Busulfan Pharmacogenetics and Drug Exposure in Cancer Patients Undergoing Hematopoietic Stem Cell Transplantation

Introduction: Busulfan, a bifunctional alkylating agent widely used in conditioning regimens before hematopoietic stem cell transplantation (HSCT), is metabolized via glutathione-S-transferases (GSTs) and cytochrome P450 (CYP) enzymes. Busulfan demonstrates wide interpatient variability in pharmacokinetics (PK); high drug exposure is associated with increased risk of toxicities (e.g. veno-occlusive disease) and low drug exposure is associated with increased risk of relapse and graft failure. Drug exposure within a narrow therapeutic range yields optimal results. PK-guided dosing is used in many patients to achieve optimal drug exposure; however, better methods to select the initial dose would improve consistency of busulfan exposure and the likelihood of achieving the target area under the concentration-time curve (AUC). Previous pharmacogenomic (PGx) studies have identified GST polymorphisms as predictors of drug exposure, however, these were primarily performed in pediatric populations and/or Asians. We performed a PGx study in adult HSCT patients to identify polymorphisms in candidate genes associated with busulfan AUC and clearance.

Methods: Buccal swabs were collected from HSCT patients scheduled to receive busulfan as part of their conditioning regimen on the day of admission (one day prior to initiating busulfan) between 04/28/14 and 06/06/2017. DNA was extracted using the GenElute™ Mammalian Genomic DNA Kit. A custom Ion AmpliSeq™ PGx Panel and various orthogonal methods were used to genotype the samples for polymorphisms in GSTA1 (135T>C), GSTM1 (null/present), GSTP1 (Ile105Val, Ala114Val), CYP2B6 (*2, *4, *5, *6, *18, *22), CYP2C8 (*2, *3), CYP2C9 (*2, *3, *8, *11), CYP2C19 (*2, *3, *17), and MTHFR (A1298C, C677T). Phenotypes were inferred according to established guidelines or literature. IV busulfan was administered once daily over 3 hours at 2.8 mg/kg (for autos) or 3.2 mg/kg (for allos) for a total of 4 doses. PK samples were obtained at 15 mins, 1 hr, 2 hrs, 3 hrs, and 5 hrs after the end of the first infusion. Non-compartmental analysis was performed to estimate PK parameters and doses 3 and 4 were adjusted to target an AUC of either 4500 or 5000 uM*min/L, depending on transplant type and conditioning regimen. The primary objective was to investigate the impact of polymorphisms on busulfan AUC, which was measured using univariate linear regression analysis adjusting for dose. Secondary objectives included a comparison of mean AUCs between phenotypes, which was measured using ANOVA, and to determine the association between SNPs and busulfan clearance using univariate linear regression adjusting for dose.

Results: A total of 36 patients were evaluable for the primary endpoint. The median age was 47 (22-73) years, 56% were male, 75% received busulfan/cyclophosphamide/etoposide, 22% received busulfan/cyclophosphamide with post-transplant cyclophosphamide, and 3% received thiotepa/busulfan/cyclophosphamide. The genotype and inferred phenotype frequencies are summarized in Table 1. The mean AUC across all patients was 4255 µM*min/L (range 2834-5989). Results from the univariate linear regression analysis identified that CYP2B6 polymorphisms were significantly associated with busulfan AUC (intermediate [IM] vs. poor metabolizer [PM], p = 0.05; normal/rapid metabolizer [NM/RM] vs. PM, p = 0.02) (Table 2). The mean AUC in CYP2B6 PMs, IMs, NM/RMs was 5242, 4277, and 4134 µM*min/L, respectively (p = 0.03 by ANOVA test). There was no association of polymorphisms in GSTA1, GSTM1, GSTP1, CYP2C8, CYP2C9, CYP2C19 and MTHFR with AUC. CYP2B6 polymorphisms were also associated with busulfan clearance (IM vs. PM, p = 0.07; NM/RM vs. PM, p = 0.03).

Conclusion: CYP2B6 polymorphisms were significantly associated with busulfan AUC and clearance; these appear to be an important determinant of drug exposure, however, GST polymorphisms did not replicate as significant, possibly due to differences in patient populations. PGx testing for CYP2B6 may provide an important tool to individualize up front dosing and improve the likelihood of achieving the target AUC earlier or to achieve target AUC in a higher proportion of the many patients who do not receive PK-guided dosing; however, our findings require further validation in larger cohorts of patients. We plan to enroll more patients and assess the relationship between PK, PGx and clinical outcomes, including toxicity and response.

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