Vorkommen und Bedeutung von Mutationen im NT5C2-Gen in der rezidivierten T-ALL des Erwachsenen

Wolf, Sebastian

Introduction: Relapse specific focal mutations in the cytosolic 5’-nucleo- tidase II gene (NT5C2) were shown to be associated with chemoresistance to purine analogues (e.g. 6-mercaptopurine) in childhood acute lymph- oblastic leukemia (ALL), in 10% of B-precursor ALL and 19% of T-ALL. Patients with acquired mutations in NT5C2, on average, relapse early typically during the phase of maintenance therapy, where 6-mercaptopurine is assumed to have a predominant role in treatment. Relapse specific mutations occur, if ever, only low frequent at diagnosis supporting the specific association of NT5C2 mutations with relapsed disease. Hotspot mutations (non-synonymous) in NT5C2 were determined in exon 9 (R238W) and exon 13 (R367Q). To date, no data are available in terms of activating NT5C2 mutations in relapsed adult T-ALL. Methods: We investigated DNA of bone marrow specimens from 60 adult patients (18–81 years of age) with relapsed/refractory T-ALL after being treated within the German Multicenter ALL Study Group (GMALL) study protocols and who subsequently received Nelarabine as salvage treatment. Matched samples of diagnosis and relapse were analysed. Sanger Sequencing of exon 9, 13 and 15 and amplicon-based targeted next generation sequencing (NGS) of all exonic regions in NT5C2 was performed (Illumina MiSeq) to screen for NT5C2 mutations. Furthermore analysis of the most common hotspot mutations in exon 9 (R238W) and 13 (R367Q) were done using allele-specific PCR in an extended cohort of 255 relapsed adult T-ALL. Positive controls, har- boring R238W and R367Q mutations, were used to analyse the reliability and sensitivity of the NGS and allele-specific PCR for NT5C2 mutation identification. Results: Expected hotspot variants R238W and R367Q in NT5C2 were detected by NGS in serial dilutions of the positive controls up to a sensitiv- ity of 2%. No NT5C2 mutations were identified with Sanger Sequencing in the matched diagnostic and relapse samples of 60 adult T-ALL patients. NGS has found 1 relapse sample being positive for R367Q. Allele-specific PCR identified 4 sample being positive for R367Q in the extended panel of 255 relapse T-ALL samples. Discussion: In contrast to childhood T-ALL, relapse-specific NT5C2 mutations are rare in adults, indicating potential differences in biological pathways between both groups. Resistance to 6- mercaptopurine may not be a crucial factor contributing to relapse in adult T-ALL, and NT5C2 mu- tational events during treatment in adult T-ALL seemingly do not drive the progression of T-ALL from primary disease to relapse.

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Wolf, Sebastian: Vorkommen und Bedeutung von Mutationen im NT5C2-Gen in der rezidivierten T-ALL des Erwachsenen. 2019.

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