6-MP tolerability varies between individuals and is influenced by genotypes affected by thiopurine metabolite enzyme activity, such as TPMT and NUDT15. Thiopurine metabolite levels may directly influence the effect of 6-MP; however, the transition range of 6-MP metabolite concentrations during maintenance therapy is unclear. Therefore, this study measured the 6-MP metabolite concentrations at multiple therapeutic time points in each patient during maintenance therapy. The 6-MP metabolite levels correlated with the 6-MP dose and the DNA-TG/TGNs ratio was affected by the NUDT15 genotype and erythrocyte TGNs levels in Japanese children with ALL.
The DNA-TG, erythrocyte TGNs, and MMPN levels positively correlated with the 6-MP dose at all sampling points. However, the variation in these 6-MP metabolite levels to dose differed in each patient. Asian patients with the NUDT15 variant required a lower 6-MP dose than those with the *1/*1 genotype during maintenance therapy [7, 10, 11]. Regarding the difference in NUDT15 variants, the DNA-TG ratio to the 6-MP dose of patients with NUDT15 variants was higher than that of patients with the *1/*1 genotype. In a previous study, erythrocyte TGNs levels correlated with the 6-MP dose at the sampling point and the total 6-MP dose in the previous month [12]. Furthermore, DNA-TG levels correlated with erythrocyte TGNs, which reached a plateau at high erythrocyte TGN levels in patients with childhood ALL with the TPMT variant in Northern Europe [13]. The present study did not include patients with the TPMT variant; however, five patients had the NUDT15 variant. The findings of this study showed that 6-MP metabolites correlated with the 6-MP dose, regardless of the genetic variant of the 6-MP metabolizing enzyme.
6-MP is metabolized by multiple enzymes, and the active metabolites (thioguanosine triphosphate [TGTP] and deoxy-TGTP) are incorporated into DNA. NUDT15 hydrolyzes TGTP to 6-thioguanosine 5′-monophosphate, and the findings of this study showed that the DNA-TG levels were higher in patients with the low-level NUDT15 variant than in those with the NUDT15 *1/*1 genotype. Interestingly, the DNA-TG/TGNs ratio significantly and inversely correlated with erythrocyte TGNs levels. RBCs do not have a nucleus, and circulating erythrocytes are only slightly affected by 6-MP metabolite levels. In contrast, WBCs are susceptible to the effects of 6-MP metabolites incorporated into DNA, which induce apoptosis. High DNA-TG levels induce cell death, and this study suggested that the DNA-TG/TGNs ratio is low at high erythrocyte TGNs levels.
Relapse-free survival is significantly associated with DNA-TG levels [3], and the NUDT15 low-activity variant may have poor outcomes. Patients with low NUDT15 activity experienced severe blood cell decrease—which may be caused by the rapid 6-MP active metabolite accumulation—and required long-term dose cessation. Therefore, NUDT15-deficient patients find it difficult to maintain sufficient DNA-TG levels in cells. In an animal model, implanted NUDT15-/- murine leukemic cells were treated with 6-MP at a tolerable dose, and the mice were completely leukemia-free for the entire experiment[8]. However, the present study was a preliminary study to confirm the relationship between the 6-MP dose and thiopurine metabolites in NUDT15 variants; therefore, the differences in outcomes in 6-MP metabolite levels were not investigated. The study results showed that DNA-TG levels positively correlated with the 6-MP dose and the erythrocyte TGNs level regulated TGNs incorporation. Therefore, NUDT15 genotypes and erythrocyte TGNs levels are predictive markers of DNA-TG level variation in Japanese patients with ALL.
In maintenance therapy, the 6-MP dose is typically adjusted according to the degree of myelosuppression while monitoring the WBC count. Patients with low NUDT15 activity receiving a 6-MP standard protocol dose inevitably experience severe myelosuppression, and the daily 6-MP dose needs to be adjusted to > 10 mg/m2 [14]. Erythrocyte TGN and MMP are associated with 6-MP-induced toxicities [5, 15]. In this study, the erythrocyte TGNs levels were associated with decreasing WBC and lymphocyte counts. Generally, the lifetime of neutrophils is shorter than that of lymphocytes and erythrocytes. Therefore, 6-MP metabolites accumulated more in lymphocytes than in neutrophils, and toxicity to lymphocytes may be associated with erythrocyte TGNs levels. Erythrocyte TGNs levels significantly correlate with neutrophil counts after 14 days of sampling [12]. However, the results of this study for the total sampling point showed no relationship between neutrophil count and 6-MP metabolite levels. These results indicated cell death at a high DNA-TG level. Only a few reports have shown thiopurine metabolite- and 6-MP-induced toxicities in NUDT15 variants. In a previous study, erythrocyte TGNs levels in hematopoietic toxicity in low-activity NUDT15 were < 171 pmol/8 × 108 in maintenance therapy for Korean children with ALL [16]. In the NUDT15 low-activity variant, DNA-TG levels were lower than those in the other variants. Therefore, DNA-TG is a principal cytotoxic metabolite [17].
In this study, aminotransferase levels were significantly associated with erythrocyte MMPN levels. This relationship aligns with that reported by Nygaard et al. [15]. Therefore, high MMPN levels induce 6-MP-related hepatotoxicity.
The limitation of this study was that the 6-MP dose was adjusted based on the patients’ 6-MP sensitivity during the prior treatment phase with 6-MP and laboratory data of WBC counts, liver enzymes, and bilirubin in peripheral blood. Blood sampling was conducted 14 days or later after the 6-MP dose adjustment.
In conclusion, the 6-MP dose correlated with DNA-TG and erythrocyte TGNs levels during maintenance therapy in Japanese children with ALL. In the low NUDT15 activity genotype, the DNA-TG/6-MP dose ratio and erythrocyte TGNs level were high; however, the DNA-TG level was lower than that in the other genotypes. The DNA-TG/TGNs ratio also differed in each patient. In summary, the NUDT15 genotype and erythrocyte TGNs level elevated the DNA-TG level in Japanese patients with ALL. Future studies will evaluate the association between thiopurine metabolites and patient outcomes in patients with the NUDT15 genotype.