Polycythemic Patients Research Articles

Exploring the Role of Polycythemia in Patients With Cyanosis After Palliative Congenital Heart Surgery.

To understand the relationship between polycythemia and clinical outcome in patients with hypoplastic left heart syndrome following the Norwood operation. A retrospective, single-center cohort study. Pediatric cardiovascular ICU, university-affiliated children's hospital. Infants with hypoplastic left heart syndrome admitted to our medical center from September 2009 to December 2012 undergoing stage 1/Norwood operation. None. Baseline demographic and clinical information including first recorded postoperative hematocrit and subsequent mean, median, and nadir hematocrits during the first 72 hours postoperatively were recorded. The primary outcomes were in-hospital mortality and length of hospitalization. Thirty-two patients were included in the analysis. Patients did not differ by operative factors (cardiopulmonary bypass time and cross-clamp time) or traditional markers of severity of illness (vasoactive inotrope score, lactate, saturation, and PaO2/FIO2 ratio). Early polycythemia (hematocrit value > 49%) was associated with longer cardiovascular ICU stay (51.0 [± 38.6] vs 21.4 [± 16.2] d; p < 0.01) and total hospital length of stay (65.0 [± 46.5] vs 36.1 [± 20.0] d; p = 0.03). In a multivariable analysis, polycythemia remained independently associated with the length of hospitalization after controlling for the amount of RBC transfusion (weight, 4.36 [95% CI, 1.35-7.37]; p < 0.01). No difference in in-hospital mortality rates was detected between the two groups (17.6% vs 20%). Early polycythemia following the Norwood operation is associated with longer length of hospitalization even after controlling for blood cell transfusion practices. We hypothesize that polycythemia may be caused by hemoconcentration and used as an early marker of capillary leak syndrome.

Polycythemia Vera Vs. Secondary Erythrocytosis Mortality Outcome in a Multiracial Inner City Cohort

BackgroundLife expectancy of patients with Polycythemia Vera is reduced compared with the general population. History of thrombosis has been found to be the main predictor of death in this disease. Factors like older age, leukocytosis and thrombosis have been shown to increase the risk of mortality. Secondary polycythemia on the other hand is largely an unknown entity. This study aims to compare the various prognostic and predictive factors for mortality between primary polycythemia vera and secondary polycythemia group.Patients and MethodsPatients >=18 years of age seen at a Montefiore outpatient clinic and/or inpatient setting from 1998 to 2014 with a diagnosis of polycythemia, either primary or secondary, who have died, were included. This was done with the help of Clinical Looking Glass software after obtaining Institutional Review Board approval. Patients were stratified by race and ethnic groups. Primary polycythemia was confirmed by evaluating each medical record in detail, lab values including erythropoietin level, JAK2 mutation status and bone marrow biopsy reports. Other laboratory results evaluated were WBC, Hg, platelets, hemoglobin, MCV, RDW and albumin.Morbidity including history of arterial/venous thrombosis and mortality data were obtained to compare survival and identify potential predictors of morbidity and mortality.All analyses were conducted using STATA statistical software. Baseline characteristics were compared using two sample tests of proportions and Student t-tests for means. Kaplan-Meier curves were constructed to compare survival by various subgroups. Hazard ratios were calculated using multivariate Cox regression models to adjust for co-variates to compare mortality risk and identify potential independent predictors of mortality.ResultsTotal of 112 patients with polycythemia, who have died within the period of 1998-2014, were identified. 64 (57%) patients had primary polycythemia and 48 (43%) patients were diagnosed with secondary erythrocytosis. The median age for diagnosis was 63 years for secondary and 72 years for primary. The commonest causes of secondary polycythemia in these patients were severe COPD, congenital heart disease and severe pulmonary hypertension.In patients with polycythemia who died within the specified period of 16 years, median time to mortality from diagnosis was significantly shorter (p value: 0.004) in secondary polycythemia (21.1 months or 634 days) compared to primary polycythemia group (42.6 months or 1277 days). Figure 1 illustrates the duration in survival from time to diagnosis between the two groups. There was no significant difference amongst the white, black and Hispanic populations in the 2 groups.23 (38%) patients in the primary polycythemia group had venous/arterial thrombosis compared to 6 (5%) in the secondary group. This difference was also significant with a p value of 0.005.As expected, erythropoietin was lower in primary compared to secondary group. Also, WBC and platelet counts were significantly higher in primary polycythemia versus secondary polycythemia group.An interesting finding was that the mean albumin was significantly lower in the secondary polycythemia (3.3) patients who died compared to the primary polycythemia patients (3.8) with a p value of 0.004.ConclusionsOur findings demonstrate that patients who die with secondary erythrocytosis have shorter time to death from diagnosis compared to primary polycythemia vera. This is most likely due to the worse prognosis associated with the underlying disease causing erythrocytosis. Even though, secondary polycythemia is not generally treated in the same way as primary polycythemia, development of increased hemoglobin should be taken as a cautionary sign for these patients as this is likely a strong predictor of mortality. Moreover, patients who died with secondary polycythemia have lower albumin levels compared to primary polycythemia vera patients. This signifies the importance of albumin levels as a prognostic factor in patients with secondary erythrocytosis. This is a novel study which compares primary and secondary polycythemia and helps to delineate various prognostic factors in these disease groups. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hydroxyurea Treatment Is Associated with Elevated Serum Erythropoietin Concentration but Suppressed Global Hypoxic Transcriptional Responses in Sickle Cell Disease

BackgroundThe level of distorted erythrocytes due to polymerization of hemoglobin S in sickle cell disease (SCD) (Science 1949;110:543) is a major determinant of the severity of hemolysis and microvascular occlusion (Lancet 2010;376:2018). Erythropoietin (EPO) is elevated in SCD due to hemolytic anemia and a related increase in hypoxia-inducible factors (HIFs) (Eur J Haematol 2007;78:183). Hydroxyurea (HU) is widely used in the treatment of SCD. HU inhibits ribonucleotide reductase (Semin Oncol 1992;19(3 Suppl 9):1-10) and promotes γ globin synthesis thereby increasing HbF-containing erythrocytes (F cells) while suppressing sickle β hemoglobin production (J Clin Invest 1984;74:652 and 2003;111:231). Increased level of F cells reduces hemolysis and ameliorates clinical complications in SCD. We and others have observed an increase in serum EPO level with HU treatment in SCD despite an increase in the hemoglobin concentration, and we hypothesized that this may be due to the known increased affinity of hemoglobin F for oxygen and related tissue hypoxia (Blood 2009;114:4639).MethodsMessenger RNA from peripheral blood mononuclear cells (PBMCs) was profiled using Affymetrix Human Exon 1.0 ST Array. Hypoxic transcriptional alteration was defined in 15 Chuvash polycythemia (CP) patients vs. 17 control individuals. CP leads to constitutive up-regulation of HIFs in the absence of anemia or hypoxia. Transcriptional alteration in SCD was determined in 13 HbSS subjects without HU treatment vs. 16 control individuals, and that induced by HU treatment was determined in 19 HbSS subjects with vs. 13 without HU treatment. For meta-analysis on serum EPO concentration, genomic DNA isolated from PBMCs was hybridized to the Illumina Human 610-Quad SNP array. Genotypes were imputed to 1000 genomes project phase 1 data. A linear regression model was applied adjusting for age, gender, hemoglobin concentration, and HU treatment.ResultsGene expression changes by HbSS highly correlated with those associated with homozygous VHLR200W (Pearson's r=0.79, Figure 1A). At 5% false discovery rate (FDR), expression levels of 377 genes were altered in both VHLR200W homozygotes and HbSS by >1.2 fold. For these hypoxic genes, the correlation of expression changes between HbSS and homozygous VHLR200W reached r=0.97 (Figure 1B). In contrast to our hypothesis, HU treatment in general suppressed expression changes induced by HbSS (r=-0.85, Figure 1C), especially for the hypoxic genes (r=-0.95, Figure 1D). In VHLR200W homozygotes, 62 of the hypoxic genes correlated with plasma EPO levels (adjusted P<0.05, n=42). These EPO-correlated genes were the most strongly up-regulated hypoxic genes in HbSS (red points in Figure 1B) and also the most strongly suppressed by HU treatment (red points in Figure 1D). Consistent with previous observations, we found that EPO was elevated by HU treatment in two SCD cohorts, and this persisted after adjusting for covariates including hemoglobin concentration which reflects hypoxic as well as inflammatory and hemolytic responses: Walk-PHaSST (β=0.49, P=2.5×10-15, n=586) and PUSH children (β=0.34, P=2.5×10-7, n=387). This observation suggests that biological signals independent of hypoxic regulation may contribute to EPO production under HU treatment. In a meta-analysis for the Walk-PHaSST and PUSH children cohorts, SNP rs60684937, located within the first intron of MAP2K6, an upstream regulator of HIF signaling (Mole Cell Biol 2005; 25:4853), was significantly associated with EPO levels at genome-wide significance (combined P=3.5×10-8). The C allele of the SNP decreased EPO levels in both Walk-PHaSST (β=-0.30, n=388) and PUSH children (β=-0.24, n=249) cohorts. This association was validated in an additional 89 SCD patients from the Howard cohort (β=-0.39, P=0.011). Further investigations are needed to determine whether the causal polymorphism affects protein function or gene regulation of the nearby genes.DiscussionOur study demonstrates a prominent release from hypoxic transcriptional responses by HU treatment in SCD despite an increase in serum EPO, a defining characteristic of an up-regulated hypoxic response. Our study hypothesizes that hypoxia-independent signals trigger EPO production in the setting of HU therapy and it identifies a potential genetic determinant in this alternative pathway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Assessment and Validation of the EQ-5D Among a Population of Myeloproliferative Neoplasm Patients

IntroductionIndividuals with the Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) have previously been shown to experience symptoms in excess to age and comorbidity matched peers. Although numerous assessments of symptom burden and quality of life have been utilized among MPN populations, including the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30), Patient Reported Outcomes Measurement Information System (PROMIS¨) fatigue scale, and Functional Assessment of Cancer Therapy-Lymphoma (FACT-Lym) and the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-10), there are currently no validated assessments of overall health. Developed by the EuroQol Group to assess heath status, the EQ-5D is an internationally validated self-assessment which may be useful in the assessment of health state among MPN patients (Annals of Medicine, 2001. 33:337-43).MethodsThe EQ-5D evaluates five dimensions of mobility, self-care, usual activities, pain/discomfort and anxiety/depression, on a scale from no problems to extreme problems and assesses patient reported health state via a numerical visual analogue scale (VAS) ranging from 0 to 100. The health state score is a composite score of these items ranging from 0 to 1.0, with higher scores indicating an improved health state. EQ-5D survey participants were essential thrombocythemia (ET) and polycythemia (PV) patients participating in the MAJIC United Kingdom (UK) trial. The MAJIC trial is a randomized, phase II study of best available therapy versus JAK inhibition in individuals with PV or ET who have been resistant or intolerant of cytoreduction with hydroxyurea /hydroxycarbamide. This study was one of the first Trials Acceleration Programme (TAP) trials and is funded by Leukaemia Lymphoma Research, with drug supplied by Novartis. Only baseline EQ-5D assessments taken at study enrollment were included in this analysis. Comparative analysis was preformed using the MPN-10 (J Clin Oncol.2012. 30:4098-103).Results:Participant Demographics: Sixty six patients with ET and 55 patients with PV completed the EQ-5D questionnaire. Median respondent age was 65 years old and half of respondents were female (54%). Average MPN duration was 10 years for ET and 9.2 years for PV.EQ-5D Scoring: Severe to extreme difficulties in the items of mobility, self-care, usual activities, pain/discomfort and anxiety/depression were common (Table 1).Patients with PV tended to have higher proportion of patients with severe to extreme difficulties in individual disease dimensions, higher VAS score, and worsened health state compared to their essential thrombocythemia counterparts, although these effects were not significant. Average VAS health score was 70.4 (+21.2) for ET and 66.5 (+19.6). When evaluating the health state composite score, the mean ET score was 0.74 (+0.32) and mean PV score was 0.66 (+0.28).EQ-5D Validation: The EQ-5D had excellent internal consistency (CronbachÕs alpha=0.87) and strong internal correlation. When evaluating external validity, strong correlations existed between the EQ-5D and the MPN-SAF TSS scales for dimensions of mobility, usual activities, pain and overall health state (many items with r >0.5 and p<0.001).Conclusions:The EQ-5D is a valid and accurate instrument for the brief assessment of health state among MPN patients. This instrument demonstrates excellent clinical utility in a large clinical study cohort and can be used in both clinical practice and trial settings. When comparing EQ-5D health state scores, ET and PV patients had worsened health than the general UK population age 60 to 69 (0.774; Med Decis Making. 2011.31(6)800-4), but similar health to individuals with other malignancies including breast cancer (0.760), prostate cancer (0.743), and lung cancer (0.689). [Display omitted] DisclosuresMesa:NS Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Incyte Corporation: Research Funding; Pfizer: Research Funding; Promedior: Research Funding; CTI Biopharma: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Pfizer: Research Funding. Harrison:CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Shire: Speakers Bureau; Gilead: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.

Unusual involvement of the portal vein in polycythemia vera-genetic perspective

Polycythaemia Vera or erythrocytosisis a medical condition with high concentration of red blood cells. The thicker blood is less able to travel through blood vessels and organs. Most symptoms of polycythaemia are related tothis sluggish blood flow. Moderate symptoms of polycythaemia include headache, blurredvision, red skin, tiredness, elevated blood pressure, dizziness, abdominal discomfort, bleeding problems, gout and itchy skin although more severe medical events like vas-occlusion, thrombosis and strokes may occur. This case reports a 50 year old female with an unusual presentation of polycythemia vera in the form of abrupt onset &one-day-old' history of massive hematemesis with otherwise unremarkable physical exam except caput medusa around umblicus. A diagnosis of Polythythaemiavera complicated by portal vein thrombosis induced portal hypertension was finally made.Patients with polycythemia vera are at high risk of vaso-occlusive events. While the etiology of events in polycythemic patients is likely to be multifactorial, hemodilution is potentially beneficial. Attending clinicians should be well aware of the unlikely associations and squeal of thrombotic events in polycythemia vera like portal venous occlusion and hypertension.

Myeloproliferative and thrombotic burden and treatment outcome of thrombocythemia and polycythemia patients.

Prospective studies indicate that the risk of microvascular and major thrombosis in untreated thrombocythemia in various myeloproliferative neoplasms (MPN-T) is not age dependent and causally related to platelet-mediated thrombosis in early, intermediate and advanced stages of thrombocythemia in MPN-T. If left untreated both microvascular and major thrombosis frequently do occur in MPN-T, but can easily be cured and prevented by low dose aspirin as platelet counts are above 350 × 10(9)/L. The thrombotic risk stratification in the retrospective Bergamo study has been performed in 100 essential thrombocythemia (ET) patients not treated with aspirin thereby overlooking the discovery in 1985 of aspirin responsive platelet-mediated arteriolar and arterial thrombotic tendency in MPN-T disease of ET and polycythemia vera (PV) patients. The Bergamo definition of high thrombotic risk and its persistence in the 2012 International Prognostic Score for ET is based on statistic mystification and not applicable for low and intermediate MPN-T disease burden in ET and PV patients on aspirin. With the advent of molecular screening of MPN patients, MPN-T disease associated with significant leukocytosis, thrombocytosis, constitutional symptoms and/or moderate splenomegaly are candidates for low dose peglyated interferon (Pegasys(R), 45 μg/mL once per week or every two weeks) as the first line myeloreductive treatment option in JAK2(V617F) mutated MPN-T disease in ET and PV patients. If non-responsive to or side effects induced by IFN, hydroxyurea is the second line myelosuppressive treatment option in JAK2(V617F) mutated ET and PV patients with increased MPN-T disease burden.

RUNX1 and NF-E2 upregulation is not specific for MPNs, but is seen in polycythemic disorders with augmented HIF signaling

AbstractOverexpression of transcription factors runt-related transcription factor 1 (RUNX1) and nuclear factor, erythroid-derived 2 (NF-E2) was reported in granulocytes of patients with polycythemia vera and other myeloproliferative neoplasms (MPNs). Further, a transgenic mouse overexpressing the NF-E2 transgene was reported to be a model of MPN. We hypothesized that increased transcripts of RUNX1 and NF-E2 might characterize other polycythemic states with primary polycythemic features, that is, those with exaggerated erythropoiesis due to augmented erythropoietin (EPO) sensitivity. We tested the expression of RUNX1 and NF-E2 in polycythemic patients of diverse phenotypes and molecular causes. We report that RUNX1 and NF-E2 overexpression is not specific for MPN; these transcripts were also significantly elevated in polycythemias with augmented hypoxia-inducible factor activity whose erythroid progenitors were hypersensitive to EPO. RUNX1 and NF-E2 overexpression was not detected in patients with EPO receptor (EPOR) gain-of-function, suggesting distinct mechanisms by which erythroid progenitors in polycythemias with defects of hypoxia sensing and EPOR mutations exert their EPO hypersensitivity.

Nitric oxide scavenging by cell-free hemoglobin may be a primary factor determining hypertension in polycythemic patients

We tested the hypothesis that hypertension associated with polycythemia vera (PV) may be related to hemoglobin released from erythrocytes (cell-free hemoglobin, fHb). We assessed hematocrit, mean arterial pressure (MAP), blood viscosity, and the level of fHb and nitrite/nitrate (NOx) in the plasma of 73 PV patients and 38 healthy controls. The effect of isovolemic erythrocytapheresis (ECP) on the considered parameters was also studied. From the whole group of PV patients a subset of subjects with normal (normotensive patients, n = 16) and elevated MAP (hypertensive patients, n = 57) can be subtracted.It was found that in comparison with healthy controls, PV patients have significantly (p ≤ 0.01) elevated Hct (0.567 vs. 0.422), blood viscosity (5.45 vs. 3.56 cP), MAP (106.8 vs. 93.8 mmHg), plasma fHb (9.7 vs. 2.8 mg/dL), and NOx levels (34.1 vs. 27.5 μM). Compared with normotensive patients, hypertensive PV patients demonstrated a higher rise in fHb (10.2 vs. 8.0) and plasma NOx levels (35.8 vs. 31.0). In PV patients, fHb positively correlates with MAP (r = 0.489), NOx levels (r = 0.461), hematocrit (r = 0.428), and viscosity (r = 0.393). Blood viscosity positively correlated with hematocrit (r = 0.894), but not with other considered parameters. In PV patients MAP poorly correlated with hematocrit, whereas the correlation between MAP and NOx altered from − 0.325 (healthy control) to + 0.268 (PV patients). ECP procedure was associated with a significant (p < 0.01) reduction of hematocrit, fHb, blood viscosity, and MAP. In the normotensive subgroup of PV patients the ECP procedure did not affect MAP. It can be concluded that accelerated scavenging of nitric oxide by fHb rather than high Hct may be a key factor determining the development of hypertension in PV patients.

RUNX1 and NF-E2 Transcriptional Upregulation Is Not Specific For Myeloproliferative Neoplasms But Is Seen In Those Polycythemic Disorders With Augmented HIFs Signaling

▪RUNX1 and NF-E2 are transcription factors that regulate hematopoietic stem cell homeostasis. It has been reported that increased RUNX1 expression in the granulocytes is present in all three classical myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis (Wang et al, Blood 2010), and that elevated NF-E2 promotes erythropoietin (EPO)-independent erythroid maturation of hematopoietic stem cells in vitro (Bogeska et al, Stem Cells Transl Med 2013). A mouse model overexpressing the NF-E2 transgene in hematopoietic cells was reported to be a new model of myeloproliferative neoplasms (Kaufmann et al, J Exp Med 2012).Polycythemic states can be divided into primary polycythemias, characterized by intrinsically hyperproliferative erythroid progenitors that are hypersensitive to EPO, and secondary polycythemias, wherein erythroid progenitors respond normally to EPO but circulating EPO is elevated or inappropriately normal for the level of increased red cell mass. Some congenital disorders including those with mutations in the hypoxia sensing pathway may share features of both primary and secondary polycythemias. We considered the possibility that increased transcripts of RUNX1 and NF-E2 might be the feature of other primary polycythemic states as well.We report a study of 19 polycythemic patients with primary or secondary polycythemia with diverse etiologies including mutations in positive and negative regulators of hypoxia sensing pathway. RUNX1 and NF-E2 transcripts were quantitated in granulocytes and BFU-E colonies by qPCR. All primary polycythemic patients had erythroid progenitors hypersensitive to or independent to EPO; all secondary polycythemic subjects had normal erythroid progenitor response to EPO.RUNX1 and NF-E2 gene transcripts were increased in granulocytes and BFU-E colonies in all PV patients, two unrelated subjects with the VHLR200W homozygous mutation (Chuvash polycythemia), one polycythemic patient homozygous for the VHLP138L exon 2 mutation, and a patient with the HIF2αM535V gain-of-function mutation. We also found upregulated expression of RUNX1 and NF-E2 in granulocytes and BFU-Es from a polycythemic patient (with no detectable EPOR, JAK2V617F or JAK2 exon 12 mutations and low level of EPO < 1 mU/mL) who was heterozygous for a SNP in exon 3 (rs147341899) in the LNK gene. We examined transcripts of RUNX1 and NF-E2 genes in granulocytes from two Croatian polycythemic patients with a homozygous VHLH191D exon 3 mutation whose erythroid progenitors were not hypersensitive to EPO and whose RUNX1, but not NF-E2, transcript was increased. We found similar results in two compound heterozygotes for VHLT124A exon 2 and VHLL188V exon 3 mutations. These two polycythemic siblings had hypersensitive erythroid colonies, increased RUNX1 transcripts and decreased NF-E2 transcripts in granulocytes. RUNX1 and NF-E2 transcripts were normal in two subjects with primary polycythemia due to the EPOR gain-of-function EPORQ434Xmutation, and in five unrelated subjects with secondary polycythemia.We next examined granulocyte transcripts of HIF-regulated genes: TFRC, SLC2A1, VEGF, BNIP3 and HK1, and found them to be increased in all PV patients and all studied polycythemic patients with VHL, HIF2α or LNK mutations, but not in polycythemic EPORQ434Xpatients or five patients with secondary polycythemia. Increased transcripts of HIF regulated genes are compatible with the previously unappreciated Warburg effect in PV (see S. Sana's Abstract at this ASH meeting).We propose that increased expression of RUNX1 and NF-E2 is not specific for myeloproliferative neoplasms but also is not universal for primary polycythemic disorders. Therefore, increased expression of RUNX1 and NF-E2 do not seem to be underlying mechanism for MPNs development but rather represent factors associated with diverse primary polycythemia states with augmented HIF signaling.(Note: KK and LL contributed equally to this work.)This work was supported by 1P01CA108671-O1A2 (NCI) Myeloproliferative Disorders (MPD) Consortium (PI Ron Hoffman) project#1 (PI Prchal) and the Leukemia & Lymphoma Society. Work by KK, LL, MH and VD was in part supported by the European Structural Funds (project CZ.1.07/2.3.00/20.0164 and CZ.1.07/2.3.00/30.0041), grant LF_2013_010 and by Czech Science Foundation (Project-P301/12/1503). Disclosures:No relevant conflicts of interest to declare.

Genetic Association Of a MAPK8 Expression Quantitative Trait Locus With Pre-Capillary Pulmonary Hypertension In Sickle Cell Disease

Sickle cell disease (SCD) is associated with pleiotropic clinical outcomes, the severity of which exhibits remarkable inter-individual variability. Investigations of the pathophysiology of SCD have focused on the adverse effects of vaso-occlusion, chronic inflammation, and hemolysis. SCD is also characterized by up-regulation of the hypoxic response to chronic anemia. We postulated that the up-regulated hypoxic response in SCD contributes to altered gene expression that might impact pulmonary hypertension, a complication associated with early mortality.To identify genes regulated by the hypoxic response and not other effects of chronic anemia, we compared expression variation in peripheral blood mononuclear cells from 13 sickle cell anemia untreated with hydroxyurea and 15 Chuvash polycythemia (CP) patients characterized by homozygous VHLR200W-induced constitutive up-regulation of hypoxia inducible factors in the absence of anemia or hypoxia. Gene expression of both cohorts was profiled on identical Affymetrix exon arrays. The degree and direction of differential gene expression were highly correlated between sickle cell anemia and CP (Spearman's ρ = 0.73 between regression coefficients of differential gene expression), suggesting that 53% of expression variation in sickle cell anemia is related to hypoxic transcriptional responses. At 5% false discovery rate (FDR), 1040 genes exhibited a >1.15 fold change in both sickle cell anemia and VHLR200W homozygotes, among which 297 were up-regulated and 743 down-regulated. Hypoxia strongly induced inflammatory response pathways but suppressed T-cell activation in sickle cell anemia. MAPK8, encoding a mitogen-activated protein kinase important for stress-induced apoptosis, T-cell differentiation and inflammatory responses, was a hypoxia down-regulated gene and played a central role in hypoxic gene regulation in sickle cell anemia according to gene network analysis.To assess the genetic contribution to hypoxic transcriptional variation among SCD patients, we mapped expression quantitative trait loci (eQTL) for the 1,040 hypoxia response genes. Association mapping with a focus on local regulatory polymorphisms in 61 SCD patients identified eQTL for 103 of the hypoxia response genes at 5% FDR. We further tested the hypothesis that these hypoxic eQTL potentially underlie heterogeneity in risk of pulmonary hypertension in an additional SCD cohort (University of Illinois cohort). In this cohort, the A allele of an eQTL of MAPK8, rs10857560, was associated with pre-capillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 and pulmonary capillary wedge pressure ≤15 mm Hg at right heart catheterization (allele frequency=0.66; OR=4.4, P=0.00037, n=238). This association was confirmed in another independent cohort (Walk-PHaSST cohort) (allele frequency=0.65; OR=7.2, P=0.0025, n=519). The homozygous AA genotype of rs10857560, which was associated with decreased MAPK8 expression, was present in all 14 identified pre-capillary pulmonary hypertension cases among the combined 757 SCD patients (P = 6 x 10-6 by the Fisher exact test). Our study demonstrates a prominent hypoxic transcription component in SCD that in part contributes to pre-capillary pulmonary hypertension. Disclosures:No relevant conflicts of interest to declare.

The Clinical Utility Of Next-Generation Sequencing In The Diagnosis Of Polycythemia

Polycythemia or erythrocytosis is a disorder characterized by expansion of the RBC mass, and can be primary or secondary. Primary polycythemia is caused by an acquired or inherited mutation, it includes polycythemia vera and familial/congenital variants. e.g. due to gain of function mutations in the erythropoietin receptor (EPOR), or mutations in the hypoxia sensing pathway. Secondary polycythemia is caused by a circulating factor stimulating erythropoiesis, usually erythropoietin (EPO). It is most often due to an EPO response to hypoxia, but can also result from an EPO-secreting tumor. Either primary or secondary polycythemias can be inherited, i.e. due to germline mutations. The hypoxic response, mediated by hypoxia inducible transcription factors (HIFs), is central to the control and development of many essential biological functions, including erythropoiesis. Mutations in this pathway, causing polycythemia, have been identified in negative regulators of HIFs, such as the von Hippel-Lindau (VHL) gene, the HIF-prolyl-hydroxylase 2 (PHD2) gene, and gain-of-function mutations of the HIF-2-alpha (HIF2A) gene. Routine diagnostic testing can be challenging with specialized testing often only available in specialized research laboratories. Comprehensive, coding region analysis of all candidate genes by selective amplification of DNA regions of interest by PCR followed by the sequencing and analysis of amplified DNA fragments involved can be daunting due to molecular heterogeneity of causative genes as well as the size of the genes involved. Targeted molecular analysis is now being developed for both acquired (somatic) mutations or inherited (germline mutations) causing acquired and inherited diseases. We developed a novel, high-throughput, sensitive sequencing assay for diagnosis of congenital causes of polycythemias and polycythemia vera. Our diagnostic panel includes 9 genes and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Custom targeted gene capture and library construction for next-generation sequencing (NGS) was performed using HaloPlex as described by the manufacturer (Agilent Technologies, Santa Clara, CA). One hundred base-pair paired-end sequencing was done on a HiSeq 2000 system (Illumina, San Diego, CA). Bioinformatic analysis was based on an “in house” pipeline using standard open-source software. A total of 10 patients with clinically suspected polycythemia, and 30 normal controls were tested in our assay. Whole blood genomic DNA was isolated from the patients and targeted gene capture performed. Mutations in the target genes were identified in 3/10 patients, two of these being novel. All identified mutations were confirmed by Sanger sequencing. In one of these patients, a child with increased RBC mass, with EPO hypersensitive BFU-E colonies, a novel pathogenic, nonsense mutation was found in exon 8 of the EPOR gene (Q434X) resulting protein truncation and absence of the C-terminal negative regulatory domain of the receptor. In a different patient with suspected primary congenital/familial polycythemia due to EPO hypersensitive BFU-E colonies, we identified a novel pathogenic mutation in exon 3 of the MPL gene. This novel mutation, a single base deletion causes a frame-shift in codon 126 (F126L) and early termination at codon 130. To the best of our knowledge, this is the first report of a loss of function mutation in the MPL gene in a patient with polycythemia. Analysis of greater cohort of polycythemic patients is now in progress. Disclosures:No relevant conflicts of interest to declare.

Are Outcomes Worse For Patients With Polycythemia Vera compared to Polycythemia Of Unknown Cause? – a Retrospective Analysis Of Single Institution Patient Cohorts

BackgroundThe JAK2 V617F mutation is present in almost 95% of patients with Polycythemia Vera (PV). Prior to the discovery of this mutation, red cell mass and plasma volume measurement to establish the presence of true erythrocytosis was required first, before the diagnosis could be confirmed using other criteria. The revised WHO criteria no longer mandate this. Besides, access to this test is restricted in our region, due to non-availability of radio-isotopes. We previously showed the utility of early JAK2 mutation screening when used with history, physical examination, oxygen saturation, complete blood count, blood film examination, splenomegaly, serum erythropoietin, phlebotomy trial, bone marrow biopsy and, when available, red cell mass studies. PatientsBetween 2002 and 2006 we screened 231 consecutive patients referred to our institution with raised hemoglobin and hematocrit for JAK2 mutation. PV was diagnosed in 27 patients. Polycythemia of unknown cause (idiopathic) was identified in 40 patients after excluding patients with transient polycythemia and secondary polycythemia due to hypoxia, sleep apnoea, excessive smoking, alcohol abuse, renal disease, androgens or inherited causes. All PV patients were assessed at 3-6 monthly intervals from diagnosis until death or to present day. All idiopathic polycythemia patients were assessed at 6-12 monthly intervals until discharge, death or to present day. Patients with idiopathic polycythemia were discharged from follow-up when their hematocrit returned to normal range without any interventions for at least 12 months. All patients received thromboprophylaxis with aspirin or vitamin K antagonists as appropriate unless contra-indicated. ResultsOf 27 PV patients, 15 were males and 13 females. The median age at diagnosis was 69 years (range 21-88 years). Leucocytosis was present at diagnosis in 14 and thrombocytosis in 15 patients. Patients were treated with phlebotomy to a target hematocrit<0.45 (18 patients), hydroxycarbamide (16 patients), alpha-interferon (1 patient) and busulfan (1 patient). Nine patients were treated with phlebotomy alone and the remainder (18 patients) started with phlebotomy and switched to cytoreductive therapy. The median follow-up was 9 years (range 7-11 years). One patient transformed to myelofibrosis and 2 patients developed acute myeloid leukemia. Thrombotic complications included stroke in 7 patients, retinal vein and mesenteric vein thrombosis in 1 patient each. Bleeding occurred in 1 patient. Eleven patients died (40%) and death was likely related to underlying PV in 4 patients. Of 40 idiopathic polycythemia patients, 30 were males and 10 females. The median age at diagnosis was 59 years (range 30-84 years). Patients with idiopathic polycythemia were treated with phlebotomy to a target hematocrit<0.50 (36 patients) or hydroxycarbamide (3 patients). The median follow-up was 6 years (2-11 years). One patient developed acute myeloid leukemia after 8 years of hydroxycarbamide therapy. Thrombosis was observed in 15 patients: stroke in 5 patients, acute coronary syndrome in 6 patients and venous thrombosis in 4 patients. Red cell indices returned to normal without any interventions in 25 patients over a median duration of 5 years (range 2-8 years). Seven patients died and death was related either to polycythemia or hydroxycarbamide in 1 patient. ConclusionDisease progression occurred in 11% (3/27) and thrombosis in 33% (9/27) of patients with PV. Idiopathic polycythemia was associated with thrombosis in 37% (15/40) of patients. Our findings indicate that thrombosis occurs with equal or higher frequency in idiopathic group when compared to PV group. These patients require a stricter target hematocrit control to <0.45. However, in the absence of red cell mass studies to confirm the presence of true erythrocytosis, it is unclear as to who will benefit from this approach. This lack of clarity is reflected in our finding that 62% eventually normalised their red cell indices without any interventions, indicating that at least a proportion of these patients had apparent polycythemia. Disclosures:Chacko:Amgen : Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees. Killick:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Transcriptomic and phospho‐proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRβ, TGFβ, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.

Acute embolic cerebral ischemia as an initial presentation of polycythemia vera: a case report

IntroductionPatients with polycythemia vera are at high risk for vaso-occlusive events including cerebral ischemia. Although unusual, acute ischemic stroke may be an initial presentation of polycythemia vera. It had been previously assumed that cerebral ischemic events were due to increased blood viscosity and platelet activation within the central nervous system arterial vessels. However, there are now a few isolated case reports of probable micro-embolic events originating from outside of the brain. This suggests unique management issues for these patients.Case presentationWe present the case of a 57-year-old right-handed Caucasian male in excellent health who presented to the Emergency Department with acute right-handed clumsiness. Hematologic investigations revealed a hyperviscous state and magnetic resonance imaging was consistent with cerebral emboli. Symptoms rapidly improved with phlebotomy and hydration.ConclusionThe etiology of stroke in polycythemic patients is likely to be multifactorial. While hemodilution has been generally discredited for general stroke management, it is potentially beneficial for patients with polycythemia vera and euvolemic hemodilution should be considered for the polycythemic patient with acute cerebral ischemia.

Characteristics of Polycythemia in Sana'a, Yemen

Background: High altitude polycythemia is one of the altitude illnesses. Sana'a region is located at high altitudewhich reaches 3600 meters above the sea level. Objectives: To determine the hematological and clinical features of polycythemic patients residingin Sana'a region and to clarify the effect of high altitude in causing polycythemia. Methods: Hematological, clinical and demographic data were obtained from 30 polycythemic patients (26 males, 4 females, aged 26 to 85 years residing Sana'a regionand referred to the National Centre of Public Health aboratories in Sana'a city for the firsttime venesection. Results:All studied samples showed high hemoglobinlevels (mean 18.85 g/dL for both sexes), normal platelet counts (228 and 267 x109/L for males and females, respectively),normal white cell counts in 96.7% of the cases (means 6.01 to 5.98 x109/L). Their clinical features showed predominance of headache, ruddy cyanosis, dyspnea and night sweating by 86.7%, 76.7%, 70% and 60%, respectively. Hypertension, joint pain, renal disease, peptic ulcer were less commonly mentioned by 36.7%, 20%, 13.3%, and10%, respectively. Hemorrhage, pruritus, splenomegaly, heart disease andliver disease ereobserved by only 6.67% each. Conclusions: Findings suggest thatsecondary polycythemia is predominant among polycythemicpatients due to the high altitude of Sana’a region. Findings needs to be confirmed bystudyinga larger sample and extended to investigate the erythropoietin level and JAK2V617F mutation for accurate diagnosis. Keywords: Polycythemia, Sana'a, Yemen

Occult leydig cell tumour and androgen-receptor positive breast cancer in a woman with severe hyperandrogenism

Leydig cell tumours represent more than 75% of all testosterone-secreting ovarian masses. These benign tumours are frequently occult or very small, but cause dramatic virilization. Chronic hyperandrogenism can also induce systemic complications, which increase morbidity and mortality risk. One of the most obvious effects of increased testosterone levels is polycythemia, a complication which induces dermatologic, osteoarticular and gastrointestinal manifestations and is associated with increased thrombotic risk. However, scientific literature reports few data concerning etiopathogenesis and management of polycythemia in patients with Leydig cell tumours. Moreover, no data are available about the effect of androgen excess on other concomitant tumours expressing androgen receptors. In this paper we report for the first time the case of a woman, with previous infertility, dramatic virilisation and chronic erythrocytosis, who was affected by an occult Leydig cell tumour and an androgen receptor positive breast cancer. This association gives us the opportunity to discuss the role of the steroid receptor expression of breast cancer in the presence of circulating androgen excess. Moreover, we demonstrate for the first time that treatment with flutamide (anti-androgen drug) is able to normalize blood cell count and haematocrit, before of achieving the definitive cure of hyperandrogenism by oophorectomy.

Yenidoğan Polisitemisi JAK2V617F Mutasyonundan Bağımsız Olabilir

Objective: Polycythemia is known as increased erythrocytosis and is linked to the erythropoiesis cascade including erythropoietin, erythropoietin receptor and intracellular signaling proteins. The Janus kinase 2 (JAK2) is the key signal transducer in the erythropoiesis cascade. A function gain mutation (V617F) at JAK2 gene has been identified in polycythemia vera in adults. On the other hand, the molecular etiology of neonatal polycythemia has not been elucidated well. Thus, the aim of this randomized controlled study was to investigate the role of JAK2 V617F mutation in the etiology of neonatal polycythemia similar to polycythemia vera. Material and Methods: Fifty-one neonates diagnosed with polycythemia according to venous hematocrit level over 65% and 26 healthy neonates as the control group were enrolled in the study in addition to 43 adult patients diagnosed with polycythemia vera. JAK2 V617F mutation analysis was performed using Real-Time PCR system. Results: All the neonatal polycythemia patients were negative for the specific mutation JAK2 V617F, as well as 26 control neonates, whereas in 31 (72%) out of 43 adult polycythemia vera patients, JAK2 V617F mutation was present. Conclusion: This is the first report showing that JAK2-V617F mutation may be an acquired somatic mutation instead of congenital and neonatal polycythemia is not related to the disrupted erythropoiesis cascade.

Anemia is Associated with Bone Mineral Density in Chronic Obstructive Pulmonary Disease

Objective: Patients with chronic obstructive pulmonary disease (COPD) often suffer from systemic co-morbidities, including anemia. However, anemia is related to multiple outcomes in COPD and in other chronic diseases, but it's impact is underestimated in COPD. The objective of the present study was to relate anemia in patients with COPD with disease-related outcomes, systemic inflammation and COPD related co-morbidities. Methods: Data of 321 patients with COPD admitted for pulmonary rehabilitation were analysed. Besides general characteristics, lung function, body composition, arterial gases and plasma haemoglobin concentration, disease-related outcomes (health-related quality of life by St. George's Respiratory Questionnaire, 6-minute walking distance, mMRC dyspnea scale, and BODE index), systemic inflammation (C-reactive protein (CRP)) and self-reported and objectified co-morbidities (low muscle mass, osteoporosis, renal failure, risk for undernutrition) were taken into account. Results: First, 20% of the patients were anemic, and 8% was polycythemic. Polycythemic patients had a lower proportion of men and a lower proportion of low muscle mass compared to the other groups. Anemic patients had higher plasma CRP levels and lower total body bone mineral density compared to the other groups. There was no difference in disease-related outcomes or other co-morbidities in the patients with and without anemia. Even after adjustment for confounders, anemia was an independent determinant for higher CRP levels and lower bone mineral density. Conclusion: Anemia is frequently present in patients with COPD and there is evidence that it is associated with lower whole body bone mineral density.

A novel EPAS1/HIF2A germline mutation in a congenital polycythemia with paraganglioma

Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2A (F374Y) (exon 9) mutation, inherited from his mother, who developed PHEO/PGL. We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2A (F374Y) may be predisposing rather than causative of PHEO/PGL. This report, in view of two other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL-promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.

Masked Polycythemia in Patients With Heart Failure: An Underappreciated Phenotype?

Masked Polycythemia in Patients With Heart Failure: An Underappreciated Phenotype?