Thrombocytosis in children and adolescents-classification, diagnostic approach, and clinical management.

Secondary thrombocytosis is a frequent secondary finding in childhood infection and inflammation. Primary hereditary thrombocytosis may be caused by germline mutations within the genes encoding key regulators of thrombopoiesis, i.e., thrombopoietin (THPO) and its receptor c-MPL (MPL) or the receptor's effector kinase Januskinase2 (JAK2). Furthermore, somatic mutations in JAK2, MPL, and in the gene-encoding calreticulin (CALR) have been described to act as driver mutations within the so-called Philadelphia-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Increasing knowledge on the molecular mechanisms and on the clinical complications of these diseases is reflected by the WHO diagnostic criteria and European LeukemiaNet (ELN) recommendations on the management of adult MPN. However, data on childhood thrombocytosis are rare, and no consensus guidelines for pediatric thrombocytosis exist. Current literature has highlighted differences in the epidemiology and molecular pathogenesis of childhood thrombocytosis as compared to adults. Furthermore, age-dependent complications and pharmacological specificities suggest that recommendations tailored to the pediatric population are necessary in clinical practice. Here we summarize literature on classification, diagnostics, and clinical management of childhood thrombocytosis.

Refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T): 2017 update on diagnosis, risk-stratification, and management.

DISEASE OVERVIEW: Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include refractory anemia with ring sideroblasts (RARS), now classified under myelodysplastic syndromes with RS (MDS-RS) and RARS with thrombocytosis (RARS-T); now called myelodysplastic/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T). DIAGNOSIS: MDS-RS is a lower risk MDS, with single or multilineage dysplasia (SLD/MLD), <5% bone marrow (BM) blasts and ≥15% BM RS (≥5% in the presence of SF3B1 mutations). MDS/MPN-RS-T, now a formal entity in the MDS/MPN overlap syndromes, has diagnostic features of MDS-RS-SLD, along with a platelet count ≥ 450 × 10(9)/L and large atypical megakaryocytes (similar to BCR-ABL1 negative MPN). MUTATIONS AND KARYOTYPE: Mutations in SF3B1 are seen in ≥80% of patients with MDS-RS-SLD and MDS/MPN-RS-T, and strongly correlate with the presence of BM RS; MDS/MPN-RS-T patients also demonstrate JAK2V617F, ASXL1, DNMT3A, SETBP1, and TET2 mutations; with ASXL1/SETBP1 mutations adversely impacting survival. Cytogenetic abnormalities are uncommon in both diseases. RISK STRATIFICATION: Most patients with MDS-RS-SLD are stratified into lower risk groups by the revised-International Prognostic Scoring System (R-IPSS). Disease outcome in MDS/MPN-RS-T is better than that of MDS-RS-SLD, but worse than that of essential thrombocythemia. Both diseases have a low risk of leukemic TREATMENT: Anemia and iron overload are complications seen in both and are managed similar to lower risk MDS and MPN. Aspirin therapy is reasonable in MDS/MPN-RS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs is uncertain.

Class prediction models of thrombocytosis using genetic biomarkers.

Criteria for distinguishing among etiologies of thrombocytosis are limited in their capacity to delineate clonal (essential thrombocythemia [ET]) from nonclonal (reactive thrombocytosis [RT]) etiologies. We studied platelet transcript profiles of 126 subjects (48 controls, 38 RT, 40 ET [24 contained the JAK2V(617)F mutation]) to identify transcript subsets that segregated phenotypes. Cross-platform consistency was validated using quantitative real-time polymerase chain reaction (RT-PCR). Class prediction algorithms were developed to assign phenotypic class between the thrombocytosis cohorts, and by JAK2 genotype. Sex differences were rare in normal and ET cohorts (< 1% of genes) but were male-skewed for approximately 3% of RT genes. An 11-biomarker gene subset using the microarray data discriminated among the 3 cohorts with 86.3% accuracy, with 93.6% accuracy in 2-way class prediction (ET vs RT). Subsequent quantitative RT-PCR analysis established that these biomarkers were 87.1% accurate in prospective classification of a new cohort. A 4-biomarker gene subset predicted JAK2 wild-type ET in more than 85% patient samples using either microarray or RT-PCR profiling, with lower predictive capacity in JAK2V(617)F mutant ET patients. These results establish that distinct genetic biomarker subsets can predict thrombocytosis class using routine phlebotomy.

MDS/MPN-RS-T justified inclusion as a unique disease entity?

Myelodysplastic syndrome/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a disease entity characterized by anemia, bone marrow dysplasia with ring sideroblasts and persistent thrombocytosis ≥450 × 109/L with proliferation of large and morphologically atypical megakaryocytes. Although initially recognized by the World Health Organization only as a provisional entity, next generation sequencing has identified recurrent somatic mutations in SF3B1, JAK2 and other genes providing further evidence of the clonal nature of this disease and the need to recognize it as a separate entity. Despite its overlapping features with MDS with ring sideroblasts and essential thrombocythemia, MDS/MPN-RS-T is characterized by specific clinical features and distinct survival outcomes. In the current review we will describe the morphological and genomic features of MDS-RS-T and the potential diagnostic challenges and distinction from other possible conditions. We will also review how the current evidence supports its recognition as an independent disorder.

Platelet Function Tests and Inflammatory Markers for the Differentiation of Primary Thrombocytosis and Secondary Thrombocytosis.

Background: The prognosis and management of primary thrombocytosis (PT) and secondary thrombocytosis (ST) are different. This study aims to evaluate the role of platelet function tests by light transmission platelet aggregometry (LTA), plasma von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity (vWF:RCo) and inflammatory markers [erythrocyte sedimentation rate (ESR), C-reactive protein (CRP)] for the differentiation between PT and ST. Methods: This prospective study was carried out in patients with platelet counts greater than 450 x 109/L. Primary outcomes were the sensitivity and specificity of platelet function tests by LTA for the differentiation of PT and ST. Secondary outcomes were sensitivity and specificity of ESR, CRP, vWF:Ag, and vWF:RCo for the differentiation of PT and ST. Results: Fifty-two patients were enrolled onto the study of which 26 (50%) had PT. The sensitivity and specificity of epinephrine, collagen, and arachidonic acid (AA) induced abnormal LTA for the differentiation of PT from ST were sensitivity of 50%, 38.5%, 26.9% and specificity of 88.5%, 100%, 100% respectively. The sensitivity and specificity of abnormal ESR, CRP, and either abnormal ESR or CRP in the differentiation of ST from PT were sensitivity of 88.5%, 80.8%, 100% and specificity of 65.4%, 61.5%, 46.2% respectively. The sensitivity and specificity of low vWF:Ag and vWF:RCo in the differentiation of PT from ST were sensitivity of 7.69%, 42.3% and specificity of 100%, 88.5% respectively. Conclusions: Abnormal platelet function determined by LTA with collagen, AA, epinephrine had high specificity ratings enabling the differentiation between PT and ST. vWF:RCo, ESR and CRP levels could be helpful in differentiating between PT and ST.

Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis.

The JAK2/V617F mutation has been noted in essential thrombocytemia. We investigated 19 cases with refractory anemia with ringed sideroblasts (RARS), including three RARS with thrombocytosis (RARS-T). Only the RARS-T patients showed this mutation. More cases need to be analyzed to determine the prevalence of the JAK2/V617F mutation in RARS-T.

A retrospective analysis of myelodysplastic syndromes with thrombocytosis: reclassification of the cases by WHO proposals.

Myelodysplastic syndromes (MDS) show occasionally thrombocytosis, common feature of myeloproliferative diseases (MPD), with the overlapping of both disorders. Classically, thrombocytosis has been associated with some MDS subtypes: refractory anaemia with ringed sideroblasts (RARS), 5q- syndrome and those MDS with 3q chromosome rearrangements. The recent WHO classification recognises an unclassifiable MDS/MPD category including some of these disorders. Our aim is to determine the frequency of presentation, subtype classification and chromosome abnormalities of MDS with thrombocytosis diagnosed in our institution. Between 1990 and 2003 we studied 317 SMD patients according to FAB and WHO revised classifications and identified 22 cases presenting thrombocytosis associated with dysplasia, that are analysed in this article.

Plasma levels of thrombin-activatable fibrinolysis inhibitor in primary and secondary thrombocytosis.

An elevated platelet count is a common finding in both hospitalized and ambulatory patients. Thrombosis and bleeding complications are more frequently observed in patients with clonal thrombocytosis than secondary thrombocytosis. The aim of this study was to investigate the behaviors of thrombin-activatable fibrinolysis inhibitor (TAFI) activity, the inhibitor of fibrinolysis, and also prothrombin time (PT), active partial thromboplastin time, and D-dimer and fibrinogen levels in 21 patients affected with clonal thrombocytemia as compared with 21 patients with reactive thrombocytosis and 21 healthy controls. In the clonal thrombocytemia group, plasma levels of TAFI activity were significantly higher than in both the reactive thrombocytosis and the control group. Plasma levels of leukocyte and platelet counts were significantly higher in the clonal thrombocytemia group than in the other two groups and also higher in the reactive thrombocytosis group than in the control group, which was also significant. Fibrinogen and D-dimer levels were higher in patients than in the control group but showed no significant difference between the clonal and secondary thrombocytosis groups. Plasma levels of PT and aPTT were higher in secondary thrombocytosis group than the clonal thrombocytosis group. The results of this study showed for the first time that TAFI activity is increased in patients with clonal thrombocytosis. These increased levels in clonal thrombocytosis can be considered a factor to explain the thrombotic tendency in myeloproliferative disorders.

Thrombocytosis and thrombocythemia.

Thrombocytosis is caused by three major pathophysiological mechanisms: (1) reactive or secondary thrombocytosis; (2) familial thrombocytosis; and (3) clonal thrombocytosis, including essential thrombocythemia and related myeloproliferative disorders. Recent work has begun to elucidate the abnormal megakaryocytopoiesis of essential thrombocythemia, which is associated with paradoxically elevated plasma levels of thrombopoietin. The clonal nature of all cases of essential thrombocythemia has been challenged. Thrombotic complications are the major causes of morbidity and mortality in this disease. Indications for platelet cytoreduction and antiplatelet therapy, as well as complications of treatment, are being clarified.

Blood thrombopoietin levels in clonal thrombocytosis and reactive thrombocytosis.

BACKGROUND: Although the distinction between clonal and reactive thrombocytosis is clinically relevant because clonal thrombocytosis has more thrombohemorragic complications, the differential diagnosis of these two entities can be difficult. Methods such as the detection of unstimulated erythroid or megakaryocyte colony growth are not readily available. Therefore, we measured blood thrombopoietin levels to determine whether these levels can be used to distinguish the two conditions. PATIENTS AND METHODS: Thrombopoietin levels were measured in 73 patients with thrombocytosis (platelet count > 500,000/microL), including 39 patients with clonal thrombocytosis (20 patients with essential thrombocythemia, 15 with agnogenic myeloid metaplasia, 1 patient with polycythemia vera, and 3 with undefined myeloproliferative disorders) and 34 patients with reactive thrombocytosis (17 with malignant tumors, 11 with inflammatory diseases, 4 with sickle cell disease, and 2 with iron deficiency anemia). Seventeen normal volunteers were used as controls. RESULTS: Thrombopoietin levels were significantly higher (P < 0.05) in patients with clonal thrombocytosis (mean +/- SD of 555 +/- 585 pg/mL), including the subgroup with essential thrombocythemia (505 +/- 459 pg/mL), than in patients with reactive thrombocytosis (290 +/- 133 pg/mL) who had similar levels as controls (201 +/- 112 pg/mL). Thrombopoietin levels in patients with clonal thrombocytosis, including essential thrombocythemia, were not correlated with platelet counts. CONCLUSIONS: Thrombopoietin levels may be helpful in distinguishing between clonal thrombocytosis and reactive thrombocytosis. Thrombopoietin is probably responsible for the elevated platelet counts in clonal thrombocytosis including essential thrombocythemia, but not in reactive thrombocytosis. High thrombopoietin levels in patients with clonal thrombocytosis cannot be explained solely by platelet megakaryocyte mass.

Mixed myelodysplastic and myeloproliferative syndromes.

Aplastic anemia, myelodysplastic syndromes (MDS) and chronic myeloproliferative diseases (MPD) are stem cell disorders. There is no clear-cut demarcation of them. Hypoplastic MDS displays features of aplastic anemia and MDS, on the other side mixed myelodysplastic and myeloproliferative syndromes (MDS-MPS) develop. In our collection of 566 MDS patients, features of myelodysplasia as well as myeloproliferation, MDS-MPS, were present in 25 patients (4.4%). Twelve patients had at the time of diagnosis megakaryocytic proliferation and thrombocythemia beside signs of MDS, and seven had myelodysplasia with granulocytic proliferation and leukocytosis. In another six patients, MDS was the first diagnosis and the proliferative phase developed later during the course of the disease. These patients can be characterized as MDS-MPS in evolution. All subjects had a variable degree of anemia. While the level of thrombocythemia has been relatively stable, the number of leukocytes has been progressive, but rarely extended beyond 100 x 10(9)/l. Ring-sideroblasts and myelofibrosis were frequent findings. Two more homogeneous MDS-MPS groups emerged in our analysis: sideroblastic anemia with thrombocythemia and a group fulfilling the criteria of Philadelphia chromosome negative and bcr-abl negative "atypical chronic myeloid leukemia (aCML)'. One patient with thrombocythemia and three with leukocytosis (23%) transformed to acute myeloid leukemia (AML). Men prevailed (12/13) in patients with leukocytosis and MDS-MPS in evolution. Of the 46% MDS-MPS patients with chromosomal aberrations, del(20)(q) is of interest.

Platelet parameters and aggregation in essential and reactive thrombocytosis.

Platelet characteristics were assessed in 15 patients with essential thrombocytosis (ET), 89 patients with reactive thrombocytosis (RT), and 23 normal controls. A platelet volume distribution width (PDW) greater than or equal to 10.5 was found in 50%, 21%, and 14% of the three groups, respectively (P = 0.01 between patients with ET and patients with RT; P = 0.02 between patients with RT and controls), reflecting an excess of extreme values at both ends of the distribution. Compared with controls, the increase in platelet number in patients with RT was about twofold throughout the platelet volume range, whereas ET was characterized by a fivefold increase in small platelets less than 7.5 fL and threefold increase in larger size platelets. Mean platelet volume (MPV) was significantly lower in patients with ET versus patients with RT and in patients with RT versus controls (mean +/- SD 7.5 +/- 1.2 vs. 8.8 +/- 0.1 and 10.2 +/- 1.8 fL, respectively, P less than 0.01). Rate of in vitro platelet aggregation greater than or equal to 50% was significantly lower in patients with ET versus patients with RT and in patients with RT versus controls (0%, 23%, and 45%, respectively, P less than 0.01). Aggregation rate was positively correlated with MPV (r = 0.54; P less than 0.0001). Aggregation rate in patients with ET was significantly lower (P = 0.01) than expected from their reduced MPV alone. Despite these group differences, the overlap of individual platelet characteristics between the three groups precludes their usefulness for diagnostic purposes.

Classification and molecular biology of polycythemias (erythrocytoses) and thrombocytosis.

In this article, polycythemic disorders are classified based on the current understanding of biology of erythropoieses and divided into primary and secondary polycythemias. Special emphasis is given to recently uncovered molecular bases of newly described congenital polycythemic disorders. This clarification of the pathophysiology of some of the congenital polycythemic states has obvious utility for more accurate diagnosis and rational prognostic determination. The molecular basis of congenital thrombocytoses is only beginning to be uncovered. In contrast, the molecular bases of polycythemia vera and essential thrombocythemia remain unknown, thus their diagnostic criteria are imprecise and their treatment remains largely empirical. The central premise of this article is that deciphering the molecular basis of human diseases leads to improved understanding of hematopoiesis, precise diagnosis, and the potential for development of a specific therapy.

Thrombocytosis with sideroblastic erythropoiesis: a mixed myeloproliferative myelodysplastic syndrome.

Some patients with haematological neoplasms have features which overlap between a myelodysplastic syndrome and a myeloproliferative disorder. Two such patients are reported, both having sideroblastic erythropoiesis and thrombocytosis and one sequentially developing features of atypical chronic myeloid leukaemia, idiopathic myelofibrosis and acute megakaryoblastic leukaemia. The prevalence of thrombocytosis among cases of refractory anaemia with ring sideroblasts may be as high as 15-20% and has implications for choice of therapy.

Diagnostic applications of haemopoietic progenitor culture techniques in polycythaemias and thrombocythaemias.

Clonogenic cultures support the proliferation of haemopoietic cells into colonies of differentiated progeny. Using these techniques it has been established that normal haemopoietic progenitors are dependent upon growth factors for their survival, proliferation and differentiation in vitro. However, progenitors from patients with polycythaemia vera (PV) generate erythroid colonies in cultures deprived of exogenous erythropoietin. These have been termed endogenous erythroid colonies (EEC). Recently, endogenous megakaryocytic colonies (EMC), those arising in assays without megakaryocyte growth factors, have been reported, particularly in patients with primary thrombocythaemia (PT). Many investigators have found an EEC positive culture to have 100% diagnostic specificity and sensitivity for PV. Similarly, EMC have been shown by some to be an unequivocal marker of PT. Accordingly, clonogenic assays for EEC and EMC have been advocated as diagnostic markers of PV and PT. However, the specificity of these assays is not universally attested to as there are some reports of EEC in patients with secondary polycythaemia and of EEC and EMC in normal subjects. Thus, for diagnostic use, EEC and EMC assays must be exhaustively validated for specificity using clinically appropriate controls. Furthermore, clonogenic culture techniques are not amenable to external quality assurance, are technically demanding and are unlikely to be available in most haematology laboratories. These considerations must be taken into account when assigning weighting to EEC and EMC assays as diagnostic criteria in myeloproliferative disorders.

[C-reactive protein in differential diagnosis of primary thrombocytosis]. / La proteína C reactiva en el diagnóstico diferencial de la trombocitosis primaria.

BACKGROUND: The most potent stimulator for the hepatic synthesis of C-reactive protein is the interleukin-6. Also interleukin-6 is endowed with thrombopoietic activity, and its seric levels increases in most of secondary thrombocytosis whereas they remain normal in chronic myeloproliferative diseases or primary thrombocytosis. The aims of the study were verify the ability of quantitation of serum C-reactive protein in the differential diagnosis of primary thrombocytosis. METHODS: Serum samples from 89 patients with thrombocytosis (> 400 x 10(9)/1) and 54 normal controls were assayed for C-reactive protein. Patients with thrombocytosis were classified in primary thrombocytosis with 27 patients (chronic myeloproliferative disease with thrombocytosis) and secondary thrombocytosis (62 cases). RESULTS: The mean C-reactive protein serum levels observed in the 27 patients with primary thrombocytosis were 13 +/- 10 mg/l, superior to normal controls (7 +/- 5 mg/l; p < 0.01). In the secondary thrombocytosis group, C-reactive protein serum levels reached a mean value of 59 +/- 34 mg/l, clearly superior to control group and the primary thrombocytosis group (p < 0.0001). No patients in primary thrombocytosis group reached a C-reactive protein value > 40 mg/l, versus 65% of patients in secondary thrombocytosis group. A normal value occurred in 67% cases of primary thrombocytosis group, but also in 17% cases of secondary thrombocytosis group. CONCLUSIONS: Quantitation of C-reactive protein could thus prove useful in the differential diagnosis between primary and secondary thrombocytosis.

[Clinical biological features of mixed myeloid diseases]. / Kliniko-biologicheskie osobennosti smeshannykh mieloidnykh zabolevanii.

AIM: To characterize patients with mixed myeloid neoplasias with proliferation of neutrophils, platelets and eosinophils. MATERIAL AND METHODS: Examination and treatment results were analysed for patients with atypical myeloid leukemia (n = 4), myelodysplastic syndrome (MDS, n = 1) and thrombocytosis, MDS and eosinophilia (n = 1). The examination included morphological, histological, cytogenetic and molecular tests. RESULTS: One patient with atypical chronic myeloid leukemia was prior diagnosed to have primarily MDS with a typical aberration of chromosome 5. Two other patients had an initial morphological picture of resistant anemia with blast excess, signs of myeloproliferation and extramedullary hemopoiesis. One and two months after the first examination they received transfusions of erythrocytic mass. Just then they were found to have splenomegaly and leukocytosis due to proliferating and maturating forms of neutrophils. The course of the disease in patients with MDS, thrombocytosis and normal karyotype and in patients with MDS, eosinophilia and combined chromosomal breaks including translocation (3;12)(q21;p13) was characterized by resistance to standard programs of polychemotherapy and transformation into acute myeloblastic leukemia. CONCLUSION: In some cases atypical CML is a stage of a natural course of MDS. Some MDS variants with eosinophilia and thrombocytosis should be referred to the group of mixed myeloid neoplasias.

Thrombocytosis in children.

In healthy pediatric subjects normal count platelet ranges between 250,000 µL and 450,000 µL. An elevated platelet count greater than 2 SD defines a condition of thrombocytosis. On a clinical level, thrombocytosis is classified "mild" at a platelet count between >500,000 µL and <700,000 µL; "moderate" at a platelet count between >700,000/µL and <900,000/µL; "severe" at a platelet count >900,000/µL; and "extreme" at a platelet count >1,000,000/µL. Thrombocytosis can be classified as primary or secondary. Primary thrombocytosis is divided into familial and essential. Primary thrombocytosis is an extremely rare clonal disease in childhood with incidence of one per million children, i.e., 60 times lower than in adults. It is classified as a myeloproliferative disorder with polycythemia vera, chronic myeloid leukaemia and myelofibrosis and may be associated with thrombotic or haemorrhagic events. Platelet count is generally above 1,000,000/µL. The median age at diagnosis is about 11 years. On the contrary, secondary or reactive thrombocytosis (RT) is very common in pediatric age, occurring in 3-13% of hospitalized children because of several causes. Generally, it is a reactive process caused by infection, chronic inflammation, iron deficiency, tissue damage, cancer, drugs and surgical or functional splenectomy. Thrombocytosis is mild in 72-86% of children, moderate in about 6-8%, and extreme in 0.5-3%. Consultation with a pediatric hematologist is required if elevation of platelet count persists, is unexplained or symptomatic. In the majority of cases no treatment is necessary, and the patient must be only closely monitored.