Liquid chromatography–tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard

Jacobsen, Jack H.; Schmiegelow, Kjeld; Nersting, Jacob

doi:10.1016/j.jchromb.2011.11.032

Cited by 51 publications

(28 citation statements)

References 21 publications

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“…The levels of DNA-TG were quantified using LC-MS/MS, as described in previous reports [16, 18–20]. DNA-TG stability was evaluated and we observed no significant variation within the 72 h window.…”

Section: Introductionmentioning

confidence: 77%

The effects of inherited NUDT15 polymorphisms on thiopurine active metabolites in Japanese children with acute lymphoblastic leukemia

Moriyama¹,

Nishii

Lin³

et al. 2017

Pharmacogenetics and Genomics

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Thiopurines (e.g., mercaptopurine [MP]) are widely used chemotherapeutic agents in the treatment of pediatric acute lymphoblastic leukemia (ALL) with dose-limiting hematopoietic toxicity. Recently, germline variants in NUDT15 have been identified as a major genetic cause for MP-related bone marrow suppression, and there is increasing interest in clinical implementation of NUDT15 genotype-guided MP dose individualization. Therefore, we sought to evaluate the effects of NUDT15 on thiopurine metabolism and identify pharmacologic markers to inform NUDT15 genotype-guided MP dosing. In 55 Japanese children with ALL, we simultaneously measured both thioguanine nucleotides (TGN) in red blood cells and DNA-incorporated thioguanine (DNA-TG) in white blood cells. TGN levels were significantly lower in patients with NUDT15 deficiency, likely due to toxicity-related MP dose reduction. In contrast, when exposed to the same dose of MP, DNA-TG accumulated more efficiently in vivo with increasing number of risk alleles in NUDT15 (P = 4.0 × 10−9). Cytosolic TGN and nuclear DNA-TG were positively correlated with each other across genotype groups (P = 6.5 × 10−4), but the ratio of DNA-TG to TGN was significantly higher in NUDT15 deficient patients (P = 3.6 × 10−9), consistent with excessive MP activation. In conclusion, our results suggest that DNA-TG is a more relevant MP metabolite than TGN to inform NUDT15 genotype-guided dose adjustments.

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Section: Introductionmentioning

confidence: 77%

The effects of inherited NUDT15 polymorphisms on thiopurine active metabolites in Japanese children with acute lymphoblastic leukemia

Moriyama¹,

Nishii

Lin³

et al. 2017

Pharmacogenetics and Genomics

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“…Cytosolic thiopurine metabolites (e.g., TGTP, TGDP, and TGMP) were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) according to a previously reported method 62 . In parallel, aliquots of MP-, TG-, or azathioprine-treated cells were processed to extract genomic DNA, and DNA-TG was measured using LC-MS/MS as described previously 63 . MP, TG, and azathioprine cytotoxicity was determined for NUDT15 knockdown and control cells by using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at the end of the 72-hour drug exposure.…”

Section: Methodsmentioning

confidence: 99%

“…In the Japanese cohort (N = 32), peripheral blood collection was scheduled at the 13 th and 41 st week of maintenance therapy. For both cohorts, genomic DNA was extracted from leukocytes within 72 hours of blood draw and DNA-TG was quantified using LC-MS/MS as described previously 63 , and detailed MP dosing history was recorded for 14 days prior to each sample collection.…”

Section: Methodsmentioning

confidence: 99%

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

et al. 2016

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Widely used as anti-cancer and immunosuppressive agents, thiopurines have narrow therapeutic indices due to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and its clinical implications remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore, and Japan, we identified 4 NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile, p.Val18_Val19insGlyVal) that resulted in 74.4%–100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across three cohorts (P=0.021, 2.1×10−5, and 0.0054, respectively; meta-analysis P=4.45×10−8, allelic effect size=−11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased its cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive thiopurine active metabolites and toxicity. Taken together, our results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy.

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“…Patients included were established on AZA therapy for more than 6 months and were in clinical remission according to the treating physician. For this cohort of patients, the incorporated dTG levels varied from 20 to 1360 mol dTG/10 6 moles dA; expressed in the units used by Jacobson et al [33] , this represents 14–940 fmol/μg DNA (median 113; Table 6 ). Within this small group of patients there was no significant positive correlation between dTG levels and drug dose (Kendall’s rank correlation coefficient P = 0.143), although further studies with a larger sample size are warranted.…”

Section: Resultsmentioning

confidence: 99%

Liquid chromatography–mass spectrometry for measuring deoxythioguanosine in DNA from thiopurine-treated patients

Coulthard

Berry

McGarrity

et al. 2016

Journal of Chromatography B

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Liquid chromatography–tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard

Cited by 51 publications

References 21 publications

The effects of inherited NUDT15 polymorphisms on thiopurine active metabolites in Japanese children with acute lymphoblastic leukemia

The effects of inherited NUDT15 polymorphisms on thiopurine active metabolites in Japanese children with acute lymphoblastic leukemia

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

Liquid chromatography–mass spectrometry for measuring deoxythioguanosine in DNA from thiopurine-treated patients

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