STEM-AMI OUTCOME TRIALcentrostudi.anmco.it/csweb/uploads/4FULL... · 2014. 6. 5. · STEM-AMI OUTCOME Trial Study Protocol / Version 2.0, April 24, 2014 1 STUDY PROTOCOL STEM-AMI

STEM-AMI OUTCOME Trial Study Protocol / Version 2.0, April 24, 2014

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STUDY PROTOCOL

STEM-AMI OUTCOME TRIAL

STem cElls Mobilization in Acute Myocardial Infarction Outcome Trial

Protocol Number G112

A national, multicentre, randomised, controlled, open label, parallel group, Phase III study

EudraCT No. 2013-001772-38

Version 2.0, April 24, 2014

Sponsor

Manzoni Hospital Lecco, via Dell’Eremo 9/11, Lecco

Centro Cardiologico Monzino IRCCS, via Parea 4, Milano

Fondazione Per il Tuo Cuore / Heart Care Foundation ONLUS, Via A. La Marmora 36,Firenze

Coordinating Center

ANMCO Research Center

Via La Marmora 34 – 50121 Firenze


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CONTENTS

SIGNATURE PAGE FOR STUDY CHAIRMAN AND CO-CHAIRMAN ...................................... 5

SIGNATURE PAGE FOR THE PRINCIPAL INVESTIGATOR .................................................... 6

LIST OF ABBREVIATION ........................................................................................... 7

1. INTRODUCTION ................................................................................................... 8

1.1 BACKGROUND INFORMATION............................................................................... 8

1.2 EXPERIMENTAL EVIDENCE ................................................................................... 9

1.3 CLINICAL DATA .................................................................................................... 9

1.4 OUR EXPERIENCE AND STUDY RATIONALE ........................................................... 9

1.5 RISK-BENEFIT ASSESSMENT................................................................................ 11

2. STUDY OBJECTIVES ......................................................................................... 12

2.1 PRIMARY OBJECTIVE ......................................................................................... 12

3. OVERALL DESIGN AND PLAN OF THE STUDY ........................................ 12

3.1 OVERVIEW ......................................................................................................... 12

3.2 ASSESSMENT OF INVESTIGATIONAL PRODUCT .................................................... 12

3.2.1 Primary efficacy endpoint ......................................................................... 12

3.2.2 Secondary efficacy endpoints .................................................................... 13

3.2.3 Safety endpoints ........................................................................................ 13

4. STUDY POPULATION ....................................................................................... 13

4.1 INCLUSION CRITERIA .......................................................................................... 13

4.2 EXCLUSION CRITERIA ......................................................................................... 13

4.3 PLANNED SAMPLE SIZE ...................................................................................... 14

4.4 PATIENT IDENTIFICATION AND RANDOMIZATION ................................................ 14

4.5 PATIENT WITHDRAWAL ...................................................................................... 14

5. STUDY TREATMENT ........................................................................................ 15

5.1 INVESTIGATIONAL TREATMENT .......................................................................... 15

5.2 CONCOMITANT MEDICATIONS ........................................................................... 15

5.3 TEMPORARY TREATMENT DISCONTINUATION ..................................................... 15

5.4 DEFINITIVE TREATMENT DISCONTINUATION ....................................................... 15

5.5 HANDLING OF PATIENTS AFTER DEFINITIVE TREATMENT DISCONTINUATION ....... 16

5.6 EXPECTED SIDE EFFECTS .................................................................................... 16

5.7 PACKAGING AND LABELING ............................................................................... 16

5.8 THE STORAGE .................................................................................................... 16

6 PARAMETERS AND METHODS OF ASSESSMENT ................................... 16

6.1 EFFICACY PARAMETERS..................................................................................... 16


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6.1.1 All cause Death ......................................................................................... 16

6.1.2 Recurrent MI ............................................................................................. 16

6.1.3 Hospitalization due to heart failure .......................................................... 18

6.1.4 Cardiovascular death ................................................................................ 18

6.1.5 Coronary Revascularization ..................................................................... 18

6.1.6 Fatal and Non-fatal stroke ........................................................................ 18

6.1.7 Hospitalization due to any cause .............................................................. 18

6.1.8 Cardiovascular hospitalization ................................................................. 19

6.1.9 Resuscitation and/or appropriate AICD therapy ...................................... 19

6.2 SAFETY ENDPOINTS ............................................................................................ 19

6.2.1 Adverse Events (AEs) ................................................................................ 19

6.2.2 Serious Adverse Events (SAEs) ................................................................. 19

6.2.3 Reporting of AEs ....................................................................................... 20

6.2.4 Other events to be treated as serious adverse events ................................ 20

7 STUDY CONDUCT .............................................................................................. 20

7.1 SCHEDULE OF OBSERVATIONS ........................................................................... 20

7.2 OBSERVATIONS BY VISIT ................................................................................... 20

7.2.1 Screening and randomization (Assessment I; day -1/0)) .......................... 20

7.2.2 In-Hospital phase (Assessment II; day 0 to 7) .......................................... 21

7.2.3 1-month visit (Assessment III; day 30) ...................................................... 21

7.2.4 6-months visit (Assessment IV; day 180) .................................................. 21

7.2.5 12-months follow up (Assessment V; day 365) ......................................... 21

7.2.6 24-months follow up (Assessment VI; day 730) ........................................ 21

8 SAMPLE SIZE AND STATISTICAL METHODS ........................................... 23

8.1 GENERAL CONSIDERATIONS .............................................................................. 23

8.2 DETERMINATION OF SAMPLE SIZE ..................................................................... 23

8.3 RANDOMIZATION ............................................................................................... 23

8.4 EFFICACY ANALYSIS .......................................................................................... 23

8.5 SAFETY ANALYSIS ............................................................................................. 24

8.6 SUBGROUP ANALYSIS ......................................................................................... 24

9 ETHICAL, LEGAL AND ADMINISTRATIVE ASPECTS ............................ 24

9.1 CASE REPORT FORMS AND SOURCE DOCUMENTS .............................................. 24

9.2 ACCESS TO SOURCE DATA ................................................................................. 24

9.3 GOOD CLINICAL PRACTICE ................................................................................ 24

9.4 INFORMED CONSENT .......................................................................................... 25

9.5 PROTOCOL APPROVAL AND AMENDMENT .......................................................... 25


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9.6 DURATION OF THE STUDY .................................................................................. 25

9.7 PREMATURE TERMINATION OF THE STUDY ........................................................ 25

9.8 CONFIDENTIALITY .............................................................................................. 25

9.9 PUBLICATION POLICY AND AUTHORSHIP OF THE PAPERS/ABSTRACTS PRODUCED

BY THE STUDY .................................................................................................... 25

BIBLIOGRAPHY ......................................................................................................... 27

APPENDIX 1: STUDY COMMITTEES .................................................................... 30

STEERING COMMITTEE ................................................................................................. 30

EXECUTIVE COMMITTEE ............................................................................................... 30

DATA SAFETY AND MONITORING COMMITTEE (DSMC) .............................................. 30

EVENT VALIDATION COMMITTEE (EVC) ...................................................................... 30

SCIENTIFIC SECRETARIAT ............................................................................................. 30

APPENDIX 2. EVENT VALIDATION COMMITTEE CHARTER ...................... 31


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Signature page for Study Chairman and Co-Chairman

Protocol:



Approved by:

Chairman

Dr. Felice Achilli

____________________________

Signature

_________________________

Date

24.04.2014

_____________

Co-Chairman

Dr. Giulio Pompilio

____________________________

Signature

_________________________

Date

24.04.2014

_____________


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Signature page for the Principal Investigator

Protocol:



I have read this protocol and I agree to carry out this trial in compliance with all protocol conditions

and with the Declaration of Helsinki

Principal Investigator

____________________________

Signature

_________________________

Date

_____________


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LIST OF ABBREVIATION

STEMI ST Elevation Myocardial Infarction

PCI Percutaneous Coronary Intervention

EF Ejection Fraction

LV Left Ventricular

SCs Stem Cells

cEPCs circulating Endothelial Progenitor Cells

BMCs Bone Marrow Cells

EDV End Diastolic Volume

ESV End Systolic Volume

LAD Left Anterior Descending

SPECT Single-Photon Emission Computed Tomography

LGE Late Gadolinium enhancement

G-CSF Granulocyte Coloning Stimulating Factor

WBC White Blood Cells

HF Heart Failure

SAEs Serious Adverse Events

AEs Adverse Events

ADRs Adverse Drug Reactions

SSAR Suspected Seriuos Adverse Reaction

SUSAR Suspected Unespected Serious Adverse Reaction

AICD Automated Implanted Cardioverter Defibrillator

ATP Appropriate AICD Therapy

MRI Magnetic Resonance Imagining

TIMI Thrombolysis in Myocardial Infarction

CABG Coronary Artery By pass Graft

ACE Angiotensin Converting enzyme

MRA Mineralcorticoid Receptor Antagonist

HIV Human Immunodeficiency Virus

IP Investigational Product

SC Steering Committee

DSMC Data Safety and Monitoring Committee

EVC Event Validation Committee

eCRF Electronic Case Report Form

PE Primary End point

SE Secondary End point

ITT Intention To Treat


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1. INTRODUCTION

1.1 Background Information

Myocardial infarction (MI) can be defined from a number of different perspectives related to

clinical, electrocardiographic, biochemical and pathologic characteristics. In MI with ST segment

elevation (STEMI), occlusive and persistent thrombosis affecting coronary arteries prevails. It is

accepted that the term MI reflects death of cardiac myocytes caused by prolonged ischemia. Because

remaining cardiomyocytes are not able to replicate, the loss of cardiomyocytes after infarction is

irreversible and the tissue defect heals by fibrotic scarring after 8 weeks. Moreover, postinfarction

geometric disarrangement of the myocytes and extracellular matrix, dilation of ventricular cavity

and compensatory hypertrophy of the remaining myocardium, a process defined ventricular

remodeling, may contribute to development of ventricular dysfunction. STEMI continues to be a

significant public health problem in industrialized countries and is becoming an increasingly

significant problem in developing countries[1]. Although the exact incidence is difficult to

ascertain, 500.000 STEMI events per year are estimated in the U.S., from the National Registry of

MI-4 [NRMI-4]. In Italy 170.000 people each year are admitted to the hospital because of acute MI

[2].

Post infarction heart failure (HF) remains a major cause of morbidity and mortality[3]. Although

prompt reperfusion of the occluded artery has significantly reduced early mortality rates[4],

contemporary reperfusion strategies using percutaneous coronary intervention (PCI) were shown to

be associated with modest improvements in global left ventricular (LV) function as evidenced by a

2% to 4% increase in LVEF (EF) six months after an acute MI[5]. In the United States, more than

three million patients, and 700.000 in Italy, have cardiac failure and its most common cause is

ischemic heart disease. Approximately 200.000 people in the United States, and 40.000 people in

Italy, each year die of this condition. Moreover, cardiac failure is the most frequent cause of

hospitalization among patients older than 65 years[6, 7]. Heart failure due to extreme myocardial

loss can be treated by two methods only: heart transplantation, severely limited by donor organ

shortage, and cardiac assist device implantation, limited by high incidence of serious complications

during long term use. So in the future additional research of new strategies for the management of

STEMI patients in the community are needed.

The major goal to improve post infarction LV function would be the stimulation of

neovascularization and the enhancement of regeneration of cardiac myocytes within the infarcted

area. Recent experimental studies suggest that bone marrow-derived progenitor cells (BMCs) or

circulating endothelial progenitor cells (cEPCs) contribute to the regeneration of infarcted

myocardium, to enhance neovascularization of ischemic myocardium, to prevent cardiomyocyte

apoptosis, to alter scar formation by reducing the development of myocardial fibrosis and, thereby,

to improve cardiac function.

Intracoronary infusion[8, 9] or intramyocardial injection[10, 11] of BMCs or cEPCs have been

shown to be effective in patients with acute MI or after MI with no other treatment option. Although

the relevance of these data published in literature, the invasiveness of BMCs collection limits its

clinical application[8-10]. Mobilization of stem cells (SCs) with granulocyte-colony stimulating

factor (G-CSF) and stem-cell factor have been studied as alternative, less invasive approaches in

animals and humans. G-CSF is a hematopoietic cytokine produced by monocytes, fibroblasts and

endothelial cells. G-CSF is known to have multiple functions in normal, steady-state hematopoiesis

such as the regulation of neutrophil production and their release from the bone marrow, neutrophil

progenitor proliferation and differentiation, and the state of functional activation of neutrophils[12].

The use of recombinant human G-CSF is nowadays used as a standard therapeutic option for

treating hematological diseases. In fact, it is routinely used to mobilize CD34+ hematopoietic stem

cells from the BM into peripheral blood, thus enabling their easier collection compared to BM


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aspirate procedure. The use of CD34+ stem cells collected by using this procedure is approved by

the American Society for Clinical Oncology[13].

1.2 Experimental evidence

Several studies demonstrated the spontaneous mobilization of cEPCs from the bone marrow to the

peripheral blood after MI; this phenomenon seems to be correlated with the regeneration of the

damaged myocardium reducing the scar size and improving myocardial perfusion and systolic

function[14-16]. The Authors demonstrated, also, a correlation between SCs mobilization and the

level of endogenous granulocyte colony stimulating factor (G-CSF)[15]. This evidence supported

research development to study the effects and the safety of G-CSF injection in experimental models

of MI[17-21], demonstrating that G-CSF administration was safe and effective in preventing LV

remodelling and dysfunction after AMI.

The first study, published by Anversa in 2001[19], conducted on 15 mice-model of large anterior

MI, demonstrated G-CSF efficacy in determining structural and functional repair of the damaged

myocardium, traducing in a dramatic increase in survival in the treated group. Same results were

described by Minatoguchi et al., in 120 rabbit of ischemia/reperfusion model, confirming G-CSF

efficacy in reducing ventricular volumes and improving LV EF[21].

Harada et al. documented, also, a dose-time dependent G-CSF effect, with the best results in the

group treated early and with high dose of cytokine[20].

1.3 Clinical data

Inspired by the exciting experimental data about bone marrow stem cells (BMCs) effectiveness in

preventing LV remodelling after acute MI, several trials were designed to demonstrate BMCs ability

promoting cardiac repair in humans after acute MI. Different approaches have been proposed such

as intracoronary injection of BMCs[8-10, 19, 22, 23] but the use of G-CSF, as a potent inducer of

stem cells mobilization in the peripheral blood, was considered a valid non-invasive alternative[24-

32]. Despite large consensus about feasibility and safety of G-CSF administration in terms of

restenosis and major cardiac adverse events[33, 34], the results about effectiveness were

controversial. Ince H et al [24, 25], in the FIRSTLINE-AMI trial, were the first describing an

improvement in cardiac performance defined as a significantly increase in LVEF (from 48±4% to

54±8%) at 4 months of follow-up which was sustained at 1 year of follow-up, in patients treated

with G-CSF after acute MI subjected to primary PCI. Later, encouraging results were documented

also by Takano[30] at 6 months of follow-up in terms of improvement in LVEF, perfusion and

infarct size evaluated by SPECT.

In the RIGENERA study[32] the investigators enrolled 41 patients with large anterior acute MI after

5 days from a successfully performed revascularization. LV function, evaluated by echo, improved

in the group of patients treated with G-CSF, whereas no significant improvement was observed in

patients with conventional therapy. At the same time an unfavourable remodelling was observed in

the controlled group when the volumes analysis was performed.

Viceversa, four randomized trials did not demonstrated any adjunctive beneficial effect of cytokines

administration at follow-up. Zohlnhofer[31], in the REVIVAL-2 trial, and Ellis[27], in a pilot dose-

escalation randomized trial, were unable to demonstrate any influence of G-CSF therapy on infarct

size and LV function after acute MI. Same conclusions were described, also, by Ripa[29], in the

STEMMI trial, and more recently by Engelmann[28], in the G-CSF-STEMI trial, when G-CSF was

administrated in the sub-acute phase.

1.4 Our experience and study rationale

Many hypothesis have been suggested to explain these controversial results. First, heterogeneity in

infarct size and site sustained the enrolment of patients with already normal or near normal (>50%)

LVEF. Only two studies used a cut-off of ventricular function as inclusion criteria (LVEF <50% in


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the RIGENERA study, 20 < LVEF <40% by Ellis) but one of these did not impose any timing

criteria and the other one evaluated LVEF, measured by echo, only 30 days after MI. Second, short

reperfusion times included patients with re-opening vessel within 2 hours from symptom onset, in

the so called “golden hours” when revascularization plays an important role in the healing process

of the damaged myocardium and the evaluation of any other additional therapy becomes very

difficult to demonstrate. Only two studies imposed a timing criteria between symptom onset and

reperfusion: STEMI >4 hours by Ellis but with short follow-up and >6 hours by Engelmann but in

the setting of sub-acute MI. Third, different imaging tools were used for evaluating cardiac function

and ventricular remodelling (echo, SPECT and MRI). Only three studies used cardiac MRI, a

technique considered the gold standard in assessing ventricular size and shape. Fourth, G-CSF

timing administration was not standardized. Definition of the “susceptible phase” is uncertain.

Three studies administered G-CSF >37 hours after acute MI or PCI. Fifth, patients with a poor

prognosis (i.e. Killip class >2 and cardiogenic shock at presentation, evidence of multivessel disease

at coronary angiogram) were often excluded from studies. As Piepoli et al suggest in a recent paper,

patients with a poor prognosis have the greatest need for therapeutic approaches and thus have the

most favourable risk-benefit ratio. Demonstration of incremental benefit, as compared with

conventional therapy, could be easier in these population[35].

Recent meta-analyses, including 385 patients from 8 randomized study, failed to demonstrate G-

CSF efficacy in ameliorate LV function, but patient subgroups evaluation revealed a significant

improvement in LV function for patients with EF <50% at baseline and with G-CSF administration

within 37 hours from revascularization. So the authors concluded that”…G-CSF may potentially

benefit acute MI patients with impaired LV function and when therapy is started early...”[34].

On the base of these speculations, in 2005, we designed a randomized, single-blind, phase II study

(STEM-AMI trial) using G-CSF administration to mobilize BMCs in the “acute phase” of anterior

MI with significant LV dysfunction, and with short reperfusion times[36]. We imposed a cut-off of

ventricular function (LVEF ≤45%) evaluated by echo within 12 hours post PCI and we enrolled

patients with a definite symptom-to-ballon time (>2 hours). In our study the median LVEF

calculated at baseline, in the treatment arm, was 39 ± 7% and the median symptom-to-ballon time

was 4,9 hours. A second important element regards G-CSF timing of stimulation. Definition of the

“susceptible phase” is uncertain: in animal studies beneficial effects were described when G-CSF

was administered before or shortly after MI. In our study, and in the FIRSTLINE only, cytokine

administration occurs very early in time (less than 10 hours from PCI), so G-CSF effects and BMCs

mobilization begin early after reperfusion, in a “susceptible phase” (within 24 hours from ischemic

injured, as Harada et al sustained)[20] favourable for stem cells engraftment.

In STEM-AMI trial, sixty consecutive patients with anterior STEMI, undergoing primary PCI, with

symptom-to-reperfusion time of 2–12 h and EF ≤45% after PCI, were randomized to G-CSF 5

mg/kg b.i.d. subcutaneously (n° 30) or placebo (n° 30) for 5 days, starting, within 12 h after PCI.

The primary endpoint was an increase from baseline to 6 months of 5% in LVEF, as measured by

MRI. Co-primary endpoint was a ≥20 mL difference in end-diastolic volume (EDV). Infarct size

and perfusion were evaluated with late gadolinium enhancement (LGE) and gated 99mTechnetium

Sestamibi single-photon emission computed tomography (SPECT). Left ventricular EDV and end-

systolic volume (ESV) increased from baseline to 6 months in the placebo group (81.7±24.4 to

94.4±26.0 mL/m2, P=0.00005 and 45.2±20.0 to 53.2±23.8 mL/m2, P=0.016) but were unchanged in

the G-CSF group (82.2±20.3 to 85.7±23.7 mL/m2, P=0.40 and 46.0±18.2 to 48.4±20.8 mL/m2,

P=0.338). There were no significant differences in EF or perfusion between groups. A significant

reduction in transmural LGE segments was seen at 6 months in the G-CSF vs. placebo groups

(4.38±2.9 to 3.3±2.6, P=0.04 and 4.2±2.6 to 3.6±2.7, P=0.301, respectively). Significantly more

placebo patients had a change in left ventricular end-diastolic volume above the median (9.3

mL/m2) when reperfusion time exceeded 180 min (median time-to-reperfusion) (P=0.0123). Since

adverse remodelling is the main predictor of prognosis, the ability of G-CSF to attenuate


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unfavourable post-infarction remodelling is expected to improve in these patients event-free

survival.

A recent review, published by Sanganalmath SK et al. [37] suggest that “the final verdict on G-CSF

therapy will emerge not from meta-analyses, but from adequately powered randomized controlled

trials with optimized study parameters, i.e., dose, duration, timing, patient population, and outcome

parameters…”.

So, on the basis of our results, and supported by this speculation we propose a multicenter

randomized controlled phase III study conducted in a large cohort of patients.

1.5 Risk-benefit assessment

G-CSF is a hematopoietic cytokine produced by monocytes, fibroblasts and endothelial cells. G-

CSF is known to have multiple functions in normal, steady-state hematopoiesis such as the

regulation of neutrophil production and their release from the bone marrow, neutrophil progenitor

proliferation and differentiation, and the state of functional activation of neutrophils[12]. The use of

recombinant human G-CSF (filgrastim, lenograstim) is nowadays used as a standard therapeutic

option for treating hematological diseases. In fact, it is routinely used to mobilize CD34+

hematopoietic stem cells from the BM into peripheral blood, thus enabling their easier collection

compared to BM aspirate procedure. The use of CD34+ stem cells collected by using this procedure

use is approved by the American Society for Clinical Oncology[13]. The proven efficacy and safety

of G-CSF, both in healthy donors and patients with haematological disease, along with favourable

results from studies of CD34+ cell transplantation in patients with MI or ischemia, suggest that G-

CSF based BMC transplantation may have an efficacy in patients with MI.

Clinical data about BMCs mobilization in acute MI, published from 2005 to 2009, did not

documented any cardiac adverse events related to G-CSF treatment[24-32, 38].

A recent meta-analysis, published by Ince et al.[33], comprising 325 patients, of whom 177 were

subjected to G-CSF treatment, did not documented any significant differences in terms of in stent-

restenosis, thrombosis, re-infarction or death between treated and control group (Table 1).

Table 1 Cardiovascular events

In-stent re-stenosis

(n)

30-day stent-

thrombosis (n)

Re-infarction (n) Death

Study G-CSF Control G-CSF Control G-CSF Control G-CSF Control

MAGIC 7 0 0 0 0 0 0 0

Valgimigli 0 1 0 0 0 0 0 0

Ince 4 5 0 0 0 0 0 0

REVIVAL-2 19 17 0 0 0 1 1 0

STEMMI 3 4 1 0 0 0 0 1

Kuethe 1 n.a. 0 0 0 0 1 0

De Lezo 0 - 0 - 0 - 0 -

Ince H et al. Heart 2008

Preliminary experimental and clinical data show that stem cells mobilization during acute MI and

their ability to become localized in myocardium are able to improve myocardial perfusion and

function in patients treated with G-CSF. At the moment, effective alternative therapies in patients

with large anterior MI and reduced left ventricular EF aren’t available.


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2. STUDY OBJECTIVES

2.1 Primary Objective

To demonstrate that G-CSF therapy in addition to state of the art treatment (pharmacological and

non pharmacological) is safe and significantly improves clinical outcome in patients with reduced

left ventricular EF (≤ 45%) after successful reperfusion for anterior acute MI.

3. OVERALL DESIGN AND PLAN OF THE STUDY

3.1 Overview

This will be a Phase III, multicentre, randomized, controlled, open label study. A total of 1530

patients with anterior STEMI undergoing primary PCI or rescue, with TIMI Flow <2 at presentation

and symptoms-to-balloon time ≥2 h and ≤12 h or ≤24 h if symptoms persist suffering from lack of

contractile recovery (EF biplane ≤45%) after successful (TIMI flow ≥2) reperfusion therapy will be

randomized on a 1:1 basis to either the active treatment or control group (i.e. 765 patients per

group). All patients will receive best standard of care throughout the trial. Patients randomized to G-

CSF will receive 5 µg/kg subcutaneously bis in die (b.i.d.) of drug for 6 days (from Day 0 to Day 5),

starting within 24 h after PCI and reperfusion.

Patients will be required to attend 6 study assessments:

1. Assessment I screening and randomization (day -1/0)

2. Assessment II in-hospital phase (day 0-7)

3. Assessment III at 1 month after discharge (day 30 ± 1 week)

4. Assessment IV at 6 months after discharge (day 180 ± 2 weeks)

5. Assessment V at 12 months after discharge (day 365 ± 2 weeks)

6. Assessment VI at 24 months after discharge follow-up (day 730 ± 4 weeks)

At the screening phase eligibility criteria will be checked. Before randomization for each

randomized patient, the investigator will have to get an informed consent dated and signed. Each

randomized patient will be registered into the website with a unique number.

At Assessment I patient will be randomized, within 24 hours after PCI, to receive G-CSF as soon as

the results of the screening exams are available and confirm the patient’s eligibility.

White blood cells count (WBC) will be recorded twice a day, after every administration, during

study treatment (from day 0 to day 5) and at discharge.

Clinical evaluation, and SAEs/ADRs collection will be conducted during all in-hospital and follow-

up duration. Follow up visits are scheduled at 1, 6 and 24 months. At 12 months a phone interview

will be conducted.

These assessment will include death, recurrence of MI, hospitalization due to heart failure, non fatal

stroke, hospitalization due to any cause, cardiovascular hospitalization, development of events

related to the use of G-CSF. In case of an event, the appropriate eCRF pages will need to be

completed.

3.2 Assessment of investigational product

3.2.1 Primary efficacy endpoint

Clinical outcome will be assessed by the composite endpoint of:

- All cause death or,

- recurrence of MI or,

- hospitalization due to heart failure.

If more than one of these events occurs, the first event will define the primary efficacy endpoint

(more details in section 6).


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3.2.2 Secondary efficacy endpoints

- All cause death,

- recurrence of MI,

- hospitalization due to heart failure,

- cardiovascular death,

- coronary revascularization,

- fatal and non fatal stroke,

- hospitalization due to any cause,

- cardiovascular hospitalization,

- resuscitation and/or appropriate AICD therapy.


3.2.3 Safety endpoints

- Incidence and severity of bleeding complications,

- incidence of malignancy,

- incidence and intensity of SAEs and ADRs.


4. STUDY POPULATION

The study population will consist of patients with anterior STEMI undergoing primary PCI or

rescue with TIMI Flow <2 at presentation and with symptoms-to-balloon time ≥2 h and ≤12 h or

≤24 h if symptoms persist) , suffering from lack of contractile recovery after successful (TIMI Flow

≥2) reperfusion (EF Echo Simpson biplane ≤45%). Patients must be able to provide written consent

and meet all the inclusion criteria and none of the exclusion criteria. A total of 1530 patients who

meet the following inclusion/exclusion criteria will be randomized (765 patients each group) at

about 60 centers in Italy in order to achieved the required number of endpoints for this study. It is

anticipated that each centre will recruit about 20-25 patients within three years.

4.1 Inclusion criteria

To be considered eligible to participate in this study, the patient must have the following conditions:

- Patients affected by acute anterior STEMI undergoing primary PCI or PCI-rescue with TIMI

Flow <2

- Time symptom-to-balloon (≥2 h and ≤12h or ≤24 h if symptoms persist),

- TIMI flow post PCI ≥2,

- Evidence of left ventricular dysfunction (EF biplane 45%) ≤24 h after revascularization,

- Men and women aged ≥18 years and ≤80 years,

- Informed consent must be signed before proceeding with any study procedure.

4.2 Exclusion criteria

Patients will be excluded from the study if they have any of the following conditions:

- Previous anterior MI,

- Recent MI (within 1 month),

- Known previous LV dysfunction (EF <45%),

- Patients with angiographic evidence of coronary anatomy not suitable for PCI, or needing

coronary artery bypass grafting (CABG),

- Valve disease requiring surgical correction,

- History of previous cardiac surgery or PCI on LAD within 6 months,

- Previous, within the past 5 years, or current documented history of leukemia,

myeloproliferative or myelodisplastic disorder,

- Previous within the past 5 years or current documented history of malignant disease


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- Haemoglobin <10 mg/dl,

- White blood cells (WBC) >25.000 mm3,

- Platelet <50.000 mm3,

- Sepsis,

- Known HIV infection,

- Immune system diseases,

- Interstitial lung disease

- Serious concomitant medical conditions (other than ischemic heart disease),

- Pregnancy and breast feeding,

- Documented alcohol and drug abuse,

- Anticipated poor compliance.

- Current participation in a clinical trial with other investigational products

- Other cell therapy.

4.3 Planned sample size

It is planned to recruit 1530 patients for this study. See Section 8 for a discussion of sample size.

4.4 Patient identification and randomization

Among patients who fulfilled all inclusion criteria and have no exclusion criteria half of them will

be randomly allocated to receive G-CSF (1:1 ratio). After verification of eligibility of a patient and

after having obtained informed consent, he/she will be randomized using a WEB-based

randomization system. Patients will be considered randomized when there is confirmation of

successful allocation of a randomization treatment number through the study treatment allocation

system. Treatment will be assigned according to a pre-defined randomization list. A unique patient

number will be assigned in the context of the study. This patient number will identify the patient

throughout the study. At the randomization, the investigator will receive the allocation code for the

6 administration treatments. The assigned treatment group will be recorded in the eCRF.

In the case of a discrepancy between the treatment allocated by central lab and treatment

administered to patient, the patient should be switched as soon as possible to the treatment allocated

by central lab for further injection.

If an enrolled patient is discontinued from the study, his or her patient identifier code will not be

reallocated.

4.5 Patient withdrawal

The patients may withdraw from the study follow-up period, before study completion if they decide

to do so, at any time and irrespective of the reason. If a patient does not come in for scheduled visit,

the Investigator should take every effort to contact the patient to identify the reason why he/she has

failed to attend the visit, and to determine his/her health status particularly related to efficacy and

safety endpoints of the study. In case of written withdrawal of consent for follow-up visits, the

Investigator will be encouraged to get information from the general practitioner, any other physician

or other medical-care provider, in order to follow the medical status of the patients. In all cases, the

reasons for withdraw must be recorded in the eCRF. Patients who do not complete the study and for

whom no endpoint data are available will be considered as lost to follow up. Patients lost to follow

up and without a primary efficacy event will be censored at the date of their last assessment visit

(where data on the components of the primary endpoint is available). Their inclusion and treatment

number must not be reused.


15

5. STUDY TREATMENT

5.1 Investigational treatment

Five (5) µg/kg of G-CSF (Filgrastim) will be administered subcutaneously in the abdomen b.i.d for

6 days (from day 0 to day 5) to the patients randomized to active treatment, starting within 24 hours

after successful PCI and reperfusion.

The preparation used in this trial is Zarzio (Sandoz), prefilled syringes containing 48 MU filgrastim

in a 0.5 ml vial. Excipients are glutamic acid, sorbitolo and polysorbate.

5.2 Concomitant Medications

Any medication the patient takes other than study drug, including herbal and other non traditional

remedies, is considered a concomitant medication.

All patients should receive best standard of care throughout the trial, in respect to current guidelines

for the management of STEMI, independently from the randomization status. These include but are

not limited to:

- Betablockers should be given to all subjects, if there is no contra-indication, or documented

intolerance,

- ACE-inhibitors or angiotensin II-receptor blockers should be given to all patients, if there is

no contra-indication, or documented intolerance,

- Statins should be given to all patients, if there is no contraindication, or documented

intolerance,

- Aspirin at a dose of 100 mg/day should be given to all patients, or documented intolerance,

- Clopidogrel (75 mg/day) or Prasugrel (10 mg/day) or Ticagrelor (90 mg b.i.s. in die) should

be given to all patients for 12 months

- Mineral corticoid receptors antagonists (MRAs) are recommended for patients with EF

<40% and clinical signs of advanced heart failure,

- Diuretics are given according to clinical signs and/or symptoms of heart failure or to treat

hypertension,

- Glycoprotein IIb/IIIa inhibitors are to be given only according to clinical indication. If use is

required due to an emergency, platelet count has to be monitored carefully,

5.3 Temporary treatment discontinuation

The investigational product should be continued whenever possible. In case of G-CSF interruption,

it should be determined if the discontinuation can be made temporarily. Re-initiation of the

treatment with G-CSF will be done under close and appropriate clinical and/or laboratory

monitoring once the Investigator will have considered that the responsibility of G-CSF in the

occurrence of the concerned event was unlikely. All temporary treatment discontinuation reasons

and date of treatment re-initiation should be recorded by the Investigator in the appropriate eCRF

page when considered as confirmed.

5.4 Definitive treatment discontinuation

WBC count >50.000/mm3 is considered the cut-off for the definitive treatment discontinuation.

The patients may withdraw from treatment if they decide to do so, at any time and irrespective of

the reason, or this may be the Investigator’s decision.

In case of a AEs, assessed as possibly related to study drug, occurs during the treatment phase, the

Investigator should evaluate the benefit/risk for pursuing with further course of treatment. In this

case the Investigator is asked to contact the study team to discuss the case.

All efforts should be made to document the reason for discontinuation in the eCRF.


16

5.5 Handling of patients after definitive treatment discontinuation

Patients will be followed up in any cases according to the study procedures as specified in this

protocol up to the scheduled date of study completion, or up to recovery or stabilization of a

followed-up SAE or ADR, whichever comes first.

All definitive treatment discontinuation should be recorded and documented by the Investigator in

the appropriate eCRF pages when considered as confirmed.

Permanent treatment discontinuation should be a last resort.

In case of a permanent discontinuation of treatment, the patient should remain in the study as long

as possible and will be followed up to 24 month exploratory visit/phone contact planned.

5.6 Expected side effects

G-CSF is nowadays used as a standard therapeutic option for treating hematological diseases. In

fact, it is routinely used to mobilize hematopoietic stem cells from the BM into peripheral blood,

thus enabling their easier collection compared to BM aspirate procedure. The use of stem cells

collected by using this procedure is approved by the American Society for Clinical Oncology (15-16)

.

Known side effects of G-CSF, reported in the pharmaceutical brochure, are listed below:

Very common (more than 1 in 10 people): abnormal laboratory test results, back pain, blood

problems, bone pain, diarrhea, fever, hair loss, headaches, infections, inflammation of the

mouth, nausea, sepsis, vomiting, weakness.

Common (more than 1 in 100 people): enlarged spleen, injection site problems, pain,

stomach pain.

Rare (more than 1 in 10,000 people): lung problems.

Very rare: (fewer than 1 in 10,000 people): allergic reactions, anaphylactic shock, Lyell's

syndrome, skin problems, splenic rupture, Sweet's syndrome, vasculitis.

5.7 Packaging and labeling

Each participating center will be supplied with an adequate number of disposable prefilled syringes

containing Filgrastim 48 MU in 0.5 ml. Each syringe will be marked by batch and expiry date.

5.8 The storage

The IP will be shipped and must be stored in a secure refrigerated area (2-8 °C).

6 PARAMETERS AND METHODS OF ASSESSMENT

6.1 Efficacy Parameters

A precise definition of these clinical events is given as follows.

6.1.1 All cause Death

Death from any cause/all-cause mortality.

6.1.2 Recurrent MI

a. For patients in whom biomarkers have been documented not to be elevated for at least 12 hours

after the last episode of ischemia related to their index presentation or in whom biomarkers have

been documented to return to normal after the index event, criteria (1) AND (2) OR criteria (3)

OR criteria (4) must be met:


17

(1) Typical cardiac biomarker of necrosis rise & fall with the following degrees of elevation

accepted as biochemical evidence of myocardial necrosis: peak troponin (I or T) or CK-MB > ULN,

or peak total CK > 2 x ULN

AND

(2) At least 1 of the following additional supportive criteria:

a. Ischemic discomfort at rest lasting ≥20 minutes, or

b. ECG changes indicative of ischemia (ST elevation ≥0.1 mV or ST depression ≥0.05 mV, or new

T-wave inversions

OR

(3) Development of new, abnormal Q waves (≥30 msec in duration and ≥1 mm depth) in ≥2

continguous precordial leads or ≥2 adjacent limb leads; or increase R amplitude in V1-V3 consistent

with posterior infarction

OR

(4) Pathologic findings of an acute MI

b. For patients in whom biomarkers from the index event remain elevated at the time of onset of the

potential new MI, criteria (1) AND (2) OR criterion (3) must be met:

(1) Cardiac biomarker re-elevation defined as increase by ≥50% over the previous value; and

documentation that the biomarker assayed was decreasing prior to the suspected new MI

AND

(2) At least 1 of the following additional supportive criteria:

a. Ischemic discomfort or equivalent (e.g. acute, unexplained dyspnea, or nausea/vomiting, or

diaphoresis) at rest lasting ≥20 minutes, or

b. ECG changes indicative of ischemia (ST elevation ≥0.1 mV or ST depression ≥0.05 mV, or new

T-wave inversions

OR



with posterior infarction, felt to be distinct from the index event

c. Definition of recurrent MI within 48 hours after PCI (i.e. study drug application qualifies as PCI)

i. For patients in whom biomarkers are documented not to be elevated for at least 12 hours after the

last episode of ischemia (including, if necessary, values obtained in the first 12 hours immediately

post-PCI) or in whom biomarkers have been documented to return to normal after the last episode

of ischemia, criteria (1) OR (2) OR (3) OR must be met:

(1) CK-MB drawn (or total CK, if CK-MB is not available) drawn at least 4 hours post-PCI that is >

3 ULN

OR



with posterior infarction

OR

(3) Pathologic findings of an acute MI

ii. For patients in whom biomarkers from index event remain elevated immediately pre-PCI,

criteria (1) AND (2) AND (3) must be met:

(1) Documentation that CK-MB drawn (or total CK, if CK-MB is not available) was decreasing

(including values obtained in the first 12 hours immediately post-PCI)

AND

(2) CK-MB drawn (or total CK, if CK-MB is not available) drawn at least 4 hours post-PCI that is >

3 ULN

AND


18

(3) An increase in CK-MB (or total CK, if CK-MB is not available) by at least 50% over the

previous value.

6.1.3 Hospitalization due to heart failure

Patients rehospitalised due to increased heart failure must have either:

1. typical clinical findings (e.g. rales, 3rd heart sound, edema on chest x-ray)

OR

2. a change in medication for the treatment of heart failure as indicted by any of the following:

a. substantial increase in dose of oral medication

b. addition or new introduction of a new class of medication

c. addition of i.v. medication (diuretics, inotropic medication, vasodilator)

6.1.4 Cardiovascular death

Death due to either:

a. MI

b. heart failure

c. sudden death

d. documented arrhythmia

e. other cardiac related problems (e.g. pericarditis, cardiac tamponade, septal or free wall cardiac

rupture)

f. pulmonary embolism

g. stroke

h. other CV causes

6.1.5 Coronary Revascularization

Coronary revascularization is defined when a patient undergo percutaneous transluminal coronary

intervention — i.e, angioplasty, stenting, atherectomy, laser ablation — or coronary artery bypass

graft.

6.1.6 Stroke

Ischemic Stroke is defined as the presence of clinical evidence of cerebral, spinal cord or retinal

focal ischemic injury based on symptoms persisting ≥ 24 hours and other etiologies excluded.

Hemorrhagic Stroke is defined as rapidly developing clinical signs of neurological dysfunction

attributable to a focal collection of blood within the brain parenchyma or ventricular system that is

not caused by trauma.

Subarachnoid hemorrhage is defined as bleeding into the subarachnoid space. Subarachnoid

hemorrhage may not cause focal deficit.

Uncertain or unknown type of stroke is an episode of acute neurological dysfunction presumed

to be caused by ischemia or hemorrhage, persisting ≥ 24 hours without sufficient evidence to be

classified as one of the above

On the basis of clinical symptoms, autopsy and/or CT/MRI, strokes will be classified as:

a) definite or probable ischemic stroke,

b) definite hemorrhagic stroke, (for both, a & b, confirmed by CT, MRI scan),

c) subarachnoid hemorrhage, (a CT scan or cerebrospinal fluid report must be available

showing evidence of bleeding in the subarachnoid space),

d) uncertain or unknown type of stroke.

Hemorrhagic stroke does not include hemorrhage secondary to cerebral infarct, into a tumor, into a

vascular malformation or post-traumatic hemorrhage.

6.1.7 Hospitalization due to any cause

All causes of hospital inpatient admission.


19

Visits to an emergency department without admission will not be regarded as hospitalization.

6.1.8 Cardiovascular hospitalization

Hospitalization due to:

a) Acute coronary syndrome

b) Heart failure

c) Arrhythmia

d) Pericardial, myocardial and endocardial disease

e) Valvular disease

f) Aortic disease

g) CV procedures

6.1.9 Resuscitation and/or appropriate AICD therapy

Resuscitation is defined when all these events occur:

- Documented cardiac arrest

- Return of spontaneous circulation (ROSC) after basic and advanced life support

Appropriate AICD therapy, documented by device interrogation, is defined by device intervention

and interruption of ventricular arrhythmia (ATP/shock).

6.2 Safety endpoints

6.2.1 Adverse Events (AEs)

An AE is defined as any untoward medical occurrence in a patient or in a clinical trial subject

administered an investigational product and which does not necessarily have a causal relationship

with this treatment.

All AEs fall into either the category “non serious” OR “ serious”.

6.2.2 Serious Adverse Events (SAEs)

A serious adverse event is any untoward medical occurrence that at any dose:

- Results in death;

- Is life-threatening (“Life-threatening” means that the patient was at immediate risk of death

at the time of the SAE; it does not refer to a SAE that hypothetically might have caused

death if it were more severe);

- Requires inpatient hospitalization or prolongation of existing hospitalization (This means

that hospital inpatient admission or prolongation of hospital stay were required for the

treatment of the AE, or that they occurred as a consequence of the event. Visits to a hospital

by ambulance without admission will not be regarded as hospitalization unless the event

fulfils any other of the serious criteria);

- Results in persistent or significant disability or incapacity (“Persistent or significant

disability or incapacity” means a permanent or significant and substantial disruption of a

person's ability to carry out normal life functions);

- Is a congenital anomaly or birth defect;

- Is an important medical event.

Medical and scientific judgment should be exercised in deciding whether expedited reporting is

appropriate in situations where none of the outcomes listed above occurred. Important medical

events that may not be immediately life-threatening or result in death or hospitalization but may

jeopardise the patient or may require intervention to prevent one of the other outcomes listed in the

definition above should also usually be considered serious. Examples of such events include allergic

bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias, or

convulsions that do not result in inpatient hospitalization, or the development of drug dependency or

drug abuse. A diagnosis of cancer/ malignant tumor during the course of a treatment should always


20

be considered as medically important. This category also includes medical events requiring

intervention to prevent permanent impairment or damage.

6.2.3 Reporting of AEs

In this study each suspected clinical endpoints events constituting primary (PE) and secondary (SE)

efficacy endpoints, regardless of when the event occurs, are not to be considered as an adverse

event. All SAEs which coincide with primary and secondary efficacy end-points will be monitored

through the data collected in the CRF.

All other SAEs reported by participants at each follow-up visit will be recorded directly on the study

computer-based data entry system. Clinic staff are to record whether any SAE is thought likely to be

due to study treatment. Other adverse events not considered serious (as defined above) will only be

recorded if they are believed to be due to study treatment (ADRs).

As recommended by regulatory authorities, every SAE suspected by the Investigator to be related to

study medication (Suspected Serious Adverse Reaction – SSAR), both expected and unexpected

(SUSAR) must be reported to the study Coordinating Center within 24 hours of learning of its

occurrence which will transfer immediately the information to the drug’s manufacturer.

Abnormal laboratory parameters per se will not be recorded as adverse events. They will only be

reported as adverse events if the deviation from the normal range is assessed as “clinically

significant” by the investigator.

Documentation supporting the clinical endpoints will be forwarded to the Coordinating

Center (ANMCO Research Center) for adjudication by the Event Validation Committee. The

required documentation is outlined in the Event Validation Committee Manual (Appendix 2).

6.2.4 Other events to be treated as serious adverse events

Exposure to drug during pregnancy or lactation.

In principle, pregnancy and the lactation period are exclusion criteria for clinical studies involving

investigational drugs, which are not directly related to the respective conditions. In the event of a

pregnancy occurring during the course of this particular study, the subject should be closely

followed-up during the entire course of the pregnancy and postpartum period.

The Sponsor must be notified without delay. Parental and neonatal outcomes must be recorded even

if they are completely normal and without AEs. Off-spring should be followed up for at least 8

weeks after delivery.

7 STUDY CONDUCT

7.1 Schedule of Observations

The schedule of assessments is provided in Table 2.

7.2 Observations by Visit

7.2.1 Screening and randomization (Assessment I; day -1/0))

- Obtain written informed consent, record date and time of informed consent

- Documented acute anterior STEMI

- Documented primary PCI/PCI-rescue (time to symptoms onset)

- TIMI flow post-PCI registration

- Evidence of left ventricular dysfunction (Echo Simpson biplane EF ≤45%and EDV

and ESV)

- Hemochrome (hemoglobin, platelets, WBC)

- Pregnancy test (if necessary)


21

- Determine patient’s eligibility for enrollment

- Randomization to study treatment group

7.2.2 In-Hospital phase (Assessment II; day 0 to 7)

- G-CSF administration

- Hemochrome (WBC count) twice a day after every administration

Please note that after approximately 72 hours following the first

administration, there will be an expected increase in WBC count in the

treatment group due to G-CSF.

- Documentation of ADRs/SAEs

- ECG

- Assessment by echo of EF (Echo Simpson biplane), EDV and ESV

7.2.3 1-month visit (Assessment III; day 30)

- Clinical Evaluation


- ECG

7.2.4 6-months visit (Assessment IV; day 180)



- ECG


7.2.5 12-months follow up (Assessment V; day 365)

- Phone interview


7.2.6 24-months follow up (Assessment VI; day 730)



- ECG



22

Table 2: Schedule of assessments

Assessments I II III IV V VI

Screening/randomization In hospital Phase Follow-Up

Time points day -1/0 day 0 day 1 day 2 day 3 day 4 day 5 day 7 1

Month

6

Mont

hs

12

Months

24

Months

Informed consent X

In- exclusion criteria X

Clinical evaluation X X X X X

ECG X X X

Primary PCI X

EF, EDV and ESV assessment X X X X

Hemochrome (*) X XX XX XX XX XX XX X

Randomization X

G-CSF (*) XX XX XX XX XX XX

ADRs/SAEs X X X X X X X X X X X

(*) only if randomized to treatment


23

8 SAMPLE SIZE AND STATISTICAL METHODS

8.1 General Considerations

In statistical analyses no missing values will be replaced. All statistical analyses will be performed

by the Biostatistics Unit of Centro Cardiologico Monzino IRCCS. Statistical analyses will be

carried out using the SPSS statistical analysis system, version 17.0. For all data collected during the

trial and reported in the eCRF describing the sample, listings of the individual raw data as well as

tables of sample characteristics and/or frequencies will first be given. Continuous data will be

summarized first by treatment group and secondly by certain study time points using descriptive

statistics (mean, median, standard deviation [SD], minimum and maximum, number of available

observations and number of missing observations). It will be done for absolute and percent changes

and if appropriate from baseline screening too. Categorical data will be summarized first by

treatment group and secondly by certain study time points. The numbers and percentages of each of

categories, the number of available observations and the number of missing observations will be

presented in frequency tables.

The statistical analysis of study results will be performed according to the CPMP guidelines for

“Biostatistical methodology in clinical trials in applications for marketing authorizations for

medicinal products” and the ICH guideline “Statistical principles for clinical trials”

8.2 Determination of Sample Size

It is estimated that in this study the event rate at 2 years will be 15% in the control group. The aim

of this study is to show that the experimental treatment leads to a reduction of events. It is assumed

that a 25% reduction in events at 2 years is clinically meaningful. This corresponds to an event rate

of 11.25%. The sample size for the final analysis is based on event rates of 15% for control and

11,25% for the experimental treatment.

For the sample size calculation the following assumptions were made:

- exponential distribution

- power 80%

- α 0.05

- Two-sided test

- accrual time 3 years

- follow-up time 2 years.

During the planning phase of this study an estimate of the number of events needed in the control

group is 188. It is planned to randomize a total of 1530 patients (i.e. 765 per group).

8.3 Randomization

A computer-generated randomization list will be provided by the ANMCO Research Center.

8.4 Efficacy Analysis

All randomized patients will be included in the ITT analysis

Normal distribution will tested by Skewness-Kurtosis test. Between group comparison for

continuous variables will performed by t test or the corresponding nonparametric alternative (Mann-

Whitney test), as appropriate. Discrete variables will be compared by chi-square or Fischer exact

test when appropriate. We will apply repeated measures analysis of variance (ANOVA) with

Newman-Keuls post-hoc analysis, or Wilcoxon test when appropriate, to study the relation between

randomly allocated treatment and changes in continuous data at baseline and follow-up time point.

Further, a two-way multivariate analysis of covariance (MANCOVA) will performed to assess

differences between the 2 groups at 6-months follow-up adjusted for baseline variables. The level of

statistical significance will be set at p<0.05.


24

8.5 Safety Analysis

All randomized patients who received at least one administration of the active treatment will be

included in the safety analysis. Adverse drug reactions (ADRs) and serious adverse events (SAEs)

will be listed summed by occurrence, severity, outcome, and causal relationship to treatment and

will be descriptively compared between the two treatment groups using Fisher’s exact test.

8.6 Subgroup analysis

The effects of the study drugs will be evaluated in the following predefined subgroups of patients:

Gender

Age (above/below the median value)

Age groups

<50 yrs

50-59 yrs

60-69 yrs

70-75 yrs

Time symptoms to balloon (within 3-6 hours vs 12 hours vs 24 hours)

TIMI flow post PCI (II vs III)

Multivessl disease (yes vs no)

Previous MI (yes vs no)

Diabetes (yes vs no)

Renal insufficiency (yes vs no)

EF at randomization (above/below the median value)

The end-point for all the subgroup analyses is the same primary end point of the study.

9 ETHICAL, LEGAL AND ADMINISTRATIVE ASPECTS

9.1 Case Report Forms and Source Documents

All relevant data collected during the study for all the patients enrolled in the study will be recorded

in eCRF. The data will be entered by the responsible Investigator or someone authorized by him

(Data Manager or Co-Investigator) in a timely manner.

The physician will confirm the completeness, correctness and plausibility of the data by his

signature. All source documents from which eCRF entries are derived should be placed in the

patient’s medical records. The original data in the eCRF will be checked against source documents

at the study site continuously by the data manager. Additions and corrections will be dated and

signed by the responsible physician or data manager.

9.2 Access to Source Data

The review of medical records will be performed in a manner to ensure that patient confidentiality is

maintained.

9.3 Good Clinical Practice

The procedures set out in this protocol are designed to ensure that the Principal Investigator abide

by the principles of the Good Clinical Practices guidelines of the ICH, and of the Declaration of

Helsinki (1996). This study also will be carried out in keeping with local legal requirements.


25

9.4 Informed Consent

The Investigator or a person designed by the Investigator, and under the Investigator’s

responsibility, should fully inform the Patient of all pertinent aspects of the Clinical Trial. Patients

will be allowed adequate time for consideration and making an informed decision. All partecipants

should be informed to the fullest extent possible about the study, in language and terms they are able

to understand. In patient willing to participate in the trial, informed consent will be obtained from

the patient (or his/her legally authorized representative) according to the regulatory and legal

requirements of Italy. This consent form must be dated and retained by the Investigator as part of the

study records. The Investigator will not undertake any investigation specifically required only for

the clinical study until valid consent has been obtained. The date and time when consent was

obtained will also be documented in the eCRF.

A copy of the signed and dated written Informed Consent Form will be provided to the patient.

9.5 Protocol Approval and Amendment

Before the start of the study, the study protocol and/or other relevant documents will be approved by

the IEC and Competent Authorities. This protocol is to be followed exactly. To alter the protocol,

amendments must be written, receive approval from the appropriate personnel, and receive IEC and

Competent Authority approval prior to implementation (if appropriate).

Administrative changes (not affecting the patient benefit/risk ratio) may be made without the need

for a formal amendment.

9.6 Duration of the study

The maximum duration of the study for each patient will be approximately 24 months. The study

will close when all patients have completed the 24-Months Follow-Up Assessment (Assessment

VI).

9.7 Premature Termination of the Study

If the Investigator or the Competent Authorities becomes aware of conditions or events that suggest

a possible hazard to subjects if the study continues, the study may be terminated after appropriate

consultation between the relevant parties.

Conditions that may warrant termination include, but are not limited to:

- The discovery of an unexpected, significant, or unacceptable risk to the patients enrolled in

the study,

- Failure to enrol patients at an acceptable rate.

9.8 Confidentiality

All study findings and documents will be regarded as confidential.

The anonymity of participating patients must be maintained. Patients will be identified on eCRF

submitted to a Data Base by their patient number, not by name. Documents not to be submitted to

the Data Base that identify the patient (e.g., the signed informed consent) must be maintained in

confidence by the Investigator.

9.9 Publication policy and authorship of the papers/abstracts produced by the study

Any publication of data collected as a result of this study will be considered a joint publication by

the investigators, Steering Committee members and personnel of the Scientific Secretariat. After the

publication of the main paper, the database will be available for further analyses to all participating

Investigators and Committee members. Requests for further analyses to support ancillary

publications must be submitted to the Steering Committee for review and approval. The Steering


26

Committee must receive a copy of any presentation, manuscript, or abstract prior to dissemination

according to the terms outlined in the protocol.

Authorship of the main paper and of the ancillary analyses will be determined according to the

current medical community rules. According to the Uniform Requirements for Manuscripts

Submitted to Biomedical Journals, designation as an author must satisfy at least two conditions. The

author must have (a) contributed substantially to the conception and design of the study, the

acquisition of data, or their analysis and interpretation and (b) drafted or provided a critical revision

of the article.

http://en.wikipedia.org/wiki/Uniform_Requirements_for_Manuscripts_Submitted_to_Biomedical_Journals

http://en.wikipedia.org/wiki/Uniform_Requirements_for_Manuscripts_Submitted_to_Biomedical_Journals


27

BIBLIOGRAPHY

1. O'Gara PT, Kushner FG, Ascheim DD et al. 2013 ACCF/AHA Guideline for the Management

of ST-Elevation Myocardial Infarction: A Report of the American College of Cardiology

Foundation/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol

2012.

2. Perugini E, Maggioni AP, Boccanelli A, Di Pasqauel G. Epidemiology of acute coronary

syndromes in Italy. G Ital Cardiol (Rome) 2010; 11: 718-729.

3. Krum H, Gilbert RE. Demographics and concomitant disorders in heart failure. Lancet 2003;

362: 147-158.

4. Lange RA, Hillis LD. Reperfusion therapy in acute myocardial infarction. N Engl J Med 2002;

346: 954-955.

5. Stone GW, Grines GL, Cox DA, et al. Comparison of angioplasty with stenting, with or without

abciximab, in acute myocardial infarction. N Engl J Med 2002; 346: 957-966.

6. McMurray JJ, Adamopoulos S, Anker SD, et al. ESC Guidelines for the diagnosis and treatment

of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of

Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in

collaboration with the Heart Failure Association (HFA) of the ESC. Eur Heart J 2012; 33: 1787-

1847.

7. Hunt SA, Abraham WT, Chin MH, et al. 2009 Focused update incorporated into the ACC/AHA

2005 Guidelines for the Diagnosis and Management of Heart Failure in Adults A Report of the

American College of Cardiology Foundation/American Heart Association Task Force on

Practice Guidelines Developed in Collaboration With the International Society for Heart and

Lung Transplantation. J Am Coll Cardiol 2009;53:e1-e90.

8. Assmus B, Schächinger V, Teupe C, et al. Transplantation of Progenitor Cells and Regeneration

Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 2002; 106: 3009-

3017.

9. Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous

intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002; 106:

1913-1918.

10. Tse HF, Kwong YL, Chan JK, Lo G, Ho CL, Lau CP. Angiogenesis in ischaemic myocardium

by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet 2003; 361:

47-49.

11. Perin EC, Dohmann HF, Borojevic R, et al. Transendocardial, autologous bone marrow cell

transplantation for severe, chronic ischemic heart failure. Circulation 2003; 107: 2294-2302.

12. Anderlini P, Przepiorka D, Champlin R, Körbling M. Biologic and clinical effects of

granulocyte colony-stimulating factor in normal individuals. Blood 1996; 88: 2819-2825.

13. American Society of Clinical Oncology. Recommendations for the use of hematopoietic colony-

stimulating factors: evidence-based, clinical practice guidelines. J Clin Oncol 1994; 12: 2471-

2508.

14. Massa M, Rosti V, Ferrario M, et al. Increased circulating hematopoietic and endothelial

progenitor cells in the early phase of acute myocardial infarction. Blood 2005; 105: 199-206.

15. Leone AM, Rutella S, Bonanno G, et al. Endogenous G-CSF and CD34+ cell mobilization after

acute myocardial infarction. Int J Cardiol 2006; 111: 202-208.

16. Leone AM, Rutella S, Bonanno G, et al. Mobilization of bone marrow-derived stem cells after

myocardial infarction and left ventricular function. Eur Heart J 2005; 26: 1196-1204.

17. Ince H, Nienaber CA. Future investigations in stem cell activation with granulocyte-colony-

stimulating factor after myocardial infarction. Nat Clin Pract Cardiovasc Med 2007; 4 (Suppl 1):

S119-22.

18. Lee SS, Naqvi TZ, Forrester J, et al. The effect of granulocyte colony stimulating factor on

regional and global myocardial function in the porcine infarct model. Int J Cardiol 2007; 116:

225-230.


28

19. Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart,

improving function and survival. Proc Natl Acad Sci U S A 2001; 98: 10344-10349.

20. Harada M, Qin Y, Takano H, et al. G-CSF prevents cardiac remodeling after myocardial

infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med 2005; 11: 305-311.

21. Minatoguchi S, Takemura G, Chen XH, et al. Acceleration of the healing process and

myocardial regeneration may be important as a mechanism of improvement of cardiac function

and remodeling by postinfarction granulocyte colony-stimulating factor treatment. Circulation

2004; 109: 2572-2580.

22. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for

angiogenesis. Science 1997; 275: 964-967.

23. Schächinger V, Erbs S, Elsässer A, et al. Improved clinical outcome after intracoronary

administration of bone-marrow-derived progenitor cells in acute myocardial infarction: final 1-

year results of the REPAIR-AMI trial. Eur Heart J 2006; 27: 2775-2783.

24. Ince H, Petzsch M, Kleine HD, et al. Preservation from left ventricular remodeling by front-

integrated revascularization and stem cell liberation in evolving acute myocardial infarction by

use of granulocyte-colony-stimulating factor (FIRSTLINE-AMI). Circulation 2005; 112: 3097-

3106.

25. Ince H, Petzsch M, Kleine HD, et al. Prevention of left ventricular remodeling with granulocyte

colony-stimulating factor after acute myocardial infarction: final 1-year results of the Front-

Integrated Revascularization and Stem Cell Liberation in Evolving Acute Myocardial Infarction

by Granulocyte Colony-Stimulating Factor (FIRSTLINE-AMI) Trial. Circulation 2005; 112 (9

Suppl): I73-80.

26. Valgimigli M, Rigolin GM, Cittanti C, et al. Use of granulocyte-colony stimulating factor

during acute myocardial infarction to enhance bone marrow stem cell mobilization in humans:

clinical and angiographic safety profile. Eur Heart J 2005; 26: 1838-1845.

27. Ellis SG, Penn MS, Bolwell B, et al. Granulocyte colony stimulating factor in patients with large

acute myocardial infarction: results of a pilot dose-escalation randomized trial. Am Heart J

2006; 152: 1051.e9-14.

28. Engelmann MG, Theiss HD, Hennig-Theiss C, et al. Autologous bone marrow stem cell

mobilization induced by granulocyte colony-stimulating factor after subacute ST-segment

elevation myocardial infarction undergoing late revascularization: final results from the G-CSF-

STEMI (Granulocyte Colony-Stimulating Factor ST-Segment Elevation MI) trial. J Am Coll

Cardiol 2006; 48: 1712-1721.

29. Ripa RS, Jørgensen E, Wang Y, et al. Stem cell mobilization induced by subcutaneous

granulocyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation

myocardial infarction: result of the double-blind, randomized, placebo-controlled stem cells in

myocardial infarction (STEMMI) trial. Circulation 2006; 113: 1983-1992.

30. Takano H, Hasegawa H, Kuwabara Y, et al. Feasibility and safety of granulocyte colony-

stimulating factor treatment in patients with acute myocardial infarction. Int J Cardiol 2007;

122: 41-47.

31. Zohlnhöfer D, Ott I, Mehilli J, et al. Stem cell mobilization by granulocyte colony-stimulating

factor in patients with acute myocardial infarction: a randomized controlled trial. JAMA 2006;

295: 1003-1010.

32. Leone AM, Galiuto L, Garramone B, et al. Usefulness of granulocyte colony-stimulating factor

in patients with a large anterior wall acute myocardial infarction to prevent left ventricular

remodeling (the rigenera study). Am J Cardiol 2007; 100: 397-403.

33. Ince H, Valgimigli M, Petzsch M, et al. Cardiovascular events and re-stenosis following

administration of G-CSF in acute myocardial infarction: systematic review and meta-analysis.

Heart 2008; 94: 610-616.

34. Abdel-Latif A, Bolli R, Zuba-Surma EK, Tleyjeh IM, Hornung CA, Dawn B. Granulocyte

colony-stimulating factor therapy for cardiac repair after acute myocardial infarction: a


29

systematic review and meta-analysis of randomized controlled trials. Am Heart J 2008; 156:

216-226.e9.

35. Piepoli MF, Vallisa D, Arbasi M, et al. Bone marrow cell transplantation improves cardiac,

autonomic, and functional indexes in acute anterior myocardial infarction patients (Cardiac

Study). Eur J Heart Fail 2010; 12: 172-180.

36. Achilli F, Malafronte C, Lenatti L, et al. Granulocyte colony-stimulating factor attenuates left

ventricular remodelling after acute anterior STEMI: results of the single-blind, randomized,

placebo-controlled multicentre STem cEll Mobilization in Acute MI (STEM-AMI) Trial. Eur J

Heart Fail 2010; 12: 1111-1121.

37. Sanganalmath SK, Abdel-Latif A, Bolli R, Xuan YT, Dawn B. Hematopoietic cytokines for

cardiac repair: mobilization of bone marrow cells and beyond. Basic Res Cardiol 2011; 106:

709-733.

38. Engelmann MG, Theiss HD, Theiss C, et al. G-CSF in patients suffering from late

revascularised ST elevation myocardial infarction: final 1-year-results of the G-CSF-STEMI

Trial. Int J Cardiol 2010; 144: 399-404.


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APPENDIX 1: STUDY COMMITTEES

Steering Committee

The Steering Committee is responsible for:

Agreement of the final Protocol

Agreeing the Data Analysis Plans

Reviewing progress of the study and, if necessary, agreeing changes to the Protocol

Reviewing new studies that may be of relevance

Review and approval of study reports and sub-study proposals

Members of the Steering Committee: Felice Achilli (Chair), Francesco Bovenzi, Maurizio

Capogrossi, Francesco Chiarella, Gualtiero Colombo, Stefano De Servi, Francesco Gentile,

Maddalena Lettino, Stefano Maggiolini, Cristina Malafronte, Attilio Maseri, Fabrizio Oliva, Luigi

Oltrona Visconti, Giulio Pompilio (Co-Chair), Serena Rakar

Executive Committee

Members of the Executive Committee: Felice Achilli (Chair), Francesco Chiarella, Francesco

Gentile, Giulio Pompilio (Co-Chair), Stefano Maggiolini, Cristina Malafronte, Luigi Oltrona

Visconti

Data Safety and Monitoring Committee (DSMC)

The roles and responsibilities of the Data and Safety Monitoring Committe will be defined by the

same DSMC with special focus on intensive monitoring of the safety aspects in the whole study

population. No specific interim analysis is foreseen for efficacy.

Members of the DSMB: Luigi Tavazzi, Tiziano Barbui, Claudio Rapezzi, Renato Urso, Filippo Crea

Event Validation Committee (EVC)

The Event Validation Committee members will not have direct contact with patients randomized

into this study.

The main roles and responsibilities of the Event Validation Committee are:

- To agree on definitions of the clinical endpoints and on standard procedures for assessing these

endpoints;

- To validate blindly the events recorded and reported by the Investigators as primary end-points

of the study.

The decisions of the Event Validation Committee will be used for health authority submissions and

publications. An Event Validation Committee manual will be pre-defined, by the members of the

Committee. The main responsibility of the Event Validation Committee is to review and validated

reported clinical events included in the primary end point in a blind manner. The Event Validation

Committee will work in accordance with the procedures as defined in the ad hoc manual

independently from the Steering Committee (SC) and the Data Safety and Monitoring Committee

(DSMC). Members of the EVC: Gianna Fabbri (coordinator), Francesco Orso, Marco Magnoni,

Aldo Maggioni

Scientific secretariat

The main role of the Scientific Secretariat located at the ANMCO Research center is to

operationally coordinate the project, provide support to the Committees and participating centres

and guard the methodological concepts of the study.

Specifically, the Scientific Secretariat has to assure the constant quality control and continuity,

necessary to ensure that projects are completed in an appropriate methodological way, on time and

within budget.


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APPENDIX 2. EVENT VALIDATION COMMITTEE CHARTER

INTRODUCTION

Multicentre, randomized, open label, blinded end point study. The purpose of the study is to

demonstrate that G-CSF therapy in addition to state of the art treatment (pharmacological and non

pharmacological) is safe and significantly improves clinical outcome in patients with reduced left

ventricular EF (≤45%) after successful reperfusion for large anterior acute MI when compared to a

placebo (saline) group.

Primary end point:

- All cause death or,

- recurrence of MI or,

- hospitalization due to heart failure

Secondary endpoints

- All cause Death,

- Recurrence of MI,

- Hospitalization due to heart failure,

- Coronary revascularization,

- Cardiovascular death,

- Fatal and non fatal stroke,

- Hospitalization due to any cause,

- Cardiovascular hospitalization,

- Resuscitation and/or appropriate AICD therapy.

ROLE AND RESPONSABILITIES

The main responsibility of the Event Validation Committee (EVC) is to review and validated

reported clinical events included in the primary and secondary end points in a blind manner.

The Event Validation Committee will work in accordance with the procedures as defined in this

manual independently from the Steering Committee (SC) and the Data and Safety Monitoring

Committee (DSMC).

The Committee will receive documentations of the events by the Coordinating Center and will meet

on a regular basis. During its first meeting the Events Validation Committee will discuss the

definitions of the events and approved this charter.

The objective for the committee is to utilize a consistent and unbiased classification system. For

each event the EVC will adjudicate whether they meet the criteria of an outcome event as defined in

this manual. The EVC will be blind regarding any information relating to randomization group.

To perform their activities the EVC members will review the outcome events forms completed by

the investigative site together with the required documentation.

The EVC will not responsible for any safety assessment of the study. The responsibility for safety

assessment will remain with the DSMC.

OUTCOME EVENT DEFINITIONS AND DOCUMENTATION

All cause death

Death will be classified as cardiovascular, non cardiovascular or unknown. Only deaths due to a

clearly documented non cardiovascular cause such as cancer, trauma, infection, respiratory failure

are non cardiovascular. Multi organ failure as a result of cardiovascular disease should be

adjudicated as a cardiovascular death.


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Cardiovascular death

Cardiovascular death includes sudden cardiac death, unwitnessed unexpected death, fatal MI, death

from heart failure, death related to invasive diagnostic or therapeutic procedures, death from stroke,

death from non cerebral embolism, arrhythmic death, death due to presumed cardiovascular cause

and death due to other cardiovascular cause.

Sudden cardiac death

Death that occurs suddenly, unexpectedly and includes the following:

Death witnessed within 60 minutes of the onset of new or worsening symptoms,

Identified arrhythmia captured on an ECG recording or witnessed on a monitor by a health

professional (lasting less than 60 min),

Cardiac arrest or successfully resuscitated cardiac arrest with death within 24 hours without

identification of non cardiac etiology,

Witnessed and instantaneous death without new or worsening symptoms.

The diagnosis of sudden death excludes subjects with a known diagnosis such as myocardial

infarction, pulmonary edema, stroke.

Unwitnessed unexpected death

Death without other cause that occurs unexpectedly and is unwitnessed. This includes patients who

are found dead where there is no other explanation.

Death from stroke

Death occurring within 30 days from a stroke without any evidence of other cause contributing to

the event (for definition see page 7 “Non fatal stroke”).

Death from non cerebral embolism

Death occurring within 7 days from a systemic arterial embolism (for definition see page 8

“Systemic arterial embolism”).

Fatal MI

Death occurring within 7 days after a documented/validated myocardial infarction (for definition see

pages 6-7 “Myocardial Infarction”) in which there is no evidence of an another cause of death.

Autopsy evidence of a recent infarct without other evidence of another cause of death.

A fatal myocardial infarction may be adjudicated for a death that has suggestive criteria for

infarct but does not meet the strict definition of MI. Suggestive criteria are:

- ECG changes indicative of acute injury,

- Abnormal markers without evolution (patient died before a subsequent lab draw),

- Other evidence of wall motion abnormality.

Death from Heart Failure/Cardiogenic shock

Death that occurs within the context of clinical symptoms/signs of heart failure (even if the terminal

event was an arrhythmia) (for definition see page 6 “Heart failure” and “Cardiogenic shock”).

Cardiogenic shock without another cause of shock (sepsis, anaphylaxis) is included.

If heart failure is secondary to MI, then MI should be listed as the primary cause of death given the

patient suffered a MI in the 7 days of death (see above).

Death related to invasive procedures

Cardiovascular death occurring within 30 days of any cardiovascular or non cardiovascular surgery

or within 7 days of angiography evaluation, angioplasty, atherectomy, stent placement, ablation or

other invasive vascular intervention, without any evidence of other cause contributing to the event.


33

Arrhythmic death

Death due to documented arrhythmia (complete A-V block, ventricular tachyarrhythmia) not

occurring suddenly or unexpectedly without any evidence of other cause contributing to the event.

An example of death from arrhythmia is a subject who is admitted with tachyarrhythmias and dies 5

hours later.

Death due to presumed cardiovascular cause

Death not attributed to the categories of cardiovascular death and not attributed to a non

cardiovascular cause.

Death from other cardiovascular cause

Refers to death due to pericardial tamponade, valve thrombosis or other causes not mentioned

above.

NON CARDIOVASCULAR DEATH

Non cardiovascular death is defined as any death clearly due to a non cardiovascular cause and

categorized as follows.

Malignancy

A documented malignant neoplasia in the absence of a new evidence of deterioration of the cardiac

disease (absence of new ischemic events, worsening HF, cerebrovascular disease etc).

Infective (including sepsis)

All causes of death from infection that cannot be classified as cardiovascular will be included in

this category.

Accidental/Trauma

Cases of death from suicide, violence or accident will be classified as a separate category with the

option of considering the three categories separately.

Other non cardiovascular cause

Other serious non cardiovascular disease in the absence of a new evidence of deterioration of the

cardiac disease (absence of new ischemic events, worsening HF, cerebrovascular disease etc).

Unknown cause of death. Not determinable cause of death. Lack of specific documentation despite

any effort to obtain it.

HOSPITALIZATIONS

Hospitalization means unplanned or elective admission to a hospital for at least an overnight stay or

resulting in death.

All hospitalizations will be classified as cardiovascular or non cardiovascular; prolongation of a non

cardiovascular hospitalization for a cardiovascular cause may be considered as cardiovascular

hospitalization if there is a clear documentation that the cardiovascular diagnosis prolonged the

hospitalization for at least two consecutive dates (overnight). Day care admission for the

management of heart failure will be considered as a hospitalization. For scheduled multiple day care

admissions a single hospitalization form will be filled reporting the number of days of admission.


34

CAUSES OF CARDIOVASCULAR HOSPITALIZATION

Heart failure

Patients presenting with at least two of the following signs or symptoms and requiring intravenous

medications (diuretics or vasodilators or inotropes) or initiation or increase in dosage (if previously

prescribed for another cause, i.e. hypertension) of loop diuretic, ACE-inhibitor/ARB therapy, or

evidence-based beta-blocker therapy.

increased dyspnea on exertion,

orthopnea,

nocturnal dyspnea,

increasing peripheral edema,

pulmonary edema,

increasing fatigue/decreasing exercise tolerance,

renal hypoperfusion (worsening renal function),

elevated jugular venous pressure,

radiological sign of CHF,,

new evidence of BNP ≥400 pg/mL, NT-proBNP 1500pg/ml

Cardiogenic shock

Cardiogenic shock is decreased cardiac output and evidence of tissue hypoxia in the presence of

adequate intravascular volume.

Hemodynamic criteria for cardiogenic shock are sustained hypotension (systolic blood pressure < 90

mm Hg for at least 30 min) and a reduced cardiac index (< 2.2 L/min/m2) in the presence of

elevated pulmonary capillary occlusion pressure (>15 mm Hg).

The diagnosis of cardiogenic shock can be made at the bedside by observing hypotension and

clinical signs of poor tissue perfusion, which include oliguria, cyanosis, cool extremities, and altered

mentation. These signs usually persist after attempts have been made to correct hypovolemia,

arrhythmia, hypoxia, and acidosis.

Myocardial infarction: Non periprocedural MI

Myocardial infarction will be diagnosed if the subject has detection of rise and/or fall of cardiac

biomarkers (preferably troponin) with at least one value above the 99th

percentile of the upper

reference limit (URL) together with evidence of myocardial ischemia with at least one of the

following:

Symptoms of ischaemia,

ECG changes indicative of new ischaemia (see ST elevation/ST depression and T wave

changes)*,

Development of pathological Q waves in the ECG,

Imaging evidence of new loss of viable myocardium or new regional wall motion

abnormality.

*[ST elevation = new St elevation at the J point in two contiguous leads with the cut off points:

≥0.2 mV in men or ≥0.15 mV in women in leads V2 – V3 and/or 0.1 mV in other leads. ST

depression and T-wave changes = new horizontal or down sloping ST depression ≥ 0.05 mV in

two contiguous leads; and/or T inversion ≥0.1 mV in two contiguous leads with prominent R-wave

or R7S ratio >1].

Myocardial infarction: Periprocedural MI

For percutaneous coronary interventions (PCI) in patients with normal baseline troponine

values, elevations of cardiac biomarkers above the 99th

percentile URL are indicative of


35

periprocedural myocardial necrosis. By convention, increases of biomarkers greater than

3x99th percentile URL have been designated as defining PCI-related myocardial infarction

For coronary artery bypass grafting (CABG) in patients with normal baseline troponin

values, elevation of cardiac biomarkers above the 99th

percentile URL are indicative of peri-

procedural myocardial necrosis. By convention, increases of biomarkers greater than 5x 99th

percentile URL plus either new pathological Q waves or new LBBB or angiographically

documented new graft or native coronary artery occlusion, or imaging evidence of new loss

of viable myocardium, have been designed as defining CABG - related myocardial

infarction.

Non fatal stroke.

Ischemic Stroke is defined as the presence of clinical evidence of cerebral, spinal cord or retinal

focal ischemic injury based on symptoms persisting ≥ 24 hours and other etiologies excluded.

Hemorrhagic Stroke is defined as rapidly developing clinical signs of neurological dysfunction

attributable to a focal collection of blood within the brain parenchyma or ventricular system that is

not caused by trauma.

Subarachnoid haemorrhage is defined as bleeding into the subarachnoid space. Subarachnoid

hemorrhage may not cause focal deficit.

Uncertain or unknown type of stroke is an episode of acute neurological dysfunction presumed

to be caused by ischemia or hemorrhage, persisting ≥ 24 hours without sufficient evidence to be

classified as one of the above

On the basis of clinical symptoms, autopsy and/or CT/MRI, strokes will be classified as:

e) definite or probable ischemic stroke,

f) definite haemorrhagic stroke, (for both, a & b, confirmed by CT, MRI scan),

g) subarachnoid haemorrhage, (a CT scan or cerebrospinal fluid report must be available

showing evidence of bleeding in the subarachnoid space),

h) uncertain or unknown type of stroke.

Haemorrhagic stroke does not include haemorrhage secondary to cerebral infarct, into a tumor, into

a vascular malformation or post-traumatic haemorrhage.

Transient Ischemic Attack (TIA)

Acute disturbance of focal neurological or monocular (amaurosis fugax) function with symptoms

lasting less than 24 hours and thought to be due to vascular mechanism.

Major haemorrhage

Major bleeding is defined as bleeding associated with any of the following:

Drop in haemoglobin of at least 5g/dl,

Significant hypotension with the need for inotropic agents,

Intraocular bleeding leading to significant loss of vision,

Requirement for a transfusion of at least of 2 units of red blood cells or equivalent whole

blood.

Minor hemorrhage

Minor bleeding is defined as any other bleeding requiring modification of the drug regimen of the

patients. Bleeding clearly due to trauma or to some other factors e.g. peptic ulcer would not be a

cardiovascular admission.

Systemic arterial embolism

Clinical history consistent with acute loss of blood flow to a peripheral artery which is supported by

evidence of embolism from surgical specimens, autopsy, angiography or other objective testing.


36

Unstable angina

Cardiac biomarkers are negative for myocardial necrosis. Clinical presentation (one of the

following) with cardiac symptoms considered to be myocardial ischemia on final diagnosis:

Rest angina,

New onset severe angina (CCS grading scale ≥III),

Increasing angina (intensity, duration and/or frequency) with an increase in severity of at

least 1 CCS class.

AND

New or worsening ST or T wave changes on ECG. ECG changes should satisfy the following

criteria for acute myocardial ischemia in the absence of LVH and LBBB:

ST elevation,

New transient (known to be less than 20 minutes) ST elevation at the J-point in two

contiguous leads with the cut-off point,

≥0.2 mV in men or ≥0.15 mV in women in leads V2-V3 and/or ≥0.1 mV in other leads,

ST Depression and T wave changes,

New horizontal or down-sloping St depression ≥0.05 mV in two contiguous leads and/or T

inversion ≥0.1 mV in two contiguous leads with prominent R-wave or R/S ratio >1,

OR

Need of urgent coronary revascularization.

Coronary revascularization

Coronary revascularization includes coronary artery grafting or angioplasty (with or without

coronary stenting) as well as other percutaneous coronary interventions designed to treat coronary

artery lesions (atherectomy, embolectomy). Non coronary cardiac interventions to treat

consequences of coronary artery disease are not to be considered as revascularization (e.g.heart

transplantation).

Resuscitation and/or appropriate AICD therapy

Resuscitated sudden death will be adjudicated when a subject experiences cardiac arrest and is

successfully resuscitated by cardioversion or cardiopulmonary resuscitation or defibrillation with

recovery of spontaneous circulation.

Other cardiovascular causes

Coronary Angiography

CABG/PTCA

Atrial/Ventricular arrhythmias

PM implantation

ICD Implantation

Biventricular pacing implantation

Pulmonary embolism

Other causes

Non cardiovascular causes

Neoplasia

Diabetes

Renal Failure

Bronchopulmonary

Sepsis

Thoracic pain

Hematological causes (thrombocytopenia, leukocytosis)


37

Other non cardiovascular causes

OUTCOME EVENT DOCUMENTATION

To authorize an accurate, independent and unbiased evaluation of deaths and of the potential

outcome events by the EVC, the investigators must submit all necessary supportive documentation.

The supportive documentation for each outcome event may depend on the event and is listed in the

following sections.

The investigator must provide the following documentation to the EVC:

Sudden death

Emergency room or hospital physician’s notes, and ECG tracing when available, or if death did not

occur in hospital, notes describing the history and circumstances of the death from the health

professional having observed the event or obtained the history from the witness of the event.

Unwitnessed unexpected death

Note describing the subject’s medical state before the event and the circumstances of the death.

Death from myocardial infarction

Hospital or emergency room physicians’ notes. Electrocardiogram, all serial cardiac enzymes and

biomarkers test results (particularly troponin, CK and CK-MB when troponin is not available, CK

and or myoglobin when troponin and CK-MB are not available).

Death from heart failure

Hospital or emergency room physicians’ notes. Chest X-ray report (when available). Echo lab report

(when available). Intravenous treatments description.

Death from stroke

Hospital or emergency room physicians’ notes. CT scan report, or MRI report or cerebrospinal fluid

results.

Death due to cardiovascular causes related to invasive diagnostic or therapeutic procedures

Summary of the procedures or surgery notes. Hospital or emergency room physicians’ notes.

Death due to other cardiovascular causes

Hospital summary or emergency room notes describing the history and circumstances of the death

from the health professional having observed the event. Radiological reports, or pertinent reports

such as ventilation-perfusion scan.

NON CARDIOVASCULAR DEATH

Death without well documented non cardiovascular etiology will be considered cardiovascular

death. Non cardiovascular death cases will require a well documented etiology and will be classified

as:

Death due to neoplasia

Histology report for cancer. Hospital discharge summary.

Death from renal failure


38

Laboratory report showing serum creatinine levels and previous values of creatinine. Hospital

discharge summary.

Death from violence, suicide, accident

Summary for suicide, accident.

Death due to infection

Hospital summary for infection.

Death From Unknown Causes

Notes describing medical status when the subject was last seen and notes describing the

circumstances surrounding the death report.

For all deaths, the following documentation should be provided by the investigator to the

EVC: narrative summary, death certificate or physician’s note attesting the participant’s death,

autopsy report (when available).

Non-periprocedural non-fatal myocardial infarction.

Electrocardiograms and serial cardiac enzymes and biomarkers test results with their respective

normal value range for CKMB or lower level of necrosis for troponin (I or T); if CKMB or troponin

are not available, serial CK, or myoglobin with their respective upper normal value should be sent.

Hospital discharge summary (when available) or investigator summary with date of the onset of

symptoms.

Periprocedural non-fatal myocardial infarction

Electrocardiograms and serial cardiac enzymes and biomarkers test results with their respective

normal value range for CKMB or lower level of necrosis for troponin (I or T); if CKMB or troponin

are not available, serial CK, or myoglobin with their respective upper normal value should be sent.

Hospital discharge summary (when available) or investigator summary with date of PCI or CABG

and date of MI.

Non-Fatal Stroke

CT scan report, MRI report, or cerebrospinal fluid report. If both the CT scan and MRI reports are

not available, the event could be adjudicated as a stroke (uncertain or unknown origin).

Coronary revascularization

Hospital discharge summary, cath lab report

Resuscitation and/or appropriate AICD therapy

Emergency Room notes, discharge summary, report from AICD, Intensive care report.

Heart Failure

A descriptive summary with signs, symptoms, BNP or NT pro-BNP report and changes in drug

therapy. Chest X-ray report (when available). Echo lab report (when available). Intravenous

treatments or initiation of evidence based therapy for heart failure description. For hospitalized

patients, hospital discharge summary. Day Hospital or emergency room notes.

Hematological disorders

Laboratory examinations., bone marrow biopsy (if available) For hospitalized patients, hospital

discharge summary


39

MANAGEMENT OF OUTCOME EVENTS

Outcome Event Review Form

The EVC member reviewing the outcome event completes the EVC Outcome form to confirm or

reject the validation of the event. The form is also used to categorize the outcome events and

establish the date of the event.

Outcome Event Query Form

This form will be used to indicate to the Investigator that the EVC member does not believe there is

enough information to adjudicate the event. The EVC member will complete this form and fax it to

the Investigator. When the additional information has been obtained, the same initial assigned EVC

member will continue the revision of the event.

PROCEDURES

1. All the documentation necessary to validate the outcome events will be collected by EVC

Coordinating Center at the ANMCO Research Center.

2. The documentation will be periodically reviewed by members of the EVC.

3. The validation of the events by each member of EVC should take no more than 2 weeks.

4. In case of agreement between the EVC member and the Investigator assessment, the EVC

Coordinating Center will allow the entry of the adjudicated event in the database. Nearly

10% of the cases will be randomly selected for a further evaluation done by another member

of EVC.

5. In case of disagreement or uncertainty, the event will be evaluated by another member of the

EVC, if one the two members of the EVC is in agreement with the investigator the event

will be adjudicated and will enter the database. The event will also be allowed to enter the

database if the two EVC members are in agreement.

6. In case of residual uncertainty, the chairman of EVC will adjudicate the event.

7. The EVC Coordinating Center will allow to enter the adjudicated event in the database,

according to the final opinion of the chairman.

8. All members of EVC must inform the EVC Coordinating Center in case of impossibility to

be compliant with the deadline (due to vacations, congresses etc). In this case, the member

of EVC will not receive the events to validate for the period of absence.

CLINICAL EVENTS COMMITTEE MEMBERS

Dr. Francesco Orso


Via La Marmora, 34 50121 Firenze

Dr. Gianna Fabbri


Via La Marmora, 34 50121 Firenze

Dr Aldo Pietro Maggioni (chairman)


Via La Marmora 34 50121 Firenze

Dr. Marco Magnoni

Casa di Cura S. PIO X - Servizio Di Cardiologia

Via Francesco Nava, 3 20159 Milano (MI)

Dr. Andrea Bacigalupo

Ospedale San Martino - Divisione di Ematologia

Largo Rosanna Benzi, 10 16132 Genova


40