NUDT15 genotype and erythrocyte thioguanosine levels affect thiopurine metabolite levels into DNA of Japanese children with acute lymphoblastic leukemia

NUDT15 genotype and erythrocyte thioguanosine levels affect thiopurine metabolite levels into DNA of Japanese children with acute lymphoblastic leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article NUDT15 genotype and erythrocyte thioguanosine levels affect thiopurine metabolite levels into DNA of Japanese children with acute lymphoblastic leukemia Yoichi Tanaka, Rintaro Ono, Miho Ashiarai, Ayako Sakurai, Atsushi Watanabe, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4634919/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2025 Read the published version in Cancer Chemotherapy and Pharmacology → Version 1 posted 9 You are reading this latest preprint version Abstract Purpose This study aimed to measure thiopurine metabolites—such as deoxythioguanosine incorporated into DNA (DNA-TG)—and erythrocyte thioguanine nucleotides (TGNs) and methyl mercaptopurine (MMPN) levels in Japanese children with acute lymphoblastic leukemia (ALL). This study also evaluated the factors that elevate thiopurine metabolites incorporated into the DNA. Methods DNA-TG, erythrocyte TGNs, and MMPN levels were measured on consecutive clinical visits in 20 Japanese patients with childhood ALL (171 sampling points) using liquid chromatography with tandem mass spectrometry. Nudix hydrolase 15 ( NUDT15 ) was genotyped using Sanger sequencing. Results Of the 20 patients, three had the NUDT15 intermediate activity genotype (*1/*2 or *1/*3), and two had a low activity genotype (*3/*3). The median DNA-TG level was 318 fmol/µg DNA. Erythrocyte TGNs and MMPN levels were 341.4 and 14,136 pmol/8 × 10 8 red blood cells, respectively. The ratio of DNA-TG/TGNs—which is the active thiopurine metabolite ratio in DNA—was higher in patients with the NUDT15 variant than in the wild type. The DNA-TG/TGNs ratio was inversely correlated with the TGNs level. Erythrocyte TGNs levels were significantly correlated with white blood cell and lymphocyte counts (p = 0.02 and 0.01, respectively), and MMPN levels were significantly correlated with lymphocyte count and aspartate and alanine aminotransferase levels (p = 0.005, 0.0004, and 0.007, respectively). Conclusion The DNA-TG/TGNs ratio differed in each patient. The NUDT15 genotype and erythrocyte TGNs level were affected and elevated the DNA-TG level in Japanese patients with ALL. DNA-incorporated deoxythioguanosine thioguane nucleotides 6-mercaptopurine NUDT15 childhood acute lymphoblastic leukemia toxicities Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction 6-Mercaptopurine (6-MP) is used to treat childhood acute lymphoblastic leukemia (ALL) [ 1 ]. The 6-MP dosage during maintenance therapy is a important determinant of event-free survival in ALL [ 2 ]. Multiple enzymatic reactions metabolize 6-MP and generate active metabolites, such as thioguanine nucleotides (TGNs) or non-active metabolites. One of the mechanisms of action of 6-MP is to incorporate TGNs into DNA and RNA, which induces apoptosis. Recently, relapse-free survival in children with ALL was associated with DNA-incorporated TGNs in a Northern European group study (NOPHO ALL2008) [ 3 ]. TGNs levels in cells were also associated with 6-MP-induced cytotoxicity, particularly myelotoxicity. Erythrocyte TGNs levels are inversely related to the white blood cell (WBC), absolute neutrophil, erythrocyte, and platelet counts[ 4 ]. Furthermore, pharmacogenetic factors also affect thioguanine metabolites. For example, genetic thiopurine S-methyl transferase (TPMT) variants have high TGNs levels after treatment with a standard 6-MP dose [ 5 , 6 ]. The standard 6-MP dose in maintenance therapy in Asian countries is 40–50 mg/m 2 , lower than that used in other countries. The 6-MP dose is adjusted by monitoring blood cell counts; however, sudden drops in blood cell counts have been reported that required therapy suspension. Nudix hydrolase 15 ( NUDT15 ) genetic variants are another factor affecting 6-MP intolerance, primarily in Asians [ 7 ]. NUDT15 dephosphorylates thioguanine triphosphate to monophosphate in cells. However, 6-MP tolerance differs in patients with the same NUDT15 genotype. Therefore, assessing 6-MP metabolite levels is required to adjust to the appropriate 6-MP dose. The enzyme deficiency genotype increases TGNs incorporation into DNA and RNA and causes toxicity [ 8 ]. Thus, appropriate 6-MP dose adjustment is needed for patients with the NUDT15 enzyme-deficient genotype. Thiopurine metabolites may directly affect the effectiveness of 6-MP; however, the concentration transition of 6-MP and its metabolites and the association between 6-MP metabolite levels and dose are unknown in the Asian population. Therefore, this study measured the amount of deoxythioguanine incorporated into DNA (DNA-TG) and erythrocyte 6-MP metabolite levels and evaluated the association between 6-MP metabolite levels and dose in Japanese children with ALL. Materials and Methods Patients and treatment The institutional ethics committees of each participating institution approved the study. Written informed consent was obtained from the parents or guardians of the patients or the patients themselves, depending on their age and conceptual ability. This study enrolled Japanese children with ALL treated with 6-MP-based maintenance therapy at St. Luke’s International Hospital, Japanese Red Cross Narita Hospital, and the University of Yamanashi Hospital. The patients received treatment between 2018 and 2021 according to the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) ALL-B12 protocol. The initial 6-MP and methotrexate (MTX) doses for maintenance therapy were 50 mg/m 2 daily and 25 mg/m 2 weekly, respectively. These dosages were adjusted to maintain the target leukocyte count at 1,500–3,000/µL. The therapy-induced toxicities were evaluated, and laboratory data were collected on whole blood cell counts and liver enzymes. Genotyping Germline DNA was extracted from peripheral blood samples collected in EDTA tubes during maintenance therapy. NUDT15 exon 1 and 3 genotypes were identified using Sanger sequencing. TPMT c.238G > C, c.460G > A, and c.719A > G genotypes were genotyped using the TaqMan probe method. Quantifying 6-MP metabolite levels The TGNs and methyl mercaptopurine nucleotide (MMPN) levels in erythrocytes and DNA-TG were determined. Samples were collected multiple times from each patient during maintenance therapy. The DNA concentration was measured using the Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). 6-MP metabolites in erythrocytes and DNA were measured using ultra-high performance liquid chromatography-tandem mass spectrometry (VantageTQD; Thermo Fisher Scientific). TGNs and MMPN levels were measured as previously described with modifications [ 9 ] Briefly, 50 µL of internal standard, including 12.5 µmol/L 2-amino-6MP 13 C 2 15 N and 6-methylmercaptopurine-d3, 250 µL of 100 mmol/L dithiothreitol, and 20 µL of 60% perchloric acid, were added to 25 µL of erythrocytes and mixed thoroughly using a vortex mixer. The mixture was placed on ice for 10 min and centrifuged at 13,000 × g at 4°C for 10 min. The supernatant was incubated at 95°C for 1 h, and the equivalent mobile phase was added after cooling. After filtration using a 0.2 µm filter, 5 µL was injected for quantification. Chromatography was conducted under gradient conditions at 30°C using a CORTECS UPLC C18 + instrument (2.1 × 50 mm, 1.6 µm; Waters Corporation, Milford, MA, USA). The flow rate was 0.5 mL/min. The runs were performed using a gradient. Buffers A and B were 2 mmol/L ammonium acetate and 0.1% formic acid in water and methanol, respectively. The gradient conditions were as follows: 0–1.8 min, 3% B; 1.8–2.3 min, 95% B; and 2.5–5 min, 3% B. DNA-TG levels were quantified as previously described with modifications. Briefly, 50 µL of DNA was extracted, which included 500–5000 ng of DNA and 1 µL of 59.1 µmol/L 6-methylmercaptopurine-d3, and incubated at 100°C for 5 min. After cooling, 5.6 µL of 10× digestion buffer (500 mM sodium acetate, 10 mM MgCl 2 pH 5.3) and 5 µL of 0.12 U/µL nuclease P1 (Sigma-Aldrich, St. Louis, MO, USA) were added and incubated at 50°C for 1 h. Subsequently, 7 µL of 500 mM Tris-HCl (pH 8.0) and 1 µL of 3 U/µL calf intestine alkaline phosphatase (TOYOBO, Osaka, Japan) were added and incubated at 37°C for 30 min. After filtration using a 0.2 µm filter, 5 µL was injected for quantification. Chromatography was conducted under gradient conditions at 30°C using a CORTECS UPLC C18 + instrument (2.1 × 50 mm, 1.6 µm; Waters Corporation). The flow rate was 0.4 mL/min. The runs were performed using a gradient. Buffers C and D contained 0.05% formic acid in water and acetonitrile, respectively. The gradient conditions were as follows: 0–0.1 min, 1% D; 1.1 min, 3% D; 2.4 min, 8% D; 4.1 min, 30% D; 4.5 min, 5% D; and 5.6 min, 1% D. Statistical analyses The thiopurine metabolite values of the genotypes were estimated using the Kruskal–Wallis test. The relationship between the 6-MP dose and thiopurine metabolite levels was estimated using Pearson’s correlation coefficient. The difference between the two groups was estimated using the Mann–Whitney U -test. Statistical analyses were performed using GraphPad Prism 9 (DotMatics, Boston, MA, USA) and R statistical software (v 4.0.2; http://www.r-project.org/ ). Statistical significance was defined as p < 0.05. Results Patients A total of 171 samples from 20 patients were collected to measure the thiopurine metabolite levels (DNA-TG, TGNs, and MMPN) during maintenance therapy (Table 1 ). The median 6-MP and MTX doses were 47.6 (1.7–86.4) mg/m 2 daily and 20.2 (9.9–41) mg/m 2 weekly, respectively. Three patients had the NUDT15 intermediate genotype (*1/*2 or *1/*3), and two patients had a low activity genotype (*3/*3). None of the patients had a TPMT variant that reduced enzyme activity. Table 1 Patient characteristics and 6-MP metabolites NUDT15 genotype All *1/*1 *1/*3 *1/*2 *3/*3 Number Age (years old) Male/Female 6-mercaptopurine dose (mg/m 2 /day) Methotrexate dose (mg/m 2 /week) Number of sampling points 6-mercaptopurine metabolites concentration DNA-TG (fmol/ugDNA) Erythrocyte TGNs (pmol/8×10 8 RBC) Erythrocyte MMP (pmol/8×10 8 RBC) 20 3 7/11 47.8 19.5 171 351.2 419.0 20773 15 3 4/11 48.2 19.6 142 344.8 457.0 24786 1 4 1/0 67.5 25.8 6 728.4 †† 149.3 † 32212 2 3 1/1 32.4 19.4 18 349.4 358.0 † 9739 †† 2 2 1/1 2.7 12.5 5 178.8 10.0 †† 50.0 †† † p < 0.05 and †† p < 0.01 (vs. *1/*1). Abbreviation; DNA-TG, thioguanine incorporate into DNA; TGNs, thioguanine nucleotides; MMPN, methyl mercaptopurine Thiopurine metabolite levels The median DNA-TG level was 351.2 fmol/µg DNA in the 171 samples (interquartile range [IQR], 248.5–481.4). The DNA-TG levels of patients with the NUDT15 variants were significantly higher than those with the *1/*1 genotype (740.5 vs. 304.0 fmol/µg DNA, p = 1.35 × 10 − 4 ). The median DNA-TG levels in individuals varied three-fold in the NUDT15 *1/*1 genotype (Fig. 1 A). The median erythrocyte TGNs level was 419.0 pmol/8 × 10 8 red blood cells (RBC; IQR, 305.6–542.0). The median TGNs levels varied among individuals (Fig. 1 B). A three-fold difference was observed for each individual with the NUDT15 *1/*1 genotype, and the number of thiopurine metabolites in the erythrocytes was lower than the limit of quantification (10 pmol/8 × 10 8 RBC) for patients with the NUDT15 deficient genotype (*3/*3). Erythrocyte TGNs levels in NUDT15 variants were lower than those in patients with the *1/*1 genotype (Fig. 1 B). The median erythrocyte MMPN levels were 20,773 pmol/8 × 10 8 RBCs (IQR, 9,742–34,319), and the amount of thiopurine metabolites in erythrocytes was lower than the limit of quantification (10 pmol/8 × 10 8 RBCs for TGNs and 50 pmol/8 × 10 8 RBCs for MMPN). The 6-MP dose significantly correlated with all three 6-MP metabolite levels in the evaluation of all points (p < 0.01) (Fig. 2 ). In patients with the *1/*1 genotype and NUDT15 variants, DNA-TG (p = 0.0006 and < 0.0001; r = 0.29 and 0.74, respectively) and MMPN levels (p < 0.0001 and 0.05 and r = 0.15 and 0.33, respectively). The regression line between the 6-MP dose and MMPN level showed a similar trend for each NUDT15 *1/*1 genotype and variant. The DNA-TG level to dose (DNA-TG/dose) ratio in patients with the NUDT15 variant was significantly higher than in those with the *1/*1 genotype (p = 0.004, Mann–Whitney U -test). The DNA-TG/dose ratio ranged from 5 to 10 in almost all patients with the NUDT15 *1/*1 genotype. Patients with NUDT15 variants and a TGNs level/dose ratio of > 15 pmol/8 × 10 8 RBCs/mg had a higher DNA-TG/dose ratio of > 10 pmol/8 × 10 8 RBCs/mg of patients with NUDT15 *1/*1. The DNA-TG/TGNs fomol/µgDNA/(pmol/8 × 10 8 RBCs) ratio correlated with the TGNs level (r = -0.67, p = 0.0011) (Fig. 3 ). In patients with the NUDT15 *1/*1 genotype, the median DNA-TG level correlated with TGNs levels (r = 0.60, p = 0.017). The ratios of the DNA-TG/TGNs levels—which show the DNA incorporation ratio—were affected by the NUDT15 genotype (Fig. 3 D, p < 0.0001 and r = -0.33), and these correlations were observed in each wild type and NUDT15 variant. The DNA-TG/TGNs ratio was 0.71 fomol/µgDNA/(pmol/8 × 10 8 RBCs) for patients with the NUDT15 *1/*1 genotype (IQR, 0.67–1.08). The DNA-TG/TGNs ratio in patients with the NUDT15 variant was significantly higher than those with the *1/*1 genotype (*1/*1, 0.72; variant, 5.07, p = 0.0015, Mann–Whitney U -test). The DNA-TG/TGNs ratio showed an inverse correlation with the TGNs level in each patient (data not shown). DNA-TG levels were weakly correlated with MMPN levels (p < 0.0001, r = 0.36) but not correlated with TGNs levels. In patients with the NUDT15 *1/*1 genotype, the DNA-TG level was weakly correlated with TGNs and MMP levels (Fig. 3 ). Thiopurine metabolite levels, blood cell counts, and hepatic enzymes The association between laboratory data and the 6-MP metabolite levels at each sampling point was evaluated (Fig. 4 ). WBC counts correlated with erythrocyte TGN levels (Fig. 4 A, p = 0.013, r = -0.19) but not with DNA-TG levels. Lymphocyte counts were weakly correlated with DNA-TG (p < 0.001, r = -0.28), erythrocyte TGNs (p < 0.0001, r = -0.36), and MMPN levels (p < 0.0001, r = -0.35). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels correlated with erythrocyte MMPN levels (Fig. 4 B, p 200 IU than in ALT < 200 IU (p < 0.0001). Discussion 6-MP tolerability varies between individuals and is influenced by genotypes affected by thiopurine metabolite enzyme activity, such as TPMT and NUDT15. Thiopurine metabolite levels may directly influence the effect of 6-MP; however, the transition range of 6-MP metabolite concentrations during maintenance therapy is unclear. Therefore, this study measured the 6-MP metabolite concentrations at multiple therapeutic time points in each patient during maintenance therapy. The 6-MP metabolite levels correlated with the 6-MP dose and the DNA-TG/TGNs ratio was affected by the NUDT15 genotype and erythrocyte TGNs levels in Japanese children with ALL. The DNA-TG, erythrocyte TGNs, and MMPN levels positively correlated with the 6-MP dose at all sampling points. However, the variation in these 6-MP metabolite levels to dose differed in each patient. Asian patients with the NUDT15 variant required a lower 6-MP dose than those with the *1/*1 genotype during maintenance therapy [ 7 , 10 , 11 ]. Regarding the difference in NUDT15 variants, the DNA-TG ratio to the 6-MP dose of patients with NUDT15 variants was higher than that of patients with the *1/*1 genotype. In a previous study, erythrocyte TGNs levels correlated with the 6-MP dose at the sampling point and the total 6-MP dose in the previous month [ 12 ]. Furthermore, DNA-TG levels correlated with erythrocyte TGNs, which reached a plateau at high erythrocyte TGN levels in patients with childhood ALL with the TPMT variant in Northern Europe [ 13 ]. The present study did not include patients with the TPMT variant; however, five patients had the NUDT15 variant. The findings of this study showed that 6-MP metabolites correlated with the 6-MP dose, regardless of the genetic variant of the 6-MP metabolizing enzyme. 6-MP is metabolized by multiple enzymes, and the active metabolites (thioguanosine triphosphate [TGTP] and deoxy-TGTP) are incorporated into DNA. NUDT15 hydrolyzes TGTP to 6-thioguanosine 5′-monophosphate, and the findings of this study showed that the DNA-TG levels were higher in patients with the low-level NUDT15 variant than in those with the NUDT15 *1/*1 genotype. Interestingly, the DNA-TG/TGNs ratio significantly and inversely correlated with erythrocyte TGNs levels. RBCs do not have a nucleus, and circulating erythrocytes are only slightly affected by 6-MP metabolite levels. In contrast, WBCs are susceptible to the effects of 6-MP metabolites incorporated into DNA, which induce apoptosis. High DNA-TG levels induce cell death, and this study suggested that the DNA-TG/TGNs ratio is low at high erythrocyte TGNs levels. Relapse-free survival is significantly associated with DNA-TG levels [ 3 ], and the NUDT15 low-activity variant may have poor outcomes. Patients with low NUDT15 activity experienced severe blood cell decrease—which may be caused by the rapid 6-MP active metabolite accumulation—and required long-term dose cessation. Therefore, NUDT15 -deficient patients find it difficult to maintain sufficient DNA-TG levels in cells. In an animal model, implanted NUDT15 -/- murine leukemic cells were treated with 6-MP at a tolerable dose, and the mice were completely leukemia-free for the entire experiment[ 8 ]. However, the present study was a preliminary study to confirm the relationship between the 6-MP dose and thiopurine metabolites in NUDT15 variants; therefore, the differences in outcomes in 6-MP metabolite levels were not investigated. The study results showed that DNA-TG levels positively correlated with the 6-MP dose and the erythrocyte TGNs level regulated TGNs incorporation. Therefore, NUDT15 genotypes and erythrocyte TGNs levels are predictive markers of DNA-TG level variation in Japanese patients with ALL. In maintenance therapy, the 6-MP dose is typically adjusted according to the degree of myelosuppression while monitoring the WBC count. Patients with low NUDT15 activity receiving a 6-MP standard protocol dose inevitably experience severe myelosuppression, and the daily 6-MP dose needs to be adjusted to > 10 mg/m 2 [ 14 ]. Erythrocyte TGN and MMP are associated with 6-MP-induced toxicities [ 5 , 15 ]. In this study, the erythrocyte TGNs levels were associated with decreasing WBC and lymphocyte counts. Generally, the lifetime of neutrophils is shorter than that of lymphocytes and erythrocytes. Therefore, 6-MP metabolites accumulated more in lymphocytes than in neutrophils, and toxicity to lymphocytes may be associated with erythrocyte TGNs levels. Erythrocyte TGNs levels significantly correlate with neutrophil counts after 14 days of sampling [ 12 ]. However, the results of this study for the total sampling point showed no relationship between neutrophil count and 6-MP metabolite levels. These results indicated cell death at a high DNA-TG level. Only a few reports have shown thiopurine metabolite- and 6-MP-induced toxicities in NUDT15 variants. In a previous study, erythrocyte TGNs levels in hematopoietic toxicity in low-activity NUDT15 were < 171 pmol/8 × 10 8 in maintenance therapy for Korean children with ALL [ 16 ]. In the NUDT15 low-activity variant, DNA-TG levels were lower than those in the other variants. Therefore, DNA-TG is a principal cytotoxic metabolite [ 17 ]. In this study, aminotransferase levels were significantly associated with erythrocyte MMPN levels. This relationship aligns with that reported by Nygaard et al. [ 15 ]. Therefore, high MMPN levels induce 6-MP-related hepatotoxicity. The limitation of this study was that the 6-MP dose was adjusted based on the patients’ 6-MP sensitivity during the prior treatment phase with 6-MP and laboratory data of WBC counts, liver enzymes, and bilirubin in peripheral blood. Blood sampling was conducted 14 days or later after the 6-MP dose adjustment. In conclusion, the 6-MP dose correlated with DNA-TG and erythrocyte TGNs levels during maintenance therapy in Japanese children with ALL. In the low NUDT15 activity genotype, the DNA-TG/6-MP dose ratio and erythrocyte TGNs level were high; however, the DNA-TG level was lower than that in the other genotypes. The DNA-TG/TGNs ratio also differed in each patient. In summary, the NUDT15 genotype and erythrocyte TGNs level elevated the DNA-TG level in Japanese patients with ALL. Future studies will evaluate the association between thiopurine metabolites and patient outcomes in patients with the NUDT15 genotype. Declarations Data availability statements The data that support the findings of this study are not openly available due to reasons of ethical reason and are available from the corresponding author upon reasonable request. Data are located in controlled access data storage at National Institute of Health Sciences. Compliance with Ethical Standards Research involving human participants All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The Ethics Committee of each institution approved the study. Informed consent Written informed consent was obtained from the parents or guardians of the patients or the patients themselves, depending on the age and conceptual ability of the patient. Statements and Declarations Acknowledgments and Funding Information This study was supported by JSPS KAKENHI (grant number JP18K06756). Conflicts of Interest The authors declare that they have no conflicts of interest. Author Contribution Yoichi Tanaka wrote the main manuscript and text. Rintaro Ono, Miho Ashiarai, Ayako Sakurai, Atsushi Watanabe, Taichiro Tsuchimochi, Takeshi Inukai and Daisuke Hasegawa were recruited patients and obtained informed consent from patients and guardians. Ruri Hanajiri editing manuscript. All authors reviewed the manuscript. References Kato M, Manabe A (2018) Treatment and biology of pediatric acute lymphoblastic leukemia. Pediatr Int 60:4–12 Relling MV, Hancock ML, Boyett JM et al (1999) Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukemia. 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J Clin Pharmacol 53:670–674. https://doi.org/10.1002/jcph.81 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2025 Read the published version in Cancer Chemotherapy and Pharmacology → Version 1 posted Editorial decision: Revision requested 08 Aug, 2024 Reviews received at journal 12 Jul, 2024 Reviews received at journal 10 Jul, 2024 Reviewers agreed at journal 30 Jun, 2024 Reviewers agreed at journal 30 Jun, 2024 Reviewers invited by journal 27 Jun, 2024 Editor assigned by journal 25 Jun, 2024 Submission checks completed at journal 25 Jun, 2024 First submitted to journal 25 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4634919","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326363882,"identity":"bed1f8f4-40e8-4d69-9dc6-ade5785f4f12","order_by":0,"name":"Yoichi Tanaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYJACxgYgwcbAw3DgA4NEAkSMB7dyHmQtB2eQpAXEZAbyEgg6yp797MGPM2oOy/EBGYdtd1jkyTewX3zAIHMHty08ecmSG44dNmbjyUs4nHtGotjgAE+xAQPPMzwOyzGQfMB2OLFNgsfgcG6bROIGBp40CQaew7i18L8x/vngH1SLJVDL/AZCWiRyzCQ3tkG1MAK1NBxgP4Zfy403ZpYz+9KBfskxONgLcthhHmaDBDx+Ye/PMb7Z881aTr79jPGHn211ifPb2x8++NiDO8SgoBmJzcxjwJDYc4CQljoUmx8wMPwgqGUUjIJRMApGDgAAKIhVFBAwYvcAAAAASUVORK5CYII=","orcid":"","institution":"National Institute of Health Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yoichi","middleName":"","lastName":"Tanaka","suffix":""},{"id":326363883,"identity":"222abb96-5fe5-4eb7-a482-e5d8d348b9be","order_by":1,"name":"Rintaro Ono","email":"","orcid":"","institution":"St. Luke’s International Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rintaro","middleName":"","lastName":"Ono","suffix":""},{"id":326363884,"identity":"a9bec69d-f1c2-4b06-a7f4-e0f26866595e","order_by":2,"name":"Miho Ashiarai","email":"","orcid":"","institution":"St. Luke’s International Hospital","correspondingAuthor":false,"prefix":"","firstName":"Miho","middleName":"","lastName":"Ashiarai","suffix":""},{"id":326363885,"identity":"b0b419c1-8983-4fe2-8460-a99fc78c5e71","order_by":3,"name":"Ayako Sakurai","email":"","orcid":"","institution":"Japanese Red Cross Narita Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ayako","middleName":"","lastName":"Sakurai","suffix":""},{"id":326363886,"identity":"39fcad63-d4ef-4cf6-a621-7fd8b996376a","order_by":4,"name":"Atsushi Watanabe","email":"","orcid":"","institution":"University of Yamanashi","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Watanabe","suffix":""},{"id":326363887,"identity":"3ad08efe-a126-486d-b111-551cef6b07b7","order_by":5,"name":"Taichiro Tsuchimochi","email":"","orcid":"","institution":"Japanese Red Cross Narita Hospital","correspondingAuthor":false,"prefix":"","firstName":"Taichiro","middleName":"","lastName":"Tsuchimochi","suffix":""},{"id":326363888,"identity":"c222fa05-ffc3-4f88-ac8a-9a957a732d53","order_by":6,"name":"Yosuke Hosoya","email":"","orcid":"","institution":"St. Luke’s International Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yosuke","middleName":"","lastName":"Hosoya","suffix":""},{"id":326363889,"identity":"531d562d-1659-408f-934a-665fae16a5bd","order_by":7,"name":"Ruri Hanajiri","email":"","orcid":"","institution":"National Institute of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ruri","middleName":"","lastName":"Hanajiri","suffix":""},{"id":326363890,"identity":"03d221e1-daa6-41c4-ae4e-8eb0cc8794cc","order_by":8,"name":"Takeshi Inukai","email":"","orcid":"","institution":"University of Yamanashi","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Inukai","suffix":""},{"id":326363891,"identity":"d0870aa8-57f1-43f5-86f2-f933920fc9e2","order_by":9,"name":"Daisuke Hasegawa","email":"","orcid":"","institution":"St. Luke’s International Hospital","correspondingAuthor":false,"prefix":"","firstName":"Daisuke","middleName":"","lastName":"Hasegawa","suffix":""}],"badges":[],"createdAt":"2024-06-25 08:45:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4634919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4634919/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00280-025-04774-9","type":"published","date":"2025-04-01T15:56:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60851364,"identity":"3ae9da84-f527-428f-9068-648e9c660379","added_by":"auto","created_at":"2024-07-22 21:00:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64197,"visible":true,"origin":"","legend":"\u003cp\u003eMedian 6-mercaptopurine (6-MP) levels in each patient. A, DNA-deoxythioguanosine (DNA-TG); B, erythrocyte thioguanine nucleotides (TGNs); C, DNA-TG/TGN ratio. The horizontal bar is the *1/*1 genotype, the black circle is the *1/*3 genotype, the open circle is the *1/*2 genotype, and the black triangle is the *3/*3 genotype of\u003cem\u003e NUDT15\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4634919/v1/6637e7e12b7c1289be99e055.jpg"},{"id":60850411,"identity":"b038d6f2-ba22-4743-bf6a-879bf9989f1c","added_by":"auto","created_at":"2024-07-22 20:52:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68224,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationships between the daily 6-MP dose and metabolite levels. A, 6-MP dose vs. DNA-TG level; B, 6-MP dose vs. TGN level; C, 6-MP dose vs. erythrocyte 6-methyl mercaptopurine (6-MMP) level. The black circle is the \u003cem\u003eNUDT15\u003c/em\u003e *1/*1 genotype, and the open circle is the \u003cem\u003eNUDT15\u003c/em\u003evariants.\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4634919/v1/852d1bafd3baed3e7c77c935.jpg"},{"id":60851363,"identity":"bd2c05c1-38c1-477e-b0e5-3a6ec9cf533a","added_by":"auto","created_at":"2024-07-22 21:00:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90130,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationships between 6-MP metabolite levels. A, TGN level vs. DNA-TG level; B, 6-MMP level vs. DNA-TG level; C, erythrocyte 6-MMP level vs. TGN level; D, erythrocyte TGN level vs. DNA-TG/TGN ratio. The black circle is the *1/*1 \u003cem\u003eNUDT15\u003c/em\u003e genotype, and the open circle is the \u003cem\u003eNUDT15\u003c/em\u003e variant.\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4634919/v1/05e1908546ca8ed075cb0b79.jpg"},{"id":60850413,"identity":"5d32cb53-130a-4412-8a0c-4ca45f21f09e","added_by":"auto","created_at":"2024-07-22 20:52:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between 6-MP metabolite levels and laboratory data. A, TGN level vs. white blood cell counts; B, 6-MMP level vs. aspartate transaminase (AST) level.\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-4634919/v1/96559538affa9363ba760087.png"},{"id":80081903,"identity":"f2d40dbc-1466-45af-9702-393250067bcd","added_by":"auto","created_at":"2025-04-07 16:00:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":909312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4634919/v1/68f85060-eaf9-4a8c-9b13-cc7f59d375c8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"NUDT15 genotype and erythrocyte thioguanosine levels affect thiopurine metabolite levels into DNA of Japanese children with acute lymphoblastic leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003e6-Mercaptopurine (6-MP) is used to treat childhood acute lymphoblastic leukemia (ALL) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The 6-MP dosage during maintenance therapy is a important determinant of event-free survival in ALL [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Multiple enzymatic reactions metabolize 6-MP and generate active metabolites, such as thioguanine nucleotides (TGNs) or non-active metabolites. One of the mechanisms of action of 6-MP is to incorporate TGNs into DNA and RNA, which induces apoptosis. Recently, relapse-free survival in children with ALL was associated with DNA-incorporated TGNs in a Northern European group study (NOPHO ALL2008) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. TGNs levels in cells were also associated with 6-MP-induced cytotoxicity, particularly myelotoxicity. Erythrocyte TGNs levels are inversely related to the white blood cell (WBC), absolute neutrophil, erythrocyte, and platelet counts[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, pharmacogenetic factors also affect thioguanine metabolites. For example, genetic thiopurine S-methyl transferase (TPMT) variants have high TGNs levels after treatment with a standard 6-MP dose [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The standard 6-MP dose in maintenance therapy in Asian countries is 40\u0026ndash;50 mg/m\u003csup\u003e2\u003c/sup\u003e, lower than that used in other countries. The 6-MP dose is adjusted by monitoring blood cell counts; however, sudden drops in blood cell counts have been reported that required therapy suspension. \u003cem\u003eNudix hydrolase 15\u003c/em\u003e (\u003cem\u003eNUDT15\u003c/em\u003e) genetic variants are another factor affecting 6-MP intolerance, primarily in Asians [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eNUDT15\u003c/em\u003e dephosphorylates thioguanine triphosphate to monophosphate in cells. However, 6-MP tolerance differs in patients with the same \u003cem\u003eNUDT15\u003c/em\u003e genotype. Therefore, assessing 6-MP metabolite levels is required to adjust to the appropriate 6-MP dose. The enzyme deficiency genotype increases TGNs incorporation into DNA and RNA and causes toxicity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, appropriate 6-MP dose adjustment is needed for patients with the NUDT15 enzyme-deficient genotype. Thiopurine metabolites may directly affect the effectiveness of 6-MP; however, the concentration transition of 6-MP and its metabolites and the association between 6-MP metabolite levels and dose are unknown in the Asian population.\u003c/p\u003e \u003cp\u003eTherefore, this study measured the amount of deoxythioguanine incorporated into DNA (DNA-TG) and erythrocyte 6-MP metabolite levels and evaluated the association between 6-MP metabolite levels and dose in Japanese children with ALL.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and treatment\u003c/h2\u003e \u003cp\u003e The institutional ethics committees of each participating institution approved the study. Written informed consent was obtained from the parents or guardians of the patients or the patients themselves, depending on their age and conceptual ability.\u003c/p\u003e \u003cp\u003eThis study enrolled Japanese children with ALL treated with 6-MP-based maintenance therapy at St. Luke\u0026rsquo;s International Hospital, Japanese Red Cross Narita Hospital, and the University of Yamanashi Hospital. The patients received treatment between 2018 and 2021 according to the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) ALL-B12 protocol. The initial 6-MP and methotrexate (MTX) doses for maintenance therapy were 50 mg/m\u003csup\u003e2\u003c/sup\u003e daily and 25 mg/m\u003csup\u003e2\u003c/sup\u003e weekly, respectively. These dosages were adjusted to maintain the target leukocyte count at 1,500\u0026ndash;3,000/\u0026micro;L. The therapy-induced toxicities were evaluated, and laboratory data were collected on whole blood cell counts and liver enzymes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGenotyping\u003c/h2\u003e \u003cp\u003eGermline DNA was extracted from peripheral blood samples collected in EDTA tubes during maintenance therapy. \u003cem\u003eNUDT15\u003c/em\u003e exon 1 and 3 genotypes were identified using Sanger sequencing. \u003cem\u003eTPMT\u003c/em\u003e c.238G\u0026thinsp;\u0026gt;\u0026thinsp;C, c.460G\u0026thinsp;\u0026gt;\u0026thinsp;A, and c.719A\u0026thinsp;\u0026gt;\u0026thinsp;G genotypes were genotyped using the TaqMan probe method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eQuantifying 6-MP metabolite levels\u003c/h2\u003e \u003cp\u003eThe TGNs and methyl mercaptopurine nucleotide (MMPN) levels in erythrocytes and DNA-TG were determined. Samples were collected multiple times from each patient during maintenance therapy. The DNA concentration was measured using the Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003cp\u003e6-MP metabolites in erythrocytes and DNA were measured using ultra-high performance liquid chromatography-tandem mass spectrometry (VantageTQD; Thermo Fisher Scientific). TGNs and MMPN levels were measured as previously described with modifications [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] Briefly, 50 \u0026micro;L of internal standard, including 12.5 \u0026micro;mol/L 2-amino-6MP\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e15\u003c/sup\u003eN and 6-methylmercaptopurine-d3, 250 \u0026micro;L of 100 mmol/L dithiothreitol, and 20 \u0026micro;L of 60% perchloric acid, were added to 25 \u0026micro;L of erythrocytes and mixed thoroughly using a vortex mixer. The mixture was placed on ice for 10 min and centrifuged at 13,000 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 10 min. The supernatant was incubated at 95\u0026deg;C for 1 h, and the equivalent mobile phase was added after cooling. After filtration using a 0.2 \u0026micro;m filter, 5 \u0026micro;L was injected for quantification. Chromatography was conducted under gradient conditions at 30\u0026deg;C using a CORTECS UPLC C18\u0026thinsp;+\u0026thinsp;instrument (2.1 \u0026times; 50 mm, 1.6 \u0026micro;m; Waters Corporation, Milford, MA, USA). The flow rate was 0.5 mL/min. The runs were performed using a gradient. Buffers A and B were 2 mmol/L ammonium acetate and 0.1% formic acid in water and methanol, respectively. The gradient conditions were as follows: 0\u0026ndash;1.8 min, 3% B; 1.8\u0026ndash;2.3 min, 95% B; and 2.5\u0026ndash;5 min, 3% B.\u003c/p\u003e \u003cp\u003eDNA-TG levels were quantified as previously described with modifications. Briefly, 50 \u0026micro;L of DNA was extracted, which included 500\u0026ndash;5000 ng of DNA and 1 \u0026micro;L of 59.1 \u0026micro;mol/L 6-methylmercaptopurine-d3, and incubated at 100\u0026deg;C for 5 min. After cooling, 5.6 \u0026micro;L of 10\u0026times; digestion buffer (500 mM sodium acetate, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e pH 5.3) and 5 \u0026micro;L of 0.12 U/\u0026micro;L nuclease P1 (Sigma-Aldrich, St. Louis, MO, USA) were added and incubated at 50\u0026deg;C for 1 h. Subsequently, 7 \u0026micro;L of 500 mM Tris-HCl (pH 8.0) and 1 \u0026micro;L of 3 U/\u0026micro;L calf intestine alkaline phosphatase (TOYOBO, Osaka, Japan) were added and incubated at 37\u0026deg;C for 30 min. After filtration using a 0.2 \u0026micro;m filter, 5 \u0026micro;L was injected for quantification. Chromatography was conducted under gradient conditions at 30\u0026deg;C using a CORTECS UPLC C18\u0026thinsp;+\u0026thinsp;instrument (2.1 \u0026times; 50 mm, 1.6 \u0026micro;m; Waters Corporation). The flow rate was 0.4 mL/min. The runs were performed using a gradient. Buffers C and D contained 0.05% formic acid in water and acetonitrile, respectively. The gradient conditions were as follows: 0\u0026ndash;0.1 min, 1% D; 1.1 min, 3% D; 2.4 min, 8% D; 4.1 min, 30% D; 4.5 min, 5% D; and 5.6 min, 1% D.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eThe thiopurine metabolite values of the genotypes were estimated using the Kruskal\u0026ndash;Wallis test. The relationship between the 6-MP dose and thiopurine metabolite levels was estimated using Pearson\u0026rsquo;s correlation coefficient. The difference between the two groups was estimated using the Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e-test. Statistical analyses were performed using GraphPad Prism 9 (DotMatics, Boston, MA, USA) and R statistical software (v 4.0.2; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.r-project.org/\u003c/span\u003e\u003cspan address=\"http://www.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eA total of 171 samples from 20 patients were collected to measure the thiopurine metabolite levels (DNA-TG, TGNs, and MMPN) during maintenance therapy (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The median 6-MP and MTX doses were 47.6 (1.7\u0026ndash;86.4) mg/m\u003csup\u003e2\u003c/sup\u003e daily and 20.2 (9.9\u0026ndash;41) mg/m\u003csup\u003e2\u003c/sup\u003e weekly, respectively. Three patients had the NUDT15 intermediate genotype (*1/*2 or *1/*3), and two patients had a low activity genotype (*3/*3). None of the patients had a \u003cem\u003eTPMT\u003c/em\u003e variant that reduced enzyme activity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient characteristics and 6-MP metabolites\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eNUDT15\u003c/em\u003e genotype\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e*1/*1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e*1/*3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e*1/*2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e*3/*3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003cp\u003eAge (years old)\u003c/p\u003e \u003cp\u003eMale/Female\u003c/p\u003e \u003cp\u003e6-mercaptopurine dose (mg/m\u003csup\u003e2\u003c/sup\u003e/day)\u003c/p\u003e \u003cp\u003eMethotrexate dose (mg/m\u003csup\u003e2\u003c/sup\u003e/week)\u003c/p\u003e \u003cp\u003eNumber of sampling points\u003c/p\u003e \u003cp\u003e6-mercaptopurine metabolites concentration\u003c/p\u003e \u003cp\u003eDNA-TG (fmol/ugDNA)\u003c/p\u003e \u003cp\u003eErythrocyte TGNs (pmol/8\u0026times;10\u003csup\u003e8\u003c/sup\u003eRBC)\u003c/p\u003e \u003cp\u003eErythrocyte MMP (pmol/8\u0026times;10\u003csup\u003e8\u003c/sup\u003eRBC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e7/11\u003c/p\u003e \u003cp\u003e47.8\u003c/p\u003e \u003cp\u003e19.5\u003c/p\u003e \u003cp\u003e171\u003c/p\u003e \u003cp\u003e351.2\u003c/p\u003e \u003cp\u003e419.0\u003c/p\u003e \u003cp\u003e20773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e4/11\u003c/p\u003e \u003cp\u003e48.2\u003c/p\u003e \u003cp\u003e19.6\u003c/p\u003e \u003cp\u003e142\u003c/p\u003e \u003cp\u003e344.8\u003c/p\u003e \u003cp\u003e457.0\u003c/p\u003e \u003cp\u003e24786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e1/0\u003c/p\u003e \u003cp\u003e67.5\u003c/p\u003e \u003cp\u003e25.8\u003c/p\u003e \u003cp\u003e6\u003c/p\u003e \u003cp\u003e728.4\u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e149.3\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e32212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e1/1\u003c/p\u003e \u003cp\u003e32.4\u003c/p\u003e \u003cp\u003e19.4\u003c/p\u003e \u003cp\u003e18\u003c/p\u003e \u003cp\u003e349.4\u003c/p\u003e \u003cp\u003e358.0\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e9739\u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e1/1\u003c/p\u003e \u003cp\u003e2.7\u003c/p\u003e \u003cp\u003e12.5\u003c/p\u003e \u003cp\u003e5\u003c/p\u003e \u003cp\u003e178.8\u003c/p\u003e \u003cp\u003e10.0\u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e50.0\u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e\u0026dagger;\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (vs. *1/*1).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eAbbreviation; DNA-TG, thioguanine incorporate into DNA; TGNs, thioguanine nucleotides; MMPN, methyl mercaptopurine\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eThiopurine metabolite levels\u003c/h2\u003e \u003cp\u003eThe median DNA-TG level was 351.2 fmol/\u0026micro;g DNA in the 171 samples (interquartile range [IQR], 248.5\u0026ndash;481.4). The DNA-TG levels of patients with the \u003cem\u003eNUDT15\u003c/em\u003e variants were significantly higher than those with the *1/*1 genotype (740.5 vs. 304.0 fmol/\u0026micro;g DNA, p\u0026thinsp;=\u0026thinsp;1.35 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e). The median DNA-TG levels in individuals varied three-fold in the \u003cem\u003eNUDT15\u003c/em\u003e*1/*1 genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The median erythrocyte TGNs level was 419.0 pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e red blood cells (RBC; IQR, 305.6\u0026ndash;542.0). The median TGNs levels varied among individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A three-fold difference was observed for each individual with the \u003cem\u003eNUDT15\u003c/em\u003e *1/*1 genotype, and the number of thiopurine metabolites in the erythrocytes was lower than the limit of quantification (10 pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e RBC) for patients with the NUDT15 deficient genotype (*3/*3). Erythrocyte TGNs levels in \u003cem\u003eNUDT15\u003c/em\u003e variants were lower than those in patients with the *1/*1 genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The median erythrocyte MMPN levels were 20,773 pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e RBCs (IQR, 9,742\u0026ndash;34,319), and the amount of thiopurine metabolites in erythrocytes was lower than the limit of quantification (10 pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e RBCs for TGNs and 50 pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e RBCs for MMPN).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 6-MP dose significantly correlated with all three 6-MP metabolite levels in the evaluation of all points (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In patients with the *1/*1 genotype and \u003cem\u003eNUDT15\u003c/em\u003e variants, DNA-TG (p\u0026thinsp;=\u0026thinsp;0.0006 and \u0026lt;\u0026thinsp;0.0001; r\u0026thinsp;=\u0026thinsp;0.29 and 0.74, respectively) and MMPN levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and \u0026lt;\u0026thinsp;0.0001; r\u0026thinsp;=\u0026thinsp;0.70 and 0.90, respectively) were also significantly correlated with the 6-MP dose; however, the TGN level was only weakly correlated (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and r\u0026thinsp;=\u0026thinsp;0.15 and 0.33, respectively). The regression line between the 6-MP dose and MMPN level showed a similar trend for each \u003cem\u003eNUDT15\u003c/em\u003e *1/*1 genotype and variant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe DNA-TG level to dose (DNA-TG/dose) ratio in patients with the \u003cem\u003eNUDT15\u003c/em\u003e variant was significantly higher than in those with the *1/*1 genotype (p\u0026thinsp;=\u0026thinsp;0.004, Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e-test). The DNA-TG/dose ratio ranged from 5 to 10 in almost all patients with the \u003cem\u003eNUDT15\u003c/em\u003e *1/*1 genotype. Patients with \u003cem\u003eNUDT15\u003c/em\u003e variants and a TGNs level/dose ratio of \u0026gt;\u0026thinsp;15 pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e RBCs/mg had a higher DNA-TG/dose ratio of \u0026gt;\u0026thinsp;10 pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e RBCs/mg of patients with \u003cem\u003eNUDT15\u003c/em\u003e *1/*1.\u003c/p\u003e \u003cp\u003eThe DNA-TG/TGNs fomol/\u0026micro;gDNA/(pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e RBCs) ratio correlated with the TGNs level (r = -0.67, p\u0026thinsp;=\u0026thinsp;0.0011) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In patients with the \u003cem\u003eNUDT15\u003c/em\u003e *1/*1 genotype, the median DNA-TG level correlated with TGNs levels (r\u0026thinsp;=\u0026thinsp;0.60, p\u0026thinsp;=\u0026thinsp;0.017). The ratios of the DNA-TG/TGNs levels\u0026mdash;which show the DNA incorporation ratio\u0026mdash;were affected by the \u003cem\u003eNUDT15\u003c/em\u003e genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and r = -0.33), and these correlations were observed in each wild type and \u003cem\u003eNUDT15\u003c/em\u003e variant. The DNA-TG/TGNs ratio was 0.71 fomol/\u0026micro;gDNA/(pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e RBCs) for patients with the \u003cem\u003eNUDT15\u003c/em\u003e *1/*1 genotype (IQR, 0.67\u0026ndash;1.08). The DNA-TG/TGNs ratio in patients with the \u003cem\u003eNUDT15\u003c/em\u003e variant was significantly higher than those with the *1/*1 genotype (*1/*1, 0.72; variant, 5.07, p\u0026thinsp;=\u0026thinsp;0.0015, Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e-test). The DNA-TG/TGNs ratio showed an inverse correlation with the TGNs level in each patient (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDNA-TG levels were weakly correlated with MMPN levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, r\u0026thinsp;=\u0026thinsp;0.36) but not correlated with TGNs levels. In patients with the \u003cem\u003eNUDT15\u003c/em\u003e *1/*1 genotype, the DNA-TG level was weakly correlated with TGNs and MMP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThiopurine metabolite levels, blood cell counts, and hepatic enzymes\u003c/h2\u003e \u003cp\u003eThe association between laboratory data and the 6-MP metabolite levels at each sampling point was evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). WBC counts correlated with erythrocyte TGN levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, p\u0026thinsp;=\u0026thinsp;0.013, r = -0.19) but not with DNA-TG levels. Lymphocyte counts were weakly correlated with DNA-TG (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, r = -0.28), erythrocyte TGNs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, r = -0.36), and MMPN levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, r = -0.35). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels correlated with erythrocyte MMPN levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, r\u0026thinsp;=\u0026thinsp;0.32 and 0.46, respectively), and erythrocyte MMPNs levels were higher in ALT\u0026thinsp;\u0026gt;\u0026thinsp;200 IU than in ALT\u0026thinsp;\u0026lt;\u0026thinsp;200 IU (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e6-MP tolerability varies between individuals and is influenced by genotypes affected by thiopurine metabolite enzyme activity, such as TPMT and NUDT15. Thiopurine metabolite levels may directly influence the effect of 6-MP; however, the transition range of 6-MP metabolite concentrations during maintenance therapy is unclear. Therefore, this study measured the 6-MP metabolite concentrations at multiple therapeutic time points in each patient during maintenance therapy. The 6-MP metabolite levels correlated with the 6-MP dose and the DNA-TG/TGNs ratio was affected by the \u003cem\u003eNUDT15\u003c/em\u003e genotype and erythrocyte TGNs levels in Japanese children with ALL.\u003c/p\u003e \u003cp\u003eThe DNA-TG, erythrocyte TGNs, and MMPN levels positively correlated with the 6-MP dose at all sampling points. However, the variation in these 6-MP metabolite levels to dose differed in each patient. Asian patients with the \u003cem\u003eNUDT15\u003c/em\u003e variant required a lower 6-MP dose than those with the *1/*1 genotype during maintenance therapy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Regarding the difference in \u003cem\u003eNUDT15\u003c/em\u003e variants, the DNA-TG ratio to the 6-MP dose of patients with \u003cem\u003eNUDT15\u003c/em\u003e variants was higher than that of patients with the *1/*1 genotype. In a previous study, erythrocyte TGNs levels correlated with the 6-MP dose at the sampling point and the total 6-MP dose in the previous month [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, DNA-TG levels correlated with erythrocyte TGNs, which reached a plateau at high erythrocyte TGN levels in patients with childhood ALL with the \u003cem\u003eTPMT\u003c/em\u003e variant in Northern Europe [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The present study did not include patients with the \u003cem\u003eTPMT\u003c/em\u003e variant; however, five patients had the \u003cem\u003eNUDT15\u003c/em\u003e variant. The findings of this study showed that 6-MP metabolites correlated with the 6-MP dose, regardless of the genetic variant of the 6-MP metabolizing enzyme.\u003c/p\u003e \u003cp\u003e6-MP is metabolized by multiple enzymes, and the active metabolites (thioguanosine triphosphate [TGTP] and deoxy-TGTP) are incorporated into DNA. NUDT15 hydrolyzes TGTP to 6-thioguanosine 5\u0026prime;-monophosphate, and the findings of this study showed that the DNA-TG levels were higher in patients with the low-level \u003cem\u003eNUDT15\u003c/em\u003e variant than in those with the \u003cem\u003eNUDT15\u003c/em\u003e *1/*1 genotype. Interestingly, the DNA-TG/TGNs ratio significantly and inversely correlated with erythrocyte TGNs levels. RBCs do not have a nucleus, and circulating erythrocytes are only slightly affected by 6-MP metabolite levels. In contrast, WBCs are susceptible to the effects of 6-MP metabolites incorporated into DNA, which induce apoptosis. High DNA-TG levels induce cell death, and this study suggested that the DNA-TG/TGNs ratio is low at high erythrocyte TGNs levels.\u003c/p\u003e \u003cp\u003eRelapse-free survival is significantly associated with DNA-TG levels [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and the \u003cem\u003eNUDT15\u003c/em\u003e low-activity variant may have poor outcomes. Patients with low NUDT15 activity experienced severe blood cell decrease\u0026mdash;which may be caused by the rapid 6-MP active metabolite accumulation\u0026mdash;and required long-term dose cessation. Therefore, \u003cem\u003eNUDT15\u003c/em\u003e-deficient patients find it difficult to maintain sufficient DNA-TG levels in cells. In an animal model, implanted \u003cem\u003eNUDT15\u003c/em\u003e-/- murine leukemic cells were treated with 6-MP at a tolerable dose, and the mice were completely leukemia-free for the entire experiment[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the present study was a preliminary study to confirm the relationship between the 6-MP dose and thiopurine metabolites in \u003cem\u003eNUDT15\u003c/em\u003e variants; therefore, the differences in outcomes in 6-MP metabolite levels were not investigated. The study results showed that DNA-TG levels positively correlated with the 6-MP dose and the erythrocyte TGNs level regulated TGNs incorporation. Therefore, \u003cem\u003eNUDT15\u003c/em\u003e genotypes and erythrocyte TGNs levels are predictive markers of DNA-TG level variation in Japanese patients with ALL.\u003c/p\u003e \u003cp\u003eIn maintenance therapy, the 6-MP dose is typically adjusted according to the degree of myelosuppression while monitoring the WBC count. Patients with low NUDT15 activity receiving a 6-MP standard protocol dose inevitably experience severe myelosuppression, and the daily 6-MP dose needs to be adjusted to \u0026gt;\u0026thinsp;10 mg/m\u003csup\u003e2\u003c/sup\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Erythrocyte TGN and MMP are associated with 6-MP-induced toxicities [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this study, the erythrocyte TGNs levels were associated with decreasing WBC and lymphocyte counts. Generally, the lifetime of neutrophils is shorter than that of lymphocytes and erythrocytes. Therefore, 6-MP metabolites accumulated more in lymphocytes than in neutrophils, and toxicity to lymphocytes may be associated with erythrocyte TGNs levels. Erythrocyte TGNs levels significantly correlate with neutrophil counts after 14 days of sampling [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the results of this study for the total sampling point showed no relationship between neutrophil count and 6-MP metabolite levels. These results indicated cell death at a high DNA-TG level. Only a few reports have shown thiopurine metabolite- and 6-MP-induced toxicities in \u003cem\u003eNUDT15\u003c/em\u003e variants. In a previous study, erythrocyte TGNs levels in hematopoietic toxicity in low-activity \u003cem\u003eNUDT15\u003c/em\u003e were \u0026lt;\u0026thinsp;171 pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e in maintenance therapy for Korean children with ALL [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the \u003cem\u003eNUDT15\u003c/em\u003e low-activity variant, DNA-TG levels were lower than those in the other variants. Therefore, DNA-TG is a principal cytotoxic metabolite [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, aminotransferase levels were significantly associated with erythrocyte MMPN levels. This relationship aligns with that reported by Nygaard et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, high MMPN levels induce 6-MP-related hepatotoxicity.\u003c/p\u003e \u003cp\u003eThe limitation of this study was that the 6-MP dose was adjusted based on the patients\u0026rsquo; 6-MP sensitivity during the prior treatment phase with 6-MP and laboratory data of WBC counts, liver enzymes, and bilirubin in peripheral blood. Blood sampling was conducted 14 days or later after the 6-MP dose adjustment.\u003c/p\u003e \u003cp\u003eIn conclusion, the 6-MP dose correlated with DNA-TG and erythrocyte TGNs levels during maintenance therapy in Japanese children with ALL. In the low \u003cem\u003eNUDT15\u003c/em\u003e activity genotype, the DNA-TG/6-MP dose ratio and erythrocyte TGNs level were high; however, the DNA-TG level was lower than that in the other genotypes. The DNA-TG/TGNs ratio also differed in each patient. In summary, the \u003cem\u003eNUDT15\u003c/em\u003e genotype and erythrocyte TGNs level elevated the DNA-TG level in Japanese patients with ALL. Future studies will evaluate the association between thiopurine metabolites and patient outcomes in patients with the \u003cem\u003eNUDT15\u003c/em\u003e genotype.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are not openly available due to reasons of ethical reason and are available from the corresponding author upon reasonable request. Data are located in controlled access data storage at National Institute of Health Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch involving human participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The Ethics Committee of each institution approved the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the parents or guardians of the patients or the patients themselves, depending on the age and conceptual ability of the patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments and Funding Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by JSPS KAKENHI (grant number JP18K06756).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYoichi Tanaka wrote the main manuscript and text. 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Cancer Res Treat 50:872\u0026ndash;882. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4143/crt.2017.283\u003c/span\u003e\u003cspan address=\"10.4143/crt.2017.283\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbbesen MS, Nersting J, Jacobsen JH et al (2013) Incorporation of 6-thioguanine nucleotides into DNA during maintenance therapy of childhood acute lymphoblastic leukemia-the influence of thiopurine methyltransferase genotypes. J Clin Pharmacol 53:670\u0026ndash;674. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcph.81\u003c/span\u003e\u003cspan address=\"10.1002/jcph.81\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-chemotherapy-and-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccap","sideBox":"Learn more about [Cancer Chemotherapy and Pharmacology](http://link.springer.com/journal/280)","snPcode":"280","submissionUrl":"https://submission.nature.com/new-submission/280/3","title":"Cancer Chemotherapy and Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"DNA-incorporated deoxythioguanosine, thioguane nucleotides, 6-mercaptopurine, NUDT15, childhood acute lymphoblastic leukemia, toxicities","lastPublishedDoi":"10.21203/rs.3.rs-4634919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4634919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThis study aimed to measure thiopurine metabolites\u0026mdash;such as deoxythioguanosine incorporated into DNA (DNA-TG)\u0026mdash;and erythrocyte thioguanine nucleotides (TGNs) and methyl mercaptopurine (MMPN) levels in Japanese children with acute lymphoblastic leukemia (ALL). This study also evaluated the factors that elevate thiopurine metabolites incorporated into the DNA.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eDNA-TG, erythrocyte TGNs, and MMPN levels were measured on consecutive clinical visits in 20 Japanese patients with childhood ALL (171 sampling points) using liquid chromatography with tandem mass spectrometry. Nudix hydrolase 15 (\u003cem\u003eNUDT15\u003c/em\u003e) was genotyped using Sanger sequencing.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOf the 20 patients, three had the NUDT15 intermediate activity genotype (*1/*2 or *1/*3), and two had a low activity genotype (*3/*3). The median DNA-TG level was 318 fmol/\u0026micro;g DNA. Erythrocyte TGNs and MMPN levels were 341.4 and 14,136 pmol/8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e red blood cells, respectively. The ratio of DNA-TG/TGNs\u0026mdash;which is the active thiopurine metabolite ratio in DNA\u0026mdash;was higher in patients with the \u003cem\u003eNUDT15\u003c/em\u003e variant than in the wild type. The DNA-TG/TGNs ratio was inversely correlated with the TGNs level. Erythrocyte TGNs levels were significantly correlated with white blood cell and lymphocyte counts (p\u0026thinsp;=\u0026thinsp;0.02 and 0.01, respectively), and MMPN levels were significantly correlated with lymphocyte count and aspartate and alanine aminotransferase levels (p\u0026thinsp;=\u0026thinsp;0.005, 0.0004, and 0.007, respectively).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe DNA-TG/TGNs ratio differed in each patient. The \u003cem\u003eNUDT15\u003c/em\u003e genotype and erythrocyte TGNs level were affected and elevated the DNA-TG level in Japanese patients with ALL.\u003c/p\u003e","manuscriptTitle":"NUDT15 genotype and erythrocyte thioguanosine levels affect thiopurine metabolite levels into DNA of Japanese children with acute lymphoblastic leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 20:52:52","doi":"10.21203/rs.3.rs-4634919/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-08T22:47:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-13T02:34:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-10T21:06:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224114990496933302736430139233456020795","date":"2024-06-30T14:56:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192658472125651810777083845303627643548","date":"2024-06-30T12:39:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-28T02:42:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-26T02:12:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-26T02:12:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Chemotherapy and Pharmacology","date":"2024-06-25T08:44:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-chemotherapy-and-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccap","sideBox":"Learn more about [Cancer Chemotherapy and Pharmacology](http://link.springer.com/journal/280)","snPcode":"280","submissionUrl":"https://submission.nature.com/new-submission/280/3","title":"Cancer Chemotherapy and Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"654f265e-c015-44fd-8f03-b95eec6c59f7","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-07T15:58:12+00:00","versionOfRecord":{"articleIdentity":"rs-4634919","link":"https://doi.org/10.1007/s00280-025-04774-9","journal":{"identity":"cancer-chemotherapy-and-pharmacology","isVorOnly":false,"title":"Cancer Chemotherapy and Pharmacology"},"publishedOn":"2025-04-01 15:56:51","publishedOnDateReadable":"April 1st, 2025"},"versionCreatedAt":"2024-07-22 20:52:52","video":"","vorDoi":"10.1007/s00280-025-04774-9","vorDoiUrl":"https://doi.org/10.1007/s00280-025-04774-9","workflowStages":[]},"version":"v1","identity":"rs-4634919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4634919","identity":"rs-4634919","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}