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Methotrexate neurotoxicity: risk factors & outcome
who experienced late MTX neurotoxicity after consolida- tion (n=53, P=0.011, Figure 2) (Cox regression P=0.036, HR 1.92 [95% CI: 1.04-3.54]). A similar number of patients had IT MTX ceased in both groups (21 of 42 in the group with early MTX toxicity; 27 of 53 who experi- enced late MTX toxicity). ALL risk-group did not vary sig- nificantly between those who experienced MTX neuro- toxicity before or after consolidation (P=0.874).
Long-term neurological outcome
Long-term neurological outcome was reported at last follow-up (median 94 months, range, 3-181 months) for 41.7% (n=522 of 1,251) of patients. There was no reported mortality due to long-term neurological problems follow- ing MTX neurotoxicity. A total of 1.3% (n=7 of 522) were diagnosed with epilepsy at last follow-up. Three out of these seven children were diagnosed with epilepsy fol- lowing symptomatic MTX neurotoxicity, and all remained in CR1.
Genome-wide association study results
There was no difference in the incidence of MTX neu- rotoxicity among the subgroup who were included and excluded from the GWAS, in relation to age, sex, timing and CTCAE grade of toxicity (Online Supplementary Table S7).
We found seven intronic SNP that were associated with MTX neurotoxicity at a significance level of P<1x10-6 (Table 3), which mapped to six genes (MBOAT-1, GIPC1, ZDHHC19, NXN, PKN1, HMGB1P37). The rare alleles at SNP near MBOAT-1 and NXN were protective (OR <1) while those at the other SNP correlated with increased risk of MTX neurotoxicity.
There were 53 additional SNP (minor allele frequency [MAF] >2%) associated with MTX neurotoxicity at a sig- nificance level of P<5x10-6, which mapped near to seven genes (Online Supplementary Table S8). Further understand- ing of SNP function and roles was determined using con- temporary online data repositories (see the Online Supplementary Appendix). Most SNP (P<5x10-6) were intronic, except for rs76301301 (3’ untranslated region [UTR] variant, P=1.34x10-6) located within GIPC1 and rs7555699 located within 2 kb upstream of a 5’ end of BMP8A (P=4.54x10-6) (Online Supplementary Table S9).
Discussion
This is the largest cohort of children treated for ALL/LBL mapped for the incidence, risk factors, and long-term impact of MTX toxicity. Independent risk fac- tors for symptomatic MTX neurotoxicity were age ≥10 years at diagnosis and >grade 3 elevation of serum AST during early therapy. Discontinuation of IT MTX, in an attempt to minimize further neurotoxicity after a first MTX neurotoxic event, was associated with an increased incidence of CNS relapse. Patients continuing IT MTX had only a small risk of MTX neurotoxicity recurrence (<13%). Regardless of subsequent IT management strat- egy, children who developed MTX neurotoxicity had an increased risk of epilepsy.
We report on independent risk factors in multivariable analysis that are associated with symptomatic methotrex- ate neurotoxicity: age ≥10 years at diagnosis and >grade 3 elevation of serum AST during induction/consolidation.
Importantly, these factors were determined in a combined cohort of Australian children treated for ALL on either BFM or COG-based therapy. AST elevation in the current series may reflect direct hepatotoxicity from methotrex- ate,17 systemic metabolic disturbances,18 release from non- hepatic sources such as erythrocytes,18 or a marker of longer serum exposure to MTX as has been shown for ALT elevation post HD MTX.17
Older age has been previously reported as a risk factor in univariate1 and recently in multivariable analysis in a smaller cohort.19 Potential reasons include reduced clear- ance of HD MTX20 or higher steady state MTX concen- tration following IV administration21 in older children. Protocols that used higher IT doses (15 mg) for children ≥9 years of age were not associated with increased neurotox- icity in our study.
The observed increase in cumulative incidence of CNS relapse following permanent cessation of IT MTX therapy requires validation in contemporaneous cohorts. In study POG 9005 (1991-1995), which had the same number of cases of acute MTX neurotoxicity (n=95), 54 patients had intrathecal therapy modified without an increase in CNS relapse.6 CNS-relapse rates in our overall cohort (n=1,251) were consistent with published rates.22,23 Treatment of CNS relapse involves exposure to additional neurotoxic agents including CNS irradiation.24 Taken together, these data suggest that clinicians should not cease IT MTX ther- apy after a first episode of MTX neurotoxicity, especially when it occurs early in therapy.
Out of the 95 patients identified with MTX central neu- rotoxicity, 53 fulfilled the Ponte di Legno Toxicity Working Group MTX stroke-like syndrome (SLS) defini- tion that was published after our study commenced, i.e., symptoms within 21 days of IT/IV MTX, characteristic clinical course and/or imaging, with exclusion of other causes.4 All patients included in the GWAS experienced MTX neurotoxicity <21 days from IT/IV MTX.
We did not identify any SNP associated with MTX neu- rotoxicity at genome-wide levels of significance, but report seven SNP at P<1x10-6 which require replication in larger, independent studies of MTX neurotoxicity. These seven SNP mapped near six genes, five of which have potential roles in neuronal cell growth, differentiation, development, or developmental delay phenotypes (MBOAT-1, GIPC1, ZDHHC19, NXN, PKN1).25-29 A 3’UTR variant in GIPC1 was also associated with MTX neurotoxicity (P=1.34x10-6).
A prospective study by Bhojwani et al.1 reported 14 chil- dren with subacute symptomatic MTX neurotoxicity and did not identify any genome-wide significant SNP (top SNP significance level P=3.65x10-6). We were unable to replicate their top SNP for symptomatic leukoen- cephalopathy. There is a critical need to validate germline risk factors for MTX neurotoxicity in a larger international study, as larger numbers will increase power of the GWAS.
There are several limitations inherent to systematic ret- rospective analyses. Collated clinical laboratory data were dependent on tests performed during routine clinical care, including AST values. Patients with missing data were censored and some variables were more complete than others. Clinical documentation regarding longer-term neu- rological function was not consistently available nor stan- dardized, reducing the cohort for analysis.
With respect to the MTX doses, analysis was performed
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