Janus kinase V617F mutation detection in patients with myelofibrosis

The existence of extramedullary hematopoiesis, over-stimulated fibroblasts in abnormal microenvironment, fibrous tissue deposits in the bone marrow, dysregulation of the normal production of blood cells, low count of platelets and subsequently, the onset of anemia, are events typical for myelofibrosis (MF). Myelofibrosis could be Philadelphia chromosome positive or negative, primary or resulting as a consequence to initially diagnosed polycythemia vera (PV) or essential thrombocytopenia (ET) [1]. According to the diagnostic criteria of the World Health Organization (WHO), the V617F mutation on the Janus kinase 2 (JAK2) gene is listed among the major criteria for primary MF (PMF), ET and PV [1]. Mutations on three genes are associated with PMF: V617F (JAK2), ins/del (CALK), W515L/W515K (MPL) [2]. Different researchers have investigated the frequency of those genetic mutations in patients with ET, PMF and PV [3, 4, 5, 6, 7, 8].

Janus kinase 2 exon 14 mutation in JAK2 is the most frequent mutation in myeloproliferative neoplasms (MPN), present in the majority of PV and ET/PMF patients (96.0 and 65.0% in ET, respectively) [9]. It is considered to be the most significant potentiator of JAK/STAT signaling pathway leading to its constitutive activation [10]. Patients with ET positive for JAK2 V617F slowly progress to PV [11], while PV and ET progress differently to secondary MF [12]. The enhanced phosphorylation of STAT1 or STAT5 following JAK2 activation promotes megakaryopoiesis or erythropoiesis.

In the current study, we performed targeted mutational analysis of 232 patients from our Hematology Ward for JAK2 V617F mutation. Thirty-eight (16.4%) patients were suspected to carry PMF, and in 20, the diagnosis was confirmed by bone marrow biopsy. Our aim was to estimate the carriers of JAK2 V617F in our cohort of patients with PMF and compare it with the frequency so far reported by other researchers.

Two hundred and thirty-two patients with different diagnoses (Figure 1) were referred for analysis to the genetic laboratory of the Clinic of Hematology, University Hospital "St.Ivan Rilski," Sofia, Bulgaria. DNA was extracted from venous blood of patients using a commercial kit (QIAamp DNA Blood Mini Kit; Qiagen Inc., Valencia, CA, USA). The procedure was executed in accordance with the manufacturer's recommendations. DNA samples were collected and analyzed for a total period of 3 years (from January 2016 to December 2018). All of the patients gave written informed consent for genetic analysis upon their acceptance at the Clinic.

Figure 1

JAK2 V617F Mutation Detection by Restriction Analysis. Previously isolated DNA samples were amplified using JAK2 codon 617 mutation specific primers (V617F) (forward primer sequence: 5'-GGG TTT CCT CAG AAC GTT-3' and reverse primer sequence: 5 '-TCA TTG CTT TCC TTT TTC-3'). Amplifications were performed for 32 cycles with Taq polymerase (Qiagen Inc.), annealing temperature of 60 °C, and standard amplification conditions. Polymerase chain reaction (PCR) of the human JAK2 exon was performed as described by Baxter et al. [13]. The amplified 460 bp fragments were enzymatically digested using BsaXI restriction enzyme (New England Biolabs® Inc., Ipswich, MA, USA). Fragments of three different sizes (241, 189 and 30 bp) resulted after digestion of the wild type allele, the mutant allele remained undigested (Figure 2). Digested fragments were separated in 2.0% agarose gel. Visualization of the restriction fragments was achieved by ethidium bromide.

Figure 2

Two hundred and thirty-two patients with chronic myeloproliferative neoplasms have been genotyped for their JAK2 V617F genotypic status. Eighty-two of 232 patients (35.4%) were diagnosed with ET, 35/232 (15.1%) PV/EV, 38/232 (16.4%) MF/OMF (osteomyelofibrosis), 70/232 (30.1%) chromic MPN and 7/232 (3.0%) with other hematological diseases (Figure 1). We performed analysis of the bone marrow biopsy samples of patients with MF/ OMF (38 individuals) and in 20 of them (52.6%) the initial diagnosis was confirmed. In six patients, the diagnosis was rejected based on the negative result of the biopsy sample (15.8%), five patients eventually proved to carry ET(13.2%),six had PV(15.8%) and one had MPN (2.6%).

The mean age of the patients in our study was 62 years (29-94 years), there was an equal distribution men:women (10 vs. 10)(Table 1). According to the JAK2 genotype status: 8/20 (40.0%) were homozygous with the wild type allele (NN) (men:women=3:5); 9/20 (45.0%) were heterozygous (MN) (men:women = 6:3) and 3/20 (15.0%) were homozygous with the mutated allele (MM) (men:women = 1:2).

Overall, in 20 patients with MF, proven by trepanobiopsy, 12 (60.0%) (group 1) carried the mutant allele and eight (40.0%) (group 2) did not. The mean age of patients from group 1 was 61.4 years; men:women ratio was 7:5. Patients in group 2 showed similar characteristics (mean age 62.4 years; men:women ratio was 3:5).

Extramedullary hematopoiesis, overstimulated fibroblasts in abnormal microenvironment, fibrous tissue deposits in the bone marrow, dysregulation of the normal production of blood cells, low count of platelets and subsequently the onset of anemia are characteristic hallmarks of MF. Generally, it is a rare hematological event, predominantly diagnosed in elderly individuals aged over 50 years [14, 15, 16]. The MF/OMF patients were also rare in our dataset (20/232 patients confirmed the diagnosis, 8.6%) and the mean age of the patients was 62 years. The gender does not affect the occurrence of the disease; there is an equal distribution men:women (10 vs. 10).

The total survival time of patients with PMF can vary substantially, as well as the clinical course of the disease and its progression. It can be diagnosed as primary or developed as a part of other hematological malignancies as PV or ET. However, the mechanisms of development of MF are not completely understood. The JAK2, together with CALR and MPL mutations, is a common triggering factor in Philadelphia chromosome-negative MPNs [17]. The so-called "driver" mutations activate the JAK-STAT signaling pathway. According to previous reports, about 85.0% of PMF patients have affected one of the three genes: JAK2 (60.0-65.0%), MPL (5.0%) or CALR (20.0-25.0%) [14]. In our study, 20 patients with MF were analyzed for JAK2 mutation by quantitative PCR; and 12 of them carried the mutant allele (60.0%), which is in accordance with the data reported by other researchers [4]. The remaining patients (12 individuals) who were accepted with an initial diagnosis of MF, five were confirmed to carry ET, six with PV and one remained an unclassified MPN. This showed that trepanobiopsy is absolutely essential for the final precision of the diagnosis.

The discovery of the genetic transformation of the JAK2 gene in the majority of our cases with MF makes possible their treatment with Jakafi (Ruloxitinib), which is an approved targeted drug for MF. It is indicated both for primary and secondary MF, either high- or intermediate-risk groups. It helps to reduce side effects such as splenomegaly, abdominal and bone pain. At the same time, it controls the blood levels of the inflammatory cytokines.

The JAK2 V617F mutation leads to constitutive activation of JAK/ STAT signaling [9]. As the JAK2 mutation is present in only about 60.0% of patients with MF, it could not be considered to be the only reason for this pathology. The triggering event of MF is not yet clarified and other mutations besides JAK2 have recently been studied in relation to MF. They include mutations in genes such as IDH1, IDH2, ASXL1, SRSF2 and EZH2 [2]. In 2019, Wilms tumor 1 (WT1) expression was investigated in 152 patients with MPNs [18]. The progression of ET and PV to MF was accompanied by increased levels of WT1 gene expression. Mutations in a tumor-suppressor gene, TP53, have also been detected in MPN, especially on older patients [19]. Some cytogenetic transformations (e.g., additional chromosome 8, inversion of chromosome 3 and rearrangements of chromosome 11) have been considered unfavorable markers for MF [20].

Even though the number of MF patients in our study are quite few (20 individuals), we could underline the slight prevalence of males in the mutant allele carriers (seven men vs. five women), while in the group of non carriers, females prevailed slightly (three men vs. five women). As for the age of the patients, the registered difference between carriers and non carriers of the JAK2 V617F mutation was 1 year (mean age of carriers:non carriers = 61.4:62.4 years). Whether the male gender and younger age are factors that contribute to JAK2 mutational status is disputable and has to be further investigated in a larger cohort of MF patients. We have not had a case in which the JAK2 mutation occurs simultaneously with other mutations, as it generally occurs as a single event. However, it could be associated with a different severity of symptoms from asymptomatic ET to MF [19]. We could speculate that other factors rather than genetic could modify the severity of the disease (for example age, gender, comorbidity, microenvironment in the bone marrow, etc.) [19].

The frequency of MF in our cohort of patients (232 individuals) was estimated to be 8.6% (20 individuals). The distribution was equal between genders and the mean age (63.95 years) approaches the mean age given by other researchers (65 years). The majority of MF patients (60.0% or 12 individuals) were JAK2 V617F mutation-positive and thereby treatable by Jakafi (Ruxolitinib). For the other 40.0% of cases (eight individuals), treatment options were limited. They included erythropoiesis-stimulating agents, hydroxyurea and immunomodulatory drugs, which have unconvincing effect in improving patients' survival.