In the discovery cohort, we estimated methotrexate clearance for 3,014 treatment courses given to 434 patients (Data Supplement Table 1). There was substantial interpatient variability in average (± standard deviation) methotrexate clearance (), which differed among the 213 patients on the Total XIIIB (133 ± 25 mL/min/m2
), the 114 patients on the Total XV low-risk (123 ± 28 mL/min/m2
), and the 107 patients on the Total XV standard/high-risk (106 ± 22 mL/min/m2
) treatment regimens (Data Supplement Fig 1; P
< 2.2 × 10−16
). The variability observed between treatment regimens is not surprising, given that dosage and the concomitant hydration and alkalinization (factors that can affect methotrexate clearance) differed (Data Supplement Table 2).22
Average methotrexate clearance differed by ancestral group (Data Supplement Fig 2; P
= .005) with African American patients having the highest clearance. As previously noted,19,22
methotrexate clearance decreased with increasing age (Data Supplement Fig 3; P
= 1.1 × 10-6
). Thus, we adjusted all analyses for age, race, sex, and treatment regimen. Using a linear mixed effect model, the intrapatient variability in methotrexate clearance was 48%, while the interpatient variability was 52%.
Fig 1. Average methotrexate clearance and linkage disequilibrium structure of the SLCO1B1 single-nucleotide polymorphisms (SNPs) in 434 children with acute lymphoblastic leukemia. The average methotrexate clearance for each of the 434 children in the discovery (more ...)
We tested the association between 398,699 SNPs and average methotrexate clearance in 434 children in the discovery cohort. The strongest association signal was on chromosome 12 (). The two top SNPs associated with methotrexate clearance were annotated to the transporter gene, SLCO1B1/OATP1B1, located on chromosome 12 (; Data Supplement Tables 3-5). These two SLCO1B1 SNPs remained significant (rs11045879, P = 1.7 × 10−10 and rs4149081, P = 1.7 × 10−9) even after correction for multiple testing. These two SLCO1B1 SNPs were in complete LD (r2 = 1) with each other () and were associated with methotrexate clearance across regimens ().
Fig 2. Genome-wide P values showing the association of single-nucleotide polymorphisms (SNPs) with methotrexate clearance in the discovery cohort of 434 children with acute lymphoblastic leukemia. Shown is the distribution of P values (as –log10 values) (more ...)
SLCO1B1 SNPs and Methotrexate Clearance
Fig 3. SLCO1B1 single-nucleotide polymorphism (SNP) rs11045879 is associated with methotrexate clearance and GI toxicity. (A) Association of SLCO1B1 SNP genotype (rs11045879) with methotrexate clearance in patients on Total XIIIB (TXIIIB), Total XV low-risk (more ...)
We also used a linear mixed effects model to identify associations between SNP genotypes and methotrexate clearance; the results were similar to those assessing average clearance, with the same two top-ranked SNPs (P < 6.8 × 10−9) in SLCO1B1 (Data Supplement Table 4). Only 6.0% of the intracourse variability in clearance was accounted for by course number, even when accounting for the premethotrexate serum creatinine (Data Supplement Table 6).
Eight additional SLCO1B1 SNPs () were associated with clearance (P < .05); these SNPs were not in LD (based on r2) with the top two SLCO1B1 SNPs () and were encompassed by multiple haplotype blocks (Data Supplement Figs 4A and B). In a gene-level analysis, SLCO1B1 had a stronger association with methotrexate clearance (P < 1 × 10−6), with a higher r2 and a lower Akaike information criterion, than any other gene (Data Supplement Tables 7 and 11).
Fig 4. Incidence of toxicity based on SLCO1B1 rs11045879 genotype. Cumulative incidence of the first episode of GI toxicity significantly differed (P = .024) by SLCO1B1 rs11045879 genotype during the continuation phase of treatment during the Total XIIIB treatment (more ...)
We genotyped a validation cohort of 206 additional patients (). Seven of the 10 interrogated SLCO1B1 SNPs remained associated (P < .05) with methotrexate clearance (). Nine of the 10 SLCO1B1 SNPs were associated with clearance (P < .05) when the two cohorts (discovery and validation) were combined (), and the strongest signal remained in SLCO1B1 (Data Supplement Fig 5).
The percentage of methotrexate clearance variability that could be explained by SLCO1B1 genotypes compared with the variability explained by factors such as race, sex, age, treatment regimen, or serum creatinine was estimated in a multivariate analysis for both the discovery and validation cohorts (). In a stepwise linear regression analysis, five SNPs were retained in the model (), such that SLCO1B1 genetic variation accounted for 9.3% of the interpatient variability in the discovery cohort and 11.3% in the validation cohort. This superseded the proportion of variability contributed by the significant factors of race, serum creatinine, and sex; only treatment regimen accounted for a higher proportion (17.9%) of the variation in clearance (). Average serum creatinine concentrations explained 3.2% and 1.9% of the variability in methotrexate clearance in the discovery and validation cohorts, respectively; in a linear mixed effects model (Data Supplement Table 6), 2.4% of interpatient and 0.4% of intrapatient variability was accounted for by serum creatinine. Treatment regimen and SLCO1B1 SNP genotypes accounted for 18.6% and 11.0% of variability in clearance, respectively. Results were similar for the first cycle only of methotrexate (Data Supplement Table 8).
Multivariate Analysis of Factors Associated With Average Methotrexate Clearance in the Discovery and Validation Cohorts
GI toxicity (grade 3 or 4 mucositis) and infection were the most common toxicities observed during the methotrexate-intensive consolidation and continuation phases of Total XIIIB therapy.26
SNPs in SLCO1B1
, rs11045879 T allele (OR, 16.4; 95% CI, 8.7 to 26.7; P
= .004) and the G allele at rs4149081 (OR, 15.3; 95% CI, 7.9 to 24.6; P
= .03; data not shown) were each associated with GI toxicity during consolidation (). The same two SNPs were also associated with GI toxicity during the continuation phase (). During the consolidation phase of Total XV, GI toxicity was less frequent (5% v
18% in Total XIIIB), and we found no association with SLCO1B1
SNP genotypes. Because all patients on Total XV had their methotrexate doses adjusted to achieve a common steady-state plasma concentration, relationships among toxicity and methotrexate exposure and SLCO1B1
SNPs may have been attenuated on this trial. We found no associations between SLCO1B1
SNP genotype and infectious toxicity in either trial (data not shown).
We also genotyped the nonsynonymous SLCO1B1 T521C (rs4149056) polymorphism in a subset of 489 patients; this subset included 387 patients from the discovery cohort and 102 patients from the validation cohort (Data Supplement Table 1). SLCO1B1 T521C was in high LD with the top SLCO1B1 SNPs rs11045879 (r2 = 0.86, discovery cohort; r2 = 0.89, validation cohort) and rs4149081 (r2 = 0.86, discovery cohort; r2 = 0.89, validation cohort). SLCO1B1 T521C was associated with methotrexate clearance in the discovery and the combined (discovery plus validation) patient cohorts (Data Supplement Table 9; P = 1.9 × 10−7 and P = 1.2 × 10−7, respectively). In addition, a marginal association (P = .07) was observed between this SNP genotype and the occurrence of GI toxicity during consolidation (Data Supplement Fig 6). In multivariate analysis, the T521C SNP was significantly associated with methotrexate clearance; however, when genotypes at T521C and at rs11045879 were allowed to compete, only the rs11045879 SNP remained in the model (Data Supplement Table 10).