Marc Ansari

Polymorphisms in multidrug resistance-associated protein gene 4 is associated with outcome in childhood acute lymphoblastic leukemia

Methotrexate and 6-mercaptopurine, important components of acute lymphoblastic leukemia treatment, are substrates for multidrug resistance-associated protein MRP4. Eight single nucleotide polymorphisms were analyzed in MRP4 gene, and 4 variants were identified as tagSNPs with frequency more than or equal to 5%. They were investigated for association with treatment responses in 275 children with acute lymphoblastic leukemia. The TC genotype of the regulatory T-1393C polymorphism was associated with better event-free survival (P = .02) and lower methotrexate plasma levels (P = .01). The CA genotype of A934C (Lys304Asn) substitution correlated in contrast with lower event-free survival (P = .02) and higher frequency of high-grade thrombocytopenia (P = .01). Gene reporter assay showed that the promoter haplotype uniquely tagged by the C-1393 allele conferred higher promoter activity compared with remaining haplotypes (P < .001). Further analyses are needed to replicate this pilot study and get closer insight into the functional effect of these polymorphisms.

Influence of GST gene polymorphisms on busulfan pharmacokinetics in children.

Busulfan (BU) is a key compound in conditioning myeloablative regimens for children undergoing hematopoietic stem cell transplantation (HSCT). There are wide interindividual differences in BU pharmacokinetics, which increase the risk of veno-occlusive disease, graft rejection and disease relapse. As BU is mainly metabolized by glutathione S-transferase (GST), it is hypothesized that functional polymorphisms in GST genes may explain in part the variability in BU pharmacokinetics. We analyzed polymorphisms in GSTA1 (C-69T, A-513G, G-631T, C-1142G), GSTM1 (deletion) and GSTP1 (A1578G, C2293T) genes in 28 children undergoing HSCT. All patients had individualized dosing based on pharmacokinetics after the first dose of intravenous BU. GSTM1-null individuals had higher drug exposure (PCmax=0.008; PAUC=0.003; PCss=0.02) and lower clearance (PCL=0.001). Multivariate regression models showed that, other than the drug dose and age, the GSTM1 genotype was the best predictor of first-dose pharmacokinetic variability. GSTM1-null patients also received lower cumulative BU doses (P=0.02). No association was found between BU exposure and major GSTA1 or GSTP1 gene variants. In children, GSTM1 polymorphism seems to modify BU pharmacokinetics after intravenous drug administration.

Pharmacogenomics in cancer treatment defining genetic bases for inter-individual differences in responses to chemotherapy.

Purpose of review Pharmacogenomics is evolving rapidly due to the expansion of genomics and proteomics, the emerging technologies, knowledge of the molecular basis of neoplasms and of drug pathways. This article will give an update on the genetic basis of variable therapeutic responses to anticancer agents in children. Recent findings The majority of recent findings concern the pharmacogenetics of key components of acute lymphoblastic leukemia treatment, 6-mercaptopurine and methotrexate. This is not surprising given that leukemia is the most common cancer affecting children, accounting for 25–35% of childhood malignancies worldwide with acute lymphoblastic leukemia comprising 80% of leukemia cases. In certain patients treatment fails due to drug resistance, rendering acute lymphoblastic leukemia the leading cause of cancer-related death in children. Most of the studies use a candidate gene approach adding a new body of evidence to existing knowledge. Recent findings relating to other childhood tumors and the potential to optimize treatment of these malignancies are briefly discussed. Summary Interindividual differences in drug responses are an important cause of resistance to treatment and adverse drug reactions. Pharmacogenetics tends to identify the genetic basis of these suboptimal responses allowing traditional treatment to be complemented by genotype-based drug dose adjustment.

Risk-stratified staging in paediatric hepatoblastoma: a unified analysis from the Children's Hepatic tumors International Collaboration

BACKGROUND Comparative assessment of treatment results in paediatric hepatoblastoma trials has been hampered by small patient numbers and the use of multiple disparate staging systems by the four major trial groups. To address this challenge, we formed a global coalition, the Childrens Hepatic tumors International Collaboration (CHIC), with the aim of creating a common approach to staging and risk stratification in this rare cancer. METHODS The CHIC steering committee-consisting of leadership from the four major cooperative trial groups (the International Childhood Liver Tumours Strategy Group, Childrens Oncology Group, the German Society for Paediatric Oncology and Haematology, and the Japanese Study Group for Paediatric Liver Tumours)-created a shared international database that includes comprehensive data from 1605 children treated in eight multicentre hepatoblastoma trials over 25 years. Diagnostic factors found to be most prognostic on initial analysis were PRETreatment EXTent of disease (PRETEXT) group; age younger than 3 years, 3-7 years, and 8 years or older; α fetoprotein (AFP) concentration of 100 ng/mL or lower and 101-1000 ng/mL; and the PRETEXT annotation factors metastatic disease (M), macrovascular involvement of all hepatic veins (V) or portal bifurcation (P), contiguous extrahepatic tumour (E), multifocal tumour (F), and spontaneous rupture (R). We defined five clinically relevant backbone groups on the basis of established prognostic factors: PRETEXT I/II, PRETEXT III, PRETEXT IV, metastatic disease, and AFP concentration of 100 ng/mL or lower at diagnosis. We then carried the additional factors into a hierarchical backwards elimination multivariable analysis and used the results to create a new international staging system. RESULTS Within each backbone group, we identified constellations of factors that were most predictive of outcome in that group. The robustness of candidate models was then interrogated using the bootstrapping procedure. Using the clinically established PRETEXT groups I, II, III, and IV as our stems, we created risk stratification trees based on 5 year event-free survival and clinical applicability. We defined and adopted four risk groups: very low, low, intermediate, and high. INTERPRETATION We have created a unified global approach to risk stratification in children with hepatoblastoma on the basis of rigorous statistical interrogation of what is, to the best of our knowledge, the largest dataset ever assembled for this rare paediatric tumour. This achievement provides the structural framework for further collaboration and prospective international cooperative study, such as the Paediatric Hepatic International Tumour Trial (PHITT). FUNDING European Network for Cancer Research in Children and Adolescents, funded through the Framework Program 7 of the European Commission (grant number 261474); Childrens Oncology Group CureSearch grant contributed by the Hepatoblastoma Foundation; Practical Research for Innovative Cancer Control and Project Promoting Clinical Trials for Development of New Drugs and Medical Devices, Japan Agency for Medical Research; and Swiss Cancer Research grant.

DNA Variants in Region for Noncoding Interfering Transcript of Dihydrofolate Reductase Gene and Outcome in Childhood Acute Lymphoblastic Leukemia

Purpose: Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. We recently reported an association of DHFR promoter polymorphisms with ALL outcome. Lower event-free survival correlated with haplotype *1, defined by A317 and C1610 alleles. Haplotype *1 was also associated higher DHFR expression. Experimental Design: Here, we analyzed adjacent 400-bp region participating in DHFR regulation as both a major promoter and a noncoding minor transcript. Results: Six polymorphisms were identified, of which five were single nucleotide polymorphisms and one was length polymorphism composed of variable number of 9-bp elements and 9-bp insertion/deletion. Haplotype analysis including all promoter polymorphisms revealed diversification of haplotype *1 into five subtypes (*1a-*1e). DNA variations of major promoter/noncoding transcript region and haplotype *1 subtypes were subsequently analyzed for the association with ALL outcome. Lower event-free survival was associated with an A allele of G308A polymorphism (P = 0.02) and with *1b haplotype (P = 0.01). This association was particularly striking in high-risk patients (P = 0.001) and was subsequently confirmed in independent patient cohort (P = 0.02). Haplotype *1b was the only haplotype *1 subtype associated with higher mRNA levels. Conclusions: The study provides a new insight into DHFR regulatory variations predisposing to an event in ALL patients. (Clin Cancer Res 2009;15(22):69318)

Association between busulfan exposure and outcome in children receiving intravenous busulfan before hematopoietic stem cell transplantation

Background and Objective: Intravenous (IV) busulfan (Bu) combined with therapeutic drug monitoring-guided dosing is associated with better event-free survival (EFS), lower transplant-related mortality. But optimal target steady-state concentration (Css) of Bu in children undergoing hematopoietic stem cell transplantation (HSCT) remains unclear. This study aimed to evaluate the relation between Css of Bu and clinical outcomes in children receiving Bu before HSCT. Methods: This study includes 75 children receiving IV Bu in 16 doses, with first dose assigned based on age. Bu first-dose pharmacokinetic parameters were estimated from Bu plasma concentrations measured at 6 time points by high-performance liquid chromatography. Doses were adjusted at the fifth dose to a target Css of 600–900 ng/mL. Cumulative incidence of overall survival (OS), EFS, transplant-related mortality, acute graft-versus host disease (aGVHD), and other toxicities in relation to Css of Bu were analyzed using Kaplan–Meier curves in univariate and Coxs proportional hazards model in multivariate analysis. Results: After the first dose, median Css was 578 (325–1227) ng/mL. Forty-one patients had Bu IV dose increased by > 10%. Neutrophil and platelet recoveries, grade 2–4 aGVHD, and nonrelapse mortality (NRM) incidences were 90%, 91%, 12%, and 13%, respectively. Relapse incidence was 33%. Incidence of veno-occlusive disease, hemorrhagic cystitis, and lung toxicities were 13%, 24%, and 7%, respectively. OS and EFS were 70% and 58%. First-dose Bu Css >600 ng/mL was associated with a higher NRM (P < 0.001) and grade 2–4 aGVHD (P = 0.04), a lower EFS (P < 0.001), and OS (P = 0.001). Conclusions: This study demonstrated a significant association between the first-dose pharmacokinetics of Bu and NRM, OS, and EFS. Bu therapeutic drug monitoring provides information that potentially influences outcomes of HSCT in pediatric patients.

Polymorphisms of ABCC5 and NOS3 genes influence doxorubicin cardiotoxicity in survivors of childhood acute lymphoblastic leukemia

Anthracyclines are efficient chemotherapy agents. However, their use is limited by anthracycline-induced cardiotoxicity (CT). We investigated the influence of polymorphisms in doxorubicin metabolic and functional pathways on late-onset CT as estimated by echocardiography in 251 childhood acute lymphoblastic leukemia (cALL) patients. Association analyses revealed a modulating effect of two variants: A-1629 T in ABCC5, an ATP-binding cassette transporter, and G894T in the NOS3 endothelial nitric oxide synthase gene. Individuals with the ABCC5 TT-1629 genotype had an average of 8–12% reduction of ejection (EF) and shortening fractions (SF; EF: P<0.0001, and SF: P=0.001, respectively). A protective effect of the NOS3 TT894 genotype on EF was seen in high-risk patients (P=0.02), especially in those who did not receive dexrazoxane (P=0.002). Analysis of an additional cohort of 44 cALL patients replicated the ABCC5 association but was underpowered for NOS3. In summary, we identified two biomarkers that may contribute to cALL anthracycline CT risk stratification.

A simplified method for busulfan monitoring using dried blood spot in combination with liquid chromatography/tandem mass spectrometry

RATIONALE Busulfan (Bu) is an important component of the myeloablative conditioning regimen prior to hematopoietic stem cell transplantation (HSCT) especially in children. Intravenously administered Bu exhibits a therapeutic window phenomenon requiring therapeutic drug monitoring. Analytical methods developed for Bu routine monitoring were aimed at using low volumes of biological fluids and development of simple procedures to facilitate the dosage adjustment. In this report, we describe a simple, rapid method for Bu measurement using dried blood spots (DBS) from only 5 μL of whole blood. METHODS Bu extracted from DBS with methanol was measured by high-performance liquid chromatography with electrospray ionization and tandem mass spectrometry in multiple reaction monitoring mode using D8-Bu as an internal standard. The method was in-house validated evaluating trueness, repeatability, within-laboratory reproducibility, specificity and the lower limit of quantification (LLOQ). RESULTS The method was linear in the calibration range of 100-2000 ng mL(-1) (r(2)>0.99) encompassing the therapeutic concentrations of Bu. A good trueness (<14%), precision (<10%), and recovery (100%) were observed during validation of the method with quality controls of 300, 600 and 1400 ng mL(-1). The LLOQ was determined as 50 ng mL(-1) and no matrix or carryover effects were observed. The validated method was applied to measure Bu levels in four children receiving infusion of Bu prior to HSCT. A good correlation was observed between the Bu levels measured by DBS and dried plasma spot (DPS) (r(2) =0.96) and between DPS and the GC/MS method (r(2) =0.92). Bu was found to be stable in DBS up to 6 h at room temperature and for 24 h at 4 °C. CONCLUSIONS The new DBS method facilitates earlier dosage adjustment during Bu therapy by its specific and simple procedure using 5 μL of whole blood.

Association of busulfan exposure with survival and toxicity after haemopoietic cell transplantation in children and young adults: a multicentre, retrospective cohort analysis

BACKGROUND Intravenous busulfan combined with therapeutic drug monitoring to guide dosing improves outcomes after allogeneic haemopoietic cell transplantation (HCT). The best method to estimate busulfan exposure and optimum exposure in children or young adults remains unclear. We therefore assessed three approaches to estimate intravenous busulfan exposure (expressed as cumulative area under the curve [AUC]) and associated busulfan AUC with clinical outcomes in children or young adults undergoing allogeneic HCT. METHODS In this retrospective analysis, patients from 15 centres in the Netherlands, USA, Canada, Switzerland, UK, Italy, Germany, and Australia who received a busulfan-based conditioning regimen between March 18, 2001, and Feb 12, 2015, were included. Cumulative AUC was calculated by numerical integration using non-linear mixed effect modelling (AUCNONMEM), non-compartmental analysis (AUC from 0 to infinity [AUC0-∞] and to the next dose [AUC0-τ]), and by individual centres using various approaches (AUCcentre). The main outcome of interest was event-free survival. Other outcomes of interest were graft failure or relapse, or both; transplantation-related mortality; acute toxicity (veno-occlusive disease or acute graft versus-host disease [GvHD]); chronic GvHD; overall survival; and chronic-GvHD-free event-free survival. We used propensity-score-adjusted Cox proportional hazard models, Weibull models, and Fine-Gray competing risk regressions for statistical analyses. FINDINGS 790 patients were enrolled, 674 of whom were included: 274 (41%) with malignant and 400 (59%) with non-malignant disease. Median age was 4·5 years (IQR 1·4-10·7). The median busulfan AUCNONMEM was 74·4 mg × h/L (95% CI 31·1-104·6), which correlated with the standardised method AUC0-∞ (r2=0·74), but the latter correlated poorly with AUCcentre (r2=0·35). Estimated 2-year event-free survival was 69·7% (95% CI 66·2-73·0). Event-free survival at 2 years was 77·0% (95% CI 72·1-82·9) in the 257 patients with an optimum intravenous busulfan AUC of 78-101 mg × h/L compared with 66·1% (60·9-71·4) in the 235 patients at the low historical target of 58-86 mg × h/L and 49·5% (29·2-66·0) in the 44 patients with a high (>101 mg × h/L) busulfan AUC (p=0·011). Compared with the low AUC group, graft failure or relapse occurred less frequently in the optimum AUC group (hazard ratio [HR] 0·57, 95% CI 0·39-0·84; p=0·0041). Acute toxicity (HR 1·69, 1·12-2·57; p=0·013) and transplantation-related mortality (2·99, 1·82-4·92; p<0·0001) were significantly higher in the high AUC group (>101 mg × h/L) than in the low AUC group (<78 mg × h/L), independent of indication; no difference was noted between AUC groups for chronic GvHD (<78 mg × h/L vs ≥78 mg × h/L, HR 1·30, 95% CI 0·73-2·33; p=0·37). INTERPRETATION Improved clinical outcomes are likely to be achieved by targeting the busulfan AUC to 78-101 mg × h/L using a new validated pharmacokinetic model for all indications. FUNDING Research Allocation Program and the UCSF Helen Friller Family Comprehensive Cancer Center and the Mt Zion Health Fund of the University of California, San Francisco.

Glutathione S-transferase gene variations influence BU pharmacokinetics and outcome of hematopoietic SCT in pediatric patients

BU is a key compound of conditioning regimens in children undergoing hematopoietic SCT (HSCT). Inter-individual differences in BU pharmacokinetics (PKs) might affect BU efficacy and toxicity. As BU is mainly metabolized by glutathione S-transferase (GST), we investigated the relationship between GSTA1, GSTM1 and GSTP1 genotypes with first-dose BU PKs, and the relationship with HSCT outcomes in 69 children receiving myeloablative conditioning regimen. GSTM1 null genotype correlated with higher BU exposure and lower clearance in patients older than 4 years (P⩽0.04). In accordance with the suggested functional role, GSTA1*A2 haplotype was associated with lower drug levels and higher drug clearance (P⩽0.03). Gene-dosage effect was also observed (P⩽0.007). GSTA1 haplotypes were associated with HSCT outcomes. Patients with two copies of haplotype *A2 had better event free survival (P=0.03). In contrast, homozygous individuals for haplotypes *B and *B1 had higher occurrence of veno-occlusive disease (P=0.009). GSTM1 null individuals older than 4 years had more frequently graft versus host disease (P=0.03). In conclusion, we showed that GST gene variants influence BU PK and outcomes of HSCT in children. A model for the dosage adjustment with the inclusion of genetic and non-genetic factors should be evaluated in a future prospective validation cohort.