The Association of Combined GSTM1 and CYP2C9 Genotype Status with the Occurrence of Hemorrhagic Cystitis in Pediatric Patients Receiving Myeloablative Conditioning Regimen Prior to Allogeneic Hematopoietic Stem Cell Transplantation

Chakradhara Rao S Uppugunduri, Flavia Storelli, Vid Mlakar, Patricia Huezo-Diaz Curtis, Aziz Rezgui, Yves Théorêt, Denis Marino, Fabienne Doffey-Lazeyras, Yves Chalandon, Peter Bader, Youssef Daali, Henrique Bittencourt, Maja Krajinovic, Marc Ansari, Chakradhara Rao S Uppugunduri, Flavia Storelli, Vid Mlakar, Patricia Huezo-Diaz Curtis, Aziz Rezgui, Yves Théorêt, Denis Marino, Fabienne Doffey-Lazeyras, Yves Chalandon, Peter Bader, Youssef Daali, Henrique Bittencourt, Maja Krajinovic, Marc Ansari

Abstract

Hemorrhagic cystitis (HC) is one of the complications of busulfan-cyclophosphamide (BU-CY) conditioning regimen during allogeneic hematopoietic stem cell transplantation (HSCT) in children. Identifying children at high risk of developing HC in a HSCT setting could facilitate the evaluation and implementation of effective prophylactic measures. In this retrospective analysis genotyping of selected candidate gene variants was performed in 72 children and plasma Sulfolane (Su, water soluble metabolite of BU) levels were measured in 39 children following treatment with BU-CY regimen. The cytotoxic effects of Su and acrolein (Ac, water soluble metabolite of CY) were tested on human urothelial cells (HUCs). The effect of Su was also tested on cytochrome P 450 (CYP) function in HepaRG hepatic cells. Cumulative incidences of HC before day 30 post HSCT were estimated using Kaplan-Meier curves and log-rank test was used to compare the difference between groups in a univariate analysis. Multivariate Cox regression was used to estimate hazard ratios with 95% confidence intervals (CIs). Multivariate analysis included co-variables that were significantly associated with HC in a univariate analysis. Cumulative incidence of HC was 15.3%. In the univariate analysis, HC incidence was significantly (p < 0.05) higher in children older than 10 years (28.6 vs. 6.8%) or in children with higher Su levels (>40 vs. <11%) or in carriers of both functional GSTM1 and CYP2C9 (33.3 vs. 6.3%) compared to the other group. In a multivariate analysis, combined GSTM1 and CYP2C9 genotype status was associated with HC occurrence with a hazards ratio of 4.8 (95% CI: 1.3-18.4; p = 0.02). Ac was found to be toxic to HUC cells at lower concentrations (33 μM), Su was not toxic to HUC cells at concentrations below 1 mM and did not affect CYP function in HepaRG cells. Our observations suggest that pre-emptive genotyping of CYP2C9 and GSTM1 may aid in selection of more effective prophylaxis to reduce HC development in pediatric patients undergoing allogeneic HSCT. Article summary: (1) Children carrying functional alleles in GSTM1 and CYP2C9 are at high risk for developing hemorrhagic cystitis following treatment with busulfan and cyclophosphamide based conditioning regimen. (2) Identification of children at high risk for developing hemorrhagic cystitis in an allogeneic HSCT setting will enable us to evaluate and implement optimal strategies for its prevention. Trial registration: This study is a part of the trail "clinicaltrials.gov identifier: NCT01257854."

Keywords: CYPs; HepaRG; acrolein; busulfan; conjugation; cyclophosphamide; induction; urothelial cells.

Figures

FIGURE 1
FIGURE 1
Hypothetical model for Hemorrhagic Cystitis occurrence in children receiving BU-CY regimen prior to allogeneic HSCT. Busulfan (BU) and cyclophosphamide (CY) metabolism predominantly occurs in the liver generating the water soluble metabolites sulfolane (Su) and acrolein (Ac), respectively. During the metabolic process BU utilizes glutathione (GSH) and activity of GSTs such as GST alpha 1 (GSTA1), GST mu1 (GSTM1) plays an important role in conjugation of BU with GSH. GSH is also essential for scavenging the CY active metabolites including Ac also catalyzed by GSTs. When CY is administered after BU, the water soluble metabolite of BU, i.e., Su could influence the activity of cytochrome P 450 enzymes (CYPs) and thus might affect the activation of CY, or on the other hand higher Su levels also represents the utilization of GSH and formation of γ-glutamyl-dehydroalanyl-glycine (EdAG) affecting the availability of reduced form of GSH. Su might also affect the function of GSTs and other enzymes in BU, CY metabolism and proteins such as Mitochondrial uncoupling proteins (UCP) encoded by SLC25A27 gene affecting cell survival. Su possibly affect the function of ADH (alcohol dehydrogenase) enzyme resulting in increased levels of hydroxy CY metabolite, resulting increased Ac formation. BU could irreversibly alkylate the glutaredoxins (Grx) and Thioredoxins (Trx) increasing the oxidative stress, thus increasing toxicity of incoming CY metabolites such as 4 hydroxy cyclophosphamide (4OH-CY); Ac, chloro acetaldehyde (CA), and Phosphoramide mustard (PM). Altered activity of CYPs due to less functional alleles in the encoding genes might have a predictable function of Su formation and CY activation. Circulating CY metabolites (4OH CY, PM) including Ac inhibit cytokine function by alkylation and also cause immunosuppression, further increasing the chances of viremia and viruria (BK and/or JC virus). Ac conjugated with GSH in the liver gets delivered to the bladder and releases free Ac which then could alkylates cysteine residues and causing toxicity. Altogether increased rate of metabolite delivery to the urinary bladder together with viral load and lack of protection due to increased oxidative stress, endothelial damage to the blood vessels, urothelial damage and enhanced viral replication results in bladder inflammation and hematuria and the condition hemorrhagic cystitis.
FIGURE 2
FIGURE 2
Sufolane levels and occurrence of Hemorrhagic cystitis. (A,B) Su levels between patients with and without HC. (C) ROC curve for Su levels as a predictive marker for HC (n = 39). A cut off of 49.43 and 36.28 ng/mL/mg/kg for HC, before dose 7 and after dose 9, respectively [before dose 7; Area = 0.75 (95% CI: 0.59–0.93; p = 0.02); after dose 9; Area = 0.74 (95% CI: 0.57–0.91), p = 0.03]. (D,E) Cumulative incidences HC. Patients with HC/total number of patients in each group. (D) Before dose 7 (E) after dose 9.
FIGURE 3
FIGURE 3
Incidence of Hemorrhagic cystitis. (A) in relation to the CYP2C9 genotype status. Group I represents patients without dysfunctional alleles, and II represents carriers of at least one dysfunctional allele. (B) In relation to GSTM1 genotype, group I represents nonNull carriers; and group II represents Null allele carriers. (C) In relation to combined CYP2C9 and GSTM1 genotypes group I represents carriers of normal functional CYP2C9 and GSTM1 genotypes and group II represents carriers of at least one dysfunctional allele in either CYP2C9 or GSTM1 or both. Number of patients with HC/total number of patients in each group and p-values are mentioned on the plots. Multivariate Cox-regression p-value and hazards ratio with 95% confidence intervals are mentioned for 3C.

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