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. 2025 Jul 16;95(1):76.
doi: 10.1007/s00280-025-04784-7.

DNA-incorporated thioguanine to detect potential non-adherence to maintenance therapy in acute lymphoblastic leukemia

Mathilde Rønne Koch  1 , Anna Sofie Buhl Rasmussen  1 , Bodil Als-Nielsen  1 , Ximo Duarte  2 , Gabriele Escherich  3 , Mats Heyman  4 , Kristi Lepik  5 , Johan Malmros  4 , Jacob Nersting  1 , Inga Johannsdottir  6 , Riitta Niinimäki  7 , Malene Johanne Petersen  1 , Heidi Segers  8   9 , Inge Margriet van der Sluis  10 , Maria Thastrup  1 , Goda Vaitkeviciene  11 , Kjeld Schmiegelow  1   12 , Linea Natalie Toksvang  13
Affiliations

DNA-incorporated thioguanine to detect potential non-adherence to maintenance therapy in acute lymphoblastic leukemia

Mathilde Rønne Koch et al. Cancer Chemother Pharmacol. .

Abstract

Purpose: Adherence to 6-mercaptopurine (6-MP)/methotrexate maintenance treatment for acute lymphoblastic leukemia (ALL) is pivotal to preventing relapse, and the 6-MP metabolite DNA-incorporated thioguanine (DNA-TG) is associated with relapse risk. In the ALLTogether-1 (A2G1) Maintenance sub-study (EU CT nr 2022-501050-11-01), DNA-TG, thioguanine nucleotides (TGN), and methylated mercaptopurine metabolites (MeMP) are analyzed regularly. Upon levels below preset limits (TGN < 50, or MeMP < 200 or < 100 nmol/mmol hemoglobin for thiopurine S-methyltransferase (TPMT) wild type and heterozygous patients, respectively), the treating physician is informed of potential non-adherence. We investigated the feasibility of using DNA-TG as the primary flagging of potential non-adherence.

Methods: We analyzed 6-MP metabolites in 3,074 blood samples from 368 children enrolled in the A2G1 Maintenance sub-study.

Results: In 6% of samples, TGN (median 212, 95% range 40-642), MeMP (median 4,959, 95% range 135-23,880) or both were below the flagging potential non-adherence limits. DNA-TG was associated with TGN (estimate = 1.72, p < 0.0001), MeMP (estimate = 1.10, p < 0.0001), and prescribed 6-MP dose (estimate = 1.083 and 1.132, p < 0.0001, for TPMT wild type and heterozygous patients) in linear effects models, and the predicted probability of treatment interruption in logistic regression models. DNA-TG was below 200 fmol TG/µg DNA (13th percentile of all measurements, median 569, 95% range 73-1,823) in all samples with both TGN and MeMP below the flagging potential non-adherence limits.

Conclusion: DNA-TG can provide a cost-effective guidance on when to measure TGN and MeMP to determine whether non-adherence should be suspected, which is an additional benefit to monitoring DNA-TG during maintenance therapy.

Keywords: Acute lymphoblastic leukemia; Adherence; DNA thioguanine; Maintenance therapy; Mercaptopurine; Therapeutic drug monitoring.

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Conflict of interest statement

Declarations. Competing interests: A liquid formulation of 6-thioguanine (6TG) is provided by Novalab to the TEAM trial ( www.clinicaltrials.gov no NCT04307576), but the patients receiving 6TG are not included in this study. Ethical approval: This study is part of the A2G Maintenance Therapy sub-study to the ALLTogether1 study (EU CT nr 2022-501050-11-01). Oral and written consent was obtained from parents/caregivers and patients when appropriate. The study was approved on October 28th, 2020, in the European voluntary harmonization process (VHP1557/SA1[VHP2019111/SA1], current amendment accepted November 12th, 2021; VHP1557/SA3[VHP2019111/SA3]), followed by individual approval in participating countries by ethics committees.

Figures

Fig. 1
Fig. 1
Consort diagram of included patients. A2G: ALLTogether, SR: standard risk, SR-Down: standard risk Down syndrome, IR-low: intermediate risk-low, IR-high: intermediate risk-high, IR-Down: Intermediate risk Down syndrome, HR: high risk, TPMT: thiopurine S-methyltransferase. NUDT15: nudix hydrolase 15. *No indication for the analyses for 251 of these patients, NUDT15 analysis only mandatory for patients of Asian ancestry in the A2G1 protocol. Created with BioRender.com
Fig. 2
Fig. 2
Distribution of methylated mercaptopurine metabolites (MeMP), thioguanine nucleotides (TGN) and DNA incorporated thioguanine nucleotides (DNA-TG) in the collected blood samples. *Below the detection limit
Fig. 3
Fig. 3
Plots of measured 6-mercaptopurine (6-MP) metabolites and prescribed doses of 6-MP with locally estimated scatterplot smoothing (LOESS, red line). Samples from one thiopurine S-methyltransferase (TPMT) deficient patient not included. Black points: samples from TPMT homozygous wild type patients, blue points: samples from TPMT heterozygous patients. Dotted line: median value. a Mercaptopurine metabolites (MeMP) and DNA incorporated thioguanine nucleotides (DNA-TG, 2,991 samples from 363 patients), b thioguanine nucleotides (TGN) and DNA-TG (2,992 samples from 363 patients), c dose of 6-MP in mg/m2/day and DNA-TG for TPMT homozygous wild type patients (2,066 samples from 317 patients, 17 samples with dose of 6-MP > 130 mg/m2/day not shown), d dose of 6-MP in mg/m2/day and DNA-TG for TPMT heterozygous patients (128 samples from 21 patients). *Value below the detection limit
Fig. 4
Fig. 4
Plot of methylated mercaptopurine metabolites (MeMP) and thioguanine nucleotides (TGN) in 2,990 blood samples from 364 patients. The corresponding measurement of DNA incorporated thioguanine nucleotides (DNA-TG) in the sample is illustrated by color. Median DNA-TG = 569 fmol TG/µg DNA. The limits for generation of a reporting back notice for MeMP and TGN are illustrated as dotted lines (MeMP limit dependent on the TPMT genotype). *Below detection limit
Fig. 5
Fig. 5
Receiver-operating-characteristic (ROC) curves, illustrating the performance of general mixed effect models to predict the probability of a prescribed treatment interruption at the time of sample collection, based on 6-mercaptopurine (6-MP) metabolite levels. a Model with thioguanine nucleotides (TGN) and DNA incorporated thioguanine nucleotides (DNA-TG) as predictors, b Model with only DNA-TG as predictor. AUC = area under the curve, pAUC = partial area under the curve at given False Positive Rate
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