Jacob Nersting

Methotrexate/6-mercaptopurine maintenance therapy influences the risk of a second malignant neoplasm after childhood acute lymphoblastic leukemia - results from the NOPHO ALL-92 study

Among 1614 children with acute lymphoblastic leukemia (ALL) treated with the Nordic Society for Paediatric Haematology and Oncology (NOPHO) ALL-92 protocol, 20 patients developed a second malignant neoplasm (SMN) with a cumulative risk of 1.6% at 12 years from the diagnosis of ALL. Nine of the 16 acute myeloid leukemias or myelodysplastic syndromes had monosomy 7 (n = 7) or 7q deletions (n = 2). In Cox multivariate analysis, longer duration of oral 6-mercaptopurine (6MP)/methotrexate (MTX) maintenance therapy (P = .02; longest for standard-risk patients) and presence of high hyperdiploidy (P = .07) were related to increased risk of SMN. Thiopurine methyltransferase (TPMT) methylates 6MP and its metabolites, and thus reduces cellular levels of cytotoxic 6-thioguanine nucleotides. Of 524 patients who had erythrocyte TPMT activity measured, the median TPMT activity in 9 patients developing an SMN was significantly lower than in the 515 that did not develop an SMN (median, 12.1 vs 18.1 IU/mL; P = .02). Among 427 TPMT wild-type patients for whom the 6MP dose was registered, those who developed SMN received higher average 6MP doses than the remaining patients (69.7 vs 60.4 mg/m2; P = .03). This study indicates that the duration and intensity of 6MP/MTX maintenance therapy of childhood ALL may influence the risk of SMNs in childhood ALL.

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

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

Prevalence of t(12;21)[ETV6-RUNX1]-positive cells in healthy neonates.

t(12;21)(p13;q22)[ETV6-RUNX1] is the most common chromosomal translocation in childhood acute lymphoblastic leukemia, and it can often be backtracked to Guthrie cards supporting prenatal initiation and high levels of circulating t(12;21)-positive cells at birth. To explore the prevalence of ETV6-RUNX1-positive cells in healthy neonates, mononuclear cells from 1417 umbilical cord blood samples were isolated within 24 hours from birth and subsequently screened for ETV6-RUNX1 transcripts using a highly sensitive real-time reverse transcription polymerase chain reaction assay. In first-run polymerase chain reaction, 14 samples were positive at levels below 10(-5), of which specific hybridization reflecting the relevant genetic region was positive in 9 cases. Repeated analyses using stored mRNA and flowcytometric sorting of a CD19(+), CD8(+), and CD19(-)/CD8(-) subpopulations from cryopreserved mononuclear cells from the same cord blood samples (mean sorted: 18 × 10(6) cells) revealed no positive findings, which demonstrates that the level and/or frequency of ETV6-RUNX1-positive cells is markedly lower than suggested in previous studies.

Mercaptopurine/Methotrexate Maintenance Therapy of Childhood Acute Lymphoblastic Leukemia: Clinical Facts and Fiction

The antileukemic mechanisms of 6-mercaptopurine (6MP) and methotrexate (MTX) maintenance therapy are poorly understood, but the benefits of several years of myelosuppressive maintenance therapy for acute lymphoblastic leukemia are well proven. Currently, there is no international consensus on drug dosing. Because of significant interindividual and intraindividual variations in drug disposition and pharmacodynamics, vigorous dose adjustments are needed to obtain a target degree of myelosuppression. As the normal white blood cell counts vary by patients’ ages and ethnicity, and also within age groups, identical white blood cell levels for 2 patients may not reflect the same treatment intensity. Measurements of intracellular levels of cytotoxic metabolites of 6MP and MTX can identify nonadherent patients, but therapeutic target levels remains to be established. A rise in serum aminotransferase levels during maintenance therapy is common and often related to high levels of methylated 6MP metabolites. However, except for episodes of hypoglycemia, serious liver dysfunction is rare, the risk of permanent liver damage is low, and aminotransferase levels usually normalize within a few weeks after discontinuation of therapy. 6MP and MTX dose increments should lead to either leukopenia or a rise in aminotransferases, and if neither is experienced, poor treatment adherence should be considered. The many genetic polymorphisms that determine 6MP and MTX disposition, efficacy, and toxicity have precluded implementation of pharmacogenomics into treatment, the sole exception being dramatic 6MP dose reductions in patients who are homozygous deficient for thiopurine methyltransferase, the enzyme that methylates 6MP and several of its metabolites. In conclusion, maintenance therapy is as important as the more intensive and toxic earlier treatment phases, and often more challenging. Ongoing research address the applicability of drug metabolite measurements for dose adjustments, extensive host genome profiling to understand diversity in treatment efficacy and toxicity, and alternative thiopurine dosing regimens to improve therapy for the individual patient.

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

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.

Cost-effective multiplexing before capture allows screening of 25 000 clinically relevant SNPs in childhood acute lymphoblastic leukemia

Genetic variants, including single-nucleotide polymorphisms (SNPs), are key determiners of interindividual differences in treatment efficacy and toxicity in childhood acute lymphoblastic leukemia (ALL). Although up to 13 chemotherapeutic agents are used in the treatment of this cancer, it remains a model disease for exploring the impact of genetic variation due to well-characterized cytogenetics, drug response pathways and precise monitoring of minimal residual disease. Here, we have selected clinically relevant genes and SNPs through literature screening, and on the basis of associations with key pathways, protein–protein interactions or downstream partners that have a role in drug disposition and treatment efficacy in childhood ALL. This allows exploration of pathways, where one of several genetic variants may lead to similar clinical phenotypes through related molecular mechanisms. We have designed a cost-effective, high-throughput capture assay of ∼25 000 clinically relevant SNPs, and demonstrated that multiple samples can be tagged and pooled before genome capture in targeted enrichment with a sufficient sequencing depth for genotyping. This multiplexed, targeted sequencing method allows exploration of the impact of pharmacogenetics on efficacy and toxicity in childhood ALL treatment, which will be of importance for personalized chemotherapy.

Incorporation of 6‐Thioguanine Nucleotides into DNA During Maintenance Therapy of Childhood Acute Lymphoblastic Leukemia—The Influence of Thiopurine Methyltransferase Genotypes

Relapse of childhood acute lymphoblastic leukemia (ALL) may reflect both inherent chemoresistance of the leukemic clone and adverse drug disposition that primarily is determined by genetic polymorphisms . In the current Nordic–Baltic ALL2008 protocol, as well as in many other contemporary ALL protocols, all post‐induction treatment phases include the thiopurines, 6‐thioguanine or 6‐mercaptopurine (6‐MP), and the latter constitutes, together with methotrexate (MTX), the backbone of maintenance therapy. 6MP has a relatively narrow therapeutic index, and because 6MP disposition varies substantially, standard dosing may lead to under treatment and relapse in some patients 2 and life‐threatening toxicity or second malignant neoplasia (SMN) in others . 6‐mercaptopurine is a prodrug and its cytotoxicity depends on conversion to 6‐thioguanine nucleotides (TGN). TGN are incorporated into DNA (DNA‐TG) and are occasionally mismatched to thymidine, causing cell death by post‐replicative mismatch repair . The enzyme thiopurine methyltransferase (TPMT) methylates 6MP and thereby reduces the amount of the drug available for TGN formation . However, the methylated metabolite methyl‐thioinosine monophosphate (Me‐TIMP) is a potent inhibitor of purine de novo synthesis, which enhances the incorporation of TGN into DNA . Nucleotide polymorphisms in the TPMT gene results in approximately 90% of Caucasians being homozygous for high TPMT activity (TPMTwild type), 10% carry one low activity allele (TPMT heterozygous) and 1 in 300 carries two low activity alleles (enzyme deficient) . 6MP/MTX maintenance therapy studies have showed far higher red blood cell TGN levels (Ery‐6TGN) and lower relapse rates in patients with low TPMT activity compared with wild‐ type patients . This is noteworthy, since patients are adjusted to the same target white blood cell count (WBC) during maintenance therapy, and TPMT low activity patients obtain only slightly lowerWBC levels than TPMT wild type (TPMT) patients during maintenance . Nevertheless, virtually nothing is known about how these two groups differ in DNA‐TG levels during 6MP based maintenance therapy. As part of Nordic/Baltic NOPHO ALL2008 pharmacology studies we here report preliminary data on the effect of low activity TPMT variants (TPMT) on the levels of the end‐point metabolite DNA‐TG and its relation to TPMT enzyme activity, Ery‐TGN, and erythrocyte levels of methylated metabolites (Ery‐ MeMP) in a cohort of children with ALL undergoing 6MP/MTX maintenance therapy.

Pharmacogenetically based dosing of thiopurines in childhood acute lymphoblastic leukemia: Influence on cure rates and risk of second cancer

Previous studies have indicated that patients with thiopurine methyltransferase (TPMT) low activity (TPMTLA) have reduced risk of relapse but increased risk of second malignant neoplasm (SMN) compared to patients with TPMT wild‐type (TPMTWT) when treated with 6MP maintenance therapy starting doses of 75 mg/m2/day. To reduce SMN risk, 6MP starting doses were reduced to 50 mg/m2/day for patients with TPMT heterozygosity in the Nordic Society of Paediatric Haematology and Oncology (NOPHO) ALL2000 protocol.

DNA-thioguanine nucleotide concentration and relapse-free survival during maintenance therapy of childhood acute lymphoblastic leukaemia (NOPHO ALL2008): a prospective substudy of a phase 3 trial

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.

Maintenance therapy of childhood acute lymphoblastic leukemia revisited-Should drug doses be adjusted by white blood cell, neutrophil, or lymphocyte counts?

6‐Mercaptopurine (6MP) and methotrexate (MTX) based maintenance therapy is a critical phase of childhood acute lymphoblastic leukemia treatment. Wide interindividual variations in drug disposition warrant frequent doses adjustments, but there is a lack of international consensus on dose adjustment guidelines.