MPL Gene Mutations Research Articles

Quantification of IGF‐1 receptor is useful in the differential diagnosis of essential thrombocytosis from reactive thrombocytosis

To make a definite diagnosis of essential thrombocytosis (ET) from reactive thrombocytosis (RT), the most reliable criteria are the presence of driver mutations, namely JAK2, CALR, or MPL gene mutations. In the absence of these driver mutations, so-called triple-negative ET, the differential diagnosis could be difficult. Although bone marrow biopsy could be helpful, it may be difficult in some cases, to do gene sequence analysis to identify other clonal marker gene mutations than the driver mutations, as only very few were found. IGF-1R quantification by flow cytometry in mononuclear cells (MNC) from peripheral blood was performed in 33 patients with ET (untreated or off treatment with hydroxyurea), 28 patients with RT, and 16 normal volunteer controls. We found IGF-1R levels were significantly elevated in ET patients compared to RT patients or controls. A cutoff value of 253 was chosen from the logistic regression to predict each patient's group, a value ≥253 meant that a patient belonged to the ET group (sensitivity 96.4% and specificity 68.6%). We suggest that adding quantification of IGF-1R in blood MNC by flow cytometry is useful in differentiating ET from RT.

Quantification of IGF-1 Receptor Is Useful in the Differential Diagnosis of Essential Thrombocytosis from Reactive Thrombocytosis

To make a definite diagnosis of essential thrombocytosis (ET) from reactive thrombocytosis (RT), the most reliable criteria are the presence of driver mutations, namely JAK2, CALR, or MPL gene mutations. In the absence of these driver mutations, so-called triple-negative ET, the differential diagnosis could be difficult; bone marrow biopsy could be helpful, but in some cases it may be difficult, to do gene sequence analysis to find other clonal marker gene mutations than the driver mutations, only very few were found. We studied IGF-1R quantification by flow cytometry in mononuclear cells (MNC) from peripheral blood, a substitute for performing endogenous erythroid colony formation (EEC), in 33 patients with ET (untreated or off treatment with hydroxyurea), 28 patients with RT, and 16 normal volunteer controls. We found IGF-1R levels significantly elevated in ET patients compared to RT patients or controls. A cutoff value of 253 was chosen from the logistic regression to predict each patient's group, a value ≥253 meant that a patient belonged to the ET group (sensitivity of 68.6% and specificity 96.4.%). Therefore, we suggest adding quantification of IGF-1R in blood MNC by flow cytometry is useful in differentiating ET from RT , we suggest this maybe added to the minor criteria for diagnosing ET. Funding Statement: JCW received grants from Celgen Corp. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of Interests: The author declares no conflicting interests with the commercial funder relating to using products, consultancy, patents, product development, or market products. The other authors declare no competing financial interests. Ethics Approval Statement: Peripheral blood was obtained from patients with written informed consent, the protocol for which was approved by the IRB of Brookdale University Hospital.

Programmed Cell Death Receptor (PD-1) Ligand (PD-L1) expression in Philadelphia chromosome-negative myeloproliferative neoplasms

Programmed Cell Death Receptor (PD-1) and its Ligand (PD-L1) pathway inhibitor therapy has been explored in the field of oncology treatment mainly for solid tumors. In hematologic malignancies, there is limited information except for Hodgkin’s lymphoma, and there is even less information regarding myeloproliferative neoplasm (MPN). Therefore, we explored this by first measuring PD-1 and PD-L1 levels (percentage of positive cells) in 63 patients with Philadelphia chromosome-negative MPN (Ph(-) MPN), including 16 MF (12 PMF, 2 post-PV-MF, 2 post-ET-MF), 29 ET, and 18 PV. We found there was no significant difference in PD-1 or PD-L1 levels between the different MPN groups but that there was a significant difference when PV, ET and MF were grouped as MPN and compared with controls, of all immune cells including CD4+, CD8+, CD14+ and CD34+ progenitor cells. We further found a higher incidence of higher expression levels (more than 50% of cells with positive expression) of PD-1 and PD-L1 (20% and 26%, respectively) in the CD34+ cells; in contrast, we found a low incidence (0.08–1.8%) in the immune cells in MPN patients. PD-1 and PD-L1 levels were also measured by MFI methods, and we obtained similar results except the measurements by percentage appeared to be more sensitive than the MFI methods. We found no correlation between PD-1 and PD-L1 expression levels and clinical features including WBC, platelet counts, hemoglobin levels, presence or absence of the JAK2, MPL, or CALR gene mutation, or splenomegaly. Since MPN represents stem cell disorders, the presence of elevated expression of PD-1 and PD-L1 in these cells suggests that the exploration of PD-1 and PD-L1 pathway inhibitor therapy may be worthwhile in Ph(-) MPN.

Common Laboratory Findings with Clinical Correlation and Follow Up in Patients with Genetically Proven Myeloproliferative Neoplasms at Age 50 and Younger

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders with shared gene mutations, excess blood production and wide range of clinicopathological changes. JAK2, CALR and MPL gene mutation study has become one of the major criteria in the diagnosis of MPNs.

Programmed Cell Death Receptor (PD-1), PD-1 Ligand (PD-L1) Expression in Philadelphia Chromosome Negative Myeloid Neoplasms

Introduction : PD-1 and PD-L1 pathway inhibitor therapy has been extensively explored in the field of oncology treatment recently, however there is no information so far about its usefulness in myeloid neoplasms (MPN). We have previously reported on PD-1 and PD-L1 expression in Philadelphia chromosome negative myeloid neoplasm (MPN), but now that we have accumulated more cases , we wish to report the final results.Materials and Methods: 63 patients with Philadelphia chromosome negative MPN in total were studied which included 16 MF (14 PMF, 2 post-PV-MF, 2 post-ET-MF), 29 ET, 18 PV, and 25 normal volunteer controls.Flow CytometryStudies: Leukocytes were obtained from whole blood after RBC lysis using RBC lysis buffer (Qiagen). To quantify surface bound PD-1 and PDL-1, the leukocytes were incubated with CD279-APC and CD274-PE, as well as CD4-FITC, CD8-FITC, CD14-PCP, or CD34-FITC (BD Biosciences) and were subjected to flow cytometric analysis.Results: PD-1 and PD-L1 positive cells were expressed as a percentage in gated CD4bright, CD8+, CD14+, or CD34+ cells. We found that 1) there was no difference in PD-1 or PD-L1 levels among different groups of MPN, but there was a significant difference when they were grouped of PV, ET and MF as MPN and compared with controls in all the immune cells including CD4+, CD8+, CD14 + and,CD34+ cells.(Representative of CD4+ shown inFig(1a). 2) High expression levels (more than 50% cell positivity) was found on the CD34+ cells with PD-1 and PD-L1 expression of 20% and 26% in MPN patients respectively ( Fig 1b,1c) . 3) We also found that there was no correlation between of the PD-1 or , PD-L1 expression levels with clinical features including such as WBC counts , platelet counts, and hemoglobin levels, , or presence or absence of the JAK2 , MPL, or CALR gene mutations or splenomegalyConclusion: We have shown elevated increased expression levels of PD1 and PDL-1 on the immune cells and high expression ( more than 50%) in the CD34+ cells , suggest that PD-1 or , PD-L1 pathway inhibitor therapy maybe worthwhile in Philadelphia chromosome negative Ph(-) MPN .. [Display omitted] DisclosuresWang:celgene: Other: clinical trial.

Detection and Diagnostic Values of JAK2, CALR, MPL Gene Mutations in 208 Cases of BCR/ABL1 Negative Chronic Myeloproliferative Diseases

To detect the JAK2, CALR and MPL gene mutations in patients with BCR/ABL1 negative chronic myeloproliferative diseases（BCR/ABL1-CMPD）and to evaluate their diagnostic value. Two hundred and eight cases of BCR/ABL1-CMPD comprising of 146 cases of essential thrombocythemia（ET）, 37 cases of polycythemia vera（PV）and 25 cases of primary myelofibrosis（PMF）from March 2012 to December 2015 were enrolled in the BCR/ABL1-CMPD, while 124 cases of secondary thrombocythemia and 73 cases of secondary polycythemia were enrolled in the control group. The genomic DNA and total RNA Were isolated from bone marrow or peripheral blood, then the exons 12 to 20 of JAK2 gene, exon 10 of MPL gene and exons 3 to 9 of CALR gene were analyzed by using DNA sequencing. among 146 ET patients, the JAK2, CALR or MPL mutations were found in: 138 cases（94.5%）including 86 cases with JAK2V617F mutation（58.9%）and 2 cases（1.4%）with exon 12 of JAK2 mutations. CALR mutations were detected in 41 cases（28.1%）, among them type 1（c.1092_1143del）in 22 cases, type 2（c.1154_1155insTTGTC）in 11 cases, and type 5（c. 1091_1142del）, type 8（c.1104_1137del）, type 41(c.1107_1137del）, type 42(c.1125_1125del）in one case respectively. In addition, 4 cases were detected withother mutations of the CALR gene（c.1107_1115del, c.1111_1144 del, c.1101 A>C, c.1112_1117del）. Moreover, 9 cases harbored MPL mutations（6.2%）. Secondly, 31 patients were detected with JAK2V617F mutation（83.8%）in 37 cases of PV, and JAK2 exon 12 mutations were found in 2 cases（5.4%）. Besides, CALR mutations were detected in 2 cases（5.4%）, including 1 case of type I, the other of novel mutation of CALR. Thirdly, 19 in 25 cases of PMF were detected with JAK2V617F mutation（76%）, 2 cases with CALR mutations（8%）. 4 patients（16%）, JAK2, CALR or MPL mutations were not detected, but among them 3 cases were found harboring other genetic abnormalities. Fourthly, no mutations of JAK2, MPL and CALR genes were detected in 124 patients with secondary thrombocytosis and 73 cases with secondary polycythemia. Combined detection of JAK2, CALR and MPL gene mutations can cover the vast majority of patients with BCR/ABL1-negative myeloproliferative neoplasms. For higher frequencies of the mutations of CALR in ET patients, CALR mutation can be used as a new diagnostic marker in ET patients with JAK2 and MPL wild type.

Progress of CALR gene mutation in myeloproliferative neoplasm

Myeloproliferative neoplasm (MPN) is a kind of clone hematopoietic stem cell disease characterized by one or more myeloid cell lines hyperproliferation, including bcr-abl gene positive chronic myeloid leukemia (CML) and bcr-abl negative MPN, the later representing polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). A big stride has been made since the discovery of JAK2 and MPL gene mutations. However, the exact genetic basis of JAK2/MPL mutation double negative in MPN patients is still unclear. It has been reported recently that a new CALR mutation is discovered in the JAK2/MPL unmutated MPN patients who show unique clinical presentations, which provides a new diagnostic and prognosis-accessing criteria. The paper reviews CALR mutation and genetic mechanism mediating MPN. Key words: Myeloproliferative neoplasm; CALR mutation; Calreticulin

Analysis of gene mutation and risk factors of thrombosis in patients with bcr-abl negative myeloproliferative neoplasms

Objective To explore the relationship between gene mutation of JAK2 V617F, JAK2(exon12), CALR, MPL and clinical features of patients with bcr-abl negative myeloproliferative neoplasms (MPN), and to analyze the risk factors of thrombosis. Methods Clinical features and laboratory tests of 115 patients with bcr-abl negative MPN were analyzed retrospectively. 34 patients with thrombosis were treated as the observation group, and 81 patients without thrombosis were treated as the control group. Results Among 71 primary thrombocythemia cases, the white blood cell count (WBC) and hemoglobin level of JAK2 V617F positive group and CALR positive group was higher than that of 4 gene mutations in negative group (F= 5.835, P= 0.005; F= 3.405, P= 0.039). The incidence of splenomegaly in JAK2 V617F positive group and CALR positive group was higher than that of 4 gene mutations in negative group (χ 2= 16.902, P= 0.000 2; χ 2= 12.658, P= 0.001). The patients'proportion of JAK2 V617F positive, high hemoglobin level (male ≥160 g/L, female ≥150 g/L), hypertension and over 60 years old in the observation group was higher than that in the control group (χ 2= 5.585, P= 0.025; χ2= 4.909, P= 0.043; χ2= 8.891, P= 0.004; χ2= 15.933, P= 0.023). Conclusion The detection of JAK2 V617F, JAK2 (exon12), CALR and MPL gene mutations is helpful to the diagnosis of bcr-abl negative MPN; JAK2 V617F positive, high hemoglobin level, hypertension, and elderly age are risk factors of thrombosis. Key words: Myeloproliferative disorders; Genes; Mutation; Thromboembolism

Latest Research Advance of Myeloproliferative Diseases Related Genes-Review

JAK2, MPL and CALR gene mutations play an important role in the onset of myeloproliferative disease(MPD). The latest researches show that the difference of ATP binding ability between the wild type JAK2 protein and mutated JAK2 protein can help us understand the pathogenesis of the MPD further, and the clinical manifestation is related to the mutation burden of the JAK2. In some ET and PMF patients, research find the expression of MPL mutation, which can affects the progress of the disease by collaborating with the JAK2 mutation. CALR mutation is a gene related with the MPD that has been found recently. The pathogenesis of the CALR is similar to that of the JAK2, while there are some features in clinical manifestation comparing with the other mutations.

Clinical significance of JAK2、CALR and MPL gene mutations in 1 648 Philadelphia chromosome negative myeloproliferative neoplasms patients from a single center

目的研究Ph染色体阴性的慢性骨髓增殖性肿瘤（MPN）患者JAK2、CALR及MPL基因突变情况，比较不同类型基因突变及突变阴性患者部分临床参数的差异。方法采用基因测序法分别检测1 648例Ph阴性MPN患者JAK2V617F、JAK2基因12号外显子、CALR基因9号外显子及MPL基因10号外显子突变情况。结果①JAK2V617F突变总检出率为82.8%（1 364/1 648），其中真性红细胞增多症（PV）为92.7%（471/508），原发性血小板增多症（ET）为78.1%（819/1 049），原发性骨髓纤维化（PMF）为81.3%（74/91）；JAK2基因12号外显子突变总检出率为0.5%（9/1 648），均为PV患者[1.7%（9/508）]；CALR基因突变总检出率为8.7%（143/1 648）,其中ET为12.6%（132/1 049），PMF为12.1%（11/91）；MPL基因突变总检出率为0.6%（10/1 648）,其中ET为0.9%（9/1 049），PMF为1.1%（1/91）。未发现两种及以上基因突变共存病例。②JAK2V617F突变组中位年龄[61（15~95）岁]高于JAK2 12号外显子突变组[49（33~62）岁]及突变阴性组[42（3~78）岁]（P值均<0.001），与CALR突变组[57（17~89）岁]、MPL突变组[59（22~71）岁]比较差异无统计学意义（P>0.05）。JAK2V617F突变阳性组白细胞计数、血红蛋白浓度均高于CALR突变阳性及阴性组（P<0.05），而与MPL突变阳性组比较仅表现为高白细胞计数（P=0.013）；CALR突变组血小板计数高于JAK2V617F突变组[966（400~2 069）×109/L对800（198~3 730）×109/L，P<0.001]。③共1 160例患者进行了有效染色体核型分析，CALR基因突变阳性、阴性患者异常核型检出率差异无统计学意义[9.8%（8/82）对7.4%（80/1 078），P=0.441]；JAK2V617F突变阳性、阴性患者异常核型检出率差异无统计学意义[7.7%（75/971）对6.9%（13/189），P=0.688]。CALR突变阳性组与JAK2V617F突变阳性组的异常核型检出率差异无统计学意义[9.8%（8/82）对7.7%（75/971），P=0.512]。7例JAK2基因12号外显子突变阳性及6例MPL基因突变阳性患者均未发现异常核型。结论基因检测使MPN的诊断及预后判断有了更可靠的依据，不同MPN亚型基因突变的发生频率、分布情况不同，不同基因突变导致独特的临床表型。

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia.

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×109/l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR.

Congenital Amegakaryocytic Thrombocytopenia Type II Presenting with Multiple Central Nervous System Anomalies.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare autosomal recessive bone marrow failure, caused by MPL gene mutations. The combination of CAMT and central nervous system abnormalities is uncommon. We describe a case with a homozygous missense MPL gene mutation and polymicrogyria, underdevelopment of the cerebellum, and multiple intracranial hemorrhages.

Mutational profiling in patients with myeloproliferative neoplasms: ready for every-day use in the clinic?

The novel second or next-generation sequencing (NGS) technique enables comprehensive mutational profiling analysis in patients with myeloproliferative neoplasms (MPN). JAK2, MPL and CALR gene mutations are successfully introduced into diagnostic practice, mutations in genes may impact outcomes such as survival and risk of leukemic transformation, therefore, molecular mutation analysis is ready for use in all patients with suspected MPN. Key words: Myeloproliferative neoplasms; Gene mutation; Diagnosis; Prognosis

Analysis of CALR, JAK2 and MPL gene mutations in BCR-ABL negative myeloproliferative neoplasms

To explore the mutational status of CALR, JAK2 and MPL genes in BCR-ABL negative myeloproliferative neoplasms (MPN) patients and the clinical features of MPN patients with these mutations. A total of 246 patients with a definite diagnosis of BCR-ABL negative MPN were enrolled from January 2009 to January 2014 into this study. Among them, there were 48 cases of polycythemia vera (PV) patients, 171 cases of essential thrombocythemia (ET) patients and 27 cases of primary myelofibrosis (PMF) patients. And CALR, JAK2 V617F, 12 exons of JAK2 and MPL W515L/K genes were amplified by PCR and sequenced directly. Clinical features were also analyzed in patients. Among 246 cases of BCR-ABL-negative MPN patients, 52 cases (21.1%) had CALR mutation, 121 cases (49.2%) JAK2 V617F mutation, 0 case (0) 12 exons of JAK2 mutation, and 2 cases (0.8%) MPL W515L/K mutation, respectively. These mutations were found existing exclusively. In PV patients, the white blood cell and platelet counts in JAK2 V617F mutated group were higher than those in wild-type JAK2 V617F group, while the level of hemoglobin was higher in wild-type JAK2 V617F group (all P<0.05). In ET patients, the white blood cell count, the level of hemoglobin, the frequency of thromboembolic events and risk stratification in JAK2 V617F mutated group were higher than those in CALR mutated group (all P<0.05). In PMF patients, the level of hemoglobin in JAK2 V617F mutated group were significantly higher than those in CALR mutated group (P<0.05). The proliferative level of bone marrow, risk of thromboembolic events and stratification are lower in CALR mutated patients than those in JAK2 V617F mutated patients. The pathogenic mechanism of mutated gene should be further investigated in future.

Isolation, Expansion and Characterization of BM-Mesenchimal Cells (MSCs) in Patients Affected By Chronic Myeloproliferative neoplasm (MPN)

Introduction MSCs constitute a pivotal cell type capable of shaping both the architecture of the microenvironment and modulating communication between the various cell types through effects on the extracellular matrix (ECM) and by secretion of various growth factors and cytokines. MSCs and hematopoietic stem cells are thought to share the same mesenchymal origin. Some data confirm that MSCs express a functional erythropoietin receptor and JAK2-transduction pathway, but their role in the development and evolution of MPN is still not well known so our aim was the isolation, expansion and characterization of MSCs in patients affected by MPN. Some data indicates that BM-MSCs of patients affected by MPN do not carry the JAK2-V617F mutation. We studied 20 patients affected by MPN with the following characteristics: M/F 12/8, median age 53years, 8 affected by PV, 8 by ET and 4 by PMF. 15 patients were positive for JAK2 V617F mutation, 1 pts for cMPL and 2 were CARL mutations carriers.Methods: MSC were isolated by bone marrow fraction by gradient separation on Lympholyte cell separation media and expanded in culture with a specific medium (MesenCult) in plastic-adherent cultures up to the second passage. DNA was extracted from MSC using QIAmp DNA Mini kit and the study of recurrent alterations (JAK2, MPL and CARL gene mutations was performed).Results: MCS were expanded in 14 on 21 patients. Flow citometry analysis confirmed the standard MSC phenotype (CD45 negative, CD73 positive, CD90 positive and CD105 positive). The molecular analysis of JAKV617F, cMPL and CARL mutations resulted negative in all analyzed samples both in patients carriers of mutations and in wild type ones.Conclusions: We conclude that common mutations markers of MPN neoplasm are absent in the mesenchymal compartment of bone marrows of patients affected by MPN and are restricted to the neoplastic clone.This research project was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.) DisclosuresNo relevant conflicts of interest to declare.

Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

Minimal morphological criteria for defining bone marrow dysplasia: a basis for clinical implementation of WHO classification of myelodysplastic syndromes

The World Health Organization classification of myelodysplastic syndromes (MDS) is based on morphological evaluation of marrow dysplasia. We performed a systematic review of cytological and histological data from 1150 patients with peripheral blood cytopenia. We analyzed the frequency and discriminant power of single morphological abnormalities. A score to define minimal morphological criteria associated to the presence of marrow dysplasia was developed. This score showed high sensitivity/specificity (>90%), acceptable reproducibility and was independently validated. The severity of granulocytic and megakaryocytic dysplasia significantly affected survival. A close association was found between ring sideroblasts and SF3B1 mutations, and between severe granulocytic dysplasia and mutation of ASXL1, RUNX1, TP53 and SRSF2 genes. In myeloid neoplasms with fibrosis, multilineage dysplasia, hypolobulated/multinucleated megakaryocytes and increased CD34+ progenitors in the absence of JAK2, MPL and CALR gene mutations were significantly associated with a myelodysplastic phenotype. In myeloid disorders with marrow hypoplasia, granulocytic and/or megakaryocytic dysplasia, increased CD34+ progenitors and chromosomal abnormalities are consistent with a diagnosis of MDS. The proposed morphological score may be useful to evaluate the presence of dysplasia in cases without a clearly objective myelodysplastic phenotype. The integration of cytological and histological parameters improves the identification of MDS cases among myeloid disorders with fibrosis and hypocellularity.

Two rare MPL gene mutations in patients with essential thrombocythemia

Two rare MPL gene mutations in patients with essential thrombocythemia

JAK2 and MPL gene mutations in V617F-negative myeloproliferative neoplasms

We report three novel mutations in JAK2 exons 12, 19 and 25 in V617F-negative patients with polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Scanning of JAK2 exons 12–25 and MPL exon 10 revealed the presence of JAK2 alterations in six and MPL W515L/K mutations in five of 34 patients with myeloproliferative disorders. Our results confirm that routine JAK2 analysis should include exon 12 mutations in polycythemia vera patients. MPL gene mutations seem to be associated with thrombocytosis, regardless of the type of myeloproliferative neoplasm.

Hereditary Thrombocythemia: Clinical Characteristics, Biological Markers and Long-Term Follow-up in 4 Families Observed in a Single Hematologic Pediatric Center

Hereditary thrombocytosis (HT) is an autosomal dominant disorder resulting from mutations involving the thrombopoietin (TPO) gene or its receptor gene, MPL. To date, 4 different mutations in the TPO gene and 2 in the MPL gene have been described. Although the clinical features of HT can resemble those of sporadic essential thrombocytemia (ET), different biological markers characterize the two conditions with different clinical and biological characteristics. The presence of JAK2 V617F and MPLW515L mutations identify familial ET (variable penetrance, clinical features and outcome similar to those of sporadic ET). The absence of JAK2 V617F mutations and the presence of MPLS505A or TPO gene mutations characterize HT (complete predominance, thrombocytosis, no organomegaly and benign clinical course). In our study, we analyzed the clinical and biological features of 17 members of 4 families (fam), diagnosed and followed at the study Institution between 1979 and 2007. The median follow-up is 79 months. Of 49 patients (pts) aged ≤20 years with a diagnosis of ET according to the PVSG or WHO criteria, we have identified 17 pts (M: 8; F: 9; median age 14 years) with HT (34.7%) through a detailed history. Peripheral blood samples were collected after informed consent from 11/17 pts: molecular analysis for the JAK2V617F, TPO (5′ untranslated region) and MPL (exon 10) gene mutations was performed. In addition, all pts were also evaluated for PRV-1 RNA overespression by qualitative RT-PCR, for endogenous erythroid colony (EECs) growth and all female patients were tested for clonality of hematopoiesis using the HUMARA polymorphism assay and the HUMARA methylation-specific PCR. The table summarizes the features, treatment and follow-up of the pts.Sex/Age at diagnosis (yrs)Plts at diagnosis (× 109/l)JAK2 V617FPRV-1EECsClonalityMPL mutationTreatmentFollow-up (FU) Plts (× 109/l)FU Duration (mo)Fam 1M/18900negnegneg-MPLS505ANOPLT≤1,000>330M/88/121,459negnegneg-MPLS505AATPLT≤500>79F/20934N.T.*N.T.N.T.N.T.N.T.AT; Anagrelide-> Vercite ->HU->IFNPLT≤500 Lost to FU184Fam 2M/18700negposneg-MPLS505AAT HUà stopC hepatitis PLT≤1,000>228M/114/12783negnegneg-MPLS505ANOPLT<1,000>39F/165/12963negposposPolyMPLS505ANOPLT≤1,500>129F/173/12695negposnegPolyMPLS505ANOPLT≤1,000>39F/92/121,230N.T.N.T.N.T.N.T.N.T.NOPLT≤1,000 Lost to FU47F/14932N.T.N.T.N.T.N.T.N.T.NOPLT≤1,000 Lost to FU42Fam 3F/149/121,060negposposPolyMPLS505AAT1 pregnancy PLT≤1,000>229M/03/121,528negpospos-MPLS505ANOPLT ≤1,500>44F/1010/12611negposnegPolyMPLS505ANOPLT<1,000>131M/103/12759N.T.N.T.N.T.N.T.N.T.NOPLT<1,000 Lost to FU49M/142,950N.T.N.T.N.T.N.T.N.T.Verciteà HUPLT<1,000 Lost to FU72F/38/121,430N.T.N.T.N.T.N.T.N.T.NOPLT<1,000 Lost to FU36Fam 4M/1911/12690negpospos-No mutationAT HUàAnagrelidePrevious splenectomy PLT<1,000>86F/178/121,118negnegnegPolyNo mutationAT, HU->Anagrelide->IFNPLT<1,000>144*N.T.=Not testedMild splenomegaly was present in 2 pts (family 4). All pts were negative for JAK2V617F and TPO gene mutations. All females were polyclonal. In 9 pts of families 1, 2 and 3 studied for MPL mutations, the MPLS505A mutation was found. No mutations within the TPO or MPL genes were found in the affected members of family 4. Overall, 6 pts received antiaggregant therapy (AT) and 5 pts were treated with hydroxyurea (HU), interferon (IFN), pipobroman (vercite) or anagrelide. No thromboembolic events occurred. To date, 2/11 evaluable pts (family 4) have required treatment because of a persistent high platelet count. Our data suggest that ET in young pts is most frequently a familial form. A detailed history and accurate biological marker studies are needed to identify, characterize and better manage HT.