Pediatric Essential Thrombocythemia Research Articles

Essential Thrombocythemia in Children and Adolescents.

Simple SummaryAmong chronic Ph-negative myeloproliferative neoplasms, essential thrombocythemia is found in children with low but increasing incidence. The diagnostic and clinical features do not completely overlap with ET of adult age. A significant number of cases, in fact, do not meet the criteria of clonality, and many cases require extensive clinical evaluation to exclude secondary, reactive forms. Therefore, histological analysis of bone marrow biopsy is necessary, and its use should be enforced. The clinical course appears to be more benign, at least within the first decades of observation, with the incidence of thrombotic events being much lower than in adults (4 % vs. 30%). Hemorrhages are mostly irrelevant. Therefore, the management should be carefully adapted to the individual patient, balancing the risk of future complications with long-term collateral effects of any drug. This review analyzes the peculiarities of the disease facing similarities and differences with adult scenarios.This paper reviews the features of pediatric essential thrombocythemia (ET). ET is a rare disease in children, challenging pediatric and adult hematologists alike. The current WHO classification acknowledges classical Philadelphia-negative MPNs and defines diagnostic criteria, mainly encompassing adult cases. The presence of one of three driver mutations (JAK2V617F, CALR, and MPL mutations) represent the proof of clonality typical of ET. Pediatric ET cases are thus usually confronted by adult approaches. These can fit only some patients, because only 25–40% of cases present one of the driver mutations. The diagnosis of hereditary, familial thrombocytosis and the exclusion of reactive/secondary thrombocytosis must be part of the diagnostic process in children and can clarify most of the negative cases. Still, many children present a clinical, histological picture of ET, with a molecular triple wild-type status. Moreover, prognosis seems more benign, at least within the first few decades of follow-up. Thrombotic events are rare, and only minor hemorrhages are ordinarily observed. As per the management, the need to control symptoms must be balanced with the collateral effects of lifelong drug therapy. We conclude that these differences concert a compelling case for a very careful therapeutic approach and advocate for the importance of further cooperative studies.

Novel Insights Regarding Pediatric Essential Thrombocythemia

Background: Essential thrombocythemia (ET) is a chronic myeloproliferative neoplasm. As it is extremely rare in children, data regarding its clinical course are scarce and pediatric treatment guidelines are lacking. Aim: To evaluate diagnosis, treatment and clinical outcome in a group of pediatric ET patients. Methods: Medical files of all pediatric patients (age 0-18 years) diagnosed with ET between January 2010 and February 2019 in three tertiary hospitals were reviewed. Study was approved by all institutional ethics committees. Diagnosis was established according to the WHO criteria of ET. All patients had undergone bone marrow biopsy (BMB) and molecular evaluation for JAK2V617F. Patients with wild type JAK2V617F were also tested for JAK2 exon 12 mutation, calreticulin (CALR) mutations and thrombopoietin receptor (MPL) mutation. Complete blood count parameters at first evaluation and follow up were collected. Lag in diagnosis, defined as the period between the time at which thrombocytosis was first noticed until diagnosis of ET was documented. Patients were evaluated for acquired von Willebrand syndrome (AVWS) by testing for von Willebrand antigen level and activity. Clinical data included any adverse events particularly those related to thrombosis or bleeding. Initial treatment strategies and any need for therapy modifications were recorded. Results: Twelve children (5 males and 7 females) followed for a median time of 27.5 months (range 4-108 months) were included. Table 1 displays their demographic and clinical data. Family history of thrombocytosis was negative in all patients. Median age at which thrombocytosis was first noted was 8 years (range 1-14.5 years). In 5/12 patients thrombocytosis was detected as an incidental finding. In 7/12 patients CBC was performed due to symptoms including headache, visual disturbances, seizure and acroparesthesia (table 1). Patients who suffered from neurological symptoms had undergone cranial MRI; all were interpreted as normal. The mean lag period between the time in which thrombocytosis was first noted until diagnosis of ET was 36 months (range: 0.1-120 months). Molecular diagnosis yielded 5/12 patients who were positive for JAK2V617F, one patient with a JAK2 exon 12 mutation and 2/12 patients with mutations involving CALR (one with type 1 and one with type 2 mutation). No subjects with CMPL mutation were detected. Four children tested negative for all mutations. Bone marrow biopsies were compatible with ET and no chromosomal aberrations were identified in our cohort. Evaluation for AVWS was performed in nine of the panties. It was diagnosed in 67% of assessed patients. Median VWF:Rco/VWF:Ag 0.18 (range: 0.01-0.76). At diagnosis treatment with Aspirin was initiated in 4/12 patients. Cytoreductive therapy with Hydroxyurea was added at diagnosis in 2/4 patients, both symptomatic at presentation. One Patient underwent plateletpheresis at presentation due to severe headache and extreme thrombocytosis. In 3/8 untreated patients, therapy was added during follow up, with either Aspirin (n=1, due to increased severity of headaches and raising platelet count) or Hydroxyurea (n=2, following TIA). During follow up period neither leukemia nor myelofibrosis evolved in our cohort. One patient developed a provoked DVT, secondary to a femoral CVL. Three patients experienced TIA during study period. Two females experienced excessive bleeding (heavy menstrual bleedings and bleeding due to a raptured corpus luteum), both diagnosed with AVWS. Conclusions: Our study suggests that pediatric hematologists should increase awareness to ET as delayed diagnosis is common. Among children with ET, AVWS may be more prevalent as compared to adults and may increase the risk of bleeding. Further collaborative multicenter studies are required for robust data collection and may facilitate future ET treatment in children. Table 1 Disclosures Kenet: Alnylam: Consultancy, Honoraria, Research Funding; CSL: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Opko Biologics: Consultancy, Honoraria, Research Funding; BPL: Research Funding. Steinberg Shemer:Emendo bio: Consultancy. Revel-Vilk:Prevail therapeutics: Honoraria, Other: Travel, Research Funding; Sanofi: Honoraria, Other: Travel, Research Funding; Pfizer: Honoraria, Other: Travel, Research Funding; Takeda: Honoraria, Other: Travel, Research Funding.

Compound Heterozygosity of Two Novel JAK2 Mutations in Hereditary Essential Thrombocythemia Implicates Important Monomer-Monomer Interactions in Thrombopoiesis Signaling

Essential thrombocythemia (ET) is a myeloproliferative neoplasm that is characterized by pathological overproduction of platelets with an increased risk of thromboembolic events, progression to myelofibrosis and leukemic transformation. Mutations of the Janus Kinase 2 (JAK2) gene, most commonly JAK2V617F, are known driving factors of ET pathogenesis. However, the molecular processes involved in JAK2 activation and regulation and the specific mechanism(s) by which JAK2 mutations disrupt hematopoiesis remain unclear. Most mutations identified in hereditary ET involve the thrombopoietin or thrombopoietin receptor gene; only 4 germline JAK2 mutations have been reported, all of which are inherited in an autosomal dominant pattern. We report the first case in which 2 novel germline JAK2 mutations, JAK2L815P and JAK2V1123G, were found in the compound heterozygous state in 2 siblings with ET. Family members who carry either one of these variants have normal blood counts. The purpose of this study was to demonstrate causality and to explore the mechanism(s) by which these JAK2 mutations, in compound heterozygous state, result in thrombopoietic dysregulation.To study signaling in the JAK2-signal transducer and activator of transcription (STAT) pathway, wild-type (WT) and mutated forms of murine JAK2 were transduced into murine pro-B cells (Ba/F3) stably expressing the thrombopoietin receptor (MPL), Ba/F3-MPL cells. Immunoblotting analysis was performed on Ba/F3-MPL cells that were starved of cytokines overnight and then treated with 10 ng/mL thrombopoietin (TPO) or vehicle for 5 minutes. At baseline (under starved conditions), JAK2L815P + JAK2V1123G Ba/F3-MPL cells demonstrated increased STAT3 and STAT5 phosphorylation compared to WT JAK2 Ba/F3-MPL cells. However, JAK2 double mutant Ba/F3-MPL cells had less JAK2, STAT1 and STAT3 phosphorylation in comparison to JAK2V617F Ba/F3-MPL cells at baseline. After TPO stimulation, both JAK2 double mutant and JAK2V617F Ba/F3-MPL cells showed increased phosphorylated JAK2, STAT1 and STAT3 compared to WT JAK2 Ba/F3-MPL cells.To investigate JAK2-STAT signaling in the patients' cells, immunoblotting analyses of platelets and megakaryocytes grown in vitro from patients' CD34 cells were performed. Our patients' platelets demonstrated increased JAK2, STAT1, STAT3 and STAT5 phosphorylation compared to platelets from a normal control but less JAK2, STAT1 and STAT3 phosphorylation compared to the platelets from a pediatric ET patient with a known JAK2V617F mutation, a similar signaling pattern to that seen in JAK2 double mutant Ba/F3-MPL cells. Similarly, in vitro megakaryocytes from the patients' CD34 cells showed increased STAT5 activation compared to those from normal volunteers in the unstimulated state.To assess cell proliferative capability, IL-3 dependent Ba/F3 cells were starved of cytokines and viable cells were counted every 24 hours for a total of 72 hours. The JAK2V617F and JAK2 double mutant Ba/F3-MPL cells easily proliferated without cytokines and had a large fold-increase at 24, 48 and 72 hours compared to 0 hours. The JAK2L815P + JAK2V1123G Ba/F3-MPL cells had a significantly higher proliferation rate compared to JAK2V617F and WT JAK2 Ba/F3-MPL cells. As expected, the number of viable parental Ba/F3 cells declined over time in the absence of cytokines.To evaluate response to ruxolitinib, a JAK1 and JAK2 inhibitor, IL-3 independent Ba/F3-MPL cells were grown under starved conditions in the presence of 0 to 1 mM ruxolitinib. After 48 hours, the number of viable Ba/F3-MPL cells was measured as a percentage of the dimethylsulfoxide (DMSO) control. The 50% inhibitory concentration (IC50) value was higher in JAK2L815P + JAK2V1123G Ba/F3-MPL cells (147+/-20 nM) compared to JAK2V617F Ba/F3-MPL cells (93+/-1 nM).In the compound heterozygous state, the novel germline JAK2 mutations, JAK2L815P and JAK2V1123G, appear to function cooperatively to increase cell proliferation through activation of the JAK2-STAT pathway. This finding implicates that within the JAK2 dimer in the MPL-JAK2-STAT pathway, the monomer-monomer interactions are important in JAK2 activation and regulation. Additional in vitro and in vivo studies are currently underway to elucidate the exact mechanism(s) of thrombopoietic dysregulation resulting in the siblings' ET and to gain further insight into JAK2 activation and regulation. DisclosuresRaj:Novartis: Speakers Bureau. Kalfa:Baxter/Baxalta/Shire: Research Funding.

Two Pediatric Cases of Essential Thrombocytopenia Characterized By Extremely Rare Mutations (CALR and MPL W515K)

Essential Thrombocythemia (ET) is an extremely rare disorder during childhood, characterized by clonal expansion of megakaryocytic and thrombocytic lineages in bone marrow, leading to a persistent increase in the number of circulating thrombocytes and thus increasing the risk for thrombotic and hemorrhagic events. Most of children with ET have JAK2 V617F mutation (1). MPL and CALR mutastions have been reported in a small proportion of adult ET patients. However, it is extremely rare in children. Herein we present two cases of pediatric ET with different rare mutations.Case 1: 15-year old female presented with Budd-Chiari syndrome and thrombocytosis (1500x109/L). Bone marrow smear and biopsy were consistent with ET. To elucidate the molecular mechanisms involved in this case of ET, we investigated candidate gene alterations. BCRABL, JAK2 V617F and calreticulin mutations were negative. Thus we analyzed codon 515 of MPL gene. The patient was found to have mutation for MPL W515K (figure 1). The patient who presented with BCS was diagnosed as ET with MPL W515K mutation. Hydroxyurea therapy at a dose of 20mg/kg/day was started. After 10 days, enoxaparin dose was reduced to single dose daily (1mg/kg/day) as a maintenance anticoagulation. The patient underwent liver transplantation at the end of the 3rd month of treatment. Warfarin treatment was started to patient after transplantation.Case 2: Nine year old female was applied to our policlinic because of thrombocytosis identified in routine control (1300x109/L). Secondary causes of thrombocytosis were excluded. Bone marrow smear and biopsy were consistent with ET. We investigated candidate gene alterations. BCRABL, JAK2 V617F and MPL mutations were negative. CALR type I mutation was identified (figure 2). Hydroxyurea treatment was successfully reduced the platelet count.Discussion: Thrombocytosis occurs in about 6-15% of children, but ET is very rare in children (2). Most of cases with pediatric ET have JAK2V617F mutation. Our first case has MPLW515K mutation. To our knowledge, this is the first pediatric patients of ET mutated W515K. The other patient has CALR mutation. This was also extremely rare mutation in children (3). We believe that our two cases will contribute to the literature.

MPL W515L mutation in pediatric essential thrombocythemia

Essential thrombocythemia (ET) is extremely rare in the pediatric population. In most patients no molecular abnormality can be found, with about 40% of pediatric patients harboring a JAK2 V617F mutation. Another recurrent mutation, involving a W to L or K transversion at MPL codon 515, has been reported in about 3-8% of adult ET patients. Herein we describe this mutation in a pediatric patient.

Two cases of pediatric essential thrombocythemia managed effectively with hydroxyurea

Thrombocytosis is common in infancy and childhood. Essential thrombocythemia (ET), a myeloproliferative disorder, is a much less common cause of thrombocytosis in childhood. We report two cases of essential thrombocythemia in 5- and 10-year-old children, who presented with platelet counts of more than 1,000,000/mm(3). Treatment is not recommended for ET in an asymptomatic patient in the absence of bleeding or thrombosis and a platelet count <1,500,000/mm(3). Our first case had platelets >1,500,000/mm(3), and a second child was symptomatic with recurrent headache. Both responded well to therapy with hydroxyurea (dose 15-30 mg/kg/day) and tolerated it well.

A Novel Mutation in MPL (Y252H) Results in Increased Thrombopoietin Sensitivity and Moderate Thrombocytosis in a Pediatric Patient with Essential Thrombocytosis

Abstract▪2817▪This icon denotes a clinically relevant abstractMyeproliferative neoplams (MPNs) constitute a group of hematopoietic malignancies that feature enhanced proliferation and/or survival of one or more myeloid lineage cells, including Essential Thrombocythemia (ET). MPN development is rare in children with an estimated annual incidence of ET of 0.09/106 children. The WHO defines ET as persistent platelet count >600 k/mcL in the absence of known cause of reactive/secondary thrombocytosis. The JAK2 V617F mutation is most commonly reported in both children and adults with ET although the reported frequency varies in pediatric populations from 0 to 36% of patients. Mutations in the thrombopoietin receptor (TPO receptor or MPL) intracellular domain, specifically W515K/L, have also been reported in both adult and pediatric ET patients. Here we report a novel mutation in the MPL extracellular domain, Y252H, causing mild thrombocytosis. The patient presented at 2 years of age with a platelet count of 765 k/mcL. During the 3-year follow-up period, she possessed platelet counts between 600–700k/mcL, without any obvious indication of reactive/secondary thrombocytosis. Because of the persistently increased platelet count, her bone marrow was evaluated and it demonstrated increased numbers of megakaryocytes with focal clustering. JAK2 mutation analysis was negative and cytogenetics did not show any clonal abnormalities. Sequencing of the MPL gene showed a missense variant at c.754 T>C resulting in a tyr252his amino acid substitution. To investigate if this Y252H mutation in MPL dysregulates TPO/MPL- mediated cell growth, we introduced it into cytokine-dependent BaF3 cells. Cells stably expressing the mutant MPL allele showed increased proliferation to TPO, in particular at low concentration, in comparison to cells expressing wildtype (WT) MPL. Upon cytokine withdrawal, BaF3 cells expressing the MPL Y252H mutant survived better than that of WT MPL. Primary bone marrow cells from this patient along with the healthy control were subjected to colony forming unit -megakaryocyte (CFU-meg) assays in response to a serial dose of TPO. The Y252H MPL bone marrow showed significantly increased megakaryocyte colonies at low dose of TPO when compared to control bone marrow (17.5 ± 2.5 colonies versus 4.75 ± 1.1 colonies at 15 ng/mL TPO, p<0.001). These results are consistent with the clinically mild thrombocytosis. In summary, our results suggest a novel MPL mutation, Y252H, results in pediatric ET. Further evaluation of the mechanisms of increased TPO sensitivity imparted by this mutation should contribute to our understanding of the molecular pathogenesis of ET. Disclosures:Lambert:Cangene: Honoraria.

JAK2V617F Mutation-positive Childhood Essential Thrombocythemia Associated With Cerebral Venous Sinus Thrombosis

Myeloproliferative diseases (MPDs) in childhood are quite rare. Although pediatric and adult MPDs exhibit similar hematologic findings, JAK2V617F mutations and clonality status of MPDs in the DNA of neutrophils are evaluated less frequently in children than in adults. Increased incidence of venous thrombosis at uncommon sites is associated with JAK2V617F mutation in MPDs and thrombotic complications are more common in essential thrombocythemia (ET). Here, we describe 6-year-old girl with clonal myelopoiesis and JAK2V617F-positive ET associated with cerebral venous sinus thrombosis. To our knowledge, this is the first report of pediatric monoclonal and JAK2V617F-positive ET with cerebral venous sinus thrombosis.

Frequency and clinical features of the JAK2 V617F mutation in pediatric patients with sporadic essential thrombocythemia

Pediatric essential thrombocythemia (ET) is a rare and heterogenous disease entity. While several recent studies have focused on the role of the JAK2 V617F mutation in pediatric ET, the frequency of pediatric ET cases with this mutation and the associated clinical features remain unclear. We examined six childhood cases who had been diagnosed with ET according to WHO criteria (onset age: 0.2-14 years) for the presence of the JAK2 V617F mutation, MPLW515L mutation and JAK2 exon 12 mutations. Two sensitive PCR-based methods were used for the JAK2 V617F genotyping. We also examined the expression of polycythemia rubra vera-1 (PRV-1), which is a diagnostic marker for clonal ET. We found that three of the six cases had the JAK2 V617F mutation and that all six cases expressed PRV-1 in their peripheral granulocytes. Neither MPL W515L mutation nor JAK2 exon 12 mutations was detected in the patients without JAK2 V617F mutation. The two patients who developed thrombocythemia during infancy were JAK2 V617F-negative. These findings suggest that the JAK2 V617F mutation is not rare in childhood sporadic ET cases, and that these cases might be older and myeloproliferative features.

JAK2 Genotyping (Real-Time Allelic Discrimination) Reveals Rare JAK2 V617F-Positive Erythroid Colonies in Pediatric Patients with Essential Thrombocythemia.

Childhood essential thrombocythemia (ET) is a very rare disease belonging to a group of Bcr-Abl negative myeloproliferative disorders. Our study involved 15 pediatric ET patients diagnosed at the age of 6–17 years. Monoclonal hematopoiesis was detected only in one patient, who is recently an adult (diagnosed at the age of 9). JAK2 V617F was detected in whole blood DNA by allele-specific (AS)-PCR and BsaXI restriction analysis. In our hands AS-PCR sensitivity reached the detection limit of one homozygous cell in 2000 normal cells. In addition, more sensitive AS-RT-PCR was performed on RNA isolated from granulocytes and platelets from peripheral blood. Only one patient was JAK2 V617F-positive as detected by these established protocols. In the clonal assay of hematopoietic progenitors, most patients showed hypersensitivity to erythropoietin (Epo) in vitro with the growth of Epo-independent erythroid colonies (EECs) in some of them. Over 300 myeloid colonies were picked and analyzed by real-time allelic discrimination assay for JAK2 V617F mutation run on a RotorGene 3000 instrument (Corbet Research, Australia) using fluorescently labeled LNA-modified probes. We have detected a few JAK2 V617F-positive EECs and CFU-GMs in 4 patients from our cohort. For example in one patient out of 7 analyzed EECs, one colony was homozygous and one heterozygous for the mutation. In the same patient out of 10 analyzed CFU-GMs one was homozygous for the mutation. In another patient out of 27 analyzed EECs only one was heterozygous. Some positive clones were confirmed by direct sequencing. Our data clearly show that some childhood ET patients posses JAK2 V617F-positive hematopoietic colonies derived from in vitro assays of committed hematopoietic progenitors. The rarity of the mutated subclones and the inability to detect them from peripheral blood using standardized protocols suggest either that the culture conditions without addition of Epo led to a great enrichment of the mutated colony growth or that the JAK2 V617F positive clones may be repressed in ET. The presence of JAK2 V617F clone in our cohort was not associated with any severe clinical symptoms. It would be interesting to follow up the patients for possible subsequent acquisition of JAK2 mutation as well as possible evolution of the disease to a clonal one or to polycythemia vera.This work was supported by Ministry of Health of the Czech Republic (Grant NR9471-3), Ministry of Education, Youth and Sports of Czech Republic (projects MSN 6198959205 and MSN 6198959216).

Pediatric Essential Thrombocythemia (ET): Evaluation of Clonality Status.

ET is a very rare disease in pediatric age and some evidence suggest that ET in childhood may be a possible variant of the ET as described in adults. Clonal assays using X chromosome inactivation patterns (XCIPs) have been used to demonstrate the clonal origin of ET. However, about half women with ET seem to have a polyclonal XCIPs and the occurrence of thrombosis seems to be higher in patients with monoclonal XCIPs. We report 7 females in pediatric age affected with ET (mean age at diagnosis 9 years). The diagnosis of ET was made in agreement with the Polycythemia Vera Study Group Criteria. The main clinical data of our children are summarized in the table. None of the children had heavy haemorrhages nor splenomegaly. HUMARA trinucleotide repeated polymorphism in granulocytes DNA amplified using two primers flanking the STR, was used to assess myeloid clonality of our patients. The PCR products will be quantified after electrophoresis with density analysis. T lymphocytes DNA of the patients were also tested for the HUMARA polymorphism as control. Monoclonal granulocytes XCIPs was demonstrated in only 1 asymptomatic child who had polyclonal T-lymphocytes, while 5 other children had polyclonal XCIPs of both granulocytes and T-lymphocytes. Omozygous XCIP of 1 chlid did not permit clonality evaluation.In conclusion, as observed in adults, also children with ET seem to be heterogeneous concerning clonality status. The percentage of polyclonal cases seem to be higher in pediatric age if compared with adults (data of the literature). The sole patient with a major thrombotic complication is polyclonal. Again pediatric ET seems to be different than adults ET: more studies are needed to clarify the biological characteristics of pediatric ETageplts x 10to9/Lclinical symptomsTPO pg/mlclonality granulocytes/T lymphocytesCV9 m945Budd Chiari syndrome30polyclonal/polyclonalGM12 y1611headache30omozygousTF10 y1038headache9polyclonal/polyclonalFE6 y3266headache + purpura15polyclonal/polyclonalBC14 y1308asymptomatic25monoclonal/polyclonalGI9 y2718asymptomatic22polyclonal/polyclonal8 y1550asymptomatic64polyclonal/polyclonal

Cultures of myeloid progenitor cells in pediatric essential thrombocythemia

Cultures of myeloid progenitor cells in pediatric essential thrombocythemia