GSTA1 diplotypes affect busulfan clearance and toxicity in children undergoing allogeneic hematopoietic stem cell transplantation: a multicenter study

Abstract

Busulfan (BU) dose adjustment following therapeutic drug monitoring contributes to better outcome of hematopoietic stem cell transplantation (HSCT). Further improvement could be achieved through genotype-guided BU dose adjustments. To investigate this aspect, polymorphism within glutathione S transferase genes were assessed. Particularly, promoter haplotypes of the glutathione S transferase A1 (GSTA1) were evaluated in vitro, with reporter gene assays and clinically, in a pediatric multi-center study (N =138) through association with BU pharmacokinetics (PK) and clinical outcomes. Promoter activity significantly differed between the GSTA1 haplotypes (p<0.001) supporting their importance in capturing PK variability. Four GSTA1 diplotype groups that significantly correlated with clearance (p=0.009) were distinguished. Diplotypes underlying fast and slow metabolizing capacity showed higher and lower BU clearance (ml/min/kg), respectively. GSTA1 diplotypes with slow metabolizing capacity were associated with higher incidence of sinusoidal obstruction syndrome, acute graft versus host disease and combined treatment-related toxicity (p<0.0005). Among other GST genes investigated, GSTP1 313GG correlated with acute graft versus host disease grade 1-4 (p=0.01) and GSTM1 non-null genotype was associated with hemorrhagic cystitis (p=0.003). This study further strengthens the hypothesis that GST diplotypes/genotypes could be incorporated into already existing population pharmacokinetic models for improving first BU dose prediction and HSCT outcomes. (No Clinicaltrials.gov identifier: NCT01257854. Registered 8 December 2010, retrospectively registered).

Keywords: busulfan; hematopoietic stem cell transplantion; pharmacogenetics; pharmacokinetics; toxicity.

Conflict of interest statement

CONFLICTS OF INTEREST H.B has acted as a consultant for Jazz Pharmaceuticals and obtained an education grant from them. H.B also acted as a consultant for Seattle Genetics. The authors declare that they have no other financial relationship(s) to disclose.

Figures

Figure 1. GSTA1 Haplotype and Reporter Gene assay of GSTA1 promoter

A. Haplotypes investigated with luciferase reporter assay. rs (reference SNP ID) numbers correspond to each SNP included for site directed mutagenesis. SNPs used for genotyping and for inferring sub-haplotypes in patients are highlighted in bold. B. Luciferase activities of the proximal promoters of GSTA1 variants (GSTA1*A1, GSTA1*A2, GSTA1*A3, GSTA1*B1a, GSTA1*B2, GSTA1*B1b) in transient transfection in HepG2 cells. Error bars represent the standard deviations. Pairwise comparisons by analysis of variance (ANOVA) between data for the GSTA1*A1 vs. any other haplotype, after Bonferroni correction *** = p < 0.001; ****= p <0.0001, ***** = p < 0.000001.

Figure 2. Pharmacokinetic parameters of BU and dose requirement in relation to GSTA1 functional diplotype groups and GSTM1 genotypes

A. Busulfan first dose clearance (CL, in ml/min/kg) against GSTA1 diplotypes B. Busulfan first dose CL in females only against GSTA1 genotypes. C. Dose requirement (ratio of adjusted to initial dose) against GSTA1 diplotypes. D. Cumulative AUC (mg.h/L) against GSTA1 diplotypes. CHU Sainte-Justine patients only were included for analysis presented in C and D. Diplotype groups II and III were combined into a single group in C and D. E. Busulfan first dose clearance in children above 4 yrs of age against GSTM1 genotypes. DELGSTM = Deleted GSTM1 gene. Number of patients and p values are depicted on the plots.

Figure 3. Incidence of SOS, aGvHD and TRT in relation to GSTA1 functional diplotype groups

Cumulative incidences of A. sinusoidal obstruction syndrome (SOS), B. acute graft versus host disease (aGvHD) 1-4, C. treatment related toxicity (TRT) including aGvHD 1-4 and D. TRT including aGvHD 2-4. Results plotted for diplotype group IV (IV+) versus groups I, II & III (IV-). E. Overall survival (OS) in relation to GSTA1 extreme diplotype status (group I vs. group IV), in patients who received busulfan-cyclophosphamide conditioning regimen. Total number of patients represented by each curve with number of patients with indicated toxicities in parenthesis, and p value are depicted on each plot; group IV associated hazard ratios are depicted below each plot. F. Association of TRT with diplotype group IV in a competing events risk analysis. IV+ and IV- indicates the presence of this GSTA1 diplotype group. Competing events for TRT incidence were: death and relapse. p values for the difference in cumulative incidence of TRT, death, and relapse, between haplotype groups (IV vs others) is 0.000003, 0.3 and 0.5, respectively.

Figure 4. Complications of HSCT in relation to GSTP1 genotypes and GSTA1 diplotypes

A. Acute GvHD 1-4 incidence according to GSTP1 c.313A>G genotypes; and B. Acute GvHD 1-4 incidence according to combinatory GSTA1-GSTP1 status. A plus sign represents the risk genotypes, which is presence of GSTP1*GG and/or GSTA1 diplotype group IV. C. Hemorrhagic Cystitis (HC) incidences in relation to GSTM1 Null and Non-null genotype.

Figure 5. Busulfan plasma exposure and clinical outcomes of HSCT

Incidences of event-free survival (EFS), overall survival (OS), and treatment related toxicity (TRT) plotted against 3 groups based on first dose steady state concentration (Css) i.e. 900 ng/mL in all patients (n = 138). Total number of patients in each group (number of patients with events) are depicted on all plots. P values are shown on the plots.

Figure 6. Treatment related toxicity (TRT) in relation to both 1st dose Css and GSTA1 groups

TRT (all cases combined) plotted against A. Busulfan Css below 900 ng/mL or B. Css above 900 ng/mL, dependent on whether patients are in GSTA1 diplotype group I, II, III (IV-) or IV (IV+). Total number of patients in each group with number of patients with TRT in brackets is depicted on all plots. Hazard ratio for group IV is depicted for plot (A) only.