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B.O. Wolthers et al.
the (rs4726576; rs10273639) risk allele, these patients are at higher risk of trypsin activation and pancreatitis.26 In a study investigating the effect of asparaginase on pancreat- ic acinar cells, asparaginase evoked intracellular calcium release from the endoplasmic reticulum mediated by the protease-activated receptor 2 (Figure 3). This elevation in calcium levels in turn activated calcium release activated calcium (CRAC) channels further increasing intracellular calcium levels, leading to decreased ATP levels, trypsin activation and necrosis.35 The pancreatitis-causing mecha- nism seems to be independent of the anti-neoplastic effect of asparaginase, and drugs inhibiting CRAC channels could thus be used to prevent AAP during asparaginase therapy, not least in patients who are re-exposed to asparaginase after having had AAP, since these patients have a ~50% risk or developing a second episode of AAP.9,13
The NFATC2 associated variant rs62228256 was most significantly associated with AAP. However, with no asso- ciation found in the replication study and no association with pancreatitis found in adult studies on non-asparagi- nase associated pancreatitis,26 the association between NFATC2 and AAP seems to have low credibility. We were not able to associate the PRSS1-PRSS2 genotype to risk of AAP-related complications, indicating that this allele only alters the risk of AAP, while the complications are a result of other factors. The validation of our PRSS1-PRSS2 top SNPs in the AALL0232 cohort strengthens the credibility of this result. The association was of similar effect size, but of borderline statistical significance, which may reflect three key issues: (i) diagnostic criteria differ between the cohorts, and cases are not completely comparable; (ii) pan- creatitis is strongly associated with asparaginase exposure, and it was a prerequisite that included controls received a significant amount of asparaginase to reduce the risk of
false negative controls; and (iii) the validation cohort was relatively small.36,37
Our results need to be judged in the light of their limita- tions. The association analyses were strongly influenced by individuals of CEU ethnicity, and we cannot determine the effects in non-CEU populations. Moreover, our results for the PRSS1-PRSS2 locus did not reach genome-wide significance with a P-value <5 x 10-8. This highlights a challenge when doing GWA studies in cohorts of limited size, as will often be the case in childhood ALL, and requires strategies for validation in independent and simi- lar cohorts. In this present study, we attempted to limit the problem of limited sample size by improving the qual- ity of phenotyping, collecting individual clinical data on AAP cases and including controls with documented com- pletion of extensive asparaginase therapy.
In conclusion, we found that children who develop AAP possess identical genetic risk variants as adults with non- asparaginase-associated pancreatitis. This may allow future preventive measures for reduction of AAP.
Acknowledgments
We thank all the researchers who scrutinized patients’ files and completed phenotype questionnaires, colleagues at Harvard Department of Biomedical Informatics for valuable insights, and organizational support from the research staff at Bonkolab, at the University Hospital Rigshospitalet. Furthermore, we thank the Bloodwise Childhood Leukaemia Cell Bank, UK, for providing samples and data for this research.
Funding
This study was funded by the Kirsten and Freddy Johansen Foundation, the Danish Childhood Cancer Foundation, the Swedish Childhood Cancer Foundation and the Danish Cancer Society (R150-A10181).
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