Evaluation of the JAK2 V617F gene mutation in myeloproliferative neoplasms cases: a one-center study from Eastern Anatolia

Introduction

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders characterized by an increased number of mature myeloid peripheral blood cells [1]. In 1951, William Dameshek defined chronic myelogenous leukemia (CML), polycythemia vera (PV), primary myelofibrosis and essential thrombocythemia as myeloproliferative disorders (MPDs) [2], [3]. Currently, the classification suggested by the World Health Organization (WHO) is most widely used and describes eight entities. According to this classification, MPNs include CML, BCR-ABL1 positive, chronic neutrophilic leukemia, PV, primary myelofibrosis (PMF), essential thrombocythemia (ET), chronic eosinophilic leukemia, not otherwise specified, mastocytosis and MPNs, unclassifiable (MPN-U) [4]. The reported incidence of MPNs worldwide is 0.5–2 cases per 100,000 individuals [5]. Diagnostic criteria include clinical features, bone marrow histopathology and genetic alterations [6], [7], [8].

Janus kinase (JAK) is a family of non-receptor tyrosine kinases [2]. JAKs perform their effect by further phosphorylating transcription factors called signal transducer and activator of transcription (STAT) in the JAK-STAT pathway. The JAK-STAT pathway is particularly important for hematopoiesis. It permits cell proliferation, cell differentiation and cell apoptosis regulation [2], [6], [8], [9]. JAKs include JAK1, JAK2, JAK3 and tyrosine kinase 2 (Tyk2). JAK3 expression is limited to hematopoietic cells, while JAK1, JAK2 and Tyk2 are expressed in mammalian cells [2], [9].

The JAK2 gene, encoded by the gene region localized in 9p24, consists of 25 exons and encodes a protein with 1132 amino acids [2]. In 2005, research groups defined the somatically acquired point mutation JAK2 in exon 14 of the JAK2 gene [10], [11], [12], [13]. In BCR-ABL1 negative MPN patients, the JAK2 V617F mutation emerges as a result of a valine to phenylalanine substitution at codon 617 [14]. The JAK2 V617F mutation was observed in 95% of patients with PV, in approximately 50–60% of patients with ET and in about 50% of patients with PMF [11], [12], [15]. Less commonly JAK2 exon 12 also result in JAK-STAT pathway activation in MPNs [16], [17].

Our study evaluated the frequency of the JAK2 V617F mutation in a group of individuals with MPNs.

Materials and methods

Results

The study group consisted of 720 patients, 174 women and 546 men, who were referred to the Inonu University Turgut Ozal Medical Center Outpatient Clinic of Hematology. The participants were diagnosed with MPNs according to the WHO criteria. The patients clinical and hematological characteristics are summarized in Table 1.

We first determined that the JAK2 V617F mutation positive rate was 22.6%, occurring in 33.3% of women and 19.2% of men (Table 2). In our study, the JAK2 V617F mutation was present in 48.6% of patients with ET, 80.5% of patients with PV, 47.5% of patients with PMF, 10% of patients with MPN-U and 0.8% of other patients (Table 3). Also, five patients (6.8%) were BCR-ABL1 negative. We found that the proportion of JAK2 V617F allele burden in patients with PV was higher than in other patients (Table 3). In our study ET (27%), PV (18.4%) and PMF (17.2%) were more common in females (p<0.001) (Table 4).

Next, we investigated the difference in hematological parameters between JAK2 V617F positive and JAK2 V617F negative cases (Table 1). There was a significant difference in white blood cell (WBC) counts (p<0.001), hemoglobin (Hb) values (p<0.001), hematocrit (HCT) values (p=0.004), red blood cell distribution widths (RDW) (p<0.001) and platelet counts (p<0.001) in the JAK2 V617F positive patients. The median WBC count was 10.6 10⁹/L (range 4.3–10.3), the median Hb value was 15.1 g/dL (range 13.6–17.2), hematocrit (HCT) levels (median 46.6%) (range 39.5–50.3) and the median RDW and platelet counts were 15.9% (range 11.8–14.3) and 450.0 10⁹/L (range 150–400), respectively. In our study, there was no difference between JAK2 V617F positive cases and JAK2 V617F negative cases in terms of red blood cell (RBC) (median 5.5 10¹²/L) (range 4.38–5.77) (p=0.579).

In addition to the JAK2 V617F mutation, we also examined the BCR-ABL1 mutation. Patients who were BCR-ABL1 mutation negative were divided into groups based on whether the JAK2 V617F mutation was positive or negative. Out of 73 patients, five (6.8%) had a positive JAK2 mutation and a negative BCR-ABL1 mutation. We analyzed the difference in hematological parameters between patients with positive JAK2 V617F mutations/negative BCR-ABL1 mutations and negative JAK2 V617F mutations/negative BCR-ABL1 mutations. However, no significant difference in terms of hematological values were found (Table 5).

In our study, at the same time, we evaluated the clinical parameters of our study groups according to the JAK2 V617F mutation status (Table 6). The number of cases diagnosed with ET is 70. JAK2 V617F mutated ET cases occurred RBC count (p<0.001), HCT level (p<0.001) and RDW count (p=0.046) were higher than JAK2 V617F negative ET cases (Table 6). When compared with PV cases (113 cases), median WBC counts, RDW counts and platelet counts were found to be significantly greater in cases with the JAK2 V617F mutation (p, 0.022, 0.003, and 0.006, respectively). However, the Hb value (p=0.037) was found to be high in the JAK2 V617F mutation negatives. When we examined PMF (61 cases) and CML (73 cases) cases for hematological parameters, there was no significant difference between positive and negative cases in terms of JAK2 V617F mutation (Table 6). Our MPN-U study group consisted of 10 cases. In the MPN-U group, the number of JAK2 V617F mutation positive cases (one case) was not sufficient and no statistical evaluation was performed. When a similar comparison was made among others cases (393 cases including 10 JAK2 V617F mutation positive, 390 JAK2 V617F mutation negative), median WBC (p=0.004) and RDW (p=0.006) was higher for the JAK2 V617F positive group and median RBC (p=0.007), Hb (p=0.007), HCT (p=0.011) was greater for the JAK2 V617F negative group. There was no significant differences among the two groups in terms of platelets count (PLT) (p=0.121) (Table 6).

Discussion

MPNs are clonal hematopoietic stem cell malignancies characterized by hypersensitivity of hematopoietic progenitors to numerous cytokines and by clonal proliferation of one or more myeloid blood cell lineages [1]. Additionally, thrombotic or hemorrhagic complications or leukemic transformations can occur [18]. JAK2 was found in the majority of MPNs patients. The JAK2 V617F assay is a very important test in the assessment of MPNs [19]. This is the first molecular JAK2 gene study of patients with MPNs from the Malatya Province in Turkey.

In our study, we detected the JAK2 V617F mutation in exon 14 of the JAK2 gene in 163 (22.6%) patients. We found that the frequency of JAK2 V617F mutations was 48.6% in patients with ET, 80.5% in patients with PV, 47.5% in patients with PMF, 6.8% in patients who were BCR-ABL1 negative and in 10% of patients with MPN-U.

Based on a study population of 174 women and 546 men, we found that the JAK2 V617F mutation was positive in 58 women (33.3%) and 105 men (19.2%). Of the female participants, 20.7% were diagnosed with ET, 18.4% were diagnosed with PV, 17.2% were diagnosed with PMF and 1.7% were diagnosed with MPN-U. PV is defined as a disease seen predominantly in men [6], [20], [21], although there are PV groups reported to be predominantly female [22], [23], [24]. Our PV group was also predominantly female. Female predominance in ET and male predominance in PMF has been reported in the literatüre [20], [25], [26]. JAK2 V617F mutation positive patients were predominantly female in our study. However, it should be noted that the number of men and women participants was not equal.

The JAK2 V617F mutation was found in the vast majority (80.5%) of patients with PV [6]. Two other studies from Turkey have declared the frequency of the JAK2 V617F mutation to be 80% and 95% of patients with PV, respectively [24], [27], [28]. Our results are compatible with the literature. We found no JAK2 V617F mutation in 22 patients with PV (19.5%). A JAK2 exon 12 mutation screening should be performed on these patients. Mutations in exon 12 of JAK2 has also been found in one-third of V617F negative PV cases [29].

Up to 50–60% of ET and PMF cases do not have mutations in JAK2 [6]. In our study, we found the JAK2 V617F mutation in 48.6% of patients with ET and in 47.5% of patients with PMF. The frequencies of the JAK2 V617F mutation in an Iranian study were 63% in patients with ET and 53.3% in patients with PMF [30]. In a study by Karkucak et al. the JAK2 V617F mutation was found in 42% of patients with ET [27]. Our results were similar to the majority of studies of the JAK2 V617F mutation in patients with ET and PMF found in the literature [27], [30], [31].

CML is from MPDs, and increased tyrosine kinase activity due to BCR-ABL1 translocation is observed in generally patients with CML. The resulting fusion gene is observed in 90% of patients with CML and differentiates this disease from other myeloproliferative diseases [4], [32]. In this study, only five (6.8%) out of 73 patients had positive JAK2 mutation/negative BCR-ABL1 mutation. Therefore, no significant difference in terms of hematological parameters was found between patients with positive JAK2 V617F mutation/negative BCR-ABL1 mutation and negative JAK2 V617F mutation/negative BCR-ABL1 mutation.

Although most of the patients with chronic MPNs can be classified as having CML, PV, ET, or PMF, it is sometimes impossible to classify patients. This is referred to as unclassified chronic MPNs [33]. Our study found that 10% of MPNs were unclassifiable.

In the present study, we also researched the relationship between JAK2 V617F and hemogram variation in MPNs. This examination provided significant information in understanding the role of JAK2 V617F in influencing hematological values. They display higher WBC counts, HCT and hemoglobin levels and they have increased RDW and platelet counts [34]. However, we did not find a significant difference between the two groups in terms of RBC values. The JAK2 V617F mutation is known to cause erythroide lineages proliferation. Our results are consistent with the literature [27], [35], [36]. Other studies have found elevated levels of different hematological parameters in patients carrying mutations [27], [35], [36], [37].

Cases with the JAK2 V617F mutation were shown to have a tendency for higher WBC counts, and higher RDW count, and lower Hb levels. In ET, several studies have found that JAK2 V617F mutated cases presented with, higher Hb level and WBC count, but lower PLT count [24], [38], [39]. In our ET group, we found that cases with the JAK2 V617F mutation were with higher Hb value, RBC level, HCT value and RDW count compared with cases JAK2 V617F mutation negative. In literature research, JAK2 V617F positive in PV patients was shown to be correlated with leukocytosis, greater cell count [40], [41]. We found WBC, PLT and RDW counts to be greater JAK2 V617F positive in PV patients. In the group of others, three cases are JAK2 V617F positive. The number of JAK2 V617F negative patients is 390. Studies on larger population groups are needed.

In conclusion these molecular assays have become a helpful tool, making the diagnosis faster and more exact. The assessment of genetic factors implicated in the improvement of MPDs helps to detect acquired mutations, such as JAK2 V617F. The demonstration of the presence of this mutation is important in terms of making a differential diagnosis. Longer follow-up and larger case populations are needed to investigate the effects of clinical and laboratory parameters in progression myeloproliferative diseases.