Incidence in Ph-Negative Myeloproliferative Neoplasms in Armenia from 2005 to 2019

Introduction: Enhancements of laboratory diagnostics and the emergence of new therapies had a significant impact on the incidence, prevalence and survival of patients with myeloproliferative neoplasms (MPN). Published epidemiology data are scarce, and multiple sources are needed to assess the disease burden. The aim of our work was to identify the patterns and trends of incidence, prevalence and survival of patients with MPN in the Republic of Armenia (RA) for the period 2005-2019. Methods: The data from Hematology Center blood diseases register, Oncological Center cancer register, as well as the data from death registration were the basis of our research. Demographic data were obtained from National Statistical Office of RA. Results: Analysis of the data obtained has shown that during the reporting period the average annual incidence of MPN was 1.84 per 100.000 inhabitants, including 2.1 for males and 1.64 for females. Analysis of incidence rates of MPN in relation to sex and age in the period under study revealed high rates in patients in groups 65 74 (8.3) and 55 64 (5.12 per 100 thousand years), respectively. According to the data obtained in the group of patients with MPN, the high annual average incidence rates are noted in primary myelofibrosis (PMF) (1.09 in 2018) and polycythemia vera (PV) (0.89 in 2016), the lowest for essential thrombocythemia (ET) (0.7 in 2016) per 100.000 population, respectively. In comparing our data to those obtained for 1966-1971 and 1998-2004 periods, one may detect a statistically significant increase in the total incidence of PMF and PV (p < 0.001). Conclusions: Analysis of the incidence rate in MPNs adjusted for age and gender shown the prevalence in group 65 74 (8.3) and in group 55 64 (5.13) per 100,000 inhabitants. The peak of incidence rate for both males and females was the age 65 74 and the male female incidence ratio in this age group was 11.3:6.2. The increasing incidence rate in MPNs in Armenia depends on the improvement of laboratory How to cite this paper: Lusine, S., Anahit, T.-G., Maria, B., Anahit, S., Alla, S. and Samvel, D. (2020) Incidence in Ph-Negative Myeloproliferative Neoplasms in Armenia from 2005 to 2019. Open Journal of Epidemiology, 10, 355-368. https://doi.org/10.4236/ojepi.2020.104029 Received: June 30, 2020 Accepted: September 6, 2020 Published: September 9, 2020 Copyright © 2020 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Improvement of laboratory diagnostics and the detection of new specific molecular abnormalities, such as Janus kinase 2 (Jak2 V617F), created the prerequisites for the revision of WHO 2008 diagnostic criteria, according to which several clonal Ph-negative neoplasms were combined into a group of chronic myeloproliferative diseases or neoplasms (MPN) [1] [2] [3] [4]. The most frequent MPN include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Less common conditions encompass systemic mastocytosis, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, and chronic neutrophilic leukemia. The origin of MPN is associated with the transformation of the hematopoietic stem cell, resulting in excessive production of mature cells of erythroid, megakaryocytic and granulocyte lineages with a relatively long course of the disease [5] [6] [7]. In contrast to chronic myeloid leukemia, these diseases are characterized by the absence of a specific genetic marker: the Philadelphia chromosome (Ph)-t (9; 22) (q34; q11) and the fusion BCR-ABL gene [1] [5]. According to the literature data, there is a certain risk of family clustering of MPN [8] [9]. In the WHO system, patients with Ph-negative CML may be correctly classified as atypical CML (aCML) or, for cases that do not fit into any of these categories, MDS/MPD-unclassified. Collectively, these entities plus systemic mastocytosis (SM) are often referred to as "atypical" or "non-classic" MPD to distinguish them from the four "classical" MPD.
PV, the most common MPD, originates from a single hematopoietic stem cell precursor. The principal molecular mechanism by which the abnormal PV clone develops and propagates is based on the ability of PV progenitor cells to form erythroid colonies in the absence of exogenous erythropoietin (Epo). In addition, the erythroid clone is hypersensitive to stimulating cytokines, such as insulin-like growth factor-1 or, to a lesser degree, interleukin-3 and granulocyte macrophage colony-stimulating factor, suggesting that this propagation occurs because of transcriptional dysregulation affecting a number of proteins involved in cytokine receptor signaling. Many pathophysiologic mechanisms of the molecular mechanisms of PV: the absence of an alternative product of the Epo re-ceptor (EpoR) gene, which inhibits EpoR-controlled erythropoiesis; overexpression of the PRV-1 gene, which belongs to the urokinase-type plasminogen activator receptor superfamily; deregulation of Bcl-x, which is an inhibitor of apoptosis; and increases of p16 protein and tyrosine phosphatase activity in red cell precursors [10].
Genome-wide scanning comparison of PV clonal and polyclonal cells from the same individuals revealed a loss of heterozygosity (LOH) in chromosome 9p, found in approximately 30% of PV patients. This LOH is not a result of a chromosomal deletion, but rather results from duplication of 1 parental allele and loss of the corresponding partner's allele, that is, uniparenteral disomy. Of note, the 9p region codes for the p16 and JAK2 tyrosine kinase gene [11]. The JAK family of protein tyrosine kinases is involved in cytokine receptor signaling, transmits the activating signal in the Epo-EpoR signaling pathway, and also is a required subunit for surface expression of cytokine receptors, including EpoR [12]. Ugo et al. have shown that inhibition of JAK2 abrogated Epo independence of erythroid progenitors [13]; this observation was followed by a description of the principal defect of the molecular basis of Ph-negative MPD, that is, a loss-of-function somatic mutation of an autoinhibitory JAK2 domain resulting from a valine-to-phenylalanine mutation at amino acid JAK2V617F. JAK2V617F leads to ongoing phosphorylation activity, which then can bind to a receptor and promote STAT recruitment and increased expression of PRV-1 [14]. This recruitment occurs in the absence of Epo [15]. This helps explain the known observed phenomenon of in vitro cytokine hypersensitivity. Furthermore, in a mouse bone marrow transplant model, murine marrow cells transduced with the JAK2 mutant allele led to a clinical phenotype consistent with PV, which included erythrocytosis, extramedullary hematopoiesis, and marrow fibrosis. This seminal work was rapidly confirmed by multiple groups, all of which demonstrated that >90% of patients with PV and lower but still significant proportions of patients with ET and IMF carry the JAK2V617F mutation [16]. However, contradictory data exist as to whether or not this mutation is classically dominant. A study of 193 patients without 9p LOH found that 66 of 193 were heterozygous and 127 did not have the JAK2V617F mutation [17], findings similar to other reports, which also confirmed that approximately 30% of PV patients were homozygous for JAK2V617F [14].
The JAK2V617F mutation also appears to play a role in IMF and ET. Approximately 50% -70% of ET patients and >50% of IMF patients have the JAK2V617F somatic mutation [18]. Presumably, this JAK2 mutation leads to a proliferation of clonal HSCs in these disorders through similar mechanisms as found in PV; however, the exact processes are not clear. Also, the fact that many of these patients do not have the JAK2V617F mutation raises other significant questions regarding etiology, specifically, what other mutations may exist that lead to these phenotypes in patients in whom the JAK2V617F mutation is not present. In this context, another important somatic mutation was identified in the transmembrane domain of the thrombopoietin receptor (cMPL) in 9% (4/45) patients with JAK2V617F-negative IMF. The murine model of this mutation, but not the wild-type MPL, led to a fully penetrant MPD characterized by marked thrombocytosis, marked splenomegaly, and increased reticulin fibrosis. Interestingly, and significantly, a small-molecule JAK inhibitor, in vitro, inhibited MPLW515L-mediated proliferation and JAK-STAT signaling. This recent work demonstrated that this mutation, MPLW515L, also activates the JAK-STAT signaling pathway, and that the likely presence of multiple other somatic and/or germ-line mutations besides JAK2V617F and MPLW515L will activate this signaling cascade and may account for the cases of JAK2V617F-negative IMF and ET. However, whether the JAK2V617F mutation is the PV disease initiating mutation is not clear; some PV JAK2V617F-negative patients have monoclonal hematopoiesis and Epo-independent erythropoiesis, and in some individual PV families there are PV subjects with and without the JAK2V617F mutation.
From a clinical point of view, patients with MPD have a more aggressive clinical course, poorer response to treatment, and shorter survival than those with typical CML. In a series of 76 cases from a single institution over a 32-year period, the median overall survival was only 2 years, with a median age at presentation of 66 years. Of those who died, one-third had transformed to myeloid blast crisis; the remainder died from other causes. Multivariate analysis indicated that age greater than 65 years, hemoglobin < 10 g/dl, and leukocyte count > 50 × 109/l were independent predictors of a poor prognosis and that these factors could be used to distinguish a high-risk group with a median survival of only 9 months from a lower-risk group with a median survival of 38 months [19].
Enhancements of laboratory diagnostics and the emergence of new therapies had a significant impact on incidence, prevalence and survival of patients with MPN. Published epidemiology data are scarce, and multiple sources are needed to assess the disease burden. The purpose of our study was to identify the patterns and trends of incidence, prevalence and survival of patients with MPN in the Republic of Armenia for the period 2005-2019.

Patients and Samples
The data from Hematology Center blood diseases register, Oncological Center cancer register, as well as the data from death registration were basis of our re- Demographic data were obtained from the National Statistical Office PA (http://www.armstat.am). The calculation of standardized indicators has been based upon the data from the Demographic compendium of Armenia from 2005 to 2019. All p values were two tailored and the level of significance was set at the level of p < 0.05. Continuous variables were shown as median and range. Categorical variables were described as frequency and percentage. Comparison between categorical variables was performed by Chisquared statistics. Comparison between categorical and continuous variables was performed by Mann-Whitney U-test or Kruskal-Wallis test, where appropriate. All parameters used for statistical analysis, except for those addressing prognosis (survival, leukemic or fibrotic transformation), were those obtained at the time of diagnosis and before any therapeutic intervention. These patients' clinical and biological characteristics are summarized in Tables 1-3. For each described parameter, we also give the number and the percentage of data available in the published population. The median age at diagnosis was 47.7 ± 2.7 years for ET patients, 58.9 ± 2.5 years for PMF patients, and 55.4 ± 3.1 years for PV patients. The percentage of female cases was also different between the three groups (63.7% in ET, 45.6% in PMF and 54.6% in PV).

Result and Discussion
The reason for the original consultation was unclear or unknown in most cases. At the time of the diagnosis, 65.9% of the ET patients, 54.4% of the PMF and 69.9% of PV were declared to be asymptomatic. Splenomegaly and hepatomegaly was the most frequent abnormal sign described in the papers: 50.5% and 45.1% of ET, 30.8% and 64.4% of PMF, 71.9% and 66.0% of PV patients had a palpable spleen.   The results of the full blood count at diagnosis are presented in Tables 1-3. For ET patients, the median leukocyte count was 10.6 × 10 9 /L, the hemoglobin concentration was 141.6 g/L and the platelet count was 1008.1 × 10 9 /L. For PMF patients, the median leukocyte count was 13.9 × 10 9 /L, the hemoglobin concentration was 107.5 g/L and the platelet count was 444.9 × 10 9 /L. For PV patients, the median leukocyte count was 13.2 × 10 9 /L, the hemoglobin concentration was 180 g/L (maximum level, 189 g/L), the maximum hematocrit was 72.5% and the platelet count was 799 × 10 9 /L. According to the International Prognostic Scoring System (IPSS) ( The average annual incidence of MPN was 1.84 per 100.000 inhabitants, including 2.10 for male and 1.64 for female. Linear analysis of the average annual incidence indicators of the MPN (Figure 1) revealed a tendency to a certain increase in the incidence in the period under study, both for male and female.
In different years, there were higher rates for male (2017, 2018, 2019), and female (2018, 2019). The overall incidence of MPN between male and female was 1.9:1.5, respectively. We also analyzed the incidence rates of patients with MPN (Table 5)  In general, there were no specific patterns of incidence depending on sex and age.
According to the data received, the increase in the incidence of MPN is par-      We tend to assume that in Armenia, as well as all over the world, with the improvement of the instrumentation of the diagnostic service, in particular with the extensive use of automated platelet count, the special attention of physicians was drawn to patients with their elevated rates, which is associated with such a sharp increase in the incidence rate MPN in recent years.
Analysis of incidence rates of MPN in relation to sex and age in the period under study revealed high rates in patients in groups 65 -74 (8.3) and 55 -64 (5.12 per 100 thousand years), respectively ( Figure 2). In both sex male and female with MPN, the incidence peak is at the age of 65 -74 years, the ratio of the incidence of male and female in this age group is 11.3: 6.2, respectively As for the indicators of ET, in previous studies in Armenia they were not covered. However, an analysis of the available literature [24] [25] revealed an increase in the incidence of ET after 2008 and other countries. Thus, in European Open Journal of Epidemiology   cases, ET 0.6 -2.1 and PMF 0.72 -1.56 cases per 100,000 population is described [27].
Taking into account the clonal markers, mainly the JAK2-V617F mutation, as well as the lower platelet count (450 × 109/L) in diagnosis, the frequency of ET after 2008 in different countries increased more than 2 times, both in male and in female [28] [29]. Reduction of the threshold value of platelets in the recommendations of WHO 2008, had little impact on the incidence rates of the PMF and PV. The JAK2 V617F mutation is found in 90% -95% of cases of PV, 50% -70% of cases of ET and 40% -50% of cases of PMF [28]. A number of authors attach great importance of screening for the JAK2 mutation in PV, which has  completely transformed the diagnostic approach to this disease: in current recommendations, when suspected of PV, screening of this mutation in peripheral blood in a set of diagnostic procedures is recommended [30]. Analysis of mor-  Figure 2). In both female and male with MPN, the incidence peak is at the age of 65 -74 years for all nosology (Figures 4-6).
Thus, for the study period, the average annual incidence of the MPN was 1.     Among thrombotic complications in patients with PV, arterial vascular thrombosis predominated-acute cerebrovascular accidents (25.8%), myocardial infarction (4.8), lower/deep vein thrombosis (DVT) of the lower extremities (3.0) and less common visceral vein thrombosis (3.5%). Thrombosis in patients with ET in 12.8% of cases is presented by stroke and myocardial infarction, in 6% DVT. In patients with PMF, in 12.1% of cases there was a stroke, in 10.5 -DVT and thrombosis of system v. portae/portal vein thrombosis, and in 3.9%myocardial infarction. The median time interval between the diagnosis of the disease and the development of thrombosis was 3 years (from 0 to 30 years). Repeated episodes of thrombotic complications were observed in 17.2% of patients undergoing thrombosis.
Interferon preparations as the first line of therapy were received by 42.5% of patients (17/40) with PV, 51.3% (20/39) with ET and 24% (6/25) with PMF. Hydroxyurea was prescribed in 30% (12/40) patients with PV, 23.1% (9/39) with ET and 44% (11/25) with PMF. In other cases, antiplatelet therapy and vascular therapy was performed. 8 patients are currently receiving targeted therapy with positive dynamics of the drug Ruxolitinib. Thrombotic complications were not observed in patients treated with Ruxolitinib therapy.

Conclusion
Analysis of the incidence rate in MPNs adjusted for age and gender shown prevalence in group 65 -74 (8.3) and in group 55 -64 (5.13) per 100,000 inhabitants. The peak of incidence rate for both males and females was the age 65 -74 and the male:female incidence ratio in this age group was 11.3:6.2. The increasing incidence rate in MPNs in Armenia depends on the improvement of laboratory diagnosis. Thrombotic complications are observed in patients with MPN in 45.3% of cases. In most cases, thrombosis is the first clinical symptom of a myeloproliferative disease, which determines the need for the introduction into clinical practice of molecular genetic testing methods among patients with thrombosis, an increase in blood levels, splenomegaly for the early diagnosis of clonal hematopoiesis and the use of a targeted drug.