1.1. Endpoints—The trial primary and secondary endpoints are listed in Table 1. The primary endpoints, as supported by the power calculations based on our previous open-label study (19), are (1) the change in myocardial scar tissue mass (g) and area (%) at the AAMs-patch transplantation site across preoperative and 6-month-postoperative CMR recordings and (2) change in NT-proBNP circulatory levels across the timeframe. Secondary endpoints focus on cardiac structural and functional parameters, feasibility, and safety.
Table 1
The AAMS2 trial endpoints
Primary |
- Change in the myocardial scar tissue by the patch transplantation site |
|
|
Preoperative vs 6-month-follow-up 5SD CMR for mass (g) and area (%) |
|
|
- Change in the plasma NT-proBNP levels across the trial period |
|
Secondary |
|
|
Efficacy |
- Measured by CMR preoperatively and at 6-month-follow-up by the patch site |
|
|
• Change in the live left ventricular wall thickness |
|
|
• Change in viable left ventricular myocardium |
|
|
• Change in the local ventricular wall systolic and diastolic function |
|
|
• Change in left ventricular ejection fraction |
|
|
- Measured by TTE preoperatively and at 3-month-follow-up by the patch site |
|
|
• Changes in systolic and diastolic function of the ventricular wall • Changes in local myocardial strains |
|
|
- Change in NYHA class at the 3- and 6-month vs. preoperative NYHA |
|
|
- Change in 6-minute walk test (preoperative vs. 6-month-follow-up) |
|
|
- Changes in the quality of life (RAND36 preoperative vs. 6-month-follow-up) |
|
Safety |
- Telemetric rhythm postoperatively |
|
|
• Incidence of VT, VF, atrial fibrillation / flutter, or other arrythmias |
|
|
- Days in need for invasive vasoactive medication postoperatively |
|
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- In-hospital infections (Transplant-related = 1; non-related = 2; no = 0) |
|
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- Days in hospital |
|
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- MACCE* during the whole trial period |
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- Anticipated SADE* (serious adverse device effects) during the trial - Other serious adverse events* and unanticipated SADE* during the trial |
|
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- Deaths and deaths due to primary cardiovascular cause |
|
|
• Death and cause (ICD-10) of death during the whole trial period |
|
Feasibility |
- Success in completing the delivery of the patch to the epicardium |
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|
• 0 = success, 1 = no success |
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|
- Waiting time for the ready micrograft transplant |
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• Time from the ready AAMs-gel to the AAMs-patch transplantation |
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- Waiting time in minutes for the heart |
|
|
• After all the anastomoses finished and before the patch transplanted |
|
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- Closing the right atrial appendage |
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|
• Closing the RAA after biopsy for preparing the AAMs-patch. |
|
|
0 = no additional suturing needed, 1 = additional suturing needed |
|
* See section 1.6 for the adverse event definitions. CMR, cardiac magnetic resonance imaging with late gadolinium enhancement; LVEF, left ventricular ejection fraction; MACCE, major adverse cardiac and cerebrovascular events; RAA, right atrial appendage; RAND36, questionnaire for quality of life; TTE; transthoracic echocardiography; VT/VF, ventricular tachycardia/fibrillation; 5SD, 5-standard deviation. |
The power analysis was carried out using SAS 9.4 TS Level 1M4 software (SAS Institute Inc., Cary, NC, USA), the POWER Procedure Wilcoxon-Mann-Whitney Test with the fixed scenario elements O'Brien-Castelloe approximation method and two-sided statistical evaluation. With a total sample size of 50 (two groups, group size 25, distribution 1:1) these parameters yield a power greater than 80% at an α of 0.05. Figure 3 presents the power analysis output graphs. A detailed consideration on the nature of the power analysis is provided at the end of Discussion.
1.2. Patient selection and enrolment—The AAMS2 trial will recruit patients with chronic ischemic HFrEF requiring surgical revascularization, and with a visible myocardial scar in preoperative CMR. Patients with an AMI within last 30 days will be excluded.
Similar to our AAMs-patch feasibility pilot (20), and the AAMs-patch open-label study (19), patients regardless of their gender will be first evaluated by an academic hospital cardiologist in Finland at either Helsinki University Central Hospital or Oulu University Hospital. The patients are then listed on the hospital’s elective surgery list. The usual waiting time on the list ranges between 2–8 weeks, thus allowing any changes to medication, as made by cardiologist on clinical grounds unrelated to the trial, to take effect before surgery. After CABG, the recruited patients are called for a clinical control visit (denoted as the 3-month follow-up) and a dedicated trial control visit (at 6–8 months postoperatively, denoted as the 6-month follow-up).
Patients meeting both the inclusion and exclusion criteria (Table 2) will be provided a consent form describing the trial and are provided with sufficient time and information to make an informed decision on participation. Before a subject undergoes any study procedure, an informed consent discussion will be conducted and written informed consent will be obtained. If either one of the following, i.e. the systolic impairment (LVEF < 50%) or ischemic scar, are not present in the preoperative CMR, the recruited patients are excluded from the trial due to screening failure (Table 2). This also applies if logistical issues prevent the CMR, typically done just days prior planned CABG, to be performed before the CABG in adequate time. The trial will be conducted following the Declaration of Helsinki on Ethical Principles for Medical Research Involving Human Subjects (22). The data collected in the trial will fulfil EU regulations for personal health data protection, including General Data Protection Regulation (GDPR).
Table 2
Eligibility |
Age: 18–75 years |
|
Sexes: All |
|
Healthy volunteers: not accepted |
Inclusion |
Informed consent obtained |
|
Ischemic heart disease requiring CABG for revascularization |
|
LVEF between ≥ 15% and ≤ 40% at recruitment (echocardiography) |
|
NYHA Class II-IV heart failure symptoms |
Exclusion |
Heart failure due to left ventricular outflow tract obstruction |
|
Expected life expectancy < 1 year |
|
Acute myocardial infarction (AMI) within last 30 days |
|
History of life-threatening and likely repeating ventricular arrhythmia or resuscitation, or an implantable cardioverter defibrillator |
|
Stroke or other disabling condition in 3 months before screening |
|
Severe valve disease or scheduled valve surgery |
|
Renal dysfunction (GFR < 45 ml/min/1.73m) |
|
Other major disease limiting life expectancy |
|
Contraindications for coronary angiogram or CMR |
|
Allergy or hypersensitivity to the fibrin glue or to the Hemopatch® |
|
A part of any special patient group* |
|
Participation in some other clinical trial |
Screening |
No visible scar or LVEF ≥ 50% in preoperative CMR |
failure |
Preoperative CMR has not been performed prior scheduled CABG |
* Pregnant, nursing, handicapped, persons in an especially vulnerable position, or those in emergency situations. CABG, coronary artery bypass grafting; CMR, cardiac magnetic resonance imaging with late gadolinium enhancement; LVEF, left ventricular ejection fraction. |
1.3 Baseline morbidity—A general baseline morbidity assessment will be carried out and involves a patient information system search for significant cardiovascular and non-cardiovascular comorbidities, electrocardiogram, structured patient interview and a case report form fill-out comprising dyspnea and angina pectoris symptom scaling, numeration of quality of life (section 1.10), traditional Framingham cardiovascular risk factors (23), supplemented with body mass index (BMI), personal history of MI, or family history of IHD, and medication list review. Data on patient medication will be curated and analyzed based on anatomical therapeutic chemical classification using the defined daily dose values enabling dosage comparisons. The CRF and trial checklists are provided as online-only supplementary files (Supplementary Files S1-9).
1.4. Randomization and blinding—Participants passing the screening failure are randomized to receive either standard CABG and collagen-based patch without the AAMs (control group) or CABG with an epicardial transplantation of an AAMs-patch (collagen-based patch + AAMs) after all the anastomoses have been completed (the AAMs-patch group). Patients are randomized to the groups using sex-stratified block randomization via openly available online tool at www.sealedenvelope.comwith block sizes 2 and 4, and stratification according to sex (female, male). Randomization is carried out by the study nurse. In this trial, we randomize 50 participants that are treated during CABG either with Hemopatch® or the AAMs-patch. Specifically, the research nurse texts the Sealed Envelope service phone number 'AAMS2' (the trial abbreviation), followed by an execution command 'randomise', and completed by the detailing patient pseudonym. Then, the research nurse receives the allocation information by a text message. This is done a day prior each patient's surgery to grant adequate time to organize practicalities required for the perioperative AAMs-patch assembly. The study nurse oversees the allocation in a double-blind manner, where the patient and the evaluating cardiologist(s), radiologist(s) and academic researchers remain blinded to the allocation. Given the nature of the treatment intervention (intraoperative AAMs preparation vs. no preparation) the operating cardiac surgeon or the study nurse are not blinded intraoperatively. However, the operating cardiac surgeon is blinded during the time of planning the anastomoses prior CABG. Moreover, all CMR and transthoracic echocardiography (TTE) measurements as well as laboratory analyses are done by persons blinded to the patients’ study group allocations. In this trial, if considered acutely mandatory to remove the epicardial patch material, it is done without consideration of the randomization. Hence, no emergency unblinding is required in this trial.
1.5. Preparation and administration of atrial appendage micrografts—A piece of the RAA is harvested at the beginning of cardiac surgery upon right atrial cannulation as a part of the heart-and-lung-machine setup. For the AAMs-patch group, the RAA tissue is then weighed and mechanically grinded to micrografts in the operating room by using the Rigeneracon blade (Rigenera-system, HBW s.r.l., Turin, Italy) as previously described (15, 19, 20). As in the open-label safety and feasibility study (19), the targeted weight of the sample tissue for AAMs preparation is around 0.6–1.0 grams. For the CABG control group, the tissue piece is stored as a sample (section 1.7). Dedicated CE-marked instrumentation kits to support tissue processing in the operating room are purchased from EpiHeart Oy (EpiHeart CMT Core kit, Helsinki, Finland).
After grinding the RAA with Rigeneracon blade and subsequent centrifugation (5 min, 400 rcf) to pellet the AAMs, the cold cardioplegia supernatant is removed. The AAMs-pellet is collected with 0,4mL of Tisseel (Baxter AG, Vienna, Austria) fibrinogen solution (diluted in 0.9% NaCl with 1:1 relation). Then, the AAMs in fibrinogen are spread onto a cooled sterile metallic dish. The spread AAMs in fibrinogen are mixed in situ with 0,2mL of Tisseel thrombin solution (diluted in 0.9% NaCl with 1:30 relation). The AAMs–fibrinogen–thrombin mixture is allowed to undergo spontaneous gelling for at least 10–15 minutes. Then, the gelled AAMs–fibrin mixture is maintained cooled (+ 6 – +8oC), covered, and sterile when waiting for transplantation. When all the anastomoses are ready, the AAMs–fibrin gel is gently lifted with spatula and transplanted onto the dry matrix sheet (Hemopatch® Sealing Hemostat 45 mm × 45 mm; catalog ref. 1506256; Baxter International Inc., Illinois, USA), thus forming the AAMs-patch (Supplementary Video S1), just prior the transplantation by the surgeon. Then, the Hemopatch® edges are moistened with sodium bicarbonate solution (4.2–8.4%) to activate the polyethylene glycol-coating of the patch. Finally, to achieve proper epicardial adherence, the AAMs-patch with the moistened patch edges is transplanted onto the epicardium of the scarred border zone of the myocardium by using a dry gauze with uniform pressure for 2 minutes. The exact transplantation site is preoperatively assessed with CMR to the ischemia-induced scar. In this trial, the AAMs patch shall not wait longer than 6 hours prior transplantation.
To standardize the effects of the patch material, the control group receives the patch without the AAMs. The place of transplanted patch is documented with a photo. The patients are carefully monitored according to clinical routine after the operation in the intensive care unit (Table 2, Supplementary File S4). Tissue processing-supporting instrumentation (EpiHeart Oy, Helsinki, Finland) usability is recorded in detail by the research nurse, and the usability outcome is included as one of the AAMS2 trial’s secondary outcomes (Table 1).
1.6. Data collection—Clinical, laboratory and drug treatment data are collected to the hospital electronic health records. Any visit related to the operation or their cardiovascular system condition as well as drug treatment changes are collected by the study investigators. This data is stored pseudonymised with the other data from the patient. In addition, the cardiovascular-related changes in medication with Anatomical Therapeutic Classification (ATC) codes and defined daily doses (DDDs) will be recorded for analysis to serve as a source data for surrogate modelling of an improved (i.e., reduced medication or dosage) or worsened (added medication or increased dosage) disease state.
1.7 Concomitant care, possible harm, and trial adherence—No concomitant care or interventions are prohibited. In this trial, based on regulatory body and company evaluations, the use of Hemopatch® is on-label (CE-marked) in the AAMs-patch-treated cohort, whilst an off-label use in the control group, where no AAMs are given. Hence, any product liability on possible participant harm(s), which can be causally verified to the use of Hemopatch® sealing the AAMs micrografts epicardially, is on the manufacturer, Baxter International Inc., Illinois, USA. Also in the control cohort, where no AAMs are being used, the liability for compensation for any participant harm(s) that can be causally linked to the Hemopatch® product itself is on the manufacturer. If the possible participant harms cannot be causally linked to the product, the liability is on the respective hospital trial investigators, all of which have an extensive insurance covering compensation for any damages in accordance with the national Patient Injury Act of Finland during the treatment and examination of patients. In Finland, these processes are centrally governed and handled by the Patient Insurance Centre.
The participant adherence is monitored by the research nurse by continuously updating an anonymized stepwise trial progression file. In case of participant- or investigator-derived deviation from the trial protocol, the participant is followed and examined per the trial protocol as extensively as possible after the deviation. In case of participant discontinuation, the study data accumulated until the date of discontinuation is included in the trial datasets and reported separately.
1.8. Adverse events—In this trial, adverse events are divided to (1) major adverse cardiac and cerebrovascular events (MACCE), (2) anticipated SADE (serious adverse device effects), (3) other SAE (serious adverse events), (4) unanticipated SADE. All the events are continuously monitored and reported to regulatory bodies. In this trial, the MACCE definition comprise death (all-cause), MI, any acute coronary revascularization, or stroke. MI is defined according to fourth universal definition on MI as either (1) Perioperative myocardial injury (i.e., type 5 MI ≤ 48 hours post-CABG, creatinine kinase muscle-brain isoenzymes [CK-MB] ≥ 10 times the upper reference) or (2) Postoperative MI (i.e., an increase in the CK-MB or troponin concentration above the upper reference limit with ischemic symptoms or signs). Stroke is indicated by neurological deficits and confirmed by a neurologist on the basis of imaging modalities with lesion(s) concordant to the clinical presentation. New revascularizations span any postoperative coronary intervention.
As a part of our overall comprehensive safety evaluation of the method, anticipated SADE are events identified by us with possible causal relationship to the AAMs-patch therapy. These include: (1) mediastinitis, (2) postoperative pericardial effusion requiring subxiphoidal drainage or resternotomy, (3) major bleeding (BARC classes 4–5)(24) from the RAA biopsy site, or (4) major postoperative arrhythmia (ventricular fibrillation, or ventricular tachycardia over 30 seconds). Mediastinitis is diagnosed according to Centers for Disease Control and Prevention (CDC) guidelines (25). The accumulated published data in mice (15), pigs (21), and human (19) on the AAMs-patch method does not indicate heightened risk for these events.
Other SAE, comprise any adverse event that has led to either death, life-threatening illness, (prolongation of) hospitalization, medical intervention to prevent life-threatening illness, or chronic disease. According to our risk assessment of the AAMs-patch therapy, these could include: myocarditis, pericardial effusion, HF exacerbation, resternotomy, atrial tachycardia or fibrillation, atrial flutter, transient ischemic attack, major bleeding (BARC 3–5) (24), acute kidney injury, or other hospitalization due to ischemic cause.
1.9. Blood samples—The AAMS2 trial assesses blood, plasma, and RAA tissue (section 1.7) samples for their RNA with a focus on their contained post-transcriptional, epitranscriptomic, modifications as described in the IHD-EPITRAN study design (www.ihd-epitran.com) (26). These modifications, as recently reviewed comprehensively by us in the field of cardiovascular diseases (27), are emerging regulators of both cardiac disease and regeneration. The schedule of sample collection is summarized in Fig. 2.
Briefly, the study blood sample set contains tubes for RNA-stabilized TEMPUS™ whole blood (3 mL × 8) as well as EDTA-stabilized blood (9 mL × 3) from which RNA-stabilized plasma (900 µL × 10) and standard plasma (500 µL × 4–10) are separated and aliquoted for storage (26). In addition, a sample for N-terminal pro-brain natriuretic peptide (NT-pro-BNP) measurement by the clinical laboratory (HUSLAB, Helsinki, Finland; NordLab, Oulu, Finland) is collected in the trial preoperatively and at both 3-month and 6-month clinical and study follow-ups, respectively.
1.10. RAA tissue samples—Since the RAA tissue sample removed during CABG in control group is not used for producing AAMs, it is collected for analyses. The initial piece of RAA tissue will be divided into two pieces and stored as previously described (Fig. 2) (26).
The RNA-stabilized RAA piece is used to profile a m6A-epitranscriptome as in IHD-EPITRAN study (26). Briefly, the RNAlater-stored (AM7021, ThermoFisher Scientific Inc., Waltham, MA, USA) RAA piece will be subjected for RNA extraction, fractionation and m6A-targeted sequencing, and bioinformatics. Overall, these produced epitranscriptomes are correlated for changes in repeated CMR, TTE, and blood sampling results.
1.11. Echocardiography—For functional and anatomical insight of the participants’ cardiac status, the participants are assessed with TTE both pre- and postoperatively (Fig. 2). Postoperative TTE recordings are done at hospital discharge (approximately one week postoperatively) and at 3-months of follow-up. The recordings are performed with prespecified acquisition methods and imaging windows by few designated cardiologists and include both anatomical and functional assessments of atria, valves, and ventricles. The presence or absence of pericardial effusion, thrombus and aneurysm is recorded. The detailed protocol is provided as a Supplementary File S10. Also, a perfusion anesthesiologist will perform transesophageal echocardiography in the operating room during anesthesia to evaluate both left and right atrial appendages and atria for blood flow velocities, possible sludge, thrombus, and anatomy before CABG. The raw data will also be exported and stored for further state-of-the-art functional analyses (myocardial imaging), such as strain and strain rate measurements.
1.12. Late gadolinium enhancement cardiac magnetic resonance imaging (CMR)—CMR is performed preoperatively to evaluate cardiac function, and myocardial anatomy, including ischemic and fibrotic areas. The preoperative CMR excludes patients without any fibrotic scar or systolic impairment (Table 2) and guides the AAMs-patch transplantation site at the end of CABG surgery (section 1.4). Its repetition at a 6-month of follow-up enables assessment of any change in either scar or vital myocardial tissue mass by the epicardial AAMs-patch transplantation site. Specifically, a whole body 1.5-T MRI scanner (In Helsinki, Siemens Sola or Avanto-fit, Siemens AG, Erlangen, Germany; in Oulu, Siemens Sola or Sola-fit, Siemens AG, Erlangen, Germany) is used for CMR image acquisition. Cardiac structure and function are evaluated with a standardized CMR protocol using electrocardiogram and respiratory gating. Short-axis cine images are used for left and right ventricular volumetric measurements. Myocardial contractility is evaluated using longitudinal, circumferential, and radial strain measurements from short- and long-axis cine images. Late gadolinium enhancement is used to measure infarction volume and mass using 5-SD semiautomatic gain estimate, as previously suggested for semiautomatic thresholding for infarction detection (28). Image post-processing is performed with Medis Suite software (Medis Medical Imaging Systems, Leiden, The Netherlands) with QMass and QStrain applications.
1.13. Quality of life assessment—Health-related quality of life (HRQoL) is measured using the RAND36 short form questionnaire (SF36) (29). The questionnaire is standardized with specified mean and standard deviation values for eight dimensions that range from physical functioning and subjective feeling of vitality and health to bodily pain. The obtained scores are compared for a Finnish cohort with any chronic disease. Also, a subjective symptom-evaluation is performed for the two cardinal symptoms of IHD and HF, angina pectoris and exertional dyspnea, with standardized classification systems develop by Canadian Cardiovascular Society (CCS) and New York Heart Association (NYHA), respectively (30, 31).
1.14. Six-minute walking test (6MWT)—To measure the general physical capacity, a 6MWT is performed for all participants preoperatively and postoperatively at the 6-month follow-up (32). The parameters included are the following: the actualized walking distance in meters, the predicted walking distance, oxygen saturation (baseline at rest and lowest during the test), and subjective related symptoms according to Borg's scale (0–10). The test is scheduled just before CABG (Fig. 2) to minimize the effects of preoperatively initiated pharmacotherapies on exertional capacity prior CABG and AAMs-patch transplantation.
1.15. Data confidentiality—In this trial, all personal or other study information collected is done by healthcare professionals committed in verbatim to the utmost standards of data safety and confidentiality. The produced data are stored in the research registry in the University Hospitals' and University of Helsinki safe network hard drives and on the servers of the Finnish IT Center for Science designed for sensitive data storage, all with an automated backup. The tailored data storage services—SD-Connect, SD-Desktop and SD-Publish—provided by Finnish CSC–IT Center for Science (https://research.csc.fi/sensitive-data) are financially supported by the Finland's Ministry of Education and Culture. These services are designed to comply with the EU General Data Protection Regulation (GDPR), which is followed throughout the trial lifetime. The accession to these registries is controlled via role-based accession rights. Only those specified in the registry description approved by the HUS Ethics Committee can access the data therein.
The participants' TTE and CMR data is stored and accessed via software fulfilling the Hospitals' data security guidelines. Case report formats are both physically stored in the safe Hospital premises with an access control and electronically in the research registry. The pseudonymized sequencing datasets with metadata will be made available upon publication principally via the long-term sensitive data storage service SD-Publish, which appoints an independent data monitoring committee to evaluate and process all applications (in writing) by other scientists interested to re-use the deposited pseudonymized research data. The principal investigators of the trial will have access to the final trial dataset. Contractual agreements will not limit this right. The identificatory data and pseudonymized data will be stored 15 years after the trial completion. Then, the pseudonymization codes and keys will be erased and the anonymized data will be principally stored in SD-Apply, or in another public repository. The anonymized group-level datasets of the trial results will be made available by submission(s) for publication(s) in esteemed journals of the field of cardiology and cardiac surgery.
1.16. Trial monitoring —The AAMS2 trial is externally monitored by the Clinical Research Institute HUCH to ensure trial subjects’ rights, safety, and well-being. A detailed monitoring plan has been formulated and is in effect with the monitor and the research group. The trial monitor regularly audits the trial data registries for appropriateness and completeness. In addition, the AAMS2 trial has a Trial Monitoring Committee with national medical professionals from the field of cardiology, cardiac surgery, and nursing to evaluate the appropriateness of the trial in a periodical manner, as each 10 patients are recruited, from a focused medical viewpoint. As per local standards and the trial size, this trial does not have a separate data monitoring committee.
1.17. Interim analysis—If considered appropriate, an interim analysis can be performed when a significant proportion of the total recruited participants (i.e., 20–30 patients) have undergone the full protocol. This consideration is undertaken jointly with the Trial Monitoring Committee. All the trial investigators can access the interim analysis results. The principal investigator makes the final decision to terminate the trial.
1.18. Statistical analyses—Comparisons between groups will be performed with the Mann Whitney U test. Ordinal variables are tested with the Chi Square test. Multiple comparisons are corrected with the Bonferroni method, significant findings are further tested groupwise using the Mann Whitney U test. Quality of life data is presented as mean and analysed with the independent samples t-test (two-sided). Analyses are performed with the IBM SPSS Statistics 27 program (IBM Corp., Armonk, NY) or equivalent. Only those participants are included in the main endpoint analyses (Table 1) that have both undergone the treatment (AAMs-patch or Hemopatch®-only) and completed the 6-month follow-up CMR imaging with late gadolinium enhancement.
1.19. Protocol amendments—Any important protocol modifications (i.e., changes to eligibility criteria, endpoints, or analyses) are communicated to trial investigators, participants, registries, and regulators as soon as practically possible by the trial coordinators. In a such unlikely case, the protocol modification is coordinated in a close mutual communication with the trial external regulatory bodies (e.g. HUS Ethical Committee, Trial Monitor, Trial Monitoring Committee, and FIMEA).