Myeloproliferative Disease: An Unusual Cause of Raynaud’s Phenomenon and Digital Ischaemia

We describe a 59-year-old female who presented with ischaemic digits, preceded by a 6-month history of Raynaud’s phenomenon affecting her fingers and toes. There were no clinical or laboratory features of primary vasculitis or connective tissue disease, Doppler imaging was normal, and bloods were unremarkable aside from a platelet count of 786 × 109/L (150–400) and white cells of 16 × 109/L (4–11). In view of the thrombocytosis a JAK2 mutation assay was requested which confirmed a JAK2 V617F mutation, suggesting essential thrombocytosis (ET) as the cause. She received treatment with hydroxycarbamide which normalised her platelet count and led to a complete resolution of her Raynaud’s symptoms. Raynaud’s phenomenon is a rare manifestation of ET. Myeloproliferative disorders such as ET should be considered in the differential diagnosis of Raynaud’s phenomenon and vasculitis.

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JAK2 V617F in 3 Patients with Venous Thromboembolism (VTE) Followed during a Long Period of Time.

Blood ◽

10.1182/blood.v108.11.4092.4092 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4092-4092

Author(s):

Jacqueline Conard ◽

Christophe Marzac ◽

Fabrice Juin ◽

Marie-Helene Horellou ◽

Ors’Antone Calendini ◽

...

Keyword(s):

Platelet Count ◽

Melting Curve ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jugular Vein Thrombosis ◽

Jak2 Mutation ◽

Vein Thrombosis ◽

Hereditary Thrombophilia ◽

Cell Counts ◽

Thrombophilia Screening

Abstract Hereditary thrombophilia and myeloproliferative disorders (MPD) are risk factors of VTE. The JAK2 V617F mutation may be found in polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis. We report three women referred to our out-patient clinic for a thrombophilia screening. JAK2 mutation was studied using wild type FRET probes and subsequent analysis of the melting curve (James et al, Leukemia2006; 20: 350–353). Patient 1, 75 year-old (yo), had pulmonary embolism (PE) and proximal deep vein thrombosis (DVT) at 68. We detected heterozygous FV Leiden (FVL) and FII 20210A mutations. In 2005, she had internal jugular vein thrombosis after surgery (platelet count > 600 G/L). ET was suspected in 2006. Patient 2, 44 yo, on oral contraception, had PE and proximal DVT at 33 after sclerotherapy, recurrent massive PE requiring emergency embolectomy and proximal DVT during pregnancy. Heterozygous FVL was detected in 1995 (platelet count was 432 G/L) and ET in 2000. Patient 3, 65 yo, with PV detected in 1995, had a proximal DVT one year later. Heterozygous FVL was detected. She is the only patient treated with hydroxyurea. Results of blood cell counts and JAK2 V617F/JAK2 WT ratios are shown in the table. Conclusion 1. JAK2 mutation was found in 3 patients with VTE, platelet count >400 G/L and hereditary thrombophilia; 2. the risk of VTE related to JAK2 needs further investigation; 3. JAK2 V617F increased over time in 2 of 3 cases; 4. its early detection might improve the diagnosis and treatment of MPD and VTE. Patient Year Platelet (G/L) Hemoglobin (g/dl) Hematocrit (%) Leukocytes (G/L) JAK2 ratio 1 2000 431 15.3 45.9 8.6 10% 2006 448 13.9 43.3 10.4 50% 2 1997 541 14.1 42.6 6.1 25% 2005 796 14.0 42.8 7.4 15% 3 1997 501 14.1 42.6 4.6 15% 2003 609 13.6 42.1 5.8 20% 2006 485 12.8 37.9 4.8 50%

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Raynaud’s phenomenon and digital ischaemia - pharmacologic approach and alternative treatment options

VASA ◽

10.1024/0301-1526/a000526 ◽

2016 ◽

Vol 45 (3) ◽

pp. 201-212 ◽

Cited By ~ 13

Author(s):

Birgit Linnemann ◽

Matthias Erbe

Keyword(s):

Quality Of Life ◽

Angiotensin Receptor Blockers ◽

Raynaud’S Phenomenon ◽

Treatment Options ◽

Raynaud's Phenomenon ◽

Tissue Loss ◽

Receptor Antagonists ◽

Skin Ulcers ◽

Digital Ischaemia

Abstract. The primary goal of therapy is to reduce the frequency and intensity of Raynaud’s attacks and to minimize the related morbidity rather than to cure the underlying condition. Treatment strategies depend on whether Raynaud’s phenomenon (RP) is primary or secondary. All patients should be instructed about general measures to maintain body warmth and to avoid triggers of RP attacks. Pharmacologic intervention can be useful for patients with severe and frequent RP episodes that impair the patient’s quality of life. Calcium channel blockers are currently the most prescribed and studied medications for this purpose. There has been limited evidence for the efficacy of alpha-1-adrenergic receptor antagonists, angiotensin receptor blockers, topical nitrates or fluoxetine to treat RP. The intravenously administered prostacyclin analogue iloprost can reduce the frequency and severity of RP attacks and is considered a second-line therapy in patients with markedly impaired quality of life, critical digital ischaemia and skin ulcers who are at risk for substantial tissue loss and amputation. Phosphodiesterase inhibitors (e.g., sildenafil) can also improve RP symptoms and ulcer healing whereas endothelin-1 receptor antagonists (e.g., bosentan) are mainly considered treatment options in secondary prevention for patients with digital skin ulcers related to systemic sclerosis. However, their use in clinical practice has been limited by their high cost. Antiplatelet therapy with low-dose aspirin is recommended for all patients who suffer from secondary RP due to ischaemia caused by structural vessel damage. Anticoagulant therapy can be considered during the acute phase of digital ischaemia in patients with suspected vascular occlusive disease attributed to the occurrence of new thromboses. In patients with critical digital ischaemia, consideration should be given to hospitalisation, optimisation of medical treatment in accordance with the underlying disease and evaluation for a secondary, possibly reversible process that is causing or aggravating the clinical symptoms.

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Einsatz des niederenergetischen Laser bei Patienten mit primärem und sekundärem Raynaud-Phänomen

VASA ◽

10.1024/0301-1526.30.4.281 ◽

2001 ◽

Vol 30 (4) ◽

pp. 281-284 ◽

Cited By ~ 10

Author(s):

Mahdi Al-Awami ◽

M. Schillinger ◽

M. E. Gschwandtner ◽

T. Maca ◽

M. Haumer ◽

...

Keyword(s):

Raynaud’S Phenomenon ◽

Challenge Test ◽

Raynaud's Phenomenon ◽

Digital Ulcers ◽

Invasive Treatment ◽

Non Invasive ◽

Level Laser ◽

Digital Blood Flow ◽

Cold Challenge ◽

Digital Ischaemia

Background: Patients who had been treated with low level laser (LLL) for their digital ulcers reported an impressive improvement of their symptoms of episodic digital ischaemia. Therefore this pilot study was performed to evaluate the efficacy of LLL as a new non-drug non-invasive treatment for patients with primary and secondary Raynaud’s phenomenon (RP). Patients and methods: Forty patients (29 female, 11 male, mean age 51 years) with active primary (28%) and secondary (72%) Raynaud’s phenomenon received 10 sessions of LLL distant irradiation during winter months. Assessment of subjective and objective parameters was performed at baseline, one week after the last session and three months later. Variations of subjective parameters as number of daily acute episodes and severity of discomfort were assessed by a coloured visual analogue scale. A standardised cold challenge test using computed thermography of continuous temperature recordings by means of infrared telethermography was used to assess the digital blood flow. Results: A significant improvement was noticed clinically and thermographically after 6 weeks and 3 months, respectively (p < 0.0001). Conclusions: These data suggest that LLL treatment has a good short and medium term effectiveness in patients with Raynaud’s phenomenon.

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The Philadelphia Chromosome in an Unusual Case of Myeloproliferative Disease

Blood ◽

10.1182/blood.v26.4.471.471 ◽

1965 ◽

Vol 26 (4) ◽

pp. 471-478 ◽

Cited By ~ 21

Author(s):

CLARK W. HEATH ◽

WILLIAM C. MOLONEY

Keyword(s):

Ankylosing Spondylitis ◽

Direct Relationship ◽

Unusual Case ◽

Philadelphia Chromosome ◽

Myeloproliferative Disorders ◽

Clinical Findings ◽

Myeloproliferative Disease ◽

Diagnostic Significance ◽

History Of

Abstract In an unusual case of myeloproliferative disease, the Ph1 chromosome was found in association with persistently elevated levels of LAP activity. Clinical findings in this case included marked thrombocytosis, basophilocytosis, absence of splenomegaly and a preceding history of untreated ankylosing spondylitis. Cytogenetic findings were compatible with the existence of the Ph1 chromosome in erythroid and megakaryocytic as well as granulocytic marrow precursors. This case illustrates the difficulties currently encountered in the clinical differentiation of myeloproliferative disorders and in interpreting the diagnostic significance of the Ph1 chromosome. The co-existence in this case of the Ph1 chromosome and elevated LAP does not support the concept of a direct relationship between Group G chromosomes and LAP activity.

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Metatarsalgia Caused by an Increase in Circulating Platelets: A Case Report

Foot & Ankle ◽

10.1177/107110078400400414 ◽

1984 ◽

Vol 4 (4) ◽

pp. 216-217

Author(s):

Leon Rosenkranz ◽

M. Michael Cataletto

Keyword(s):

Platelet Count ◽

Case Report ◽

Essential Thrombocythemia ◽

Foot Pain ◽

Myeloproliferative Disease ◽

Disease Reduction ◽

History Of ◽

Work Up

A patient with a year-long history of metatarsalgia was found to have an elevated platelet count due to essential thrombocythemia, a benign myeloproliferative disease. Reduction of the platelet count with chemotherapy eliminated the foot pain. The authors recommend that a platelet count be part of the work-up of patients with metatarsalgia when the etiology of the foot pain is unclear.

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Distinct Hemostatic Profile of Leukocytes in Essential Thrombocythemia (ET) Carrying the JAK2 V617F Mutation.

Blood ◽

10.1182/blood.v106.11.378.378 ◽

2005 ◽

Vol 106 (11) ◽

pp. 378-378 ◽

Cited By ~ 1

Author(s):

Anna Falanga ◽

Marina Marchetti ◽

Donatella Balducci ◽

Alfonso Vignoli ◽

Laura Russo ◽

...

Keyword(s):

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Specific Marker ◽

Control Groups ◽

Jak2 Mutation ◽

Mutation Carriers ◽

Mixed Aggregates ◽

Platelet Aggregates ◽

And Control

Abstract The pathogenesis of the thrombotic diathesis of patients with ET is not completely clarified. Activation of polymorphonuclear leukocyte (PMN) occurs in these patients and is associated with a hypercoagulable state and increased number of circulating platelet/PMN aggregates. Further, increased procoagulant Tissue Factor (TF) expression in ET PMN is also reported. Recently, a gain-of-function JAK2 mutation (V617F) has been described in a high proportion of bcr/abl-negative myeloproliferative disorders. Specifically, in subjects with ET the mutation is present in about 50% of cases and retrospective data suggest an association with a higher rate of complications, including thrombosis. Aim of this study was to evaluate whether ET patients carrying the JAK2 mutation possess PMN with different hemostatic characteristics compared to ET subjects without JAK2 mutation and healthy controls. Twenty ET patients, 10 with and 10 without JAK2 mutation (median age: 50 years, range 32–61; platelet count: median 782 x 109/L, range 474–1,565 x 109/L), not receiving cytoreductive therapy (classified as low risk group), were included in the study; 16 age matched healthy subjects acted as controls. Expression of CD11b, TF and fibrinogen on PMN surface as well as PMN-platelet mixed aggregates (defined as the percentage of CD11b-positive PMN co-expressing a platelet-specific marker, i.e. CD42b or CD62P) were evaluated by whole blood flow-cytometry in both basal condition and after in vitro PMN stimulation by f-MLP. In washed isolated PMN samples the level of TF-mRNA was determined by RT-PCR. In basal conditions, significantly (p<0.01) increased levels of PMN/platelet aggregates (both CD42 and CD62P pos. PMN) compared to controls were found, independently from JAK2 mutation. Differently, PMN from JAK2 mutation carriers expressed significantly higher surface TF (18.2±9 %pos. cells) and fibrinogen (12.3±7 %pos. cells) antigens compared to non-carrier (TF: 11.6±5 %; fibrinogen: 7.2±2 %pos. cells) and control subjects (TF: 11.1±3 %; fibrinogen: 7.1±3 %pos. cells). In vitro stimulation with f-MLP increased the proportion of platelet/PMN aggregates (CD11b/CD42 and CD11b/CD62P) in all ET patients and controls. However, in JAK2 mutation carriers the levels of both CD62P and CD42b positive PMN were significantly greater than those of non-carrier ET patients and controls (p<0.05). Similarly, TF and fibrinogen levels on stimulated PMN surface were more elevated in the JAK2 mutation positive group compared to both the mutation negative and control groups. The analysis of PMN TF-mRNA showed a significantly higher expression in the whole ET patient group compared to controls, independently from the presence of JAK2 mutation. In conclusions, these data indicate that the expression of JAK2 mutation in ET patients may confer to PMN a different hemostatic phenotype in terms of increased interaction with platelets and increased expression of surface TF and fibrinogen, suggesting a new link of this mutation with a prothrombotic status.

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Myeloid Blasts in Transformed JAK2-V617F Positive Myeloproliferative Disorders Are Frequently Negative for the JAK2-V617F Mutation.

Blood ◽

10.1182/blood.v108.11.375.375 ◽

2006 ◽

Vol 108 (11) ◽

pp. 375-375 ◽

Cited By ~ 4

Author(s):

Alexandre Theocharides ◽

Marjorie Boissinot ◽

Richard Garand ◽

François Girodon ◽

Soon-Siong Teo ◽

...

Keyword(s):

De Novo ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

Jak2 Mutation ◽

Allelic Ratio ◽

Pcr Products ◽

Blast Cells ◽

V617f Mutation ◽

De Novo Aml

Abstract Acute myeloid leukemia (AML) is a common complication of myeloproliferative disorders (MPDs). The role of the JAK2-V617F mutation in this process is unknown. We performed a retrospective analysis of DNA samples from MPD patients with secondary AML. We analysed DNA samples taken at the time of transformation to AML from 54 MPD patients (24 PV, 21 ET, 9 IMF). In addition, DNA samples taken at diagnosis of MPD were obtained in 21 of these patients. DNA was extracted from bone marrow or peripheral blood films, purified granulocytes or frozen cells. FACS sorting of blast cells, T cells and neutrophils was performed in some of the samples. The allelic ratio of JAK2-V617F was determined by allele-specific quantitative PCR (AS-PCR). We obtained AS-PCR data on 52/54 samples taken at the time of transformation (96%), whereas 2 samples did not yield PCR products: 24/52 samples were negative for JAK2-V617F (46%) and 28/52 were positive (54%). For 14/24 negative patients (58%) we had additional DNA samples taken at the time of MPD diagnosis and interestingly, 5 of these 14 patients (36%) were positive for JAK2-V617F at this earlier time point before AML transformation. This suggests that in these patients the JAK2-V617F positive clone was lost during the evolution to AML. Furthermore, comparison of the JAK2-V617F allelic ratios with the percentage of blast cells in patient samples positive at transformation revealed 8/28 cases where the JAK2-V617F allelic ratio was markedly lower than the percentage of blasts, e.g. 8%T-allele and 52% myeloid blast cells. In these patients a JAK2-V617F negative AML clone most likely co-exists with a JAK2-V617F positive MPD clone. To address the question whether the AML clone arose independently from the JAK2-V617F clone, we analyzed loss of heterozygosity on chromosome 9p (9pLOH) in one informative patient who displayed a high allelic ratio of mutant JAK2 at diagnosis (94%T). The CD15+ cells from this patient showed 9pLOH at diagnosis, as demonstrated with two independent microsatellite markers. In contrast, the FACS sorted blast cells at the time of transformation contained both parental alleles in the 9p region and were JAK2-V617F negative by AS-PCR. This excludes the possibility that the AML clone lost the JAK2V617F in the process of undergoing mitotic recombination at a stage heterozygous for JAK2-V617F. Analysis of additional patients is under way. In summary, we found in a cohort of 54 MPD patients, 13 patients initially positive for JAK2-V617F that transformed into JAK2-V617F negative AML. Although not confirmed in the one patient analyzed, we cannot exclude that other patients the JAK2-V617F positive MPD clone lost the JAK2 mutation during the process of transformation. Alternatively, the AML clone could have developed de novo from a JAK2-V617F negative progenitor or stem cell. The latter model has difficulties explaining the high incidence of de novo AML (8/54 patients), unless the JAK2-V617F negative progenitor already carried an as yet unknown mutation and was part of the MPD clone.

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Correlation of JAK2 V617F Mutation Status and Cytogenetic Findings in Myeloproliferative Disorders.

Blood ◽

10.1182/blood.v108.11.3633.3633 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3633-3633

Author(s):

Guoxian Sun ◽

Frank Buccini ◽

Elizabeth Fuentes ◽

James Weisberger

Keyword(s):

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Chromosome Abnormalities ◽

Jak2 V617f Mutation ◽

Homozygous Mutation ◽

Jak2 Mutation ◽

Mutation Status ◽

Trisomy 9 ◽

V617f Mutation ◽

Trisomy 9P

Abstract Detection of JAK2 V617F mutation is quickly becoming a front-line screening test for suspected myeloproliferative disorders (MPDs), as the mutation shows high frequency and specificity in non-CML MPDs, PV, ET or CIMF. Routine cytogenetics can detect chromosome abnormalities in approximately 20% of MPDs and is very helpful in establishing or confirming the presence of aberrant clonality, although chromosome changes are often numerical gains and losses, deemed non-specific. To see if there is correlation between JAK2 mutation and karyotypes, we studied 57 consecutive patients with clinically and morphologically confirmed diagnosis of non-CML MPDs. JAK2 V617F mutation performed using allele-specific PCR (sensitive to 10% using pyrosequencing) was found in 72% of patients (41/57), whereas clonal chromosome abnormalities were observed in 15.8% (9/57). There was no correlation between JAK2 mutational status and karyotypes. In 41 patients positive for the JAK2 mutation, 6 were cytogenetically abnormal and 35 normal. In 16 patients negative for the mutation, 3 showed abnormal karyotypes and 13 had normal karyotypes (X2 test, p>0.5). Among 6 patients with both JAK2 mutation and an abnormal karyotype, JAK2 mutation was seen in >50% of each sample in 4 patients, consistent with a homozygous mutation. Interestingly, in two cases, one with PV and trisomy 9 and another with MPD unclassifiable and trisomy 9p resulting from an unbalanced translocation between chromosomes 9p and 13, JAK2 mutation was present in >65% of each sample. Trisomy 9 and trisomy 9p are common abnormalities in MPDs, particularly in PV, seen in over 20% of cytogenetically abnormal cases. JAK2 gene is located on 9p24. Mitotic recombination is considered the most likely cause of loss of heterozygosity (LOH) and thus mutant homozygosity which is undetectable at the cytogenetic level. However, in cases with trisomy 9 or 9p, the JAK2 allele genotypes may be G/T/T and/or T/T/T as well as the usual G/T and/or T/T. Our observations suggest that trisomy 9 or 9p should be taken into consideration when interpreting JAK2 mutation status and that further molecular studies are needed to delineate the implication of trisomy 9 or 9p in potential mutant allele selective advantage and clonal evolution in JAK2 mutation positive MPDs.

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Cultivation of Megakaryocytes On a Collagen Matrix: A More Sensitive and Reproducible Test for the Diagnosis of Patients with Essential Thrombocythaemia.

Blood ◽

10.1182/blood.v114.22.4970.4970 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4970-4970

Author(s):

Adrian Emanuel Schmidt ◽

Patricia Darlington ◽

Lucie Kopfstein ◽

Elisabeth Ischi ◽

Elisabeth Oppliger Leibundgut ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Negative Predictive Value ◽

Predictive Value ◽

Healthy Subjects ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Essential Thrombocythaemia ◽

Jak2 Mutation ◽

Spontaneous Proliferation

Abstract Abstract 4970 Background Essential thrombocythaemia (ET) is one of the chronic myeloproliferative neoplasms (MPN), along with polycythaemia vera (PV), primary myelofibrosis (PMF) and chronic myeloid leukaemia (CML). Their common feature is excessive proliferation of a certain stem or progenitor cell in the bone marrow; in the case of ET, the megakaryocytic lineage is affected. Clinical manifestations include thrombotic events and haemorrhage. Diagnosis of ET according to new WHO-criteria requires a sustained high platelet count, bone marrow biopsy showing proliferation of the megakaryocytic lineage with large and mature morphology, demonstration of JAK2 V617F (although only present in about 50% of patients with ET) or another clonal marker and explicit exclusion of other myeloid and myeloproliferative neoplasms as well as signs of reactive thrombocytosis. Additionally, spontaneous proliferation of megakaryocytes obtained from peripheral blood can be detected in in vitro culture assays. Presently, we use agar as a matrix for megakaryocyte cultivation, although this assay has never been validated in connection with ET. The identification of megakaryocytic colonies grown on agar can sometimes be quite difficult. Our aims were therefore to technically evaluate the use of a collagen based matrix and to investigate its suitability to identify patients with ET. Patients and Methods We have examined 63 patients (26 with ET, 21 with PV, 8 with myelofibrosis [MF; including PMF and post-ET/PV-MF], 6 with secondary or idiopathic erythrocytosis and 2 with secondary thrombocytosis; mean age=59.8, male=33, female=30, mean platelet count 457 G/l) and 5 healthy subjects. Following informed consent, both clinical and laboratory data was collected. Medication intake, phlebotomies, smoking habits and regular haemogram results were noted in order to recognise possible confounding factors influencing laboratory results. Results of megakaryocyte cultivation on both agar and collagen matrixes were recorded, considering both spontaneous growth and growth stimulated by megakaryocyte derived growth factor (MDGF). Results Based on our collagen culture results we were able to define 2 or more spontaneously grown megakaryocyte colonies as the most optimal cut-off for the identification of patients with MPN (sensitivity 71%, specificity 100% with positive and negative predictive values of 100% and 45%, respectively). Compared to the agar culture results (where a specificity and a positive predictive value of 100% were demonstrated at a cut-off value of ≥ 10 CFU-Mega) we found a higher accuracy and better reproducibility. In addition, we observed an improved negative predictive value (45% with collagen versus 25% with agar cultures) reducing false negative results. Healthy subjects and patients with secondary thrombocytosis showed no significant spontaneous megakaryocyte proliferation. In patients with MF, we observed strong spontaneous and MDGF-stimulated growth of megakaryocytic colonies. At a cut-off value of ≥ 50 CFU-Mega (after stimulation with MDGF), the collagen assay showed a sensitivity of 100% and a specifity of 70% for this special form of MPN, resulting in a negative predictive value of 100%. We found no confounding clinical or laboratory parameters such as medication intake (particularly cytoreductive treatment with hydroxyurea) or phlebotomies influencing our culture results, and no significant effect of the Jak2-V617F mutation on the growth behaviour of megakaryocytic colonies. Conclusion The results of this ongoing study imply that the collagen based assay is more sensitive, specific, time efficient and user friendly regarding the detection of spontaneous proliferation of megakaryocytes than the currently used agar based culture assay. In addition, the collagen based assay also has the great advantage that it allows isolation of single megakaryocytic colonies for further analyses, for example PCR-based identification of a JAK2 mutation. Furthermore, the collagen based assay facilitates the diagnosis of patients with MPN, especially in cases where conventional diagnostic criteria are lacking, such as in ET without a JAK2 mutation. Ultimately, the new assay may well be able to detect transformation from PV/ET to MF. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v118.21.2817.2817 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2817-2817

Author(s):

Michele Lambert ◽

Jing Jiang ◽

Wei Tong

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Bone Marrow Cells ◽

Novel Mutation ◽

Missense Variant ◽

Jak2 V617f ◽

Jak2 Mutation ◽

Hematopoietic Malignancies ◽

Healthy Control ◽

Pediatric Populations

Abstract Abstract 2817 Myeproliferative 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.

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