Ifosfamide metabolism and DNA damage in tumour and peripheral blood lymphocytes of breast cancer patients

Abstract

Purpose: This study was designed to determine individual variation in the metabolism of ifosfamide (IF) and any influence this may have on the degree of DNA damage produced in both peripheral blood lymphocytes (PBL) and in tumour tissue. Methods: The pharmacokinetics and metabolism of IF and also of doxorubicin (DOX) were determined in patients receiving IF/DOX neoadjuvant chemotherapy for the treatment of advanced breast cancer. The DNA-damaging effects of this regimen were measured using the comet assay in PBL and in breast tumour tissue obtained by fine needle aspirate. Parallel in vitro studies were carried out in order to establish if DNA damage caused by IF metabolites or DOX was predictive of cytotoxicity in breast cancer cell lines. Results: The median AUC, half-life and clearance of IF were found to be 291 μM · min, 5.2 h and 66 ml/min per m², respectively. A high degree of interpatient variability (up to sevenfold) was observed in the metabolism of both IF and DOX and also in their metabolites. Treatment-related changes in the amount of DNA damage were observed in both PBL and tumour cells. That in PBL peaked 48 h after the end of IF infusion (median 17% damaged cells at 48 h compared to 4% damaged before treatment). DNA damage in tumour cells was not elevated above low pretreatment values (median 1.5% damaged cells) until 3 weeks after IF and DOX treatment (median 30% damaged cells), by which time damage in PBL showed almost complete resolution to basal levels. The DNA damage in PBL determined 24 h after the start of chemotherapy was found to be related to the AUC of 4-hydroxyifosfamide (4OHI; P=0.05). The amount of damage in either tissue did not significantly correlate with clinical response or toxicity, but lower amounts of damage were observed in the tumour cells 3 weeks after treatment in those patients that subsequently relapsed, compared to those that remained disease free. DNA damage (more than 20% damaged cells) was observed after exposure to active IF metabolites at concentrations equal to or greater than the IC₅₀ in MCF-7 and MDA-MB231 cell lines. At concentrations of 4OHI similar to those determined in vivo, an equivalent level of DNA damage was observed in PBL and in cell lines and was associated with significant growth inhibition. DNA damage induced by DOX was not predictive of cytotoxicity. Conclusion: Systemic DNA damage appeared to be related to levels of the active metabolite, consistent with the results of in vitro investigations of DNA damage. Further studies are warranted to substantiate this observation and to explore the relationship between metabolism, DNA damage and antitumour activity.