JAK2 V617F Mutational Burden and Clinical Findings Relationship in Myeloproliferative Neoplasms

Background: Philadelphia-negative chronic myeloproliferative neoplasms(MPN) are associated with various genetic abnormalities. JAK2 V617F mutation is the most common one and important for diagnosis. We aimed to evaluate JAK2 mutation status and clinical parameters relationship of the MPN patients referred to our clinic. Methods and Results: We evaluate 143 JAK-2 positive patients diagnosed with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelobrosis (PMF). JAK2 mutational burden was higher in PV and PMF than ET. Laboratory ndings were different in MPN groups and higher –lower JAK2 mutational burden groups. JAK2 mutational burden was correlated with spleen size and LDH level, particularly in PMF. There was no signicant difference in age, gender, jak2 mutation burden and laboratory ndings in patients with and without thrombosis and bleeding. Common treatment protocols were acetylsalicylic acid (ASA) + hydroxyurea, ASA and ASA + phlebotomy and others respectively. JAK2 mutational burden, mean age and LDH level were higher signicantly in the patients treated with ASA+ hydroxyurea than the patients treated with ASA. Conclusion: We speculate that if the spleen size in MPN is as large as the massive splenomegaly and the LDH level is high, the JAK2 mutation burden may tend to be higher. This relationship is more pronounced for PMF.


Introduction
Philadelphia-negative chronic myeloproliferative neoplasms (Ph-negative MPNs) consist of polycythemia vera (PV), essential thrombocythemia (ET) and primary myelo brosis (PMF) mainly [1]. Ph-negative MPNs are associated with various genetic abnormalities. Janus kinase 2 (JAK2), a protein tyrosine kinase gene, is one of these abnormalities. It is associated with cellular growth and proliferation. The most common genetic mutation found in Ph-negative MPNs is the JAK2 V617F mutation in which phenylalanine replaces valine as a result of a point mutation in Codon 617. This mutation increases tyrosine phosphorylation activity resulting in hypersensitivity of hematopoietic progenitor cells to growth factors [2,3]. JAK2 gene is located on the short arm of chromosome 9 [4].
The JAK2 V617F mutation occurs in approximately half of ET and PMF patients and in 90% of PV patients. Other common genetic abnormalities in MPNs are mutations in CALR and MPL. JAK2, CALR and MPL mutations have been among WHO's MPN diagnostic criteria [5].
Testing for JAK2 V617F mutation in MPN should be performed routinely for diagnosis as well as for prognosis. In addition, it is thought that the mutation burden may be associated with complications related to the disease [1,6]. The clinical course of myeloproliferative diseases is known to be quite heterogeneous. It has been stated that JAK2V617 positivity is associated with clinical heterogeneity [7][8][9].
ASA, phlebotomy and hydroxyurea are recommended treatment choices for the patients. When hydroxyurea cannot be used due to its side effects, interferon-alpha, JAK-2 inhibitors(ruxolitinib) or busulfan are recommended [10]. Herein,in this study we aimed to evaluate JAK2 mutation status and clinical parameters relationship of the MPN patients referred to our clinic.

Methods
The patients who were detected for JAK-2 mutation between 2019-2020 in our clinic were screened.. We identi ed 143 JAK-2 positive patients among 844 patients who were examined (

Statistical analysis
The statistical analysis of laboratory results and patients' data were evaluated with SPSS version 20.
Categorical data were compared with the Pearson Chi-square test. Kolmogorov-Smirnov/Shapiro-Wilk test was used to determine the distribution of continuous variables, normally distributed data were presented as mean and standard deviation (SD) and continuous data that did not show normal distribution were presented as median and interquartile range (IQR). Mann-Whitney U-test and Kruskal-Wallis tests were performed for independent samples. Statistical signi cance will be considered as p < 0.05.

Results
There When laboratory ndings were evaluated according to diagnosis, WBC, RBC, Hgb, Htc, neu, erythropoietin and JAK2 mutational burden showed signi cant differences between groups. Htc, RBC, Hgb values were higher in PV group compared to ET and PMF groups (p < 0.05) .WBC and neu were higher in PV group than ET group (p < 0.05) .plt was higher in ET group than PV and PMF groups (p < 0.05)( Table 2). JAK2 mutational burden was different signi cantly in terms of diagnosis. (p < 0.05). (Fig. 1) According to JAK2 mutational burden, two groups were formed as ≥ 50% and < 50%. 92(64.3%)patients were in < 50% mutational burden group and 51(35.7%) patients were in ≥ 50% mutational burden group. When laboratory ndings were evaluated according to these groups, WBC, Hgb, neu, plt, LDH values showed signi cant differences between groups. WBC, neu, LDH levels, spleen size ( Fig. 1)and median age were higher in the high mutational burden group (p < 0.05). Gender was not a signi cant factor(p > 0.05). (Table 3). Those with a spleen size ≥ 160mm in the USG were considered massive splenomegaly and those 130-160mm were considered mild splenomegaly. Laboratory ndings were evaluated according to mild and massive SM groups and Htc, Hgb, plt, LDH, lymp, JAK2 mutational burden values showed signi cant differences between groups(p < 005). RBC, Htc, Hgb, plt, lymp were higher in the mild splenomegaly group and LDH, JAK2(%) were higher in the massive splenomegaly group. Gender, age and thrombosis were not a signi cant factors (p > 0.05) ( We also evaluate the treatment protocols of the patients. Most of the patients were treated with acetylsalicylic acid(ASA) + hydroxyurea (59.4%) and 64.7% of the patients using this treatment were PV patients, 22.4% were ET, 12.9% were PMF patients. The second most common treatment option were ASA (14.7%) and ASA + phlebotomy (14.7%). 71.4% of the patients with treated with ASA were ET, 23.8% were PV, 4.8% were PMF patients. 100% of the patients with treated with ASA + phlebotomy were PV patients. The other treatment protocols were ASA + ruxolitinib in 4.2%, ASA + Interferon-α(IFN-α) in 3.5%, ruxolitinib in 1.4%, clopidogrel + hydroxyurea in 0.7%, hydroxyurea in 0.7% and allogenic bone marrow transplantation in 0.7%. Htc and lymp level were higher and age was lower signi cantly in the patients treated with ASA + phlebotomy than the patients treated with ASA + hydroxyurea. JAK2 mutational burden, mean age and LDH level were higher signi cantly in the patients treated with ASA + hydroxyurea than the patients treated with ASA.
There were studies evaluating JAK2 mutational burden in MPN patients similar to our study. In the study of Tefferi et al., JAK2 heterozygous and homozygous PV patients were compared and no statistical difference was found in age, gender, leukocyte or platelet count or thrombosis or bleeding cases at the time of diagnosis. However, in homozygous patients, a signi cantly higher hemoglobin level at the time of diagnosis, increased pruritus, higher rate of brotic transformation were found [9]. In the study of Zhou et al., JAK2V617F mutational burden was found to be signi cantly higher in PV compared to ET and PMF. It was reported that the JAK2 mutational burden was positively correlated with WBC and plt counts in ET patients and in PV patients with WBC and RBC counts [17]. In the study of Liu et al., high to low JAK2 mutation burden was determined in patients with PV, PMF and ET, respectively. Patient age and WBC in PV, WBC in ET and Hgb, WBC, plt count in PMF were signi cantly correlated with higher mutation burden [18]. In our study, Htc, Hgb, RBC were higher signi cantly in PV group comparing to ET and PMF groups, consistent with previous studies [15,17] and WBC, neu were higher signi cantly in PV group comparing to ET group. JAK2 mutational burden was higher signi cantly in PV and PMF group comparing to ET group. The levels of lymp, LDH, uric acid were not different in the different diagnosis groups. Eritropoetin was higher signi cantly in PMF group. In addition WBC, neu, LDH and age were higher signi cantly in JAK2(≥ 50%) mutational burden group and plt, Hgb were higher in JAK2 (< 50%) mutational burden group. Contrast to our study. Vanucchi et al indicated that age, Htc, WBC levels were not different in terms of diagnosis of PV or ET. In various studies, LDH levels were not different in JAK2 mutant patients [1,7,14,20] while in another studies, LDH was different consistent with our study [21] .
In the study of Kittur et al., it was stated that JAK2 mutational burden was associated with splenomegaly and male gender in ET patients [11].There were various studies that stated splenomegaly rate was correlated with JAK2 mutation in ET patients [1,12,15,16] In our study, massive splenomegaly and JAK2 mutational burden were associated but gender was not associated similiar to other various studies [13,22]. In our massive splenomegaly group JAK2 mutational burden, LDH level were higher and plt, neu, lymp, Hgb, Htc and RBC counts were lower signi cantly than mild splenomegaly group. We speculate that patients with massive splenomegaly may have a higher JAK2 mutational burden.
There is con icting information in the literature about the relationship between the JAK2 mutation and thrombosis. Various studies stated that JAK2 mutation was associated with thrombosis in ET patients [4,7,11,13,16]. According to another study, MPN patients with an allele burden of ≥ 50% JAK2, had increased the risk of vascular complications and these patients account for most of PV and a small proportion of ET [4]. In the contrary, thrombosis or bleeding risk was not found as associated with the JAK2 mutation in myeloproliferative diseases in various studies [1,9,14,19].In our study, there was no signi cant difference between the groups with and without thrombosis in terms of age, gender, JAK2 mutational burden and laboratory parameters except lymp count. lymp count was higher signi cantly in the group without thrombosis.
Low and high risk patient groups are de ned for PV and ET patients [23]. Acetylsalicylic acid is recommended in low risk patients to prevent venous and arterial thrombosis. Phlebotomy is performed in both high and low risk groups to keep the hematocrit values below 45%. Patients over 60 years old and /or the presence of arterial or venous vascular complications are considered high-risk group [23]. Lowdose ASA, phlebotomy and hydroxyurea are recommended for patients in the high risk category. Hydroxyurea regulates the plt count and reduces the risk of vascular complications. When hydroxyurea cannot be used due to its side effects, Interferon-α(IFN-α), JAK-2 inhibitor or Busulfan are used [10].In our study, consistent with literature, ASA + hydroxyurea, ASA + phlebotomy and ASA were the common treatment options. The patients treated with ASA + hydroxyurea were older than the patients treated with ASA and ASA + phlebotomy.
21 of the patients with thrombosis were treated with acetylsalicylic acid + hydroxyurea, three with ASA + IFN-α, one with ASA + ruxolitinib and one with clopidogrel + hydroxyurea.
In conclusion, the patients with a higher JAK2 mutational burden were older and had higher WBC, neu, LDH levels and massive splenomegaly probability.
The most commonly used treatment in MPN patients were ASA + hydroxyurea in our patients (59.4%).
JAK2 relationship with vascular complications such as thrombosis and bleeding were not signi cant. There were signi cant differences in laboratory parameters and JAK2 mutational burden according to diagnoses of PV, ET and PMF. PMF patients had the highest JAK2 mutational burden, LDH levels and massive splenomegaly rates. We speculate that if the spleen size in MPN is as large as the massive splenomegaly and the LDH level is high, the JAK2 mutation burden may tend to be higher. This relationship is more pronounced for PMF.