1.1 Study aims

1. Assessment of the prognostic value of the coronary atherosclerosis and the CAC score before chemotherapy for the occurrence of the cardiovascular toxicity
2. Evaluation of IL-6, MPO and TNF-alpha for use in the diagnosis of cardiotoxicity during oncological treatment.
3. Influence of studied parameters, i.e. presence of atherosclerosis, echocardiographic parameters and biomarker levels on the prognosis of patients undergoing cardiotoxic chemotherapy

2.1 Study structure

The ANTEC Trial is a single-center, prospective, observational trial evaluating the impact of atherosclerosis in the coronary arteries determined by computed tomography on the risk of cardiotoxicity related to anthracycline-based chemotherapy. The trial is being carried out in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. The conduct of the study is approved by an Independent Ethics Committee at the Maria Sklodowska-Curie National Research Institute of Oncology in Warsaw (the date of approval– 28 October 2021, approval no. 80/2021) and all participants will provide written informed consent before study entry. The registration identifier on clinicaltrials.gov is NCT05118178.

2.2 Study patients

The study will include patients with cancer, diagnosed and qualified for further systemic treatment with high-dose anthracycline chemotherapy at the Maria Sklodowska-Curie National Research Institute of Oncology in Warsaw. Patients must give informed and voluntary consent to participate in the study and meet all the inclusion criteria and none of the exclusion criteria listed in Table 1.

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Table 1. Inclusion and exclusion criteria.

https://doi.org/10.1371/journal.pone.0288146.t001

2.3 Study visits and follow up

Approximately 80 patients are planned to be included in the study. Each patient will have 5 program visits every 3 months for 12 months. At the first visit, the patient will be informed about the type of the proposed follow-up, its course and planned tests. After signing the consent to participate in the study, patients will undergo planned procedures according to the schedule shown in Table 2.

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Table 2. Schedule of Activities (SoA).

https://doi.org/10.1371/journal.pone.0288146.t002

Clinical variables such as: age, gender, cardiovascular risk factors (hypertension, diabetes, hyperlipidemia, obesity, smoking status), alcohol use, previous medical history, concomitant heart failure therapy, statins or other lipid lowering medications will be documented at baseline and then checked at each visit. Lipid levels and diabetes status at each visit will be determined using a fasting blood sample and lipid panel at that visit.

Cancer diagnosis and current treatment (including cumulative dose of anthracyclines) will be collected and documented at each visit.

In all patients, a single coronary computed tomographic angiography (CCTA) will be performed at the beginning of the study, with calculation of coronary artery calcium (CAC) score according to the Agatston method. Contrast cardiac CT scanning for CAC quantification will be performed with a 64-slice CT scanner (Revolution Evo, GE Healthcare, Waukesha, USA) with prospective ECG triggering. A tube voltage of 120kVp will be used. Images will be reconstructed with a slice thickness of 0.625 mm and CACS was quantified using the Agatston method with dedicated software by a well-trained researcher.

At each visit, transthoracic echocardiography will be performed by using a Philips EPIQ Elite instrument (Philips Healthcare, Andover, Massachusetts). The following echocardiographic parameters will bez assessed: left atrium diameter, interventricular septum diameter, posterior wall thickness, left ventricle (LV) end-diastolic diameter, LV end-systolic diameter, LV ejection fraction, and mitral inflow with the use of Doppler echocardiography. Diastolic function will be evaluated by mitral inflow E/A pattern, E/A ratio, annular tissue Doppler curves and E/E’ ratio. Images will be digitally stored for offline analysis on custom software (Q-Station, Philips Healthcare, Andover, Massachusetts). Left ventricular, two-dimensional peak systolic global longitudinal strain (GLS) will be analysed by an offline semi-automated speckle tracking imaging technique from the three standard apical views. Analyses will be performed by one board-certified physician, who will be blinded to all clinical characteristics.

Blood samples will be collected et each visit and analyzed by the central laboratory (The Maria Sklodowska-Curie National Research Institute of Oncology). Laboratory tests will include: alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, creatinine, glucose, magnesium, potassium, sodium, uric acid, morphology, glycated hemoglobin, lipid profile, d-dimers, fibrinogen and hs-CRP.

Blood samples to determine plasma levels of high-sensitivity troponin T (hs-cTnT) and N-terminal natriuretic pro-peptide (NTproBNP) will be processed in a central laboratory using standardized methods. New atherosclerosis biomarkers (IL-6, MPO, TNF-alpha) will be assessed every 6 months using Quantikine ELISA kit R&D Systems, Inc., USA & Canada.

2.4 Primary and secondary endpoints

3.1 Data acquisition and quality control

All measurement will be performed according to routine diagnostic standards. The critical measurements (CCTA, ECHO) will be reviewed and coded by a trained member of the study team to ensure comparability of the results. Electronic case report forms (eCRFs) will be developed to capture the study subject data in secure internal environment. The clinical data will be entered manually and laboratory data will be extracted from the Hospital Information System (HIS). The data will be pseudonimised using study subject identification number, with the list of linking these numbers to the patient identification number used in the HIS stored separately. To ensure data quality validation rules will be implemented in eCRFs to preclude entry of inconsistent or out-of-range values. Data will be assessed periodically to identify missing and outlying values to be verified with the medical notes available in HIS by the study team.

3.2 Sample size calculations and statistical analysis

The estimated group size was determined for the one-sided Pearson chi-square test for two proportions. We considered an alternative hypothesis of an approximately 2-fold increase in the incidence of cardiotoxicity with the presence of atherosclerotic lesions versus null hypothesis of no effect of atherosclerotic lesions. In particular, we assumed that the incidence of cardiotoxicity in the population with and without atherosclerotic lesions will vary from 60% to 75% and from 30% to 40% respectively and that the atherosclerotic lesions of various severity will be present in 30% to 40% of patients. With additional standard assumptions of 0.05 statistical significance and 80% power and assuming the estimated sample size varies from 54 to 80 individuals, thus the desired sample size of 80 was established.

Handling of missing data will depend on the proportion of cases affected by missing values. Complete case analysis will be performed if any missing values occur in less than 5% of cases. Otherwise, imputation through full conditional specification (FCS) methods are planned, accounting for the model of interest.

Statistical analysis of the results obtained in this trial will focus on the comparison of groups determined on the basis of the presence of atherosclerotic lesions, and will include a comparison of the characteristics of these groups using for comparisons of numerical variables, the Wilcoxon test, and for categorical variables the Fisher or chi-square tests. The Cochran-Armitage test on the existence of a trend for an ordinal scale variable will be used to assess the effect of the severity of atherosclerotic lesions on the incidence of cardiotoxicity.

Cumulative incidence of cancer therapy-related cardiovascular toxicity (primary endpoint) will be estimated using competing risk approach with death from other causes as the competing event. Fine and Gray’s proportional subhazards model will be used to estimate incidence by group and evaluate the impact of atherosclerotic lesions controlling for potential confounders. This model may be used in combination with missing data imputation, if necessary, with substantive model compatible fully conditional specification (SMC-FCS) imputation [24].

For time-to-event secondary endpoint (all-cause mortality) we will use the Kaplan-Meier estimates with Long-rank test to compare the survival curves. In addition, cox proportional hazard model will be used for multivariate analysis if sufficient number of events is observed. Changes in numerical variables (ejection fraction, biomarkers’ concentration) by atherosclerotic lesions status will be investigated by two-way repeated measures ANOVA.

4.1 Possible limitations

As the expected frequency of cardiotoxicity according to the new 2022 guidelines is not well established there is a risk of inappropriate assumptions for power calculations, although we adopted rather conservative assumptions. In addition, as this is an observational study confounding by other cardiotoxicity risk factors is possible. If feasible, this will be accounted for in the multivariate analysis.