Formation of cyclophosphamide specific DNA adducts in hematological diseases

Background: Fanconi anemia (FA) patients are hypersensitive to DNA alkylating agents and require lower doses than non-FA patients to minimize serious toxicity. The mechanism by which hypersensitivity occurs is thought to be due to the inability of these individuals to effectively repair drug-induced interstrand DNA-DNA crosslinks. We recently developed a highly sensitive assay for cyclophosphamide specific interstrand DNA-DNA crosslinks (G-NOR-G) and are able to quantify and compare formation of these adducts in the blood of patients. Therefore we sought to determine whether FA patients have higher in vivo exposure to the cyclophosphamide specific interstrand DNA crosslink, G-NOR-G, relative to patients without FA. Procedure: Cyclophosphamide interstrand DNA crosslinks were measured with the first dose of cyclophosphamide in FA and non-FA patients receiving a cyclophosphamide based preparative regimen prior to hematopoietic cell transplantation (HCT). FA patients received a lower cyclophosphamide dose than the non-FA patients (5-10mg/kg/day vs. 50-60mg/kg/day). Results: Despite the lower cyclophosphamide dose and lower plasma concentrations in FA patients, they had G-NOR-G amounts similar to the non-FA patients (area under the curve (AUC) _0-∞, 99.8 vs. 144.9 G-NOR-G adducts/10 ⁶nucleotideshour, respectively, P=0.47). When G-NOR-G AUC was normalized for cyclophosphamide plasma concentrations, FA study subjects produced 15-fold higher adducts than non-FA patients (P=0.05). Conclusions: FA patients are hypersensitive to DNA alkylating agents possibly as a result of greater formation of cyclophosphamide specific interstrand DNA crosslinks and/or diminished capacity for DNA repair. Identification and quantification of these adducts may be important determinant of cyclophosphamide related toxicity.