Serum Epo Levels Research Articles

Anemia after kidney transplantation: Does its basis differ from anemia in chronic kidney disease?

Although similar factors play a role in both PTA and anemia in patients with CKD, additional risk factors exist in the pathogenesis of PTA. The present study aimed at comparing anemia and inflammation-related parameters between RTx recipients and CKD patients and elucidating the risk factors of PTA. This single-centered, cross-sectional study consisted of 68 participants: 48 were in the RTx group and 20 were in the CKD group. The CKD patients were comparable to the RTx recipients in terms of age, gender, and eGFR. Serum levels of EPO, hepcidin, and IL-6 were measured by enzyme-linked immunosorbent assays. The ratio of EPO/Hb was calculated to estimate endogenous EPO resistance. The prevalence of anemia was 46% in the RTx group and 30% in the CKD group (P=.23). RTx recipients had significantly lower Hb (P=.04), higher EPO (P<.001), and ferritin levels (P=.001), and higher EPO/Hb ratios (P<.001); however, CKD patients showed a higher frequency of absolute iron deficiency (P=.008). Neither hepcidin nor IL-6 levels differed between the two groups. Hb level of RTx recipients was correlated with only eGFR (r=.437, P=.002) but not with any of the transplantation-related factors, while Fe level was the only parameter to be correlated with Hb level of CKD patients (r=.622, P=.01). In the present study comparing GFR-matched RTx and CKD patients, lower GFR level appears to be the factor most strongly associated with anemia, and endogenous EPO resistance is among the contributing factors to PTA.

Serum erythropoietin concentration and its correlation with stage of diabetic retinopathy

BackgroundErythropoietin (EPO) is one of the systemic angiogenic factors, and its role in ocular angiogenesis and in diabetic retinopathy (DR) is not yet fully understood. The latest research data reveal a possible correlation of higher erythropoietin concentrations in the blood and in the eye with the development of more advanced stages of DR.The main aim of this work was to examine the possible influence of serum concentrations of erythropoietin on the development of diabetic retinopathy in patients with diabetes mellitus type 2.MethodsThe research involved 90 patients examined at the University Eye Clinic of the Clinical Center of Vojvodina, Novi Sad, Serbia. The first group comprised 60 patients with diabetes mellitus lasting for 10 years or more, with diabetic retinopathy. The second, control group consisted of 30 healthy individuals. In the first group of 60 patients with diabetes, 30 of them had non-proliferative diabetic retinopathy (NPDR), and 30 had proliferative diabetic retinopathy (PDR). Laboratory EPO serum levels were determined, and they were correlated to the stage of DR. Concentration of EPO was assessed by ELISA method.ResultsThe highest average concentration of EPO in serum (9.95 mIU/ml) was determined in the group of people with diabetes with PDR. The lowest average concentration of EPO in the serum (6.90 mIU/ml) was found in the control group. The average concentration of EPO in serum in the group of patients with diabetes with NPDR was 7.00 mIU/ml. The EPO concentration in serum was elevated in the group of PDR, and it was directly proportional to the level of the clinical stadium of PDR, being significantly higher in the moderate and severe subgroup of PDR comparing to the control healthy subjects, NPDR and mild PDR (p = 0.007).ConclusionsSignificantly elevated serum concentration of EPO in the advanced stages of DR, and positive correlation between EPO serum concentration and clinical stages of PDR, suggest that erythropoietin represents an important growth factor from blood, which plays a significant role in retinal ischemia and angiogenesis in diabetic retinopathy, especially in the proliferative stage of this disease.

Combined Treatment with Lenalidomide and Epoetin Alfa Leads to Durable Responses in Patients with Epo-Refractory, Lower Risk Non-Deletion 5q [Del(5q)] MDS: Final Results of the E2905 Intergroup Phase III Study - an ECOG-ACRIN Cancer Research Group Study, Grant CA180820, and the National Cancer Institute of the National Institutes of Health

Combined Treatment with Lenalidomide and Epoetin Alfa Leads to Durable Responses in Patients with Epo-Refractory, Lower Risk Non-Deletion 5q [Del(5q)] MDS: Final Results of the E2905 Intergroup Phase III Study - an ECOG-ACRIN Cancer Research Group Study, Grant CA180820, and the National Cancer Institute of the National Institutes of Health

Effects of different doses of erythropoietin in patients with myelodysplastic syndromes: A propensity score-matched analysis.

BackgroundErythropoiesis‐stimulating agents effectively improve the hemoglobin levels in a fraction of anemic patients with myelodysplastic syndromes (MDS). Higher doses (HD) of recombinant human erythropoietin (rhEPO) have been proposed to overcome suboptimal response rates observed in MDS patients treated with lower “standard doses” (SD) of rhEPO. However, a direct comparison between the different doses of rhEPO is lacking.MethodsA cohort of 104 MDS patients treated with HD was retrospectively compared to 208 patients treated with SD in a propensity score‐matched analysis to evaluate hematological improvement‐erythroid (HI‐E) rate induced by the different doses of rhEPO. The impact of rhEPO doses on survival and progression to leukemia was also investigated.ResultsOverall HI‐E rate was 52.6%. No difference was observed between different rhEPO doses (P = .28) in matched cohorts; in a subgroup analysis, transfusion‐dependent patients and patients with higher IPSS‐R score obtained a higher HI‐E rate with HD, although without significant impact on overall survival (OS). Achievement of HI‐E resulted in superior OS. At univariate analysis, a higher HI‐E rate was observed in transfusion‐independent patients (P < .001), with a lower IPSS‐R score (P < .001) and lower serum EPO levels (P = .027). Multivariate analysis confirmed that rhEPO doses were not significantly related to HI‐E (P = .26). There was no significant difference in OS or progression to leukemia in patients treated with HD vs SD.ConclusionSD are substantially equally effective to HD to improve anemia and influencing survival in MDS patients stratified according to similar propensity to be exposed to rhEPO treatment.

PB2224 POLYCYTHEMIA VERA: A DIAGNOSTIC CHALLENGE

Background:In patients referred to hematology for investigating the etiology of the increased hemoglobin concentration and/or hematocrit, the first step is to find out whether there is a secondary cause of the polycythemia. Serum erythropoietin levels and JAK‐2 mutation screening are recommended after it is decided that polycythemia is not caused by a secondary cause. World Health Organization International Agency for Research on Cancer (WHO/IARC) has made major changes in 2016 on the diagnostic criteria for polycythemia vera. One of the major changes is lowered treshold hemoglobin/hematocrit levels. These values are use not only as diagnostic criteria, but also as the initiation of screening diagnostic tests for polycythemia. Thus, there has been a significant increase in the number of patients admitted to outpatient hematology clinics.Aims:Which patients should be investigated? Are there any preliminary characteristics or should all the patients be requested to JAK‐2 mutation or erythtropoetin leves ? 2‐ Which test should be requested first: serum erytropoetin level test or JAK mutation test? 3‐ When compared to WHO 2008, do the new diagnostic hb/htc criteria allow more patients to be diagnosed as polycthemia vera?Methods:243 Patients who applied to our clinic between 2016 and 2018 years, for high hemoglobin and/or hematocrit values according to WHO 2016 criteria, were analyzed retrospectively. All patients were tested for serum EPO levels and JAK‐2 exon 14 mutation status, and evaluated for the secondary causes of polycytemia. Normal values of lab for serum EPO levels are 3,5‐16,4 UI/L. All statistical analysis was made by SPSS version 22. Categorical variables were compared using χ2 or Fishers exact test.Results:243 patients were included in the study. Demographic data and laboratory results of the patients are summarized in Table‐1. 24 of 243 patients (%9,8) had positive JAK‐2 exon 14 mutation and diagnosed polycytemia vera. 30 of 243 (%12,3) had low serum Epo levels. (≤3,5 UI/L). 18 of 24 JAK‐2 positive patients had low EPO levels so there was a significant correlation between low serum EPO levels and JAK‐2 mutation status (p&lt;0,001). There was no relationship between leukocytosis, thrombocytosis and JAK‐2 status. (p = 0,34). Age was another major factor related to JAK‐2 status. 20 of 24 JAK positive patients’ ages were 50 and above (p&lt;0,001). 12 of these 20 patiens had a low Epo level, while 8 of them had a normal serum EPO values. (p&lt;0,001). 4 of 24 JAK positive patients whose age was younger than 50 years, had a low EPO levels (p&lt;0,001). Therefore, all of patentse diagnosed P.Vera below 50 years had a supressed EPO levels. When compared to P.Vera diagnosed patients who met the previous WHO‐2008 criteria (Hb ≥ 18,5‐Htc ≥ %55) (8/39, %20,5) and patients who had Hb values between 16,5‐18,4 g/dl (according to WHO‐2016 criteria)(16/196, %8,1), no significant difference was observed (p = 0,09) between two groups.Summary/Conclusion:This analysis concluded that in patients younger than 50 years old, the investigation of polycythemia must start with EPO levels. If EPO levels are suppresed, then JAK mutation testing can be performed. After the age of 50, both tests should be performed at the same time. If we had examined the patients according to WHO 2008, we couldn’t have diagnosed P.Vera for 16 patients whom hb levels were under 18,5 g/dl. On the other hand, we wouldn’t have tested 196 patients. Therefore, in population based patient series, the preliminary characteristics for further investigation to polyctemia must be established for cost effectiveness and labor.image

SUBCLINICAL POLYCYTHEMIA VERA PRESENTING AS EXTENSIVE THROMBOSIS DUE TO MASSIVE TRANSFUSION

SESSION TITLE: Critical Care 1 SESSION TYPE: Med Student/Res Case Report PRESENTED ON: 10/09/2018 03:45 PM - 04:45 PM INTRODUCTION: Massive Transfusion Protocol (MTP) is used in patients with hemorrhagic shock. Iatrogenic polycythemia secondary to massive transfusion is exceedingly rare. We present first reported case of undiagnosed subclinical polycythemia vera (PV) which became manifest with extensive thrombosis, after massive transfusion. CASE PRESENTATION: 66-year-old male with history of hypertension and ischemic strokes, presented to the Emergency Department with hypovolemic shock due to lower gastrointestinal bleeding. Initial bloodwork showed a Hb of 6.2 gm/dL, Hct 19.1%, platelets-282 x 109/L, WBC 6.6 x 109/L, INR 2.7, aPTT 83.7s. He received 8 units of PRBC, 6 units of fresh frozen plasma, 6 units of platelets as well as human prothrombin complex concentrate to reverse coagulopathy. Patient was admitted to ICU and intubated for urgent endoscopy, which revealed an esophageal ulcer and gastritis. He was started on proton pump inhibitor and repeat labs showed Hb of 18 gm/dL, Hct 53.8%, platelets-546 x 109/L with INR of 1. On admission day 3, patient developed pulmonary infiltrate and BAL grew MRSA treated with vancomycin. Due to worsening ventilation, a CT scan was done which showed bilateral acute pulmonary embolism with right heart strain. Doppler venous ultrasound revealed deep vein thrombosis of both upper and lower extremities. He had Hb of 19.6 gm/dL, Hct 59.9%, platelets-450 x 109/L. Reviewing his history, he was noted to have baseline Hb of 14 to 16 gm/dL, Hct 40%, with one instance of Hb of 18 gm/dL, Hct 56. The previous cerebrovascular accident was found to be secondary to a carotid artery thrombus. Hematology was emergently consulted and anticoagulation with low intensity heparin was initiated inspite of GI bleeding. He underwent multiple bedside phlebotomies until his Hct was below 50%. CT abdomen showed mild splenomegaly. Serum erythropoietin initially was normal, but was low on repeat testing. Janus kinase 2 (JAK2) V617F mutation was present, confirming diagnosis of primary polycythemia. Upon discharge, he is to receive ASA, hydroxyurea and anticoagulation for the venous thrombosis. DISCUSSION: PV is a chronic myeloproliferative neoplasm that is associated with a JAK2 mutation in most cases. An elevation of hemoglobin/hematocrit>16.5/49 in men and 16/48 in women, a subnormal serum EPO level, and a JAK2 V617F mutation constitute the diagnostic criteria for PV. Thrombotic complications are a major cause of morbidity and mortality in polycythemia vera with a reported incidence of 12–39%1. CONCLUSIONS: Sustained elevation of Hb/Hct >16.5/49 after massive transfusion should raise the possibility of PV, prompting workup and a heightened concern for thrombosis. Recognition of subclinical polycythemia is extremely important in critical care setting as they share the same vascular risk as overt PV including risk of arterial and venous thrombosis. Reference #1: Cerquozzi S, Barraco D, Lasho T, Finke C, Hanson CA, Ketterling RP, Pardanani A, Gangat N, Tefferi A. Risk factors for arterial versus venous thrombosis in polycythemia vera: a single center experience in 587 patients. Blood Cancer J. 2017 Dec 27;7(12):662 DISCLOSURES: No relevant relationships by Paula Adamson, source=Web Response No relevant relationships by Shahla Bari, source=Web Response No relevant relationships by Donnie Carter, source=Web Response No relevant relationships by Marilyn Foreman, source=Web Response No relevant relationships by Swathi Sree Nutakki, source=Web Response

Effects of erythropoietin on fibroblast growth factor 23 in mice and humans.

Erythropoietin (EPO) has been reported as a novel determinant of fibroblast growth factor 23 (FGF23) production; however, it is unknown whether FGF23 is stimulated by chronic exposure to EPO or by EPO administration in nonpolycystic chronic kidney disease (CKD) models. We analyzed the effects of chronic EPO on FGF23 in murine models with chronically high EPO levels and normal kidney function. We studied the effects of exogenous EPO on FGF23 in wild-type mice, with and without CKD, injected with EPO. Also, in four independent human CKD cohorts, we evaluated associations between FGF23 and serum EPO levels or exogenous EPO dose. Mice with high endogenous EPO have elevated circulating total FGF23, increased disproportionately to intact FGF23, suggesting coupling of increased FGF23 production with increased proteolytic cleavage. Similarly, in wild-type mice with and without CKD, a single exogenous EPO dose acutely increases circulating total FGF23 out of proportion to intact FGF23. In these murine models, the bone marrow is shown to be a novel source of EPO-stimulated FGF23 production. In humans, serum EPO levels and recombinant human EPO dose are positively and independently associated with total FGF23 levels across the spectrum of CKD and after kidney transplantation. In our largest cohort of 680 renal transplant recipients, serum EPO levels are associated with total FGF23, but not intact FGF23, consistent with the effects of EPO on FGF23 production and metabolism observed in our murine models. EPO affects FGF23 production and metabolism, which may have important implications for CKD patients.

Genetic basis of congenital erythrocytosis.

Congenital Erythrocytosis (CE) represents a rare and heterogeneous clinical entity. It is caused by deregulated erythropoiesis where red blood cell overproduction results in elevated hemoglobin and hematocrit levels. CE may either be primary or secondary to elevated erythropoietin concentrations. The only known form of primary CE is caused by mutations in the EPOR gene. Secondary CE can be a consequence of tissue hypoxia, being caused by congenital defects such as hemoglobin variants with increased oxygen affinity, due to mutations in the α- or β-globin genes (HBB, HBA2, HBA1), or due to mutations in the BPGM gene. Secondary CE can also result from defects in the components of the oxygen-sensing pathway (PHD2, HIF2α and VHL). The family history and the quantification of serum EPO are mandatory to define the best diagnostic strategy regarding molecular studies. Based upon the serum EPO level, P50 and familial data, it is possible to establish a diagnostic algorithm. Despite recent important discoveries in the molecular pathogenesis of CE, in about 70% of the patients the genetic causes remain to be identified. Clinical data on patients suffering from CE are sparse. This fact is conditional upon the effective way to predict the disease evolution, the establishment of the best management and the genetic counselling. The number and phenotypic variability of patients that remain without an identified etiology suggest that other genes have to be implicated. Studies by next generation sequencing methodologies are already being performed and it is expected the identification of other genes involved in the pathophysiology of the CE.

The Plasticity of CD4+CD25+FOXP3+CD127low T Cells in Patients with Metastatic Renal Cell Carcinoma in the Course of Interferon-Alpha Immunotherapy.

Aims To examine changes in subpopulation of CD4+CD25+Foxp3+CD127low T lymphocytes (Treg) and their association with the efficiency of the IFN-α therapy. Materials and Methods Pts with mRCC who had undergone nephrectomy were treated with IFN-α at a dose of 6 × 106 U/day three times a week (n = 18). An immunophenotypic analysis of lymphocytes in peripheral blood expressing CD4, CD25, CD127, and Foxp3 antigens could be performed in 18 pts before, 2 weeks, and 2 mo after IFN-α therapy and 22 normal volunteers. Blood samples were collected at baseline and 2 mo after treatment start. Serum levels of TGF-β1, IL-17A, and Epo were measured by ELISA. Results PR was achieved in 3 (16.6%) pts who received first-line therapy. Long-lasting SD (≥6 months) was noted in 6 (33.3%) pts. The median progression free survival (PFS) was 4 mo (95% CI: 2-NE). The study of the population of Treg indicated that there were no significant differences in the groups depending on the effect (p = 0.71). In one patient, the reduction of Treg cells was associated with increased TGF-β and IL-17 levels, whereas in other two pts the increase in Treg cells was associated with decreased TGF-β and IL-17 levels. The endogenous levels of Epo did not show significant correlation with response to IFN-α immunotherapy. In the patient subgroup with an initial value of MCH > 31 pg, the median PFS was not achieved, but in the subgroup with an initial value of MCH < 31 pg, the median PFS was 2 months (p = 0.032). Conclusions In our study, we have described functional plasticity of Treg cells, which prevents them from being used as a prognostic marker. The conversion of Treg cells into Th17 can serve as a basis for the development of a new specific immunotherapeutic method in oncology after confirmation in the experiment in vitro. Given the small dataset, the results will need further validation.

Bone Marrow Histology Characteristics in MPL515 Mutated Thrombocythemia with Various Degrees of Myelofibrosis: A Cross Sectional Follow-up Study in Eight Cases

MPLW515l/K mutated Essential Thrombocythemia (ET) usually present with increased platelet counts around 1000 x 109/L as the only abnormal laboratory finding with normal values for hemoglobin and leukocytes and minor splenomegaly on palpation only in a few. Early stage MPL515 mutated ET show the presence of clustered small and giant megakaryocytes with pronounced deeply lobulated nuclei, which are not seen in JAK2V617F positive ET, prodromal PV, and classical PV. Indicating that MPL515 mutated ET has no clinical, laboratory and bone marrow features of prodromal PV. Clustering of large mature large to giant megakaryocytes with pronounced hyper lobulated nuclei in a normocellular bone marrow is the hallmark of JAK2-wild type MPL515 mutated thrombocythemia. Bone marrow histology in MPL515 mutant patients revealed isolated megakaryocytic proliferation in a normocellular bone marrow at diagnosis with a reduction of erythropoiesis. MPL515 Thrombocythemia do not have PV features at diagnosis, do not evolve into PV during follow-up, have normal LAP score, serum EPO and ferritin levels. In contrast to JAK2V617F positive ET, no spontaneous endogenous erythroid colonies (EEC) was found in any of evaluated MPLW515L cases. Spontaneous megakaryocyte growth in culture with an overall normal response to thrombopoietin (TPO) has been demonstrated in two MPL515 mutated cases.

A predictive model of response to erythropoietin stimulating agents in myelodysplastic syndrome: from the Canadian MDS patient registry.

Prediction of response to erythropoietin stimulating agents (ESAs) in anemic MDS patients is often based on the Nordic score. We wished to validate the Nordic score (IWG 2006 response criteria) in a larger cohort and determine if other variables such as IPSS/IPSS-R, ferritin, LDH, and a novel European ESA response score (Santini 2013) were of prognostic importance. We analyzed 208 ESA-treated MDS patients (WHO 2008 criteria) from a prospective registry. Ninety-four and 93% had lower risk scores by IPSS (low/int -1) and IPSS-R (low/very low), respectively. Erythroid response was achieved in 94 patients (47%); responses were similar with erythropoietin (50%) and darbepoetin (39%; p=0.2). The Nordic and European scores were both validated on univariate analysis. Variables independently predictive of response in multivariate analysis were low-risk IPSS score (OR 0.1, p=0.0016) and serum EPO level <100mIU/mL (OR 8.7, p<0.0001). We propose a new ESA response score, consisting of (a) IPSS low score (1 point) and (b) serum EPO levels <100mIU/ml (2 points), yielding scores ranging from 0 to 3, with response rates varying from 17 to 81%. The Nordic score has validity but we observed lower than the expected response rates in the best risk group. Our proposed scoring system appears more discriminating but needs validation.

Role of serum erythropoietin level in patients with absolute erythrocytosis

PRV is a myeloproliferative disorder with an acquired genetic mutation involving the stem cell. Erythropoietin (Epo) is a hemopoietic growth factor which provides some guidance to the diagnosis of PRV. The study was done to evaluate the diagnostic significance of erythropoietin level in PRV and its role in pre and post treated PRVpatients.The study includes (47) patients diagnosed as PRV including (31) males and (16) females with age range (20- 77 years). The criteria for diagnosis of polycythemia is PCV &gt; 52% and/ or Hb ≥18.5 gm/dl for male and PCV &gt;48%and/ or Hb ≥16.5 gm/dl for female.The investigations which were done for all new patients including CBC, blood films and serum Epo, in addition to the BM examination for new indicated cases of PRV. Follow up of old PRV (31) patients were done by PCV and serum Epo. We found thatthe mean Epo was 9.82mIU/ml. There was no statistical significant correlation between Epo mean and mean of other parameters.There was a statistical significance between pre and post treated cases regarding mean PCV and Epo. The acceptible EPO cutoff level to define PRV of (4.9) with sensitivity (71.4), specificity (86.7) and(p&lt;0.001). So, we conclude that the serum Epo level was a simple reliable test for diagnosis of PRV with a cutoff value of (4.9) andthere was a significant difference between treated and untreated cases of PRV regarding PCV and Epo.

Circulating autoantibodies to endogenous erythropoietin are associated with chronic hepatitis C virus infection-related anemia

Chronic hepatitis C virus (HCV) infection is associated with autoimmune phenomena and is often complicated by anemia. Circulating autoantibodies to endogenous erythropoietin (anti-EPO) have been detected in patients with chronic viral infections and were correlated to anemia. The present study aimed to determine anti-EPO prevalence in patients with chronic HCV infection and investigate its possible association with anemia. Ninety-three consecutive patients (62 males and 31 females) with chronic HCV infection, who had never received antiviral therapy or recombinant EPO, were enrolled in the study. Circulating anti-EPO were detected in the serum by using an ELISA assay. Quantitative determination of serum EPO levels was done by radioimmunoassay. HCV RNA viral load measurement and genotype sequencing were also performed. Circulating anti-EPO were detected in 10.8% of HCV-infected patients and the prevalence of anti-EPO was significantly higher in patients with anemia (19.4% vs 5.3%, P=0.040) compared to that in those without anemia. Compared to anti-EPO negative cases, anti-EPO positive patients had higher frequency of anemia (70.0% vs 34.9%, P=0.030), lower EPO concentrations (median 16.35 vs 30.65 mU/mL, P=0.005), and higher HCV RNA viral load (median 891.5X103 vs 367.5X103 IU/mL, P=0.016). In multivariate regression analysis the presence of anti-EPO remained an independent predictor of anemia (adjusted OR: 14.303, 95% CI: 1.417-36.580, P=0.024). EPO response to anemia was less prominent among anti-EPO positive patients (P=0.001). Circulating anti-EPO are detected in a significant proportion of treatment-naive HCV-infected patients and are independently associated with anemia, suggesting a further implication of autoimmunity in the pathophysiology of HCV-related anemia.

Comparison of the effect of intermittent hypoxic training vs. the live high, train low strategy on aerobic capacity and sports performance in cyclists in normoxia.

The aim of the study was to compare the effect of intermittent hypoxic training (IHT) and the live high, train low strategy on aerobic capacity and sports performance in off-road cyclists in normoxia. Thirty off-road cyclists were randomized to three groups and subjected to 4-week training routines. The participants from the first experimental group were exposed to normobaric hypoxia conditions (FiO2 = 16.3%) at rest and during sleep (G-LH-TL; n=10; age: 20.5 ± 2.9 years; body height 1.81 ± 0.04 m; body mass: 69.6 ± 3.9 kg). Training in this group was performed under normoxic conditions. In the second experimental group, study participants followed an intermittent hypoxic training (IHT, three sessions per week, FiO2 = 16.3%) routine (G-IHT; n=10; age: 20.7 ± 3.1 years; body height 1.78 ± 0.05 m; body mass: 67.5 ± 5.6 kg). Exercise intensity was adjusted based on the lactate threshold (LT) load determined in hypoxia. The control group lived and trained under normoxic conditions (G-C; n=10; age: 21.8 ± 4.0 years; body height 1.78 ± 0.03 m; body mass: 68.1 ± 4.7 kg; body fat content: 8.4 ± 2.4%). The evaluations included two research series (S1, S2). Between S1 and S2, athletes from all groups followed a similar training programme for 4 weeks. In each research series a graded ergocycle test was performed in order to measure VO2max and determine the LT and a simulated 30 km individual time trial. Significant (p<0.05) improvements in VO2max, VO2LT, WRmax and WRLT were observed in the G-IHT (by 3.5%, 9.1%, 6.7% and 7.7% respectively) and G-LH-TL groups (by 4.8%, 6.7%, 5.9% and 4.8% respectively). Sports performance (TT) was also improved (p<0.01) in both groups by 3.6% in G-LH-TL and 2.5% in G-IHT. Significant changes (p<0.05) in serum EPO levels and haematological variables (increases in RBC, HGB, HCT and reticulocyte percentage) were observed only in G-LH-TL. Normobaric hypoxia has been demonstrated to be an effective ergogenic aid that can enhance the exercise capacity of cyclists in normoxia. Both LH-TL and IHT lead to improvements in aerobic capacity. The adaptations induced by both approaches are likely to be caused by different mechanisms. The evaluations included two research series (S1, S2). Between S1 and S2, athletes from all groups followed a similar training programme for 4 weeks. In each research series a graded ergocycle exercise test was performed in order to measure VO2max and determine the lactate threshold as well as a simulated 30 km individual time trial.

Combined Treatment with Lenalidomide (LEN) and Epoetin Alfa (EA) Is Superior to Lenalidomide Alone in Patients with Erythropoietin (Epo)-Refractory, Lower Risk (LR) Non-Deletion 5q [Del(5q)] Myelodysplastic Syndrome (MDS): Results of the E2905 Intergroup Study-an ECOG-ACRIN Cancer Research Group Study, Grant CA180820, and the National Cancer Institute of the National Institutes of Health

▪Background: Treatment with rhu-Epo ameliorates anemia in a subset of LR-MDS patients, however, effective salvage therapy is limited. LEN promotes erythroid lineage competence and expansion of primitive erythroid precursors in vitro. In the MDS-002 and MDS-005 trials, treatment with LEN improved erythropoiesis, yielding RBC transfusion-independence in 26% of azanucleoside-naïve, transfusion-dependent (TD) LR, non-del(5q) MDS patients for a median of 10.2 and 7.75 months, respectively. We previously reported that LEN restores Epo-responsiveness in MDS progenitors by inducing formation of lipid rafts enriched for signaling competent JAK2/Epo-receptor complexes and excluding large isoforms of the JAK2/lyn kinase-phosphatase CD45 (McGraw K, et. al. PLoS One 2014; Basiorka A, et. al. Cancer Res 2016). In a pilot study of Epo-refractory MDS patients, addition of EA yielded erythroid responses in 28% of patients who were unresponsive to LEN alone, suggesting that LEN may overcome resistance and augment response to rhEpo (Komrokji R, et. al. Blood 2012). To test this hypothesis, we performed a randomized phase III trial comparing treatment with LEN to LEN+EA in LR non-del(5q) MDS patients who were refractory to, or not candidates for treatment with rhEpo.Methods: Patients with Low or Intermediate-1 (Int-1) risk IPSS MDS with hemoglobin <9.5 g/dL who were unresponsive to rhEpo treatment or were TD (>2 units/mo) with serum Epo >500mU/mL were eligible for study. Patients were stratified by serum Epo level and prior rhEpo (EA vs. darbepoetin vs. none) then randomized to treatment with LEN 10 mg/d x21d q4wk (Arm A) or LEN + EA 60,000U SC/wk (Arm B). Primary endpoint was IWG 2006 major erythroid response (MER) rate after 4 cycles. Arm A non-responders were offered cross-over to combined therapy. Secondary endpoints included analysis of response biomarkers.Results: Between April 2009 and May 2016, 248 patients were enrolled and 195 were randomized and will be included in the primary analysis. Interim analysis of 163 patients (Arm A, 81; B, 82) accrued before July 2015 showed that the study met predefined stopping criteria. Baseline characteristics were balanced between arms. Median age was 74 years (range, 47-89) receiving a median of 2 RBC units/mo (0-8). Overall, 64 (39%) patients had Low IPSS risk and 90 (55%) Int-1 risk. Among these, 150 received prior rhuEpo (92%) and 27, azanucleosides (17%). In an ITT analysis, MER rate was significantly higher with combination therapy, Arm B 25.6% (n=21) vs. Arm A 9.9% (n=8) (P=0.015). Among 116 patients evaluable at week 16, 33.3% (20/60) and 14.3% (8/56) achieved MER, respectively (P=0.018), with a median response duration of 25.4 months vs. not reached in Arm A responders. Response to combined treatment was associated with baseline CD45-isoform distribution in erythroid precursors. Patients achieving MER had a significantly lower CD45 RA+RB:RO ratio (median, 1.51) compared to non-responders (median, 4.21; P=0.04), favoring homo-dimerization of the short CD45-RO isoform and inhibition of phosphatase activity. MER rate in Arm B patients with a low isoform ratio (< median) was 72.7% vs. 18.2% in the high ratio group (P=0.03). Thirty-four Arm A non-responders crossed over to combination-therapy with only 1 MER. There was no difference in the frequency or distribution of >Grade 3, non-hematologic AEs.Conclusions: LEN restores sensitivity to rhEpo in Epo-refractory LR-non-del(5q) MDS patients to yield durable and significantly higher rates of erythroid response to combination treatment without added toxicity. Erythroid CD45 isoform profile may serve as a response biomarker for selection of candidates for combination therapy. DisclosuresBennett:Celgne: Membership on an entity's Board of Directors or advisory committees. Altman:Syros: Honoraria; Janssen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau. Schiffer:Teva: Other: DSMB member; BMS: Research Funding; Ariad: Research Funding; Pfizer: Other: DSMB member.

Discovery of the First Pathogenic Human EPO Mutation Provides Mechanistic Insight into Cytokine Signaling

Congenital hypoplastic or Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder characterized by a paucity of red blood cells and their precursors in the bone marrow. The majority of cases of DBA are due to haploinsufficient mutations in ribosomal protein genes and in rare cases result from GATA1 mutations. However, nearly half of the DBA cases do not have an identified genetic etiology. While analyzing whole exome sequencing data from a cohort of over 450 patients with a clinical diagnosis of DBA, we encountered the case of a male child of a first cousin consanguineous union who was diagnosed with DBA as an infant and remained transfusion dependent. The patient responded to corticosteroid therapy for a year as a toddler, but this treatment was discontinued due to side effects. The patient subsequently remained transfusion dependent and at 6 years of age an allogeneic bone marrow transplant from a matched maternal aunt was performed. Surprisingly, despite achievement of robust donor chimerism, the patient remained transfusion dependent. Unfortunately the patient developed severe graft-versus-host disease and died of resultant complications. Both the potential recessive nature of the mutation, given parental consanguinity, and the lack of anemia correction following transplant made this case extremely unusual. Thus we evaluated this patient’s whole exome sequencing data. We identified a homozygous recessive mutation in the erythropoietin gene (EPO), which resulted in an R150Q substitution in the mature EPO protein. This mutation was absent from a cohort of 60,706 individuals depleted for Mendelian disease and fit the model of complete penetrance in the family. The R150Q mutation was expected to disrupt the high-affinity binding site to the EPO receptor (EPOR). However, we found by producing recombinant proteins that the EPO R150Q mutation only reduced the EPOR binding affinity by 3-fold. Surprisingly, the patient had an over 100-fold elevation in their serum EPO levels, suggesting that this mutation did not cause disease through altered affinity. Rather we observed altered EPOR binding kinetics by this mutant ligand. There was a slightly increased on-rate with a much faster dissociation rate (t1/2 of 10 seconds for the mutant vs. 6 minutes for the wild type). Using human erythroid cells and primary hematopoietic stem and progenitor cells, we could show that this mutant ligand never reached the same efficacy as the wild type (WT) EPO in promoting erythroid differentiation and proliferation. To better characterize this abnormal activity, we examined downstream signaling responses. We found identical phosphorylation of STAT5 at maximally potent concentrations of the WT (1 nM) and R150Q mutant (100 nM) EPO. By surveying a broad array of >120 phosphorylation events using intracellular flow cytometry, we demonstrated that maximal levels of STAT3 and STAT1 phosphorylation were reduced by 30% and 25%, respectively, with the R150Q (100 nM) compared to WT (1 nM) EPO. To determine the mechanistic basis for variation in downstream effector activation by the R150Q mutant ligand, we used inhibitors of both the JAK2 kinase and the SHP1/2 phosphatases that are respectively up- and downstream of STAT phosphorylation. While SHP1/2 inhibition did not alter STAT phosphorylation, JAK2 inhibition by ruxolitinib more potently inhibited STAT1/3 phosphorylation compared to STAT5. Interestingly, treatment with a low dose of ruxolitinib (40 nM) reduced erythroid proliferation to the same extent at maximally potent concentrations of the WT or R150Q EPO, demonstrating that the impairment in signaling by the R150Q EPO was primarily due to reduced JAK2 activity. Finally, we utilized single molecule fluorescent imaging of EPOR dimerization at the intact cell surface to directly show that the kinetically-biased R150Q EPO has a reduced ability to promote productive dimerization as compared to the WT EPO, even at maximally potent concentrations. Collectively, our results demonstrate how the R150Q mutant EPO - the first pathogenic mutation in EPO identified in humans - results in biased agonism of EPOR signaling through reduced receptor dimerization and consequently impaired JAK2 activation. More broadly our findings reveal how variation of cytokine-receptor binding kinetics can be used to tune downstream responses, which has broad implications for modulating the activity of numerous hematopoietic cytokines. DisclosuresNo relevant conflicts of interest to declare.

Ex Vivo Gene Therapy Approach By Targt Technology for the Treatment of β-Thalassemia Intermedia

β-Thalassemia intermedia or non-transfusion dependent thalassemia (NTDT) is a genetic blood disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload mediated by down-regulation of hepcidin. Using Hbbth3/+animals affected by NTDT, we have shown that iron restriction improves anemia and reduces splenomegaly by reducing the formation of hemichromes and improving the lifespan of red blood cells (RBC; Gardenghi et al, JCI, 2010; Guo et al, JCI, 2013; Casu et all Blood 2016). These findings emphasize the strong correlation between erythropoiesis, RBC synthesis and iron metabolism. In theory, agents that can stimulate erythropoiesis (such as erythropoietin or EPO) could improve anemia by increasing the production of RBC and, under conditions of reduced iron absorption, correct the iron overload by consuming the excess of iron stored in organs. Here we used a new ex vivo technology called TARGT (Transduced Autologous Restorative Gene Therapy) for prolonged production and secretion of therapeutic agents. Primary dermal fibroblasts transduced with helper dependent adenovirus expressing mouse Epo were embedded in Matrigel and implanted in the dorsal area of 2-3 months old Hbbth3/+and wild-type (WT) C57BL/6 mice. Three groups were generated for each genotype using different amounts of genetically modified fibroblasts (10e5, 5x10e5 and 1x10e6 transduced cells), while one group received no transduced fibroblasts as control. Complete blood count and serum harvest for Epo levels measurement were performed one week after implantation and then every ten days until the end of the experiment, at six weeks. In WT mice, already one week after fibroblasts implantation, hemoglobin (Hb) levels raised 2/3 g/dL, showing erythrocytosis. At the end of the treatment animals showed significantly increased Hb levels (18.5±1.7 g/dL, 19.4±0.5 g/dL and 19.8±0.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 13.5±0.8 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases in the range of 27% to 39%.In Hbbth3/+mice Hb levels rose about 3 g/dl already one week after fibroblasts implantation and remained elevated until the end experiment (8.7±0.3 g/dL, 10.7±1.0 g/dL and 10.6±1.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 7.7±0.7 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases in the range of 7% to 27%. At the end of the treatment, serum Epo levels were markedly increased at all doses both in Hbbth3/+and WT animals (more than 50% and up to 90%) when compared with their respective controls.However, as expected, stimulation of erythropoiesis led to worsening of splenomegaly and suppression of hepcidin, likely preventing the beneficial effect of erythroid-mediated consumption of stored iron in Hbbth3/+animals. Here we postulated that, in presence of agents that increase erythropoiesis such as Epo, some level of iron restriction is required to improve the anemia, prevent exacerbation of the splenomegaly and, concurrently, decreased iron overload. As a proof of principle of the ability of this technology to improve anemia and decrease iron overload, TARGTEPO was combined with low iron diet or iron chelation. We expect that decreased iron intake will improve the quality of the RBC, while the larger number of RBC produced should utilize more stored iron, reversing the iron overload in Hbbth3/+animals. Preliminary results at three weeks from the beginning of the experiment indicated that in WT implanted-animals the combination of TARGTEPO with low iron diet significantly reduced Hb levels (-40%), HTC (-42%), RBC number (-38%) and reticulocytes (-80%) when compared to animals overexpressing Epo and receiving normal iron diet or iron chelation. This indicates that in WT animals, after 3 weeks, the stored iron is insufficient to support the increased erythropoiesis. In contrast, in Hbbth3/+animals this drop in Hb was not seen, suggesting that this effect is delayed due to the highest level of stored iron. Complete characterization of these models and their parameters is in progress. These preliminary results suggest that the TARGTplatformcould be utilized, in combination with drugs that limit iron intake, to improve anemia and decrease iron overload in NTDT. Additional experiments are in progress to test this hypothesis. DisclosuresCasu:Ionis Pharmaceuticals: Research Funding; Medgenics LLC: Research Funding; Merganser Biotech: Research Funding. Neil:Medgenics: Employment. Miari:Medgenics: Employment. Shapir:Medgenics: Employment.

Higher Versus Standard Doses of Recombinant Erythropoietin for the Treatment of Anemia in Patients with Myelodysplastic Syndromes: Results of a Retrospective Survey from the Italian Registry of Myelodisplastic Syndromes (FISM)

▪Introduction: Erythropoietic stimulating agents (ESAs) have not yet received FDA nor EMA approval to treat anemia of myelodysplastic syndromes (MDS), but ESAs are in use in this clinical setting since more than 25 years. Early studies, using various but usually "standard" doses of recombinant EPO (rEPO) (30-40.000 IU weekly), showed a disappointing overall response-rate of no more than 15-25%. In recent years most studies reported a response rate of more than 50%, provided MDS patients were selected according to favorable clinical variables determined during the years ( ie low transfusion requirement, absence of blasts, endogenous EPO levels < 500). Higher response rates with respect to early studies are probably due to higher, not weight-adjusted doses of rEPO (60-80.000 UI weekly). Nevertheless, a direct comparison between the two different schedules of rEPO treatment is still lacking, and the superiority of the so called higher doses of rEPO to standards regimens is inferred only by results of meta-analysis comparing studies performed at different times with various schedules and heterogenous cohorts of MDS patients.Objectives: We aimed at clarifying whether the erythroid response rate differed in patients exposed to higher doses versus standard doses of rEPO.Methods: Within the framework of the Italian Network of regional MDS registries established by Fondazione Italiana Sindromi Mielodisplastiche (FISM) a cohort of 103 MDS patients (pts) with anemia treated with higher doses of rEPO (40.000 IU twice a week, H) for at least 3 months within 6 months from diagnosis were identified; a second cohort of 206 pts, similar for clinical parameters known to influence response to rEPO (i.e. Hb concentrations at the time of starting treatment, IPSS score, transfusion-dependency, endogenous Epo levels at diagnosis and time of treatment from diagnosis) and treated with standard doses (40.000 IU weekly, S) were compared to the first cohort. Univariate and multivariate analysis were performed as appropriate in order to identify factors influencing clinical response to treatment.Results: Characteristics of subjects were: median Hb pre-treatment 8.9 g/dL in H cohort and 9.1 g/dL in S cohort, IPSS score Intermediate-2/high in 5% of H cohort and 8% of S cohort, transfusion-dependency in 25% of H cohort and 26% of S cohort, median EPO at diagnosis 79 IU in H cohort and 69 IU in S cohort. According to IWG 2006 criteria, after 3 months of rEPO treatment the overall erythroid response-rate among all the pts in the two cohorts was 53% (163 out of 309 pts). No difference in erythroid response-rate was found between MDS pts treated with higher doses (49 (48%) responders out of 103 cases) when compared to cases treated with standard doses (114 (55%) responders out of 206 pts, p= 0.23). As expected, IPSS score, transfusion-dependency and EPO serum levels at diagnosis were statistically associated with response. In particular, at multivariate analysis, significantly lower response-rates to rEPO were associated with transfusion-dependency (yes vs no, OR= 0.59 (95%CI: 0.44-0.79, p<0.001), higher endogenous serum EPO levels at diagnosis (>500 vs <=500, OR= 0.36 (95%CI: 0.19-0.68, p=0.002) and higher IPSS score (intermediate 2/ high vs, intermediate 1 / low, OR=0.42 (95%CI: 0.24-0.74, p=0.003).Conclusions: Our data, although derived by a retrospective analysis, seem to indicate that standard doses of rEPO are at least as effective as higher-doses for correcting anemia in MDS patients; in this clinical scenario, a standard-doses rEPO treatment allows for a consistent reduction of costs without precluding to achieve a durable erythroid response to rEPO. Prospective, randomized studies addressing this point are necessary to definitely address this topic. DisclosuresAngelucci:Novartis oncology, celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Finelli:Celgene: Research Funding; Celgene: Other: Speaker fees; Novartis: Other: Speaker fees. Oliva:Celgene: Consultancy, Honoraria, Speakers Bureau. Santini:Janssen: Consultancy, Honoraria; Onconova: Other: advisory board; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Other: advisory board; Astex: Other: advisory board; Novartis: Consultancy, Honoraria.

Racial Disparities in Patients with Myelodysplastic Syndrome (MDS): Clinical and Molecular Depiction

▪BackgroundLittle is known about racial disparities amongst patients with MDS. The incidence of MDS is higher in Caucasians, who represent the majority of patients included on clinical trials. We explored the differences in disease features and outcomes between Caucasians, African Americans (AA), and Hispanics.MethodsUsing our MDS database, patients with a diagnosis of MDS were identified and categorized into the aforementioned racial groups. We compared baseline characteristics, risk stratification, treatment, and overall survival (OS). Data on recurrent somatic mutations (SM) by next generation sequencing was available for 349 patients and compared between the groups.ResultsAmong 2538 MDS patients, 63 (2%) patients were AA and 107 (4%) were Hispanic. Table-1 summarizes baseline characteristics. In Hispanic patients, 42% were <60 years, 30% had t-MDS, and the mean platelet (plt) count was lower. In AA patients, the mean hemoglobin (hgb) was lower and the serum EPO was higher. More Hispanic and AA females were diagnosed with MDS compared to Caucasians.A higher percentage of Hispanic patients underwent allogenic stem cell transplant (33% versus 13% in Caucasian and 10% in AA) (p= <.005). There was no difference in treatment with hypomethylating agents or lenalidomide. Only 1 AA and 7 Hispanic patients were enrolled on clinical trials, compared to 113 Caucasians. Among patients evaluable for response, none of the 7 AA patients treated with erythroid stimulating agents (ESA) had a hematologic improvement, compared to 10% among Hispanics and 39% in Caucasians.The median OS was 37, 30, and 47 months (mo) among Caucasian, AA, Hispanic (p=0.022). Among patients with low/int-1 risk IPSS, the median OS was 55, 45, and 81 mo respectively (p=0.1), and among int-2/high risk IPSS, the median OS was 17, 17, 26 mo respectively (p=0.1). There was no difference in the rate of AML transformation.Data on 349 patients who had recurrent somatic mutation (326 Caucasian, 8 AA, and 15 Hispanic) were reviewed. The most common somatic mutations in Caucasian patients were TET-2 30%, ASXL-1 18%, SF3B1 14%, DNMT3A 14%, SRSF2 11% and p53 11%. In AA, the most common SM were IDH1 25%, EZH2 25%, DNMT3A, ASXL-1, TP53, ETV6, NRAS, in13% each. The most common SM in Hispanic patients were SRSF2 20%, RUNX-1 20%, TET-2, IDH-2, UA2F1, ASXL-1 and p53 in 13% for each. No splicing mutation or TET-2 was observed among AA patients. There was a higher trend for IDH-1 mutation among AA (25%) vs 6% in Caucasian and none in Hispanic (p=.07).ConclusionWe observed differences in several baseline characteristics between races. Hispanic patients were younger, had more therapy related MDS and thrombocytopenia. AA patients had higher serum EPO levels and decreased response to ESAs. Hispanic patients had better overall survival, especially in lower risk disease. Preliminary data suggests potential racial differences in recurrent somatic mutations observed in MDS. [Display omitted] [Display omitted] DisclosuresSweet:Novartis: Consultancy, Speakers Bureau; Pfizer: Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Incyte Corporation: Research Funding; Karyopharm: Honoraria, Research Funding. Komrokji:Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Perinatal Asphyxia and Erythropoietin and VEGF: Serial Serum and Cerebrospinal Fluid Responses

Background: Infants with neonatal encephalopathy (NE) of hypoxic-ischaemic origin are at risk of oxidative and ischaemia-reperfusion injury, which may induce abnormal inflammatory responses involving excessive cytokine production and release in serum and cerebrospinal fluid (CSF). Systemic inflammation is found in infants with NE, and we therefore were interested in cytokines associated with hypoxia, including vascular endothelial growth factor (VEGF) and erythropoietin (Epo). Objective: To investigate the relationship between Epo, VEGF levels, brain injury and outcome in a group of term infants exposed to perinatal asphyxia (PA) compared to controls. Methods: Serum and CSF biomarkers associated with hypoxia (VEGF, Epo) were serially measured using multiplex immunoassays over days 1-4 in term infants exposed to PA including infants with NE and controls. Results were compared to severity of encephalopathy, MR brain imaging and mortality. Results: Ninety-four infants had 247 serum samples collected (n = 12 controls, 82 exposed to PA with 34 CSF samples), and 4 infants died. Controls had significantly lower serum Epo levels on days 1 and 2 compared to those exposed to PA (p = 0.02). Grade II/III NE was significantly associated with elevated day 2 Epo and decreased day 1 VEGF (p < 0.05; day 2 Epo AUC = 0.74, cut-off 10.05 IU/ml). Elevated serum Epo was associated with severely abnormal MRI. Mortality was associated with elevated day 3 Epo and decreased day 1 VEGF. CSF levels were all after hypothermia and were not significantly associated with outcome. Conclusion: Serum Epo and VEGF may be markers of severity of hypoxia-ischaemia and brain injury as they are closely related to hypoxic exposure.