Additive effects of TPMT and NUDT15 on thiopurine toxicity in children with acute lymphoblastic leukemia across multiethnic populations

Abstract

Background: Thiopurines such as mercaptopurine (MP) are widely used to treat acute lymphoblastic leukemia (ALL). Thiopurine-S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15) inactivate thiopurines, and no-function variants are associated with drug-induced myelosuppression. Dose adjustment of MP is strongly recommended in patients with intermediate or complete loss of activity of TPMT and NUDT15. However, the extent of dosage reduction recommended for patients with intermediate activity in both enzymes is currently not clear.

Methods: MP dosages during maintenance were collected from 1768 patients with ALL in Singapore, Guatemala, India, and North America. Patients were genotyped for TPMT and NUDT15, and actionable variants defined by the Clinical Pharmacogenetics Implementation Consortium were used to classify patients as TPMT and NUDT15 normal metabolizers (TPMT/NUDT15 NM), TPMT or NUDT15 intermediate metabolizers (TPMT IM or NUDT15 IM), or TPMT and NUDT15 compound intermediate metabolizers (TPMT/NUDT15 IM/IM). In parallel, we evaluated MP toxicity, metabolism, and dose adjustment using a Tpmt/Nudt15 combined heterozygous mouse model (Tpmt+/-/Nudt15+/-).

Results: Twenty-two patients (1.2%) were TPMT/NUDT15 IM/IM in the cohort, with the majority self-reported as Hispanics (68.2%, 15/22). TPMT/NUDT15 IM/IM patients tolerated a median daily MP dose of 25.7 mg/m2 (interquartile range = 19.0-31.1 mg/m2), significantly lower than TPMT IM and NUDT15 IM dosage (P < .001). Similarly, Tpmt+/-/Nudt15+/- mice displayed excessive hematopoietic toxicity and accumulated more metabolite (DNA-TG) than wild-type or single heterozygous mice, which was effectively mitigated by a genotype-guided dose titration of MP.

Conclusion: We recommend more substantial dose reductions to individualize MP therapy and mitigate toxicity in TPMT/NUDT15 IM/IM patients.

Figure 1.

Association of TPMT and NUDT15 metabolizer status with MP toxicity. TPMT and NUDT15 metabolizer status of 1768 patients with acute lymphoblastic leukemia was determined on the basis of the genotype of functionally characterized TPMT and NUDT15 variants, that is, TPMT *2, *3B, and *3C and NUDT15 *2, *3, and *9. Data represent the median, minimum, and maximum values and interquartile ranges of individual MP dosages. ***P < .001, according to the Wilcoxon rank-sum test conducted between patients who were single intermediate metabolizer for TPMT or NUDT15 and patients compound intermediate metabolizers. MP = mercaptopurine; NUDT15 = Nudix hydrolase 15; TPMT = thiopurine-S-methyltransferase.

Figure 2.

Adjustment of MP dosage in TPMT/NUDT15 IM/IM patients across cohorts. A) The dose intensity (%) of MP was used to compare the dosage adjustment in compound intermediate metabolizer patients (TPMT/NUDT15 IM/IM) across cohorts. Data represent the median, minimum, and maximum values and interquartile ranges of individual dose intensity. B) A meta-analysis was conducted to evaluate the homogeneity of clinical practices in adapting the MP dosage in TPMT/NUDT15 IM/IM patients versus patients who were single IM (TPMT IM or NUDT15 IM). The estimates and standard errors of independent regression analyses from each cohort were pooled using a random effect model. *P < .05, according to the Wilcoxon rank-sum test conducted between single IM patients and TPMT/NUDT15 IM/M patients. CI = confidence interval; IM = intermediate metabolizer; MP = mercaptopurine; NM = normal metabolizer; NUDT15 = Nudix hydrolase 15; TPMT = thiopurine-S-methyltransferase; UNOP = Unidad Nacional de Oncología Pediátrica.

Figure 3.

Mercaptopurine toxicity and metabolism in Tpmt/Nudt15 compound heterozygous mice. A) Tpmt/Nudt15 genotype-modified mice were treated with MP in drinking water. Hematopoietic toxicity was evaluated after blood collection after 5, 10, 17, and 30 days of treatment with MP administered in drinking water (N = 10) (left panel). Concentration of DNA-thioguanine (DNA-TG) in bone marrow was measured after 5 days of MP on a separated group of mice (N = 5) (right panel). B) Mice treated with 20 mg/kg of MP were followed up and were euthanized when body weight loss exceeded >20%, or they became moribund. C) Neutrophils were measured on day 10 of treatment with MP 20 mg/kg (median and interquartile range [IQR]). D) DNA-TG accumulation in bone marrow cells after 5 days of MP 20 mg/kg (median and IQR). Panel A was generated on Biorender.com. LC-MS/MS = liquid chromatography coupled to tandem mass spectrometry; MP = mercaptopurine; Nudt15 = Nudix hydrolase 15; Tpmt = thiopurine-S-methyltransferase. *P < .05, **P < .01, ***P < .001, according to the Wilcoxon rank-sum test conducted between single heterozygous mice (Tpmt^+/- or Nudt15^+/-) and compound heterozygous mice (Tpmt^+/-/Nudt15^+/-).

Figure 4.

Pharmacogenetics-guided dose adjustment of mercaptopurine in mice. MP toxicity was evaluated in Tpmt^+/-/Nudt15^+/- after a 75% dose reduction (5 mg/kg) and was compared with one of the single heterozygous mice (Tpmt^+/- or Nudt15^+/-) treated with a half dose (10 mg/kg) and wild-type mice treated with a full dose (20 mg/kg). A) Mice overall survival was monitored for 70 days, and they were euthanized when body weight loss exceeded >20% or they became moribund. B) Neutrophils were measured on day 10 of treatment (median and interquartile range [IQR]). C) DNA-TG concentrations were measured after 5 days of treatment to evaluate MP metabolism and toxicity (median and IQR). MP = mercaptopurine; Nudt15 = Nudix hydrolase 15; Tpmt = thiopurine-S-methyltransferase. *P < .05, **P < .01, according to the Wilcoxon rank-sum test conducted between single heterozygous mice (Tpmt^+/- or Nudt15^+/-) and compound heterozygous mice (Tpmt^+/-/Nudt15^+/-).