Adherence to 6-mercaptopurine (6-MP)/methotrexate maintenance treatment for acute lymphoblastic leukemia (ALL) is pivotal to preventing relapse, and the 6-MP metabolite DNA-incorporated thioguanine (DNA-TG) is associated with relapse risk. In the ALLTogether-1 (A2G1) Maintenance sub-study (EU CT nr 2022-501050-11-01), DNA-TG, thioguanine nucleotides (TGN), and methylated mercaptopurine metabolites (MeMP) are analyzed regularly. Upon levels below preset limits (TGN < 50, or MeMP < 200 or < 100 nmol/mmol hemoglobin for thiopurine S-methyltransferase (TPMT) wild type and heterozygous patients, respectively), the treating physician is informed of potential non-adherence. We investigated the feasibility of using DNA-TG as the primary flagging of potential non-adherence. We analyzed 6-MP metabolites in 3,074 blood samples from 368 children enrolled in the A2G1 Maintenance sub-study. In 6

Acute graft-versus-host disease (aGvHD) with gastrointestinal (GI) involvement is a significant cause of transplant-related morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Although elevated plasma Regenerating islet-derived 3α (REG3α) and reduced citrulline levels have been associated with GI aGvHD, their kinetics and predictive value in pediatric HSCT remains unclear. We measured plasma REG3α and citrulline levels weekly from pre-conditioning to day +30 post-transplant in 117 children (1-18 years) undergoing myeloablative, matched-donor HSCT. Patients developing GI aGvHD (n = 16, 14%) exhibited significantly increased REG3α levels from day +7 and onward, while citrulline levels were consistently reduced from before conditioning and during the first 2 weeks post-HSCT, particularly in those with lower GI aGvHD (all p < 0.05). No influence of GI infections was observed. In age-, sex-, diagnosis- and donor type-adjusted analyses, increasing REG3α (day +7) and decreasing citrulline (day 0) levels were independently associated with increased odds of lower GI aGvHD (OR = 2.25 (1.36-3.70), and OR = 15.43 (1.72-138.15) per doubling/5 μmol/L decrease, p < 0.05). Both markers showed notable predictive value, with a sensitivity and specificity of 0.86 and 0.79 for REG3α and 0.80/0.83 for citrulline. Early post-HSCT increases in REG3α and reductions in citrulline may serve as prognostic biomarkers for GI aGvHD, particularly for identifying lower GI involvement, supporting their use for risk-stratified immunosuppressive interventions.

Chemotherapy-induced mucositis increases the risk of blood stream infections (BSI) due to translocation of bacteria across the intestinal epithelium. Our study investigated if quantitative measures of intestinal mucositis severity, including plasma citrulline (a marker of functional enterocytes) and CCL20 (an intestinal immune homeostatic chemokine), could identify patients at risk of BSI. A total of 106 children with ALL undergoing induction treatment (NOPHO ALL 2008) were included and information regarding BSI episodes was collected from the patients' medical records. Twenty-seven patients (25%) developed BSI during induction. Patients with BSI had a larger decrease in citrulline after chemotherapy than patients without BSI, and nearly all BSI episodes (25/27) occurred in the group of patients exhibiting a drop in citrulline (OR = 6.4 [95% CI: 1.4-29.3], P = .008). Patients who developed BSI had higher plasma CCL20 levels on days 8, 15 and 22 than patients without BSI (all P < .05), and elevated CCL20 levels on day 8 increased the risk of subsequent BSI (OR = 1.57 [1.11-2.22] per doubling of CCL20 level, P = .01) in a multivariable logistic regression analysis. These findings suggest that children with ALL who develop BSI during chemotherapy are characterised by more severe intestinal mucositis, as measured by plasma citrulline and CCL20. These markers may be useful in early risk stratification to guide treatment decisions.

Journal Article Next-Level Natriuretic Peptide Measurement Get access Jacob Nersting, Jacob Nersting Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Search for other works by this author on: Oxford Academic Google Scholar Dijana Terzic, Dijana Terzic Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Search for other works by this author on: Oxford Academic Google Scholar Lasse Holst Hansen, Lasse Holst Hansen Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Search for other works by this author on: Oxford Academic Google Scholar Jens P Goetze Jens P Goetze Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Address correspondence to this author at: Department of Clinical Biochemistry, Section 3014, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Tel +45-3545-2202; e-mail JPG@dadlnet.dk. https://orcid.org/0000-0001-6356-3829 Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 69, Issue 4, April 2023, Pages 313–315, https://doi.org/10.1093/clinchem/hvad013 Published: 21 February 2023 Article history Received: 14 January 2023 Accepted: 26 January 2023 Published: 21 February 2023

Immune-checkpoint inhibitors (ICI) are highly effective in reinvigorating T cells to attack cancer. Nevertheless, a large subset of patients fails to benefit from ICI, partly due to lack of the cancer neoepitopes necessary to trigger an immune response. In this study, we used the thiopurine 6-thioguanine (6TG) to induce random mutations and thus increase the level of neoepitopes presented by tumor cells. Thiopurines are prodrugs which are converted into thioguanine nucleotides that are incorporated into DNA (DNA-TG), where they can induce mutation through single nucleotide mismatching. In a pre-clinical mouse model of a mutation-low melanoma cell line, we demonstrated that 6TG induced clinical-grade DNA-TG integration resulting in an improved tumor control that was strongly T cell dependent. 6TG exposure increased the tumor mutational burden, without affecting tumor cell proliferation and cell death. Moreover, 6TG treatment re-shaped the tumor microenvironment by increasing T and NK immune cells, making the tumors more responsive to immune-checkpoint blockade. We further validated that 6TG exposure improved tumor control in additional mouse models of melanoma. These findings have paved the way for a phase I/II clinical trial that explores whether treatment with thiopurines can increase the proportion of otherwise treatment-resistant cancer patients who may benefit from ICI therapy (NCT05276284).

Background Methotrexate is widely used in inflammatory diseases during the patients' reproductive years. The effect on male fertility and sperm DNA integrity is largely unknown. We evaluated sperm DNA integrity and basic semen parameters according to the World Health Organization (WHO) in male patients with inflammatory diseases treated with methotrexate. Methods Semen samples from 14 patients on low-dose maintenance methotrexate were compared with samples from 40 healthy volunteers. Further, 5 patients delivered samples on and off methotrexate therapy for paired comparison. Sperm DNA fragmentation index (DFI), concentration, motility, and morphology were evaluated. Blood sex hormones and methotrexate levels were measured in blood and semen. Results DNA fragmentation index in methotrexate-treated patients was comparable with that in healthy volunteers (DFI, 11.5 vs 15.0; P = .06), and DFI did not change significantly on and off methotrexate in the paired samples (DFI, 12.0 vs 14.0; P = 0.35). Sperm concentration, motility, and morphology did not differ between men treated with methotrexate and healthy volunteers. Sperm progressive motility increased off therapy compared with on therapy (65.0% vs 45.0%, P = .04), but all fluctuations in progressive motility were within the WHO reference interval. All methotrexate polyglutamates1-5 were detected in blood, but only methotrexate polyglutamate1 in semen. Serum testosterone was unaffected by methotrexate therapy. Conclusions Patients treated with low-dose methotrexate have a sperm quality comparable with that of healthy volunteers, and methotrexate treatment does not increase sperm DNA fragmentation. This study does not support cryopreservation of semen before treatment initiation nor a 3-month methotrexate-free interval prior to conception.

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 ( wm 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 wm 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 wm 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.

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. DNA-TG levels were measured in leucocytes (DNA-TGTotal), polymorph nucleated granulocytes (neutrophils, eosinophils, basophils [DNA-TGPMN]) and mononucleated cells (lymphocytes, monocytes [DNA-TGMNC]) in 1013 samples from 52 patients on ALL maintenance therapy (951 samples during therapy and 62 samples after therapy discontinuation, respectively). Median DNA-TGTotal, DNA-TGPMN and DNA-TGMNC during maintenance therapy were 539, 563 and 384 fmol/µg DNA, respectively. DNA-TGPMN displayed more pronounced fluctuation than DNA-TGMNC (range 0–3084 [interquartile range IQR 271–881] versus 30–1411 [IQR 270–509] fmol/µg DNA). DNA-TGTotal was more strongly correlated with DNA-TGPMN (rS = 0.95, p < 0.0001) than DNA-TGMNC (rS = 0.73, p < 0.0001). DNA-TGPMN correlated less with DNA-TGMNC (rS = 0.64, p < 0.0001) and to a much lesser extent with absolute neutrophil count (rS = 0.35, p < 0.0001). Following discontinuation of therapy, DNA-TGPMN was rapidly eliminated, and not measurable beyond day 22 after discontinuation, whereas DNA-TGMNC was slowly eliminated, and five patients demonstrated a measurable DNA-TGMNC more than 365 days after therapy discontinuation. Fluctuations in DNA-TGTotal are predominantly caused by corresponding fluctuations in DNA-TGPMN, thus DNA-TGTotal measures recent TGN incorporation in these short-lived cells. Measurement of DNA-TGTotal at 2–4 weeks intervals provides a reliable profile of DNA-TG levels.

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. 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/m2 for TPMT heterozygous and wildtype patients and was adjusted to a target WBC of 1.5 − 3.0 × 109/L. 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). TPMT geno- and phenotype were not associated with relapse in non-high risk NOPHO ALL2008.

Background Socioeconomic differences in survival among children with acute lymphoblastic leukemia (ALL) have been reported in high-income countries and there is an unmet need for strategies to identify vulnerable patient subgroups. Reported differences in survival for children from families with different socioeconomic positions seem to arise when starting maintenance therapy. This could reflect reduced physician's compliance or family adherence to maintenance therapy. Methods This nationwide cohort study with extensive monitoring of systemic methotrexate (MTX)/6-mercaptopurine (6MP) dosing and metabolite levels, retrospectively investigated 173 Danish children treated according to The Nordic Society for Pediatric Hematology and Oncology ALL2008 protocol from 2008 to 2016. Results Significantly lower prescribed doses of MTX and 6MP were seen in the children in families with short parental education (short vs. medium vs. higher education: mMTX: 13.8, 16.2, and 18.6 mg/m(2)/week; p < .01; m6MP: 47.4, 64.9, and 66.1 mg/m(2)/day; p = .03) or parents unemployed/not in workforce (unemployed/not in workforce vs. mixed vs. at work: mMTX: 15.0, 19.9, and 17.2 mg/m(2)/week; p < .01; m6MP: 54.8, 72.0, and 65.1 mg/m(2)/day; p < .01). When assessing family adherence by analyzing MTX and 6MP metabolite levels, including per prescribed dose of MTX and 6MP, we found no significant differences by levels of parental education, affiliation to work market, or income (p > .05 for all comparisons). Conclusions These results indicate that inferior physician compliance to protocol recommendations on drug dosage rather than families' adherence to therapy may contribute to the association between socioeconomic position and cure rates in childhood ALL, although precise mechanisms remain to be explored.

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.

Bone marrow minimal residual disease (MRD) is the strongest predictor of relapse in children with acute lymphoblastic leukemia (ALL). 6-mercaptopurine (6MP) in ALL therapy has wide inter-individual variation in disposition and is strongly influenced by polymorphisms in the thiopurine methyltransferase (TPMT) gene. In 952 patients treated according to the NOPHO ALL2008 protocol, we explored the association between thiopurine disposition, TPMT genotypes and MRD levels after consolidation therapy with 6MP, high-dose methotrexate (HD-MTX), asparaginase, and vincristine. The levels of the cytotoxic DNA-incorporated thioguanine were significantly higher on day 70-79 in G460A/A719G TPMT heterozygous (TPMT (HZ)) compared to TPMT wild type (TPMT (WT)) patients (mean: 230.7 vs. 149.7 fmol/mu g DNA, p = 0.002). In contrast, TPMT genotype did not associate with the end of consolidation MRD levels irrespective of randomization of the patients to fixed dose (25 mg/m(2)/day) or 6MP escalation (up to 50 or 75 mg/m(2)/day) during consolidation therapy.

BACKGROUND:Interprofessional education in childhood cancer is a multifaceted field involving multiple healthcare professionals with general and specialised knowledge and skills. Complex treatment, care and rehabilitation require continuous professional development and maintenance of healthcare professionals' competencies in their field of expertise. However, limited knowledge exists in comparing interprofessional and monoprofessional education. Only a few randomised studies have evaluated the effectiveness and efficiency of interprofessional education. The objective of this single-centre, investigator-initiated cluster randomised trial is to study the effect of interprofessional versus monoprofessional case-based learning on healthcare professionals' knowledge of gastrointestinal side effects and attitudes towards team collaboration.METHODS:This study will randomise healthcare professionals to participate in either the experimental interprofessional group or the control monoprofessional group of case-based learning. The topic of the case-based intervention will be gastrointestinal side effects, one of six categories identified in a three-round Scandinavian Delphi study as relevant for interprofessional education in childhood cancer. The primary outcome is the self-reported questionnaire Assessment of Interprofessional Team Collaboration Scale. Secondary outcomes are measured by the self-reported questionnaires Readiness for Interprofessional Learning Scale Questionnaire, Safety Attitudes Questionnaire, and knowledge will be evaluated using a multiple-choice quiz. Participants will receive the self-reported questionnaires about 2 weeks before and 1 month after the intervention. On the day of the intervention, participants will answer a multiple-choice quiz before and after the case-based learning. Linear mixed models will be used to compare differences between the two groups in mean scores postintervention, adjusting for preintervention scores.DISCUSSION:This study will provide insight into the differences between interprofessional and monoprofessional case-based learning and how it affects healthcare professionals' knowledge of gastrointestinal side effects and attitudes towards team collaboration.TRIAL REGISTRATION:The intervention was registered at Clinical Trials.gov : NCT04204109 on December 102,019 and with the National Committee on Health Research Ethics: H-19087506 December 112,019 and the Danish Data Protection Agency: P-2019-637 October 152,019.

Relapse-enriched somatic variants drive drug resistance in childhood acute lymphoblastic leukemia. We used digital droplet-based polymerase chain reaction to establish whether relapse-enriched mutations in emerging subclones could be detected in peripheral blood samples before frank relapse. Although limitations in sensitivity for some probes hindered detection of certain variants, we successfully detected variants in NT5C2 and PRPS1 at a fractional abundance of 0.005% to 0.3%, 41 to 116 days before relapse. As mutations in both these genes confer resistance to thiopurines, early detection protocols using peripheral blood could be implemented to preemptively alter maintenance therapy to extinguish resistant clones before overt relapse.

Methotrexate (MTX) during maintenance therapy is essential for curing acute lymphoblastic leukemia (ALL), but dosing strategies aiming at adequate treatment intensity are challenged by interindividual differences in drug disposition. To evaluate genetic factors associated with MTX metabolism, we performed a genome-wide association study in 447 ALL cases from the Nordic Society for Pediatric Haematology and Oncology ALL2008 study, validating results in an independent set of 196 patients. The intergenic single-nucleotide polymorphism rs1382539, located in a regulatory element of DHFR, was associated with increased levels of short-chain MTX polyglutamates (P = 1.1 × 10-8) related to suppression of enhancer activity, whereas rs35789560 in FPGS (p.R466C, P = 5.6 × 10-9) was associated with decreased levels of long-chain MTX polyglutamates through reduced catalytic activity. Furthermore, the FPGS variant was linked with increased relapse risk (P = .044). These findings show a genetic basis for interpatient variability in MTX response and could be used to improve future dosing algorithms.

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. 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 (Cmax), time to maximum plasma concentration (Tmax), and terminal half-life (T½). 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 Cmax,Tmax and T½ (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. 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.

Intestinal mucositis is a common side effect of chemotherapy leading to diarrhea, abdominal pain and increased risk of infections. The intestinal microbiota has been recognized as a key regulator of mucosal immune responses. Therefore, we hypothesized that intestinal microbial changes would be associated with enterocyte loss and systemic inflammation during induction treatment for childhood acute lymphoblastic leukemia (ALL). We prospectively included 51 children newly‐diagnosed with ALL treated in Denmark in 2015–2018. Plasma C‐reactive protein (CRP), plasma citrulline (marker of functional enterocytes mass) measurements and fecal samplings were performed on treatment Days 1, 8, 15, 22 and 29. Moreover, intestinal mucositis was scored by a trained nurse/physician. Fecal samples in patients and 19 healthy siblings were analyzed by 16S rRNA gene sequencing (V3–V4 region). Bacterial alpha diversity was lower in patients compared to siblings. It decreased from Day 1 to Days 8–22 and increased on Day 29. Shannon alpha diversity index was correlated with CRP on Days 15–29 (rho = −0.33−0.49; p < 0.05) and with citrulline on Days 15 and 29 (although with p values <0.06, rho = 0.32–0.34). The abundance of unclassified Enterococcus species (spp.) was correlated with CRP on Days 22–29 (rho = 0.42–0.49; p < 0.009), while the abundance of unclassified Lachnospiraceae spp. was correlated with citrulline on days 8–15 (rho = 0.48–0.62, p < 0.001). Systemic inflammation, enterocyte loss and relative abundance of unclassified Enterococcus spp. reached a peak around Day 15. In conclusion, specific changes in the microbiota were associated with the severity of enterocyte loss and systemic inflammation during chemotherapy.

Methotrexate polyglutamates (MTXpg) facilitate incorporation of thioguanine nucleotides into DNA (DNA-TG, the primary cytotoxic thiopurine metabolite and outcome determinant in MTX/6-mercaptopurine treatment of childhood ALL). We hypothesized that mapping erythrocyte levels of MTXpg with 1–6 glutamates and their associations with DNA-TG formation would facilitate future guidelines for maintenance therapy dosing.

BACKGROUND AND AIMS:Sperm DNA integrity, concentration, and motility are suspected to be altered by thiopurines (azathioprine [AZA] and 6-mercaptopurine [6-MP]). We investigated the impact of thiopurines on semen quality in men with inflammatory bowel disease [IBD], by a comprehensive panel of semen analyses.METHODS:Semen from 40 men with IBD, in remission on AZA/6-MP therapy, was prospectively collected and compared with samples from 40 healthy volunteers. Paired samples [off and on AZA/6-MP] were obtained from a subset of IBD patients, and blood and semen were collected to determine 6-MP transmission to the ejaculate. Sperm DNA fragmentation was evaluated via sperm chromatin structure assay [SCSA] and Comet analysis. Conventional World Health Organization [WHO] parameters, i.e. semen volume and sperm concentration, motility, and morphology, were assessed. Additionally, we measured thioguanine nucleotide [TGN] incorporation in sperm cell DNA.RESULTS:Sperm DNA fragmentation levels did not differ between men with IBD on AZA/6-MP and healthy volunteers when evaluated by SCSA [p = 0.23] and Comet analysis [p = 0.72]. IBD patients on AZA/6-MP had significantly lower total and progressive sperm motility than healthy volunteers [48.5% versus 64.5%, p = 0.0003; 27.4% versus 43.3%, p = 0.0004; respectively], with no differences in concentration, volume, or morphology. The same trend was observed in the 10 paired samples. TGN incorporation was not detectable in sperm DNA, but 6-MP was detected in seminal plasma and correlated to blood levels [rs = 0.79, p = 0.02].CONCLUSIONS:Thiopurines do not increase sperm DNA fragmentation but may impair sperm motility in this IBD cohort. Our findings support existing epidemiological data that thiopurine therapy is safe during preconception and should not be abandoned.