Patients with primary myelofibrosis (PMF) have a poorer prognosis than those with other subtypes of myeloproliferative neoplasms (MPNs). To investigate the relationship between gene mutations and the prognosis of Japanese PMF patients, we analyzed mutations in 72 regions located in 14 MPN-relevant genes (CSF3R, MPL, JAK2, CALR, DNMT3A, TET2, EZH2, ASXL1, IDH1/2, SRSF2, SF3B1, U2AF1, and TP53) utilizing a target resequencing platform. In our cohort, ASXL1 mutations were more frequently detected in both overt and prefibrotic PMF patients than other mutations. The frequency of ASXL1 mutations was slightly higher among overt PMF patients than among prefibrotic PMF patients (44.6% vs 25.0%, FDR = 0.472). Decision tree classification algorithms revealed that ASXL1, EZH2, and SRSF2 mutations were associated with a poor prognosis for overt PMF. Overall survival was significantly shorter in patients harboring ASXL1, EZH2, or SRSF2 mutations than in those without these mutations (p = 0.03). These results suggest that, as reported in Western countries, MIPSS70 is applicable to Japanese PMF patients and ASXL1, EZH2, and SRSF2 mutations may be utilized as surrogate markers of a poor prognosis.

Keywords: Gene mutations; Myeloproliferative neoplasms; Primary myelofibrosis; Prognostic factors; Target resequencing.

Objective: to determine the frequency of major somatic mutations in the JAK2, MPL and CALR genes in the genomeof patients with Ph-negative myeloproliferative neoplasms that occur in individuals who have been exposed to ionizing radiation as a result of the Chornobyl accident.

Materials and methods: Molecular genetic analysis of genomic DNA samples isolated from blood was performed in90 patients with Ph-negative myeloproliferative neoplasia (MPN) with a history of radiation exposure and 191patients with spontaneous MPN utilizing allele-specific polymerase chain reaction (PCR).

Results: The presence of major mutations in the genes JAK2, CALR and MPL was revealed in patients with MPN witha history of radiation exposure with a frequency 58.9 % (53 of 90), 12.2 % (11 of 90), and 0 % respectively, and without exposure with frequency 75.4 % (144 of 191), 3.1 % (6 out of 191) and 1.6 % (3 out of 191) respectively.Mutations JAK2 V617F in patients with spontaneous MPN were observed in each clinical form: polycythemia vera (PV),essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations were detected exclusively inpatients with PMF and ET, significantly more often in groups with a radiation exposure history (18.9 % and 33.3 %,vs. 4.2 % and 6.5 %) than without one. At the same time, the occurence of MPL mutations was determined only inpatients with spontaneous MPN in 1.6 % of casees. Triple negative mutation status of genes JAK2, MPL and CALR prevailed in the group of patients with MPN with a history of radiation exposure and was 27.8 %, against 16.2 % inpatients without radiation exposure (p = 0.05).

Conclusions: Genomic research of patients with Ph-negative MPN revealed features of molecular genetic damage inthose patients who were exposed to IR as a result of the Chornobyl accident and those with spontaneous MPN. Thedata obtained by determining of JAK2, MPL and CALR genes mutational status in the genome of patients with MPN isnecessary to expand the understanding of the mechanism of leukogenesis, especially caused by radiation.

Meta: vyznachyty chastotu osnovnykh somatychnykh mutatsiĭ geniv JAK2, MPL ta CALR u genomi khvorykh na Ph-negatyvnimiieloproliferatyvni neoplaziï, shcho vynykaiut' v osib, iaki zaznaly diï ionizuiuchoï radiatsiï vnaslidok avariï na ChAES.Materialy i metody. Provedeno molekuliarno-genetychnyĭ analiz zrazkiv genomnoï DNK, vydilenykh z krovi 90khvorykh na Ph-negatyvni miieloproliferatyvni neoplaziï (MPN) z radiatsiieiu v anamnezi i 191 khvorogo na spontanniMPN metodom alel'-spetsyfichnoï polimeraznoï lantsiugovoï reaktsiï.Rezul'taty. Vyiavleno naiavnist' osnovnykh mutatsiĭ v genakh JAK2, MPL i CALR u khvorykh na MPN z radiatsiĭnym anamnezom z chastotoiu 58,9 % (53 iz 90), 12,2 % (11 z 90), ta 0 % vidpovidno, ta bez takogo – 75,4 % (144 zi 191), 3,1%(6 zi 191) ta 1,6% (3 zi 191) vidpovidno. Mutatsiï JAK2 V617F u khvorykh na spontanni MPN sposterigalysia v kozhniĭnozologichniĭ formi: spravzhniĭ politsytemiï (SP), esentsial'niĭ trombotsytemiï (ET) i pervynnomu miielofibrozi(PMF). Mutatsiï CALR vyiavlialysia vykliuchno u khvorykh na PMF ta ET, dostovirno chastishe u grupakh z radiatsiĭnymanamnezom (18,9 %, ta 33,3 %, proty 4,2 % ta 6,5 %), nizh bez takogo. Vodnochas chastota mutatsiĭ MPL vyznachenatil'ky u khvorykh na spontanni MPN – u 1,6 %. Potriĭno negatyvnyĭ mutatsiĭnyĭ status geniv JAK2, MPL ta CALR prevaliuvav u grupi khvorykh na MPN z radiatsiĭnym anamnezom i skladav 27,8 %, proty 16,2 %, bez takogo (r = 0,05).Vysnovky. Doslidzhennia genomu khvorykh na Ph-negatyvni MPN vyiavyly osoblyvosti molekuliarno-genetychnykhposhkodzhen' u tykh khvorykh, iaki zaznaly vplyvu ionizuiuchoï radiatsiï vnaslidok avariï na ChAES i tykh, khto zakhvorivspontanno. Otrymani dani mutatsiĭnogo statusu geniv JAK2, MPL ta CALR v genomi khvorykh na MPN ie neobkhidnymy dliarozshyrennia uiavlen' shchodo mekhanizmu leĭkogenezu, osoblyvo sprychynenogo radiatsiieiu.

Keywords: JAK2 V617F; MPL and CALR; essential thrombocythemia; ionizing radiation; myeloproliferative neoplasia; polycythemia vera; primary myelofibrosis.

Myeloproliferative neoplasms - Update on diagnosis and treatment Abstract. Myeloproliferative neoplasms are hematopoietic stem cell disorders presenting as chronic leukemias with excessive production of mature, myeloid blood cells. Driver mutations in JAK2, CALR or MPL mediate constitutive activation of JAK2 signaling, a common hallmark of the classical Philadelphia chromosome-negative MPN including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Dysregulated hematopoiesis primarily presents with polyglobulia in PV, thrombocytosis in ET and progressive bone marrow fibrosis and increased, atypical megakaryocytes in PMF. The molecular characterization of MPNs has advanced our understanding of their pathogenesis and has facilitated diagnosis. Implementation of genetic markers enables improved prognostication, particularly in myelofibrosis. In recent years, we have seen an encouraging increase in therapeutic options for MPN including the approval of a first JAK inhibitor now followed by other agents of this class, as well as refined forms of interferon alpha. Combination therapies as well as novel therapeutic approaches are increasingly studied and hold promise for the future. Hematopoietic stem cell transplantation, which is still the only treatment option with a curative potential, is increasingly available also for elderly patients with MPN.

Essential thrombocytosis (ET) is a chronic myeloproliferative neoplasm characterized by the presence of thrombocytosis and it can be complicated by thrombotic and/or hemorrhagic events. Treatment options include low-dose aspirin and cytoreductive agents such as hydroxyurea. In cases of extreme thrombocytosis, therapeutic thrombocytapheresis can be a useful procedure. We present a case of a 61-year-old-man previously diagnosed with CALR-mutated ET, who develop acute myeloid leukemia. When recovering after induction chemotherapy, he developed an extreme thrombocytosis up to 2337 × 10⁹ /L regardless hydroxyurea was started. Two therapeutic trombocytapheresis were performed and anagrelide was added to cytoreductive regimen. Platelet count stabilized around 570 × 10⁹ /L. Both procedures were performed with the Spectra Optia Apheresis System version 11.3 (Terumo BCT) and we decided to use a higher collection preference and lower collection speed than manufacturer's recommendations. Platelet count decreased from 2380 × 10⁹ /L to 1035 × 10⁹ /L in the first procedure and from 1813 × 10⁹ /L to 768 × 10⁹ in the second procedure. Platelet collection efficiency was calculated to be 110.3% and 86.1% in the first and second thrombocytapheresis, respectively. Therapeutic thrombocytapheresis with Spectra Optia is a safe and efficient therapy to treat patients with primary thrombocytosis while effect of cytoreductive agents is attained. Platelet collection efficiency was calculated to be higher than previously reported. We suggest that changes in technical parameters such as a deeper aspiration point and/or lower collection speed may increase procedure's efficiency.

The present study assessed the criteria for initiating cytoreduction and response to conventional therapies in 1446 patients with essential thrombocythemia (ET), 267 (17%) of which were CALR-mutated. In low risk patients, time from diagnosis to cytoreduction was shorter in CALR-positive than in the other genotypes (2·8, 3·2, 7·4 and 12·5 years for CALR, MPL, JAK2V617F and TN, respectively, P < 0·0001). A total of 1104 (76%) patients received cytoreductive treatment with hydroxycarbamide (HC) (n = 977), anagrelide (n = 113), or others (n = 14). The estimated cumulative rates of complete haematological response (CR) at 12 months were 40 % and 67% in CALR and JAK2V617F genotypes, respectively. Median time to CR was 192 days for JAK2V617F, 343 for TN, 433 for MPL, and 705 for CALR genotypes (P < 0·0001). Duration of CR was shorter in CALR-mutated ET than in the remaining patients (P = 0·003). In CALR-positive patients, HC and anagrelide had similar efficacy in terms of response rates and duration. CALR-mutated patients developed resistance/intolerance to HC more frequently (5%, 23%, 27% and 15% for JAK2V617F, CALR, MPL and TN, respectively; P < 0·0001). In conclusion, conventional cytoreductive agents are less effective in CALR-mutated ET, highlighting the need for new treatment modalities and redefinition of haematologic targets for patients with this genotype.

Keywords: calreticulin mutation; essential thrombocythaemia; genotype; haematological response; myeloproliferative neoplasms; therapy.

OBJECTIVE

To explore the prevalence of CARL gene mutations and the mutation types in patients with essential thrombocythemia (ET), and to compare the patients clinical characteristics of CALR mutation with JAK2 V617F, MPL W515K mutation patients and triple negative group.

METHODS

The mutations of CALR gene at extron 9 and MPL W515K in 150 ET patients were detected by PCR amplification followed by direct sequencing of genomic DNA, the JAK2 V617F mutation by using allele specific PCR.

RESULTS

(1)The CALR mutations were found in 38 patients (25.3%) of 150 ET patients. A total of 4 types of CALR mutations were identified (type Ic.1092_1143del52bp, n=17; type II c.1154_1155insTTGTC, n=16; type III c.1094_1139del46bp, n=4; type IV c.1103_1136del34bp, n=1). (2)The incidence of JAK2 V617F and MPL W515K was 61.3% (92/150) and 2.7% (4/150), respectively. The frequency of CALR mutation was 70.4% (38/54) in 54 ET patients without JAK2 V617F and MPL W515K mutations. The co-occurrence of any two kinds of gene mutations was not detected. (3)The hemoglobin level and leukocyte counts of patients with CARL mutations were significantly lower than that in patients with JAK2 V617F mutations (P<0.05). The median age of patients with CALR mutation was significantly higher than that of triple negative patients (59 years vs 29.5 years, P<0.01). Cytogenetic analysis was performed in 147 patients, and there were 4 abnormal karyotype cases. CALR mutation incidence was significantly higher in abnormal karyotype cases than that in normal ones (75% vs 24.5%, P=0.019).

CONCLUSIONS

The incidence of CALR mutations is high in ET patients without JAK2 V617F and MPL W515K mutations, and is associated with abnormal karyotype. CARL-mutated cases showed a significantly lower leucocyte and hemoglobin levels compared with JAK2 V617F mutated cases.

To review the updated trends of national practice and outcomes in transplanting myelofibrosis, we retrospectively evaluated 142 patients who underwent allogeneic hematopoietic stem cell transplantation for primary (n=94) or secondary (n=48) myelofibrosis (MF) at Australian/New Zealand transplant centers between 2006 and 2017. Median follow-up was 51.8 months (range: 3.1-148). Median age at allo-SCT was 56 years (range: 26-69). Fifty-two percent had HLA-identical sibling donors and 45% had matched unrelated donors (UD). Conditioning was predominantly reduced intensity (83%). Before transplant, 16% had splenectomy or splenic irradiation and 54 patients (38%) received JAK inhibitors. JAK2 mutation testing was performed in 66.9% of patients whilst other mutations CALR, MPL, ASXL1, SRSF2, U2AF1Q57, EZH2 and IDH1/2 were rarely tested (1.4-8.4%). Only 4.2% of patients had next generation sequencing mutation analysis. Median time to neutrophil engraftment was 19 days (range: 10-43) and median time to platelet engraftment was 27 days (range: 13-230). The cumulative incidences of grade II-IV acute graft-versus-host disease (GvHD) were 21.4% at 100 days and that of extensive chronic GvHD at 5 years was 18.1%. Overall survival (OS) was 67% at 1 year and 57% at 5 years. GvHD-free, relapse-free survival was 54% at 1 year and 42% at 5 years. The cumulative incidence of non-relapse mortality (NRM) was 16% at 100 days and 25% at 1 year. In multivariate analysis, age ≥ 65 years and use of an UD were significant unfavourable risk factors for OS and NRM. Use of an UD increased the incidence of acute GvHD whereas antithymocyte globulin/ alemtuzumab lowered the risk of both acute GvHD and chronic GvHD. Pretransplant splenectomy/splenic irradiation had a positive influence on time to engraftment. There have been no improvements in MF allo-SCT outcomes in Australasia in the last decades with low uptake of molecular genomic technology due to limited funded access.

Craniosynostosis, a condition in which the cranial sutures prematurely fuse, can lead to elevated intracranial pressure and craniofacial abnormalities in young children. Currently surgical intervention is the only therapeutic option for patients with this condition. Craniosynostosis has been associated with a variety of different gene mutations and chromosome anomalies. Here we describe three cases of partial deletion of chromosome 19p. Two of the cases present with syndromic craniosynostosis while one has metopic ridging. A review of the genes involved in the rearrangements between the three cases suggests several gene candidates for craniosynostosis. CALR and DAND5, BMP regulators involved in osteoblast differentiation, and MORG1, a mediator of osteoclast dysregulation may play a role in abnormal cranial vault development. Additionally, CACNA1A, a gene that when mutated is associated with epilepsy and CC2D1A, a gene associated with nonsyndromic mental retardation may contribute to additional phenotypic features seen in the patients we describe. In addition, these findings further support the need for genetic testing in cases of syndromic craniosynostosis.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineages. The current study demonstrates that three driver mutations were detected in 82.6% of 407 MPNs with a mutation distribution of JAK2 in 275 (67.6%), CALR in 55 (13.5%) and MPL in 6 (1.5%). The mutations were mutually exclusive in principle except in one patient with both CALR and MPL mutations. The driver mutation directed the pathologic features of MPNs, including lineage hyperplasia, laboratory findings and clinical presentation. JAK2-mutated MPN showed erythroid, granulocytic and/or megakaryocytic hyperplasia whereas CALR- and MPL-mutated MPNs displayed granulocytic and/or megakaryocytic hyperplasia. The lineage hyperplasia was closely associated with a higher mutant allele burden and peripheral cytosis. These findings corroborated that the lineage hyperplasia consisted of clonal proliferation of each hematopoietic lineage acquiring driver mutations. Our study has also demonstrated that bone marrow (BM) fibrosis was associated with disease progression. Patients with overt fibrosis (grade ⩾2) presented an increased mutant allele burden (P<0.001), an increase in chromosomal abnormalities (P<0.001) and a poor prognosis (P<0.001). Moreover, among patients with overt fibrosis, all patients with wild-type JAK2/CALR/MPL (triple-negative) showed genomic alterations by genome-wide microarray study and revealed the poorest overall survival, followed by JAK2-mutated MPNs. The genetic-pathologic characteristics provided the information for understanding disease pathogenesis and the progression of MPNs. The prognostic significance of the driver mutation and BM fibrosis suggests the necessity of a prospective therapeutic strategy to improve the clinical outcome.

Objective: To investigate the effect of other gene mutations outside the fusion gene on the first complete remission (CR₁) induced by one course of induction chemotherapy in patients with core binding factor-associated acute myeloid leukemia (CBF-AML).

Methods: DNA was extracted from bone marrow or peripheral blood samples of newly diagnosed CBF-AML patients admitted to the Hematology Department of the Second Hospital of Shanxi Medical University from January 2015 to January 2019. Next-generation sequencing was used for detection of 34 kinds of hematologic malignancy-related gene mutations in patients with CBF-AML, the effect of related gene mutations on the first complete remission (CR₁) rate in one course of induction chemotherapy was analyzed by combineation with clinical characteristics.

Results: 34 kinds of genes in bone marrow or peripheral blood of 43 patients were detected by high throughput sequencing and the gene mutations were detected in 16 out of 34 genes. The mutation rate of KIT gene was the highest (48.8%), followed by NRAS (16.3%), ASXL1 (16.3%), TET2 (11.6%), CSF3R (9.3%), FLT3 (9.3%), KRAS (7.0%). The detection rates of mutations in different functional genes were as follows: genes related with signal transduction pathway (KIT, FLT3, CSF3R, KRAS, NRAS, JAK2, CALR, SH2B3, CBL) had the highest mutation frequency (72.1% (31/43); epigenetic modification gene mutation frequency was 30.2% (13/43), including ASXL1, TET2, BCOR); transcriptional regulation gene mutation frequency was 7.0% (3/43), including ETV6, RUNX1, GATA2). Splicing factor related gene mutation frequency was 2.3% (1/43), including ZRSR2). The CR₁ rate was 74.4% after one course of induction chemotherapy. At first diagnosis, patients with low expression of WT1 (the median value of WT1 was 788.9) were more likely to get CR₁ (P=0.032) and the RFS of patients who got CR₁ after one course of induction chemotherapy was significantly longer than that of patients without CR₁ [7.6 (2.2-44.1) versus 5.8 (1-19.4), (P=0.048)]. The rate of CR₁ in the signal transduction pathway gene mutation group was significantly lower than that in non-mutation group (64.5% vs 100%) (P=0.045), while the level of serum hydroxybutyrate dehydrogenase (HBDH) was significantly higher than that in non-mutation group [(418 (154-2702) vs 246 (110-1068)] (P=0.032). There was no difference in CD56 expression between the two groups (P=0.053), which was limited to the difference between (≥20%) expression and non-expression. (P=0.048).

Conclusion: CBF-AML patients with signal transduction pathway gene mutation are often accompanied by high HBDH level and CD56 expression, moreover, the remission rate induced by one course of treatment is low.

题目: 信号传导通路基因突变对CBF-AML患者一个疗程诱导缓解率的影响及临床特征分析.

目的: 研究融合基因外其他基因突变对核心结合因子相关急性髓系白血病（CBF-AML）患者1个疗程诱导化疗达首次完全缓解的影响.

方法: 采集2015年1月至2019年1月在山西医科大学第二医院血液科收治的43例初发CBF-AML患者的骨髓或外周血标本，提取DNA，通过高通量测序检测34种常见血液肿瘤基因突变情 况；结合其临床特征，分析相关基因突变对1个疗程诱导化疗完全缓解（CR₁）率的影响.

结果: 43例初发CBF-AML患者初发时骨髓或外周血DNA均通过二代测序（NGS）检测了34种基因，其中16种基因存在突变，KIT基因突变率最高（48.8%），其次为NRAS（16.3%），ASXL1（16.3%），TET2（11.6%），CSF3R（9.3%），FLT3（9.3%）, KRAS（7.0%）。不同功能基因突变检出率依次为：信号传导通路基因（KIT、FLT3、CSF3R、KRAS、NRAS、JAK2、CALR、SH2B3、CBL）突变发生频率最高，为72.1%（31/43）；表观遗传修饰基因突变频率 为30.2%（13/43，包括ASXL1、TET2、BCOR）；转录调节基因突变频率为7.0%（3/43，包括ETV6、RUNX1、GATA2）；剪接因子相关基因为2.3%（1/43，包括ZRSR2）。经1个疗程诱导化疗CR₁率为74.4%。初诊时WT1低表达（表达量临界值选取WT1中位值788.9）患者经1个疗程诱导化疗更易获得CR₁（P=0.032），且1个疗程获CR₁ 组患者的RFS显著长于未获CR₁组患者[7.6（2.2-44.1）对5.8（1-19.4），（P=0.048）]。信号传导通路基因突变组的1个疗程诱导治疗CR₁率明显低于非信号传导通路基因突变组（64.5%对100%，P=0.045），但血清羟丁酸脱氢酶（HBDH）水平显著高于非突变组[418（154-2702）对246（110-1068），P=0.032]，2组CD56表达情况总体无差异（P=0.053），仅限于≥20%表达与不表达间存在差异（P=0.048）.

结论: 存在信号传导通路基因突变的CBF-AML患者常伴较高的HBDH水平和CD56表达，1疗程诱导缓解率低.

Myeloproliferative neoplasms including essential thrombocythemia (ET) is usually caused by somatic mutations in multiple genes, including the JAK2 (most frequently), CALR gene, and MPL. In rare cases, the disease is caused by other mutations such as THPO or TET2 gene; however, around 10-15% with ET might have triple-negative mutations. Here we present 2 cases of ET who were asymptomatic on diagnoses, but found to have extremely high platelet counts as never reported earlier. The management and treatment plan can be a challenging step. The objective is to draw attention to the early introduction of thrombocytapheresis in the management of such patients given its notable outcomes.

Keywords: Cytoreduction; Essential thrombocythemia; Myeloproliferative neoplasm; Thrombocytapheresis.

Calreticulin (CALR) is a chaperone protein that localizes primarily to the endoplasmic reticulum (ER) lumen where it is responsible for the control of proper folding of neo-synthesized glycoproteins and for the retention of calcium. Recently, mutations affecting exon 9 of the CALR gene have been described in approximately 40% of patients with myeloproliferative neoplasms (MPNs). Although the role of mutated CALR in the development of MPNs has begun to be clarified, there are still no data available on the function of wild-type (WT) CALR during physiological hematopoiesis. In order to shed light on the role of WT CALR during normal hematopoiesis, we performed gene silencing and overexpression experiments in Hematopoietic Stem Progenitor Cells (HSPCs). Our results showed that CALR overexpression is able to affect physiological hematopoiesis by enhancing both erythroid and megakaryocytic (MK) differentiation. In agreement with overexpression data, CALR silencing caused a significant decrease in both erythroid and MK differentiation of human HSPCs. Gene expression profiling (GEP) analysis showed that CALR is able to affect the expression of several genes involved in HSPCs differentiation towards both the erythroid and MK lineages. Moreover, GEP data also highlighted the modulation of several genes involved in ER stress response, unfolded protein response (UPR), DNA repair and of several genes already described to play a role in MPN development, such as pro-inflammatory cytokines and hematological neoplasms-related markers. Altogether, our data unraveled a new and unexpected role for CALR in the regulation of normal hematopoietic differentiation. Moreover, by showing the impact of CALR on the expression of genes involved in several biological processes already described in cellular transformation, our data strongly suggest a more complex role for CALR in MPN development that goes beyond the activation of the THPO receptor and involves ER stress response, UPR and DNA repair.

Genomic characterization of patients with myeloproliferative neoplasms (MPN) may lead to better diagnostic classification, prognostic assessment, and treatment decisions. These goals are particularly important in myelofibrosis (MF). We performed target Next Generation Sequencing for a panel of 255 genes and Chromosome Microarray Analysis (CMA) in 27 patients with MF. Patients were classified according to genomic findings and we compared the performance of a personalized prognostication system with IPSS, MIPSS70 and MIPSS70 + v2. Twenty-six patients presented mutations: 11.1% had single driver mutations in either JAK2, CALR or MPL; 85.2% had mutations in non-restricted genes (median: 2 per patient). CMA was abnormal in 91.7% of the 24 cases with available data. Copy-Number-Neutral Loss-of-Heterozygosity was the most common finding (66.7%). Del13q was the most frequent copy number variation, and we could define a 2.4 Mb minimally affected region encompassing RB1, SUCLA2 and CLLS2 loci. The largest genomic subgroup consisted of patients with mutations in genes involved with chromatin organization and splicing control (40.7%) and the personalized system showed better concordance and accuracy than the other prognostic systems. Comprehensive genomic characterization reveals the striking genetic complexity of MF and, when combined with clinical data, led, in our cohort, to better prognostication performance.

Keywords: Cancer genes; Developing countries; Myeloproliferative neoplasms; Primary myelofibrosis; Prognostic factors.

Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. ABSTRACT: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.

In 2008, WHO made the JAK2V617F gene mutation as one of the specific molecular diagnostic markers of BCR/ABL-negative myeloproliferative neoplasms (MPN). In 2013 two research teams demonstrated that whole genome sequencing technology (WGS) was used to detect calreticulin gene mutation in essential thrombocythaemia (ET) and primary myelofibrosis (PMF) patients with JAK2V617F⁻ and MPL⁻ mutations. In this review, the relationship of CALR gene mutation with MPN is briefly summarized.

Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders, including polycythemia vera, essential thrombocytosis, and primary myelofibrosis, characterized by constitutive activation of JAK/STAT signaling. JAK2, MPL, and CALR mutations are considered "driver mutations" and are directly implicated in the disease pathogenesis by activation of JAK2/STAT signaling. In addition to these driver mutations, several other mutations in epigenome regulatory and RNA splicing molecules have been found. This genetic information, especially regarding driver mutations, is essential for the diagnosis of MPN. Furthermore, assessment of non-driver mutations is also becoming increasingly important for disease risk assessment and treatment strategy definition.

Mutations in the JAK2 gene are thought to underlie the development of chronic myeloproliferative neoplasms (cMPN). Indeed, ≥95% of polycythemia vera patients, and half or more of essential thrombocythemia and primary myelofibrosis (PMF) patients, harbor the JAK2V617F mutation. Besides the JAK2V617F mutation, the JAK2 exon 12 deletion, the MPLW515L/K, and CALR mutation have been discovered and shown to be involved in the pathogenesis of these diseases. Based on these advancements in the study of cMPN, the JAK2 inhibitor was developed as a new therapy for PMF. Moreover, recent advancements in our ability to diagnose cMPN have paralleled the development of large clinical trials for patients with cMPN. This article provides explanatory information from these large clinical trials that is useful for the actual clinical practice of caring for patients with cMPN in Japan.

Myeloproliferative neoplasm(MPN) is clonal hematopoietic stem cell disorder characterized by abnormal proliferation and expansion of one or more myeloid lineages. BCR-ABL-negative MPN includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutations of JAK2, CALR and MPL genes are involved in the pathogenesis of MPN that provided a more complete molecular diagnostic standard for MPN. More and more new mutated genes related to prognosis of MPN were discovered in the past few years, at same time it was found that cytokines were also involved in the genesis and development of MPN. These provide a theoretical basis for the diagnosis, risk stratification and treatment management of MPN. In this article, the mechanisms of MPN-related cytokines and mutated genes in the genesis and development of disease and prognosis characteristics are summarized.

Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders, including polycythemia vera, essential thrombocytosis, and primary myelofibrosis. The JAK2V617F mutation was identified in 2005, followed by the discovery of the JAK2 exon12, MPNW515 mutation, and CALR mutation. About 90% of patients with BCR/ABL negative MPNs have been shown to have one of these driver mutations. In addition, mutations in epigenetic regulators and RNA splicing genes were found to co-exist with driver mutations and to play critical roles in the disease progression of MPNs. Currently, evaluations of these gene mutations are essential for the diagnosis of MPNs, and are also necessary for estimating the clinical course and the risk of disease progression. Guidelines for the management of MPNs were based on the results of large clinical trials. Furthermore, recent advancements in understanding the pathogenesis of MPNs are anticipated to promote the development of MPN-targeted therapies such as JAK2 inhibitors. Clinical trials for patients with PMF and PV have confirmed the efficacies of JAK2 inhibitors.

The classical Philadelphia chromosome-negative myeloproliferative neoplasms consist of three main pathological and clinical entities with the recurrent JAK2 V617F mutation present in ∼98% of patients with polycythemia vera and ∼50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The recent discovery of mutations within the CALR gene in up to 80% of JAK2 V617F-negative ET and PMF patients compels employment of CALR mutational analysis for the molecular diagnosis of these diseases. Evidence derived from a retrospective audit of JAK2 V617F testing provides a framework for the proposal of a diagnostic algorithm that incorporates CALR mutation assessment. It is recommended that relevant clinical details, including a provisional morphological and clinical diagnosis, are provided and that exclusion of alternative causes of erythrocytosis, leucocytosis, or thrombocytosis is required before molecular testing. It should be acknowledged that over-requesting such investigations can impact on the clinical predictive value of these tests when considering the disease specificity of such mutations, with adherence to the diagnostic algorithm necessary to ensure appropriate use of resources.

The identification of mutually exclusive somatic mutations shared among myeloproliferative neoplasm (MPN) subtypes has provided a powerful tool for studying disease evolution. Clinical features, gene mutations, and survival over 18 years were analyzed in MPN patients. One hundred thirty-eight MPN patients were subcategorized according to MPN subtypes: essential thrombocythemia (ET, n = 41), polycythemia vera (PV, n = 56), primary myelofibrosis (PMF, n = 10), and MPN unclassified (MPN-U, n = 31). Patient characteristics included clinical parameters, overall survival, and mutational status of the JAK2, CALR, and MPL genes. We compared hematologic and clinical features of JAK2V617F-ET vs. CALR-mutated ET vs. JAK2V617F-PV patients. JAK2V617F-patients had higher values of erythrocytes, hemoglobin, and hematocrit compared to CALR-mutated patients (p < 0.05). The mutant allele burden in JAK2V617F-PV and JAK2V617F-ET patients directly correlated with erythrocyte, hemoglobin, and hematocrit values, but it inversely correlated with platelet count. Thus, mutant allele burden was an indicator of the clinical phenotype in JAK2V617F-MPN patients. OS was not affected by the mutational status. In general, mutated JAK2, CALR, and MPL genes left specific hematological signatures.