Clinical Significance of NT5C2 Mutations in Children with First Relapse of B-Cell Precursor Acute Lymphoblastic Leukemia
Authors: Hof Jana , Szymansky Annabell , von Stackelberg Arend , Eckert Cornelia , Kirschner-Schwabe Renate 
Published: October 2019

Abstract
The ubiquitous cytosolic 5´nucleotidase II (NT5C2) dephosphorylates purine nucleotide monophosphates and has an important role in cellular purine metabolism. Increased levels of nucleotidase activity have been correlated with resistance to nucleoside analog drugs that are commonly used in the treatment of children with acute lymphoblastic leukemia (ALL). Recently, activating mutations of NT5C2 have been identified in relapsed childhood ALL. NT5C2 mutations were present in 20% and 10% of children with relapsed T-cell ALL and with relapsed B-cell precursor ALL, respectively. In vitro studies showed that NT5C2 mutations conferred an increased resistance to purine analog drugs to ALL cell lines. However, the predictive and prognostic value of NT5C2 mutations for response and outcome of patients has remained elusive and systematic studies are warranted.
Therefore, we studied the presence of NT5C2 mutations in 259 children with first relapse of B-cell precursor ALL. The patient cohort was a representative subset the German relapse trial ALL-REZ BFM 2002. NT5C2 exons 9, 13, 15 and 16 were sequenced according to Sanger in leukemic samples taken at diagnosis of first relapse. The clinical significance of NT5C2 mutations was assessed by comparing clinical parameters and survival differences between patients with and without NT5C2 mutation, and by multivariate Cox regression modelling.
We identified NT5C2 mutations in 5.8% (15/259) of patients with first relapse of B-cell precursor ALL. This confirms the overall lower NT5C2 mutation rate in children with relapsed B-cell precursor ALL compared to those with relapsed T-cell ALL. Sixty percent (9/15) of the patients with NT5C2 mutation showed a G to A transition in codon 367 in exon 13. This mutation substitutes arginine with glutamine (p.R367Q) and represents a mutation hot spot in relapsed T-cell ALL. Our study reveals that p.R367Q is the predominant site of mutation also in relapsed ALL of B-cell precursor lineage. Patients with NT5C2 mutation significantly more often presented with a very early relapse within 18 months after initial diagnosis (P<0.001) compared to patients with wildtype NT5C2. Likewise, the median time of first remission was significantly shorter in patients with NT5C2 mutation (1.5 years versus 2.83 years, P<0.001). Regarding outcome after relapse treatment, patients with NT5C2 mutation showed a significantly reduced event-free (0.143±0.094 vs. 0.483±0.032; P=0.003) and overall survival rate (0.284±0.121 vs. 0.569±0.033; P=0.007) compared to patients with wildtype NT5C2. The predominant second event in relapse patients with NT5C2 mutation was the occurrence of a second relapse. Accordingly, the cumulative incidence of second relapse was significantly increased in patients with NT5C2 mutations compared to patients with wildtype NT5C2 (0.643±0.140 vs. 0.302±0.030; P=0.001). Multivariate analysis including time of relapse and site of relapse as established risk stratification factors in relapsed ALL revealed that NT5C2 mutation is an independent predictor for the occurrence of a second relapse (P=0.002). Surprisingly, mutation of NT5C2 was not associated with response to relapse treatment. Response was assessed by histological examination and by PCR-based sensitive detection of minimal residual disease at different time points during treatment. However, patients with NT5C2 mutation showed a similar proportion of responding and non-responding patients than relapsed children with wildtype NT5C2.
We conclude that mutation of NT5C2 can serve as predictor for the occurrence of a second relapse independent of response to relapse treatment in children with relapsed B-cell precursor ALL.

Disclosures
No relevant conflicts of interest to declare.