Cyclophosphamide is a prodrug metabolized by several enzymes to the toxic metabolite phosphoramide mustard to elicit its therapeutic effect. Polymorphisms in drug-metabolizing enzymes or drug transporters may affect pharmacokinetics of cyclophosphamide or its active metabolites, which could affect treatment efficacy or toxicity. We investigated the effect of polymorphisms in eleven pharmacogenes on cyclophosphamide treatment-related toxicity, with a particular focus on CYP2B6 based on prior evidence of the effect of CYP2B6 polymorphisms on cyclophosphamide pharmacokinetics and toxicity risk.[5, 6, 10, 12] Our primary analyses found no evidence of a decrease in toxicity in patients with reduced CYP2B6 activity. In an exploratory, statistically uncorrected secondary analysis, carriers of the ALDH1A1 rs8187996 variant had lower odds of cyclophosphamide toxicity or treatment modification due to toxicity.
A prior pharmacogenetic analysis that reported that patients with breast cancer who carried ALDH1A1 rs8187996 had higher hematological toxicity risk when receiving doxorubicin/cyclophosphamide (AC). This statistically uncorrected secondary analysis of the prospective SWOG 0221 clinical trial was the basis for inclusion of this polymorphism within our analysis. However, our results suggest that carriers of this variant have lower odds of cyclophosphamide toxicity, which is in the opposing direction and should not be considered replication. Aldehyde dehydrogenase (ALDH) enzymes, including ALDH1A1 and ALDH3A1, are responsible for inactivating the intermediate metabolite aldophosphamide to carboxyphosphamide. In silico analyses indicate that ALDH1A1 variants could affect aldophosphamide metabolism, however, clinical pharmacokinetics studies have not investigated the effect on aldophosphamide, carboxyphosphamide, or phosphoramide mustard concentrations to our knowledge and no effect has been detected on concentrations of the parent cyclophosphamide compound or the upstream metabolite 4-hydroxycyclophosphamide. The discrepant findings from pharmacogenetic analyses with toxicity, combined with the lack of studies investigating an association with concentrations of active cyclophosphamide metabolite concentrations, do not support a clinically useful pharmacogenetic association, though further investigation is warranted.
CYP2B6, CYP2C9, CYP2C19, and CYP3A4 activate cyclophosphamide to 4-hydroxycyclophosphamide. Several prior studies have reported that patients carrying reduced-activity polymorphisms in these drug metabolizing enzymes have lower bioactivation of cyclophosphamide,[5–7] leading to our hypothesis that patients with reduced metabolic activity would have lower risk of cyclophosphamide toxicity. Our study could not identify any effect of CYP2B6 metabolic phenotypes on cyclophosphamide-induced toxicity. A prior study from Tsuji et al. reported that carriers of the reduced-activity CYP2B6*6 allele had lower risk of severe neutropenia, which is consistent with the reduced cyclophosphamide activation in these patients.[5, 12] Our inability to replicate this association may be due to differences in our endpoint, which included all cyclophosphamide toxicity, not just neutropenia. However, retrospective pharmacogenetic analyses of large prospective clinical trials have also been unable to replicate this association. This inconsistent replication suggests that this association, if it is real, can only be identified in certain patient cohorts, potentially based on their cyclophosphamide dose or the other components of their combination chemotherapy regimen. We were also unable to replicate other previously reported associations with cyclophosphamide toxicity for patients who carry variants in other non-CYP pharmacogenes including GSTP1,[8, 9] ERCC1, ABCB1, and ABCC1.
Our results indicate that patients who inherit germline variants in ALDH1A1 may have lower risk of cyclophosphamide toxicity. Validation of this association in independent cohorts of cyclophosphamide-treated patients would warrant investigation into cyclophosphamide dose individualization to optimize therapeutic outcomes. Interestingly, ALDH1A1 overexpression has also been implicated in tumor resistance to cyclophosphamide treatment, indicating that germline ALDH1A1 variants may affect both toxicity and efficacy of cyclophosphamide treatment, and both would need to be considered when adjusting treatment. Further work is needed to confirm the effect of ALDH1A1 polymorphisms on cyclophosphamide metabolism and treatment outcomes to warrant translational studies that can use this information to optimize clinical outcomes in cyclophosphamide-treated patients.
This study had several limitations that should be considered. First, retrospective collection of toxicity data may contribute to errors in classifying outcome events. Second, this study had a modest small sample size, which may have caused insufficient power to identify some true associations. Also, assuming dominant genetic effects and that all variants in a gene had consistent directions of effect may have prevented identification of some associations. Lastly, we could not demonstrate the mechanisms underlying the putative association between ALDH1A1 rs8187996 and cyclophosphamide toxicity due to the lack of pharmacokinetic data for these patients.
In conclusion, patients who carry ALDH1A1 rs8187996 may have lower risk of cyclophosphamide-induced toxicity. Confirmation of this associations in independent cohorts of cyclophosphamide-treated patients is necessary to justify translational studies evaluating the effect of genotype-guided cyclophosphamide dosing on treatment toxicity and efficacy, which may optimize therapeutic outcomes in patients with cancer.