RNA as Prognostic Biomarkers in Patients With Acute Coronary Syndrome
Conditions: Coronary Syndrome
Sponsor: IRCCS Policlinico S. Donato

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RNA as prognostic biomarkers in patients with acute 
coronary syndrome 
Sponsor IRCCS POLICLINICO SAN DONATO 
 
 PI: Dr. Luca Testa  
 
Co-I: Dr.Fabio Martelli 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Introduction and Rational 
Ischemic heart disease is the most frequent cause of death in the world and is the disease. heart disease, 
which has the highest incidence and prevalence in industrialised countries [1, 2]. It includes several. clinical 
pictures caused by anatomical and/or functional pathologies of coronary arterial vessels capable of. 
determine the development of myocardial ischemia. Within ischemic heart disease is possible. distinguish 
chronic and acute coronary syndromes. Acute coronary syndromes (ACS). include two main categories, 
distinguished on the basis of the electrocardiographic picture:. - Acute coronary syndromes without 
persistent overshooting of the ST tract (NSTE. coronary syndromes - not ST elevation), which include 
myocardial infarction without above the ST tract vellement (NSTEMI) and unstable angina (AU); 
pathologically the syndromes. Coronary NSTE are characterized by the subocclusion of a vessel, with 
necrosis or ischemia of the. successor layers in case of NSTEMI or UA respectively.. - Acute coronary 
syndromes with persistent ST elevation (coronary syndromes. STE - ST elevation); physiopathology, are 
caused by a complete occlusion of a vessel which, in the absence of reperfusion, causes a full-thickness 
necrosis. In patients with NSTE coronary syndromes, therapy is guided by stratification of risk and is aimed 
primarily at facilitating the resolution of the thrombus and avoiding its evolution towards a thrombus 
occlusive, in addition to the control of the ischemic-anginal picture. It is therefore necessary to set 
promptly a drug therapy that includes an antiaggregant therapy (single or in association), an anticoagulant 
therapy and drugs that reduce myocardial oxygen demand (e.g. beta-blockers). Patients should then be 
subjected, with different timing depending on the estimated ischemic risk, coronarography and possible 
myocardial revascularization. All patients with STEMI should receive, unless contraindications, therapy 
Binding antifungal with acetylsalicylic acid and a P2Y12 inhibitor (Prasugrel or Ticagrelor). The addition of 
an anticoagulant (non-fractionated heparin, enoxaparina, fondaparinux, bivalirudin).The primary 
therapeutic goal in patients with STEMI, however, is the re-channeling of the vessel occluded as early as 
possible. This can be achieved by two types of treatment, one pharmacological (fibrinolysis) and one 
mechanical (primary angioplasty). Primary angioplasty (with stent implant) is the treatment of choice for 
reperfusion coronary because it is much more effective than thrombolysis in obtaining the reunalization of 
the vessel occluded. 
Despite thrombolytic therapy, angioplasty (PCI) and progress in recent decades have significantly improved 
the prognosis of patients after a heart attack with or withoutincrease in the ST tract, the overall mortality 
rate is 8% to one month and 12% a year after discharge [3]. It has also been estimated that, following a 
myocardial infarction, about 40% of STEMI patients and 30% of NSTEMI patients develop a remodelling of 
the left ventricle (VS) resulting in contractile dysfunction within 12 months [4, 5].  
Previous studies Randomized clinicians identified a left ventricle ejection fraction (FEVS) < 45 as cut-off to 
define a left ventricular systolic dysfunction [6]. The development of a Left ventricular dysfunction is one of 
the largest independent predictors of mortality after heart attack myocardial [3, 7]. In contrast to patients 
with acute myocardial infarction, patients with AU, have a preserved FEVS (>60%), an excellent prognosis 
and a low rate of evolution towards ventricular dysfunction at one year ( 5-7%) [8]. The AU, characterized 
by ischemia in the absence of cardiomyocytic necrosis, with the advent of high sensitivity tests for the 
dosage of troponins thistle accounts for 10% of all ACS [8]. 
Many circulating proteins, such as Creatine Kinase (CK-MB), cardiac troponin measured with high sensitivity 
test (hs-cTn), type B Natriuretic Peptide (NT-proBNP), the binding protein fatty acids (H-FABP), lactate 
dehydrogenase (LDH) have been studied for their potential as biomarkers and used (hs-ctn and NT-proBNP) 
in the diagnosis and prognosis of dysfunction ventricular [7]. LV pathological remodeling is characterized at 
the molecular level by a alteration of the expression of different transcripts be they encoding (mRNA) or 
non-coding (ncRNA) [9, 10]. In particular, circulating RNA, present both in the plasma and within the cells 
blood, is highly stable and easily measurable even if present at very low concentrations. 

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In addition, the potential of circulating RNA for use as biomarkers has been known for some time 
cardiovascular [11-13]. 
Objective of the project 
We propose to carry out an observational, prospective study that involves the evaluation of predictive 
circulating biomarkers of ventricular dysfunction (FE <45%) in patients with ACS.In particular our aim will be 
to check if there are transcripts circulating in the blood peripheral, coding or not for proteins (mRNAs or 
ncRNAs), differently modulated in patientswith ACS (ACS_signature) submitted to PCI, distinguished on the 
basis of the evolution towards dysfunction ventricular. Our study will be based on transcriptomic analysis of 
the entire genome. 
Objectives 
Primary objective: 
Detect circulating transcripts encoding proteins (mRNAs) or not (ncRNAs) (ACS_signature) predictive 
ventricular dysfunction in patients with ACS undergoing PCI.  
Secondary objective:  
Evaluate the association of the ACS_signature with possible adverse events at 12 months and its prognostic 
ability in the prediction of adverse events additional to the use of standard clinical parameters. Adverse 
events are defined: death from cardiovascular causes, re infarction, non-fatal stroke, new coronary 
revascularization, arrhythmias, development of heart failure and new hospitalizations for cardiovascular 
causes. 
Study population 
378 patients with a first episode of ACS (STEMI, NSTEMI and UA) will be recruited to PCI. 
Criteria for Inclusion: 
1. Age >18 years  
2. ACS patients (first episode), defined according to ESC 2017 guidelines [14] 
3. Indication for treatment of percutaneous revascularization 
4. Informed consent to enrol in the study 
 
 
Exclusion Criteria 
1. Severe valvulopathy or other conditions requiring cardiac surgery 
2. Previous cardiac surgeries including coronary bypass 
3. Total chronic occlusion  
4. Patients with known hypersensitivity or contraindication to one of the following drugs:  
- heparin  
- aspirin,  

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- clopidogrel,  
- ticlopidine,  
- sirolimus, 
- everolimus.  
5. Any contraindication to medicated stent implantation (DES) 
6. Patients with previous documented myocardial infarction; 
7. FEVS <30% before PCI 
8. Patients in cardiogenic shock 
9. Patients with evolved STEMI (> 48 h from onset of symptoms/Q-waves to ECG) or undergoing to 
fibrinolysis 
10. Patients with known previous cardiomyopathy with FEVS < 40% 
11. Patients with malignant neoplasm or systemic disease with a "quoad vitam" prognosis less than 1 year 
12. Patients with known active infectious diseases; 
13. Patients who are unable to give valid informed consent at the time enlistment 
14. Pregnant women 
 
Study design 
This study, observational, prospective, monocentric will rely on the recruitment of patients consecutive 
with acute coronary syndrome (STEMI, NSTEMI) treated with angioplasty. I am 4 years and 6 months for 
recruitment and 12 months thereafter for completion of the follow-up, followed by a period of three 
months to complete the data analysis. Recruitment will be divided into two phases, functional to the 
subsequent molecular analysis: one of discovery and one of validation, partially overlapped temporally. 
Based on literature and experience data previous [5], we expect the number of patients who will develop 
ventricular dysfunction at 12 months from the PCI will be higher among the STEMI than the NSTEMI. A 
number of NSTEMI patients higher, so the number of patients who will develop ventricular dysfunction  
will be similar between these two groups. In addition, 100 patients diagnosed with AU, in which a low rate 
of evolution towards left ventricular dysfunction, will form a group of further control that will be tested 
after the validation phase, more relevant and homogeneous compared to healthy patients. 
 
 
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9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
1.1. RECLUTAMENTO - DISCOVERY 
1.2. FOLLOW UP - DISCOVERY
2.1. RECLUTAMENTO - VALIDAZIONE
2.2.FOLLOW UP - VALIDATION
3.1. EXP ACTIVITY PROFILING: POTENTIAL ACS_SIGNATURE
3.2. EXP ACTIVITY VALIDATION: VALIDATED ACS_SIGNATURE
3.3. CODE BROKEN, CORRELATIONS AND STAT ANALYSIS
MONTHS

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Assessments 
Pre-procedure 
Post-procedure 
(12-24 h) 
6 months 
12 months 
Medical History* 
x 
 
x 
x 
Recruitment criteria 
x 
 
 
 
Blood draw ACS_SIGNATURE 
x 
x 
x 
x 
Blood count* 
x 
x 
x 
x 
 Blood draw Tn-hs e CK-MB* 
x 
x 
 
 
ECG* 
x 
x 
x 
x 
Echocardiography * 
x 
x 
x 
x 
Angiography * 
x 
 
 
 
Endpoints  
 
 
x 
x 
*  routine clinical evaluations 
Patients will be enrolled after diagnosis of ACS and before coronary surgery and will be treated according to 
the guidelines provided for the treatment of acute coronary syndrome and for post-PCI. Unless special 
contraindications, all patients will receive optimal medical therapy that includes: 
- Dual therapy for 12 months 
- Statins  
- beta-blockers,  
- ACE inhibitors, 
- Mineralocorticoid receptor antagonists. 
Follow-up visits including echocardiographic examination will be performed at 6 and 12 months from 
myocardial revascularization procedure. The evaluation of the FEVS will be carried out by 3D 
echocardiography that, as has been documented, shows a proven concordance with the MRI [15]. This 
method has also shown high reproducibility, with low intra- and inter-operator variability, and increased 
diagnostic accuracy. During the echocardiographic examination, in addition to FEVS data, other function 
indices will be evaluated systolic left ventricle such as, for example, telesistolic volumes, telediastolic, 
parameters tissue Doppler derivatives and myocardial strain indices (global Longitudinal strain). 
The study will also need to take samples that will be made with the following scheme: 1) pre operative, 
before PCI, 2) post-operative 1 taken at 12 - 24h from PCI, 3) post-operative2 -6 months after PCI and 4) 
post-operative3-12 months after PCI. 
Pre-operative withdrawal will be performed in the recovery room of the hemodynamics room after 
diagnosis of ACS. The post-operative withdrawal.1 will be performed at the Department of Cardiology 
12/24 hours after the procedure. Post-operative.2 and post-operative.3 withdrawals will be performed 
outpatient during the planned echocardiographic checks (6 and 12 months respectively).  

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In pre-operative and post-operative samples.1 the complete blood count will be performed and Tn-hs, NT-
proBNP and LDH will be dosed RNA analysis will be divided into two phases: in the discovery phase, the 
analysis of the transcriptome by RNA sequencing. The aim is to identify a potentialACS_signature in blood 
mononucleate cells (PBMCs) of a subset of patients who eventually of the follow up of the discovery phase, 
show ventricular dysfunction (n= 40) or function conserved ventricular (n= 40). An equal number of STEMI 
and NSTEMI patients will be analysed. 
In the validation phase, the ACS_signature will be measured with independent technique (qPCR) in all 
patients enrolled in the study, to obtain a Validated ACS-Signature. 
 
Study procedures 
They will be taken at each time-point: 1) 3 ml of whole blood in a tube containing stabilizers RNA (type PAX-
gene or Tempus), 2) 4 ml in a tube containing EDTA from which the plasma that will be frozen at -80,5° C 
pending the extraction of RNA, and 3) 4 ml in a tube containing EDTA from which PBMCs will be isolated. 
The withdrawals in point 2 and 3 must be processed within two hours of the withdrawal, while the 
withdrawal in tube with RNA stabilizer can be stored at room temperature for up to 3 days, at +4 killed C 
for 7 days and at -20 killed C/-80 killed C indefinitely without altering the integrity of nucleic acids.Plasma 
and whole blood RNA will be extracted by silica columns, while RNA from PBMCs will be extracted by Trizol. 
The integrity of the extracted RNA will be evaluated by running on capillary gel type Bioanalyzer.  
On RNA extracted from PBMCs derived from pre-operative sampling in the discovery group, it will be 
analysis of the entire transcriptome by RNA sequencing (platform Illumina), in order to identify a potential 
ACS_signature consisting of transcripts differentially expressed in the two groups of patients: those with 
left ventricular dysfunction and those without ventricular dysfunction. 
In the validation phase, the potential ACS_signature will then be validated by qPCR in all STEMI and NSTEMI 
patients enrolled in the study, including those who will submit a degree intermediate ventricular 
dysfunction (LVEF 45-49%). They will also be analysed separately two subgroups of patients diagnosed with 
STEMI and NSTEMI. 
RNA that will pass validation (Validated ACS_Signature), will be further tested also in the AU control group 
and all other timepoints, to assess the time trend potential biomarkers. In addition, the expression of the 
Validated ACS-Signature will be evaluated in the plasma and whole blood, to assess biodistribution and 
translational potential. Patient recruitment, withdrawals, patient data collection and all clinical procedures 
requests will be made by the Clinical, Interventional and Intensive Care Cardiology Unit Coronary Surgery 
(UTIC) of the IRCCS Policlinico San Donato. The collection and conservation of blood and pre-analytic 
treatment will be carried out at the BioCor Biobank of the IRCCSPoliclinico San Donato, while RNA 
measurements will be carried out in the Laboratory of Molecular Cardiology of IRCCS Policlinico San 
Donato. The study of modulated transcripts by RNA Sequencing with library preparation, sequencing and 
analysis bioinformatics will be carried out at the Center for Translational Genomics and bioinformatics, of 
the San Raffaele Hospital. 
 
 
Sample size 
Based on literature [4, 5] it is expected that about 40% of STEMI patients and 30% of STEMI patients 
NSTEMI patients, after a first episode of myocardial infarction, experience dysfunction of the left ventricle. 

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Based on these data, we expect recruiting in 18 months 278 patients with ACS performing PCI, 100 will 
experience ventricular dysfunction (Table 1). 
Table 1.                 Patient recruitment and follow up 
Study phase 
Total number of 
patients 
Dysfunction LV 
expected 
  
STEMI 
NSTEMI 
STEMI 
NSTEMI 
DISCOVERY 
49 
75 
20 
22 
VALIDATION 
61 
93 
31 
28 
TOTAL STEMI + 
NSTEMI 
110 
168 
50 
50 
 
The number of patients to be recruited will be 378, of which 110 STEMI, 168 NSTEMI and 100 AU, as 
control. By preventing 100 events in total, respectively 50 per group, you can enterabout 6/7 variables for 
each model of multivariate logistic regression (STEMI NSTEMI).Experimental RNA measurement activities 
shall follow the distribution shown in Table 2. 
Table 2.                         Experimental activity 
 
DISCOVERY 
VALIDATION 
 
STEMI 
NSTEMI 
STEMI 
NSTEMI 
LV DYSFUNCTION 
20 
20 
50 
50 
NO LV 
DYSFUNCTION 
20 
20 
60 
118 
TOTAL 
40 
40 
110 
168 
 
 
 
For discovery activities using RNA-sequencing, based on literature data [16], we assume a sample size of 40 
patients (20 per group) to evaluate the 40 transcriptsthat differentiate patients with ventricular dysfunction 
and not ventricular dysfunction, out of a total of 17,500 tests, considering a power of 94.0 %, a value of FDR 
equal to 0.02. For the activities of validation using qPCR, 95% power is obtained by analyzing 50 samples 
per group evaluating differences between the averages of 2,2 times, with α= 0,01 and σ= 1,4. 

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Statistical methods 
The characteristics of patients will be presented with the average, median, standard deviation and range 
interquartile for continuous and frequency variables and % for nominal and categorical variables. The 
Shapiro-Wilks will be used to check if distributions are Gaussian. For distributions normal, statistical 
analysis will be carried out with parametric tests while for non-normal distributions a non-parametric 
statistical analysis will be carried out. Logistic regression models will be used to assess the association 
between gene characteristics and clinical outcome. In particular, the power discriminatory (c statistics) will 
be expressed in terms of area under the curve in a Receiver Operating Characteristics (ROC) analysis. 
Different cut-off levels will be explored for each of which sensitivity, specificity, negative predictive power 
and positive predictive power will be evaluated. The added prognostic value of ACS_signature in relation to 
variables will also be assessed traditional clinics including NTproBNP, Tn-hs and CK-MB using the prediction 
curve. 
 
Ethical aspects 
The study will be conducted according to GCP, local laws and rules of the World Medical Association 
Declaration of Helsinki. 
 
Publication policy 
The data collected are the property of IRCCS Policlinico San Donato and will be the subject of publication. 
 
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