Arterial Function and Atherosclerosis in Essential Thrombocythemia

The aim of the study is to examine (a) whether patients with JAK2 V617F positive ET in comparison to age-and sex-matched, apparently healthy control subjects show more advanced progression of arterial stiffness, pulse-wave velocity and coronary calcium score in a 4 year observation period, and (b) whether the burden of JAK2 V617F mutation correlates with the measured vascular parameters. All subjects will be examined twice. The first visit already took place between the years 2014 - 2015 and the second visit will take place between 2018-2019. All participants will have signed their informed consent before entering the study. Each visit will consist of completing a structured questionnaire (on personal and family medical history, risk factors for CVD and medication), physical examination, donating a blood sample for laboratory tests and undergoing carotid ultrasound and coronary calcium measurement oft the extent of coronary artery calcification. At the first and the second examination the JAK2 V617F allele burden, i.e. the percentage of mutated alleles, will be determined from genomic DNA in peripheral blood.

1. Patients and control subjects

   Patients are selected from the database of the Department of Haematology at University Medical Centre Ljubljana, Slovenia, who were diagnosed with JAK2 V617F positive ET between 2011 and 2014. There were 124 JAK2 positive ET patients, among those 61 did not have a personal history of clinically manifest atherosclerotic vascular disease (myocardial infarction, angina pectoris, peripheral arterial disease, aortic disease, transient ischemic attack or ischemic stroke). 40 patients (14 male and 26 female) with JAK2 V617F positive ET without clinically apparent cardiovascular disease signed the informed consent and 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.

   The control group is selected among healthy employees of the University Medical Centre Ljubljana and their relatives. It is matched with the patient group for age and sex distribution and classical risk factors for cardiovascular disease. 42 individuals (16 male and 26 female) participated in the first examination and at least 38 (14 male and 24 female) subjects are expected to paticipate in the second examination.
2. Baseline measurements

   Each participant will complete a questionnaire about family history of cardiovascular diseases, personal medical history, smoking status and medications. A physical examination will be performed, including measurements of height, weight, waist circumference, systolic and diastolic blood pressure. Blood will be sampled for laboratory analysis of complete blood cell count, electrolytes, serum lipids, liver function tests, urea, creatinine, and inflammatory markers.

   The 10-year risk of coronary heart disease (CHD) and general cardiovascular disease (CVD) will be calculated using the Framingham risk equation.
3. 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 also control subjects. A SNP specific primer selectively amplifies the JAK2 V617F allele which is detected with a real-time qPCR instrument that quantifies the PCR products. The JAK2 V617F allele burden will be calculated and expressed as the percentage of JAK2 V617F mutated alleles throughout the whole JAK2 record. Analysis will be done done in laboratory of the Department of Haematology at University Medical Centre Ljubljana for all samples.
4. 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.
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-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 Coronary Artery Calcium Burden

   The calcium burden of coronary arteries is measured with a Biograph M 128-row PET-CT scanner (Siemens, Erlangen, Germany). Scanning is done from the base to the apex of the heart. A non-contrast protocol with sequential prospective ECG triggering is used. The rotation time is 0.33 sec, with a tube voltage of 120 kV, CARE Dose 4D and slice thickness 3 mm, with no slice overlap. Post-processing is done on the Syngo Leonardo workstation. The coronary calcium burden is expressed as the Agatston score. Evaluation of the dataset of every study subject is done three times and the average value is used for further analysis.
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.

Eligibility Criteria:
Inclusion Criteria:

* patients with JAK2 V617F positive essential thrombocythemia
* age-and sex-matched apparently healthy control subjects

Exclusion Criteria:

* personal history of any atherosclerotic vascular disease (myocardial infarction, angina pectoris, peripheral arterial disease, aortic disease, transient ischemic attack or ischemic stroke)
* chronic kidney disease stage 3 and above
* known cancer
* chronic inflammatory disease
* autoimmune disease
* pregnancy