Myeloproliferative Diseases Patients Research Articles

Risk of second primary malignancy in patients with primary myelofibrosis: A SEER database study.

e19079 Background: Primary myelofibrosis (PMF) carries a poorer prognosis compared to other BCR-ABL-negative myeloproliferative diseases (MPD), and there is increased risk of early mortality due to blast transformation, thrombosis, bleeding complications, and progression of disease. Prior studies report greater incidence of second primary malignancy (SPM) among MPD patients, although the true risk in PMF has not been elucidated. We performed a large database study to evaluate the risk of SPM in PMF patients and analyzed the effects of sociodemographic factors on the risk of SPM. Methods: We used the Surveillance, Epidemiology, and End Results (SEER) database to identify all patients with a histologic diagnosis of PMF from 2009 to 2018. SPM was defined as any subsequent malignancy that developed at least 1 year after the diagnosis of PMF. Using multiple primary standardized incidence ratio (SIR) session of the SEER*Stat software (version 8.3.9), we calculated SIR and absolute excess risk (AER) of SPM for the entire cohort of PMF and also stratified based on age, sex, race, marital status, receipt of chemotherapy, follow-up duration, and year of diagnosis. We generated the 95% confidence intervals (CI) and p-values assuming Poisson distribution of the observed incidences of SPM. Results: A total of 5,273 patients with PMF were included in the analysis, of which 342 patients (6.4%) developed SPM. SPM occurred at SIR of 1.97 (95% CI 1.77-2.18, p<0.05) and AER of 151.87 per 10,000 population. A significantly higher incidence of melanoma (SIR 1.96, 95% CI 1.14-3.14, p<0.05), lymphoma (SIR 3.45, 95% CI 2.31-4.96, p<0.05), and leukemia (SIR 26.87, 95% CI 22.69-31.59, p<0.05) was observed. There was no statistically significant difference in SIR based on sex, race, marital status, follow-up duration, and receipt of chemotherapy. The risk was significantly higher in patients ≤60 years vs patients >60 years (SIR 2.34, 95% CI 1.89-2.86 vs SIR 1.86, 95% CI 1.65-2.10, p 0.01) and for PMF diagnosed after 2009 vs ≤2009 (SIR 2.64, 95% CI 2.26-3.07 vs SIR 1.61, 95% CI 1.40-1.85, p<0.001). Conclusions: Patients with PMF are at a high risk of developing SPM, especially leukemia and lymphoma. Data suggests higher incidence of SPM in patients aged ≤60 years and in the decade after 2009. The impact of ruxolitinib, which was approved in 2011, on the incidence of SPM deserves further study. [Table: see text]

2970 Non-Cirrhotic Portal Hypertension in Myeloproliferative Disease: Left and Right-Sided Component of Portal Hypertension

INTRODUCTION: NCPH is the presence of portal hypertension (PH) in the absence of cirrhosis. While Schistosomiasis is the commonest cause in West, myeloproliferative diseases (MPD) characterized by cellular proliferation of one or more hematologic cell lines resulting in extra-medullary hematopoiesis predominantly in the spleen and occasionally in liver may cause PH. Pathogenesis of NCPH in MPD revolves around the concept of left sided portal hypertension. This study presents a cohort of Indian patients of NCPH with MPD. METHODS: 260 patients with NCPH were evaluated and 9 MPD was diagnosed based on 2008 WHO-criteria. Cirrhosis was ruled out by fibroscan and liver biopsy. All patients underwent a pro coagulant screen which included protein-C, protein-S, factor-V leiden, antithrombin D, anti phospholipid antibody, prothrombin gene deficiency and Jak-2 mutation study. Routine PH evaluation and Fibroscan and liver biopsy was done in all cases. RESULTS: Out of 9 patients 4 (44.4%) each had polycythemia-rubra-vera (PRV) and myelofibrosis while 1 had essential thrombocytosis. Median age was 50 ± 7 yrs with 8 male and 1 female patient. 5 (55.5%) presented with symptomatic splenomegaly, 1 (11.1%) with ascites and 3 (33%) with GI bleed and comprised of 64.3% in PRV. Splenomegaly was universally present and hepatomegaly in 44.4%. Isolated gastric varices was present in 3 and gastroesophageal varices in 4 and isolated esophageal varices in 2 patients. Except for Jak-2 mutation found positive in 100% cases of PRV and 66.7% cases of MF, none of the other markers were positive. None, except 1 patient on liver biopsy showed sinusoidal deposits. 8/9 patients underwent endoscopic therapy for varices and were given prophylaxis with carvedilol. Mean follow up was 40 months and none had recurrent bleed or worsening of liver function due to PH. CONCLUSION: True incidence MPD causing PH is under-recognised. Our results are similar to other studies from Asia. Unlike other studies, we did not find portal vein thrombosis. MPD patients had huge splenomegaly leading to increased blood flow through the splenic vein leading to left sided portal hypertension. Myeloid metaplasia in liver can also leading to right-sided PH. Whether beta blockers would help as primary or secondary prophylaxis without this component is inconclusive. However, in our cohort of patient they were helpful. Rescue therapy like splenectomy, JAK 2 inhibitors, TIPS etc may be reserved for the refractory cases.

Peripheral and bone marrow CD34+ cell levels on chronic myeloproliferative disease

ABSTRACTPurpose: The aim of the present study was to examine the relationship between peripheral CD34+ and bone marrow CD34+ levels and the clinicopathologic characteristics and laboratory parameters of myeloproliferative disease (MPD) patients.Patients and methods: A total of 103 MPD patients were enrolled in this study. We examined the relationship between bone marrow CD34+ and peripheral CD34+ levels and the patients’ clinicopathologic and laboratory parameters.Results: There were no significant correlations between the peripheral CD34+ levels and the JAK-2 V617F mutation, thrombosis, white blood cells (WBC), lactate dehydrogenase (LDH), transferrin saturation (TS), ferritin, or bone marrow cellularity. In addition, there were no significant correlations between bone marrow CD34+ levels and the JAK-2 V617F mutation, thrombosis, WBC, LDH, TS, ferritin, or bone marrow cellularity (P > 0.05). We did not identify any significant relationship between peripheral CD34+ and bone marrow CD34+ levels (P > 0.05). However, there were significant correlations between peripheral CD34+ levels and bone marrow fibrosis (P < 0.001), between bone marrow CD34+ levels and constitutional symptoms (P < 0.05), and between bone marrow CD34+ levels and bone marrow fibrosis (P < 0.001).Conclusion: We did not find any significant relationship between the clinicopathologic and laboratory characteristics and peripheral and bone marrow CD34+ cells from bone marrow fibrosis patients. There was also no significant relationship between bone marrow CD34+ cells and peripheral CD34+ cells. Some peripheral CD34+ cells may originate from the spleen rather than the bone marrow, which may given us different result of some parameters.

Correlation between JAK2-V617F mutations and variation of peripheral blood cells among BCR/ABL-negative MPD patients

To assess the correlation between JAK2-V617F mutation and complete blood counts among patients with BCR/ABL-negative myeloproliferative diseases (MPD). One hundred and ninety one patients were recruited. Retrospectively, their laboratory data were analyzed for the counts of red blood cells (RBC), white blood cells (WBC) and platelets (PLT). And the incidence of JAK2-V617F mutation was determined. There was significant difference in the incidence of JAK2-V617F mutation between patients with different cell counts (P< 0.01). The incidence of JAK2-V617F mutation has increased with the counts of RBC and PLT, which was the highest (92.86%) among those featuring simultaneous increase in all three series. The incidence of JAK2-V617F mutation seems to be strongly associated with variation of peripheral blood cell counts among patients with BCR/ABL-negative MPD. Variation of peripheral blood cells, particularly RBC, may be correlated with the rate of JAK2-V617F mutation.

Abstract DDT01-03: Discovery of BMS-911543, a highly selective JAK2 inhibitor, as a clinical candidate for the treatment of myeloproliferative disease and other malignancies

Abstract Myeloproliferative diseases (MPDs) are a subset of myeloid malignancies that are characterized by the expansion of a multipotent hematopoietic stem cell. Chronic MPDs can be classified into two categories, those harboring the BCR-ABL oncogene and those that are negative. This later category of neoplasms encompasses polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Recent discovery of activating mutations in the tyrosine kinase gene, JAK2 and constitutive activation of JAK2-STAT pathway, in large number of MPD patients has ignited considerable interest in MPD and has highlighted JAK2 as a therapeutic intervention point for drug discovery efforts. However, high-sequence homology with other JAK family members has posed a major challenge to design selective JAK2 inhibitors. Given that other JAK family members are involved in the regulation of immune function, it is important to maintain selectivity for JAK2 over these family members in order to mitigate the risks associated with undesired immunosuppression. Several JAK2 inhibitors with varying selectivity profiles are currently being evaluated in preclinical testing as well as in clinical trials for the treatment of MPD. Additionally, emerging genetic and pharmacologic evidence suggest that inhibition of the JAK2-STAT pathway may be an important therapeutic intervention point in other hematological malignancies as well as in certain solid tumors. We report here the discovery and characterization of BMS-911543, a functionally selective small molecule inhibitor of the Janus kinase family (JAK) member, JAK2. BMS-911543 is a potent and reversible inhibitor of JAK2 with a biochemical Ki of 0.48 nM. It has over 65-, 74- and 350-fold selectivity against the other JAK family members, TYK2, JAK3 and JAK1, respectively. Importantly, examination of &amp;gt; 450 other kinases in competition binding assays and in selected biochemical kinase assays did not reveal significant inhibitory activity for this JAK2 inhibitor, highlighting its high degree of biochemical selectivity for JAK2. Functionally, BMS-911543 displayed potent antiproliferative and pharmacodynamic (PD) effects in mutated JAK2-expressing cell lines dependent upon JAK2-STAT signaling and had little activity in cell types dependent upon other pathways such as JAK1 and JAK3. Further, single agent antiproliferative activity was not observed for BMS-911543 in a variety of solid tumor cell lines dependent upon other signaling pathways. In contrast, BMS-911543 was evaluated in colony growth assays using primary progenitor cells isolated from patients with JAK2V617F-positive myeloproliferative disease (MPD) and resulted in an increased antiproliferative response in MPD cells as compared with those from healthy volunteers. Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2-pSTAT signaling in multiple species with durable and potent pathway suppression observed after a single oral dose. Additionally, BMS-911543 was evaluated for effects in a JAK2V617F-expressing SET-2 xenograft model system and displayed a minimally effective dose of &amp;lt;2 mg/kg on pSTAT5 pathway suppression, which lasted up to 8 hours. To test the hypothesis that a JAK2 selective inhibitor would have less effect on immune system function, BMS-911543 was compared to pan-JAK inhibitors in a mouse model of immunosuppression. At low dose levels active in JAK2-dependent PD models, no effects were observed on antigen-induced IgG and IgM production for BMS-911543 whereas a pan-JAK family inhibitor showed pronounced effects at all dose levels tested. The mechanistic selectivity of BMS-911543 to pan-JAK family inhibitors was extended through comparative analysis of these inhibitors in whole genome gene expression profiling experiments performed in sensitive and resistant cell types. In this comparison, BMS-911543 modulated a distinct subset of transcriptional changes as compared to pan-JAK inhibitors in clinical testing, thereby defining a minimal set of transcriptional changes underlying the pharmacologic effects of JAK2 inhibition. Collectively these results define the mechanistic basis for a differential therapeutic index between selective JAK2 and pan-JAK family inhibition pre-clinically and suggest a therapeutic rationale for the further characterization of BMS-911543 in patients with MPD and in other malignancies reliant upon constitutively active JAK2 signaling.

Usefulness of Real-time Semi-quantitative PCR, JAK2 MutaScreenTM Kit for JAK2 V617F Screening

Real-time PCR for quantification of JAK2 V617F has recently been introduced and used to evaluate the importance of mutant allele burden in both diagnosis and disease progression in myeloproliferative diseases (MPDs). We evaluated the usefulness of JAK2 MutaScreen kit that uses a real-time semiquantitative PCR method and has been designed to screen JAK2 V617F mutant allele burden. Forty MPD patients were included in this study. We screened JAK2 V617F and determined the mutant allele burden using JAK2 MutaScreen kit. The mutant allele burden was estimated by six-scaled standards of JAK2 V617F mutant allele (2%, 5%, 12.5%, 31%, 50%, and 78%). For evaluation of test performance, an allele-specific PCR (AS-PCR) was carried out in all samples by using Seeplex JAK2 Genotyping kit. We assessed the clinical differences in distinct disease entities of MPDs according to JAK2 V617F mutant allele burden. JAK2 V617F mutation was detected in 30 cases, including 10 of 11 cases (91%) of polycythemia vera (PV), 13 of 20 cases (65%) of essential thrombocythemia (ET), and 2 of 3 cases (67%) of chronic idiopathic myelofibrosis (CIMF). The concordance rate between the two tests was 95% (38/40). JAK2 V617F mutant allele burden was greater than 50% in 17 cases, and 10 of them (59%) were PV. In contrast, mutant allele burden was less than 50% in 13 cases and 11 of them (85%) were ET. JAK2 MutaScreen kit that utilizes a real-time semi-quantitative PCR method is a useful tool for diagnosing MPDs precisely. It can be used to assess the grade of mutant allele burden as well as to screen JAK2 V617F simultaneously.

Plasma levels of JAK2 mRNA in patients with chronic myeloproliferative diseases with and without V617F mutation: implications for prognosis and disease biology.

The association of V617F JAK2 expression levels with disease behavior has not been studied in patients with nonchronic myelogenous leukemia (CML) myeloproliferative disease (MPD). We found plasma levels of total JAK2 mRNA to be higher in patients with non-CML MPD (n=175) than in CML patients (n=45) and normal controls (n=58) (each P<0.001). Overall survival was studied in 68 patients and showed positive correlation with levels of total and mutant JAK2 mRNA in patients with the V617F mutation, but not those without the mutation. These findings suggest that total JAK2 expression levels play a role in the biology of the disease in V617F-positive patients, and a therapy aiming at downmodulating the expression of the total JAK2 mRNA should be considered. In conclusion, we studied JAK2 total and V6217F mutant mRNA levels in plasma. We show high levels of JAK2 expression in MPD patients and these levels correlate with survival.

Screening JAK2 Exon 12 in JAK2 V617F Negative Patients with Myeloproliferative Disorders Reveals a New Splice Site Mutation

Background. The identification of the somatic mutation V617F in exon 14 of the Janus kinase 2 gene (JAK2) has simplified the diagnosis of many patients affected with typical chronic myeloproliferative diseases (MPDs). In patients without the V617F the molecular basis of MPD are still unclear but, recently, mutations in exon 12 of the JAK2 gene have been identified in a minority of patients, associated with a selective increase in erythropoiesis resulting in polycythemia vera (PV) or idiopathic erythrocytosis (IE) (Scott et al, NEJM 2007).Aim. To determine the JAK2 exon 12 mutational status in a group of JAK2 V617F negative patients with PV or IE.Methods. Genomic DNA was extracted from peripheral blood leukocytes from 85 MPD patients (PV or IE) included in the present study. All the samples were tested for JAK2 V617F mutation by allele specific polymerase chain reaction (ASO-PCR) and those negative for V617F mutant allele were subjected to real time quantitative PCR (RQ-PCR) using hybridization probes. Subsequently, all JAK2 V617F negative samples by both ASO-PCR and RQ-PCR where subjected to direct sequencing to exclude JAK2 exon 12 mutations.Results. JAK2 V617F was positive in 78 (91.7%) of the 85 patients, however, in two of these patients the V617F mutation was only detected upon subjecting genomic DNA to RQ-PCR which revealed low levels of the mutant allele, 2.65 % and 3.98%. Analysis of the JAK2 exon 12 in the seven JAK2 V617F negative patients detected three previously described mutations: a duplication (V536-1546) and two deletions (H539-K540del+542K and R541-E543 delinsK)and a previously unreported splice site mutation detected in intron 12 (IVS12nt6 T-C). To our knowledge this was the first description of intronic mutations in the JAK2 gene. No mutations were detected in the remaining 3 (3.6%) patients.Conclusions. In this cohort study JAK2 mutations were observed in 82 (96,5%), of the 85 patients, of these 78 (95.12%) had the V617F allele. In two cases V617F was detected at low levels (2.65% and 3.98%) only by RQ-PCR, highlighting the need for sensitive techniques to detect somatic mutations. One of the patients, a young male with erythrocytosis and low serum Epo levels, revealed a previously unreported splicing mutation (IVS12 nt6: T-C). This study illustrates the heterogeneity at the DNA level in PV patients which may assist in better understanding the genotype and phenotype relationship in MPD patients and assist in further delineating the role of JAK2 in these disorders.

Comparison of JAK2V617F prevalence in African American and caucasian patients with myeloproliferative disease (MPD)

18001 Background: Prevalence of JAK2V617F in patients with MPD has been mainly estimated from cohorts of Caucasians (CAUC). Little information is available from African Americans (AA) with MPD. The Washington DC VA Medical Center population has approximately 75% AA patients providing an opportunity to compare the frequency of JAK2 V617F between the two groups. Methods: DNA extracts from the peripheral blood neutrophils of 33 male MPD patients (15 AA, 18 CAUC) attending the Hematology clinic in the last 18 months were tested by using DNA sequencing for JAK2V617F Results: Overall, the prevalence of MPD in this predominantly male veteran population appears to be approximately fourfold greater among CAUC than in AA. Distribution of the MPD subtypes and the frequency of JAK2V617F are shown in Table 1. There was no difference in the proportion of JAK2 V617F in the individual subtypes of MPD between the AA and CAUC patients. When all MPD groups were combined, there was a trend for a higher proportion of AA patients to have JAK2V617F (p=.06). Conclusions: These results find no significant difference in the prevalence of JAK2V617F among the MPD subtypes between AA and CAUC patients in this cohort. The trend to a higher incidence of the mutation in the AA group when the subtypes were combined (60% vs. 27%) likely represents the higher but non-significant difference in the AA ET group (44% vs. 20%) and the smaller numbers of patients in the other subsets of both groups. Additional studies are needed to expand and validate these observations, which may have potentially important implications for treatment. No. of JAK2 V617F patients/total (%) African- American (n = 15) Caucasian (n = 18) Chi-square p value Polycythemia Vera 3/4 (75%) 1/2 (50%) – EssentialThrombocythemia 4/9 (44%) 3/15 (20%) NS Primary Myelofibrosis 2/2 (100%) 1/1 (100%) – Total 9/15 (60%) 5/18 (27%) 0.06 No significant financial relationships to disclose.

Overexpression of JAK2 mRNA in Chronic Myeloproliferative Disease and Its Implication on Mutation Testing and Biology of Disease.

The V617F point mutation in the JAK2 gene is considered the most important molecular abnormality that characterizes patients with non-chronic myeloid leukemia (CML)-myeloproliferative disease (MPD). While it is well-documented that the V617F mutation leads to continuous activation of the JAK2-STAT5 pathway, the levels of JAK2 mRNA expression is not well defined in myeloproliferative diseases. We quantified the levels total JAK2 mRNA as well as V617F mutant mRNA in the plasma of patients with non-CML MPD using RT/PCR, and compared the results with clinical findings and disease behavior. Patients with non-CML MPD (n = 175) had significantly higher levels (P <0.0001) of JAK2 mRNA (median, 1843 pg/ul) than patients with CML (median, 71.53 pg/ul) and normal controls (median, 34.34 pg/ul). In contrast, there was no significant difference (P = 0.15) in DNA levels (copies of JAK2 DNA/ul plasma) between CML patients and non-CML MPD patients. There was no significant difference in JAK2 mRNA levels in the MPD group between patients with or without the V617F mutation. There was no significant difference in JAK2 mRNA levels between patients with polycythemia vera, essential thrombocythemia, or agnogenic myeloid metaplasia, or unclassified MPD (P = 0.09). Survival data was available for only 68 patients; JAK2 mRNA levels in these patients did not correlate with overall survival. In contrast, mutant (V617F) JAK2 mRNA levels in the plasma correlated negatively with overall survival in continuous fashion (P = 0.04) when all patients (with and without the mutation) were considered, as well as when only patients with the mutation were considered (P = 0.03). In contrast, when we quantified levels of mutant JAK2 DNA, 4 of the 68 patients who showed mutation using RNA were negative for the V617F mutation by DNA analysis, and the levels of mutant JAK2 DNA did not correlate with survival. This data suggests that JAK2 mRNA is overexpressed in all patients with non-CML MPD, and levels of mutant JAK2 mRNA may correlate with clinical behavior. Furthermore, when testing for JAK2 mutation, RNA rather than DNA should be used.

The Investigation of JAK2 V617F Mutation in Chinese Patients with Hematological Malignancies.

Myeloproliferative Diseases (MPD) are a spectrum of pathogenetically related disorders of varying clinical manifestations, characterized by neoplastic expansion of relatively mature granulocyte, erythroid, megakaryocyte, or monocyte and eosinocyte lineage cells. Recently a novel point mutation affecting the Janus tyrosine kinase 2 (JAK2 V617F) was identified as pathogenetically mechanisms by multiple competing groups. To investigate its prevalence and clinical significance in Chinese patients with hematological malignancies, we introduced Allele-specific PCR (AS-PCR) combined with sequence analysis to screen JAK2 V617F mutation.A total of 98 Chinese MPD patients and 120 additional hematological malignancies including AML, ALL, MDS were analyzed for the JAK2 V617F mutation. 98 MPD patients were referred for PV (n=57), ET (n=18), IMF (n=12), HES (n=2) and CML (n=9). 2 ml peripheral blood samples of MPD and 5–10 ml bone marrow samples of AML, ALL, MDS at the time of initial diagnosis were obtained with informed consent and genomic DNA was isolated presently. In addition, peripheral blood samples from 20 healthy donors were also collected as control. All samples were first screened by AS-PCR. The positive samples were subsequently confirmed by sequence analysis.The results showed that JAK2 V617F mutation was detected in 43 of 57 PV patients (75.4%), 7 of 18 ET patients (38.9%) and 5 of 12 IMF patients (41.7%). None of the AML, ALL, MDS, CML was found JAK2 V617F. There is no statistical difference of JAK2 V617F positive ratio between PV, ET and IMF. Furthermore, the mutation was not detected in the HES patient and 20 healthy controls. There is no other mutations and polymorphisms throughout exon 12 of JAK2.To our knowledge, this is the first report of JAK2 V617F mutation in a number of Chinese patients with hematological malignancies especially BCR/ABL-negative MPD. The incidence of JAK2 V617F of our study is a little lower compared with other publications especially in PV patients. The main reason may be firstly attributed to ethnic difference. In addition, with more MPD patients introduced and more sensitive methods such as ARMS-PCR or Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) applied, more JAK2 V617F mutation will be identified.

Platelet and Coagulation Activation in Myeloproliferative Diseases (MPD), and Relationship to JAK2(V617F) Mutation Status and Clonality.

Patients with MPD have an increased risk of thrombosis. Previous reports suggest an association between clonality and thrombosis in ET. The contribution of the JAK2 mutation to thrombotic risk is unclear. In our cohort of patients with MPD (n=122) (PRV n = 54, ET n = 64, IMF n= 4), we compared coagulation and platelet activation markers (D-Dimers, thrombin antithrombin complexes (TAT), prothrombin fragments 1+2 (F1+2), soluble E-selectin(sE-selectin) and soluble P-selectin(sP-selectin)) between MPD patients and hypertensive controls. sP-selectin was significantly increased in patients with MPD (p=<0.001). The JAK2 mutation status was determined in our cohort by ARMS PCR. Of the total MPD cohort, 59% were JAK2 positive, (76% of PRV, 45% of ET, and 50% of IMF). Coagulation activation markers were compared in JAK2 positive and JAK2 negative patients. sP-selectin levels were highly significantly elevated in JAK2 positive patients compared to JAK2 negative (p= 0.002), or controls (p<0.001).There was no significant difference in platelet count between JAK2 positive and negative patients (p=0.19). The clonality of the MPD was determined in 54 female patients using an x-chromosome inactivation pattern (XCIP) assay (HUMARA). A significant proportion of “polyclonal” patients, as defined by XCIPs were positive for the JAK2 mutation. We therefore calculated the proportion clonality of samples and found no correlation between proportion clonality and any coagulation or platelet activation marker.Our results show an increase in platelet activation, as determined by sP-selectin levels, in patients with MPD compared to controls. Furthermore, platelet activation was a feature of patients positive for the JAK2 mutation when compared with wild type patients and controls. The role of the JAK2 mutation as a risk factor for thrombosis in MPD is still unclear but our study indicates that the presence of the mutation may be linked to platelet activation in MPD. Whether this translates into a higher clinical thrombosis risk requires further evaluation in a large prospective study.

Myeloproliferative Disease in the Pathogenesis and Survival of Budd-Chiari Syndrome.

Budd-Chiari syndrome (BCS) is a rare disorder caused by obstruction of the hepatic veins or the suprahepatic inferior vena cava. Myeloproliferative diseases (MPD) are the most important aetiological factor in BCS. The aim of this study was to evaluate multifactorial aetiology in BCS patients with MPD, to assess potential added value of the recently discovered JAK2 mutation in the diagnostics of MPD in BCS, and to determine the survival of MPD patients in BCS. All patients referred to our university hospital with primary, non-malignant BCS between January 1980 and January 2006 were included in this study (n=40). Median age was 28.4 years (18.4–53.3), 26 patients (65%) were female and in nine patients (23%) additional portal vein thrombosis (PVT) was present. Overall mean follow-up was 7.1 ± 6.9 years. Only two patients were lost to follow-up, of which one MPD patient. In addition to standard MPD work-up according to WHO criteria, JAK2 mutation analysis was performed in 17 patients. MPD was present in 33% of the patients: Polycythemia Vera (n=6), Essential Thrombocythemia (ET) (n=6) and unclassifiable MPD (n=1). JAK2 mutation was identified in seven out of the 17 tested BCS patients (41%). In two patients suspect for ET, but who failed to meet WHO criteria, JAK2 mutation analysis led to the diagnosis of MPD. In 38% of patients with MPD additional pro-thrombotic factors were present. Survival rates in patients with MPD at 1, 5, and 10 years remained constant at 92% (95% CI, 78%–100%). Survival rates in patients without MPD at 1, 5, and 10 years were 89% (95% CI, 77%–100%), 64% (95% CI, 44%–85%) and 53% (95% CI, 28%–79%), respectively. There was no significant difference in survival between the two (P=0.18). Additional PVT was only present in patients without MPD in our population, which may account for the slightly lowered survival of these patients, since more extensive thrombosis in the splanchnic area is associated with poorer survival. In addition, three patients with and one patients without MPD underwent liver transplantation, which may have affected survival rate in favour of MPD patients. In conclusion, we report MPD in 33% of our BCS patients. In 38% of the patients with MPD, additional pro-thrombotic factors were present. These results enforce the necessity of extensive screening for additional pro-thrombotic conditions. Our study clearly confirms the importance of JAK2 mutation analysis in patients with BCS. Long term follow-up did not show a significant difference in survival between patients with and without MPD.

The Clinical Implications of JAK2V617F Mutation in 137 Chinese Patients with Myeloproliferative Diseases.

A single acquired mutation in the JAK2 gene has recently been described in the classic myeloproliferative diseases(MPDs) including 65–97% of patients with polycythemia vera (PV), 23–57% of those with essential thrombocythemia (ET) and 35–57% of those with idiopathic myelofibrosis (IMF). The understanding of the molecular mechanisms has been considered a point mutation in exon 12 of the JAK2 gene, a key hematopoietic regulator, resulting in substitution of valine for phenylalanine at amino acid position 617 in the JH2 domain. In order to clarify the frequency and some clinical implication of JAK2V617F mutation in the Chinese patients with MPDs, we investigated this mutation in 137 Chinese patients aged 15 to 65 years. There are 57 patients with PV, 68 with ET, and 12 with IMF and the diagnosis of MPDs was defined according to published criteria. Genomic DNA samples obtained from all three MPDs and twenty cases of healthy people as negative control and the HEL, an erythroleukemic cell line, as positive control was screened by allele-specific PCR and gel electrophoresis for JAK2V617F mutation described by Baxter EJ et al.The results indicated that 65.69% of heterozygous JAK2V617F mutation was shown in 137 patients of over all MPDs, 73.68% in PV (42/57), 58.82% in ET (40/68), 66.67% in IMF (8/12). Sequence analysis of PCR products from selected patients confirmed existence of both mutant and wild-type alleles in patients with JAK2V617F mutation. A higher prevalence was observed in older patients with MPDs, and the WBC count of ET patients with JAK2V617F mutation is higher than that of ET patients without JAK2V617F mutation, and the difference has a statistical significance (P<0.05). The HGB concentration and platelet number of MPDs patients with JAK2V617F mutation were higher than those without JAK2V617F patients, but the difference did not achieve a statistical significance(P>0.05).Cytogenetic analyses were performed in 115 of the 137 MPDs patients. The chromosomal karyotype was normal in 108 of the 115 patients. Among them, JAK2V617F mutation was detected in 74 patients (68.51%) as compared with 5 of the 7 patients with karyotypic abnormalities (71.4%). Three patients with detective bcr/abl low expression assayed by FISH and real-time PCR techniques, two have JAK2V617F mutation. Our datas suggest that JAK2V617F mutation also occurs in a significant percentage of Chinese patients with the myeloproliferative diseases.

Association of Plasma Adiponectin Concentrations with Chronic Lymphocytic Leukemia and Myeloproliferative Diseases

Adiponectin, an adipocyte-secreted hormone, is an important negative regulator in the immune system and hematopoiesis. In this study, we investigated the association of adiponectin levels with chronic lymphocytic leukemia (CLL) and myeloproliferative diseases (MPDs). We measured adiponectin levels in 19 patients with CLL and 30 patients with MPD (chronic myelogenous leukemia, 15; polycythemia vera, 9; myelofibrosis, 4; essential thrombocythemia, 2). The data were (chronic myelogenous leukemia, 15; polycythemia vera, 9; myelofibrosis, 4; essential thrombocythemia, 2). The data were compared with results from a control group of healthy volunteers who were matched according to age, sex, and body mass index. The adiponectin levels in patients with CLL were lower than in the controls (4.71 +/- 1.33 microg/mL versus 16.61 +/- 3.91 microg/mL; P <.001). They were also significantly lower in patients with MPD than in the controls (8.95 +/- 1.33 microg/mL versus 16.16 +/- 4.77 microg/mL; P <.001). In addition, we compared the adiponectin levels of MPD patients who were treated with interferon (IFN) to the levels of patients who were not treated with IFN. Adipnectin levels were significantly higher in IFN-treated patients (11.03 +/- 1.39 microg/mL versus 6.87 +/- 1.79 microg/mL; P <.001). These results suggest that lymphopoiesis and myelopoiesis negatively influence adiponectin levels. Adiponectin may be related to inflammatory cytokine release. IFN therapy appears to have a positive influence on adiponectin secretion by suppressing inflammatory cytokines. Future studies are needed to prove causality and to provide insight about this hormone's mechanism of action and its potential role regarding the etiology and progression of CLL and MPD.

Fatigue, the Un-Addressed Curse of Myeloproliferative Diseases (MPD): Results of an International Internet Based QOL Survey of 830 MPD Patients.

Background:Little objective data exists regarding the impact that myeloproliferative disorders (MPDs) (essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis with myeloid metaplasia (MMM)) have on quality of life.Methods:An Internet based survey of MPD patients regarding clinical course, co-morbidities (through the Charlson Co-Morbidity Index), fatigue (through two validated fatigue instruments (FACT-AN and Brief Fatigue Inventory (BFI)), and exercise tolerance (Godin Leisure-Time Questionnaire).Results:Patient Demographics: A total of 830 MPD patients (median age 56; 60.8% male) from a broad geographic distribution (77% USA: 50 states, 30 countries, 5 continents) responded to the survey. Self reported diagnoses were PV (N=272; 33%), ET (N=221; 27%), or MMM (N=329; 40%) for a median of 5 years (range (0–53).Clinical History: 708 patients (87%) had undergone treatment for their MPD (drugs 84.6%, phlebotomy 49.5%, other 16%). A history of thrombotic (n=195, 24%) or hemorrhagic events (N=214; 26%) were frequently reported. Blood tests (available in 597 a median 1.65 months prior to survey) showed 37% were anemic (7% transfusion dependent). Additionally, 347 (44%) had splenomegaly, painful in 21%.Symptoms: Frequent symptoms reported included fatigue (90%), pruritus (57%), night sweats (54%), bone pain (48%), undesired weight loss (15%), and/or fevers (15%). 41% felt restricted in their activities, and 89 (11%) were medically disabled secondary directly to their MPD. 82% of patients felt their fatigue was linked to their MPD. Additionally 69% curtail social activity because of fatigue, and 41% need some degree of assistance with activities of daily living. Charlson co-morbidity index demonstrated 722 (87%) had minimal co-morbidities strengthening the association between fatigue and their MPD. The burden of fatigue decreased from MMM to PV to ET respectively (p=0.0002). Results from both fatigue assessment tools (FACT-AN and BFI) were highly correlated. Fatigue (FACT-AN fatigue subscale) strongly correlated with multiple MPD disease features including: anemia, bone pain, constitutional symptoms, pruritus, splenic pain, hemorrhagic events (all p<0.0001) and thrombosis(p=0.01). Additionally, currently smoking (as opposed to being a prior smoker) was highly associated with increased fatigue (p<0.0001). Interestingly, even in “asymptomatic” patients (those without anemia, splenomegaly, or thrombohemorrhagic complications (n=187)) 73% (n=128; 40% PV, 32% ET, 27% MMM; p=N.S.) described moderate to severe MPD associated fatigue. Reported physical activity was diminished (Godin Leisure Score) between MPD patients and published norms, with 38% describing minimal to no exercise. Overall, 86% stated fatigue was the major obstacle to exercise.Conclusions: Fatigue is an extremely common problem in patients with MPDs despite current therapies, and a minimal burden of co-morbidities in the majority of patients. Fatigue is also common in patients lacking advanced MPD disease features, or thrombohemorrhagic complications. Clearly, direct attempts to improve fatigue (through pharmacologic and non-pharmacologic interventions) are necessary.

Potential role for platelet apheresis for post‐liver transplant thrombocytosis complicating portal vein thrombosis

Except for patients with underlying myeloproliferative diseases (MPD), thrombocytosis is rarely encountered in cirrhotic patients after liver transplantation. Although the long-term control of primary thrombocytosis is important for the prevention of graft thrombosis in MPD patients, the threshold for intervention and best mode for the control of persistent reactive thrombocytosis after liver transplantation is unclear. We present two patients with extreme reactive thrombocytosis (over 1,000 x 10(9)/l) due to intra-abdominal sepsis after liver transplantation. Furthermore, both patients suffered from bleeding problems as well as ongoing venous thrombosis of the graft. Rapid control of the platelet count was achieved using platelet apheresis. The use of cell separation procedures may be a relatively rapid, reversible, and reasonably safe way to control platelet counts peri-operatively in liver transplant recipients.

Accelerated telomere length shortening in granulocytes: a diagnostic marker for myeloproliferative diseases.

The telomere in mature myeloid cells derived from abnormal progenitor cells of myeloproliferative diseases (MPDs) may shorten more rapidly than that in T lymphocytes, which are considered to be derived from normal clones. To test this hypothesis, we measured telomere lengths in granulocytes and T lymphocytes from patients with MPDs and compared them with those from normal individuals. Granulocytes and T lymphocytes were separated from the peripheral blood of 65 patients with MPDs (25 chronic myelogenous leukemia [CML], 16 polycythemia vera, 19 essential thrombocythemia, 5 chronic idiopathic myelofibrosis) and 35 normal individuals. Genomic DNA from each cell fraction was subjected to Southern blot hybridization to determine the mean telomere length. Telomere lengths in granulocytes from patients with MPDs were significantly shorter than those from normal individuals (vs CML, p = 0.002; vs other MPDs, p < 0.0001). However, there was no statistical difference in telomere length in T lymphocytes between MPD patients and normal individuals (vs CML, p = 0.35; vs other MPDs, p = 0.85). DeltaTRF (terminal restriction fragment) in patients with MPDs, which is defined as the difference in telomere length between granulocytes and T lymphocytes, was significantly longer than that in normal individuals. The results support the disease theory that MPDs result from extensive proliferation of myeloid progenitor cells, leading to accelerated telomere length shortening in mature granulocytes. An increase in DeltaTRF over the standard value (>1.74 kb) may be useful for discriminating leukocytosis due to MPDs from reactive leukocytosis.

Platelet distribution width for differential diagnosis of thrombocytosis

Differential diagnosis of thrombocytosis is not always obvious. The routine clinical chemistry laboratory classically provides only limited help in distinguishing between reactive thrombocytosis (RT) and autonomous thrombocytosis, where platelet production escapes normal regulatory processes, and which is seen in myeloproliferative diseases (MPD) such as essential thrombocythemia and polycythemia vera. We explored the clinical use of platelet distribution width (PDW) in the differential diagnosis of thrombocytosis. During a 3-month period, 250 patients presenting with a platelet count > 500 x 10(9)/L were studied; 174 were classified as having RT, 42 had a diagnosis of MPD, and 34 patients were excluded because they had a hemopathy different from MPD, and either did or did not present a known etiologic factor for RT. First, we determined that in the RT group the value of PDW was closely linked to both mean platelet volume (MPV) and platelet count (PLT) (PDW = 79.5-0.005 PLT -3.5 MPV; r = 0.848, R2 = 0.720). Therefore a new parameter, PDWresidual was defined (PDWresidual = PDWobserved -PDWexpected). Second, the discrimination between reactive and autonomous thrombocytosis obtained with PDWresidual was compared with that obtained with either PDW, MPV, or PLT. PDWresidual provided much more powerful than each of the other parameters used separately: 76% of MPD patients had a PDWresidual above the 95th percentile value of the RT population and none of the MPD patients had a PDWresidual below the 50th percentile. Thus, the combined interpretation of PLT, MPV, and PDW through the use of a PDWresidual appears highly useful in the differential diagnosis of thrombocytosis. Also, through simple modeling, more information can be drawn from parameters such as PDW that hitherto were mostly discarded as being without clinical interest.

Safety and efficacy of picotamide, a dual anti-thromboxane agent, in patients with thrombocytosis and a previous thromboembolic event: a 1-year observational study.

Patients with chronic myeloproliferative disease are at increased risk of both thromboembolic and haemorrhagic complications. Cerebral thrombosis is a common cause of death in myeloproliferative disease patients. Picotamide is a new anti-platelet drug sharing a dual anti-thromboxane activity: inhibition of thromboxane A2 synthase and thromboxane A2 receptor antagonism. Picotamide inhibits in vitro and ex vivo platelet aggregation induced by different agonists. Interestingly, in vitro studies show that picotamide is able to increase prostacycline biosynthesis. In the clinical setting, picotamide treatment induces only a slight prolongation of bleeding time. The safety and efficacy of picotamide long-term treatment in 15 patients with essential thrombocytosis and a positive history of previous thromboembolic events was evaluated. After 12-month treatment with picotamide no patients suffered from thrombotic events and only one minor and transient bleeding episode was observed. This observational long-term trial shows that picotamide treatment in patients with thrombocytosis at high risk of thrombotic events is safe and well tolerated. Picotamide did not increase the risk of bleeding in these patients, while at the same time, no thrombotic events were observed during the 1-year treatment.