Search

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.

IntroductionThromboembolism is a serious toxicity of acute lymphoblastic leukemia treatment, and contributes to substantial morbidity and mortality. Several single nucleotide polymorphisms have been associated with thromboembolism in the general population; however, their impact in patients with acute lymphoblastic leukemia, particularly in children, remains uncertain.Materials and methodsWe collected constitutional DNA and prospectively registered thromboembolic events in 1252 patients, 1–45 years, with acute lymphoblastic leukemia included in the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol in the Nordic and Baltic countries (7/2008–7/2016). Based on previously published data and a priori power calculations, we selected four single nucleotide polymorphisms: F5 rs6025, F11 rs2036914, FGG rs2066865, and ABO rs8176719.ResultsThe 2.5 year cumulative incidence of thromboembolism was 7.1% (95% confidence interval (CI) 5.6–8.5). F11 rs2036914 was associated with thromboembolism (hazard ratio (HR) 1.52, 95%CI 1.11–2.07) and there was a borderline significant association for FGG rs2066865 (HR 1.37, 95%CI 0.99–1.91), but no association for ABO rs8176719 or F5 rs6025 in multiple cox regression. A genetic risk score based on F11 rs2036914 and FGG rs2066865 was associated with thromboembolism (HR 1.45 per risk allele, 95%CI 1.15–1.81), the association was strongest in adolescents 10.0–17.9 years (HR 1.64).ConclusionIf validated, a F11 rs2036914/FGG rs2066865 risk prediction model should be tested as a stratification tool for prevention of thromboembolism in patients with acute lymphoblastic leukemia. Introduction: Thromboembolism is a serious toxicity of acute lymphoblastic leukemia treatment, and contributes to substantial morbidity and mortality. Several single nucleotide polymorphisms have been associated with thromboembolism in the general population; however, their impact in patients with acute lymphoblastic leukemia, particularly in children, remains uncertain. Materials and methods: We collected constitutional DNA and prospectively registered thromboembolic events in 1252 patients, 1–45 years, with acute lymphoblastic leukemia included in the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol in the Nordic and Baltic countries (7/2008–7/2016). Based on previously published data and a priori power calculations, we selected four single nucleotide polymorphisms: F5 rs6025, F11 rs2036914, FGG rs2066865, and ABO rs8176719. Results: The 2.5 year cumulative incidence of thromboembolism was 7.1% (95% confidence interval (CI) 5.6–8.5). F11 rs2036914 was associated with thromboembolism (hazard ratio (HR) 1.52, 95%CI 1.11–2.07) and there was a borderline significant association for FGG rs2066865 (HR 1.37, 95%CI 0.99–1.91), but no association for ABO rs8176719 or F5 rs6025 in multiple cox regression. A genetic risk score based on F11 rs2036914 and FGG rs2066865 was associated with thromboembolism (HR 1.45 per risk allele, 95%CI 1.15–1.81), the association was strongest in adolescents 10.0–17.9 years (HR 1.64). Conclusion: If validated, a F11 rs2036914/FGG rs2066865 risk prediction model should be tested as a stratification tool for prevention of thromboembolism in patients with acute lymphoblastic leukemia.

Reports of persistent symptoms after hospitalization with COVID-19 have raised concern of a “long COVID” syndrome. This study aimed at determining the prevalence of and risk factors for acute and persistent symptoms in non-hospitalized patients with polymerase chain reaction (PCR) confirmed COVID-19. We conducted a cohort study of non-hospitalized participants identified via the Danish Civil Registration System with a SARS-CoV-2-positive PCR-test and available biobank samples. Participants received a digital questionnaire on demographics and COVID-19-related symptoms. Persistent symptoms: symptoms > 4 weeks (in sensitivity analyses > 12 weeks). We included 445 participants, of whom 34% were asymptomatic. Most common acute symptoms were fatigue, headache, and sneezing, while fatigue and reduced smell and taste were most severe. Persistent symptoms, most commonly fatigue and memory and concentration difficulties, were reported by 36% of 198 symptomatic participants with follow-up > 4 weeks. Risk factors for persistent symptoms included female sex (women 44% vs. men 24%, odds ratio 2.7, 95% CI 1.4–5.1, p = 0.003) and BMI (odds ratio 1.1, 95% CI 1.0–1.2, p = 0.001). In conclusion, among non-hospitalized PCR-confirmed COVID-19 patients one third were asymptomatic while one third of symptomatic participants had persistent symptoms illustrating the heterogeneity of disease presentation. These findings should be considered in health care planning and policy making related to COVID-19.

Asparaginase-associated pancreatitis is a life-threatening toxicity to childhood acute lymphoblastic leukemia treatment. To elucidate genetic predisposition and asparaginase-associated pancreatitis pathogenesis, ten trial groups contributed remission samples from patients aged 1.0−17.9 years treated for acute lymphoblastic leukemia between 2000 and 2016. Cases (n=244) were defined by the presence of at least two of the following criteria: (i) abdominal pain; (ii) levels of pancreatic enzymes ≥3 x upper normal limit; and (iii) imaging compatible with pancreatitis. Controls (n=1320) completed intended asparaginase therapy, with 78% receiving ≥8 injections of pegylated-asparaginase, without developing asparaginase-associated pancreatitis. rs62228256 on 20q13.2 showed the strongest association with the development of asparaginase-associated pancreatitis (odds ratio=3.75; P=5.2x10 -8 ). Moreover, rs13228878 (OR=0.61; P=7.1x10 -6 ) and rs10273639 (OR=0.62; P=1.1x10 -5 ) on 7q34 showed significant association with the risk of asparaginase-associated pancreatitis. A Dana Farber Cancer Institute ALL Consortium cohort consisting of patients treated on protocols between 1987 and 2004 (controls=285, cases=33), and the Children’s Oncology Group AALL0232 cohort (controls=2653, cases=76) were available as replication cohorts for the 20q13.2 and 7q34 variants, respectively. While rs62228256 was not validated as a risk factor (P=0.77), both rs13228878 (P=0.03) and rs10273639 (P=0.04) were. rs13228878 and rs10273639 are in high linkage disequilibrium (r 2 =0.94) and associated with elevated expression of the PRSS1 gene, which encodes for trypsinogen, and are known risk variants for alcohol-associated and sporadic pancreatitis in adults. Intra-pancreatic trypsinogen cleavage to proteolytic trypsin induces autodigestion and pancreatitis. In conclusion, this study finds a shared genetic predisposition between asparaginase-associated pancreatitis and non-asparaginase-associated pancreatitis, and targeting the trypsinogen activation pathway may enable identification of effective interventions for asparaginase-associated pancreatitis.

Publisher Copyright: © 2021 Lippincott Williams and Wilkins. All rights reserved. Asparaginase-associated pancreatitis (AAP) frequently affects children treated for acute lymphoblastic leukemia (ALL) causing severe acute and persisting complications. Known risk factors such as asparaginase dosing, older age and single nucleotide polymorphisms (SNPs) have insufficient odds ratios to allow personalized asparaginase therapy. In this study, we explored machine learning strategies for prediction of individual AAP risk. We integrated information on age, sex, and SNPs based on Illumina Omni2.5exome-8 arrays of patients with childhood ALL (N=1564, 244 with AAP aged 1.0 to 17.9 y) from 10 international ALL consortia into machine learning models including regression, random forest, AdaBoost and artificial neural networks. A model with only age and sex had area under the receiver operating characteristic curve (ROC-AUC) of 0.62. Inclusion of 6 pancreatitis candidate gene SNPs or 4 validated pancreatitis SNPs boosted ROC-AUC somewhat (0.67) while 30 SNPs, identified through our AAP genome-wide association study cohort, boosted performance (0.80). Most predictive features included rs10273639 (PRSS1-PRSS2), rs10436957 (CTRC), rs13228878 (PRSS1/PRSS2), rs1505495 (GALNTL6), rs4655107 (EPHB2) and age (1 to 7 y). Second AAP following asparaginase re-exposure was predicted with ROC-AUC: 0.65. The machine learning models assist individual-level risk assessment of AAP for future prevention trials, and may legitimize asparaginase re-exposure when AAP risk is predicted to be low.

BackgroundReports of persistent symptoms after hospitalization with COVID-19 have raised concern of a “long COVID” syndrome. This study aimed at characterizing acute and persistent symptoms in non- hospitalized patients with polymerase chain reaction (PCR) confirmed COVID-19.MethodsCohort study of 445 non-hospitalized participants identified via the Danish Civil Registration System with a SARS-CoV-2-positive PCR-test and available biobank samples for genetic analyses. Participants received a digital questionnaire on demographics and COVID-19-related symptoms. Persistent symptoms: symptoms >four weeks (in sensitivity analyses >12 weeks).Results445 participants were included, of whom 34% were asymptomatic. Most common acute symptoms were fatigue, headache, and sneezing, while fatigue and reduced smell and taste were reported as most severe. Persistent symptoms, most commonly fatigue and memory and concentration difficulties, were reported by 36% of 198 symptomatic participants with follow-up >four weeks. Risk factors for persistent symptoms included female sex (women 44% vs. men 24%, odds ratio 2.7, 95%CI:1.4-5.1, p=0.003) and BMI (odds ratio 1.1, 95%CI:1.0-1.2, p=0.001).ConclusionAmong non-hospitalized PCR-confirmed COVID-19 patients one third were asymptomatic while one third of symptomatic participants had persistent symptoms illustrating the heterogeneity of disease presentation. These findings should be considered in future health care planning and policy making related to COVID-19.

Symptomatic venous thromboembolism (VTE) occurs in five percent of children treated for acute lymphoblastic leukemia (ALL), but whether a genetic predisposition exists across different ALL treatment regimens has not been well studied. Methods: We undertook a genome-wide association study (GWAS) meta-analysis for VTE in consecutively treated children in the Nordic/Baltic acute lymphoblastic leukemia 2008 (ALL2008) cohort and the Australian Evaluation of Risk of ALL Treatment-Related Side-Effects (ERASE) cohort. A total of 92 cases and 1481 controls of European ancestry were included. Results: No SNPs reached genome-wide significance (p &lt, 5 &times, 10&minus, 8) in either cohort. Among the top 34 single-nucleotide polymorphisms (SNPs) (p &lt, 1 &times, 6), two loci had concordant effects in both cohorts: ALOX15B (rs1804772) (MAF: 1%, p = 3.95 &times, 7) that influences arachidonic acid metabolism and thus platelet aggregation, and KALRN (rs570684) (MAF: 1%, p = 4.34 &times, 7) that has been previously associated with risk of ischemic stroke, atherosclerosis, and early-onset coronary artery disease. Conclusion: This represents the largest GWAS meta-analysis conducted to date associating SNPs to VTE in children and adolescents treated on childhood ALL protocols. Validation of these findings is needed and may then lead to patient stratification for VTE preventive interventions. As VTE hemostasis involves multiple pathways, a more powerful GWAS is needed to detect combination of variants associated with VTE.

The antileukaemic drug 6-mercaptopurine is converted into thioguanine nucleotides (TGN) and incorporated into DNA (DNA-TG), the active end metabolite. In a series of genome-wide association studies, we analysed time-weighted means (wm) of erythrocyte concentrations of TGN (Ery-TGN) and DNA-TG in 1009 patients undergoing maintenance therapy for acute lymphoblastic leukaemia (ALL). In discovery analyses (454 patients), the propensity for DNA-TG incorporation (wmDNA-TG/wmEry-TGN ratio) was significantly associated with three intronic SNPs in NT5C2 (top hit: rs72846714; P = 2.09 × 10−10, minor allele frequency 15%). In validation analyses (555 patients), this association remained significant during both early and late maintenance therapy (P = 8.4 × 10−6 and 1.3 × 10−3, respectively). The association was mostly driven by differences in wmEry-TGN, but in regression analyses adjusted for wmEry-TGN (P < 0.0001), rs72846714-A genotype was also associated with a higher wmDNA-TG (P = 0.029). Targeted sequencing of NT5C2 did not identify any missense variants associated with rs72846714 or wmEry-TGN/wmDNA-TG. rs72846714 was not associated with relapse risk, but in a separate cohort of 180 children with relapsed ALL, rs72846714-A genotype was associated with increased occurrence of relapse-specific NT5C2 gain-of-function mutations that reduce cytosol TGN levels (P = 0.03). These observations highlight the impact of both germline and acquired mutations in drug metabolism and disease trajectory.

Objectives This randomized controlled trial tested the hypothesis that children with non-high risk acute lymphoblastic leukemia could benefit from individualized 6-mercaptopurine increments during consolidation therapy (NCT00816049). Primary and secondary end points were end of consolidation minimal residual disease (MRD) positivity and event-free survival. Methods 392 patients were randomized to experimental and 396 to standard therapy. Patients allocated to standard therapy received oral 6-mercaptopurine (25 mg/m2/day) from days 30 to 85, while the experimental arm received stepwise increments of additional 25 mg/m2/day beginning on days 50 and/or 71 unless dose-limiting myelosuppression had occurred. Results In the experimental arm, 166 patients (42%) received one dose increment, and 62 (16%) received two. 57 of 387 (15%) in the experimental arm were MRD positive at end of consolidation versus 77 of 389 (20%) in the control arm (P=0.08). Five-year probability of event-free survival was 0.89 (95% CI: 0.85–0.93) in the experimental arm versus 0.93 (0.90–0.96) in the control arm (P=0.13). The median accumulated length of 6-mercaptopurine treatment interruptions was 7 (IQR 2–12) in the experimental arm versus 4 (IQR 0–10) in the control arm (P=0.002). Conclusion This study found no benefit from individualized 6-mercaptopurine increments compared to standard therapy. This article is protected by copyright. All rights reserved.

Introduction: Thromboembolism (TE) is a major toxicity of acute lymphoblastic leukemia (ALL) treatment and contributes to substantial morbidity and mortality. Several germline DNA variants have been associated with TE in adults without cancer. A meta-analysis of genome-wide association studies on TE by Germain et al. (Am J Hum Genet 2015) identified single nucleotide polymorphisms (SNPs) in 8 genes that contribute to increased risk of TE in adults (ABO,F2, F5,F11, FGG,PROCR,TSPAN15, and SLC44A2). De Haan et al. (Blood 2012) found that SNPs in 5 of these (F5, F2, F11, FGG, and ABO) predicted TE almost as well as a 31 SNP-based risk score. However, the impact of these SNPs in patients with cancer, particularly in children, remains uncertain. Materials and methods: The Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL2008 protocol for children and adults (1-45 years) included a 3-drug induction (vincristine, doxorubicin, glucocorticosteroid) followed by exposure to asparaginase (1,000 IU/m.sq. i.m.) from week 5 until week 33 (details of therapy in Toft, Leukemia 2018). We collected germline DNA and prospectively registered TE events on 1482 children and adults diagnosed with ALL and treated according to the ALL2008 protocol in seven Nordic and Baltic countries (7/2008-7/2016) (Rank, Blood 2018). Inclusion criteria for TE were i) symptomatic venous or arterial TE confirmed by imaging or by autopsy for TE diagnosed post mortem or ii) asymptomatic TE confirmed by imaging due to other non-TE symptoms and treated with systemic anticoagulation. Based on previously published data and a priori power calculations, we selected and genotyped 5 SNPs: F5 rs6025 (risk allele frequency (RAF) 0.05), F11 rs2036914 (RAF 0.52), FGG rs2066865 (RAF 0.22), and ABO SNPs rs8176719 (RAF 0.39) and rs2519093 (RAF 0.24). Three SNPs (F5 and the two ABO SNPs) were found by imputation, which was done on a subset of patients with European ancestry and included in the NOPHO ALL2008 protocol (N = 1229). We constructed genetic risk scores using a combination of the SNPs. Results: During the ALL treatment period 107 of 1482 patients developed TE (2.5-year cumulative incidence 7.3%, 95% confidence interval (CI) 5.9-8.5). In multivariate Cox regression analysis controlling for age, gender, presence of mediastinal mass and enlarged lymph nodes (N = 1192, whereof TE events 84), we found statistically significant associations with TE development for SNPs F5 rs6025 (hazard ratio (HR) 2.96, 95% CI 1.59-5.48), F11 rs2036914 (HR 1.62, 95% CI 1.18-2.24), and FGG rs2066865 (HR 1.40 95% CI 1.01-1.95), whereas there were no significant associations with ABO SNPs rs8176719 (HR 0.98, 95% CI 0.64-1.51) or rs2519093 (HR 1.06, 95% CI 0.65-1.73). An unweighted 3 SNP risk score based on SNPs F5, F11, and FGG was associated with TE development (HR 1.59, 95% CI 1.27-1.98) (Figure 1). Twenty-six of 217 patients with ≥3 risk alleles developed TE (12.0%, 95% CI 8.1-17.2), compared to 62 of 1007 patients with 18 years) the proportion who developed TE was quite high in both the group with ≥3 risk alleles (23.5%, 95% CI 7.8-50.2) and with Conclusion: Based on the strong association between F5 rs6025, F11 rs2036914, and FGG rs2066865 and TE development, not least for adolescents, future preventive measures for TE should target adolescents with ≥3 risk alleles as well as any adults ≥18 years. Disclosures No relevant conflicts of interest to declare.

Asparaginase (ASP)-associated pancreatitis (AAP) occurs during acute lymphoblastic leukemia treatment. Among 1285 children (1.0-17.9 years) diagnosed during July 2008-December 2014 and treated according to the Nordic/Baltic ALL2008 protocol, 86 (cumulative incidence=6.8%) developed AAP. Seventy-three cases were severe (diagnostic AAP criteria persisting >72 h) and 13 mild. Cases were older than controls (median: 6.5 vs 4.5 years; P=0.001). Pseudocysts developed in 28%. Of the 20 re-exposed to ASP, 9 (45%) developed a second AAP. After a median follow-up of 2.3 years, 8% needed permanent insulin therapy, and 7% had recurrent abdominal pain. Germline DNA on 62 cases and 638 controls was genotyped on Omni2.5exome-8-v1.2 BeadChip arrays. Overall, the ULK2 variant rs281366 showed the strongest association with AAP (P=5.8 × 10(-7); odds ratio (OR)=6.7). Cases with the rs281366 variant were younger (4.3 vs 8 years; P=0.015) and had lower risk of AAP-related complications (15% vs 43%; P=0.13) compared with cases without this variant. Among 45 cases and 517 controls

Background: Asparaginase-associated pancreatitis (AAP) is a well-known toxicity of childhood acute lymphoblastic leukemia (ALL) therapy. Recent multi-trial group phenotyping of 465AAP caseshas documented severe complications to AAP, including 8% risk of needing assisted mechanical ventilation, 26% risk of developing pancreatic pseudocysts and 9% risk of developing persisting diabetes (Wolthers et al. Lancet Oncology, 2017) . Investigation of host genome variation associated with AAP has been limited by varying phenotype definition, inclusion criteria and small study sizes. Objectives and Methods: To investigate genetic variants associated with risk of developing AAP, this genome-wide association study reports data on 1544 children (1.0−17.9 years) from 10 ALL trial groups treated with ALL from January 2000−January 2016. The Ponte di Legno toxicity working group consensus definition (Schmiegelow et al. Lancet Oncology, 2016) was used to diagnose AAP: At least two of i) amylase, pancreatic amylase, or pancreatic lipase >3x upper normal limit (UNL), ii) abdominal pain, iii) imaging compatible with AAP. Controls included children treated for ALL with verified completion of intended asparaginase therapy, 78% of whom (1024/1320) received at least 8 injections of PEG-asparaginase without developing AAP. Germline DNA obtained after clinical remission was genotyped on Illumina Infinium Omni2.5exome-8 BeadChip arrays. Association analyses were done in PLINK and annotation in Ensembl. Results: Of 1564 patients passing genotype quality control, 244 had AAP. 205 of 244 (84%) of cases and 1185/1320 (90%) of controls were of European ancestry. Median age was 8.1 years (IQR 4.3−13.1) and 5 (IQR: 3−9) for cases and controls, respectively. After filtering, 1401908 single nucleotide polymorphisms (SNPs) with a minor allele frequency above 1% were analyzed. In logistic regression analysis, adjusting for age and ancestry, the variant rs62228256 (reference allele=C, minor allele=T (C>T)) on 20q13.2 had the strongest association to AAP (OR=3.75; 95% CI 2.33−6.04; p=5.2∙10-8). rs62228256 is located in a non-coding region without known regulatory effects. rs13228878 (A>G; OR=0.61; 95% CI 0.5−0.76; p=7.1∙10-6) and rs10273639 (C>T; OR=0.62; 95% CI 0.5−0.77; p =1.1∙10-5) were among the top 30 SNPs most significantly associated to AAP. They are in high linkage disequilibrium (R2=0.94) and located in the PRSS1-PRSS2 locus on chromosome 7. The rs13228878 A risk allele was not associated with level of amylase (p=0.1) or lipase (p=0,68) at diagnosis of AAP, age at diagnosis of AAP (p=0.63), or risk of pseudocysts (p=0.78). Using identical diagnostic criteria for pancreatitis, the major C allele in rs10273639 has been associated with pancreatitis risk in adults (Whitcomb et al. Nature Genetics, 2012; Masson et al. Gut, 2017) with identical risk allele and similar odds ratios. PRSS1 and PRSS2 encode cationic and anionic trypsinogen, respectively. rs10273639 is an expressive quantitative locus for PRSS1 and the C risk-variant is associated with elevated expression of trypsinogen in pancreatic tissue. Gain of function mutations in PRSS1, known from hereditary pancreatitis, lead to increased autoactivation, increased intra-acinar trypsin levels, and increased risk of auto-digestion leading to pancreatitis. Further investigation of previously validated SNPs known to regulate trypsin activation gave the following results for associations with AAP; rs17107315 in pancreatic secretory trypsin inhibitor (SPINK1; OR=2.87; 95% CI 1.36−5.8; p=4∙10-3), rs10436957 in chymotrypsin C (CTRC ; OR=0.69; 95% CI 0.53−0.89; p=5∙10-3) and rs4409525 in Claudin-2 (CLDN2 ; OR=1.41; 95% CI 1.08−1.83; p=1∙10-2). In total, 207 out of 244 cases were homozygous for the risk allele in rs13228878 (n=104), rs17107315 (n=1), rs10436957 (n=165) and/or rs4409525 (n=16). However, no significant additive effect of having more than one risk allele was found. Conclusion: Children who develop AAP possess the same pancreatitis risk variants as adults with non-asparaginase associated pancreatitis. This shared genetic disposition may facilitate research into pathogenesis and identification of effective interventions towards AAP. Disclosures No relevant conflicts of interest to declare.