Serum Epo Levels Research Articles

A Study of Some Immunological and Hematological Aspects in Children with Renal Disease

This study was carried out between September 17, and December 25, 2023. It included 62 pediatric patients at the age range (1-12) years of both genders: 26 with chronic renal failure (CRF) and 36 with nephrotic syndrome (NS), who were outpatients and in-patients in the dialysis unit in both Al-Mansour pediatric teaching hospital and Child's central teaching hospital. The control group consisted of 26 children. Blood samples were collected from patients and controls. Immunological tests of interleukin-6 (IL-6) and erythropoietin (Epo) measurement were done by using ELISA technique. Biochemical tests, including blood urea and serum creatinine, were applied to evaluate renal function. Hematological aspects referred to anemia presence or not; by measuring PCV and Hb. The results explained significant increase in serum IL-6 levels (P<0.001), significant decrease in serum Epo levels (P<0.001), significant increase in both urea and creatinine concentrations (P<0.001) in serum and significant decrease in both PCV and Hb (P<0.001) in NS and CRF patients in comparison with the control group

Impact of Mutational Burden and IPSS-M on Response to ESAs in Lower Risk Myelodysplastic Neoplasms

INTRODUCTION Lower risk myelodysplastic neoplasms (LR-MDS), as defined by IPSS-R, are frequently presenting with anemia which is primarily treated with erythropoiesis stimulating agents (ESAs). However, response rates are suboptimal due to primary resistance or subsequent loss of response, leading to transfusion dependency and poor quality of life. In 2022 a new prognostic system has been developed considering number and type of somatic mutations in MDS (IPSS-M), showing a stronger prognostic power in MDS. AIMS We aimed to assess whether IPSS-M risk score could predict ESA response in IPSS-R LR-MDS patients. METHODS In this multicentric European study we evaluated baseline IPSS-M score in 397 LR-MDS cases from 14 different centers (Italy, Germany, France and Spain) and correlated with ESA response assessed with IWG criteria. Mutational data were obtained by different next generation sequencing (NGS) panels targeting genes recurrently mutated in myeloid neoplasia and necessary to calculate IPSS-M. RESULTS Median age of patients was 74 years with male preponderance (54.5%). Serum EPO levels (sEPO) were available in 277/397 cases (mean 157U/L, <200 U/L in 80% of the cases). When transfusion history (TH) was known (252/397), 44.6% of cases were transfusion dependent (TD). The majority of the cases received erythropoietin-α and darbepoetin-α with response rate of 52.6% (HI-E) according to IWG criteria and a median duration of response of 30 months (data from 165 responders with follow up information). We observed that IPSS-M score, but not the number of mutations, was significantly lower in ESA responders compared to non-responders (median -1,08 vs -0,63 respectively , p<0.0001). The very low (VL) IPSS-M risk group was enriched in ESA responders (74 vs 26% p=0.036), while groups from moderate low (ML) to very high (VH) were more enriched with non-responders (37.5 vs 62% p=0.001; 45.4 vs 54.6% p=0.045; 29.6 vs 70,4% p=0.0017; 25 vs 75% p=0.021 respectively, Fig.1). In cases with known TH (n=252), non-TD (NTD) ESA responders had lower IPSS-M scores (mean -1,17) compared to i) NTD ESA non-responders (mean -0,61 , p<0.0001), ii) TD ESA responders (mean -0,93, p=0,025) and iii) TD ESA non-responders (mean -0,54, p<0.0001). As observed in other settings, 17% of the cases (n=67) were re-stratified to higher risk IPSS-M categories (8%, 13,4% and 44,3% of IPSS-R very low, low and intermediate risk respectively). Among upstaged cases, most were ESA non-responders especially in the moderate high (MH, 67%), high (H, 61%) and very high-risk (VH, 100%) groups. Only 4% of cases (n=17) were down-staged, all from IPSS-R intermediate risk group, and showed response rates similar to IPSS-R intermediate cases who remained ML or MH. Finally, IPSS-M score, sEPO and TD correlated with response in multivariate logistic regression analysis ( p=0.005, p=0.004 and p=0.014 respectively), but only IPSS-M score and TD were predictive (OR 0.51 and 0.34 respectively). Based on this model, we designed a ROC curve (AUC 0.79) where a cut-off of 0.46 showed a sensitivity of 0.9 and specificity of 0.67 in discriminating responders from non-responders (Fig.2). CONCLUSIONS To our knowledge, this is the first report correlating IPSS-M with ESA response in a large number of LR-MDS cases. IPSS-M scores exhibited predictive power, with lower values in ESA responders compared to non-responders and in NTD compared to TD cases. We also observed that higher IPSS-M categories were inversely correlated with ESA response. A fraction of cases with scarce ESA response was upstaged to IPSS-M higher risk groups. The number of mutations did not correlate with ESA response, while IPSS-M score, which considers the prognostic weight of specific mutations, showed predictive significance. In the multivariate analysis IPSS-M score and TD were strong predictors of ESA response, irrespective of baseline sEPO, potentially defining at diagnosis LR-MDS for whom novel agents like luspatercept could be considered as first line therapeutic option.

The Role of Erythropoietin in Preventing Anemia in the Premature Neonate.

Recombinant human erythropoietin (rhEPO) treatment is an alternative to red blood cell (RBC) transfusions in neonates presenting anemia of prematurity (AOP). This study assesses the impact of early rhEPO administration on AOP (any stage) incidence, as well as the incidence of individual AOP stages and RBC transfusions. Out of 108 preterm neonates, 49 were administered rhEPO and compared to the remaining group using univariate and multivariate analyses. Univariately, gestational age (GA), birth weight (BW), hemoglobin (Hb), hematocrit (HCT), RBC levels, and iron administration were significantly associated with AOP (p < 0.05 each); however, only the latter remained significant following multivariate analysis (AOR: 2.75, 95% CI, 1.06-7.11). Multinomial analysis revealed rhEPO treatment was associated with a near three-fold reduction in moderate AOP incidence (OR: 0.36, 95% CI, 0.15-0.89). Furthermore, ANCOVA revealed positive correlations between rhEPO administration and 21-day Hb (p < 0.01), HCT (p < 0.05), and EPO (p < 0.001) levels. The results confirm previously reported benefits of rhEPO treatment, such as reduced moderate AOP incidence and increased Hb, HCT, and serum EPO levels.

B-293 Non-renal Sources of Erythropoietin: Complications for Interpreting Erythropoietin Assays

Abstract Background Erythropoietin (EPO) is a glycoprotein that stimulates the production of red blood cells, in response to systemic hypoxia. Generally, the kidney is thought to be the sole producer of EPO. Emerging data shows Epo mRNA expression can occur in non-renal tissues, yet the significance of this remains uninvestigated. Clinically, EPO assays are primarily used to identify the underlying cause of anemia and polycythemia. Interestingly, EPO serum levels are elevated following a myocardial infarct (MI); the source is always assumed to be kidney with no further investigations into non-renal sources of EPO. Here we investigate the heart as a novel source of serum EPO following a MI. We hypothesize that the heart, not the kidney, produces EPO in response to organ-specific hypoxia (i.e., MI). Methods To assess which organs are producing EPO following a MI, we examined EPO mRNA expression. mRNA levels correlate strongly with EPO protein synthesis, as EPO is exclusively regulated at the transcription level. To induce a MI, CD1 mice were subjected to ligation of the lateral anterior descending artery. Left ventricle and kidney tissues were collected at 24 h, 4 weeks and 18 weeks following MI or sham surgery; tissue was snap frozen and stored at −80°C for qPCR analysis. Hematocrit was measured at 2-, 4-, and 18-weeks following MI or sham surgery. Results 24 h following a MI, the left ventricle showed a ∼100-fold increase in EPO mRNA expression in comparison to sham group (P = 0.0012); the kidney showed no significant differences in EPO mRNA between MI and sham group. Hematocrit was measured from the carotid artery. At 2- and 4- weeks following surgery, hematocrit in MI group was significantly elevated in comparison to baseline hematocrit levels (Baseline: 40.8% ± 4, 2-week: 47.8% ± 3.1, and 4-week: 46% ± 2.6 (P = 0.0003, P = 0.0103, respectively). Whereas there were no significant differences in hematocrit between baseline and the sham group. Conclusion Here we show that following a MI, the heart, not the kidney, produces EPO. Subsequently, there is an increase in hematocrit as a result of cardiac-specific EPO production. Further, perturbations in EPO serum levels are not disease specific. In cases where EPO assays are conducted on MI patients, interpretation of EPO levels should be cautioned as it may not be exclusively diagnostic of the underlying cause of anemia or other bone marrow disorders. Therefore, an alternative diagnosis may be warranted for elevated EPO levels in this specific patient population.

Hydrophobic constituents of Polygonum multiflorum roots promote renal erythropoietin expression in healthy mice.

The roots of Polygonum multiflorum Thunberg (Polygonaceae) are used as a crude drug Kashu that is considered to improve blood deficiency based on a Kampo concept. Kashu has been included in Kampo formulas, such as Tokiinshi, which is used to treat eczema and dermatitis with itchiness by inhibiting inflammation and facilitating blood circulation in the skin. However, the effects of P. multiflorum roots on erythropoiesis are unclear. Previously, we isolated six phenolic constituents from an ethyl acetate (EtOAc)-soluble fraction of P. multiflorum root extract and identified them as (E)-2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside [(E)-THSG], emodin, emodin-8-O-β-D-glucopyranoside, physcion, physcion-8-O-β-D-glucopyranoside, and catechin. To examine whether P. multiflorum roots facilitate erythropoiesis, the EtOAc-soluble fraction was orally administered to healthy ICR mice. When compared with mice fed a standard diet alone (Controls), the mice fed a diet including the EtOAc-soluble fraction exhibited significantly higher serum erythropoietin (Epo) levels. The renal Epo mRNA levels in EtOAc-soluble fraction-administered mice were significantly higher than those in the control mice. Then, we administered roxadustat, which is a drug to treat the patient suffering with renal anemia by specifically inhibiting hypoxia-inducible factor prolyl hydroxylases. Roxadustat slightly increased renal Epo mRNA levels in healthy mice. Administration of (E)-THSG, a major constituent, significantly increased serum Epo levels. It is likely that (E)-THSG may facilitate the process to convert inactive renal Epo-producing cells to active Epo-producing cells. Collectively, it is implied that (E)-THSG in the EtOAc-soluble fraction of P. multiflorum roots may primarily improve blood deficiency of Kampo concept by promoting erythropoiesis.

Chronic intermittent hypobaric hypoxia improves iron metabolism disorders via the IL-6/JAK2/STAT3 and Epo/STAT5/ERFE signaling pathways in metabolic syndrome rats

AimOur previous study demonstrated that chronic intermittent hypobaric hypoxia (CIHH) improved iron metabolism disorder in obese rats through the downregulation of hepcidin. This study aimed to observe the molecular mechanism of CIHH in improving iron metabolism disorders, especially by Janus kinase/signal transducer and activation of the transcription (JAK/STAT) signaling pathway in metabolic syndrome (MS) rats. MethodsSix-week-old male Sprague-Dawley rats were randomly divided into four groups: CON, CIHH (subjected to hypobaric hypoxia simulating 5000-m altitude for 28 days, 6 h daily), MS (induced by high fat diet and fructose water), and MS+CIHH. The serum levels of glucose, lipid metabolism, iron metabolism, interleukin-6 (IL-6), erythropoietin (Epo) and hepcidin were measured. The protein expressions of JAK2, STAT3, STAT5, bone morphogenetic protein 6 (BMP6), small mothers against decapentaplegic 1 (SMAD1) and hepcidin were examined. The mRNA expressions of erythroferrone (ERFE) and hepcidin were analyzed. ResultsThe MS rats displayed obesity, hyperglycemia, hyperlipidemia, iron metabolism disorder, increased IL-6 and hepcidin serum levels, upregulation of JAK2/STAT3 signaling pathway, decreased Epo serum levels, downregulation of STAT5/ERFE signaling pathway in spleen, upregulation of BMP/SMAD signaling pathway in liver, and increased hepcidin mRNA and protein expression compared to CON rats. All the aforementioned abnormalities in MS rats were ameliorated in MS + CIHH rats. ConclusionsCIHH improved iron metabolism disorders, possibly by inhibiting IL-6/JAK2/STAT3 and activating Epo/STAT5/ERFE signaling pathway, thus downregulating hepcidin in MS rats.

Erythropoietin and Erythropoietin Receptor Levels and Their Diagnostic Values in Drug-naïve Patients with Generalized Anxiety Disorder.

Generalized anxiety disorder (GAD) is a prevalent psychiatric disorder. Diagnosis of GAD depends on subjective complaints of patients, thus the need for biological markers is constantly emerging. In this study, we aimed to the investigate diagnostic values of Erythropoietin (Epo) and its receptor (EpoR) levels in drug-naïve patients with GAD. This study included 45 newly diagnosed drug-naive patients with GAD, aged and sex-matched 30 healthy controls. Medical histories were obtained, and physical examinations and laboratory tests were conducted. Also, the Hamilton Anxiety Rating Scale (HAM-A) was used for all participants. Serum Epo and EpoR levels were measured by ELISA. HAM-A score was significantly higher in GAD patients versus the controls (p < 0.05). While the levels of Epo in patients with GAD were lower than the control patients, EpoR levels were increased in these patients (p < 0.05). Epo/EpoR ratios were significantly lower in the patients with GAD than in the control subjects (p < 0.05). A positive significant correlation was observed between the EpoR level and the HAM-A score (r = 0.755, p < 0.001). However, there was a negative significant correlation between Epo levels and HAM-A score (r = -0.749, p < 0.001). Receiver operator characteristic curve analysis showed high diagnostic performance for Epo and EpoR, areas under curves were 0.901 and 0.912, respectively. This is the first report to investigate the association between serum Epo and EpoR levels in GAD patients. Our results reveal possible diagnostic value of Epo and EpoR. Moreover, Epo therapy may be a good choice for GAD treatment.

JAK2 unmutated erythrocytosis: 2023 Update on diagnosis and management.

JAK2 unmutated or non-polycythemia vera (PV) erythrocytosis encompasses a heterogenous spectrum of hereditary and acquired entities. Foremost in the evaluation of erythrocytosis is the exclusion of PV through JAK2 (inclusive of exons 12-15) mutation screening. Initial assessment should also include gathering of previous records on hematocrit (Hct) and hemoglobin (Hgb) levels, in order to streamline the diagnostic process by first distinguishing longstanding from acquired erythrocytosis; subsequent subcategorization is facilitated by serum erythropoietin (Epo) measurement, germline mutation screening, and review of historical data, including comorbid conditions and medication list. Hereditary erythrocytosis constitutes the main culprit in the context of longstanding erythrocytosis, especially when associated with a positive family history. In this regard, a subnormal serum Epo level suggests EPO receptor mutation. Otherwise, considerations include those associated with decreased (high oxygen affinity Hgb variants, 2,3-bisphosphoglycerate deficiency, PIEZO1 mutations, methemoglobinemia) or normal oxygen tension at 50% Hgb saturation (P50). The latter include germline oxygen sensing pathway (HIF2A-PHD2-VHL) and other rare mutations. Acquired erythrocytosis commonly results from central (e.g., cardiopulmonary disease, high-altitude habitat) or peripheral (e.g., renal artery stenosis) hypoxia. Other noteworthy conditions associated with acquired erythrocytosis include Epo-producing tumors (e.g., renal cell carcinoma, cerebral hemangioblastoma) and drugs (e.g., testosterone, erythropoiesis stimulating agents, sodium-glucose cotransporter-2 inhibitors). Idiopathic erythrocytosis is an ill-defined terminology that presumes the existence of an increased Hgb/Hct level without an identifiable etiology. Such classification often lacks accounting for normal outliers and is marred by truncated diagnostic evaluation. Current consensus treatment guidelines are not supported by hard evidence and their value is further undermined by limited phenotypic characterization and unfounded concerns for thrombosis. We are of the opinion that cytoreductive therapy and indiscriminate use of phlebotomy should be avoided in the treatment of non-clonal erythrocytosis. However, it is reasonable to consider therapeutic phlebotomy if one were to demonstrate value in symptom control, with frequency determined by symptoms rather than Hct level. In addition, cardiovascular risk optimization and low dose aspirin is often advised. Advances in molecular hematology might result in better characterization of "idiopathic erythrocytosis" and expansion of the repertoire for germline mutations in hereditary erythrocytosis. Prospective controlled studies are needed to clarify potential pathology from JAK2 unmutated erythrocytosis, as well as to document the therapeutic value of phlebotomy.

JAK 2 NEGATIVE POLYCYTHEMIA VERA IN A TERTIARY CARE CENTER IN WEST UP POPULATION IN INDIA :A RARE PRESENTATION OF 5 CASES

Introduction: Polycythaemia vera (PV) belongs to the group of myeloproliferative neoplasms with an excessive increase in hemoglobin levels.(1). It has been seen that JAK2 V617F mutation present in 80-90% of MPNs and JAK2 exon12 mutations are seen in 4%-5% cases of MPNs like PV.As per an Indian data the JAK2V617F mutational frequency in Indian population was 81.8% for PV, which was different from that reported in western literature as more than 95% for PV.Activation of JAK2 by either point mutation or fusion protein causes activation of the JAK-STAT pathway. While mutations in JAK2 are reported in numerous MPN phenotypes, exon 12 mutations specifically result in erythrocytosis due to increased EPO signalling. In India, the incidence of JAK2-negative polycythemia is relatively high, reaching up to 18%. (2,3) Case series: Case 1-A 21-year male presented to the hematology OPD with complains of paresthesias and off and on headache for the past 6 months. He was on multiple symptomatic medications but to no relief. On routine investigations it was seen that he had a persistent hemoglobin level fluctuating between 17.5gm/dl and 18.5 gm/dl. He has iron deficiency for which he is on single oral iron tablet. No definite organomegaly was seen. In hematology OPD he was investigated for polycythemia vera keeping in mind the WHO criterias.His Serum EPO levels are within normal range,JAK2 V617F/exon 12mutation analysis is negative.A bone marrow aspiration and biopsy study done shows Hypoplastic marrow for age with erythroid predominance. In view of clinical suspicion of JAK 2 negative PV he is being treated with ecosprin, phlebotomy every 3 months and supplemental iron orally. The patient is clinically relieved of his symptoms of peripheral neuropathy. His last hemoglobin value is stable at 16.5gm/dl.Case 2-A 32 -year male presented to hematology OPD with mild headache and weakness for 6 months. A routine CBC investigation showed a high hemoglobin level of 18.4 gm/dl. Mildly raised RBC count was also seen. A subnormal serum EPO levels of 3.7 mIU/Ml (4.3-29.0 mIU/ML) was seen. JAK 2V617F/exon 12 mutation analysis was negative. The patient has been started on ecosprin and phlebotomy every 3 months and is symptomatically relieved.Case 3- A 29-year male presented to hematology OPD with paresthesias and weakness for 3 months. A routine CBC investigation showed a high hemoglobin level of 19.7 gm/dl. Mildly raised RBC count was also seen. Rest all parameters were within normal limits. On examination no hepatosplenomegaly or lymphadenopathy was seen. A low serum EPO levels of &lt;1 mIU/Ml (4.3-29.0 mIU/ML) was seen. JAK 2V617F/exon12 mutation analysis was negative. The patient has been started on ecosprin and phlebotomy every 3 months and is symptomatically relieved. Case4-33 year male presented to OPD with fatigue and paresthesias.CBC showed increased hemoglobin of 18gm/dl.Patient was a non-smoker.Vitamin B12 was&gt;1000gm/dl.Bone marrow showed mildly predominant myeloid series.JAK2 mutation profile was negative but the patient responded to phlebotomy.Case5:A 28 year female had recurrent headaches however MRI and other investigations were normal. A serum EPO level done on suspicion of PV showed subnormal ranges.Bone marrow was reported as reactive.JAK2 mutation analysis was negative however patient responds to ecosprin and hyroxyurea. Conclusion-All five cases that presented to the department had been undiagnosed before they reached our setup. The unique feature of all these cases was negativity for both JAK2V617F as well as JAK2 exon 12 mutation despite its clinical behaviour being that of PV .These cases are being actively managed as low risk polycythemia vera and since the start of the treatment comprising of phlebotomy once every 3 months their hemoglobin and hematocrit concentrations are maintained.No cytoreductive therapy is being given .Such unique presentations need to be reported and discussed extensively.

JAK 2 NEGATIVE POLYCYTHEMIA VERA IN A TERTIARY CARE CENTER IN WEST UP POPULATION IN INDIA :A RARE PRESENTATION OF 5 CASES

Introduction: Polycythaemia vera (PV) belongs to the group of myeloproliferative neoplasms with an excessive increase in hemoglobin levels.(1). It has been seen that JAK2 V617F mutation present in 80-90% of MPNs and JAK2 exon12 mutations are seen in 4%-5% cases of MPNs like PV.As per an Indian data the JAK2V617F mutational frequency in Indian population was 81.8% for PV, which was different from that reported in western literature as more than 95% for PV.Activation of JAK2 by either point mutation or fusion protein causes activation of the JAK-STAT pathway. While mutations in JAK2 are reported in numerous MPN phenotypes, exon 12 mutations specifically result in erythrocytosis due to increased EPO signalling. In India, the incidence of JAK2-negative polycythemia is relatively high, reaching up to 18%. (2,3) Case series: Case 1-A 21-year male presented to the hematology OPD with complains of paresthesias and off and on headache for the past 6 months. He was on multiple symptomatic medications but to no relief. On routine investigations it was seen that he had a persistent hemoglobin level fluctuating between 17.5gm/dl and 18.5 gm/dl. He has iron deficiency for which he is on single oral iron tablet. No definite organomegaly was seen. In hematology OPD he was investigated for polycythemia vera keeping in mind the WHO criterias.His Serum EPO levels are within normal range,JAK2 V617F/exon 12mutation analysis is negative.A bone marrow aspiration and biopsy study done shows Hypoplastic marrow for age with erythroid predominance. In view of clinical suspicion of JAK 2 negative PV he is being treated with ecosprin, phlebotomy every 3 months and supplemental iron orally. The patient is clinically relieved of his symptoms of peripheral neuropathy. His last hemoglobin value is stable at 16.5gm/dl.Case 2-A 32 -year male presented to hematology OPD with mild headache and weakness for 6 months. A routine CBC investigation showed a high hemoglobin level of 18.4 gm/dl. Mildly raised RBC count was also seen. A subnormal serum EPO levels of 3.7 mIU/Ml (4.3-29.0 mIU/ML) was seen. JAK 2V617F/exon 12 mutation analysis was negative. The patient has been started on ecosprin and phlebotomy every 3 months and is symptomatically relieved.Case 3- A 29-year male presented to hematology OPD with paresthesias and weakness for 3 months. A routine CBC investigation showed a high hemoglobin level of 19.7 gm/dl. Mildly raised RBC count was also seen. Rest all parameters were within normal limits. On examination no hepatosplenomegaly or lymphadenopathy was seen. A low serum EPO levels of &lt;1 mIU/Ml (4.3-29.0 mIU/ML) was seen. JAK 2V617F/exon12 mutation analysis was negative. The patient has been started on ecosprin and phlebotomy every 3 months and is symptomatically relieved. Case4-33 year male presented to OPD with fatigue and paresthesias.CBC showed increased hemoglobin of 18gm/dl.Patient was a non-smoker.Vitamin B12 was&gt;1000gm/dl.Bone marrow showed mildly predominant myeloid series.JAK2 mutation profile was negative but the patient responded to phlebotomy.Case5:A 28 year female had recurrent headaches however MRI and other investigations were normal. A serum EPO level done on suspicion of PV showed subnormal ranges.Bone marrow was reported as reactive.JAK2 mutation analysis was negative however patient responds to ecosprin and hyroxyurea. Conclusion-All five cases that presented to the department had been undiagnosed before they reached our setup. The unique feature of all these cases was negativity for both JAK2V617F as well as JAK2 exon 12 mutation despite its clinical behaviour being that of PV .These cases are being actively managed as low risk polycythemia vera and since the start of the treatment comprising of phlebotomy once every 3 months their hemoglobin and hematocrit concentrations are maintained.No cytoreductive therapy is being given .Such unique presentations need to be reported and discussed extensively.

Luspatercept for Treatment of Lower Risk Myelodysplastic Syndromes: Real World Data Replicates Medalist Study Results and Confirms Activity Among Hypomethylating Agents and Lenalidomide Treated Patients

Introduction: Luspatercept, TGF-B fusion trap protein, is a first in class erythroid maturating agent approved by FDA for treatment of red blood cell transfusion dependent (RBC-TD) lower risk myelodysplastic syndromes (LR-MDS) with ring sideroblasts (RS) based on the MEDALIST clinical trial. Red blood cell transfusion independence (RBC-TI) was observed in 38% of 153 patients (pts) treated with luspatercept compared to 13% of 76 pts in the placebo group. We present to our knowledge first real-world data (RWD) in a large cohort of pts treated at our center with luspatercept for LR-MDS. More importantly, we address impact of prior therapies on response namely hypomethylating agents (HMA) as the original clinical trials excluded HMA treated pts. Methods We identified all pts treated at Moffitt Cancer Center with luspatercept since the FDA approval. Baseline RBC transfusion burden (TB) was defined as: non- transfusion dependent (NTD) (0 units in 8 weeks prior luspatercept), low TB (LTB) (1-5 units /8weeks) and high TB (HTB) (≥ 6 units /8 weeks). A hematological response (HI) was defined as an (i) objective Hgb increase of > 1.5 g/dl in NTD, and (ii) RBC-TI with Hgb increase of 1.5 g/dl, or RBC-TI without Hgb 1.5 g/dl increase, or >50% reduction in RBC transfusion burden among RBC-TD. Results Between February 2020 and December 2021, 114 pts were treated with luspatercept. Table-1 summarizes baseline characteristics in comparison to Medalist trial, the median age was 70 years. Seventy-three pts (67%) had MDS-RS subtype, 91% were intermediate or lower risk MDS by R-IPSS. SF3B1 mutation (MT) was detected in 71% (80/112). The mean Hgb level was 7.90 g/dl and 47% were RBC HTB transfusion dependent. The median serum erythropoietin (epo) level at time of referral was 122 U/L (19% > 500 U/L) (n=77). Majority had prior erythroid stimulating agents (ESA) (89%) with 29% hematological response to prior ESA, 59 pts (53%) had prior HMA therapy, and 42 patients (47%) had prior lenalidomide (len). The overall HI rate to luspatercept was 39.5% (45/114). Table-2 summarizes detailed responses. HI correlated with baseline RBC-TB where 69% (9/13) NTD, 47% (21/47) LTB and 25% HTB (13/53) achieved HI respectively, p=.004. Almost 55% of pts needed dose escalation, 62 pts received 1.33 mg/kg and 63 pts received 1.75 mg among whom 40% (25 pts) demonstrated response to higher doses. Based on RBC-TB, 75% (6/8) NTD patients achieved HI with highest dose escalation, 40% (10/25) LTB pts achieved HI with dose escalation and 30% (9/30) HTB pts achieved response with dose escalation. Among responders, the median duration of response was 15.6 months (2.6-27.3). As off last follow up, 56 pts (49%) discontinued treatment. Reason of discontinuation was lack of response in 47 pts, loss of response in 4 patients and adverse events in 5 pts (58 pts are still on therapy). The most common captured adverse events were fatigue (n=8), GI (n=4), shortness of breath and arthralgia (1 each). The response rates were higher among SF3B1 MT pts compared to wild type, 48% (34/80) compared to 16% (5/32), p=.002. The response rate among pts with RS subtypes versus non-RS were 49% versus 28% respectively, p=.05. Among 14 pts with MDS/MPN-RS-T the HI rate was 79%. HI for pts with serum epo < 500 U/L, was 48% (30/62) compared to 13% 2/15) for those with serum epo > 500, p=.01. Majority of pts with high serum epo level were RBC HTB at baseline 67% (10/15). The HI rate was 30% (18/60) after HMA failure compared to 50% (27/54) in HMA naïve pts, (p=.03). More pts post HMA failure were RBC-HTB 62% compared to 30% in HMA naïve. The luspatercept HI among RBC-LTB HMA failure pts was 39% compared to 54% in HMA naïve LTB, however, it was 24% among HTB HMA failure compared to 25% among HMA naïve HTB. The HI rate was 33% (15/46) for len failure pts compared to 43% (29/67) naïve patients, (p=.3). In multivariable analysis both SF3B1 mutation status and baseline RBC TB independently correlated with luspatercept response but not HMA failure. Conclusions Our RWD confirms luspatercept clinical benefit and safety profile observed in the MEDALIST trial. Responses are dose dependent with 1/3 of patients who were dose escalated responding. Low baseline RBC-TB dependency and SF3B1 MT correlated with higher response rates. Luspatercept retained activity after HMA or lenalidomide failure however a trend of lower responses observed correlated with RBC-HTB among those patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ASXL1 mutations with serum EPO levels predict poor response to darbepoetin alfa in lower-risk MDS: W-JHS MDS01 trial

Darbepoetin alfa (DA) is used to treat anemia in lower-risk (IPSS low or int-1) myelodysplastic syndromes (MDS). However, whether mutations can predict the effectiveness of DA has not been examined. The present study aimed to determine predictive gene mutations. The primary endpoint was a correlation between the presence of highly frequent (≥ 10%) mutations and hematological improvement-erythroid according to IWG criteria 2006 by DA (240 μg/week) until week 16. The study included 79 patients (age 29–90, median 77.0 years; 52 [65.8%] male). Frequently (≥ 10%) mutated genes were SF3B1 (24 cases, 30.4%), TET2 (20, 25.3%), SRSF2 (10, 12.7%), ASXL1 (9, 11.4%), and DNMT3A (8, 10.1%). Overall response rate to DA was 70.9%. Multivariable analysis including baseline erythropoietin levels and red blood cell transfusion volumes as variables revealed that erythropoietin levels and mutations of ASXL1 gene were significantly associated with worse response (odds ratio 0.146, 95% confidence interval 0.042–0.503; p = 0.0023, odds ratio 0.175, 95% confidence interval 0.033–0.928; p = 0.0406, respectively). This study indicated that anemic patients who have higher erythropoietin levels and harbor ASXL1 gene mutations may respond poorly to DA. Alternative strategies are needed for the treatment of anemia in this population. Trial registration number and date of registration: UMIN000022185 and 09/05/2016.

Epo/EpoR signaling in osteoprogenitor cells is essential for bone homeostasis and Epo-induced bone loss

High erythropoietin (Epo) levels are detrimental to bone health in adult organisms. Adult mice receiving high doses of Epo lose bone mass due to suppressed bone formation and increased bone resorption. In humans, high serum Epo levels are linked to fractures in elderly men. Our earlier studies indicated that Epo modulates osteoblast activity; however, direct evidence that Epo acts via its receptor (EpoR) on osteoblasts in vivo is still missing. Here, we created mice lacking EpoR in osteoprogenitor cells to specifically address this gap. Deletion of EpoR in osteoprogenitors (EpoR:Osx-cre, cKO) starting at 5 weeks of age did not alter red blood cell parameters but increased vertebral bone volume by 25% in 12-week-old female mice. This was associated with low bone turnover. Histological (osteoblast number, bone formation rate) and serum (P1NP, osteocalcin) bone formation parameters were all reduced, as were the number of osteoclasts and TRAP serum level. Differentiation of osteoblast precursors isolated from cKO versus control mice resulted in lower expression of osteoblast marker genes including Runx2, Alp, and Col1a1 on day 21, whereas the mineralization capacity was similar. Moreover, the RANKL/OPG ratio, which determines the osteoclast-supporting potential of osteoblasts, was substantially decreased by 50%. Similarly, coculturing cKO osteoblasts with control or cKO osteoclast precursors produced significantly fewer osteoclasts than coculture with control osteoblasts. Finally, exposing female mice to Epo pumps (10 U·d−1) for 4 weeks resulted in trabecular bone loss (−25%) and increased osteoclast numbers (1.7-fold) in control mice only, not in cKO mice. Our data show that EpoR in osteoprogenitors is essential in regulating osteoblast function and osteoblast-mediated osteoclastogenesis via the RANKL/OPG axis. Thus, osteogenic Epo/EpoR signaling controls bone mass maintenance and contributes to Epo-induced bone loss.

Diagnostic Performance of Erythropoietin and Erythropoietin Receptors Levels in Children with Attention Deficit Hyperactivity Disorder.

ObjectiveAttention deficit hyperactivity disorder (ADHD) is a heterogeneous, highly heritable, a common childhood neurobehavioural disorder resulting from complex gene-gene and gene-environment interactions. The erythropoietin (Epo)/erythropoietin receptors (EpoR) system turned out to have additional important functions in nonhematopoietic tissue. In this study, we aimed to investigate the levels of Epo and and EpoR, and also their diagnostic values in children with ADHD.MethodsA total of 70 children were included in the study, 35 drug-naive patients with ADHD (age 6−12 years; male/female 20/15) and 35 healthy controls (age 6−12 years; male/female 22/13). Serum Epo and EpoR levels was determined using a commercial sandwich enzyme-linked immunosorbent assay kit.ResultsThe results indicated that the levels of Epo decreased in patients with ADHD compared to control (p < 0.05). On the other hand, EpoR levels increased in these patients (p < 0.05). Furthermore, the ratio of Epo/EpoR was significantly lower in ADHD patients than controls (p < 0.05). Receiver operator characteristic curve analysis showed high diagnostic performance for Epo and EpoR, areas under curve were 0.980 and 1.000, respectively.ConclusionThis is the first report to investigate the association between serum Epo and EpoR levels in ADHD patients. Our results indicated that Epo may play a role in the etiology of ADHD, and Epo therapy may be beneficial in these disorders if given in addition to the routine treatment of children with ADHD. Furthermore, our results reveal possible diagnostic value of Epo and EpoR.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

The Impact of Diabetes Mellitus As Comorbid Condition, in Patients with Lower-Risk Myelodysplastic Syndromes (MDS)— a Study of the European Leukemianet-Based MDS (EUMDS) Registry

Background: Myelodysplastic syndrome is mainly a disease of the elderly, while the prevalence of Diabetes Mellitus (DM) in the aged population is about 25%.Aim: We sought to investigate the impact of DM as comorbid condition on the diagnosis of MDS.Patients and methods: We investigated the clinical and laboratory features at time of MDS diagnosis of 431 DM patients, occurring among a total of 2114 MDS patients registered in the EUMDS registry, and compared them with the non-DM patients. Data were collected by 145 centers located in 16 European countries and Israel.Results: The overall incidence of DM at registration visit was 23%. Higher frequency of DM was observed among MDS patients in Israel (40.3%), Portugal (26.3%) and the Netherlands (25.7%) and lower in Denmark (9.3%), Serbia (10.5%) and Romania (13.6%). Diabetic MDS patients were more commonly male (65.6% vs 60.2%, p=0.01), with higher frequency in Portugal (80%), United Kingdom (76.3%) and Greece (72.9%) and lower in Romania (33.3%), Germany (45.5%) and Israel (54.1%). Higher incidence of DM was encountered among patients with MDS-RA (24.3%, more prominently in the Netherlands, Israel and Spain) and lower among those with RCMD-RS (15.4%). Ring sideroblasts were less commonly encountered among DM patients (24.9% vs 29.5%, p: n.s.) and DM patients had a greater proportion of high MDS-CI risk than non-DM (9% vs 4.8%, p<0.001). The Performance Status (Karnofsky scale) was worse in DM than in non-DM patients (“able” persons 69.1% vs 79.4%, p<0.01). Moreover, DM patients were besides antidiabetic treatment more commonly receiving antiplatelet, anticoagulant and antihypertensive drugs, as well as proton pump inhibitors. No significant difference in the prognostic categorization distribution according to IPSS, WPSS or IPSS-R, between DM and non-DM patients was observed. DM patients were more anemic at diagnosis of MDS (Hb: 10.1±1.7 vs 10.3±1.9 g/dl, p=0.03), had lower MCV (95.5±9.6 vs 98.1±10.6 fl, p<0.01), higher WBC count (5.6±3.3 vs 5.0±3.0 x 109/l, p<0.001), and higher absolute neutrophil count (3.3±2.5 vs 2.8±2.2 x 109/l, p<0.001). No significant difference in baseline platelet counts and in the percentages of patients, exhibiting 0, 1, 2 or 3 cytopenias, between DM and non-DM patients was noted. However, DM patients had higher mean serum creatinine levels (102±65 vs 89±35 μmol/l, p<0.001) and among them there was a significant negative correlation between Hb and serum creatinine levels (r= -0.131, p=0.02). All three iron status parameters (serum iron, transferrin saturation and serum ferritin levels were significantly decreased among DM-patients, implying a possible influence of DM on iron metabolism. Serum EPO levels were not significantly different between the two groups, nevertheless, considering that diabetic MDS patients were more anemic, it could be assumed that they might exhibit somewhat blunted response, probably as a result of some degree of impairment of their renal function. Finally, patient-reported Quality of Life, using the EQ-5D tool was significantly lower among DM-, compared to non-DM patients (0.70±0.24 vs 0.75±0.22, p<0.001).Conclusions: Diabetes Mellitus may modulate the common presenting features of MDS, and since some degree of normocytic, normochromic anemia is commonly associated with this disease, special attention is warranted to discriminate the real MDS patients among the diabetic patient population. DisclosuresFenaux:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Sanz:Gamida Cell: Research Funding. Germing:Janssen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Almeida:Novartis: Consultancy; Celgene: Consultancy; Bristol Meyer Squibb: Honoraria; Alexion: Honoraria; Servier: Consultancy. Guerci-Bresler:BMS: Speakers Bureau; Incyte: Speakers Bureau; Pfizer: Speakers Bureau; Novartis: Speakers Bureau.

Dyserythropoiesis Evaluated By Red Score and Hepcidin/Ferritin Levels Predicts Response to Epoetin Zeta in Lower Risk MDS

▪INTRODUCTION: Erythropoiesis stimulating agents (ESA) are generally the first line of treatment of anemia in lower risk myelodysplastic syndrome (MDS) patients (pts) with a recent approval of epoetin alfa in this indication in EU. Predictive factors of response to ESA (E Hellström, BJH 2003, S Park et al, Blood 2008, V Santini et al, Blood 2013; O Kosmider et al., Haematologica 2016) include low RBC transfusion requirement, low serum EPO level, serum ferritin (SF) level<300ng/ml, low IPSS-R, and ≤2 somatic mutations. In a clinical trial using a biosimilar (epoetin zeta) in lower risk MDS pts, we investigated new biomarkers using flow cytometry (Ogata and Red Scores) (Mathis et al, Leukemia 2013), somatic mutations, GDF-15 and hepcidin levels.METHODS: Main inclusion criteria for this multicenter prospective trial involving 16 French centers from 2013 to 2017 were: (1) IPSS low or int-1 MDS (2) ESA naive, (3) with Hb level< 10g/dL, transfusion dependent (TD) or not (4) without unstable cardiovascular disease, renal failure, folates, vitamin B12 or iron deficiency or active inflammatory status. Pts received epoetin zeta 40000 IU/week for at least 12 weeks and erythroid response (HI-E, IWG 2006 criteria) was assessed at W12. sEPO level, iron homeostasis markers, Ogata and Red Score (RS), GDF15, hepcidin, and molecular analysis by NGS on a 39-gene panel were determined at baseline (T0), and Ogata and RS were reassessed at W12.RESULTS: 70 pts were included. Median age was 78 years (range 57-93), M/F: 31/39. At inclusion, WHO classification was 22 RCMD, 19 RCUD, 14 RARS, 4 RAEB, 6 CMML, 2 del 5q-, 3 unclassifiable, IPSS was 43 low (61.4%), 27 int-1 (38.5%), and R-IPSS was 13 very low (18.5%), 47 low (67%), 9 int (13%) and high (0.7%). Twenty (28.5%) pts were TD (median of 2 RBC/ 8weeks). ECOG was ≥1 in 30 pts.Median Hb level was 9.5g/dL (IQR 8.9-10), Transferrin Saturation (TS) 39% (IQR 28-56), SF 411 ng/ml (IQR 258-831), sEPO level 49 U/L (IQR 25-122). Median Ogata score was 1 (IQR 1-2) at T0 and 2 (IQR 1-2) at W12. Median Red score (RS) was 7 (IQR 5-7) at T0 (with 90% of pts having RS ≥3), and 5 (IQR 4.75-7) at W12.Median GDF15 level was 1971 pg/ml (IQR 1236-2860). GDF15 was lower in IPSS low vs int-1 pts, and correlated with TS (p<0.003). Hepcidin level was 27 ng/ml (IQR 14-42; normal 1.3-26.4). Median hepcidin/ferritin ratio (HFR) was 5.5 (IQR 1.9-11.85). HFR was inversely correlated with MCV, serum iron, TS, GDF15, and RS at T0 and at W12, and with the number of mutations.Before treatment, NGS was performed in 68 pts. Most frequent mutations involved SF3B1 (n=26), TET2 (n=18), ASXL1 (n=14), SRSF2 (n=9), DNMT3A (n=9), U2AF1 (n=7), IDH2 (n=6), and EZH2 (n=6). No, 1, 2, 3, 4 or 5 mutations were detected in 12, 12, 26, 10, 7 and 1 pts respectively, and 26% of pts had >2 mutations.The erythroid response (HI-E) rate was 47.6%. Median duration of response was 13 months. Three pts progressed to AML. Twelve pts had died. Median OS was 41 mo.Responders had significantly lower baseline RS (median 5 in responders vs 7 in non-responders, p=0.01), higher HFR (median 9 vs 4.8, p=0.04), lower IPSS (p=0.01). GDF15 tended to be lower in responders (1822 pg/mL) vs non-responders (2180 pg/mL; p=0.47). Age, sex, ECOG, blood cell count (ANC, platelets, Hemoglobin), number and type of mutations, IPSS-R, SF, sEPO level, T0 and W12 Ogata score, previous RBC transfusions, decrease of RS and of Ogata score between T0 and W12 were not significantly associated with response. In multivariate analysis, only RS≥5 at T0 was significantly associated with worse HI-E (p= 0.007, OR 19, 95% IC: 2.1-476). HI-E was 39% in pts with RS≥5 vs 75% in pts with RS<5.CONCLUSION: Erythroid response (HI-E) to epoetin zeta was 48% in lower risk MDS and was correlated with higher baseline hepcidin/ferritin ratio and lower Red score at T0. In multivariate analysis, only Red Score≥5 was associated with worse HI-E, in this population with very low median sEPO level. DisclosuresMaloisel:Pfizer/Hospira: Research Funding. Drenou:Alexion: Consultancy, Honoraria. Fenaux:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astex: Honoraria, Research Funding. Laribi:MUNDIPHARMA: Research Funding; NOVARTIS: Honoraria, Research Funding; ROCHE: Research Funding; TEVA: Research Funding; HOSPIRA: Research Funding; AMGEN: Honoraria; TAKEDA: Honoraria, Research Funding. Pegourie:Takeda, Novartis, Janssen, BMS: Consultancy. Wagner Ballon:Alexion: Consultancy, Honoraria. Park:Celgene: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira/Pfizer: Research Funding.

A Gain-of-Function Mutation in EPO Gene Causes Familial Erythrocytosis

Mutations in the erythropoietin-receptor gene (EPOR) are well known causes of inherited forms of primary erythrocytosis that is characterized by low serum erythropoietin (Epo) levels. Secondary erythrocytosis with elevated Epo serum levels is mainly caused by mutations in genes involved in oxygen sensing pathway. Epo is the primary regulator of erythropoiesis, but mutations in the EPO gene have so far not been described as a cause or erythrocytosis. Here we studied a Norwegian family with autosomal dominant erythrocytosis and elevated Epo serum levels.We performed genome-wide linkage analysis using SNP arrays on 16 informative family members including 10 affected individuals and identified a co-segregating region on chromosome 7q22.1 with a LOD score of 3.3. Targeted sequencing of all 215 genes within the co-segregating region revealed only a heterozygous single base deletion in exon 2 of EPO as the sole candidate gene mutation. ThisΔG deletion creates a frame-shift that truncates the Epo signal peptide and generates a novel peptide, terminating after additional 51 amino acids. Thus, contrary to the expectation based on the erythrocytosis phenotype, the EPO ΔG deletion was predicted to be a loss-of-function mutation.Since we had no access to patient's kidney or liver tissues where Epo is produced, we used the CRISPR technique to introduce the ΔG mutation into the EPO gene into Hep3B cells, a human hepatoma cell line that expresses EPO . We obtained two Hep3B single-cell derived clones (ΔG1 and ΔG4) that were homozygous for the EPO ΔG mutation and measured Epo protein in the culture supernatants. We found that these supernatants contained 8-10 times more Epo than parental Hep3B cells or controls, as determined by ELISA (Figure 1A), and were also capable of stimulating the growth of an Epo-dependent cell line (BaF3- EpoR), demonstrating biological activity (Figure 1B). Thus, the ΔG deletion introduced into the EPO gene locus in Hep3B cells behaved as a gain-of-function mutation.To decipher the mechanism of how the ΔG mutation was able to produce the excess of Epo protein, we searched for alternative EPO mRNAs by 5'-RACE. In addition to the expected transcripts originating from the physiologic promoter (P1) upstream of exon 1, we discovered two types of alternative mRNAs that initiate in intron 1 of EPO. Both initiatied from a putative alternative promoter (P2) in intron 1 that contains two GATA motifs. In addition to Hep3B ΔG clones, P2 transcripts were also detected in parental Hep3B cells and also in mRNA from normal kidney and liver.To determine which transcripts are capable of producing Epo, we transfected HEK293 cells with cDNAs representing P1 or P2 transcripts with or without the ΔG mutation. Supernatants of cells transfected with P2 ΔG transcripts contained more Epo than cells transfected with the wildtype P1 cDNA. These supernatants also stimulated the growth of Epo dependent BaF3- EpoR cells and supported erythroid colony formation of progenitors from human peripheral blood, demonstrating that P2 ΔG transcripts produce biologically active Epo protein.Analysis of the P2 mRNA sequences shows that the normally non-coding P2 transcripts through the ΔG single base deletion connect to the Epo coding sequence creating a functional signal peptide with a novel N-terminus, but retaining the physiologic Epo signal peptide cleavage site. These P2 ΔG transcripts are more abundant in liver and produce an excess of biologically active Epo by escaping the oxygen sensing regulation. Our data demonstrate for the first time mutation in EPO as a cause of familial erythrocytosis and provide an explanation of how the ΔG results in a gain-of-function mutation (Figure 1C).Figure 1 legend: A) Production of Epo protein by He3B cells carrying the ΔG mutation. Supernatants of parental Hep3B or CRISPR-modified Hep3B clones were collected and Epo protein was measured by Epo ELISA. The levels of Epo are given as a mean ± SD (n=3), p < 0.001 = *** as determined by one-way ANOVA. B) Biological activity of Epo produced by parental Hep3B or CRISPR-modified Hep3B cells measured by a proliferation assay with BaF3-EpoR cell line. BaF3-EpoR cells were grown in serial dilutions of supernatants and cell numbers were assessed by Cell Titer-Glo Assay, (n=3). Serial dilutions of 2.5 IU/ml of recombinant human Epo were used for reference. C) Model and proposed mechanism for EPO overproduction by a single nucleotide deletion in exon 2 of the EPO gene. [Display omitted] DisclosuresWaage:Celgene, Takeda: Honoraria. Skoda:Novartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Consultancy, Speakers Bureau.

MO560IS SERUM ERYTHROPOIETIN CORRELATED WITH IRON METABOLISM AND INFLAMMATION IN NON-DIALYSIS CHRONIC KIDNEY DISEASE PATIENTS WITH ANEMIA?

Abstract Background and Aims Both the relative erythropoietin (Epo) deficiency and its relationship with serum hemoglobin (Hb) are widely postulated in chronic kidney disease (CKD), but the influence of chronic inflammation and iron status on serum Epo levels is still a matter of debate, with yet divergent reported results. Therefore, we aimed to assess the determinants of serum Epo in non-dialysis CKD patients. Method Fifty-two adults with CKD and anemia (defined as Hb &amp;lt;12g/dL), in stable clinical condition, never treated with erythropoiesis-stimulating agents (ESA) entered this cross-sectional, single-center study. Diabetes mellitus, active infectious and inflammatory diseases, malignancy, anemia of other causes than CKD, current immunosuppressive therapy, iron supplementation and blood transfusions in the previous six months were exclusion criteria. The subjects were mostly men (56%), elderly (two thirds over 60 years), with advanced CKD [71% in CKD stages G4-G5, median estimated glomerular filtration rate – eGFR 14.5 (95%CI 16 to 25) mL/min], moderate anemia [Hb 9.8 (95%CI 9.2 to 9.9) g/dL], and mild to moderate inflammation [C-reactive protein 6 (95%CI 9.2 to 18.4) mg/L]. Serum Epo was assessed by ELISA (Abcam® 119522). Complete blood count, reticulocyte index, peripheral blood smear, bone marrow aspiration (Perls’ stain), serum ferritin, and transferrin saturation, were used to investigate anemia and iron metabolism. Parameters of kidney disease (CKD etiology, eGFR and proteinuria), demographic data (age, gender), C-reactive protein, serum albumin, and serum hepcidin-25 (Hep-25, Bachem® commercial ELISA kit) were also analyzed. Results The median serum Epo of the whole cohort was 4.8 (95%CI 5.1 to 9.9) mU/mL. According to median Epo, subjects were clustered in Group 1 (below median, G1) and Group 2 (above median, G2). Estimated GFR and serum Hep-25 were lower in G1 than in G2 [10.6 (95%CI 9.7 to 20.8) vs. 26 (95%CI 19.1 to 32.8) mL/min, p=0.004, and 62.6 (95%CI 51.0 to 85.1) vs. 95.4 (95%CI 77.0 to 108.5) ng/mL, p=0.03, respectively]. All the other investigated parameters were similar in the two groups. In bivariate analysis (Spearman rank correlation), serum Epo was positively associated only with eGFR (rs=0.40, p=0.003). Marginal associations with the percentage of bone marrow sideroblasts, as marker of the iron available for erythropoiesis (rs=0.25, p=0.08), erythrocyte mean corpuscular hemoglobin concentration (rs=−0.26, p=0.07), and reticulocyte index (rs=0.24, p=0.09) were observed. Conversely, serum Epo was not related to hemoglobin, indices of iron stores (e.g. serum ferritin and iron content in bone marrow macrophages), inflammation and nutritional status (e.g. C-reactive protein and serum albumin). In a model of multiple linear regression which explained 14% of serum Epo variation, eGFR was the only determinant: Beta 0.14 (95%CI 0.05 to 0.23), p=0.004. Also, a binary logistic multiple regression model predicting serum Epo lower or higher than the median retained the eGFR as an independent predictor, while serum hepcidin showed only borderline significance: Conclusion Kidney function is the main determinant of endogenous erythropoietin level in moderately anemic patients with advanced CKD, ESA or iron naive, while serum hepcidin-25 seems to exert a limited influence.

Secondary Erythrocytosis Is Phenotypically Distinct from Polycythemia Vera but Associated with Comparable Rates of Thrombosis at Diagnosis

Background: Since lowering the hemoglobin (Hb) and hematocrit (Hct) diagnostic thresholds for polycythemia vera (PV) to 165 g/L and 49% in men and 160 g/L and 48% in women respectively, by the World Health Organization (WHO) in 2016 (Blood. 2016;127:2391), hematologists have witnessed an increase in referrals for erythrocytosis, a significant proportion of which are found to have secondary erythrocytosis (SE). This has yielded novel challenges as comprehensive data on SE and reports comparing SE vs PV are scarce. Further, data regarding thrombosis in SE have been inconsistent (Clin Appl Thromb Hemost. 2013;19:363; J Clin Anesth. 1991;3:99; Ann Hematol. 2016;95:233). Methods: This multicentric retrospective study included PV patients enrolled in a pan-provincial registry of the chronic myeloid leukemia and myeloproliferative neoplasms Quebec Research Group (GQR LMC-NMP). SE patients were recruited from the Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada. PV diagnosis was per WHO 2016 criteria (Blood. 2016;127:2391). SE was defined as sustained elevation in Hb and/or Hct above WHO thresholds for PV with negative JAK2V617F testing and non subnormal serum erythropoietin (Epo) levels. Epo levels were drawn in therapy-naïve patients within 3 months of diagnosis using a standard immune-enzymatic assay (reference 3-30 mIU/mL). Endogenous erythroid colony assays used standard methods. JAK2 mutation screen and risk-stratification for PV were according to convention. Standard statistical methods were used to assess variables across i) SE and PV groups, and ii) subjects having experienced a thrombotic event vs no thrombosis. JMP® Pro 13.0.0 software was used for all analyses (SAS Institute, Cary, NC, USA). Results: Among 102 informative cases of erythrocytosis, 36 (35%) were SE and 66 (65%) were PV. Median age was significantly lower in the SE (57 years old; range 19-76) vs PV group (63.5 years old; range 20-89) (p=0.005), with a preponderance of males (75% in SE vs 45% PV; p=0.004). Median serum Epo levels were normal in the SE cohort (10.3 mIU/mL; range &amp;lt;1-148) vs subnormal in PV patients (2.3 mIU/mL; range &amp;lt;1-14.1) (p&amp;lt;0.0001). At diagnosis, SE vs PV patients had significantly lower baseline platelets (191 vs 417 x 109/L; range 125-476 vs 120-995), leukocyte counts (7.2 vs 10 x 109/L, range 4.1-15.1 vs 4.5-20.5) and lactate dehydrogenase (LDH) (182 vs 247 U/L, range 126-316 vs 157-874) (p&amp;lt;0.0001). Further, SE vs PV cohorts displayed less frequent palpable splenomegaly (6% vs 23%; p=0.02), higher median BMI (33.4 vs 26; p=0.02), and an excess of active smokers (31% vs 12%; p=0.03) (Table 1). The most prevalent etiology of erythrocytosis in the SE cohort was idiopathic, reported in 50% of cases (n=18), followed by smoking/chronic obstructive pulmonary disease in 22.2% (n=8), smoking plus additional factors in 8.3% (n=3), sleep apnea, polycystic kidney disease, or a combination of the latter two in 5.6% each (n=2 each) and post kidney transplant in 2.7% (n=1) (Table 1). Nearly as many SE (n=11, 31%) as PV patients (n=26, 39%) were exposed to bone marrow sampling (p=0.37) and SE patients were frequently managed with phlebotomy (42% vs 76% PV, p=0.0007) and aspirin therapy (56% vs 93% PV, p&amp;lt;0.0001) (Table 1). SE and PV populations had similar rates of thrombosis at/prior to diagnosis including events in 9 (25%) SE and 19 (29%) PV patients (p=0.68), both predominantly arterial (n=7 and n=16 respectively; p=0.58) (Table 2). History of thrombosis at any time in both groups clustered significantly with older age (p=0.001), hypertension (p&amp;lt;0.0001), diabetes (p=0.01), obesity (p=0.01) and hyperlipidemia (p&amp;lt;0.0001) (Table 3). Conclusions: SE patients are significantly younger, more likely to be male, active smokers, and obese, with normal-high Epo, normal LDH, and exceedingly rare leukocytosis and thrombocytosis, discriminating them phenotypically from PV patients. Importantly, rates of thromboembolic events prior to/at diagnosis are comparable in SE vs PV, suggesting that SE may not be benign from a thrombosis standpoint. Classic cardiovascular risk factors significantly cluster with thrombosis risk, emphasizing the importance of controlling these variables in both cohorts. Finally, a significant portion of SE patients are subject to bone marrow sampling and, though controversial, treatment with phlebotomy and aspirin, highlighting the need for formal studies to guide management in this population. Disclosures Szuber: Novartis: Honoraria. Busque:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Assouline:Takeda: Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding. Olney:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.