5'Nucleotidase (NT5C2) Genotype Influences Leukemic Blast Concentration of Ara-CTP in Pediatric Patients with Acute Myeloid Leukemia.

Cytarabine (ara-C) is one of the most widely used chemotherapeutic agents in the treatment of acute myeloid leukemia (AML). Inter-patient variability in clinical response following ara-C treatment has been correlated with leukemic cell concentration of ara-C triphosphate (ara-CTP), the active metabolite of the drug. NT5C2 is an inactivating enzyme in the activation pathway of ara-C, and catalyzes conversion of ara-C monophosphate to ara-C. We identified genetic variations in NT5C2 and investigated the association of NT5C2 genetic variations with its mRNA expression and with intracellular ara-CTP accumulation. The coding exons of NT5C2 were resequenced using genomic DNA from EBV-transformed B-lymphoblastoid HapMap cell lines derived from 90 CEPH (representing 30 trios with European ancestry) and 90 YRI (30 trios with African ancestry) sample. Thirty-nine genetic variants (one insertion-deletion and 38 SNPs), including three nonsynonymous SNPs (Thr3Ala, Lys47Arg, Gln136Arg), were identified. The strongly linked SNPs (R² > 0.8) in CEPH and YRI were grouped into ten distinct groups. NT5C2 mRNA expression levels were determined in all samples and was significantly associated with ethnicity (p = 8.5 × 10^-6), with subjects of African ancestry having significantly higher NT5C2 mRNA expression as compared to the subjects with European ancestry (Figure A). Several NT5C2 SNPs were associated with its mRNA expression in both the ethnic groups.

View largeDownload slide

View largeDownload slide

Close modal

To determine the clinical implication of NT5C2 SNPs, we investigated selected germline NT5C2 SNPs in pediatric AML patients treated on the St Jude AML 97 protocol (n=55). Patients were randomly assigned to receive ara-C as either a short daily infusion (500 mg/m²/dose intravenously over 2 hrs daily for 5 days) or a continuous infusion (500 mg/m²/day as a continuous infusion over 5 days). Bone marrow was collected at the end of the ara-C infusion on day1 for patients receiving the short daily infusion (n=27), and at 10 hrs after the start of the infusion for those receiving the continuous infusion (n=28). Ara-CTP levels in leukemia cells were analyzed by HPLC. Intracellular accumulation of ara-CTP was significantly higher when given as short daily infusion, as compared to continuous infusion (0.56 ±0.5 vs. 0.33± 0.31 nmol ara-CTP/2×10⁷leukemic cells; p = 0.014). The inter-patient variability for blast ara-CTP concentration was 40-fold in the short infusion arm and 101-fold in continuous infusion arm. Karyotypes, gender, age, and ethnicity had no significant influence on the leukemic ara-CTP levels. NT5C2 Group 1 SNPs were found to be significantly associated with intracellular ara-CTP levels in leukemic blasts from ara-C-treated Pediatric AML Patients (Figure B). These results suggest that genetic variation in NT5C2 could influence its activity and/or expression and may predict the variability observed in intracellular levels of the ara-C active metabolite, ara-CTP, which in turn can influence treatment response.

No relevant conflicts of interest to declare.