Ann Lab Med.  2021 Mar;41(2):145-154. 10.3343/alm.2021.41.2.145.

Quantification of Thioguanine in DNA Using Liquid Chromatography-Tandem Mass Spectrometry for Routine Thiopurine Drug Monitoring in Patients With Pediatric Acute Lymphoblastic Leukemia

Affiliations
  • 1Department of Laboratory Medicine and Genetics, Sungkyunkwan University School of Medicine, Seoul, Korea
  • 2Department of Laboratory Medicine, Green Cross Laboratories, Yongin, Korea
  • 3Department of Laboratory Medicine and Genetics, Samsung Medical Center, Seoul, Korea
  • 4Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
  • 5Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
  • 6Department of Pediatrics, Korea University Guro Hospital, Korea University College of Medicine, Seoul, Korea
  • 7Department of Clinical Pharmacology and Therapeutics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
  • 8Department of Health Science and Technology, Samsung Advanced Institute of Health Science and Technology, Sungkyunkwan University, Seoul, Korea

Abstract

Background
We developed an assay to measure DNA-incorporated 6-thioguanine (DNATG) and validated its clinical applicability in Korean pediatric patients with acute lymphoblastic leukemia (ALL) in order to improve individualized thiopurine treatment and reduce the life-threatening cytotoxicity.
Methods
The DNA-TG assay was developed based on liquid chromatography-tandem mass spectrometry, with isotope-labeled TG-d3 and guanine-d3 as internal standards. This method was applied to 257 samples of pediatric ALL patients. The DNA-TG level was compared with erythrocyte TG nucleotide (RBC-TGN) level in relation to the TPMT and NUDT15 genotypes, which affect thiopurine metabolism, using Spearman’s rank test and repeated measure ANOVA.
Results
For DNA-TG quantification, a linearity range of 10.0-5,000.0 fmol TG/µg DNA; bias for accuracy of –10.4% –3.5%; coefficient of variation for intra- and inter-day precision of 3.4% and 5.8% at 80 fmol TG/µg DNA and of 4.9% and 5.3% at 800 fmol TG/µg DNA, respectively; and recovery of 85.7%–116.2% were achieved without matrix effects or carry-over. The median DNA-TG level in the 257 samples was 106.0 fmol TG/µg DNA (interquartile range, 75.8–150.9). There was a strong correlation between DNA-TG and RBC-TGN levels (ρ = 0.68,ρ < 0.0001). The DNA-TG/RBC-TGN ratio was significantly higher in NUDT15 intermediate metabolizers (*1/*2 and *1/*3) than in patients with wildtype alleles (ρ < 0.0001).
Conclusions
This simple and sensitive method for measuring DNA-TG level can improve therapeutic drug monitoring for thiopurine treatment.

Keyword

DNA-incorporated 6-thioguanine; Liquid chromatography-tandem mass spectrometry; Therapeutic drug monitoring; Thiopurine; TPMT; NUDT15
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