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Pancreatitis in childhood ALL
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 (r2=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 pan- creatitis in adults. Intra-pancreatic trypsinogen cleavage to proteolytic trypsin induces autodigestion and pancreatitis. In conclusion, this study finds a shared genetic predisposition between asparagi- nase-associated pancreatitis and non-asparaginase-associated pancreatitis, and targeting the trypsino- gen activation pathway may enable identification of effective interventions for asparaginase-associ- ated pancreatitis.
Introduction
Intensification of chemotherapy for childhood acute lymphoblastic leukemia (ALL) has generated 5-year sur- vival rates greater than 90%, but has been associated with an increase in therapy-related toxicity.1 Asparaginase is a key drug in the treatment of childhood ALL and there is growing interest in its use as an anti-metastatic agent in breast cancer.2 Asparaginase depletes the body of the non- essential amino acid asparagine through deamidation of asparagine into aspartic acid and ammonia,3 and targets protein synthesis in malignant lymphoblasts by impairing the ability to synthesize asparagine.4,5 Pancreatitis associ- ated with asparaginase therapy (AAP) is a frequent toxici- ty affecting 4−10% of children treated on contemporary ALL protocols, and is associated with severe complica- tions.6–9 In addition, re-exposure to asparaginase after AAP is associated with a high risk (~50%) of a second episode of AAP, and thus AAP often entails truncation of asparag- inase therapy, thereby decreasing the patients’ chance of survival.4,5,9 The mechanism(s) by which asparaginase causes pancreatitis are elusive, thus hampering attempts to identify patients with an altered risk of AAP.10 The Ponte di Legno toxicity working group, therefore, initiated a study with three main purposes: (i) to define diagnostic consensus criteria for AAP;11 (ii) to describe the phenotype of AAP in patients across multiple ALL trial groups;9 and (iii) to explore genotype-phenotype associations, using a genome-wide approach, to identify patients with altered risk of AAP.9,11 Genome-wide association (GWA) studies are agnostic by design, reporting phenotype-genotype associations without prior hypotheses and often including speculative mechanisms. Replication of GWA study results are thus a requisite for credibility Accordingly, this study presents results from the largest AAP GWA study so far, with a strong focus on investigating previously validat- ed variants associated with non-asparaginase-induced pancreatitis and replicating top results in similar childhood ALL cohorts.
Methods
Study design and participants
Ten international childhood ALL trial groups (Online Supplementary Table S1) contributed to the discovery cohort. Post- remission DNA was collected from June 2015 to January 2017, three groups collected DNA on AAP cases only while seven
groups did so on both cases and controls (Online Supplementary Figure S1). The database containing phenotype data was approved by the regional ethical review board of The Capital Region of Denmark (H-2-2010-022), the Danish Data Protection Authorities (j.nr.: 2012-58-0004), and by relevant regulatory authorities in all participating countries. Genotype data were stored at the Technical University of Denmark’s server Computerome.12
Children (aged 1.0–17.9 years) with newly diagnosed ALL between January 2000 and January 2016 were eligible, irrespective of ethnicity. Pancreatitis was defined as asparaginase-associated if diagnosed within 50 days of the last injection of native E. coli asparaginase or polyethylene glycolated E. coli asparaginase (PEG- asparaginase) and cases fulfilled the Ponte di Legno toxicity work- ing group consensus definition for AAP: i.e., at least two of (i) amylase, pancreatic amylase, or pancreatic lipase ≥3 x upper nor- mal limit; (ii) abdominal pain; and (iii) imaging compatible with imaging compatible with pancreatitis. All controls received the planned amount of asparaginase therapy in their respective proto- cols, with more than 78% (1024/1320) receiving at least eight injections of PEG-asparaginase without developing AAP. A subset of 62 AAP cases was previously included in a Nordic Society of Pediatric Hematology and Oncology (NOPHO) GWA study.13 These samples were genotyped on identical genotyping arrays as the remaining cohort, and raw genotyping data on these patients were pooled with those of the remaining cohort prior to quality control, and association analyses were done in one cohort.
Genotyping
Post-remission DNA was genotyped by Aros Applied Biotechnology A/S (Aarhus, Denmark) on Illumina Omni2.5exome-8 BeadChip arrays using the human genome assembly (GRCh37) for reference. Quality control was performed using the PLINK tool,14 and single nucleotide polymorphisms (SNPs) were annotated in Ensembl Variant Effect Predictor GCRCh37.15 Alleles given are refSNP alleles according to dbSNP (not necessarily the alleles supplied by the Illumina map).16
Quality control
Quality control was performed according to previously pub- lished criteria17 (Online Supplementary Figures S2-4), excluding indi- viduals with: (i) a discordance in number of X chromosomes between geno- and phenotypes; (ii) missing data on >3% of SNPs; (iii) excess heterozygosity between autosomal SNPs; and (iv) high relatedness between samples. SNPs were excluded based on: (i) missing data on >2% of individuals (call rate); (ii) Hardy-Weinberg equilibrium; (iii) minor allele frequency <0.01; (iv) difference in call rate between cases and controls (Fisher exact test P<1.10-5); and (v) duplicated genomic position.
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