Patients with the same phenotypical cancer can have different responses to a given treatment regimen due to genetic alterations. This is also true of animals with cancer, such as dogs. Monitoring biomarkers of efficacy is a strategy for stratifying patients to predict outcome and allow for personalization of treatment regimens. The majority of the currently used chemotherapy agents act by interfering with cell replication, an overactive process in rapidly dividing cancer cells, through the formation of genotoxic DNA modifications (adducts). Adducts from DNA anticancer drugs are good candidate biomarkers due to their specificity and stability in DNA. We developed a high-resolution/accurate mass (HRAM) LC-MS3 targeted and untargeted DNA adductomic workflow to screen and monitor anticancer drug-induced adducts. The workflow is based on data-dependent triggered MS2 fragmentation events for the most abundant full scan ions, and confirmation of DNA adduct identities by triggered MS3 fragmentation by accurate mass monitoring of neutral losses of common features from DNA or the drug. Initial screening is performed in drug-exposed purified DNA, followed by confirmation of adducts in drug-exposed cells or biopsies from drug-treated patients. Relative quantitation of the detected adducts is achieved by using a mixture of isotope-labeled adducts as internal standards, obtained by exposing the drug to 15N-labeled DNA. We applied the workflow to study the DNA modifications induced by doxorubicin (DOX), a component of the first-line treatment for high-grade canine lymphoma. Exposure of purified DNA with DOX in the presence of formaldehyde revealed the formation of several DNA modifications, among them the previously reported monoadduct resulting from reaction of DOX and formaldehyde with the N2 position of deoxyguanosine. These DNA modifications will now be targeted in blood cells or lymph node samples of dogs undergoing therapy with DOX to investigate the relationship between DNA adduct levels and therapy response.

High-resolution/accurate mass DNA adductomics to screen for doxorubicin-induced adducts as biomarkers of therapeutic efficacy

256th National Meeting (2018)

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