Arterial Function and Atherosclerosis in Patients With JAK2 V167F Positive Essential Thrombocythemia
Conditions: Atherosclerosis
Sponsor: University Medical Centre Ljubljana

 
 
 
 
 
 
 
 
 
 
 
 
Date: January 26, 2019 
 
 
 
 
 
Study Protocol and Statistical Analysis Plan 
 
Arterial Function and Atherosclerosis in Patients With JAK2 V167F Positive 
Essential Thrombocythemia   
 
 
 
Principal Investigator:  
Aleš Blinc, MD, PhD,  
 
Professor of Medicine,  
 
Head, Department of Vascular Diseases 
 
 
Funding:  
Project No. TP20180038, financed by the Fund for Tertiary Research and 
 
Development of the University Medical Centre Ljubljana   
 
 
Division of Internal Medicine 
Department of Vascular Diseases 
 
Zaloška cesta 7 
1525 Ljubljana 
T 01/522 80 32 
F 01/522 80 70 
E ales.blinc@kclj.si   
W www.kclj.si 

 
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1. Overview and Aim of the Study  
 
We will study patients with JAK2 V617F positive essential thrombocythemia (ET) without clinically 
apparent atherosclerotic vascular disease in comparison to age-and sex-matched, apparently healthy control 
subjects and measure changes in vascular function and morphology at baseline and after a 4-year observation 
period. In addition, the JAK2 V617F allele burden, i.e., the percentage of mutated alleles in leukocytes, will be 
determined and correlated with vascular function and morphology. The study protocol has been approved     by 
the Committee for Medical Ethics of the Republic of Slovenia, approval number: 0120-428/2017/4.  
  
Our aim is to test (a) whether changes in vascular function and morphology in a 4 year observation 
period differ between patients with JAK2 V617F  positive ET in comparison with control subjects, and whether (b) 
the JAK2 V617F allele burden correlates with vascular function and morphology. 
  
 
2. Patients and Control Subjects 
 
Patients with JAK2 V617F positive ET have been selected from the database of the Department of 
Hematology of the University Medical Centre Ljubljana, Slovenia, after having given their informed consent. Only  
patients without a personal history of clinically manifest atherosclerotic vascular disease (myocardial infarction, 
angina pectoris, peripheral arterial disease, aortic disease, transient ischemic attack or ischemic stroke) were 
eligible. The control group has been selected among apparently healthy employees of the University Medical 
Centre Ljubljana and their relatives. The groups have been matched for age and sex distribution and classical 
risk factors for cardiovascular disease. ). Forty patients (14 male and 26 female) with JAK2 V617F positive ET 
without clinically apparent cardiovascular disease were enrolled in the study in 2014 - 2015 for the first 
examination and 36 (12 male and 24 female) of them are expected to participate also in 2018-19. Forty two 
control subjects (16 male and 26 female) participated in the first examination in 2014-2015 and at least 38 (14 
male and 24 female) subjects are expected to participate in the second examination. 
 
Inclusion criteria for patients are: clinically confirmed ET with genetically confirmed JAK2 V617F 
mutation, age above 18 years, absence of clinically suspected or overt atherosclerotic vascular disease. Fort the 
control subjects, the inclusion criteria are  being  apparently healthy, matching the patients in age and sex, age 
above 18 years, and absence of clinically suspected or overt atherosclerotic vascular disease. 
 
Exclusion criteria for both groups are: clinically suspected or overt atherosclerotic vascular disease, 
chronic kidney disease stage 3 and above, known cancer, chronic inflammatory disease, autoimmune disease, 
and pregnancy. 
 
All subjects will be examined twice, the first time in 2014 - 2015 and the second time in 2018 – 2019. All 
participants will have signed the informed consent form before enrolment. The subjects will complete a structured 
questionnaire about personal and family medical history, risk factors for cardiovascular disease and their 
medication. They will be physically examined and will donate blood samples for laboratory tests. Ultrasound 
examination of the extracranial carotid arteries will be done, and echo-tracking of the common carotid arterial wall 
will be used to assess the β-stiffness index and to estimate the pulse wave velocity. EndoPat plethysmography will 
be used to assess the endothelial function via the reactive hyperemia response of the digital arteries. Coronary 
artery calcium scanning will be performed to evaluate the extent of coronary artery calcification.  
 
3. Baseline Measurements 
 
Every participant will be examined physically, including  measurements of height, weight, waist 
circumference, systolic and diastolic blood pressure. Blood will be taken for laboratory analyses - electrolytes, 
complete blood cell count, serum lipids, liver function tests, urea, creatinine, inflammatory markers and DNA 

 
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isolation for measurement of JAK V617F allele burden. A structured questionnaire about family history of 
cardiovascular diseases, personal medical history, smoking status and medications will be completed. The 10-year 
risk of coronary heart disease (CHD) and general cardiovascular disease (CVD) will be calculated using the 
Framingham risk equation.  
 
4. Vascular Imaging  
 
4.1. Carotid Artery Ultrasound Examination 
 
For examination of the extracranial carotid arteries an ultrasound machine Aloka Prosound α7 (Hitachi 
Aloka Medical, Ltd., Japan) will be used with a linear vascular probe working at a frequency of 5-13 MHz. The 
common, internal and external carotid arteries on both sided will be examined. At each examination, the 
measurements will be done twice and their average values will be calculated. The ultrasound examination will be 
done by the same ultrasonographer at both visits of all participants.  
 
The intima-media thickness will be measured 2 cm proximal to the bulb of common carotid artery on both 
sides. Screening of the extracranial carotid arteries for atherosclerotic plaques will be performed, with a plaque 
definition of a  focal lesion, exceeding the intima-media thickness for at least 50% or reaching an absolute thickness 
of at least 1.5 mm in two orthogonal projections. Scoring of atherosclerotic plaques is done according to the 
methodology from the Rotterdam Study. The presence of at least one plaque in each segment of the extracranial 
carotid arterial bed, (divided into the common carotid artery and the bulb, the internal carotid artery and the external 
carotid artery) on either sides is scored 1 point. Thus, the carotid plaque score ranges from 0 (absence of plaques) 
to 6 (plaques present in all segments on both sides).  
 
Echo-tracking of the common carotid artery will be used to assess arterial stiffness expressed by the β-
stiffness index and the pulse wave velocity. Measurements will be done at the common carotid artery 2 cm proximal 
to the bulb on both sides. The  β-stiffness index will be calculated as: β = ln (P_max / P_min) / [(D_max – D_min / 
D_min)], where P_max = the systolic blood pressure, P_min = the diastolic pressure; D_max = the maximum 
arterial diameter and D_min = the minimal arterial diameter. Pulse wave velocity (PWV) will be calculated as: PWV 
= √ ((β x P_min) / 2ρ)); ρ = 1050 kg/m³. 
 .  
4.2. Measurement of the Coronary Artery Calcium Burden  
 
The  calcium burden of coronary arteries will be measured with a Biograph M 128-row PET-CT scanner 
(Siemens, Erlangen, Germany). A non-contrast protocol with sequential prospective ECG triggering will be used. 
Post-processing will be done on the Syngo Leonardo workstation. The coronary calcium burden will be expressed 
as the Agatston score.  
 
5. Assessment of Endothelial Function of the Digital Arteries  
 
 
Endothelial function of the digital arteries will be measured by digital plethymography with EndoPat 2000, 
Itamar Medical REF, Caesarea, Israel, Software Version 3.3.x and expressed as the Reactive Hyperaemia Index 
(RHI) and the Augmentation Index (AI). Changes in arterial tone are elicited by creating a downstream hyperemic 
response induced by a standard 5-minute occlusion of the brachial artery (using a blood pressure cuff, inflated to 
60 mmHg above the arterial blood pressure). When the cuff is released, the surge of blood flow causes 
endothelium-dependent flow mediated dilatation (FMD) which is manifested as reactive hyperemia. A post-

 
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occlusion to pre-occlusion ratio is calculated by EndoPAT software and the and  A) are determined. The results 
are normalized to a heart rate of 75/min.  
 
6. Measurement of the JAK2 V617F/G1849T Allele Burden  
 
The ipsogen JAK2 MutaQuant Kit, Qiagen (ZDA) (Ref: No. 673523) will be used for the detection and 
quantification of JAK2 V617F/G1849T allele in genomic DNA extracted from peripheral blood of patients and of 
control subjects. JAK2 V617F allele burden will be calculated and expressed as a percentage of JAK2 V617F 
mutated alleles throughout the whole JAK2 record.  
 
 
 
 
7. Statistical Analysis 
 
 
All sets of data will be tested for normality of distribution using the normal-quintile plot, calculating the 
correlation coefficient and checking it for the critical value that would warrant rejection of normal distribution with 
an α-error probability of 0.05. 
 
 
Normally distributed data will be presented as mean and standard deviation, while non-normally 
distributed data will be presented as median and range between the 1st and 3rd quartile. 
 
 
Differences between subjects with ET and control subjects at the first and second examination will be 
tested by the chi-square test for discrete variables, for normally distributed continuous variables by the paired 
Student's t-test for independent samples, and for non-normally distributed continuous variables by the Mann-
Whitney test for independent samples. P-values of < 0.05 will be considered significant with a correction for 
multiple comparisons. 
 
 
Changes in vascular function and morphology in the 4-year observation  period will be compared 
between the two groups by the log rank test. P-values of < 0.05 will be considered significant. 
 
 
The association between the parameters of vascular function / morphology and the  JAK2 V617F allele 
burden will be assessed by the Pearson correlation coefficient.