Identification of new candidate biomarkers to support doxorubicin treatments in canine cancer patients

Abstract

Background Both human and veterinary cancer chemotherapy are undergoing a paradigm shift from a “one size fits all” approach to more personalized, patient-oriented treatment strategies. Personalized chemotherapy is dependent on the identification and validation of biomarkers that can predict treatment outcome and/or risk of toxicity. Many cytotoxic chemotherapy agents, including doxorubicin, base their mechanism of action by interaction with DNA and disruption of normal cellular processes. We developed a high-resolution/accurate-mass liquid chromatography-mass spectrometry DNA screening approach for monitoring doxorubicin-induced DNA modifications (adducts) in vitro and in vivo. We used, for the first time, a new strategy involving the use of isotope-labeled DNA, which greatly facilitates adduct discovery. The overall goal of this work was to identify doxorubicin-DNA adducts to be used as biomarkers to predict drug efficacy for use in veterinary oncology. Results We used our novel mass spectrometry approach to screen for adducts in purified DNA exposed to doxorubicin. This initial in vitro screening identified nine potential doxorubicin-DNA adduct masses, as well as an intense signal corresponding to DNA-intercalated doxorubicin. Two of the adduct masses, together with doxorubicin and its metabolite doxorubicinol, were subsequently detected in vivo in liver DNA extracted from mice exposed to doxorubicin. Finally, the presence of these adducts and analytes was explored in the DNA isolated from dogs undergoing treatment with doxorubicin. The previously identified nine DOX-DNA adducts were not detected in these preliminary three samples collected seven days post-treatment, however intercalated doxorubicin and doxorubicinol were detected. Conclusions This work sets the stage for future evaluation of doxorubicin-DNA adducts and doxorubicin-related molecules as candidate biomarkers to personalize chemotherapy protocols for canine cancer patients. It demonstrates our ability to combine in one method the analysis of DNA adducts and DNA-intercalated doxorubicin and doxorubicinol. The last two analytes interestingly, were persistent in samples from canine patients undergoing doxorubicin chemotherapy seven days after treatment. The presence of doxorubicin in all samples suggests a role for it as a promising biomarker for use in veterinary chemotherapy. Future studies will involve the analysis of more samples from canine cancer patients to elucidate optimal timepoints for monitoring intercalated doxorubicin and doxorubicin-DNA adducts and the correlation of these markers with therapy outcome.

Cryopreservation does not change the performance and characteristics of allogenic mesenchymal stem cells highly over-expressing a cytoplasmic therapeutic transgene for cancer treatment

“…Additionally, further assessment of therapeutic efficacy by histopathologic evaluation is not possible with canine patients due to the invasive nature of the sample collection procedures and awaits post-mortem analysis. One option is the use of liquid biopsies for serial treatment monitoring, but this is currently limited by the lack of clinically suitable biomarkers for canine cancers [67,68]. Another limitation is the availability of canine patients with similar afflictions, Table 2 Patient history and treatment details (11-year-old, Male, Toy Poodle); series of events in chronological order…”

Characterization and quantitation of busulfan DNA adducts in the blood of patients receiving busulfan therapy

“…Whole-blood DNA extraction was performed using Qiagen DNA extraction kits (Qiagen, Valencia, CA) by the manufacturer’s instructions with slight modifications as previously reported. 41 DNA amounts were estimated by UV and subsequently determined by HPLC analysis of dGuo in enzymatic hydrolysates as described below. DNA samples (10–50 μg in 200 μL of Tris buffer) were spiked with 30 fmol of [ 15 N 5 ] N 7G-Bu-OH and 50 fmol of [ 15 N 5 ] N 7G-Bu- N 7G as internal standards and subjected to enzymatic hydrolysis as described below.…”

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

“…Whole blood DNA extraction was performed immediately after the sample collection. Whole blood DNA extraction was performed using Qiagen DNA extraction kits (Qiagen, Valencia, CA) by the manufacturer’s instructions with slight modifications, as previously reported . DNA amounts were estimated by UV and subsequently determined by high-performance liquid chromatography (HPLC) analysis of dG in enzymatic hydrolysates as described below.…”

“…Whole blood DNA extraction was performed using Qiagen DNA extraction kits (Qiagen, Valencia, CA) by the manufacturer's instructions with slight modifications, as previously reported. 5 DNA amounts were estimated by UV and subsequently determined by high-performance liquid chromatography (HPLC) analysis of dG in enzymatic hydrolysates as described below. DNA samples (10−50 μg in 200 μL of buffer) were spiked with 30 fmol of [ 13 Cyclophosphamide Quantification in Patient Plasma.…”

“…The amount of adducts produced depends on drug absorption and metabolism and the patient’s genetics . Studies of DNA adducts support the existence of a fine equilibrium between DNA damage and therapy outcome. − …”