Thrombotic Incidents in Patients with Myelofibrosis Suggest to be Independent of JAK2 V617F Mutational Status

in Abstract Introduction: Myelofibrosis (MF) belongs to a group of conditions known as Philadelphia-negative myeloproliferative neoplasms (MPN). Bleeding or various vascular complications could be the main causes of morbidity and mortality in patients with MF. MPN-related thrombosis is a multifactorial process and in the case of myelofibrosis, little is known. The risk factors for thrombotic complications in MF have been rarely assessed. Aim: The purpose of this study was to investigate the incidence of thrombotic events in MF and the role of JAK2 V617F mutation as a risk factor for thrombotic incidents in patients with MF. Materials and methods: In our study of 37 patients, 35% had thrombotic events in the past. All patients were admitted to the Clinic of Hematology, St Ivan Rilski University Hospital, Sofia, Bulgaria between 2016 and 2019 and diagnosed based on the WHO criteria of 2016. Results: The majority of patients (23, 62%) proved positive for JAK2 (Janus kinase) V617F mutation carrying one (16, 70%) or two (7, 30%) mutated alleles. Thirteen of the patients (35%) had a thrombotic event in the past and 9 of them (69%) were carriers of JAK2 V617F mutation. Fourteen patients of those without thrombotic history (24, 58%) were also carriers of JAK2 V617F mutation. Conclusions: As a whole, we did not find a statistically significant difference between JAK2 V617F mutation and the frequency of thrombotic events. Rendering an account to the possible life-threatening complications, treatment decisions should be undertaken upon possible antithrombotic prevention in MF.


INTRODUCTION
Myelofibrosis is a myeloproliferative neoplasm (MPN) characterized by proliferation of clonal hematopoietic stem cells and presence of extra fibrous tissue in the bone marrow of the patient. [1] JAK2 (V617F) is one of the three well-known driver mutations for the development of MF, together with point mutation in MPL and ins/deletion in CALR genes. It has been proven in several studies that JAK2 mutation prevails in patients with MF. According to different sources, this percentage is between 50% and 60% of the patients. [2] Generally, the prognosis of MF is poor -between five and six years -and increases if treatment with Janus kinase (JAK) inhibitors is initiated or appropriate usage of bone marrow transplantation. Thrombotic events (venous and arterial thrombosis) in MPN could additionally decrease the lifetime of patients. That is why it is important to investigate whether patients with MF are prone to develop thrombotic episodes and what the factors increasing this risk are.
Venous or arterial thromboses are the main complications of BCR-ABL negative myeloproliferative neoplasms, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). [3] JAK2 V617F mutational status has been commented as an independent prognostic factor for fatal and nonfatal cardiovascular complications in patients with myelofibrosis (MF). [4] The link between the risk for the occurrence of thrombotic events in patients with MF and the carriership of JAK2 V617F mutation is still under investigation.
Our hypothesis was that JAK2 V617F mutation could be a risk factor for thrombotic events in patients with myelofibrosis. What is known at present is that the persistence of this mutation is usually low in ET (with incidence rates of 23%-57%), higher in PV (frequently over 50%, usually 65-97%), and 35-57% in idiopathic MF. [5] AIM The aim of this study was to investigate the incidence of thrombotic events in patients with MF and to clarify whether JAK2 V617F mutation could be considered as a single risk factor for thrombotic incidents in patients with MF.

Patients and ethical consent
Thirty-seven patients were diagnosed with myelofibrosis (MF) between May, 2015 and December, 2019 at the Clinic of Hematology, St Ivan Rilski University Hospital, Sofia, Bulgaria based on the criteria of WHO 2016. [6] Written informed consent was obtained from the patients for their anonymized personal information to be published in this article.

Detection of JAK2 V617F by RFLP analysis
All patients were subjected to targeted DNA analysis for the detection of JAK2 V617F mutation by RFLP procedure which has been previously described. [2] The presence of fragments of three different sizes (241, 189 and 30 bp) on 2% agarose gel certifies the presence of the wild type allele, while the mutant allele remains undigested.

History of thrombotic events and statistical analysis
The history of thrombotic events after setting the diagnosis was collected and summarized from the medical documents of the patients (thrombosis of the arteries/veins). The data were compared to the mutational status of the patients and the results were statistically analyzed by chi-square test. P-value less than 0.05 was accepted as a statistically significant difference between the analyzed groups.
The history of thrombotic events was recorded for all patients ( Table 1). The thrombotic events observed in our patients included mainly portal vein thrombosis (PVT) and brain or splenic infarction. Interestingly, post-ET myelofibrotic patients did not show any thrombotic complications (Table 1). Around 35% (13 out of 37) of the myelofibrotic patients had confirmed positive thrombotic history. They were distributed approximately evenly by their sex -seven were males (53%) and six (47%) were females. Table 1 presents some clinical characteristics of the patients as age, sex, JAK2 mutational status, the presence of thrombotic incidents, and treatment of the specific patient, if any.
The mean values of the clinical parameters of thrombocytes (Thr), hemoglobin (Hg) and hematocrit (Hct) for patients with or without thrombotic events are shown in Table 2. The levels of thrombocytes, Hg and Hct are slightly higher in patients with thrombotic incidents compared to non-thrombotic patients. In our study, we did not find a statistical significance between the levels of Hg and Hct and the presence of JAK2 V617F mutation in homozygous or heterozygous state (p=0.173 and p=0.695, respectively); however, it was correlated with the level of Thr (p=0.098).

DISCUSSION
The three major types of myeloproliferative neoplasms (myelofibrosis, essential thrombocythemia and polycythemia vera) have differences in terms of bleeding and throm-   bosis, although they also share common features. [7][8][9][10] Bleeding and thrombotic events are accepted as common complications of myelofibrosis and contribute significantly to the morbidity and mortality of the condition. A few studies only have evaluated the frequency of these events, their characteristics, and their prognostic impact. According to Devendra et al. [11] , thrombotic events in MF are about as common as in essential thrombocythemia but less common than in polycythemia vera, while bleeding events are relatively more common in MF than in ET or PV. In a study of 155 patients, 11.6% had thrombotic events during a median follow-up of 4.2 years. [7] In a study of 707 patients with PMF, thromboses were diagnosed in 7.2% patients. A subsequently larger number of patients (35%) in our study showed positive history of thrombotic/vascular events, independently from the sex and the JAK2 mutational status. The pathogenesis of vascular events in MF requires further investigation. Some authors state that age >60 and prefibrotic PMF are consistently associated with higher risk of thrombosis while thrombocytosis and JAK2 positivity are not associated with risk of bleeding. [11] The link between MF with other hereditary or acquired thrombophilic states is virtually unknown. The pro-thrombotic role of JAK2 V617F mutation in PV or ET is well established and is included in the thrombotic risk model for ET [12] , but remains to be clarified in patients with MF. More recently, JAK2 V617F mutation has been found to carry an increased risk of thrombotic complications. [13] The majority of patients in our study with MF have one or both alleles mutated for JAK2 V617F mutation (23/37 or 62%). 69% of patients with JAK2 mutation have previous thrombotic event, while 58% of JAK2 negative patients are non-thrombotic. Even if there is an observed difference between the groups, a statistical significance has not been found. Those results need to be further confirmed by including more patients and increasing the statistical power of the analysis.
The role of JAK2 V617F mutation in thrombosis in patients with MPN has been recognized. A mutated JAK2 may not only increase the platelet number but also alter the platelet function, thereby playing a role in thrombogenesis. JAK2 V617F mutation has been reported to cause changes in the process of platelet formation from megakaryocytes in a knock-in mouse model of ET. [14] The platelets were found to be prothrombotic and demonstrated enhanced reactivity to different agonists. [14] The levels of thrombocytes, Hg and Ht are slightly higher in patients with thrombotic incidents compared to non-thrombotic patients. However, we have found no statistical significance among the groups.
The pathogenesis of thrombosis in MPN patients is complex. Disease related factors, such as an increase in blood cell counts (i.e., leukocytosis, erythrocytosis, and thrombocytosis), and more importantly, presence of JAK2 mutation, can interact with non-disease patient related factors such as age, previous history of thrombotic events, obesity, hypertension, hyperlipidemia, and presence of thrombophilic defects. [15] In our study, we did not find statistical significance also between the levels of Hg and Ht and the presence of JAK2 V617F mutation in homozygous or heterozygous states (p=0.173 and p=0.695, respectively); however, it was correlated with the Thr level (p=0.098). The idea that patients with MPN-related cerebral venous thrombosis (CVT) should be treated with long-term anticoagulation with VKAs (vit. K antagonists) is presented by some authors. [15] Based on our results, we think that patients with MF, irrespectively of their JAK2 mutational status, are more prone to develop thrombotic complications and have to be treated with caution, including some anticoagulants in the regular therapeutic regimes.

CONCLUSIONS
Patients with myelofibrosis are prone to developing thrombotic complications independently of their JAK2 mutational status. Even in a cohort of small size (myelofibrosis is a rare event with an incidence of approximately 1.5:100 000 in USA) (https://rarediseases.org/rare-diseases/primary-myelofibrosis), the frequency of a thrombotic incidence is not significantly related to JAK2 V617F mutation. Rendering an account to the possible life-threatening complications of such events, treatment decisions should be undertaken upon possible antithrombotic prevention in MF. The thrombotic risk assessment should be based on a combination of clinical risk factors (patient, disease-related, and treatment-related) and a panel of biological markers extending beyond the full blood count.