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[EN] Background White blood cell count (WBC) as a measure of extramedullary leukemic cell survival is a well-known prognostic factor in acute lymphoblastic leukemia (ALL), but its biology, including impact of host genome variants, is poorly understood. Methods We included patients treated with the Nordic Society of Paediatric Haematology and Oncology (NOPHO) ALL-2008 protocol (N = 2347, 72% were genotyped by Illumina Omni2.5exome-8-Bead chip) aged 1-45 years, diagnosed with B-cell precursor (BCP-) or T-cell ALL (T-ALL) to investigate the variation in WBC. Spline functions of WBC were fitted correcting for association with age across ALL subgroups of immunophenotypes and karyotypes. The residuals between spline WBC and actual WBC were used to identify WBC-associated germline genetic variants in a genome-wide association study (GWAS) while adjusting for age and ALL subtype associations. Results We observed an overall inverse correlation between age and WBC, which was stronger for the selected patient subgroups of immunophenotype and karyotypes (rho(BCP-ALL )= -.17, rho(T-ALL )= -.19; p < 3 x 10(-4)). Spline functions fitted to age, immunophenotype, and karyotype explained WBC variation better than age alone (rho = .43, p << 2 x 10(-6)). However, when the spline-adjusted WBC residuals were used as phenotype, no GWAS significant associations were found. Based on available annotation, the top 50 genetic variants suggested effects on signal transduction, translation initiation, cell development, and proliferation. Conclusion These results indicate that host genome variants do not strongly influence WBC across ALL subsets, and future studies of why some patients are more prone to hyperleukocytosis should be performed within specific ALL subsets that apply more complex analyses to capture potential germline variant interactions and impact on WBC.

Central nervous system (CNS) involvement in childhood acute lymphoblastic leukemia (ALL) implicates enhanced intrathecal chemotherapy, which is related to CNS toxicity. Whether CNS involvement alone contributes to CNS toxicity remains unclear. We studied the occurrence of all CNS toxicities, seizures, and posterior reversible encephalopathy syndrome (PRES) in children with ALL without enhanced intrathecal chemotherapy with CNS involvement (n = 64) or without CNS involvement (n = 256) by flow cytometry. CNS involvement increased the risk for all CNS toxicities, seizures, and PRES in univariate analysis and, after adjusting for induction therapy, for seizures (hazard ratio [HR] = 3.33; 95% confidence interval [CI]: 1.26-8.82; p = 0.016) and PRES (HR = 4.85; 95% CI: 1.71-13.75; p = 0.003).

Background White blood cell count (WBC) as a measure of extramedullary leukemic cell survival is a well-known prognostic factor in acute lymphoblastic leukemia (ALL), but its biology, including impact of host genome variants, is poorly understood. Methods We included patients treated with the Nordic Society of Paediatric Haematology and Oncology (NOPHO) ALL-2008 protocol (N = 2347, 72% were genotyped by Illumina Omni2.5exome-8-Bead chip) aged 1-45 years, diagnosed with B-cell precursor (BCP-) or T-cell ALL (T-ALL) to investigate the variation in WBC. Spline functions of WBC were fitted correcting for association with age across ALL subgroups of immunophenotypes and karyotypes. The residuals between spline WBC and actual WBC were used to identify WBC-associated germline genetic variants in a genome-wide association study (GWAS) while adjusting for age and ALL subtype associations. Results We observed an overall inverse correlation between age and WBC, which was stronger for the selected patient subgroups of immunophenotype and karyotypes (rho(BCP-ALL )= -.17, rho(T-ALL )= -.19; p < 3 x 10(-4)). Spline functions fitted to age, immunophenotype, and karyotype explained WBC variation better than age alone (rho = .43, p << 2 x 10(-6)). However, when the spline-adjusted WBC residuals were used as phenotype, no GWAS significant associations were found. Based on available annotation, the top 50 genetic variants suggested effects on signal transduction, translation initiation, cell development, and proliferation. Conclusion These results indicate that host genome variants do not strongly influence WBC across ALL subsets, and future studies of why some patients are more prone to hyperleukocytosis should be performed within specific ALL subsets that apply more complex analyses to capture potential germline variant interactions and impact on WBC.

Obesity is associated with poor outcomes in childhood acute lymphoblastic leukaemia (ALL). We explored whether severe treatment-related toxicity and treatment delays could explain this observation. This study included 1 443 children aged 2 center dot 0-17 center dot 9 years with ALL treated with the Nordic Society of Pediatric Haematology and Oncology (NOPHO) ALL2008 non-high-risk protocol. Prospective treatment-related toxicities registered every three-month interval were used. Patients were classified according to sex- and age-adjusted international childhood cut-off values, corresponding to adult body mass index: underweight, <17 kg/m(2); healthy weight, 17 to <25 kg/m(2); overweight, 25 to <30 kg/m(2); and obese, >= 30 kg/m(2). Obese children had a higher incidence rate ratio (IRR) for severe toxic events {IRR: 1 center dot 55 [95% confidence interval (CI) 1 center dot 07-2 center dot 50]}, liver and kidney failures, bleeding, abdominal complication, suspected unexpected severe adverse reactions and hyperlipidaemia compared with healthy-weight children. Obese children aged >= 10 years had increased IRRs for asparaginase-related toxicities compared with healthy-weight older children: thromboses [IRR 2 center dot 87 (95% CI 1 center dot 00-8 center dot 21)] and anaphylactic reactions [IRR 7 center dot 95 (95% CI 2 center dot 15-29 center dot 37)] as well as higher risk for truncation of asparaginase [IRR 3 center dot 54 (95% CI 1 center dot 67-7 center dot 50)]. The high prevalence of toxicity and a higher risk of truncation of asparaginase may play a role in the poor prognosis of obese children aged >= 10 years with ALL.

Central nervous system (CNS) toxicity is common at diagnosis and during treatment of pediatric acute lymphoblastic leukemia (ALL). We studied CNS toxicity in 1, 464 children aged 1.0-17.9 years, diagnosed with ALL and treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol. Genome-wide association studies, and a candidate single-nucleotide polymorphism (SNP; n=19) study were performed in 1, 166 patients. Findings were validated in an independent Australian cohort of children with ALL (n=797) in whom two phenotypes were evaluated: diverse CNS toxicities (n=103) and methotrexate-related CNS toxicity (n=48). In total, 135/1, 464 (9.2%) patients experienced CNS toxicity for a cumulative incidence of 8.7% (95% confidence interval: 7.31-10.20) at 12 months from diagnosis. Patients aged ≥10 years had a higher risk of CNS toxicity than had younger patients (16.3% vs. 7.4%; P<0.001). The most common CNS toxicities were posterior reversible encephalopathy syndrome (n=52, 43 with seizures), sinus venous thrombosis (n=28, 9 with seizures), and isolated seizures (n=16). The most significant SNP identified by the genome-wide association studies did not reach genomic significance (lowest P-value: 1.11x10-6), but several were annotated in genes regulating neuronal functions. In candidate SNP analysis, ATXN1 rs68082256, related to epilepsy, was associated with seizures in patients <10 years (P=0.01). ATXN1 rs68082256 was validated in the Australian cohort with diverse CNS toxicities (P=0.04). The role of ATXN1 as well as the novel SNP in neurotoxicity in pediatric ALL should be further explored.

Central nervous system (CNS) toxicity is common at diagnosis and during treatment of pediatric acute lymphoblastic leukemia (ALL). We studied CNS toxicity in 1 464 children aged 1.0-17.9 years, diagnosed with ALL and treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol. Genome-wide association studies (GWAS), and a candidate single-nucleotide polymorphism (SNP; n=19) study were performed in 1 166 patients. Findings were validated in an Australian independent cohort of children with ALL (n=797) where two phenotypes were evaluated: diverse CNS toxicities (n=103) and methotrexate-related CNS toxicity (n=48). In total, 135/1 464 (9.2%) patients experienced CNS toxicity with cumulative incidence of 8.7% (95% CI: 7.31-10.20) at 12 months from diagnosis. Patients aged ≥10 years had higher risk of CNS toxicity than younger patients (16.3% vs 7.4%; p <0.001). The most common CNS toxicities were posterior reversible encephalopathy syndrome (n=52, 43 with seizures), sinus venous thrombosis (n=28, 9 with seizures), and isolated seizures (n=16). The most significant SNPs identified by GWAS did not reach genomic significance (lowest P-value: 1.11e-06), but several were annotated in genes regulating neuronal functions. In candidate SNP analysis, ATXN1 rs68082256, related to epilepsy, was associated with seizures in patients <10 years (P=0.01). ATXN1 rs68082256 was validated in the Australian diverse CNS toxicities cohort (P=0.04). The role of ATXN1 as well as the novel SNPs in neurotoxicity in pediatric ALL should be further explored.

Objectives: Children with acute lymphoblastic leukemia (ALL) have a tendency to gain weight during treatment. As overweight and obesity associate with health problems, prophylactic interventions are warranted. Therefore, it is important to identify the children most prone to gain weight. Methods: Patients aged 2.0–17.9 years at ALL diagnosis were identified from the NOPHO ALL2008 registry. Registry data was complemented with height and weight at the end of therapy from questionnaires. Body mass index (BMI) was classified according to international age- and sex-adjusted International Obesity Task Force BMI cut-offs. BMI values were transformed into standard deviation scores (SDS) to calculate the difference in BMISDS during treatment. Results: Data on BMI change were available for 765 children. Overweight and obesity doubled during treatment: 9.7% were overweight and 2.1% obese at diagnosis and 21.8% and 5.4% at the end of therapy, respectively. The mean BMISDS change was +0.64. Younger (2.0–5.9 years) and healthy weight children were most prone to become overweight (mean change in BMI SDS +0.85 and + 0.65, respectively). Conclusions: Younger children (2.0–5.9 years) with healthy weight at diagnosis were most prone to becoming overweight and therefore are an important group to target while considering interventions.

Asparaginase is essential in treating acute lymphoblastic leukaemia (ALL). Asparaginase-related hypersensitivity causes treatment discontinuation, which is associated with decreased event-free survival. To continue asparaginase treatment after hypersensitivity, a formulation of asparaginase encapsulated in erythrocytes (eryaspase) was developed. In NOR-GRASPALL 2016 (NCT03267030) the safety and efficacy of eryaspase was evaluated in 55 patients (aged 1–45 years; median: 6.1 years) with non-high-risk ALL and hypersensitivity to asparaginase conjugated with polyethylene glycol (PEG-asparaginase). Eryaspase (150 u/kg) was scheduled to complete the intended course of asparaginase (1–7 doses) in two Nordic/Baltic treatment protocols. Forty-nine (96.1%) patients had asparaginase enzyme activity (AEA) ≥100 iu/l 14 ± 2 days after the first eryaspase infusion [median AEA 511 iu/l; interquartile range (IQR), 291–780], whereas six of nine (66.7%) patients had AEA ≥100 iu/l 14 ± 2 days after the fourth infusion (median AEA 932 iu/l; IQR, 496–163). The mean terminal half-life of eryaspase following the first infusion was 15.3 ± 15.5 days. Few asparaginase-related adverse events were reported; five patients (9.1%) developed clinical allergy associated with enzyme inactivation. Replacement therapy was successfully completed in 50 patients (90.9%). Eryaspase was well tolerated, and most patients had AEA levels above the therapeutic target after the first infusion. The half-life of eryaspase confirmed that a 2-week schedule is appropriate.

Purpose Osteonecrosis is a burdensome treatment-related toxicity that is mostly diagnosed during or soon after 6-mercaptopurine (6MP)/methotrexate (MTX) maintenance therapy for acute lymphoblastic leukemia (ALL), possibly indicating a pathogenic role of these drugs. Methods We prospectively registered symptomatic osteonecrosis during treatment of 1234 patients aged 1.0-45.9 years treated according to the Nordic Society of Hematology and Oncology (NOPHO) ALL2008 protocol. MTX/6MP metabolites were measured as part of the NOPHO ALL2008 maintenance therapy study. Results After a median follow-up of 5.6 years [interquartile range (IQR) 3.6-7.5], 68 patients had been diagnosed with symptomatic osteonecrosis. The cumulative incidence was 2.7% [95% confidence interval (CI) 1.6-3.8%] for patients aged < 10 years, 14.9% (95% CI 9.7-20.2%) for patients aged 10.0-17.9 years, and 14.4% (95% CI 8.0-20.8%) for patients aged >= 18 years. The median time from diagnosis of ALL to diagnosis of osteonecrosis in these age groups was 1.0 year (IQR 0.7-2.0), 2.0 years (IQR 1.1-2.4), and 2.2 years (IQR 1.8-2.8), respectively (p = 0.001). With 17,854 blood samples available for MTX and 6MP metabolite analysis, neither erythrocyte levels of 6-thioguanine (TG) nucleotides (p > 0.99), methylated 6MP metabolites (p = 0.37), MTX polyglutamates (p = 0.98) nor DNA-TG (p = 0.53) were significantly associated with the hazard of osteonecrosis in Cox models stratified by the three age groups and adjusted for sex. Conclusion Maintenance therapy intensity determined by 6MP and MTX metabolites was not associated with the risk of developing osteonecrosis in the NOPHO ALL2008 cohort.

Purpose Osteonecrosis is a burdensome treatment-related toxicity that is mostly diagnosed during or soon after 6-mercaptopurine (6MP)/methotrexate (MTX) maintenance therapy for acute lymphoblastic leukemia (ALL), possibly indicating a pathogenic role of these drugs. Methods We prospectively registered symptomatic osteonecrosis during treatment of 1234 patients aged 1.0-45.9 years treated according to the Nordic Society of Hematology and Oncology (NOPHO) ALL2008 protocol. MTX/6MP metabolites were measured as part of the NOPHO ALL2008 maintenance therapy study. Results After a median follow-up of 5.6 years [interquartile range (IQR) 3.6-7.5], 68 patients had been diagnosed with symptomatic osteonecrosis. The cumulative incidence was 2.7% [95% confidence interval (CI) 1.6-3.8%] for patients aged < 10 years, 14.9% (95% CI 9.7-20.2%) for patients aged 10.0-17.9 years, and 14.4% (95% CI 8.0-20.8%) for patients aged >= 18 years. The median time from diagnosis of ALL to diagnosis of osteonecrosis in these age groups was 1.0 year (IQR 0.7-2.0), 2.0 years (IQR 1.1-2.4), and 2.2 years (IQR 1.8-2.8), respectively (p = 0.001). With 17,854 blood samples available for MTX and 6MP metabolite analysis, neither erythrocyte levels of 6-thioguanine (TG) nucleotides (p > 0.99), methylated 6MP metabolites (p = 0.37), MTX polyglutamates (p = 0.98) nor DNA-TG (p = 0.53) were significantly associated with the hazard of osteonecrosis in Cox models stratified by the three age groups and adjusted for sex. Conclusion Maintenance therapy intensity determined by 6MP and MTX metabolites was not associated with the risk of developing osteonecrosis in the NOPHO ALL2008 cohort.

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/m2 for TPMT heterozygous and wildtype patients and was adjusted to a target WBC of 1.5 - 3.0 × 109/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.

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/µg 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/m2/day) or 6MP escalation (up to 50 or 75 mg/m2/day) during consolidation therapy.