Exploratory analysis of 1,936 SNPs in ADME genes for association with busulfan clearance in adult hematopoietic stem cell recipients
Marloes H. ten Brink
1, Jesse J. Swen
1, Judith A.M. Wessels
1, Tahar van der Straaten
1, Erik W.A. Marijt
2, Peter A. von dem Borne
2, Juliette Zwaveling
1and Henk-Jan Guchelaar
11)Department of Clinical Pharmacy and Toxicology 2)Department of Hematology of the Leiden University Medical Center, Leiden, The Netherlands
m.h.ten_brink@lumc.nl
Results
Conclusion
• This is the first study using an exploratory pharmacogenetic approach in 225 genes involved in ADME to explain the interindividual variability in busulfan clearance.
• GSTA5 haplotype (and thus GSTA1) was significantly correlated with busulfan clearance, both in the exploratory and replication cohort.
• No additional genetic markers involved in drug metabolism and transport appear to be associated with busulfan clearance.
Introduction
• Busulfan is used in preparative regimens prior to hematopoietic stem cell transplantation (HSCT).
• Busulfan has a narrow therapeutic index and exposure is related to outocome.
• There is significant interpatient variability in busulfan pharmacokinetics (PK).
• To date, only polymorphisms in genes encoding for glutathione-S-
transferases were studied, which could only explain a small portion of the variability in PK.
• We hypothesize that a set of genetic variants may be more important to explain interindividual differences in busulfan clearance than a single gene.
Objective
• To identify biomarkers for busulfan clearance by interrogating 1,936 variants in 225 ADME related genes
.
Discussion
• This is the first study using an exploratory pharmacogenetic approach in 225 genes involved in ADME to explain the interindividual
variability in busulfan clearance.
• We applied an explorative approach, with the advantage of the possibility of discovering new pathways and genetic variants
• A large number of SNPs were evaluated in the screening cohort, which introduces the potential problem of multiple testing and an increase in the risk of finding false-positive relations.
• To minimalize this risk, the replication cohort was used to confirm the findings from the exploratory cohort.
• Our results confirm the previously identified association between GSTA1 haplotype and busulfan clearance. Unfortunately this variant can only explain 6.5% of the variation in busulfan clearance.
• No additional genetic markers involved in drug metabolism and transport appear to be associated with busulfan clearance.
Manhattan plot of –Log (P-values) from linear regression analysis of individual genetic variants and busulfan clearance. Blue line indicating a P- value of 0,05 and the red line indicating a corrected p-value for 225 genes.
Methods
• 62 adult patients receiving busulfan iv prior to their HSCT were included in screening cohort and were genotyped using the Drug Metabolizing Enzymes and Transporters (DMET) array, including 1,936 genetic variants in 225.
• Busulfan serum levels were measured at 2.5 and 4.0 hours after the start of the first infusion (2hr) on the first day of treatment with a validated high-performance liquid chromatography assay.
• Busulfan clearance was estimated with a limited sampling (t=2.5, 4 hrs) PK model.
• Associations of busulfan clearance with SNPs and haplotypes were initially tested by linear regression analysis with the polymorphism in the additive model.
• SNPs with a MAF > 10% and candidate markers from the initial univariate analysis with p<0.05 (SNPs or haplotypes) were analyzed in the multivariate linear regression analysis.
• Top SNPs and haplotypes were replicated in an independent cohort of adult patients undergoing HSCT (N=85).
Exploratory
Cohort
Replication Cohort
Number of patients 62 78
Age in years
(mean ±s.d.) 53 ±11 57 ±10 p=0.04
Sex
Male 34 51
p=0.20
Female 28 27
Disease
AML 11 28
CML 3 2
CLL 5 7
ALL 0 1
MM 22 18
NHL 10 18
CMML 2 1
AA 7 2
β-thl 0 1
SCA 2 0
Busulfan clearance
(L/hr/kg)
Volume of distribution
(L)
0.19 ±0.05 47.6±16.6
0.17 ±0.05 51.0±15.4
p=0.16 p=0.21
Conditioning regime
Flu Bu
+(ATG or A-mab) 59 74
p=0.55
Flu Bu CY 2 3
Bu Cy A-mab 1 1
• In the exploratory cohort 7 variants (3 SNPs and 4 haplotypes) explained 64% (adjusted R2) of variance in
busulfan clearance (p<0.001).
• These genetic variants, located in GSTA5, CYP2C19, CYP39A1 (2
haplotypes), ABCB4, SLC22A4 and
SLC7A8, were replicated in the second cohort.
• The GSTA5 (rs4715354 and rs7746993) haplotype remained significantly
associated with busulfan clearance (p=0.025) .
Flow chart of genotyping results in exploratory cohort
Patient Characteristics
Abbreviations: AML = acute myeloid leukemia, CML = chronic myeloid leukemia, CLL = Chronic lymphocytic leukemia, ALL = acute lymphoblastic leukemia , MM = Multiple Myeloma , NHL = Non-Hodgkin lymphoma , CMML = chronic myelomonocytic leukemia, AA = aplastic anemia, β-thl = β-thalassemia , SCA = sickle cell anemia. Flu = fludarabine, Bu = busulfan, Cy = cyclofosfamide, ATG = anti-thymocyte globuline, A-mab = alemtuzumab
References:
Grochow LB, et al. Cancer Chemother.Pharmacol. 1989., 2. Slattery JT, et al. Blood. 1997., 3. Sissung TM, et al.
Pharmacogenomics. 2010.
