The goal of this observational study is to learn about the long-term effects of anthracycline chemotherapy on inflammation, oxidative stress, and heart function in adult women with breast cancer. The main questions it aims to answer are: 1. Do inflammatory cytokine levels change after anthracycline chemotherapy and remain altered many years after treatment? 2. Are long-term markers of oxidative stress and antioxidant capacity associated with changes in heart structure or function after anthracycline exposure? This study does not include a comparison group. All participants were previously treated with anthracycline-based chemotherapy as part of their standard cancer care. Participants will: 1. Provide blood samples for the measurement of inflammatory cytokines and oxidative stress-related biomarkers 2. Undergo a clinical cardiovascular evaluation 3. Receive a transthoracic echocardiogram to assess heart function, including measures of systolic and diastolic function and myocardial deformation 4. Participate in a long-term follow-up assessment approximately 10 years after their initial cancer treatment
1. Study design and population. This is a prospective, observational translational study including adult patients with breast cancer undergoing anthracycline-based chemotherapy at a single tertiary-care center. Patients are evaluated longitudinally to assess subclinical cardiovascular alterations associated with anthracycline exposure. All participants are managed according to standard oncologic and cardiologic care pathways. 2. Echocardiographic assessment. Transthoracic echocardiography is performed by experienced cardiologists following current American Society of Echocardiography (ASE) recommendations. Studies are acquired at predefined time points, including baseline (prior to anthracycline exposure) and long-term follow-up. Left ventricular systolic function is assessed using biplane left ventricular ejection fraction (LVEF) calculated by the modified Simpson method. Diastolic function parameters include transmitral inflow velocities, tissue Doppler-derived mitral annular velocities, E/e' ratio, and left atrial volume index (LAVI). Left ventricular global longitudinal strain (GLS) is assessed at long-term follow-up using semi-automated speckle-tracking techniques. Right ventricular-pulmonary artery coupling is explored using the Tricuspid Annular Plane Systolic Excursion (TAPSE)/Pulmonary Artery Systolic Pressure (PASP) ratio. All measurements are performed offline, and segments with inadequate image quality are excluded from analysis. 3. Blood sample collection and processing. Peripheral venous blood samples are collected under standardized conditions at baseline (pre-anthracycline), early after chemotherapy exposure, and at long-term follow-up. Samples are obtained using chilled anticoagulant-containing tubes, centrifuged according to protocol, aliquoted, and stored at -80 °C until biochemical analyses are performed. All samples are processed under identical experimental conditions to minimize analytical variability. 4. Oxidative stress and antioxidant parameters. Plasma antioxidant capacity is assessed using the Ferric Reducing Ability of Plasma (FRAP) assay at predefined time points. Activities of antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, are determined in erythrocyte lysates using commercially available assay kits according to manufacturers' instructions. Lipid peroxidation and intracellular redox status are evaluated using established biochemical methods. Results are normalized to protein concentration when applicable. 5. Inflammatory and proinflammatory cytokines. Circulating cytokines and growth factors are quantified in plasma samples using multiplex bead-based immunoassays. Measurements are performed at baseline and early after anthracycline exposure following standardized manufacturer protocols. Analyte concentrations are calculated based on standard curves generated for each biomarker. 6. Data integration and quality control. Clinical, echocardiographic, and biochemical data are collected using predefined case report forms. Data quality is ensured through consistency checks and verification procedures. All laboratory analyses and imaging measurements are performed blinded to clinical outcomes. 7. Exploratory analyses Echocardiographic parameters are integrated with biochemical markers of oxidative stress and inflammation for exploratory mechanistic analyses aimed at identifying associations between myocardial deformation indices and biological signatures of anthracycline-related cardiotoxicity.
Study Type
OBSERVATIONAL
Enrollment
17
Change in left ventricular ejection fraction from baseline to 10-year follow-up
Assessment of left ventricular systolic function by biplane Simpson method using transthoracic echocardiography. Left ventricular ejection fraction (LVEF) was measured at baseline (7 days before the first cycle of anthracycline chemotherapy) and at the 10-year follow-up.
Time frame: From baseline (7 days before the first anthracycline chemotherapy cycle) to 10 years after completion of chemotherapy
Change in left ventricular filling pressure (E/e' ratio) from baseline to 10-year follow-up
Assessment of left ventricular diastolic function using the average E/e' ratio obtained by transthoracic echocardiography. Measurements were performed at baseline (7 days before the first cycle of anthracycline chemotherapy) and at the 10-year follow-up.
Time frame: From baseline (7 days before the first anthracycline chemotherapy cycle) to 10 years after completion of chemotherapy
Left ventricular global longitudinal strain at 10-year follow-up
Assessment of left ventricular global longitudinal strain (LVGLS) using speckle-tracking echocardiography at the 10-year follow-up. Baseline LVGLS measurements were not available; therefore, only long-term values were assessed.
Time frame: 10 years after completion of chemotherapy
Left atrial volume index at 10-year follow-up
Assessment of left atrial volume index (LAVI) by transthoracic echocardiography as an indicator of long-term left atrial remodeling after anthracycline exposure.
Time frame: 10 years after completion of chemotherapy
Right ventricular-pulmonary arterial coupling (TAPSE/PASP ratio) at 10-year follow-up
Assessment of right ventricular-pulmonary arterial coupling using the tricuspid annular plane systolic excursion (TAPSE) to pulmonary artery systolic pressure (PASP) ratio obtained by transthoracic echocardiography.
Time frame: 10 years after completion of chemotherapy
Plasma antioxidant capacity measured by ferric reducing ability of plasma assay
Assessment of systemic antioxidant capacity using the ferric reducing ability of plasma (FRAP) assay. Plasma samples were obtained at baseline (7 days before the first anthracycline chemotherapy cycle), on day 3 after the first chemotherapy cycle , and at the 10-year follow-up.
Time frame: Baseline (7 days before the first anthracycline chemotherapy cycle), day 3 after the first anthracycline chemotherapy cycle (cycle length: 21 days), and 10 years after completion of chemotherapy.
Erythrocyte antioxidant enzyme activity
Assessment of erythrocyte antioxidant enzyme activity by measuring superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities using standardized commercial assays. These enzymes were evaluated as complementary indicators of endogenous antioxidant defense mechanisms.
Time frame: Baseline (7 days before the first anthracycline chemotherapy cycle), day 3 after the first anthracycline chemotherapy cycle (cycle length: 21 days), and 10 years after completion of chemotherapy.
Markers of oxidative stress and intracellular redox status
Assessment of oxidative stress by measuring plasma 8-isoprostane concentrations and intracellular redox status using the reduced-to-oxidized glutathione (GSH/GSSG) ratio.
Time frame: Baseline (7 days before the first anthracycline chemotherapy cycle), day 3 after the first anthracycline chemotherapy cycle (cycle length: 21 days), and 10 years after completion of chemotherapy.
Inflammatory cytokine profile measured by multiplex immunoassay
Assessment of the systemic inflammatory cytokine profile using a validated MILLIPLEX® multiplex bead-based immunoassay based on Luminex® xMAP® technology. Plasma cytokines were measured simultaneously as a single multiplex biomarker panel comprising inflammatory cytokines and chemokines, including interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), interferon gamma (IFN-γ), and additional analytes included in the assay. This outcome represents the overall inflammatory biomarker profile generated by a single multiplex assay rather than multiple independent outcome measures.
Time frame: Baseline (7 days before the first anthracycline chemotherapy cycle), and day 3 after the first anthracycline chemotherapy cycle (cycle length: 21 days).
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