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

• Choi R
• Chun MR
• Park J
• Lee JW
• Ju HY
• Cho HW
• Hyun JK
• Koo HH
• Yi ES
• Lee SY

• KMID: 2512679
• DOI: http://doi.org/10.3343/alm.2021.41.2.145

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

Figure

• 

  Fig. 1 Multiple reaction monitoring transitions for measurements of DNA-TG level using LC-MS/MS, presented as % intensity vs. time (min). Abbreviations: DNA-TG, DNA-incorporated 6-thioguanine; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

• 

  Fig. 2 Association between DNA-TG and RBC-TGN levels and their relationship with 6-MP dose (mg/day/m2) according to TPMT and NUDT15 variant alleles. (A) Association between DNA-TG and RBC-TGN levels, with regression lines for all 257 measurements at steady state (bold straight solid line) and according to subgroups (light straight solid line for normal metabolizers, stiff-dotted line for NUDT15 intermediate metabolizers, including *1/*2 and *1/*3, and dash-dotted line for measurements in subjects with NUDT15 indeterminate alleles, including *1/*5 and *1/*6). (B) DNA-TG/RBC-TGN ratio by subgroup (C) Association between 6-MP dose and DNA-TG levels. (D) DNA-TG/RBC-TGN ratio with regression line (straight line) and 95% confidence interval (dashed lines) for all results. Abbreviations: DNA-e-TG, etheno-thioguanine DNA; RBC-TGN, erythrocyte 6-thioguanine nucleotides; 6-MP, mercaptopurine; DNA-TG, DNA-incorporated 6-thioguanine; TPMP, thiopurine S-methyltransferase.

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