Subclonal NT5C2 mutations are associated with poor outcomes after relapse of pediatric acute lymphoblastic leukemia

Abstract

Activating mutations in cytosolic 5'-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2 mutations in relapsed ALL, we analyzed NT5C2 in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2 mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2 mutations compared with those without (19% and 25% vs 53%, P < .001). However, subclonal, but not clonal, NT5C2 mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P = .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P < .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2 mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2 mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2 mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2 mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.

Graphical abstract

Figure 1.

NT5C2 mutations in relapsed pediatric BCP-ALL. (A) Schematic representation of the NT5C2 protein/gene. Mutations identified by Sanger sequencing and/or targeted next generation in relapses of 455 patients with BCP-ALL are described at the protein level. Multiple circles in the same amino acid position account for multiple patients with the same variant. NT5C2 exons are indicated by alternating light and dark gray boxes. Variant frequency refers to variant allele frequency for NGS-detected mutations and allelic peak ratio for Sanger-detected mutations. (B) Performance of the ASQ-PCR assays developed for sensitive detection of NT5C2 mutations p.R39Q and p.R367Q. The graphs represent exemplary amplification plots of standard dilution series of NT5C2 mutation-positive patient samples (blue) as well as of a mutation-negative DNA control (red). The distance between the 1E-03 dilution and the mutation-negative DNA control is larger than 3 C_T values for both assays as required by the guidelines for minimal residual disease detection in leukemia.(C) Range of NT5C2-mutant clone frequencies determined by ASQ-PCR for all p.R39Q and p.R367Q mutations. For this graphic, NT5C2-mutant clone frequencies were categorized into different quantitative levels in log₁₀ steps. The bar graphs represent the percentage of subclonal NT5C2 p.R39Q and p.R367Q mutations with a given quantitative level. The absolute number of mutations is displayed within the bar graphs. (D) Classification of relapsed patients with NT5C2 mutations after compilation of sequencing and ASQ-PCR data into 2 main groups: patients with clonal NT5C2 mutation (dark-red) and patients with subclonal NT5C2 mutation(s) only (light red). Approximately one-third of relapses harbored >1 NT5C2 mutation.

Figure 2.

Association of NT5C2 mutations with recurrent genetic alterations in relapsed BCP-ALL. (A) Distribution of recurrent genetic alterations in relapses with wild-type NT5C2 in comparison with relapses with NT5C2 mutation. Bars represent the percentage of relapses with a given genetic alteration within each category. P values below the .05 level of significance are depicted in bold. (B) Distribution of recurrent genetic alterations in relapses with clonal and with subclonal NT5C2 mutations only in comparison with relapses with wild-type NT5C2. Colored bars (yellow and blue) and black dots highlight the presence of a given genetic alterations in a relapse sample, whereas white bars indicate cases with no data. del, deletion; mut, mutation; rearr, rearrangement; sub, subclonal NT5C2 mutation; wt, wild-type. B-other* represents BCP-ALL relapses lacking the established cytogenetic abnormalities listed below.

Figure 3.

Survival outcomes of patients after first relapse of BCP-ALL by NT5C2 mutation status. (A) Event-free survival (EFS). (B) Overall survival (OS). (C) Competing risk analysis of cumulative incidence of relapse (CIR) at 10 years. (D) Competing risk analysis of cumulative incidence of treatment related death (CID) at 10 years. cens, censored.

Figure 4.

Evolutionary trajectories of NT5C2 mutations after first relapse. (A) The Sankey diagram illustrates the fate of 46 NT5C2 mutations from 34 first relapses in follow-up samples taken at the time of nonresponse to treatment or second relapse of the patients. All 46 mutations were tracked sensitively by p.R39Q, p.R238W, p.R367Q, p.K404N, or p.P414S ASQ-PCR. (B) Quantitative levels of NT5C2 mutations (red dots) at first relapse and at the end of relapse induction treatment in 16 patients with clonal and in 14 patients with subclonal mutations only. All NT5C2 mutations were tracked sensitively by p.R39Q, p.R238W, p.R238L, p.R367Q, p.K404N, or p.P414S ASQ-PCR. Minimal residual disease levels (blue dots) were assessed by immunoglobulin/ T-cell receptor (Ig/TCR) gene rearrangement quantification and are given as a measure of the overall leukemic burden in the patients at the end of induction treatment. Pt. ID, patient identifier.