsDNA breaks. Germinal ATM inactivation leads to neurodegenerative syndrome Ataxia telangiectasia and heterozygous mutations to predisposition for breast cancer and lymphoid tumors, particularly chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In CLL, ATM defects (11q- and/or mutation) are at diagnosis the most frequent aberrations (20% of patients) associated with negative prognosis. Regarding MCL, hallmark translocation t(11;14) is commonly accompanied by TP53 mutation and even more frequently by ATM inactivation (40-56% of patients), but information about clinical impact is still limited.

Aims: Our aims were: (1) to introduce next-generation sequencing (NGS) mutation analysis of ATM in CLL and MCL samples, (2) to correlate disclosed mutations with the ATM functional status in CLL.

Methods: The following samples were selected for NGS analysis: (a) 16 CLL samples according to the ATM functional status (determined by functional test reported by us previously) including 15 dysfunctional or borderline samples and one sample with normal ATM function; (b) 14 initial MCL samples that were chosen randomly. Three of the 16 CLL samples had known ATM mutation status and served as controls. Primers for all coding exons (n=62) and adjacent splicing sites were designed for multiplex PCR setting. Some exons had to be divided to obtain amplicons of similar length (350±50 bp). The amplicon library per sample was prepared through 15 multiplex PCR reactions, purification and pooling. Nextera XT kit (Illumina) was used for library tagmentation and normalization and sequencing ran on MiSeq instrument (Illumina). Final data was analyzed by software CLC Genomic Workbench and Annovar. The median coverage was 4025 and cut-off value for variant frequency was set to 5%.

Results: We identified 5 CLL patients with presumably pathogenic ATM mutations. Two of these samples were positive controls, in which NGS confirmed mutations previously identified by Sanger sequencing and moreover detected one additional minor mutation. The third mutated sample harbored frameshift alteration p.2613_2616del (allelic frequency (AF) 83%) and the next one manifested non-synonymous variation p.1570A (AF 33%) together with silent alteration V245V (AF 18%) leading to partially aberrant skipping of exon 10. The last mutated sample exhibited missense and frameshift mutation leading to substitution p.I323K (AF 88%) and p.A1211fs (AF 5%), respectively. All remaining samples were wild-type according to NGS output. Importantly, all three new samples with ATM mutation(s) showed dysfunction in functional test. On the other hand, there were 9 samples with predicted ATM dysfunction that do not harbor any pathogenic alteration in analyzed region. Concerning MCL samples, we identified 6 ATM mutations in 4 patients, namely p.Q2522H (AF 41%), p.Y2833fs (AF 38%), p.Q2730L (AF 32%), p.R2871T (AF 32%), p.E1724G (AF 15%), p.E590fs (AF 7%). In most of samples we detected several common polymorphisms that should not significantly affect ATM stability and function.

Summary/Conclusion: We implemented NGS analysis enabling effective and sensitive ATM mutation detection. ATM dysfunction can be connected with mutations in coding region but possibly with other defects in non-coding regions or in some other part of ATM-p53 pathway as well. Supported by grants NT/13493-4, NT/13519-4, CEITEC CZ.1.05/1.1.00/02.0068 and SuPReMMe CZ.1.07/2.3.00/20.0045.

Keywords: ATM, Chronic lymphocytic leukemia, Mantle cell lymphoma, Mutation analysis

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