Polycythemia Vera Study Group Criteria Research Articles

Development and Validation of a Multiple Factor-Based Prognostic Score System of Thrombosis in Polycythemia Vera (MFPS-PV)

Introduction: Thrombosis is an important cause of death in patients with polycythemia vera (PV). The conventional two-tiered risk stratification is based on the Polycythemia Vera Study Group (PVSG) criteria and considered only age≥60 years and previous thrombosis as thrombotic risk factors. In the current study, we integrated genetic and clinical information into thrombosis risk stratification and aimed to improve the prediction power of thrombosis in the 2016 WHO-defined PV and propose risk-adapted treatment strategies according to the new model. Methods: Patients were eligible for inclusion if 18 years or older with a confirmed diagnosis of PV according to the 2016 WHO criteria. Clinical and laboratory data were obtained from institutional databases and direct contact with patients. Bone marrow mononuclear cells were collected from the included patients and sent for next-generation sequencing (NGS). A multivariate Cox proportional hazard regression analysis was conducted to identify independent risk factors for thrombosis-free survival (TFS). Hazard ratio (HR)-based risk score allocation was employed to obtain a multifactorial prediction model. Results: The study involved 372 patients in the training cohort and another 195 patients in the external validation cohort. The median follow-up of the training cohort was 43 months (range 3-454) (Table 1). The median age at diagnosis was 53 years (range 18-86) and 59.4% were male. Altogether, 136 patients (36.6%) suffered from thrombosis, including 52 patients (14.0%) with thrombosis before diagnosis, 68 patients (18.3%) at diagnosis, and 68 patients (18.3%) after diagnosis. Mutations in genes encoding for epigenetic modifiers were more frequent in patients with thrombosis than in patients without thrombosis after diagnosis (45.6% vs. 20.1%, p<0.001). Specifically, significant differences were noted in the frequencies of ASXL1 (16.2% vs. 5.9%, p=0.004), DNMT3A (25.0% vs. 4.6%, p<0.001) and BCOR/ BCORL1 (13.2% vs. 4.6%, p=0.017) mutations between patients with and without thrombosis after diagnosis. Multivariate analysis indicated that age≥60 years (HR 2.493, p=0.001), cardiovascular risk factors (HR 4.041, p<0.001), at least one high-risk mutation for thrombosis (mutations in DNMT3A, ASXL1, and BCOR/BCORL1) (HR 4.049, p<0.001) and previous thrombosis (HR 5.576, p<0.001) were independent risk factors for thrombosis. After assigning HR-weighted scores to each risk factor mentioned above (1, 1.5, 1.5 and 2 points respectively), a multiple factor-based prognostic score system of thrombosis was developed, classifying patients into low-risk (≤1 point), intermediate-risk (1.5-2.5 points), and high-risk (≥3 points) groups. Patients in the three groups had significantly different TFS (p<0.001) and remained consistent during external validation (Figure 1). Finally, a risk-adapted therapeutic strategy was established based on the MFPS-PV. TP and low-dose antiplatelet therapy are recommended for all patients to achieve a targeted HCT level lower than 45%. Low-risk patients are not mandatory for cytoreduction and only require low-dose antiplatelet therapy. Intermediate-risk patients are advised for antiplatelet therapy, with cytoreduction added if they have two thrombotic risk factors, previous thrombosis, or age≥60 years. High-risk patients are recommended antiplatelet and cytoreductive combination therapy. Conclusion: The MFPS-PV, integrating genetic and clinical characteristics for the first time, can significantly predict thrombosis and has great therapeutic implications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Contemporary Patients with Polycythemia Vera, Rates of Thrombosis and Risk Factors Delineate a New Clinical Epidemiology

BackgroundCurrent risk stratification in Polycythemia Vera (PV), is designed to estimate the likelihood of thrombotic complications. In ECLAP large prospective study of 1638 PV cases diagnosed by Polycythemia Vera Study Group (PVSG) criteria, risk factors were age over 65 years and prior thrombotic events. After the JAK2V617F discovery, diagnosis has undergone substantial revision by the World Health Organization (WHO). Consequently, current risk factors determination should be described in patients diagnosed by the most recent recommendations and requires a large sample size with reliable and sufficiently long clinical course documentation. Accordingly, the current study includes 1545 patients with strictly WHO-defined PV diagnosed and followed-up in seven centers from Italy, Austria and the United States, belonging to the International Working Group for Myeloproliferative Neoplasm (MPN) Research and Treatment (IWG-MRT).Objective and design1) Describe the rate of thrombosis in this population;2) Report the sites and frequency of recurrences after a first arterial or venous event;3) Identify separately risk factors for arterial versus venous thrombosis and develop practically useful prognostic models.Results1. Presenting featuresThe characteristics of the cohort recruited reflected well the heterogeneity of epidemiologic and clinical features that are found in the routine clinical practice of PV. Arterial and venous thrombosis history before or at diagnosis was documented in 246 (16%) and 114 (7.4%) patients, respectively. The frequencies of arterial and venous thrombosis were lower than those recorded in the ECLAP4 (27% and 11% respectively) but comparable to those reported in the recent Cyto-PV2 (arterial 17%, venous 12%) studies. The discrepancy might be partly related to earlier diagnosis in the post-ECLAP JAK2mutation era.2. Thrombotic events after diagnosisMedian follow-up was 6.9 years and treatment included aspirin (84%) and cytoreductive agents (73%), in addition to phlebotomy. Post-diagnosis total major thrombosis rate was 2.62% pt/yr, lower than reported in the ECLAP trial (4.4% pt/yr) but comparable to the Cyto-PV study (2.7% pt/yr) where management of cardiovascular risk factors was more intensive than in ECLAP.Arterial or venous thrombosis occurred in 184 (12%, rate: 1.59 % pts/yr) and 137 (9%, rate: 1.05 % pts/yr) patients, respectively. In addition, prior arterial events were associated in 75% of cases with subsequent arterial events and in the remaining 25% with venous thrombosis including splanchnic vein events. Moreover, patients with prior venous events had recurrences in venous district in 61% of cases and in the arterial district in 39%.3. Risk factors for arterial versus venous thrombosis and prognostic modelsIn multivariable analysis, arterial thrombosis was predicted by previous arterial event (HR 1.7, 95% CI 1.2-2.4), and hypertension (HR 1.6, 95% CI 1.2-2.2) while predictors of venous events were previous venous thrombosis (HR 2.6, 95% CI 1.5-4.4), and age ≥ 65 years (HR 1.7, 95% CI 1.2-2.5). We used the above-mentioned risk factors in order to develop prognostic models allowing to identify patients at different probability to develop arterial or venous thrombosis. For arterial thrombosis, the absence of previous arterial thrombosis and hypertension defined lower-risk (rate 1.27 % pts/yr) while the presence of one or both identified higher-risk patients (rate 2.04 % pts/yr). Previous venous event and/or age ≥ 65 years identified patients at higher risk of venous (rate 1.62 % pts/yr) thrombosis and, if absent, patients at lower risk (rate 0.78 % pts/yr).ConclusionThe overall rate of thrombosis was half than in ECLAP and should be considered in future clinical trials. Of note, patients with a prior venous thrombosis had a more frequent subsequent recurrence in the venous district but a proportion of them (40%) had also arterial events; conversely, recurrences after previous arterial event were also registered in the leg and non-leg veins in 30 % of cases, indicating that in PV, venous and arterial thrombosis may share some common pathogenetic mechanisms. These findings may have practical implications and suggests antiplatelet therapy and anticoagulant therapy as the favorite choice to prevent arterial and venous thrombosis, respectively, but also suggests studies of antithrombotic drug combination. DisclosuresNo relevant conflicts of interest to declare.

Decrease in JAK2V617F allele burden is not a prerequisite to clinical response in patients with polycythemia vera

Although reduction in the JAK2(V617F) allele burden (%V617F) has been suggested as a criterion for defining disease response to cytoreductive therapy in polycythemia vera, its value as a response monitor is unclear. The purpose of this study is to determine whether a reduction in %V617F in polycythemia vera is a prerequisite to achieving hematologic remission in response to cytoreductive therapy. We compared the clinical and hematologic responses to change in %V617F (molecular response) in 73 patients with polycythemia vera treated with either interferon (rIFNα-2b: 28, Peg-rIFNα-2a: 18) or non-interferon drugs (n=27), which included hydroxyurea (n=8), imatinib (n=12), dasatinib (n=5), busulfan (n=1), and radioactive phosphorus (n=1). Hematologic response evaluation employed Polycythemia Vera Study Group criteria, and molecular response evaluation, European Leukemia Net criteria. Of the 46 treated with interferon, 41 (89.1%) had a hematologic response, whereas only 7 (15.2%) had a partial molecular response. Of the 27 who received non-interferon treatments, 16 (59.3%) had a hematologic response, but only 2 (7.4%) had a molecular response. Median duration of follow up was 2.8 years. Statistical agreement between hematologic response and molecular response was poor in all treatment groups. Generally, hematologic response was not accompanied by molecular response. Therefore, a quantitative change in %V617F is not required for clinical response in patients with polycythemia vera.

Thrombin Generation In Patients with Essential Thrombocythemia Treated with Anagrelide

AbstractAbstract 5050 Introduction:Thrombosis may be serious and life threatening in patients with essential thrombocythemia (ET). Bleeding is usually from the gastrointestinal tract, and is, in most cases, generally associated with a platelet count greater than 1 million/μL. The mechanism by which thrombocythemia produces hemorrhage or thrombosis is not well defined. Several defects have been described, including a decrease in platelet aggregation or hyperaggregation, a decrease in von Willebrand ristocetin cofactor activity and high molecular weight von Willebrand factor multimers. Some reports show patients with an acquired deficiency of antithrombin, protein C, and acquired APC resistance, probably due to a reduction in free protein S levels. More recently the presence of JAK2V617F mutation and baseline leukocyte (WBC) count have been considered as independent predictors of major thrombosis in ET. Aim of the study:To assess the thrombin generation by the calibrated automated thrombogram method in plasma from ET patients treated with anagrelide. This assay reflects the net results of the procoagulant and anticoagulant forces operating in plasma. The area under the thrombin generation curve (also known as the endogenous thrombin potential (ETP) is a good overall indicator of prothrombotic and hemorrhagic tendency. It could be hypothezised, that platelet-lowering treatment could diminish haemostasis disturbances in ET. Material and methods:The study group consisted of ten ET patients (4 males and 6 females; age range, 24–70 years), diagnosed according to the Polycythemia Vera Study Group criteria. All patients have been treated with anagrelide for mean 4 years (range 1 – 7 years), without antiplatelet drugs. At the time of enrollment their mean platelet number was 344 000/μL (210 000 – 535 000), WBC count 8 600/μL (3 300 – 9 800), and hematocrit 38 (33-47). One patient had a history of splenic vein thrombosis, another had a gastrointestinal bleeding before the start of treatment. Four ET patients (40%) were positive for the JAK2V617F. Ten healthy subjects (4 males and 6 females; age range, 30–70 years) without history of thrombohemorrhagic events, acted as a control group. Thrombin generation (TG) was determined by calibrated automated thrombography (Technothrombin TGA-Technoclone). Thrombin generation curves were described in terms of lag time, peak height, time to peak, slope and ETP. Whole blood thromboelastometry with TEM-ROTEM delta - Pentapharm GmbH has been performed as well. Results:Thrombin generation (ETP) in platelet free plasma was significantly increased in patients with ET (2235 ± 376 nM/min) in comparison with controls (1631 ± 257 nM/min; p=0,0082), the TG lag time, peak, time to peak and slope were also abnormal. Patients with ET showed markedly increased values of INTEM coagulation time-CT, clot formation time-CFT and increased maximum clot firmness, when compared to results of the control group (Table). There were no differences in the EXTEM values. Conclusion:Our results suggest that patients with essential thrombocythemia, in spite of a long-lasting anagrelide treatment, still exhibit an increased thrombin generation as well as formation of a clot with an increased firmness.INTEMCT (sec)CFT (sec)MCF (mm)A10 (mm)A15 (mm)A20 (mm)ET n=10182,1 ± 16,1 p = 0,001145,4 ± 6,34 p = 0,000770,5 ± 2,83 p = 0,00765,6 ± 3,44 p = 0,00268,5 ± 3,17 p = 0,00469,9 ± 3,03 p = 0,007Contr. n=10161,7 ± 11,862,3 ± 9,5464,1 ± 4,9368,5 ± 3,6862,2 ± 3,9363,5 ± 4,55 Disclosures:No relevant conflicts of interest to declare.

Essential thrombocythemia in patients with platelet counts below 600x109/L: Applicability of the 2008 World Health Organization diagnostic criteria revision proposal

The World Health Organization (WHO) diagnostic criteria as well as the Polycythemia Vera Study Group (PVSG) criteria define platelet counts above 600x10 9 /L as the threshold for essential thrombocythemia (ET) diagnosis [1,2]. It has been argued that such threshold excludes a number of patients with actual ET with platelet counts below 600 x 10 9 /L [3-5]. Recently, a proposal for revision of the WHO diagnostic criteria for ET has been published, which includes the combination of histological bone marrow study and testing of the JAK2 mutation to facilitate the diagnosis of ET with borderline thrombocytosis [6,7]. The aim of this study was to evaluate the applicability of the proposal of the WHO revised diagnostic criteria in patients presumed to have ET with platelet counts below 600 x 10 9 /L. Additionally, clinical and laboratory features of this group were compared to the group with platelet counts above 600 x 10 9 /L to assess any differences between both groups. Finally, clinical and laboratory features of JAK2 positive patients were compared to JAK2 negative patients to confirm in our series the differences previously described in the literature [8,9].

Thrombotic complications of polycythemia vera

Background: Polycythemia vera (PV) is an uncommon clonal disorder of stem cells. The literature regarding the thrombotic complications of this disorder in the developing countries is scarce. The present study was undertaken retrospectively to look at the association of thrombotic complications of PV with various patient characteristics.Materials and methods: All the patients diagnosed to have PV from January 1986 to December 2005 according to Polycythemia Vera Study Group criteria were included. Their clinical characteristics, laboratory parameters, clinical complications such as thrombosis and myelofibrosis, treatment modalities, malignancies and deaths, if any were noted. The various characteristics of patients who had developed thrombosis were compared statistically with those of patients without thrombosis.Results: Out of the 9550 patients seen during this period, 32 patients fulfilled the inclusion criteria. The median age at the time of diagnosis was 56·5 years. Eight patients developed thrombotic complications; out of which four had arterial and four had venous thrombosis. There was no statistically significant difference in the mean hemoglobin, white cell count (WBC), platelet count and RBC mass in patients with thrombosis as compared to patients without thrombosis. These parameters were also not statistically significant when patients with arterial thrombosis were compared with patients with venous thrombosis.Conclusions: PV is an uncommon disorder when compared with other hematological disorders in northern India. There was no relationship between the development of thrombotic complications and hemoglobin, RBC mass or platelet count. Twenty patients received hydroxyurea with a median follow-up of 57 months, none developed acute leukemia.

Sequence Analysis of MPL Exon 10 of ET and MF JAK2 V617F Negative Patients Reveals a New Mutation

Background. Diagnosis and classification of myeloproliferative disorders was greatly enhanced by reports describing JAK2V617F mutation in Polycythemia Rubra Vera (PRV) patients and approximately 50% of Essential Thrombocythemia (ET) and Myelofibrosis (MF) patients. More recently mutations mapping to the juxtamembrane region of the thrombopoietin receptor MPL were described in 5–10% of the patients with MF and 1% of the patients with ET. The reported data imply the Exon 10 MPL mutations lead to receptor activation in the absence of thrombopoietin binding and constitutive phosphorylation of JAK2, STAT3, STAT5, AKT and ERK. However, thus far only mutations mapping to codons 515 and 505 have been published. The aim of this study was to determine the frequency of JAK2V617F and MPL exon 10 mutations in MF and ET patients seen at the Hematology Department, Centro Hospitalar de Coimbra.Methods. Sixty three patients with ET and 12 with MF, diagnosed according to the Polycythemia Vera Study Group (PVSG) criteria, were studied. Genomic DNA was extracted from peripheral blood samples collected into EDTA. Samples were tested for JAK2 V617F mutation by allele specific polymerase chain reaction (ASO-PCR). Subsequently these samples were screened for MPL exon 10 mutations by direct sequencing.Results. 45 (71.4%) of the 63 patients with ET and 6 (50%) of the 12 MF patients had JAK2V617F mutation. Of the remaining 6 MF patients 3 had MPL mutations; two with W515L and one with W515K amino acid substitutions. Direct sequencing failed to detect exon 10 MPL mutations in 17 of the 18 ET JAK2 V617F negative patients. The remaining patient had a previously unreported MPL mutation mapping to codon 524 where C was substituted by T, predicting a replacement of arginine residue by cysteine (R524C).Conclusions. JAK2 V617F mutation was detected in 71% of the ET patients and in 50% of the MF. MPL mutations were found in 2 out of 12 MF patients (17%) and in one of the 63 ET patients (1.6%). The MPL codon 515 mutations are reported to activate mpl protein independent of ligand binding. The prevalence of MPL mutations among MF patients is higher than the described in previous reports, however this maybe due to the small number of patients studied. Whilst there is genotype and phenotype correlation between MPD and MPL mutations described the precise functional role in the development of MPD remain unclear. Similarly, whether the previously unreported MPL R524C mutant allele presented here leads to activation of mpl protein like codon 515 mutations is unclear. But nonetheless such mutations assist in accurate diagnosis and classification of Philadelphia negative MPD patients with the view to optimization of clinical management of these patients.

Phase II open label trial of imatinib in polycythemia rubra vera

Polycythemia rubra vera is a chronic myeloproliferative disorder characterized by panmyelosis with the resultant potential for thrombosis, myelofibrosis, and acute leukemia. Treatment has rested on phlebotomy and hydroxyurea. In 2002, we reported two patients who were unable to tolerate hydroxyurea but responded to imatinib mesylate (Gleevec). These patients have remained in complete hematologic remission on imatinib since 1999. As a result we began a phase II, open label trial of imatinib in patients with polycythemia vera. Patients meeting the Polycythemia Vera Study group criteria for the diagnosis of polycythemia vera, either naïve or intolerant to prior treatment were allowed to enroll. Initial therapy was begun with imatinib mesylate at 400 mg a day and two dose escalations, one to 600 and second to 800 mg a day, were allowed for patients not achieving a target hematocrit of 44 or less; or a platelet count of less than 600,000/mm(3). Twenty patients were enrolled, 15 achieved complete hematologic remission within 12 weeks and ten remain on study. Six patients remain in remission on 400 mg a day and four on 500 mg a day. Gastrointestinal or cutaneous toxicities were primarily grade I or II. All patients were negative for bcr/abl. Imatinib mesylate is capable of producing hematologic remission in the majority of patients with polycythemia vera and provides another option for patient management, particularly in those intolerant to hydroxyurea.

Bone marrow pathology in essential thrombocythemia: interobserver reliability and utility for identifying disease subtypes

The role of histopathology in the diagnosis of essential thrombocythemia (ET) is controversial, and there has been little attempt to quantitate interobserver variability. Diagnostic bone marrow trephine biopsy specimens from 370 patients with ET by Polycythemia Vera Study Group (PVSG) criteria were assessed by 3 experienced hematopathologists for 16 different morphologic features and overall diagnosis according to the World Health Organization (WHO) classification. Our results show substantial interobserver variability, particularly for overall diagnosis and individual cellular characteristics such as megakaryocyte morphology. Reticulin grade was the dominant independent predictor of WHO diagnostic category for all 3 hematopathologists. Factor analysis identified 3 independent factors likely to reflect underlying biologic processes. One factor related to overall and lineage-specific cellularity and was significantly associated with JAK2 V617F status (P < .001), a second factor related to megakaryocyte clustering, and a third was associated with the fibrotic process. No differences could be discerned between patients labeled as having "prefibrotic myelofibrosis" or "true ET" in clinical and laboratory features at presentation, JAK2 status, survival, thrombosis, major hemorrhage, or myelofibrotic transformation. These results show that histologic criteria described in the WHO classification are difficult to apply reproducibly and question the validity of distinguishing true ET from prefibrotic myelofibrosis on the basis of subjective morphologic criteria. This study was registered at http://isrctn.org as #72251782 and at http://eudract.emea.europa.eu/ as #2004-000245-38.

JAK2 Mutations are present in all cases of polycythemia vera

The JAK2V617F point mutation has been described in 65 to 97% of patients with polycythemia vera (PV). Previously, 17 phenotypical cases of PV from a large group of patients from three institutions were initially reported as JAK2V617F negative. These cases were re-analyzed in detail to determine the cause, if any, of the JAK2V617F negativity. Reasons for this initial negativity included inaccurate clinical diagnosis, relatively insensitive laboratory techniques, insufficient material for re-testing, and possible treatment effect. It was predicted that when tested appropriately, a JAK2V617F mutation would be found in all new cases of PV. We developed a highly sensitive amplification refractory mutation system assay (ARMS) for the detection of JAK2V617F. The analytic sensitivity of the assay is 0.05-0.1% as defined by mixing JAK2V617F-containing HEL cells with normal peripheral blood mononuclear cells. At this level of sensitivity, the assay has a false positive rate of less than 1% (we found 1 positive case in 300 individuals without myeloid disorders). Pyrosequencing involves synthetic nucleotide extension by DNA polymerase. Unlike ARMS, the PCR pyrosequencing method reliably detects JAK2V617F only when the mutant allele is more than 5%. The sensitivity of the ARMS assay is therefore approximately 50 times greater than the pyrosequencing method. Of 105 PV cases diagnosed by Polycythemia Vera Study Group criteria, pyrosequencing detected the mutation in 96 cases. The ARMS technique detected JAK2V617F alleles in 8 of the remaining 9 cases. Of the 8, 2 had been treated by phlebotomy only, 4 had been on interferon for a median of 13 years, and 2 on imatinib for 3 years. In the last patient, who was negative by both assays, the JAK2 exon 12 deletion (E543-D544) was detected. This patient had been on imatinib for 3 years and remains in complete remission. We conclude that a sensitive assay is required to detect the JAK2 mutations in patients with polycythemia vera, especially in patients whose JAK2 mutation load might be affected by treatment.

JAK2 Mutations Are Present in All Cases of Polycythemia Vera.

The JAK2V617Fpoint mutation has been described in 65 to 97% of patients with polycythemia vera (PV). Previously, 17 phenotypical cases of PV from a large group of patients from three institutions were initially reported as JAK2V617Fnegative. These cases were re-analyzed in detail to determine the cause, if any, of the JAK2V617Fnegativity. Reasons for this initial negativity included inaccurate clinical diagnosis, relatively insensitive laboratory techniques, insufficient material for re-testing, and possible treatment effect. It was predicted that when tested appropriately, a JAK2V617Fmutation would be found in all new cases of PV. We developed a highly sensitive amplification refractory mutation system assay (ARMS) for the detection of JAK2V617F. The analytic sensitivity of the assay is 0.05-0.1% as defined by mixing JAK2V617F-containing HEL cells with normal peripheral blood mononuclear cells. At this level of sensitivity, the assay has a false positive rate of less than 1% (we found 1 positive case in 300 individuals without myeloid disorders). Pyrosequencing involves synthetic nucleotide extension by DNA polymerase. Unlike ARMS, the PCR pyrosequencing method reliably detects JAK2V617Fonly when the mutant allele is more than 5%. The sensitivity of the ARMS assay is therefore approximately 50 times greater than the pyrosequencing method. Of 105 PV cases diagnosed by Polycythemia Vera Study Group criteria, pyrosequencing detected the mutation in 96 cases. The ARMS technique detected JAK2V617Falleles in 8 of the remaining 9 cases. Of the 8, 2 had been treated by phlebotomy only, 4 had been on interferon for a median of 13 years, and 2 on imatinib for 3 years. In the last patient, who was negative by both assays, the JAK2 exon 12 deletion (E543-D544) was detected. This patient had been on imatinib for 3 years and remains in complete remission. We conclude that a sensitive assay is required to detect the JAK2 mutations in patients with polycythemia vera, especially in patients whose JAK2 mutation load might be affected by treatment.

The 2001 World Health Organization and Updated European Clinical and Pathological Criteria for the Diagnosis, Classification, and Staging of the Philadelphia Chromosome-Negative Chronic Myeloproliferative Disorders

The clinical criteria according to the Polycythemia Vera Study Group (PVSG) do not distinguish between essential thrombocythemia (ET), thrombocythemia associated with early-stage polycythemia vera (PV) and prefibrotic chronic idiopathic myelofibrosis (CIMF). The criteria only classify the advanced stage of PV with increased red cell mass. The classification of myeloproliferative disorders (MPDs), proposed by the World Health Organization (WHO) in 2001, is a compromise of the clinical PVSG and WHO bone marrow criteria, and excludes early stages of ET and PV. The updated European clinical and pathological criteria combine the WHO bone marrow criteria with established and new clinical, laboratory, biological, and molecular MPD markers. This allows clinicians and pathologists to diagnose early-stage MPD and to differentiate ET, PV, and prefibrotic chronic idiopathic myelofibrosis (CIMF). Depending on laboratory tests and diagnostic criteria used, the population of the MPD patients defined as ET, PV, and CIMF are heterogeneous at the clinical, laboratory, and biological and pathological levels. The recent discovery of the JAK2 V617F mutation, which is the cause of a distinct trilinear MPD in its manifold clinical manifestations during long-term follow-up, increases the specificity of a positive JAK2 V617F polymerase chain reaction (PCR) test for the diagnosis of MPD (near 100%), but only half of the ET and CIMF patients according to the PVSG (sensitivity 50%) and the majority of PV patients (sensitivity 95%) are JAK2 V617F positive. A comparison of the laboratory features of JAK2 V617-positive and JAK2 wild-type ET patients clearly showed that JAK2 V617-positive ET is characterized by higher values for hemoglobin, hematocrit, and neutrophil counts; lower values for serum erythropoietin (EPO) levels, serum ferritin, and mean corpuscular volume; and by increased cellularity of the bone marrow in biopsy material. This indicates that JAK2 V617-positive ET patients, diagnosed according to the PVSG criteria, represent a "forme fruste of PV" consistent with early PV mimicking ET (JAK2 V617F trilinear MPD). In contrast, the JAK2 wild-type ET patients had significantly higher platelet counts and usually had a clinical picture of ET with normal serum EPO levels, PRV-1 expression, and leukocyte alkaline phosphatase score, and a typical WHO ET bone marrow picture. The clinical and pathological data on JAK2 V617F-positive MPD patients suggest that the JAK2 V617F mutation defines one disease entity with several sequential steps of ET, PV, and secondary myelofibrosis during long-term follow-up, and that the wild-type JAK2 MPDs may represent another distinct entity with a related but different molecular etiology. MPD-specific markers such as serum EPO, endogenous erythroid colony formation (EEC), and JAK2 V617F have high specificities, but the sensitivities are not high enough to detect the early stages of the MPDs, ET, PV, and prefibrotic CIMF. Bone marrow histopathology in addition to clinical, laboratory, biological, and molecular markers, including the JAK2 V617 PCR test, serum EPO, PRV-1, EEC, LAP score, peripheral blood parameters, and spleen size on echogram will detect the early stages of MPD and allows diagnostic differentiation of the three primary MPDs (ET, PV, and CIMF) in both JAK2 V617F-positive and JAK2 wild-type MPD patients.

Clinicopathological Criteria for Differential Diagnosis of Thrombocythemias in Various Myeloproliferative Disorders

Thrombocythemias with the presenting or developing complications of thromboembolic and hemorrhagic episodes may be encountered at strikingly different incidences in each subtype of chronic myeloproliferative disorders. A critical reappraisal of the Polycythemia Vera Study Group (PVSG) criteria for essential thrombocythemia (ET) reveals that differentiation is explicitly focused on the exclusion of chronic myeloid leukemia and polycythemia vera (PV), but not on prodromal stages of chronic idiopathic myelofibrosis (CIMF) or latent (initial) PV. Consequently, it may be assumed that most series of patients with so-called ET include a considerable fraction of patients with the latter entities. The diagnostic impact of bone marrow (BM) histopathology was recognized by the World Health Organization classification, which emphasizes for the first time positive criteria for ET. The need of a more accurate ET diagnosis is obvious, in particular regarding therapeutic strategies and outcome (i.e., progression into myelofibrosis and blastic crisis). Conversely, early CIMF with accompanying thrombocythemia mimicking (true) ET is characterized by a higher rate of evolution into myelofibrosis and fatal complications. A scrutinized discrimination of thrombocythemias resulting in a clear-cut diagnosis of true versus false ET is warranted by a professional evaluation of BM biopsies in ongoing and prospective clinical trials.

Essential thrombocythemia or chronic idiopathic myelofibrosis? A single-center study based on hematopoieticbone marrow histology

We reviewed a large series of patients with essential thrombocythemia diagnosed on the basis of the Polycythemia Vera Study Group criteria, and reclassified them by evaluating their major morphologic features and clinical course using the World Health Organization classification. The morphologic review of the bone marrow biopsies of 116 patients (44 males and 72 females; aged 19 – 83 years, median 55 years; median follow-up 121 months) led to 22 cases (19%) being classified as essential thrombocythemia (ET), 24 (21%) as chronic idiopathic myelofibrosis (CIMF)-0, 44 (37%) as CIMF-1, 13 (12%) as CIMF-2, 9 (8%) as latent phase polycythemia vera, and four (3%) as chronic myeloproliferative disorder, unclassifiable. There was a significant difference in the median age of the ET and fibrotic CIMF patients (54.7 ± 13.55 vs. 59.13 ± 15.05 years; P = 0.03). Histologic analysis showed that the simultaneous presence of loose clusters of large/giant megakaryocytes and nuclear hyperlobulation was significantly different between the ET and the prefibrotic CIMF (P<0.01) and fibrotic CIMF patients (P<0.01), and that the association of dense clusters of megakaryocytes with maturation defects and bulbous nuclei also distinguished the prefibrotic CIMF (P<0.05) and fibrotic CIMF patients (P<0.001) from those with ET. The association of cellularity, granulocytic proliferation and reticulin fibers was helpful in distinguishing prefibrotic from fibrotic CIMF (P<0.001).

A Prospective Analysis of Adverse Events in a Phase 2 Trial of Imatinib in the Treatment of Polycythemia Vera.

Imatinib has recently been demonstrated to be an effective treatment in Polycythemia Vera. Initial response rates are high, approaching 80%. Patients enrolled in our trial, Novartis CST 1571B (US 144), were asked to complete a questionnaire identifying side effects of imatinib known from previous trials in chronic myeloid leukemia and GIST. Questionnaires were completed weekly for the first month, biweekly for the second month, and monthly for the remainder of the study. Mean time on study is now 1.1 years. Target enrollment was 20 patients. Mean age was 54 years; male to female ratio was 1.3: 1. Fourteen patients were Caucasian and 2 African American. Twelve patients were naïve to treatment and 4 patients were previously treated with hydroxyurea. Two dose escalations were allowed sequentially to 600mg and 800mg daily based on response using the Polycythemia Vera Study Group Criteria. Toxicity was graded according to the NCI Common Toxicity Criteria, version 2.0. Patient reported toxicities are expressed as the total number of events reported during the 120 week study period. Diarrhea was the most common reported toxicity at 53 events. Periorbital edema (33), pruritis (39), fatigue (29), arthralgias (20), headache (18), nausea (19), reflux (38), and dry mouth (15) were the next most commonly reported events on study. Other toxicities reported less commonly were chest pain (3), depression (4), cough (5), insomnia (6), weight gain (3), taste changes (7), and depigmentation (3). Most toxicities were mild at grade 1 and resolved spontaneously within 30 to 40 weeks from the time of treatment initiation. The exceptions to this were diarrhea, periorbital edema, and reflux esophagitis, which persisted in a number of patients throughout the study. The severity of gastrointestinal side effects was dose dependent. Previous studies of imatinib induced toxicity in patients with chronic myeloid leukemia have reported cutaneous manifestations as the most common event. Our study demonstrates the toxicities of imatinib in patients with polycythemia vera are generally mild and self limited, although resolution may take up to 35 weeks. In contrast with studies of imatinib and chronic myeloid leukemia, gastrointestinal toxicity was more common and tended to persist, but did not result in patient withdrawal from study. These differences from our findings may be the result of several factors: the use of a patient reported adverse events scale, small trial size, or differences in drug metabolism in a related myeloproliferative disorder.

Hematocrit-Driven Referrals for Measurement of RBC Mass. Is the Natural History of Polycythemia Vera Changing?.

Red blood cell mass (RCM) elevation is the sine qua non for diagnosis of polycythemia vera (pvera). The Polycythemia Vera Study Group (PVSG) employed therapeutic phlebotomy to lower the HCT to <55% as initial therapy. In the current era of frequent blood testing, RCM is usually requested based only on a high HCT often <55%. Thus the natural history of pvera may be changing if it is being diagnosed at an earlier stage. We reviewed 101 consecutive patients referred in 2002–2003 for RCM testing to characterize RCM requests from a large community referral base. There were 61 men and 40 women with mean HCT<55% (Table 1). 20 had lung disease or were smokers, 3 presented with arterial or venous thrombosis, 7 had cancer, 2 had renal cysts. HCT of men was higher than HCT of women if RCM was normal, but HCT were equivalent between genders if RCM was elevated. HCT was higher in men (p=0.0010) and women (p<0.0001) if RCM was elevated versus normal. HCT elevation was similar in secondary polycythemia vs pvera in men (51.0±4.02 vs 54.3±6.23; p=0.3397) or women (50.5±4.80 vs 49.3±2.12; p=0.4762).Table 1HCT of patients referred for RCM measurementGenderWhole GroupNormal RCMHigh RCMnHCTnHCTnHCTMale6148.3 (47.8) ±4.894546.9 (47.2) ±4.01652.1 (51.3) ±5.16Female4045.0 (43.8)± 4.723243.3 (42.5) ±3.691050.0 (50.0) ±3.84p value0.0001<0.00010.4932HCT shown as Mean (Median) ±SDWe examined whether, despite a lower presenting HCT, those with normal RCM demonstrated PVSG criteria that justified referral to our Nuclear Medicine department. Only 7 of the 75 patients with normal RCM met at least 2 of the PVSG “B” criteria or had splenomegaly, thus might have been diagnosed with pvera had RCM been high (Table 2).Table 22 PVSG “B” criteria or splenomegaly and normal RCMGenderWBC ≥12KPLT ≥400KB12 ≥900≥ LAP 100SplenomegalyM14.4413640158“normal”M12.1585133653“normal”M9.4428113968“normal”M8.6132134315 cmM8.231249218617 cmF18.258271923“normal”F6.3226318“enlarged”F7.114681315.3 cmOverall, 10 of 40 women and 16 of 61 men had an elevated RCM: 4 women and 5 men with polycythemia vera; 6 women and 11 men with secondary polycythemia. One other man was diagnosed with polycythemia vera on the basis of a borderline elevated RCM (33.8 ml/kg), normal O2 Sat on room air, popliteal artery thrombosis, and 3 “B” criteria. Serum erythropoietin (EPO) was low at 2.6 mU/ml (ref range 4–16). He was receiving therapeutic phlebotomy for hereditary hemochromatosis when his RCM was measured. He was the only patient with pvera whose EPO was low (three others had normal EPO levels). The 11 patients with secondary polycythemia who had EPO measured had normal levels. In summary, the HCT was the primary criterion for RCM testing for 2/3 of these patients. Only 5 presented with HCT>55%; mean HCT was ~50% in patients with elevated RCM. The bottom quartile HCT of women with elevated RCM in our patient population was 48.7%. This is a sensitive “cut-off” for finding an elevated RCM (p<0.0001, Chi square with Yates correction). We conclude that patients are referred for RCM testing when a high HCT is found, but at HCT far below the original PVSG parameters. Therefore polycythemia vera is now diagnosed earlier and in mostly asymptomatic patients. A normal EPO level does not rule out a diagnosis of pvera. A HCT < 48.7% may not warrant the measurement of RBC mass.

Successful outcome with anagrelide in pregnancy

Sir, Anagrelide is an oral imidazo-quinazoline compound initially approved as a platelet-lowering agent in essential thrombocytosis (ET) by the Food and Drug Administration in 1997 [1]. It reduces platelet production, and at higher doses inhibits platelet aggregation, making it an ideal drug for use in ET [2, 3]. Furthermore, the shortand mediumterm benefits and side effects of anagrelide are now well established [4]. Although the drug is well tolerated by patients, experience with this drug during pregnancy in humans is limited [4–6]. Moreover, use of anagrelide in ET needs to be reviewed and one needs to be more cautious as it can lead to significant cardiotoxicity [7] and thrombotic complications [8]. Here we report the successful outcome of two patients with ET (Polycythemia Vera Study Group criteria) who inadvertently became pregnant while on anagrelide.

Anagrelide: analysis of long-term efficacy, safety and leukemogenic potential in myeloproliferative disorders

Appropriate treatment for nonreactive thrombocytosis resulting from a myeloproliferative disorder (MPD) is surrounded by controversy. Although few doubt the association of thrombocytosis with increased risk for life-threatening events such as thrombosis or hemorrhage, or the association between clonal myeloproliferation and the progression to acute leukemia or myelofibrosis, controversy exists regarding the timing and nature of appropriate therapeutic intervention. Studies have shown that treatment with myelosuppressive agents such as chlorambucil, busulfan, radiophosphorus ( 32P), and hydroxyurea reduces the platelet count. However, investigators have also identified an increased risk of drug-related leukemic transformation. An ideal cytoreductive treatment for long-term use should minimize thrombosis and avoid long-term complications, especially acute leukemia (AL). Anagrelide, an imidazoquinolin, inhibits megakaryopoiesis and more selectively reduces platelet production in humans. A retrospective analysis of an open-label, multicenter, international trial reviewing 3660 anagrelide-treated patients was performed to assess efficacy and long-term safety, specifically potential for increased leukemogenicity. The study included MPD patients with thrombocytosis diagnosed according to Polycythemia Vera Study Group (PVSG) criteria. Of all patients enrolled, 81% had previously received other myelosuppressive agents; of these, 33% were transferred from the original agent to anagrelide due to toxicity and 31% were transferred because of poor platelet control. Over 45% of patients were symptomatic due to thrombocythemia, most commonly vascular sequelae (25%). Dosage was titrated to achieve a platelet count <600 × 10 9 L −1 and ideally between 130 and 450 × 10 9 L −1. The safety cohort of 3660 patients, including 2251 with essential thrombocythemia (ET), 462 with polycythemia vera (PV), and 947 with chronic myeloid leukemia (CML) and other MPDs, was analyzed to establish the incidence of leukemic transformation in patients with ET and PV. From the Safety Population, 12.8% (467/3660) of patients were treated with anagrelide as the sole cytoreductive agent (naïve patients). Acute leukemia/myelodysplasia developed in 2.1% of ET patients (47/2251) with a maximum follow-up of 7.1 years. Of the PV patients, 2.8% developed acute leukemia/myelodysplastic syndrome (13/462), with a maximum follow-up of 7.0 years. ET and PV patients who transformed to AL had all been previously exposed to other cytotoxics; there were no ET or PV patients in the study who transformed to AL exposed solely to anagrelide. With maximum follow-up over 7 years, anagrelide achieved platelet control in over 75% of MPD patients and did not increase the conversion to acute leukemia during the treatment duration analyzed. Longer follow-up is required to confirm these important observations regarding the long-term safety of anagrelide.

Imatinib Mesylate Therapy for Patients (pts) with Polycythemia Vera (PV).

Imatinib is a potent inhibitor of tyrosine kinases, including abl, c-kit, and platelet-derived growth factor receptor (PDGFR). A role for PDGF in PV has been suspected, and direct inhibition of PV colonies with imatinib has been reported. We initiated a phase II trial of imatinib in pts with PV diagnosed by the Polycythemia Vera Study Group criteria. Pts were treated with imatinib at 400 mg daily; if no significant response after 1 month (mo) of therapy, the dose could be escalated to 800 mg daily. Hydroxyurea could be used only during the first mo of therapy, but anagrelide was allowed at any time. 9 patients have been treated (6 male, 3 female). Median age was 51 years (yrs) (range 28 to 72 yrs); median duration of PV was 8 years (range 1/4–35 years). ECOG performance status was 0 for 2 pts and 1 for 7 pts. Most common symptoms included fatigue (n=7), intractable pruritus (n=5) and headache (n=4). 4 pts had splenomegaly, 1 had hepatomegaly, and 1 had prior splenectomy. All patients were dependent on repeated phlebotomies. All pts had diploid cytogenetics except one who had 20q− (prior therapy with 32 P). FISH and PCR confirmed absence of Bcr-Abl translocation. After a median follow-up of 4 mo (range, 2 to 37 mo), 3 pts have ≥50% reduction in phlebotomy requirements. These include one pt with complete remission defined as hemoglobin &lt; 14 g/dl, no phlebotomies and complete resolution of splenomegaly. Pruritus improved significantly in 3 pts; fatigue improved in 3 pts, headache and visual symptoms improved in one pt each. The dose of imatinib was increased to 800 mg in 3 pts. Grade 3 diarrhea and liver dysfunction occurred in 1 pt requiring dose reduction to 300 mg daily. Grade 2 toxicities included edema (n=2), rash (n=1), bone pain (n=1) and diarrhea (n=1). We conclude that imatinib has activity in PV, leading to improved symptoms and decreased phlebotomy requirements.

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