Department of Laboratory Medicine and Genetics, Sungkyunkwan University School of Medicine, Seoul, Korea.
Department of Laboratory Medicine, Green Cross Laboratories, Yongin, Korea.
Ann Lab Med. 2021 Mar 1;41(2):145-154. doi: 10.3343/alm.2021.41.2.145.
We developed an assay to measure DNA-incorporated 6-thioguanine (DNA-TG) 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.
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 and genotypes, which affect thiopurine metabolism, using Spearman's rank test and repeated measure ANOVA.
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, <0.0001). The DNA-TG/RBC-TGN ratio was significantly higher in NUDT15 intermediate metabolizers ( and ) than in patients with wild-type alleles (<0.0001).
This simple and sensitive method for measuring DNA-TG level can improve therapeutic drug monitoring for thiopurine treatment.
为了改善个体化硫嘌呤治疗并降低危及生命的细胞毒性，我们开发了一种测量 DNA 整合的 6-硫代鸟嘌呤（DNA-TG）的检测方法，并在韩国儿科急性淋巴细胞白血病（ALL）患者中验证了其临床适用性。
基于液相色谱-串联质谱法，我们开发了 DNA-TG 检测方法，使用同位素标记的 TG-d3 和鸟嘌呤-d3 作为内标。该方法应用于 257 例儿科 ALL 患者的 257 个样本中。通过 Spearman 秩检验和重复测量方差分析，将 DNA-TG 水平与影响硫嘌呤代谢的 和 基因型相关的红细胞 TG 核苷酸（RBC-TGN）水平进行比较。
对于 DNA-TG 的定量，线性范围为 10.0-5000.0 fmol TG/μg DNA；准确度偏差为-10.4%至 3.5%；在 80 fmol TG/μg DNA 时，日内和日间精密度的变异系数分别为 3.4%和 5.8%，在 800 fmol TG/μg DNA 时，分别为 4.9%和 5.3%；回收率为 85.7%-116.2%，无基质效应或拖尾。257 个样本中 DNA-TG 的中位数为 106.0 fmol TG/μg DNA（四分位距，75.8-150.9）。DNA-TG 与 RBC-TGN 水平之间存在很强的相关性（ρ=0.68，<0.0001）。NUDT15 中间代谢物（ 和 ）的 DNA-TG/RBC-TGN 比值明显高于野生型等位基因患者（<0.0001）。
该方法用于测量 DNA-TG 水平，操作简单且灵敏度高，可改善硫嘌呤治疗的治疗药物监测。
