Primary Erythrocytosis Research Articles

A Unique Clinical Approach to Diagnose a Rare Case of Polycythemia

Primary familial and congenital polycythemia (PFCP) is a rare autosomal dominant disorder caused by hypersensitivity of erythropoetin receptor leading to increased rate of erythropoesis at any given serum erythropoetin level. Thrombosis contributes to severe morbidity and mortality in cases of primary erythrocytosis due to any cause. Primary erythrocytosis or autonomous production of excess erythrocytes occurs mostly due to myeloproliferative neoplasms like polycythemia vera (PV) but can rarely occur in familial conditions also. Even though it does not come under myeloproliferative neoplasm, it can still lead to severe morbidity and early mortality due to increased chances of thrombotic events. However, unlike polycythemia vera, leukemic transformation and myelofibrosis is not seen. Here, we present case of a 33 year old male who presented with both arterial and venous thrombosis and was then diagnosed as non – clonal primary erythrocytosis which was presumably familial as his son was also found to have erythrocytoisis. Diagnosing this fatal condition is essential ; as PFCP has autosomal dominant inheritance and early recognition in family can prevent further vascular events in other family members as well.

Extreme erythrocytosis in a cat reverts after short-term treatment with hydroxyurea and the cat remains healthy for years.

This case report describes a cat with severe erythrocytosis (Hct, 80%), which after initial treatment with hydroxyurea has gone into remission for over 3 years. A 1-year-old neutered male American Maine Coon crossbred cat. A 1-year-old neutered male American Maine Coon crossbred domestic cat was presented with acute neurologic signs, systolic heart murmur, and extreme erythrocytosis (Hct, 80%; normal interval, 30% to 48%). There were no clinical signs of dehydration, and several diagnostic tests for absolute erythrocytosis did not identify an underlying cause. A presumptive diagnosis of primary erythrocytosis (polycythemia vera [P vera], a myeloproliferative disease) was made. Repeated phlebotomies were declined by the owner, and thus the cat was treated with oral hydroxyurea. The neurologic signs, heart murmur, and erythrocytosis resolved within 2 months (Hct, 41%). Treatment with hydroxyurea was continued for 2 years and then discontinued. The Hct remained in the normal range (between 37% and 44%) during a 3-year observation period. This case illustrates the challenges of determining a precise cause of erythrocytosis. The extreme erythrocytosis reverted after treatment with hydroxyurea and did not recur even after drug withdrawal, suggesting an undefined singular or multifactorial cause of the erythrocytosis rather than a primary absolute erythrocytosis, such as P vera. The reversibility of this cat's erythrocytosis suggested that in select cases the discontinuation of treatment is warranted.

Whole Exome Sequencing Reveals Novel Variants in Unexplained Erythrocytosis.

Erythrocytosis is characterized by an increase in red cells in peripheral blood. Polycythemia vera, the commonest primary erythrocytosis, results from pathogenic variants in JAK2 in ∼98% of cases. Although some variants have been reported in JAK2-negative polycythemia, the causal genetic variants remain unidentified in ∼80% of cases. To discover genetic variants in unexplained erythrocytosis, we performed whole exome sequencing in 27 patients with JAK2-negative polycythemia after excluding the presence of any mutations in genes previously associated with erythrocytosis (EPOR, VHL, PHD2, EPAS1, HBA, and HBB). We found that the majority of patients (25/27) had variants in genes involved in epigenetic processes, including TET2 and ASXL1 or in genes related to hematopoietic signaling such as MPL and GFIB. Based on computational analysis, we believe that variants identified in 11 patients in this study could be pathogenic although functional studies will be required for confirmation. To our knowledge, this is the largest study reporting novel variants in individuals with unexplained erythrocytosis. Our results suggest that genes involved in epigenetic processes and hematopoietic signaling pathways are likely associated with unexplained erythrocytosis in individuals lacking JAK2 mutations. With very few previous studies targeting JAK2-negative polycythemia patients to identify underlying variants, this study opens a new avenue in evaluating and managing JAK2-negative polycythemia.

Polyneuropathy and primary erythrocytosis in a cat

AbstractA 22‐month‐old, male, neutered, domestic longhair cat presented with a 2‐month history of progressive paraparesis and severe muscle atrophy; neuromuscular dysfunction was suspected on neurological exam. A manual packed cell volume was 73%, and serial measurements following fluid therapy remained high. Investigations for secondary causes of erythrocytosis were unremarkable, so a diagnosis of primary erythrocytosis was made. Electromyography showed spontaneous activity within all tested muscles of the pelvic limbs. Motor nerve conduction velocity for the sciatic‐tibial nerve was prolonged, and the compound muscle action potential measured at the hip was reduced. Phlebotomy achieved a reduction of the packed cell volume to 52%, and oral hydroxyurea was started. An improvement in the degree of paraparesis was seen 3 weeks after discharge, and this continued until the cat was normal at 4 months. Fluctuations in the packed cell volume necessitated adjustments in hydroxyurea dosage, but no relapse of paraparesis occurred during the 18‐month follow‐up period.

Erythropoietin Levels in Patients with Polycythemia Vera and Secondary Erythrocytosis

BACKGROUND: Polycythemia Vera (PV) is a myeloproliferative neoplasm characterized by uncontrolled proliferation of erythrocytes with frequent co-stimulation of myeloid and megakaryocyte progenitor cells. Prior to the utilization of molecular testing, subnormal or low erythropoietin (EPO) levels were an important component in the diagnosis of PV. In 2005, the somatic JAK2 V617Fmutation was discovered to be present in greater than 95% of patients with PV. In turn, the 2008 WHO diagnostic criteria revision added the JAK2 mutation to the major criteria. The current WHO criteria for the diagnosis of PV designates the major criteria to be a hemoglobin greater than 16.5 g/dL in men and 16 g/dL in women (or a hematocrit greater than 49% in men and 48% in women), a bone marrow biopsy demonstrating hypercellularity and trilineage panmyelosis, and the presence of a JAK2 V617F or exon 12 mutation. A subnormal EPO level is a minor criterion. Based on these, PV can be diagnosed by the presence of all three major criteria, or the first two major criteria with a subnormal EPO level. Subnormal EPO levels have been reported to be found in only around 30% of patients otherwise meeting the diagnostic criteria for PV. The sensitivity and specificity of a subnormal EPO level in the diagnosis of PV has been shown to be 64% and 96%, respectively. The diagnostic value of the EPO level is confounded by the co-occurrence of comorbidities such as obstructive sleep apnea, chronic obstructive pulmonary disease, tobacco use, and obesity. As a result, controversies exist as to the role of EPO in the diagnosis of PV. In this study, we aimed to describe the EPO level in patients with primary erythrocytosis (or PV) and secondary erythrocytosis in a Los Angeles safety-net hospital where these comorbidities are prevalent. We also examined the prevalence of these comorbidities in both patient populations. METHODS: We performed a retrospective chart review of all patients aged greater than 18 years who presented to Harbor-UCLA Medical Center between 2015 and 2021 with the diagnosis of erythrocytosis using the International Classification of Disease-specific codes. We excluded patients who were erroneously given the diagnosis code for erythrocytosis when no erythrocytosis was documented or patients who had one documented hemoglobin level above the normal reference range with spontaneous resolution and no further work up. Patient demographics, baseline characteristics, laboratory values, bone marrow biopsy results, molecular testing, and comorbidities were recorded. RESULTS: There was a total of 141 patients who met the inclusion criteria (Table 1). Of these, 12 (8.5%) had the diagnosis of primary erythrocytosos or PV. Of those diagnosed with PV, all 12 (100%) had the JAK2 V617Fmutation. The average hemoglobin and hematocrit of patients with PV and secondary erythrocytosis was 17.8 g/dL and 54% and 18.1 g/dL and 57%, respectively. The average EPO for those with primary and secondary erythrocytosis was 3.4 mIU/mL and 23.7 mIU/mL, respectively (normal 2.6-18.5 mIU/mL). Of those with primary polycythemia, 6/11 (55%) had a subnormal EPO level at diagnosis. The average age of those with PV and secondary erythrocytosis was 69 years and 53 years, respectively. The average BMI for those with PV and erythrocytosis was 27.5 and 32.5, respectively. In those with secondary erythrocytosis versus PV, the most common comorbidities were pulmonary disease (58.3% versus 41.7%), smoking (36.7% versus 41.7%), heart disease (23.0% versus 25%), kidney disease (21.6% versus 25%), and testosterone use (5.0% versus 0%) (Table 2). CONCLUSIONS: In this study, we found that most patients with PV had a low EPO level at diagnosis with over half having a subnormal EPO level. In contrast, very few patients with secondary erythrocytosis had a subnormal EPO level. The prevalence of comorbidities was common in both primary and secondary erythrocytosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PB2251: TWO POLYCYTHEMIA VERA PATIENTS WITH A DIAGNOSIS OF CONCOMITANT HEREDITARY HEMOCHROMATOSIS

Background: Hereditary Hemochromatosis (HH) is an inherited iron metabolism disorder characterized by excessive iron accumulation in the liver, heart, pancreas and other endocrine organs. Although it is often associated with mutations in the HFE gene, it can also be caused by mutations associated with HJV, TFR-2 and ferroportin. JAK2 V617F (+) Polycythemia Vera (PV) is the most common cause of acquired primary erythrocytosis and has been reported very rarely in patients with HH. Aims: We presented two polycythemia vera patients with a diagnosis of concomitant hereditary hemochromatosis. Methods: Presence of HFE gene mutation and jak-2 V617F mutation was determined by PCR test Results: Case 1: N.G, a 67-year-old male patient with a history of hypertension, insulin-dependent diabetes mellitus and cerebrovascular accident was diagnosed with PV in another center in 2007. The patient was JAK2 V617F (+) and the tests performed in our laboratory after phlebotomies were EPO: 7.8 mIU/ml, ferritin: 34 ng/dl, transferrin saturation: 49%. Bone marrow aspiration biopsy revealed hypercellularity with erythroid and megakaryocytic hyperplasia. Although Hb/Hct levels were within the targeted range with hydroxyurea 500 mg 2x1 treatment, he was followed up with frequent phlebotomies due to persistent pruritus. After phlebotomy, there was a significant improvement in his pruritus. He was using clopidogrel as antiaggregant treatment. During follow-up, there was deterioration in blood glucose regulation and an increase in insulin requirement. In June 2018, he developed widespread hyperpigmentation on his skin. Due to unregulated diabetes, hyperpigmentation, and high ferritin levels despite frequent phlebotomy, he was examined for HH. He was found to be heterozygous positive for the HFE gene H63D mutation. The patient is still being followed up with HU 2X1 and phlebotomy without any problem. Case 2: E.H, a 57-year-old male patient with a history of hypertension and type 2 diabetes mellitus was diagnosed with PV in 2015. He was JAK2 V617F(+) and at diagnosis EPO:3.7 mIU/ml, ferritin:1403 ng/ml, transferrin saturation:63%. Bone marrow aspiration biopsy revealed hypercellularity and erythroid hyperplasia. Since he was a low-risk patient under the age of 60 and had no history of thrombosis, he was followed up with ASA 100 mg/day and intermittent phlebotomies. Similar to the first case, persistent pruritus was present despite the normal Hb/Hct level. Since hyperferritinemia was not expected in polycythemia vera and the patient was diabetic, he was examined for HH. In the HFE gene analysis, the H63D mutation was found to be heterozygous. Due to the frequent need for phlebotomy, the patient is considered to start treatment with hydroxyurea, and the follow-up still continues. Summary/Conclusion: The association of HH and PV has been rarely reported in the literature, and its clinical significance is not clearly known. In the presence of unexplained high ferritin levels in PV, even if the clinical findings are unclear, screening for HH may be recommended as an appropriate approach. The role of HFE genotypes in the development of myeloproliferative diseases is unknown. Clinical data accumulation is needed to elucidate iron metabolism and prognostic features in PV patients with HFE mutations. Keywords: polycythemia vera, hereditary hemochromatosis, JAK2 V617F, HFE mutation

New directions in the pathogenesis of primary erythrocytosis in IgAN.

Immunoglobulin A nephropathy (IgAN) is the most common biopsy-proven glomerulonephritis in the world.1 The disease is caused by reduced number of O-glycans in the hinge region of IgA1 (Gd-IgA1) and sequential formation of IgG auto-antibodies that link Gd-IgA1 forming circulating immune complexes that deposit in the mesangial area of glomeruli.2 IgAN may be associated with other diseases3 as well as idiopathic polycythemia/erythrocytosis observed in some IgAN patients referenced in Table 1.

Rs779805 Von Hippel-Lindau Gene Polymorphism Induced/Related Polycythemia Entity, Clinical Features, Cancer Association, and Familiar Characteristics.

Increased red blood cell count may result from primary erythrocytosis (polycythemia vera), but it is often due to secondary causes with increased erythropoietin levels. Secondary erythrocytosis may also be congenital due to different gene mutations of hemoglobin, hemoglobin stabilization proteins, EPO receptors, or oxygen sensing pathways. Von Hippel- Lindau gene mutation causes altered tissue oxygen sensation in VHL disease, usually with normal hemoglobin. Germline VHL mutations associate with classical VHL disease and represent genetic susceptibility for pheochromocytoma. VHL polymorphisms are mostly considered an innocent phenomenon. Still, some data indicate that these polymorphisms are not always harmless and can occur with prostate, renal, and colon cancer or even with isolated erythrocytosis. Seventy-eight patients referred to our department with elevated hemoglobin were screened for VHL mutations. There were no classical somatic VHL mutations. However, we found heterozygous (GA) or homozygous (AA) rs779805 VHL c.-195G>A polymorphism accompanied by erythrocytosis. These patients are Jak-2 negative, with normal or elevated EPO levels, sometimes with family accumulations and often phlebotomy needs, and in some cases with malignancies in the family. No other cause of erythrocytosis was found. We use phlebotomy regularly, and for those with cardiovascular risk factors, we recommend aspirin.

Causes of erythrocytosis and its impact as a risk factor for thrombosis according to etiology: experience in a referral center in Mexico City.

BackgroundThrombotic events are well documented in primary erythrocytosis, but it is uncertain if secondary etiologies increase the risk of thrombosis. This study aimed to determine the causes of erythrocytosis and to identify its impact as a risk factor for thrombosis.MethodsData were obtained from patients with erythrocytosis between 2000 and 2017 at a referral hospital in Mexico City. Erythrocytosis was defined according to the 2016 WHO classification. Time to thrombosis, major bleeding, or death were compared among groups of patients defined by the etiology of erythrocytosis using a Cox regression model, adjusting for cardiovascular risk factors.ResultsIn total, 330 patients with erythrocytosis were studied. The main etiologies of erythrocytosis were obstructive sleep apnea (OSA) in 29%, polycythemia vera (PV) in 18%, and chronic lung disease (CLD) in 9.4% of the patients. The incidence rate of thrombosis was significantly higher in patients with PV and CLD than that in patients with OSA (incidence rates of 4.51 and 6.24 vs. 1.46 cases per 100 person-years, P=0.009), as well as the mortality rate (mortality rates of 2.72 and 2.43 vs. 0.17 cases per 100 person-years, P=0.003).ConclusionThe risk of thrombosis in CLD with erythrocytosis was comparable to that in patients with PV. Further larger-scale studies are needed to confirm these findings and evaluate the benefits of preventive management of COPD with erythrocytosis similar to PV.

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.

Karakteristike bolesnika sa sekundarnom eritrocitozom u odnosu na bolesnike sa policitemijom verom

Introduction: Erythrocytosis represents elevated hemoglobin and hematocrit levels above the range of normal values. Primary erythrocytosis - polycythemia vera, is characterized by increased erythrocyte production, due to a disorder at the level of the multipotent stem cell in the bone marrow. On the other hand, secondary erythrocytosis (SE) is the result of bone marrow stimulation by an external factor. Aim: The aim of our study was to determine parameters which are significant in differentiating SE from primary erythrocytosis - polycythemia vera (PV). Materials and methods: This is a retrospective study involving 108 patients with SE and 111 patients with PV, who were diagnosed and treated at the Clinic of Hematology of the Clinical Center of Serbia (CCS), in the period: December 2005 - November 2018.From the patient records, the following data were extracted: demographic characteristics, laboratory parameters, spleen size, total red cell mass, serum erythropoietin (EPO) level, and spontaneous growth of the BFU-E colony. Results: Patients with SE were younger, with a predominance of the male gender and with significantly higher serum EPO values than patients with PV. Patients with PV had significantly higher values of BFU-E, leukocyte and platelet count, spleen size, and LDH level than patients with SE. Total red cell mass analysis did not show a differential diagnostic significance. Conclusion: Findings of normal spleen size, normal leukocyte and platelet count, normal serum LDH level, and elevated EPO, in patients, refer to the diagnosis of secondary erythrocytosis, while the findings of splenomegaly, leukocytosis, thrombocytosis, elevated serum LDH level, decreased EPO, and the presence of spontaneous BFU-E colony speak in favor of the diagnosis of polycythemia vera.

Protocolo diagnóstico y terapéutico de las poliglobulias

Polycythemia or erythrocytosis refers to an increase in hemoglobin and hematocrit concentration in peripheral blood. The erythopoietin, produced in 95% of renal cells, is in charge of erythropoiesis regulation due to hypoxia and is elevated in secondary erythrocytosis. The most important primary erythrocytosis is polycythemia vera, a chronic myeloproliferative neoplasm that produces erythrocytosis independently of erythropoietin due to a mutation in erythroid progenitor cells. A diagnosis is established based on an increase in hemoglobin above 16.5g/dl in men and 16g/dl in women. An etiological diagnosis is fundamental for an appropriate management of the patient.

Investigation and management of erythrocytosis.

KEY POINTS Erythrocytosis refers to an erythrocyte count above the sex-specific normal range and can be subclassified into relative erythrocytosis, caused by a reduction in plasma volume (hemoconcentration), or absolute erythrocytosis, caused by increased erythrocyte mass. Primary erythrocytosis

Molecular Analysis in Patients with Idiopathic Erythrocytosis

Background: Erythrocytosis corresponds to an increase in the red blood count, hemoglobin (Hb) concentration and hematocrit (Htc) above the reference range adjusted to age, sex and living altitude. Clinical and molecular data on patients suffering from primary erythrocytosis are sparse. Due to its rare incidence, primary erythrocytosis frequently represents a challenge for the diagnosis. The management of such patients is demanding as no clear standards of treatment have been defined. Objective: We aimed to analyze the clinical and molecular features of patients with idiopathic erythrocytosis at our clinic. Methods: We evaluate in our internal registry patients encoded as erythrocytosis (primary or secondary) which were currently in follow-up. All data was obtained from the clinical records of the patients. For patient selection, we focused on those in which a JAK2 determination was available. We identified 102 patients from them 78 patients were JAK2 positive and were excluded in this analysis. 24 patients were initially selected as possible idiopathic erythrocytosis, of which 8 patients were excluded due to possible secondary causes. Patients in whom the diagnosis of primary erythrocytosis was suspected were studied with a 13 genes NGS panel. The analyzed genes included EPOR (exons 7,8), VHL (orf including exon 1'), EGLN1, EPAS1, EPO (including several regulatory regions), JAK2 (exons 12-16), BPGM, HBB, HBA1, HBA2, HIF3A, OS9, SH2B3 (somatic). Only gene variants of the categories pathogenic, likely pathogenic and variant of unknown significance (VUS) according to ACMG guidelines 2015 are mentioned. Results: We identified 16 patients (15%) that did not fulfill the criteria for polycythemia vera nor had a secondary underlying cause of polycythemia and were diagnosed as idiopathic erythrocytosis. The median age was 32 years (r 20-76) and 81% (13/16) were males. The median Hb-value was 173 g/l (r 142-192), Htc 50% (r 43-65%). The median erythrocyte concentration was 5.87 G/l (r 5.18-6.57 G/l). No thromboembolic complications occurred in any of these patients either before or after diagnosis. Eleven patients (69%) were initially treated with venesection, which was interrupted during the follow-up except in two cases. The median follow-up in our clinic was 43 months (r 1 - 132). The median follow up since the interruption of venesection was 23 months (r 12-106). There was no evolution to leukemia, a myeloproliferative disorder or any kind of tumor. In the results of the molecular analysis we found that the EPO: c.-1306C&gt;A, rs1617640 reference allele was never homozygous. Indeed, all 16 patients showed an alternative allele: nine patients were heterozygous and 8 homozygous. EPO c.*772G&gt;T, rs551238 was similarly distributed: no patients homozygous for the reference allele, 8 heterozygous and 8 patients homozygous for the alternative allele. In 5 patients other gene variants were also detected: in two patients EGLN1: c.1088T&gt;G, p.(Leu363Arg) (the nucleotide is highly conserved, VUS, likely pathogenic), the other three were VHL: c.340+648T&gt;C (reported as quite frequent, so more likely VUS, but it is within the alternate exon 1 region described by Lenglet et al. thus further studies are recommended), EPO: c.-1206C&gt;A (not rare, VUS), EPO: c.*656G&gt;A (downstream variant, very rare, unknown significance) respectively. No other mutations were identified. Conclusion: In our clinic idiopathic erythrocytosis showed a population of patients predominantly constituted of young men, had an indolent evolution, neither presented with thrombotic complications before or after the diagnosis, nor with a hematological transformation. The high prevalence of EPO gene variant underlines the role of this association. The five patients with combined mutations did not show a different clinical pattern. The use of NGS to evaluate patients with unclear erythrocytosis might allow an improvement in clinical management avoiding unnecessary interventions. Disclosures Jalowiec: Novartis: Other: Travel grant; Amgen: Other: Travel grant; Pfizer: Other: Travel grant.

Dissecting Primary Erythrocytosis Among Polycythemia Patients Referred to an Indian Armed Forces Hospital.

Referrals for evaluation of polycythemia cases have increased since the hemoglobin (Hb) thresholds for diagnosis of Polycythemia Vera (PV) have been lowered by WHO. The current study enrolled patients of age > 18years from the Indian Armed Forces or their family members with polycythemia from November 2016 to October 2018. After exclusion of secondary causes, 49 patients were diagnosed as Primary Erythrocytosis (PE). The patients were classified into two groups: PV and Idiopathic Erythrocytosis (IE) and a systematic comparison of clinical and laboratory features of the two groups was done. The prevalence of PV in PE was 20.4% (10 of 49) while the rest 39 (79.6%) had IE. Seven PV patients had JAK2 V617F mutation, one had JAK2 Exon12 mutation, and two were JAK2 negative PV. Nine of 10 (90%) PV patients had Hb > 18.5g/dl, while only 21 of 39 (53.8%) IE patients had Hb > 18.5g/dl (p = 0.06). None of the JAK2 mutated patients had Hb < 18.5g/dl. We conclude that PV is more prevalent in patients of PE with Hb > 18.5g/dl. Most patients with Hb between 16.5-18.5g/dl would still be classified as IE. We advocate the need for further studies evaluating the utility of investigating all patients of PE with the revised WHO Hb threshold as well as studies on genetic profile of IE patients from India.

A guideline for the management of specific situations in polycythaemia vera and secondary erythrocytosis: A British Society for Haematology Guideline.

The previous BSH guideline for the management of erythrocytosis was published in 2005 (McMullin et al, 2005) and amended in 2007 (McMullin et al, 2007). Here, we re‐evaluate the literature formulate guidance on the management of specific situations encountered in polycythaemia vera (PV) and the management of the other types of secondary erythrocytosis. Recommendations for the diagnostic pathway of investigation of an erythrocytosis, risk stratification and management of PV are in the accompanying guideline (McMullin et al, 2018). We review evidence and outline guidance on management of acute thrombotic events and secondary prevention of thrombosis in PV. The unusual thrombotic events, splanchnic vein and cerebral vein thromboses are discussed and haemorrhage. The specific situations of surgery and pregnancy and guidance on management of pruritus are included. The evidence for the management of other causes of erythrocytosis, including idiopathic erythrocytosis, congenital erythrocytosis, hypoxic pulmonary disease and post‐transplant erythrocytosis, is reviewed and recommendations made.

Feline primary erythrocytosis: a multicentre case series of 18 cats.

A retrospective multicentre case series of feline primary erythrocytosis (PE) was evaluated. The aim was to gain better understanding of disease presentation and progression to guide management and prognostication. Case records were assessed for evidence of increased packed cell volume (PCV; >48%), sufficient investigation to rule out relative and secondary erythrocytosis, and follow-up data for at least 12 months or until death. Eighteen cats were included in the case series. No significant trends in signalment were noted. Seizures and mentation changes were the most common presenting signs (both n = 10). Median PCV was 70% (median total protein concentration of 76 g/l) with no other consistent haematological changes. Sixteen cats survived to discharge. Phlebotomy was performed initially in 15/16 surviving animals and performed after discharge in 10/16. Hydroxyurea was the most common adjunctive therapy, used in 10/16 cats. Of the 16 patients surviving to discharge, 14 patients were still alive at the conclusion of the study (survival time >17 months post-discharge), with the two non-survivors having lived for 5 years or more after diagnosis. PCV, when stabilised, did not correlate with resolution of clinical signs. In contrast to perceptions, feline PE was generally well managed via a combination of phlebotomy and medical therapy, with evidence of prolonged survival times. The use of hydroxyurea enabled cessation or repeat phlebotomies.

Retrospective Study of High Hemoglobin Levels in 56 Young Adults.

BackgroundErythrocytosis is a frequent request for consultation in the hematologic field. The diagnostic approach is well established in the general population but in a young adult, finding the etiology of erythrocytosis can be a real diagnostic challenge.MethodsThis is an observational retrospective unicentric study made at the Sherbrooke University Hospital Center, over a period of 20 years (1995 - 2015). Every patient aged between 16 and 35 years old with a significant elevation of hemoglobin or hematocrit was included (hemoglobin > 185 g/L and/or hematocrit > 0.52 in men; hemoglobin > 165 g/L and/or hematocrit > 0.48 in women).ResultsTotally, 426 patients met the inclusion criteria (over a total of 113,453 complete blood counts) but only 56 entered the study for investigations. The majority of patients were of male gender, 43% of the patients were obese, 59% were smokers and 38% used excess alcohol or recreational drugs. Twenty-five patients had the diagnosis of absolute erythrocytosis. Seven patients had the diagnosis of relative erythrocytosis and no cause could be identified in 24 patients. No primary erythrocytosis was found in this cohort. Among the 25 patients with secondary erythrocytosis, hypoxia was the most frequent etiology identified. Less than half of the patients in the cohort had long term follow-up. Search for JAK2 mutation and serum EPO dosage were performed in 17.9% and 23.2% of cases respectively. Seven patients were treated with aspirin and five patients had phlebotomies.ConclusionsThis retrospective study reveals an actual clinical management that is often discordant with the current recommendations and a frequent lack of follow-up after initial investigations. Harmonization of management of erythrocytosis appears to be highly desirable.

New pathogenic mechanisms induced by germline erythropoietin receptor mutations in primary erythrocytosis.

Primary familial and congenital polycythemia is characterized by erythropoietin hypersensitivity of erythroid progenitors due to germline nonsense or frameshift mutations in the erythropoietin receptor gene. All mutations so far described lead to the truncation of the C-terminal receptor sequence that contains negative regulatory domains. Their removal is presented as sufficient to cause the erythropoietin hypersensitivity phenotype. Here we provide evidence for a new mechanism whereby the presence of novel sequences generated by frameshift mutations is required for the phenotype rather than just extensive truncation resulting from nonsense mutations. We show that the erythropoietin hypersensitivity induced by a new erythropoietin receptor mutant, p.Gln434Profs*11, could not be explained by the loss of negative signaling and of the internalization domains, but rather by the appearance of a new C-terminal tail. The latter, by increasing erythropoietin receptor dimerization, stability and cell-surface localization, causes pre-activation of erythropoietin receptor and JAK2, constitutive signaling and hypersensitivity to erythropoietin. Similar results were obtained with another mutant, p.Pro438Metfs*6, which shares the same last five amino acid residues (MDTVP) with erythropoietin receptor p.Gln434Profs*11, confirming the involvement of the new peptide sequence in the erythropoietin hypersensitivity phenotype. These results suggest a new mechanism that might be common to erythropoietin receptor frameshift mutations. In summary, we show that primary familial and congenital polycythemia is more complex than expected since distinct mechanisms are involved in the erythropoietin hypersensitivity phenotype, according to the type of erythropoietin receptor mutation.

EPO Signaling Triggers Erythrocytosis By Expanding Erythrocytes and Also Subsets of Macrophages

Activated EPO receptor (EPOR) signaling (EPO-EPOR-JAK2-STATs) leads to erythrocytosis, which is defined as an absolute increase in red cell mass (RCM). Erythrocytosis is classified as primary versus secondary. Primary erythrocytosis is defined by erythroid cell-intrinsic EPO-independent mechanism due to constitutively activated EPOR signaling by gain-of function EPOR or JAK2mutations. Hypoxia inducible factor-2α (HIF-2α) is a master regulator of EPO gene expression. A defect in the oxygen-sensing VHL-PHD2-HIF-2α signaling pathway due to stress conditions or mutations leads to excess EPO production and secondary erythrocytosis (erythroid cell extrinsic EPO-dependent mechanism). It has also been suggested that a subset of macrophages play a critical role in the late stage erythropoiesis as an important erythroid cell-extrinsic factor. The role of EPOR signaling for these macrophages in the erythrocytosis context has not been fully understood.To determine the intrinsic or extrinsic regulationof erythrocytosis in vivo, we used two different mouse models of both primary and secondary erythrocytosis. We used JAK2 V617F mutant knock-in heterozygous mice (Jak2V617F mice) as a primary erythrocytosis model. To model the EPO-induced secondary erythrocytosis, we generated a liver-specific inducible human HIF-2αdouble-point-mutant (DPM) (P531A and N847A) transgenic mouse. Using liver specific Alb-Cre and Rosa26-loxP-Stop-loxP (LSL) rtTA driver, stable HIF-2α protein can be induced in a doxycycline dependent manner. We used wild-type mice for the control.Both Jak2V617F mice and doxycycline-treated Alb-Cre/LSL-rtTA-GFP/DPM (Alb-Cre/DPM) mice quickly developed a similar degree of erythrocytosis (control; Hb, 13.0 ± 1.8 g/dl; Ht, 46.2 ± 7.3%: Jak2V617F mice; Hb, 22.0 ± 0.7 g/dl; Ht, 79.7 ± 3.3%: Alb-Cre/DPM mice; Hb, 20.7 ± 2.2 g/dl; Ht, 71.4 ± 8.1%) and splenomegaly. The plasma EPO level in Jak2V617F mice was significantly decreased, while the level of EPO in Alb-Cre/DPM mice was significantly increased in comparison to the control. While the two mouse models developed similar degrees of erythrocytosis, EPO-dependent early erythroid precursors were dramatically increased in the bone marrow (BM) and spleen (SP) from Jak2V617F mice, but only modestly increased in Alb-Cre/DPM mice. This suggests that the sensitivity of the EPO-EPOR signaling pathway in erythroid cells could differ depending on whether activation is cell-intrinsic or extrinsic, and a non-erythroid population may cooperate with erythroid cells to achieve a similar degree of erythrocytosis.CD169+Vcam1+ macrophages have been shown to play a critical role in stress erythropoiesis. Thus, we analyzed the macrophage population in BM and SP. Although the frequencies of total BM macrophages did not change, CD169+Vcam1+ subpopulation, as well as total BM erythrocytes, was significantly increased in the BM macrophage population from Alb-Cre/DPM mice compared to those from the control or Jak2V617F mice. The frequencies of total SP macrophages, CD169+Vcam1+ SP macrophages, and total SP erythrocytes from both Jak2V617F mice and Alb-Cre/DPM mice were increased compared to those from the control. Notably, the increase of CD169+Vcam1+ subpopulation in the SP macrophages from Alb-Cre/DPM mice was prominent. Our results indicate that Jak2V617F could mediate cell-intrinsic activation of EPOR signaling in macrophages and induce CD169+Vcam1+ population primarily in SP but not in BM; while excessive EPO could mediate cell-extrinsic activation of EPOR signaling in macrophages and induce CD169+Vcam1+ population in both BM and SP.Taken together, using primary and secondary erythrocytosis models, we demonstrated that; 1) Jak2V617F and highly elevated EPO expression could activate EPOR signaling intrinsically or extrinsically, respectively, in both erythrocytes and macrophages, 2) both erythrocytes and macrophages cooperate to achieve an absolute increase in RCM and this could be the common underlying mechanism for erythrocytosis, 3) Extrinsic EPOR activation due to high EPO appears to induce CD169+Vcam1+ macrophages more potently than intrinsic EPOR activation in both BM and SP. Further studies are needed to understand the mechanism of how EPOR signaling induces this macrophage subpopulation, which could be a potential common therapeutic target for both primary and secondary erythrocytosis besides JAK2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.