Thiopurines e.g. mercaptopurine (MP) are widely used as chemotherapeutic agents in the treatment of pediatric acute lymphoblastic leukemia 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 the 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 acute lymphoblastic leukemia, 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 because of 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). Cytosolic TGN and nuclear DNA-TG were correlated positively with each other across genotype groups (P=6.5×10), but the ratio of DNA-TG to TGN was significantly higher in NUDT15-deficient patients (P=3.6×10), 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.
Summary Background Adjustment of mercaptopurine and methotrexate maintenance therapy of acute lymphoblastic leukaemia by leucocyte count is confounded by natural variations. Cytotoxicity is primarily ...mediated by DNA-incorporated thioguanine nucleotides (DNA-TGN). The aim of this study was to establish whether DNA-TGN concentrations in blood leucocytes during maintenance therapy are associated with relapse-free survival. Methods In this substudy of the NOPHO ALL2008 phase 3 trial done in 23 hospitals in seven European countries (Denmark, Estonia, Finland, Iceland, Lithuania, Norway, and Sweden), we analysed data from centralised and blinded analyses of 6-mercaptopurine and methotrexate metabolites in blood samples from patients with non-high-risk childhood acute lymphoblastic leukaemia. Eligible patients were aged 1·0–17·9 years; had been diagnosed with non-high-risk precursor B-cell or T-cell leukaemia; had been treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol; and had reached maintenance therapy in first remission. Maintenance therapy was (mercaptopurine 75 mg/m2 once per day and methotrexate 20 mg/m2 once per week, targeted to a leucocyte count of 1·5–3·0 × 109 cells per L). We measured DNA-TGN and erythrocyte concentrations of TGN nucleotides, methylated mercaptopurine metabolites, and methotrexate polyglutamates. The primary objective was the association of DNA-TGN concentrations and 6-mercaptopurine and methotrexate metabolites with relapse-free survival. The secondary endpoint was the assessment of DNA-TGN concentration and 6-mercaptopurine and methotrexate metabolites during maintenance therapy phase 2. Findings Between Nov 26, 2008 and June 14, 2016, 1509 patients from the NOPHO ALL2008 study were assessed for eligibility in the DNA-TGN substudy, of which 918 (89%) of 1026 eligible patients had at least one DNA-TGN measurement and were included in the analyses. Median follow-up was 4·6 years (IQR 3·1–6·1). Relapse-free survival was significantly associated with DNA-TGN concentration (adjusted hazard ratio 0·81 per 100 fmol/μg DNA increase, 95% CI 0·67–0·98; p=0·029). In patients with at least five blood samples, erythrocyte concentrations of TGN, methylated mercaptopurine metabolites, and methotrexate polyglutamates were associated with DNA-TGN concentration (all p<0·0001). Interpretation Our results suggest the need for intervention trials to identify clinically applicable strategies for individualised drug dosing to increase DNA-TGN concentration, and randomised studies to investigate whether such strategies improve cure rates compared with current dose adjustments based on white blood cell counts. Funding Danish Cancer Society, Childhood Cancer Foundation (Denmark), Childhood Cancer Foundation (Sweden), Nordic Cancer Union, Otto Christensen Foundation, University Hospital Rigshospitalet, and Novo Nordic Foundation.
Purpose
Methotrexate (MTX)/6-Mercaptopurine (6MP)-based maintenance therapy is crucial to cure childhood acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated by incorporation of thioguanine ...nucleotides (TGN) into DNA (DNA-TG) with higher levels in leucocytes being associated with reduced relapse risk. To further understand the dynamics of DNA-TG formation, we measured DNA-TG levels in leucocyte subsets during maintenance therapy and in the months following its discontinuation.
Methods
DNA-TG levels were measured in leucocytes (DNA-TG
Total
), polymorph nucleated granulocytes (neutrophils, eosinophils, basophils DNA-TG
PMN
) and mononucleated cells (lymphocytes, monocytes DNA-TG
MNC
) in 1013 samples from 52 patients on ALL maintenance therapy (951 samples during therapy and 62 samples after therapy discontinuation, respectively).
Results
Median DNA-TG
Total
, DNA-TG
PMN
and DNA-TG
MNC
during maintenance therapy were 539, 563 and 384 fmol/µg DNA, respectively. DNA-TG
PMN
displayed more pronounced fluctuation than DNA-TG
MNC
(range 0–3084 interquartile range IQR 271–881 versus 30–1411 IQR 270–509 fmol/µg DNA). DNA-TG
Total
was more strongly correlated with DNA-TG
PMN
(
r
S
= 0.95,
p
< 0.0001) than DNA-TG
MNC
(
r
S
= 0.73,
p
< 0.0001). DNA-TG
PMN
correlated less with DNA-TG
MNC
(
r
S
= 0.64,
p
< 0.0001) and to a much lesser extent with absolute neutrophil count (
r
S
= 0.35,
p
< 0.0001). Following discontinuation of therapy, DNA-TG
PMN
was rapidly eliminated, and not measurable beyond day 22 after discontinuation, whereas DNA-TG
MNC
was slowly eliminated, and five patients demonstrated a measurable DNA-TG
MNC
more than 365 days after therapy discontinuation.
Conclusion
Fluctuations in DNA-TG
Total
are predominantly caused by corresponding fluctuations in DNA-TG
PMN
, thus DNA-TG
Total
measures recent TGN incorporation in these short-lived cells. Measurement of DNA-TG
Total
at 2–4 weeks intervals provides a reliable profile of DNA-TG levels.
Thiopurines are S-substituted antimetabolites that are widely used in the treatment of hematological malignancies and as immunosuppressants. Because of extensive inter-individual variation in drug ...disposition and the significant toxicity associated with thiopurine therapy, there is a need for improved individualized treatment. We here present a fast and sensitive method for quantifying the pharmacological end-point of thiopurines, 6-thioguanine (TG) in chromosomal DNA. Purine nucleobases are released from DNA, etheno-derivatized with chloroacetaldehyde, separated by HILIC and quantified by tandem mass spectrometry using endogenous chromosomal guanine as internal standard. The method is linear up to at least 10
pmol TG/μg DNA and the limit of detection and quantification are 4.2 and 14.1
fmol TG/μg DNA, respectively. The matrix (DNA) had no effect upon quantification of TG. SPE recovery was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45–1190
fmol/μg DNA) with intra- and inter-day RSDs of less than 11%. The method uses 2
μg DNA/sample, which can easily be obtained from these patients.
Purpose
Mercaptopurine (6MP) is essential to cure childhood acute lymphoblastic leukemia (ALL). A liquid 6MP formulation was recently introduced to facilitate oral 6MP administration, especially to ...children. Its approval and bioequivalence with 6MP tablet were based on comparative pharmacokinetics in 60 healthy adults. Due to potential pharmacokinetic differences between healthy adults and children with ALL, we compared pharmacokinetics of tablet and liquid 6MP formulations in children with ALL.
Methods
Pharmacokinetics of 50 mg 6MP tablet (Puri-Nethol
®
) and 20 mg/ml 6MP liquid suspension (Xaluprine
®
) were compared in a non-blinded, random order, single-dose, cross-over study in 16 children with ALL (eight males). 6MP was administered after a 12 h fast, and 6MP plasma concentrations measured consecutively over seven hours post-dose. Pharmacokinetic outcomes were as follows: Area under the curve (AUC), maximum plasma concentration (
C
max
), time to maximum plasma concentration (
T
max
), and terminal half-life (
T
½
).
Results
Liquid 6MP formulation resulted in a 26% lower AUC (
p
= 0.02) compared with tablet (median 1215 vs. 1805 h × nmol/l). No significant differences were observed for
C
max,
T
max
and
T
½
(
p
= 0.28,
p
= 0.09,
p
= 0.41, respectively). Based on criteria declared by the World Health Organization the results did not establish non-inferiority of liquid 6MP formulation compared with 6MP tablet.
Conclusion
Non-inferiority of liquid 6MP formulation compared with 6MP tablet was not demonstrated. Yet, maintenance therapy doses are adjusted by degree of myelosuppression and not by 6MP dose. Thus, in spite of a lower bioavailability, a liquid 6MP formulation is still desirable in a clinical setting, especially for children. However, if shifting between 6MP formulation is indicated, dose adjustments should be anticipated to maintain equivalent treatment intensity in children with ALL.
The study is registered on clinicaltrials.gov (NCT01906671). Date of registration: 24.07.13.
Methotrexate/6-mercaptopurine maintenance therapy improves acute lymphoblastic leukemia (ALL) outcome. Cytotoxicity is mediated by DNA incorporation of thioguanine nucleotides (DNA-TG). We ...investigated the association of DNA-TG to relapse risk in 1 910 children and young adults with non-high risk ALL. In a cohort-stratified Cox regression analysis adjusted for sex, age, and white cell count at diagnosis, the relapse-specific hazard ratio (HRa) per 100 fmol/μg increase in weighted mean DNA-TG (
DNA-TG) was 0.87 (95% CI 0.78-0.97; p = 0.013) in the 839 patients who were minimal residual disease (MRD) positive at end of induction therapy (EOI), whereas this was not the case in EOI MRD-negative patients (p = 0.76). Validation analysis excluding the previously published Nordic NOPHO ALL2008 pediatric cohort yielded a HRa of 0.92 (95% CI 0.82-1.03; p = 0.15) per 100 fmol/μg increase in
DNA-TG in EOI MRD-positive patients. If also excluding the United Kingdom cohort, in which samples were taken non-randomly in selected patients, the HRa for the EOI MRD-positive patients was 0.82 (95% CI 0.68-0.99; p = 0.044) per 100 fmol/μg increase in
DNA-TG. The importance of DNA-TG as a biomarker for maintenance therapy intensity calls for novel strategies to increase DNA-TG, although its clinical value may vary by protocol backbone.
Survival of children with relapsed acute lymphoblastic leukemia is poor, and understanding mechanisms underlying resistance is essential to developing new therapy. Relapse-specific heterozygous ...deletions in
, a crucial part of DNA mismatch repair, are frequently detected. Our aim was to determine whether
deletion results in a hypermutator phenotype associated with generation of secondary mutations involved in drug resistance, or if it leads to a failure to initiate apoptosis directly in response to chemotherapeutic agents. We knocked down
in mismatch repair proficient cell lines (697 and UOCB1) and showed significant increases in IC50s to 6-thioguanine and 6-mercaptopurine (697: 26- and 9-fold; UOCB1: 5- and 8-fold)
, as well as increased resistance to 6-mercaptopurine treatment
No shift in IC50 was observed in deficient cells (Reh and RS4;11). 697 MSH6 knockdown resulted in increased DNA thioguanine nucleotide levels compared to non-targeted cells (3070
1722 fmol/μg DNA) with no difference observed in mismatch repair deficient cells. Loss of MSH6 did not give rise to microsatellite instability in cell lines or clinical samples, nor did it significantly increase mutation rate, but rather resulted in a defect in cell cycle arrest upon thiopurine exposure.
knockdown cells showed minimal activation of checkpoint regulator CHK1, γH2AX (DNA damage marker) and p53 levels upon treatment with thiopurines, consistent with intrinsic chemoresistance due to failure to recognize thioguanine nucleotide mismatching and initiate mismatch repair. Aberrant
adds to the list of alterations/mutations associated with acquired resistance to purine analogs emphasizing the importance of thiopurine therapy.
Purpose
6-mercaptopurine(6MP)/methotrexate maintenance therapy is essential to reduce relapse of childhood acute lymphoblastic leukemia (ALL). Common germline variants in
TPMT
cause low activity of ...thiopurine methyltransferase (TPMT) and higher 6MP metabolite (TGN) levels. Higher levels of TGNs incorporated into DNA (DNA-TG) and low TPMT activity have previously been associated with a lower relapse risk. We explored if TPMT geno- or phenotype was associated with DNA-TG levels and relapse rate in NOPHO ALL2008.
Methods
TPMT
genotype, repeated phenotyping, and DNA-TG measurements were collected in 918 children with non-high risk ALL (NOPHO ALL2008 maintenance therapy study). Maintenance therapy started with 6MP at 50 and 75 mg/m
2
for
TPMT
heterozygous and wildtype patients and was adjusted to a target WBC of 1.5 − 3.0 × 10
9
/L.
Results
Of 918 patients, 78 (8.5%) were
TPMT
heterozygous and 903 had at least one TPMT measurement (total 3063). Mean TPMT activities were higher with wildtype than heterozygous
TPMT
(
N
= 752, 16.6 versus 9.6 U/mL ery.,
p
< 0.001). The 5-year cumulative incidence of relapse was 6.4% and 6.0% for
TPMT
heterozygous and wildtype patients, and there was no association between genotype and relapse rate (
N
= 918, hazard ratio = 1.01, 95% confidence interval CI 0.40 − 2.54,
p
= 0.98). Although
TPMT
heterozygous patients had higher DNA-TG (
N
= 548, median 760.9 interquartile range (IQR) 568.7 − 890.3 versus 492.7 IQR 382.1 − 634.6 fmol/µg,
p
< 0.001), TPMT activity was not associated with relapse rate (
N
= 813; hazard ratio = 0.98 per one U/mL ery. increase in TPMT activity, 95% CI 0.91 − 1.06,
p
= 0.67).
Conclusion
TPMT geno- and phenotype were not associated with relapse in non-high risk NOPHO ALL2008.
Maintenance therapy containing methotrexate and 6-mercapto - purine is essential to cure acute lymphoblastic leukemia (ALL). Cytotoxicity is elicited by incorporation of thioguanine nucleotides into ...DNA (DNA-TG), and higher leukocyte DNA-TG is associated with increased relapse-free survival. As 6-thioguanine provides 6- fold higher cytosolic levels of thioguanine nucleotides than does 6- mercapto purine, we added low-dose 6-thioguanine to methotrexate/6- mercapto purine maintenance therapy to explore if this combination results in significantly higher DNA-TG. The target population of the "Thiopurine Enhanced ALL Maintenance therapy" (TEAM) study was 30 patients with non-high-risk ALL, aged 1-45 years on methotrexate/6-mercaptopurine maintenance therapy receiving no other systemic chemotherapy. Incremental doses of 6-thioguanine were added to methotrexate/6-mercaptopurine maintenance therapy (starting 6-thioguanine dose: 2.5 mg/m2/day, maximum: 12.5 mg/m2/day). The primary endpoint was DNA-TG increments. Thirty-four patients were included, and 30 patients completed maintenance therapy according to the TEAM strategy. Of these 30 patients, 26 (87%) tolerated 10.0-12.5 mg/m2/day as the maximum 6-thioguanine dose. TEAM resulted in significantly higher DNA-TG levels compared to those in both TEAM patients before their inclusion in TEAM (on average 251 fmol/mg DNA higher 95% confidence interval: 160-341; P<0.0001), and with historical patients receiving standard methotrexate/6-mercapto - purine maintenance therapy (on average 272 fmol/mg DNA higher 95% confidence interval: 147-398; P<0.0001). TEAM did not increase myelotoxicity or hepatotoxicity. In conclusion, TEAM is an innovative and feasible approach to improve maintenance therapy and results in higher DNA-TG levels without inducing additional toxicity. It may therefore be an effective strategy to reduce the risk of ALL relapse through increased DNA-TG. This will be tested in a randomized ALLTogether-1 substudy.
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