Assessment of a PCR-based assay to detect TP53 adducts after exposure to DNA alkylating agents

Abstract

Cyclophosphamide is an alkylating agent used in the treatment of cancer and some auto-immune disorders. However, there is substantial inter-individual variability in patient outcome. Cyclophosphamide is a prodrug, which requires bioactivation by CYP enzymes to produce 4-hydroxycyclophosphamide (4-OHCP), the precursor of phosphoramide mustard, which alkylates DNA. Pharmacogenetic variation in the activity of the two main CYP enzymes that catalyse this reaction (CYP2B6 and CYP2C19) have been extensively studied and appear to influence outcomes. However, the multiple detoxification pathways and inherited differences in the activity of DNA repair enzymes may also play a role. It is difficult to measure the unstable 4-OHCP and phosphoramide mustard in patient samples. However, it may be possible to measure the amount of DNA interstrand cross-links (ICLs) formed in patients. A PCR-based assay that quantifies the number of ICLs formed after exposure to the related DNA alkylating compound, melphalan has been reported (Stefanou et al. 2012). This assay used the long-range PCR amplification of a 6.8 kb sequence of TP53. The aim of this thesis was to determine whether this PCR-based assay could also detect ICLs formed after exposure of gDNA to 4-OHCP. Modifications of the published assay were required, including alternative DNA polymerases and amplification of additional sequences (1.6 kb and 0.6 kb). The modified assay could detect ICLs formed by melphalan, with a similar lower limit of detection (16 ICL/106 nucleotides) and linearity as the published assay. 4-OHCP-induced ICLs were also detectable using the modified 6.8 kb assay. However, longer incubation times (20 h vs 1 h) were required to achieve detectable ICLs with 4-OHCP compared with melphalan. Experiments were undertaken to assess whether this was due to incomplete chemical conversion of 4-OHCP to phosphoramide mustard. However, the literature suggests that rate of ICL formation is much faster with melphalan. In conclusion, the work in this thesis demonstrates that the PCR-based assay can detect 4-OHCP-induced ICLs. This was a proof-of-principle study and future work is required to further optimise and validate this assay, prior to its use as a biomarker to aid the investigation of the inherited and clinical factors that influence response to cyclophosphamide treatment.