Long-term Use of Anagrelid in Essential Thrombocythemia Treatment of a Child Under 12 Months

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5072-5072
Author(s):  
Pawel Laguna ◽  
Anna Klukowska ◽  
Katarzyna Pawelec ◽  
Michal Matysiak
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Jak2 V617f ◽  
Biochemical Tests ◽  
Results Of Treatment ◽  
Life Threatening ◽  
Laboratory Investigations ◽  
Chronic Myeloproliferative Disorder

Abstract Abstract 5072 Essential thrombocythemia (ET) is currently classified as a chronic myeloproliferative disorder. Anagrelide is a novel platelet lowering agent that has recently been approved for use n ET. We present the case of ET in an 8 –month- old girl, who was treated with Anagrelid for 13 months. The girl was admitted to hospital because of elevated platelet count. She had stomach pains periodically. On admission the child's condition was good but on palpation her stomach was sore and her spleen was enlarged. Laboratory investigations showed an elevated platelet count (1. 5m) and hypercalemia (6. 3mEq/l). Thrombopoetin was normal. Acesan was added to the treatment. A morphology performed after 1 month revealed further elevation of platelets (up to 2. 65m). On the basis of laboratory investigations, bone-marrow biopsy and trepanobiopsy, we eliminated an oncological disease and infectious diseases of connective tissues. On bone-marrow investigation, acquired mutation of JAK2 (V617F) tyrosine kinase was diagnosed, which confirmed ET. On account of the growing number of platelets, which was life - threatening, we decided to administer anagrelid, starting with a dose of 0. 25 mg per day. The number of platelets decreased to 1. 4m, so the dose was increased to 0. 25 mg twice daily after 3 weeks. At present, after 37 months of anagrelide treatment, the number of platelets in the child ranges between 400 and 650. The patient visits our, department for monthly check-up (morphology, biochemical tests, ECG and echocardiography). In international literature there is no information on the use of Anagrelid in ET treatment of children under 12 months old. However, on the basis of our observations and initial results of treatment, it seems that the above protocol for Anagrelid administration is safe for infants of this age. Disclosures: No relevant conflicts of interest to declare.

Anagrelid In Essential Thrombocythaemia Treatment of a Patient Under 12 Months

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4680-4680
Author(s):  
Pawel Laguna ◽  
Michal Romiszewski ◽  
Anna Klukowska ◽  
Katarzyna Pawelec ◽  
Michal Matysiak
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Conflicts Of Interest ◽  
Jak2 V617f ◽  
Essential Thrombocythaemia ◽  
Biochemical Tests ◽  
Results Of Treatment ◽  
Life Threatening ◽  
Laboratory Investigations ◽  
Chronic Myeloproliferative Disorder

Abstract Abstract 4680 Essential thrombocythaemia (ET) is a chronic myeloproliferative disorder. It occurs mostly in adults and rarely in young children. We present the case of ET in an 8 –month- old girl, who was treated with Anagrelid for 13 months. The girl was admitted to the hospital because of elevated platelet count. She had stomach pains periodically. On admission the child's condition was good but on palpation her stomach was sore and her spleen was enlarged. Laboratory investigations showed an elevated platelet count (1.5m) and hypercalaemia (6.3mEq/l). Thrombopoetin was normal. Acesan was added to the treatment. A morphology performed after 1 month revealed further elevation of platelets (up to 2.65m). On the basis of the laboratory investigations, bone-marrow biopsy and trepanobiopsy, we eliminated an oncological disease and infectious diseases of connective tissues. On bone-marrow investigation, acquired mutation of JAK2 (V617F) tyrosine kinase was diagnosed, which confirmed ET. On account of the growing number of platelets, which was life - threatening, we decided to administer Anagrelid, starting with a dose of 0.25 mg per day. The number of platelets decreased to 1.4m, so the dose was increased to 0,25 mg twice daily after 3 weeks. At present, after 13 months of Anagrelid treatment, the number of platelets in the child ranges between 400 and 650. The patient comes to our, department for monthly check-up of morphology, biochemical tests, ECG and echocardiography. In international literature there is no information on the use of Anagrelid in ET treatment of children under 12 months old. However, on the basis of our observations and initial results of treatment, it seems that the above protocol for Anagrelid administration is safe for infants of this age. Disclosures: No relevant conflicts of interest to declare.

JAK2 V617F-Negative and MPL W515K/L-Negative Essential Thrombocythemia: A High Resolution SNP Array Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5258-5258
Author(s):  
Carla AL Assaf ◽  
Els Lierman ◽  
Timothy Devos ◽  
Carlos Graux ◽  
Johan Billiet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Snp Array ◽  
Jak2 V617f ◽  
Common Mutation ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Array Data

Abstract Background JAK2 V617F is the most common mutation in essential thrombocythemia (ET), occurring in approximately 50 % of cases. Second to JAK2 V617F is MPL W515K/L, accounting for about 10 % of cases. The molecular cause of the remaining ET cases is still largely unknown. Aims We sought to investigate JAK2 V617F-negative and MPL W515K/L-negative ET for regions of copy number variations (CNV) and loss of heterozygosity (LOH). Methods We studied blood or bone marrow samples from a series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET cases. They were subjected to 2.7M SNP array by Affymetrix, which has 2,761,979 copy number markers including 400,103 SNP markers. The array data were analyzed for recurrent CNVs with Array Studio (OmicSoft), and for individual CNVs or recurrent LOHs (≥3 Mbs) with the Chromosome analysis suite (ChAS, Affymetrix). Results Only 8 recurrent gains were identified, in 5/64 patients. Interestingly, the most common gain, occurring in 5 cases was a gain of chr7 q22.3, including the gene encoding Nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is known to be overexpressed in several cancers such as multiple myeloma. It catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway. It is also required for cell growth and survival. We checked in the 5 patients for NAMPT amplification by quantitative PCR (qPCR) on genomic DNA in comparison to controls and by normalizing to ALB and RPPH1. We were able to validate the gain in 2/5 patients. The gain in these 2 patients was demonstrated to be acquired by qPCR of NAMPT in buccal swab DNA. Other recurrent gains involved regions of chromosomes 2, 5, 7, 12, 13, and 22. These gains included, amongst others, LCP1 on chr13 q14.3 and CYTIP on chr2 q24.1, occurring in 2/64 and in 3/64 respectively. We also checked for non-recurrent gains and losses in our cohort. This analysis generated a total of 8 CNVs in 6 different patients, comprising 5 regional gains in chromosomes 2, 8, 12, and 15 and 3 regional losses in chromosomes 5, 8 and 11. The array data were also analyzed for recurrent LOHs on ChAS, yielding 17 recurrent copy neutral LOHs (CN-LOH) in 35 patients (circos plot). The most common CN-LOH region was on chromosome 3 appearing in 8 patients. Other CN-LOH regions involved chromosomes 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, and 17 and they occurred in 2-5/64 patients. However, as small regions of CN-LOH can be constitutional, we suspect that most of these CN-LOH regions are not acquired. The largest region of CN-LOH observed was 12 Mbs in size. Conclusions Previous studies in unselected series of BCR-ABL1-negative myeloproliferative neoplasms have shown that copy number alterations are rare in ET as well as in polycythemia vera. In this series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET patients we found recurrent gains not reported previously in the database of genomic variants in only 8% of patients, and small areas of CN-LOH in ∼55% of cases. However, most of the latter probably are constitutional. Our SNP array study provides further evidence that gains, losses or CN-LOH of small genomic regions do not play an essential role in the pathogenesis of the majority of JAK2 V617F-negative and MPL W515K/L-negative ET. However, the low frequency of megakaryocytes and unknown level of clonal involvement of the myeloid compartment in JAK2 V617F-negative and MPL W515K/L-negative ET bone marrow remain a caveat. Next generation sequencing technology is expected to bring new insights on the molecular pathogenesis of this elusive ET subset. Circos plot showing the recurrent CN-LOHs Left half represents a total of 35 patients carrying recurrent CN-LOHs and the right half represents the chromosomes and their associated properties. Right outermost layer depicts 1+log-gene density (min, 1; max 42) where cancer, OMIM and other genes are colored in red, blue and green respectively. Right middle and innermost layers designates SNP density (blue, values < 0,02; gray values, <0,06; red values >0,06; max scale 0,013) and absolute SNP numbers (min, 1; max, 12074) per windows of 50kb. Each 5Mb distance is marked with a tick underneath the innermost layer. Links from each chromosome are colored differently. Regions that are more confined on the same chromosome are least transparent and regions that are shared by more patients are drawn on top of lesser links. Disclosures: No relevant conflicts of interest to declare.

Extramedullary Hematopoiesis and Splenic Vein Thrombosis, a Unique Presentation Of Pre-Clinical Essential Thrombocythemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5257-5257 ◽  
Author(s):  
Abdulraheem Yacoub ◽  
Abigail Brockman
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Liver Biopsy ◽  
Essential Thrombocythemia ◽  
Extramedullary Hematopoiesis ◽  
Common Finding ◽  
Thrombotic Risk ◽  
Vein Thrombosis ◽  
Life Threatening

Abstract Essential thrombocythemia (ET) is of the BCR-ABL-Negative myeloproliferative neoplasms (MPN). The incidence of ET is approximately 2.5 in every 100,000 person per year. However, given the good prognosis, associated long life expectancy, and increasing detection in younger populations,ET is associated with a higher prevalence rate estimated to be 24 in every 100,000 person per year. ET is characterized by thrombocytosis, vasomotor symptoms, and a variable but increased risk of thrombosis and bleeding. Half of all ET patients will have a positive JAK2 and/or MPL mutation(s). Extramedullary hematopoiesis (EMH) is not a common finding in ET. Nonetheless, ET and other MPNs are associated with the mobilization of CD34+ cells into the peripheral blood. This process can ultimately lead to the seeding of extramedullary sites with primitive hematopoietic capacity, resulting in EMH within the spleen and liver, as well as a variety of other organs. Herein we describe a case that presented with life-threatening thrombosis and was found to have hepatic EMH several months prior to a clinical and pathologic diagnosis of ET. Case description A 22 year-old woman presented 10 days post Cesarean section with abdominal pain and hematemesis. Abdominal imaging showed hepatomegaly, splenomegaly, along with splenic and portal vein thrombosis. The patient underwent an emergency surgical splenectomy due to severe portal hypertension and endoscopic evidence of gastric variceal bleeding. A random liver biopsy was also performed intra-operatively. The splenectomy resulted in resolution of the GI bleeding and the varices normalized on follow up. Her platelet count was normal at the time of operation, but post-splenectomy her platelet count peaked at 1,217 K/ µL. Extensive testing did not unravel any identifiable inherited and/or acquired hypercoaguable factors. Subsequently anticoagulation therapy was recommended for 6 months. On pathology review, the spleen histology showed congestion, but otherwise no diagnostic abnormalities were noted. The liver biopsy showed evidence of EMH but did not identify any liver parenchymal disease. On subsequent follow up, the patient had persistent and marked thrombocytosis for over a year. A bone marrow biopsy was performed which showed a hypercellular bone marrow and megakaryocytic hyperplasia with a few large forms. There was no dysplasia or significant reticulin fibrosis. JAK2 mutation and BCR-ABL translocation were negative. Hydroxyurea and aspirin were started due to high risk of thrombosis. Discussion We report this unique case in which there was evidence of extramedullary hematopoiesis, along with pathologic and life threatening visceral thrombosis several months before the patient met criteria for diagnosis of ET. This supports the notion that neoplastic cells can mobilize and seed other organs early in the course of MPNs, including ET. Thrombotic risk in MPNs can also occur in the preclinical phase of MPNs as has been suggested in other reports. We also conclude that the demonstration of EMH in individuals with no preexisting hematologic neoplasm should warrant close follow up and assessment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Thrombocytapheresis in Patient with Essential Thrombocythemia: A Case Report

2020 ◽  
Vol 13 (2) ◽  
pp. 675-679
Author(s):  
Afra M. Elhassan ◽  
Arwa Alsaud ◽  
Mohamed A. Yassin ◽  
Mahmood Aldapt ◽  
Lubna Riaz ◽  
...  
Keyword(s):  
Platelet Count ◽  
Case Report ◽  
Essential Thrombocythemia ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Favorable Outcome ◽  
Who Criteria ◽  
Icu Admission ◽  
Calr Mutation ◽  
Clonal Abnormalities

Essential thrombocythemia (ET) is one of the myeloproliferative neoplasms, characterized by persistent thrombocytosis, platelets >450,000/μL, and evident clonal abnormalities like JAK2 V617F, MPL, CALR mutation and not fulfilling WHO criteria for MDS, CML, PV, and IDA. Here we report a 24-year-old female who presented with headache and was found to have thrombocytosis with a platelet count of 2,141 × 103/μL, diagnosed as ET as per WHO criteria 2008; she required ICU admission and thrombocytapheresis with a favorable outcome.

scholarly journals In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation-and chemotherapy- induced bone marrow suppression

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 778-784 ◽  
Author(s):  
A Ohwada ◽  
S Rafii ◽  
MA Moore ◽  
RG Crystal
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Adenovirus Vector ◽  
Bone Marrow Suppression ◽  
Single Administration ◽  
Platelet Recovery ◽  
Platelet Production ◽  
Life Threatening ◽  
Bone Abnormalities

Abstract Thrombopoietin (TPO, c-mpl ligand) has emerged as a major hematopoietic cytokine stimulating megakaryocyte proliferation, endomitosis, and platelet production. This study shows that a single administration of an adenovirus (Ad) vector encoding TPO (AdCMV.TPO) abrogates thrombocytopenia induced in mice by carboplatin and irradiation. Normal Balb/c mice receiving the vector had increased platelet counts peaking at 7 days and returning to baseline by day 15. Mice rendered pancytopenic with 500 rads and 1.2 mg of carboplatin had a nadir platelet count of five percent of the baseline. Mice receiving AdCMV.TPO 3 days before receiving irradiation and chemotherapy achieved a platelet nadir fourfold higher, and had significant reduction in duration of thrombocytopenia, than mice receiving the control Ad vector. Introduction of AdCMV.TPO the same day of chemotherapy and irradiation was equally effective in acceleration of platelet recovery, but administration of AdCMV.TPO 3 days after chemotherapy-radiation had little effect on platelet recovery. At 30 days after therapy bone marrow and spleen of mice treated with AdCMV.TPO were populated with a large number of polyploid megakaryocytes, but there was no evidence of circulating megakaryocytes in the liver or lungs and no pathologic bone abnormalities such as osteosclerosis or myelofibrosis. These observations suggest that an Ad vector may be an excellent delivery system to provide adequate TPO production to maintain platelet levels in circumstances associated with life-threatening thrombocytopenia.

Familial Essential Thrombocythemia Showing Spontaneous Megakaryocyte Colony Formation and Acquired JAK2 Mutations without a Germline Mutation in C-MPL or TPO.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4655-4655
Author(s):  
Jillian R. Higgs ◽  
Irene Sadek ◽  
Paul E. Neuman ◽  
Vincent Ing ◽  
Jason N. Berman ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Peripheral Blood ◽  
Colony Formation ◽  
Family Members ◽  
Jak2 V617f ◽  
Related Disorder ◽  
Myeloproliferative Diseases ◽  
Sporadic Cases ◽  
Chronic Myeloproliferative Disorder ◽  
Genetic Events

Abstract Essential thrombocythemia (ET) is a chronic myeloproliferative disorder, characterized by increased proliferation of megakaryocytes and elevated platelet counts that usually occurs sporadically. We report a family with 7 affected individuals in three generations, including one individual with a phenotype resembling polycythemia vera, a related disorder. Megakaryocyte (CFU-MK) colony formation occurred in the absence of added cytokines in cultures of peripheral blood from affected family members. Some reports of familial ET have identified mutations in the genes coding for thrombopoietin (TPO), the cytokine that primarily regulates platelet production and its receptor, c-MPL. In this family, the MPL gene was excluded by linkage analysis. Although TPO levels were elevated in most affected family members and evidence for linkage was found between the disease and TPO (𝛉 = 0.0, Zmax = 3.0), a TPO mutation was not identified by DNA sequencing. The JAK2 V617F mutation that has been associated with 50% of sporadic cases of ET was identified as a somatic mutation in the peripheral blood of the two most clinically affected family members. These patients also had the most elevated TPO levels, raising the possibility of an association between JAK2 and TPO production. Further study of familial myeloproliferative diseases will help elucidate the initiating genetic events underlying ET.

Cytoreductive Therapeutic Approach in the Essential Thrombocythemia (ET) Patients of the Registro Italiano Trombocitemia (RIT): Preliminary Data

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5248-5248
Author(s):  
Luigi Gugliotta ◽  
Alessia Tieghi ◽  
Anna Candoni ◽  
Monia Lunghi ◽  
Gianluca Gaidano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Therapeutic Approach ◽  
Jak2 V617f ◽  
Bone Marrow Biopsies ◽  
Thrombotic Risk ◽  
Significant Difference ◽  
Cytoreductive Treatment ◽  
Wbc Count

Abstract Background: the Registro Italiano Trombocitemia, that is a GIMEMA project, has been activated to registry Italian Essential Thrombocythemia (ET) patients, to improve the diagnosis appropriateness (WHO criteria), to verify the prognostic value of the clinical and biological parameters, to evaluate the compliance to the therapeutical Italian guidelines (1), and to create a network for activation of new studies. Objective: this analysis is mainly devoted to describe the ET patients registered in the RIT and to evaluate the therapeutic approach adopted in the 102 participating hematological centers. Material and methods: two thousand and fifteen ET patients have been registered after the written informed consent was obtained, and data validation by various expert panels is in progress. This preliminary report considers 1785 patients, diagnosed mainly (1078, 60.4%) since the publication in the year 2004 of the ET therapy Italian guidelines (1). Results: the patients, 678 (38%) males and 1107 (62%) females, showed at diagnosis: age 60.3 ± 16.8 years with higher values in males than in females (61.7 ± 15.3 vs. 59.4 ± 17.7, p&lt;0.05), being the patients below 40 years 14% and those over 70 years 33% of cases; PLT count (109/L) 846 ± 309 with lower values in males than in females (813 ± 261 vs. 866 ± 334, p&lt;0.002), and with values 1001–1500 and over 1500 in 16% and 4% of cases, respectively; WBC count (109/L) 9.1 ± 2.9, without difference by sex, and with values 12–15 in 10% and over 15 in 3% of cases; Hgb (g/dL) 14.2 ± 1.6 with higher values in males than in females (14.8 ± 1.5 vs. 13.8 ± 1.5, p&lt;0.001), and with values over 16.5 in 8.5% of males and 2.7% of females, respectively; splenomegaly in 488 (27%), echo-documented in 324 cases (18%); history of hemorrhage and thrombosis in 90 (5%) and 325 (19%) of cases, respectively; disease-related symptoms in 41% and general thrombotic risk factors in 93% of cases, respectively. The WHO 2001 diagnostic criteria were reported for 33% of cases observed before the year 2004 and for 53 % of cases observed since the year 2004. Detailed data at diagnosis were reported as follows: bone marrow biopsy in 1087 cases (61%) with a frequency of 51% and 68% before and since the year 2004, respectively; bcr-abl study in 1045 cases (59%); cytogenetics in 828 cases (46%) with karyotype abnormalities in 27 patients (3%). The JAK2 V617F mutation, searched in 574 cases (32%), was observed in 320 of them (56%). The patient follow-up was 4.5 ± 4.5 years with a total of 5245 pt-yrs. During the follow-up the hemorrhagic events were 5.7% (1.3/100 pt-yrs), being the major events 1.9% (0.4/100 pt-yrs); the thrombotic complications were 14.9 % (3.3/100 pt-yrs), resulting the major arterial 9.4% (2.1/100 pt-yrs), the major venous 3.5% (0.8/100 pt-yrs) and the minor thrombosis 2% (0.4/100 pt-yrs). An antiplatelet treatment, almost always with low dose aspirin, was performed in 75% of the patients, without significant difference in the cases diagnosed before and since the 2004. A cytoreductive treatment was done with use of Hydroxyurea (HU, 64%), Interferon alpha (IFN, 16%), Anagrelide (ANA, 15%), Busulfan (BUS, 4%), and Pipobroman (PIPO, 2 %). In the ET patients diagnosed since the year 2004 respect those diagnosed before, it was observed a decrease in the use of all the cytoreductive drugs, particularly BUS (−62%), IFN ((−62%), and ANA ((−68%). The use of the cytoreductive drugs was related to the patient mean age (years): BUS (76), PIPO (72), HU (67), ANA (53), IFN (48). In the patients diagnosed since the 2004 as compared with those before 2004, the mean age of the treated patients increased for BUS (from 69 to 81 yrs, p&lt;0.001) and for HU (from 64 to 69 yrs, p&lt;0.001) while it decreased for IFN (from 49 to 46 yrs, p&lt;0.05). Conclusion: in the analyzed patients of the ET Italian registry the diagnosis appropriateness resulted improved in the cases observed since the year 2004 respect those observed before, with an increase of bone marrow biopsies from 51% to 68% of patients. Moreover, in accord with the ET therapy Italian guidelines, the use of the cytoreductive drugs was less frequent in the patients diagnosed since the year 2004 than before (particularly for BUS, IFN, and ANA) and the more safe molecules IFN and ANA were preferentially deserved to the younger patients.

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

Blood ◽  
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.

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

Blood ◽  
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.

Analysis of JAK2 V617F Mutation and Its Clinical Significance in Patients with Thrombocythemia and Other Myeloproliferative Neoplasms in Chinese

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4687-4687
Author(s):  
Yue Xu ◽  
Changxin Yin ◽  
Han He ◽  
Lingling Shu ◽  
Fuqun Wu ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Jak2 Mutation ◽  
Western Countries ◽  
Arms Pcr ◽  
V617f Mutation

Abstract Abstract 4687 JAK2 mutation is commonly found in Philadelphia-negative myeloproliferative neoplasms (MPNs). In Western countries, this mutation is found in approximately 96 percent of people with polycythemia vera, half of individuals with essential thrombocythemia or primary myelofibrosis. We used the method of amplification refractory mutation PCR (ARMS-PCR) to investigate MPN patients in China. We focused our study on patients with essential thrombocythemia (ET). ARMS-PCR was used to detect JAK2 V617F mutation in the bone barrow (BM) or peripheral blood of 37 MPN patients, which consisting of 7 ET, 5 polycythemia vera (PV), 5 chronic myeloid leukemia (CML), 5 chronic idiopathic myelofibrosis (CIMF), as well as 15 suspected MPNs. 17 cases of JAK2 V617F mutation (45.9%) were found in 37 patients, including 4 ET (57.1%), 4 PV (80.0%), 3 CIMF (60.0%), 6 suspected MPNs (40.0%). We did not find JAK2 V617F in the patients with CML. Our results indicated that the frequency of JAK2 V617F mutation in bcr/abl-negative MPNs in Chinese is similar to that in MPN patients in Western countries. At the same time, ARMS-PCR can distinguish the mutation is heterozygous or homozygous. Most patients were heterozygous for JAK2 but only a few were homozygous. In conclusion, our study showed that JAK2 V617F mutation frequency in Chinese MPN patients is similar to that in patients with this disorder in the West. It is the major molecular genetic abnormality in bcr-abl negative MPN and it can be used for diagnosis of MPN in China. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document