Liquid Chromatography–Mass Spectrometry Screening of Cyclophosphamide DNA Damage In Vitro and in Patients Undergoing Chemotherapy Treatment

Abstract

DNA alkylating drugs have been used as frontline medications to treat cancer for decades. Their chemical reaction with DNA leads to the blockage of DNA replication, which impacts cell replication. While this impacts rapidly dividing cancerous cells, this process is not selective and results in highly variable and often severe side effects in patients undergoing alkylating-drug based therapies. The development of biomarkers to identify patients who effectively respond with tolerable toxicities vs patients who develop serious side effects is needed. Cyclophosphamide (CPA) is a commonly used chemotherapeutic drug and lacks biomarkers to evaluate its therapeutic effect and toxicity. Upon administration, CPA is metabolically activated and converted to phosphoramide mustard and acrolein, which are responsible for its efficacy and toxicity, respectively. Previous studies have explored the detection of the major DNA adduct of CPA, the interstrand DNA–DNA cross-link G-NOR-G, finding differences in the cross-link amount between Fanconi Anemia and non-Fanconi Anemia patients undergoing chemotherapy treatment. In this study, we take advantage of our DNA adductomic approach to comprehensively profile CPA’s and its metabolites’ reactions with DNA in vitro and in patients undergoing CPA-based chemotherapy. This investigation led to the detection of 40 DNA adducts in vitro and 20 DNA adducts in patients treated with CPA. Moreover, acrolein-derived DNA adducts were quantified in patient samples. The results suggest that CPA-DNA damage is very complex, and an evaluation of DNA adduct profiles is necessary when evaluating the relationship between CPA-DNA damage and patient outcome.

DNA Adducts in Cancer Chemotherapy

“…More recently, Balbo and colleagues introduced an adductomic approach based on high-resolution mass spectrometry . Utilizing this method, DNA adducts formed by the adducting drugs have been examined thoroughly, facilitating the establishment of DNA adduct formation profiles. ,,, With the structural information provided by the mass spectra, specific adducts of interest (e.g., those with high abundance and/or occurrence in multiple individual samples) can be synthesized and quantified . These findings hold promise for the potential use of such adducts as biomarkers to predict the clinical outcome of the adducting drugs.…”

“…133 A recent DNA adductomic study identified 40 DNA adducts induced by cyclophosphamide, including known adducts (e.g., Acr-dGuo 133 and 134, N7G-NOR-OH 127, N7G-NOR-Cl 128, and N7G-NOR-N7G 129) and unreported adducts like Acr-dAdo, NOR-FapyG 130, N7G-NOR-FapyG 131. 134 Furthermore, a mass spectrometry-based proteomics approach identified DNA−protein cross-links, including Cys-NOR-N7G 132, in human fibrosarcoma cells (HT1080) treated with the phosphoramide mustard 40. 135 5.1.4.…”