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Prevalence of MPL (W515K/L) Mutations in Patients with Negative-JAK2 (V617F) Myeloproliferative Neoplasm in North-East of Iran.

Background and Objective: Janus kinase 2 (JAK2) and Myeloproliferative Leukemia (MPL) mutations are confirmatory indicators for Myeloproliferative Neoplasm (MPN). The current study was performed to determine the frequency of MPL mutation in MPN patients without JAK2 mutation, in order to assign MPL mutation frequency in North-East of Iran. Methods: Total of 105 negative JAK2 cases including 5 Myeloproliferative Disorders (MPD), 15 Polycytemia Vera (PV) and 15 Essential Thrombocytosis (ET) who referred to Qaem Medical Center were assigned to this study. ARMS-PCR was carried out for measuring MPL mutations. Results: A significant difference was observed between MPL mutant and non-mutant groups from overview of MPL mutation (P=0.00001). From the total studied population, 14.28% were ET cases and 4.71% of them had splenomegaly. About 66.66% had thrombocytosis and 33.33% of all the individuals had leukocytosis according to WHO criteria, and 4.76% of non-MPL mutant individuals had splenomegaly (P=1). This mutation was reported in 4-6% of ET and PMF individuals. In this research, 4.76 % of studied individuals had MPL (W515L/K) mutation, which were diagnosed with ET. Conclusion: Generally, the presence of JAK2 and MPL mutations are the most important criteria for MPN diagnosis. The obtained frequency of MPL mutation was similar to previous studies. Despite the high frequency of JAK2 and Philadelphia abnormality, MPL mutation was rare in myeloprolifrative disorders. Further studies are suggested to investigate its prognostic effects for these diseases.


Introduction
Myeloproliferative neoplasms (MPNs) are defined as diseases which occur due to unlimited reproduction of mature blood cells. It is associated with hematopoietic stem cells changes due to deregulation of signaling pathways (1, 2). Moreover, some of the current myeloproliferative disorders such as Essential Thrombocythemia (ET), Polycythemia Vera (PV), Primary Myelofibrosis (PMF) and Chronic Myeloid Leukemia (CML) (2) are accompanied with thrombotic and hemorrhagic complications (3); they may be transformed to acute leukemia in 4-6% of cases (2).
Some molecular changes such as translocations and mutations are known as infinitive markers for MPN diagnosis. The Janus kinase 2 (JAK2) mutations are confirming indicators of MPN approved by the World Health Organization (WHO) (4); It is a member of Jak Tyrosine kinase which is an important factor in cell proliferation. This mutation was detected in 2005 and introduced for classical MPN categorization, which is as important as the Philadelphia chromosome abnormality. Jak2 (V617F) is located in exon 14 of Jak2 gene. It can be applied for negative BCR-ABL MPN detection (5). This marker was positive in 50-60% of ET and PMF and 90% of PV patients (5,6). Generally, proliferative disorders are acquired; however, some cases are hereditary such as heredity thrombocytosis (HT) (7).
In addition to JAk2 (V617F), autosomal recessive MPL (Myeloproliferative Leukemia) mutation, affects normal regulation of hematopoietic genes. This oncogenic gene belongs to hematopoietic family (8), which is situated in 1p34 position and encodes thrombopoetin receptor. For the first time, MPL (W515L/K) mutation was discovered in PMF negative BCR-ABL patients in 2006 (9). Furthermore, the most current type of MPL mutations in PMF and ET cases occurred in Tryptophan 515 (10). Different types of MPL mutations were MPL515L, MPL515K, MPL515R and MPL515A in which, Leucin, Lysine, Argenine and Alanine are the substitutes of Tryptophan in the situation of 515 MPL gene (11). In Iranian population, the frequency of MPL mutation was 1.7% among Philadelphia negative myeloproliferative disorders (12). Although several studies reported the frequency and prevalence of MPLW515L/K mutations in MPN patients; there were no report in the cited field for North-East of Iran. Consequently, this research was performed to determine the frequency of MPL mutation in MPN patients with no JAK2 mutation.

Materials and Methods
This study was performed in Cancer Molecular Pathology Research Center, Qaem Hospital, Mashhad University of Medical Sciences. One hundred and five patients were entered in this prospective casecontrol study; they were suspected of having ET, PV and Myeloproliferative disorder (MPD). They were negative from point of JAK2 mutation. All studied individuals were evaluated for MPN. Medical histories and laboratory reports of the studied subjects were examined and two Hemato-pathologists reviewed the archived slides from 2013 to 2015. The disease was confirmed or rejected by molecular studies, slide evaluation and clinical information. Five cases were MPD, 15 were PV and 15 were ET. All data were ob-tained from recorded background files; these histories included hematologic indices, clinical signs such as lymphadenopathy, hepatomegaly, splenomegaly and complete blood counts (CBC). Since data were extracted from available evidence files in the research center, no separate sampling was done from patients in this study, and archived samples were used for the test.
DNA was extracted as follows; after acquiring medical history, 2 ml of blood was taken and transferred to Ethylenediaminetetraacetic acid (EDTA-K2) tubes as anticoagulant. Extraction was carried out by QIAamp DNA Mini Kit (Qiagen, Germany) and conforming ficoll gradient centrifugation method. The extracted DNA concentration was evaluated by Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo, Finland). The approved DNA concentration range was 100-200 ng/µl. The samples with concentrations lower than 20 ng/µl were excluded from this research.

Results
One hundred and five JAk2 negative cases that referred to Qaem Medical Center were entered in this study. About 66.66 % (70) were male and 33.33% (35) were female. Five (4.76%) out of 105 from the studied population were MPL mutant. Moreover, a significant difference was seen between MPL mutated and non-mutated groups from overview of MPL mutation (P=0.00001). The mean ages of all studied individuals were 50.8±19.57. Female and male mean ages were 52.5±23.75 and 50.07±18.85, respectively. From the total studied population, 14.28% were ET and 4.71% of them had splenomegaly. In addition, 66.66% had thrombocytosis and 33.33% of whole individuals had leukocytosis according to WHO criteria. Thrombocytosis and leukocytosis were reported in all mutated cases. Moreover, 40.00% and 65.00% of non-mutant group members had leukocytosis (P=0.013) and thrombocytosis, respectively (P=0.16). Platelet count was meaningfully higher in both groups in comparison with normal individuals (P<0.05). Leukocyte and erythrocyte counts were higher and lower in MPL mutated group according to the standard population, respectively. No splenomegaly was reported in MPL mutant group. About 4.76% of non-MPL mutant individuals had splenomegaly (P=1). Statistical analysis of hematologic parameters by Mann-withney test are displayed in Table 1.
Exact fisher test was applied to distinguish among the statistical analysis of some myeloproliferative disease containing PV, ET and MPD. Related information is described in Table 2.

Discussion
MPL is an oncogene which belongs to the hematopoietic family. Different MPL mutations affect normal hematopoiesis mechanisms, thus, it can be reported in many hematopoietic diseases. More than 95 MPL mutations have been discovered, which affect the treatment process and intervention (14,15). JAK2 is only positive in 50% of ET and PMF patients; MPL is another helpful marker for MPN suspicious cases (11). Generally, JAK2 and MPL mutations are the most important criteria for MPN diagnosis (16).
This study was designed to assign MPL mutation frequency in North-East of Iran. This mutation was reported in 4-6% of ET and PMF individuals. Molecular mechanism of negative JAK2 and MPL were vague and these two mentioned mutations were fruit-ful for disease process assessment (2). Other molecular markers such as TET2, ASXL1, IDH1, IDH2 and c-CBL have been suggested for MPN evaluation, but their prognostic values were undetermined (2,4). This mutation has been observed in ET and PMF cases but not in other disorders such as CML, PV, AML and Myelodysplastic syndrome (MDS) (3). However, Akpınar et al. reported MPL W515L/K in 4.8% of negative JAK2 cases (17). Furthermore, in an investigation to determine the frequency of different mutations in Chinese primary Myelofibrosis subjects, Xia et al. revealed that 6.7% of their studied cases, who were negative for Jak2V617F, carried MPLW515L/K mutation (18). However, compared to the mentioned studies, in the current research a total of 4.76 % of the studied individuals had MPL (W515L/K) mutation (19), which did not have JAK2 mutation. Thrombo-cytosis and leukocytosis were apperceived in 100% of MPL positive cases in our study. Siemiątkowska reported that MPL mutation was associated with thrombocytosis, which confirmed this research finding (20).

Conclusion
Despite high frequency of JAK2 and Philadelphia abnormalities, MPL mutation is rare in myeloproliferative disorders. Our findings were similar to other studies regarding the incidence of MPL mutation. However, our suggestion is to conduct more studies in larger population, patients follow up and mutant cases survival rate determination.