Author s response to reviews

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1 Author s response to reviews Title: Effects of four antiplatelet/statin combined strategies on immune and inflammatory responses in patients with acute myocardial infarction undergoing pharmacoinvasive strategy: Design and rationale of the B and T Types of Lymphocytes Evaluation in Acute Myocardial Infarction (BATTLE-AMI) study:study protocol for a randomized controlled trial Authors: Francisco Fonseca (fahfonseca@terra.com.br) Maria Izar (mcoizar@terra.com.br) Ieda Maugeri (imaugeri@unifesp.br) Otavio Berwanger (oberwanger@hcor.com.br) Lucas Damiani (lucas@estatikos.com.b) Ibraim Pinto (ibraim.pinto@grupofleury.com.b) GIlberto Szarf (gszarf@gmail.com) Carolina França (carolufscar24@gmail.com) Henrique Bianco (henriquetria@uol.com.br) Flavio Moreira (flaviotocci@yahoo.com) Adriano Caixeta (adriano.caixeta@einstein.br) Claudia Alves (cmralves@me.com) Aline Lopes (alinslopes@gmail.com) Aline Klassen (aline.klassen@gmail.com) Marina Tavares (marina.fm.tavares@gmail.com) Henrique Fonseca (har.fonseca@yahoo.com.br) Antonio Carvalho (tillacarvalho@gmail.com) Version: 1 Date: 31 Oct 2017 Author s response to reviews:

2 Manuscript TRLS-D Effects of four antiplatelet/statin combined strategies on immune and inflammatory responses in patients with acute myocardial infarction undergoing pharmacoinvasive strategy: Design and rationale of the B and T Types of Lymphocytes Evaluation in Acute Myocardial Infarction (BATTLE-AMI) study: study protocol for a randomized controlled trial Response to reviewers : I found your protocol article most interesting to read and well-written. I am a biostatistician with some experience in the design and analysis of trials of cardiac interventions, and this is no doubt reflected in the focus of my review. I do believe that the technical aspects of the protocol are well described and adequately discussed in your manuscript. I also believe, however, that the analytic strategy requires substantially more detail, explanation and clarification, that is in the current draft of the manuscript. In your revision, please be consistent throughout the paper. For example, Figure 3 refers to 30- day LVEF as primary outcome and infarcted mass as the secondary outcome. If this is the case, it will clarify the protocol to the reader if you refer to them as such starting with the Abstract forward. Abstract The abstract reads well, but I believe should include the number of patients (300, 75 per arm) and the timepoints for evaluation of the primary outcomes (which I believe is 4 weeks post- STEMI for LVEF) and secondary outcomes (infarcted mass and 30 days and/or 6 mos for cmri). You may also wish to modify the abstract further (for example, clarifying how the four treatment arms are to be compared) based on consideration of my comments on the main article, immediately below. Authors: We agree with your comments, and the abstract was modified as you suggested. BACKGROUND: Early reperfusion of the occluded coronary artery during acute myocardial infarction is considered crucial for reduction of infarcted mass and recovery of ventricular function. Effective microcirculation and the balance between protective and harmful lymphocytes may have roles in reperfusion injury and may affect final ventricular remodeling. METHODS/DESIGN: BATTLE-AMI is an open-label, randomized trial comparing the effects of four therapeutic strategies (rosuvastatin/ticagrelor, rosuvastatin/clopidogrel, simvastatin plus ezetimibe/ticagrelor, or simvastatin plus ezetimibe/clopidogrel) on infarcted mass and left ventricular ejection fraction (blinded endpoints) in patients with ST-segment elevation myocardial infarction submitted to fibrinolytic therapy prior to coronary angiogram (pharmacoinvasive strategy). All patients (n=300, 75 per arm) will be followed-up for 6 months. The effects of treatment on subsets of B and T lymphocytes will be determined by flowcytometry/elispot and will be correlated with the infarcted mass, left ventricular ejection

3 fraction, and microcirculation perfusion obtained by cardiac magnetic resonance imaging. The primary hypothesis is that the combined rosuvastatin/ticagrelor therapy will be superior to other therapies (particularly for the comparison with simvastatin plus ezetimibe/clopidogrel) for the achievement of better left ventricular ejection fraction at 30 days (primary endpoint) and smaller infarcted mass (secondary endpoint) at 30 days and 6 months. The trial will also evaluate the improvement in the immune/inflammatory responses mediated by B and T lymphocytes. Omics field (metabolomics and proteomics) will help to understand these responses by molecular events. DISCUSSION: BATTLE-AMI is aimed to a) evaluate the role of subsets of lymphocytes on microcirculation improvement and b) how the choice of statin/antiplatelet therapy may affect cardiac remodeling after acute myocardial infarction with ST elevation. TRIAL REGISTRATION: ClinicalTrials.gov. identifier: NCT Registered on 28 September SPONSOR: São Paulo Research Foundation (FAPESP) and through an investigator-initiated grant from AstraZeneca (ESR ). Background Well-written, and even compelling, although quite technical in some areas. As you go through the revisions requested below, I would ask you to consider any minor revisions you can make in this section to make this section more understandable to the protocol reader without technical expertise. (I am such a reader. I was able to "grasp" the hypotheses of the trial as discussed in the "Microcirculation" section and in Figure 2; I did have more trouble figuring out how/whether the hypotheses about B and T lymphocytes discussed and shown in Figure 1 relate to the four treatment arms. Please clarify if possible/relevant). Authors: We added the suggested information to clarify how the therapies may afffect imune/inflammatory responses and we added a reference [15]. Subsets of B cells Experimental studies suggest an important role for B cells in atherogenesis. Initial evidence came from splenectomized ApoE-/- mice developing more severe atherosclerosis compared to shamtreated mice, followed by an impressive attenuation of atherosclerosis after transfer of splenic B cells [9]; however, subsequent studies showed conflicting results, and the current understanding suggests a differential role according to B cell subsets [10,11]. The same theory might be

4 postulated for acute MI. We hypothesized that an increased number of human B CD11b- cells may decrease infarcted mass after reperfusion of an occluded coronary artery. In addition, an increased number of B2 cells, which are considered proatherogenic, may increase the infarcted mass [12]. Other possible players such as highly inflammatory T cells, lymphocyte-derived microparticles, extracellular vesicles, and exosomes may also reach the damaged myocardium, affecting the clotting process [8], oxidative stress [13] and microcirculation [14], thus influencing the recovery of the ischemic tissue [14]. We previously reported that hyperlipidemic subjects treated with rosuvastatin presented better immune responses (higher titers of antioxldl) than those receiving simvastatin/ezetimibe, thus suggesting that the choice of lipid lowering therapy may have possible beneficial role in the acute phase of myocardial infarction [15]. Figure 1 summarizes the study hypothesis regarding B and T lymphocytes. Reviewer # 1 Methods Please clarify: will the randomization be carried out after the patient arrives in the teaching hospital? Authors: We added this information. After obtaining written informed consent in the teaching hospital and prior to coronary angiography, eligible patients will be randomized using a central computerized system in the first 24 hours to 1 of the 4 open-label treatment assignments in a 1:1:1:1 ratio: rosuvastatin/ticagrelor, rosuvastatin/clopidogrel, simvastatin plus ezetimibe/ticagrelor, or simvastatin plus ezetimibe/clopidogrel. Study design Minor point: you state "4 weeks" on line 16 and "30 days" on line 48. For clarity, please refer to this consistently (based on rest of manuscript, likely "30 days") throughout. Authors: We agree. For the whole manuscript we refer this time point as 30 days. The study design of BATTLE-AMI is shown in Figure 3. The primary objective of the study is to compare the effects of the different antiplatelet/statin therapies in left ventricular ejection fraction (LVEF) obtained by cardiac magnetic resonance imaging (cmri) 30 days post-stemi.

5 The first paragraph of "Study design" gives a nice, succinct summary of what the primary, secondary, and other objectives of the trial are. In the second paragraph, please clarify further how the sample size was derived. You mention an F test comparing the four groups, and you described power for a comparison between the rosuvastatin/ticagrelor and "the other 3 groups". As you are using an alpha level of 0.05/3, this leads me to believe that you will be comparing the rosuvastatin/ticagrelor arm with each of the other 3 arms separately, perhaps using something like Dunnett's test (perhaps not). Is this assumption correct? Do you expect (based on Figure 2) adequate power for the rosuvastatin/ticagrelor versus simvastatin/clopidogrel comparison, perhaps, and less power for the other comparisons? Again, this should be clarified. Authors: We agree that these comparisons should be clarified. Our main hypothesis is that rosuvastatin/ticagrelor will be superior than simvastatin plus ezetimibe/clopidogrel, for the cardiac magnetic resonance parameters. Additionally, the pilot study indicated that the ezetimibe/clopidogrel was similar to simvastatin plus ezetimibe/ticagrelor and rosuvastatin/clopidogrel combinations regarding the primary endpoint, and we decided to compare those other groups (rosuvastatin/clopidogrel and simvastatin plus ezetimibe/ticagrelor) to explore possible mechanisms for the primary and secondary objectives. Thereafter, our sample size derivation was done considering that rosuvastatin/ticagrelor will be superior equally against the other three groups. The alpha level fixed in 0.05/3 (1.67%) was considered to adjust the global alpha error in 5% using Dunnet s test approach (many-to-one comparisons). You specify that a data safety monitoring board exists and that they will examine data 3 times during the trial. Please detail the board's activities a bit. Will they be reviewing safety data only? Will any sort of interim analysis of efficacy outcome data, with the possibility of early termination (of the trial or of a treatment arm) if there is strong evidence of efficacy, be performed? If so, what types of monitoring boundaries will be employed? Authors: Thank you for the comment. We added more information regarding the data safety monitoring board. Considering the study s exploratory nature and small sample size we believe that interruption by efficacy is not necessary. Consequently we did not intend to evaluate the primary endpoint in those interim analyses. Study organization

6 The steering committee of the study is composed of representatives from the Universidade Federal de São Paulo (UNIFESP, São Paulo, Brazil), Instituto Dante Pazzanese de Cardiologia (IDPC, São Paulo, Brazil), and Research Institute, Hospital do Coração (HCor, São Paulo, Brazil). An independent data safety monitoring board was constituted by representatives of the Heart Institute (InCor, São Paulo, Brazil) and is planned to analyze data 3 times during the study to evaluate only safety measures (serious adverse events, such as bleeding, acute kidney injury, new myocardial infarction and mortality). Based on those parameters, the board may consider remove an intervention arm or study s early termination by safety reasons. Those comparisons will consider Haybetto-Peto boundary (p < 0.001). Reviewer # 1 Under laboratory analyses, the technical procedure is quite well described. What analysis approach(es) will be used to compare the 4 groups across the 3 visits? Cross-sectional? Repeated measures? Here or at an appropriate point in the protocol discussion, please also consider outlining how the relationships of B/T lymphocyte subsets with LVEF will be assessed. I repeat this same point for the cmri and angiographic analysis sections - again, the methods are well explained (and I like the good discussion of quality control measures), but could you describe the key analyses that will be carried out for these measures, and perhaps very briefly (re)state what you would expect to find in these analyses if the agents work as postulated? Authors: We agree and added the requested information Second, the trial will evaluate the role of subsets of lymphocytes in the final infarcted mass and ventricular remodeling, exploring B and T cells phenotypes. For this analysis, the absolute number of subtypes of lymphocytes will be correlated with cmri parameters as well as with those parameters obtained in the coronary angiogram. We expect that increased number of lymphocytes of more inflammatory phenotypes will be related to reperfusion injury, thus influencing the final infarcted mass and left ventricular ejection fraction at 30 days postmyocardial infarction. Third, the effects of microbiota on the cmri parameters and lymphocytes differentiation will be examined. We expect that the presence of a pathological microbiota may contribute to lymphocyte phenotype differentiation. Finally, the relationship among microparticles derived from endothelium, platelets, and leukocytes with the cmri parameters will be addressed. This analysis will test the relevance of these new vascular biomarkers in the severity of coronary heart disease and possible influences of the treatments. On behalf of the authors, I would like to thank for the comments made by the reviewer, addressing very important topics, and contributing for the improvement of the amended manuscript. We hope that we have answered all aspects pointed by the reviewer and making our contribution suitable for publication on Trials.

7 Sincerely Francisco A H Fonseca MD, PhD Correponding author