Congenital Polycythemia Research Articles

Von Hippel- Lindau syndrome-related congenital polycythemia and response to belzutifan.

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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.

Familial Polycythemia Associated with JAK2 Mutation Previously Known As Variant of Unknown Significance

Introduction Mutations in the Janus Kinase 2 gene (JAK2) are commonly encountered in patients with myeloproliferative neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia. JAK2 mutations particularly V617F is associated with PV in 98% cases. Gain of function mutations in the JAK2 exon 12 are reported to be associated with isolated erythropoiesis. Atypical mutations in JAK2 including (c.3323A>G, p.N1108S) were reported in patients with MPN who tested negative for the established driver mutations including exon 14 JAK 2, exon 9 CALR and exon 10 MPL genes. Primary familial congenital polycythemia (PFCP) is a rare disease inherited in an autosomal dominant fashion caused by hypersensitivity of EPO receptor. In such cases, an EPO receptor mutation is only found in 12-15% of affected patients. Due to associated increased risk of thrombosis, bleeding, leukemic involvement and myelofibrosis in PV, bone marrow evaluation is essential in this patient population. In patients with polycythemia, the goals of treatment are to maintain appropriate level of hemoglobin/hematocrit and to reduce severity of symptoms. We present a family affected by polycythemia and leukocytosis associated with an atypical heterozygous variant of JAK2 [c.3323A>G (p.Asn1108Ser)] currently reported as benign or variant of unknown significance (VUS) in literature. Methods Case series, retrospective electronic medical record review with institutional board review exemption Results We report a case series of three Caucasian family members affected with JAK2 VUS mutation (mother and two sons). The proband is a 17-year-old male who presented to an outside facility at the age of 14 with complaints of fatigue, headache, and intense pruritis after hot showers. Initial workup was significant for marked erythrocytosis and reticulocytosis without evidence of abnormal cells on peripheral film. Bone marrow evaluation showed trilineage hematopoiesis with no morphological features of a myeloproliferative neoplasm. Genetic testing was negative for JAK2 V617F and exon 12 mutations. However, whole exome sequencing (WES) revealed a heterozygous mutation of JAK2 gene, c.3323A>G (p.Asn1108Ser). Additional investigation on proband's younger brother, who complained of similar symptoms, also revealed the same JAK2 variant. The proband's mother reported non-specific symptoms including headache, flushing, and occasional blurry vision that she previously attributed to hypertension. Her labs were significant for leukocytosis, monocytosis and thrombocytosis in the presence of normal red blood cell count and WES showed the same mutation. Bone marrow evaluation of the brother and the mother showed normal trilineage hematopoiesis and was negative for malignancy (Table 1). Proband's father is reportedly negative for the aforesaid mutation and is clinically asymptomatic. The proband and his brother are currently managed with phlebotomy every 3 to 4 weeks to maintain hematocrit below 45%, leading to improvement in symptoms. Cytoreductive therapies have been discussed but not initiated. Proband's mother is managed by an adult hematologist. Scheduled phlebotomies with close surveillance is recommended for clinical practice along with interval research sequencing. Conclusion Our case series reports a family with clinical features and laboratories suggestive of a familial form of polycythemia, WES detected the presence of an atypical JAK2 mutation: c.3323A>G, (p.Asn1108Ser). The phenotypical and clinical significance of this mutation has been considered non canonical per reports and classified as likely benign by reputable commercial genetic testing laboratories. However, other reports identify this JAK2 variant as having a significant increased prevalence in patients with MPNs and AML compared to the general population. It may result in a gain of function of the JAK2 protein and subsequent development of MPNs. Enrollment in research sequencing protocols is encouraged in such patients to closely follow the pathologic potential and spectrum of genetic variants rarely reported in literature. Additional research to determine the pathogenicity of this genetic variant is highly warranted.

Real-World Experience of Ropeginterferon-Alfa Treatment of PV and ET - Two Centers Experience

BACKGROUND AND AIM: Interferon alpha is an effective treatment for Polycythemia Vera (PV) and Essential Thrombocythemia (ET) and is the only treatment known to induce sustained molecular responses by decreasing JAK2 mutation allelic burden. Long-acting pegylated interferons are better tolerated. Ropeginterferon-alfa is a monopegylated interferon with a longer half-life and is the first interferon to be FDA-approved for the treatment of PV. It is not yet approved for ET, but clinical trials are ongoing. Given the similar disease biology between PV and ET and the efficacy of pegylated interferon-alfa in both PV and ET, this study aims to look at a real-world experience of treating PV and ET with ropeginterferon-alfa since its approval. METHODS: We conducted a retrospective chart review of all our PV and ET patients treated with ropeginterferon-alfa at Huntsman Cancer Institute, University of Utah (SLC cohort), and at the University of North Carolina at Chapel Hill (UNC cohort), since the drug's approval for PV from November 2021 through June 2023. The complete hematologic response (CHR) was defined as the normalization of blood counts within the reference range of the local lab standards. Prior to ropeginterferon-alfa, the participants were managed with therapeutic phlebotomies, or cytoreduced with either hydroxyurea, pegylated interferon-alpha 2a ( Pegasys), JAK2 inhibitors, or were newly diagnosed and previously untreated. RESULTS: A total of 120 patients (76 in SLC cohort and 44 in UNC cohort) were included in this study. Of those, 75 (62.5%) were diagnosed with PV, 37 (31%) with ET, and 1 with congenital polycythemia due to germline JAK2 mutation. Twenty-three (19%) patients had a prior history of thrombosis. JAK2 mutation was present in 100 (83%) patients, while 9 (7.5%) were CALR mutated, 1 with MPL mutation, and 1 with triple-negative ET. Extended myeloid gene panel sequencing was available in 64 (53.3%) patients and 35 of them (54.6%) patients had additional somatic mutations. Most common somatic mutations include TET2 (11 patients) and DNMT3A (8 patients) and CBL (3 patients), among others. Of the cohort, 15 (12%) patients discontinued ropeginterferon-alfa due to various adverse events, which included: fatigue, joint aches, skin rash, and increased anxiety and depression. Of the 105 patients who are on ropeginterferon-alfa, 9 patients were previously untreated patients while 96 (91.4%) were pretreated with other therapies and switched to ropeginterferon-alfa. In many patients, the switch to ropeginterferon-alfa was done because of the increased frequency and aggressivity of squamous cell skin cancers while on hydroxyurea treatment. All of them started ropeginterferon-alfa at a dose of either 50 mcg or 100 mcg every two weeks and were increased at an increment of 50 mcg to achieve a goal of CHR. As most of the patients were switching from prior therapies, 51 (49%) were already in CHR at the time of initiating ropeginterferon-alfa. The median ropeginterferon-alfa dose to maintain CHR was 50mcg in the SLC cohort and 100mcg in the UNC cohort. (See table for more details). We have 6 patients with DNMT3A mutation, 4 (66.6%) have achieved CHR at a median ropeginterferon-alfa dose of 100 mcg (range 50 mcg - 150 mcg), and 2 patients are undergoing dose titration. Of the 11 patients with TET2 mutation, 7 (63.3%) achieved CHR at a median ropeginterferon-alfa dose of 150 mcg (range 50 mcg - 500 mcg). The analysis of JAK2 V617F and CALR allelic burden, transcripts of inflammatory, prothrombotic, and hypoxia-regulated (HIFs) genes, and evaluation of females for reversal of clonal to polyclonal myelopoiesis is ongoing. CONCLUSION: Ropeginterferon-alfa is effective in PV and ET, however, the dose that achieves CHR is highly individualized, necessitating an incremental titration approach. The patients already in CHR from prior therapy needed relatively lower doses of ropeginterferon-alfa to maintain CHR. Clinical trials are ongoing with an alternate accelerated dosing scheme and results are awaited.

Abstract IA013: A zebrafish model for VHL and hypoxia signaling

Abstract he von Hippel-Lindau (VHL) tumor suppressor gene encodes an adaptor protein that regulates an array of transcription-dependent and -independent cellular and physiological processes. Mutations in this gene cause VHL disease, congenital polycythemia, renal cell carcinoma (RCC), and several other sporadic tumor types. Although different animal models for VHL disease have been adapted, the zebrafish VHL model offers distinct opportunities. Zebrafish vhl mutants develop key aspects of the human disease condition, including activation of the hypoxia-inducible factor (HIF) signaling pathway, polycythemia, excessive neovascularization, macular edema, and pronephric abnormalities resembling early stages of RCC. The zebrafish vhl model offers a platform for the identification of genetic pathways, modifiers, and interactors involved in the development of VHL-associated neoplasms. Vhl mutants represent a unique and clinically relevant in vivo model for studying genotype-phenotype correlations and the identification of prognostic biomarkers. The amenability of zebrafish for chemical genetic screens will not only be helpful to identify novel therapeutic agents but may also reveal novel processes that require regulation by VHL. Citation Format: Ellen van Rooijien, Stefan Schulte-Merker, Ive Logister, Emile Voest, Rachel Giles. A zebrafish model for VHL and hypoxia signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr IA013.

Elevated erythroferrone distinguishes erythrocytosis with inherited defects in oxygen-sensing pathway from primary familial and congenital polycythaemia.

Congenital erythrocytoses represent a heterogenous group of rare defects of erythropoiesis characterized by elevated erythrocyte mass. We performed molecular-genetic analysis of 21 Czech patients with congenital erythrocytosis and assessed the mutual link between chronic erythrocyte overproduction and iron homoeostasis. Causative mutations in erythropoietin receptor (EPOR), hypoxia-inducible factor 2 alpha (HIF2A) or Von Hippel-Lindau (VHL) genes were detected in nine patients, including a novel p.A421Cfs*4 EPOR and a homozygous intronic c.340+770T>C VHL mutation. The association and possible cooperation of five identified missense germline EPOR or Janus kinase 2 (JAK2) variants with other genetic/non-genetic factors in erythrocytosis manifestation may involve variants of Piezo-type mechanosensitive ion channel component 1 (PIEZO1) or Ten-eleven translocation 2 (TET2), but this requires further research. In two families, hepcidin levels appeared to prevent or promote phenotypic expression of the disease. No major contribution of heterozygous haemochromatosis gene (HFE) mutations to the erythrocytic phenotype or hepcidin levels was observed in our cohort. VHL- and HIF2A-mutant erythrocytosis showed increased erythroferrone and suppressed hepcidin, whereas no overproduction of erythroferrone was detected in other patients regardless of molecular defect, age or therapy. Understanding the interplay between iron metabolism and erythropoiesis in different subgroups of congenital erythrocytosis may improve current treatment options.

Expression of Jak2 R1063H Leads to Altered Epor/Tpor Signaling, Changes in HSC Fitness and Represents Increased Risk of Thrombosis

Non-canonical weakly activating germline JAK2 variants may predispose progenitor cells to acquire JAK2 V617F during the evolution of myeloproliferative neoplasms (MPN) (Lanikova et al. Blood 2016;128(18):2266) and represent risk factors for MPN transformation into AML (Benton et al. Cancer 2019;125:1855). We have previously described and functionally characterized the JAK2 R1063H germline variant, cooperating with the JAK2 E846D variant in a case of hereditary MPN with erythrocytosis and megakaryocytic atypia (Kapralova et al. Blood 2016;128:1418) or with the acquired JAK2 V617F causing increased JAK2 signaling and higher disease severity in patients with essential thrombocythemia (Mambet et al. Blood 2018;132:2695). The mouse model bearing Jak2 R1063H mutation was created using microinjection of synthesized ssODNs containing the R1063H mutation, sgRNA mRNA and Cas9 protein into C57BL6/N-derived zygotes. In order to intensify a possible effect of the mutation, we bred and analyzed the knock-in Jak2 R1063H homozygous mice. The mice are born in normal (expected) frequency and display increased platelets (PLT) counts when compared to wild-type (wt) mice during life-span. Despite increased PLT counts, the plasma thrombopoietin level is normal whereas erythropoietin levels are increased without marked erythrocytosis. The preliminary co-immunoprecipitation experiments with human erythropoietin and thrombopoietin receptors (EPOR/TPOR) and JAK2 variants suggest increased TPOR and decreased EPOR binding with JAK2 R1063H kinase, possibly explaining the hematological phenotype. Also experiments with humanized mouse model of EPOR (Divoky et al. Proc Natl Acad Sci USA 2001;98:986) crossed with Jak2 R1063H mice supported these findings, where anemia due to hypoactivation of wt human EPOR signaling is more severe in Jak2 R1063H mice than in a control group and mild thrombocytosis is still present in the double mutant mice. The Jak2 R1063H model displays increased number of long-term hematopoietic stem cells (HSC) suggesting increased cycling in this population at young age (3 months) and shift to increased short-term HSC and multipotent progenitors with aging (12 months). The immunophenotypically-defined myeloid progenitor cells (LK, Lin-/Sca1-/cKithigh) are statistically increased in Jak2 R1063H mice due to disproportionately increased megakaryocytic/erythroid progenitors (MEP) over other myeloid progenitors at 3 months of age. The LK compartment is compromised in old Jak2 R1063H mice due to substantial myeloid bias by disproportionately increased CMP cells and marked decreased MEP cells, explaining the more pronounced anemia of aging in these mice. Some of these features are reminiscent of aging-associated myeloid and megakaryocytic bias, increased LKs, and relative exhaustion of progenitors with an erythroid lineage-priming program in aged bone marrow (BM) of a mouse model of primary familial and congenital polycythemia (PFCP) due to gain-of-function EPOR (Kralova et al, Am J Hematol 2022; doi: 10.1002/ajh.26658). However, the gain-of-function EPOR-introduced changes into the BM microenvironment are not associated with augmented hypoxia signaling and systemic inflammation and do not represent permanent challenges that would contribute to an increased incidence of myeloid malignancies in PFCP. In this regard, it will be interesting to compare these characteristics with the possible predisposing potential of Jak2 R1063H; further analysis of proliferative history of Jak2 R1063H HSC populations and expression profiling are ongoing. In the cohort of Jak2 R1063H mice, we also observed a relatively high frequency of sudden death in mice with median overall survival of 215 days (5/15) in comparison with the control group (0/15). We therefore measured the levels of D-dimers as a marker of thrombosis and found it statistically increased in the Jak2 R1063H group. Interestingly, in a WES study of patients with venous thromboembolism, the JAK2 R1063H was identified in one case, being considered a probable disease-causing variant (Lee at el. Blood Adv 2017;1:1224). In conclusion, our results confirm a direct effect of germline Jak2 R1063H mutation on EpoR/TpoR signaling but also on HSC biology, suggesting that the variant may represent a risk factor for BM remodeling in the process of myeloid cell transformation. Grant support: Czech Health Research Council NU21/03/00338.

PSUN375 What Blindness and Paraganglioma Have in Common? A Rare Case of Paraganglioma Syndrome

Abstract Background Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors with an annual incidence in the U.S. of 500-1600 cases per year. Approximately 30-35% of PPGLs harbor germline mutations, while 35-40% are due to somatic mutations. Recent studies have identified the association between somatic mutations of the hypoxia-inducible factor 2 alpha (HIF2A) gene and a new syndrome of PPGLs associated with congenital polycythemia. Patients present with recurrent PPGLs, congenital malformation of intracranial veins, malformation of macula and retina, and type I Chiari malformations. Case A 44-year-old man with history of in-utero stroke, congenital blindness, polycythemia and spinal stenosis had an incidental para-aortic lesion found during workup of back pain. He experienced episodic palpitations associated with diaphoresis, headache and uncontrolled hypertension for the past eight year. There was no family history of neuroendocrine tumors. Dedicated abdominal computer tomography with contrast showed a right adrenal enhancing lesion measuring 1.4×1.2 cm and a conglomerate of heterogenous enhancing periaortic lesions measuring up to 5 cm in the mid-abdomen. Biochemical work up revealed plasma free normetanephrine of 27.5 nmol/L (0.00-0.89) and plasma free metanephrines of 0.49 nmol/L (0.00-0.49). Gallium 68 DOTATATE scan showed multiple avid retroperitoneal lesions measuring up to 4.4 cm and an avid right adrenal nodule. He underwent right adrenalectomy and para-aortic lesion resection with perio-operative alpha blockade. Pathology confirmed synchronous pheochromocytoma (1.5 cm, Ki-67< 1%, with capsular and periadrenal fat invasion) and paraganglioma (9.3 cm, Ki-67 < 1%, with lymphovascular invasion and one out of nine lymph nodes positive for metastatic disease). Post-operatively, his metanephrines normalized and a six-months positron emission tomography scan showed no evidence of disease. Genetic testing has been delayed. Discussion This case illustrates a rare syndrome of PPGLs, congenital blindness, and polycythemia, likely due to somatic mutations of HIF2a. HIF is an alpha/beta heterodimer. HIF2a becomes a transcription factor after dimerization with HIF2b. Any mutations that stabilize HIF protein leads to transcription of hypoxia inducible genes resulting in angiogenesis, polycythemia, and tumorigenesis. In our patient, we suspect the stroke in-utero may have been related to abnormal intracranial angiogenesis and/or polycythemia. Recognition of this syndrome and lifelong surveillance is imperative due to risk of recurrence of PPGL. Targeted therapies such as a HIF2a inhibitor have been proposed and trials are ongoing. Our patient remains disease free on radiographic and biochemical surveillance. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Characterization of Iron Metabolism in Mouse Model of Congenital Polycythemia with Human Gain-of-Function Erythropoietin Receptor

Hepcidin (encoded by HAMP), the peptide hormone regulating systemic iron homeostasis, controls iron flux to plasma and thus ensures adequate iron supply for erythropoiesis. Increased erythropoietic activity negatively regulates hepcidin synthesis by the hormone erythroferrone (ERFE) produced by erythroid precursors in response to erythropoietin (EPO). Here we evaluated iron metabolism in a mouse model of polycythemia with augmented erythropoiesis and low EPO levels bearing the gain-of-function polycythemia-causing human EPO receptor gene (mtHEPOR) (Divoky PNAS 2002). The mtHEPOR embryos developed polycythemia around embryonic day (ED) 17.5, followed by unexpected but transient correction of polycythemia in perinatal life (postnatal day 7 - PD7) and reappearance of a polycythemic phenotype at 3-6 weeks of age. (Human patients with the gain-of-function polycythemia-causing human EPOR mutations are also born non-polycythemic and then develop polycythemia within a few weeks of neonatal life). The mtHEPOR mice had reduced Epo levels, augmented EPOR signaling and prolonged activation of Stat5 in erythroblasts when compared to mEpoR mice (Divoky JMolMed 2016). To assess whether developmental changes of erythron in this mouse model correlate with changes in iron metabolism, we determined temporal changes of Erfe and Hamp expression and body iron stores.We found that mtHEPOR mice have increased expression of Erfe at ED 17.5 (7.5-times) and PD7 (4-times) compared to mEpoR controls when normalized to a housekeeping gene beta-actin and independently to erythroid marker glycophorin A. Therefore Erfe in mtHEPOR mice would be increased both because of a higher number of erythroid precursors and because each precursor is producing more Erfe by increased transcription. This corresponds to reduced expression of Hamp in the liver of ED17.5 (4-times) and PD7 mtHEPOR mice (15-times) compared to mEpoR littermates. However, perinatally (PD1), Erfe expression dramatically decreased in mtHEPOR mice (7-times) concomitantly with increased Hamp expression (45-times), and both these factors reached expression levels detected in mEpoR at PD1. Expression of Erfe and Hamp between mtHEPOR and mEpoR mice was also not different in older mice (7 months of age), even though mtHEPOR mice were markedly polycythemic with persistent erythropoietic activity, markedly increased splenic erythropoiesis (the weight of spleens of control animals was 100-180 mg compared to 200-350 mg of mtHEPOR mice), and augmented EPOR-Stat5 signaling. However, mtHEPOR and mEpoR mice differed in transferrin saturation (TSAT) and tissue iron stores. The spleens of mtHEPOR mice had markedly increased iron deposits compared to mEpoR controls and stainable iron could only be detected in the livers of mtHEPOR mice but not in the livers of mEpoR controls. TSAT was elevated in mtHEPOR mice (77±3.2%) when compared to mEpoR (54.7±8.0%), consistent with the iron overload in mtHEPOR mice. Nevertheless, Hamp expression was comparable between mtHEPOR and mEpoR mice. This shows that Hamp expression was unexpectedly normal in respect to the presence of iron overload in mtHEPOR mice at 7 months of age.With further aging (mice 14 months and older), Erfe continued to increase and Hamp continued to decrease in mEpoR mice. In contrast, there was a trend towards decreased Erfe expression while Hamp expression became significantly elevated (2-times) in mtHEPOR mice. These changes were accompanied by a 2.4-fold decline in TSAT in both genotypes, which suggests that aged mEpoR mice develop iron deficiency while mtHEPOR mice remain iron-replete likely due to mobilization of iron from high iron stores.We show that the polycythemic phenotype, as well as Erfe and Hamp expression, in mtHEPOR mice undergoes dynamic changes during ontogenesis. We conclude that further research will be needed to understand the development of iron overload and changes in Erfe and Hamp expression in mtHEPOR mice characterized by erythroid hyperplasia and low Epo levels.Acknowledgment: BK, VD, and MH were supported by the Czech Science Foundation (projects GA15-13732S and GA17-05988S), KONTAKTII project LH15223, and in part by project LTAUSA17142. BK and JS contributed equally to this work. JTP and MH act as equivalent co-senior authors. DisclosuresNo relevant conflicts of interest to declare.

Congenital erythrocytosis

Erythrocytosis, or increased red cell mass, may be labeled as primary or secondary, depending on whether the molecular defect is intrinsic to the red blood cells/their precursors or extrinsic to them, the latter being typically associated with elevated erythropoietin (EPO) levels. Inherited/congenital erythrocytosis (CE) of both primary and secondary types is increasingly recognized as the cause in many patients in whom acquired, especially neoplastic causes have been excluded. During the past two decades, the underlying molecular mechanisms of CE are increasingly getting unraveled. Gain-in-function mutations in the erythropoietin receptor gene were among the first to be characterized in a disorder termed primary familial and congenital polycythemia. Another set of mutations affect the components of the oxygen-sensing pathway. Under normoxic conditions, the hypoxia-inducible factor (HIF), upon hydroxylation by the prolyl-4-hydroxylase domain protein 2 (PHD2) enzyme, is degraded by the von Hippel-Lindau protein. In hypoxic conditions, failure of prolyl hydroxylation leads to stabilization of HIF and activation of the EPO gene. CE has been found to be caused by loss-of-function mutations in VHL and PHD2/EGLN1 as well as gain-of-function mutations in HIF-2α (EPAS1), all resulting in constitutive activation of EPO signaling. Apart from these, globin gene mutations leading to formation of high oxygen affinity hemoglobins also cause CE. Rarely, bisphosphoglycerate mutate mutations, affecting the 2,3-bisphosphoglycerate levels, can increase the oxygen affinity of hemoglobin and cause CE. This narrative review examines the current mutational spectrum of CE and the distinctive pathogenetic mechanisms that give rise to this increasingly recognized condition in various parts of the world.

Primary familial and congenital polycythemia; The forgotten entity

Primary familial and congenital polycythemia (PFCP) is a rare autosomal dominant disorder caused by hypersensitivity of erythropoietin receptor of erythroid progenitors leading to increased rate of erythropoiesis at any given serum erythropoietin level. The hallmark of this disorder is isolated erythrocytosis with the absence of splenomegaly and lack of secondary causes of polycythemia. In this short review, we will shed light on various aspects of PFCP with special focus on molecular pathogenesis and diagnostic approach.

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.

New Insights into Mechanisms of Erythropoietin Receptor Mutations in Primary Familial and Congenital Polycythemia

▪Primary Familial and Congenital Polycythemia (PFCP) is a non-malignant pathology of the erythroid lineage, characterized by an isolated increase of the red cell mass without evolution into myelofibrosis or acutisation. Around twenty constitutive non-sense and missense mutations located in the exon 8 of the erythropoietin receptor (EPOR) gene have been described so far. They all lead to the truncation of the C-terminal part of the protein and the loss of several cytoplasmic tyrosines. The erythropoietin (EPO) hypersensitivity of the PFCP erythroid progenitors is usually explained by the disappearance of these negative signaling regulation and internalization domains (Figure 1a). Nonetheless, relatively few functional studies have been carried out. We therefore investigated the mechanism of EPOR mutations in PFCP.We identified and extensively studied a new constitutive heterozygous frameshift EPOR mutation, p.Gln434Profs*11, which generates a new 11 amino acid (AA) C-terminal tail and a STOP codon at position 444, leading to the truncation of 63 AA of the wild-type receptor (Figure 1c). The primary progenitor cells displayed a major hypersensitivity to EPO, similar to Polycythemia Vera (PV) patients, as well as a spontaneous and persistent JAK2 and STAT5 phosphorylation, compared to the control cells. To study the mechanism of this new EPOR mutant, Ba/F3 cells were transduced with different retroviruses encoding either the HA-tagged wild-type EPOR (EPOR WT)or a truncated receptor at position 444, p.Gln444* (EPOR STOP) or the frameshift EPOR p.Gln434Profs*11mutation (EPOR FS), identical to the patient's mutation (Figure 2). As observed in primary cells, EPOR FS conferred a spontaneous STAT5 phosphorylation and a 4- to 5-fold EPO hypersensitivity to Ba/F3 cells (IC50 of 0.003 U/mL vs 0.01 U/mL) compared to both EPOR WT and EPOR STOP. As expected, the loss of negative regulatory domains in the C-terminal part of the receptor induced a persistent STAT5 activation in EPOR FS and EPOR STOP Ba/F3 cells. Moreover, EPOR FS was more stable (half-life of 120 minutes vs 60 minutes) and displayed a higher level of localization at the cell surface (more than 2-fold), compared to EPOR WT and EPOR STOP. However, no modification of the EPOR FS internalization pattern was observed during 125I-EPO labeling experiments and cytometry analysis. Furthermore, a dileucine motif, known to be a potential clathrin-dependent endocytosis site, is lost in the new C-terminal tail of EPOR FS mutant, yet its abrogation in EPOR WT and EPOR STOP did not modify the phenotype of Ba/F3 cells. Therefore, unlike previous reports, the major EPO hypersensitivity induced by EPOR p.Gln434Profs*11 cannot be explained by the receptor truncation, but rather by the appearance of a new C-terminal tail that confers spontaneous signaling.We wondered if this model could be extended to other EPOR mutations already described in PFCP (Figures 1a-b and 2). We therefore measured the impact on Ba/F3 cells proliferation of the frameshift EPOR p.Pro438Metfs*6 and its non-sense mutant counterpart, p.Pro443*, which retains the tyrosine at position 426, a binding site for the negative signaling regulators SOCS3 and CIS. EPO-hypersensitivity (4- to 5-fold) was only induced by EPOR p.Pro438Metfs*6, suggesting a common mechanism for the frameshift EPOR mutations. Interestingly, two proximal non-sense mutations, EPOR p.Glu399* and p.Glu425*, lacking 7 of the 8 cytoplasmic tyrosines that compose EPOR negative regulatory and internalization domains, were also able to confer a high EPO hypersensitivity to Ba/F3 cells.To our knowledge this is the first extensive functional study of EPOR mutations in PFCP. We highlighted that this pathology is much more complex than expected, since different mechanisms are involved in the EPO hypersensitivity phenotype, according to the type of EPOR mutation. Indeed, extensive truncations are sufficient by themselves to confer the EPO hypersensitivity phenotype due to the loss of all negative regulatory and internalization domains, whereas more distal truncations induced by frameshift mutants confer EPO hypersensitivity because of the appearance of a new C-terminal tail. The latter, by increasing EPOR stability at the cell surface, may cause pre-activation of both receptor and JAK2, constitutive signaling and hypersensitivity to EPO close to that of JAK2V617F-positive PVs. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Retrospective Study of High Hemoglobin Levels in 56 Young Adults

▪Introduction. Erythrocytosis is a common reason for a hematology consultation. Most of the patients are over 50 years of age. The diagnostic approach is already well established, but determining the etiology of erythrocytosis in the young adult may be a real diagnostic challenge. Hereditary causes are often first suspected, but the real etiologic panorama of erythrocytosis in this population is still poorly understood.Methods. This Canadian single-center retrospective observational study was conducted in a Québec university hospital over a period of 20 years (1995 - 2015). Data were collected by a single investigator in a standardized database. From a computer-generated query over a chosen period of time, all patients between 16 and 35 years of age with high levels of hemoglobin or hematocrit were selected (hemoglobin > 185 g/L and/or hematocrit > 0.52 in men; hemoglobin > 165 g/L and/or hematocrit > 0.48 in women). Files were analyzed after approval from the scientific and ethical committees.Results. 426 patients fulfilled the above criteria (on a total of 113 453 complete blood counts conducted in the same age range during the analysis period), but only 56 benefitted from further investigations. Only those cases were included in the study. The male-to-female ratio was 5 : 3. Among them, 59% were smokers, 11% had a high alcohol intake and 27% were using drugs on a regular basis. Obesity was present in 43% of patients (morbid obesity in nearly one third of cases). More than half of the patients had one visit or less with a specialist for investigation of their erythrocytosis. Among those who had a medical follow-up, the median length is 42 months. Erythropoietin was measured in 23% of cases and the JAK2 mutation was screened in 18% of cases. The diagnosis of an acquired polycythemia was made in 41% of cases: hypoxemia (n= 13), renal disorders (n = 6) and drug-induced (n= 4). No case of myeloproliferative syndrome was identified. Two cases were associated with congenital polycythemia (high oxygen affinity hemoglobinopathy). Diagnosis of relative polycythemia was made in slightly over 10% of cases (n= 7). In 24 patients, no conclusive cause could be identified. One third of those patients (n= 8) were hospitalised for mental health decompensation or self-induced intoxication when high hemoglobin levels were detected. When JAK2 mutation testing was not available, the complete blood count was further analyzed and it did not provide any evidence for myeloproliferative syndrome. One in five patients received treatment for their polycythemia, whatever the cause: aspirin (n= 7) and phlebotomy (n= 5). There has been no reported patient death associated with polycythemia or its treatment. Only two major complications occurred: thrombosis (n =1) and bleeding (n= 1).Conclusion. This is the first study to specifically analyze erythrocytosis in young adults. Despite the retrospective design, it demonstrates patient management often inconsistent with the actual recommendations and a lack of follow-up after initial investigations. Congenital polycythemia deserves to be systematically evoked in this age range, but represents only a small part of cases. Acquired polycythemia dominates the etiologies whereas myeloproliferative syndromes were not depicted in this study. Harmonisation of patient care for young adults with erythrocytosis is therefore highly desirable. DisclosuresNo relevant conflicts of interest to declare.

Truncating Erythropoietin Receptor Rearrangements in Acute Lymphoblastic Leukemia

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL invivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

Relative polycythemia in acute pancreatitis: a series of case report

Polycythemia is an unusual manifestation of acute pancreatitis. When polycythemia does occur, it is almost invariably due to intravascular fluid depletion, contraction of Plasma volume, Gaisbock’s Syndrome, Primary congenital and familial polycythemia, polycythemia vera, Hypoxia as in high altitude, pulmonary cyanotic congenital heart diseases, hypoventilation syndromes like sleep apnea, Smoker’s Polycythemia, carbonmonoxide intoxication caused by industrial exposure, Erythropoietin secreting tumors, post renal transplantation erythrocytosis and congenital polycythemias. This case series report presents four patients with acute pancreatitis and relative polycythemia due to reduction in plasma volume as a result of intravascular volume depletion, as demonstrated by Haemoglobin and Hematocrit during acute pancreatitis and the significance of this association is discussed. Among the case series, the case 1 was relative polycythemia with acute pancreatitis presented as pseudocyst, whereas the case 2, 3 and 4 presented as relative polycythemia with necrotizing pancreatitis. The clinical course of the patient suggested that the relative polycythemia developed subsequent to the clinical onset of acute pancreatitis. It is believed that this represents the first recorded case series in the English Literature of acute pancreatitis with Relative Polycythemia due to reduction in plasma volume as a result of intravascular volume depletion. Four Patients presented with Relative polycythemia during acute pancreatitis was found.

Delayed hemoglobin switching and perinatal neocytolysis in mice with gain-of-function erythropoietin receptor.

Mutations of the truncated cytoplasmic domain of human erythropoietin receptor (EPOR) result in gain-of-function of erythropoietin (EPO) signaling and a dominantly inherited polycythemia, primary familial and congenital polycythemia (PFCP). We interrogated the unexplained transient absence of perinatal polycythemia observed in PFCP patients using an animal model of PFCP to examine its erythropoiesis during embryonic, perinatal, and early postnatal periods. In this model, we replaced the murine EpoR gene (mEpoR) with the wild-type human EPOR (wtHEPOR) or mutant human EPOR gene (mtHEPOR) and previously reported that the gain-of-function mtHEPOR mice become polycythemic at 3~6weeks of age, but not at birth, similar to the phenotype of PFCP patients. In contrast, wtHEPOR mice had sustained anemia. We report that the mtHEPOR fetuses are polycythemic, but their polycythemia is abrogated in the perinatal period and reappears again at 3weeks after birth. mtHEPOR fetuses have a delayed switch from primitive to definitive erythropoiesis, augmented erythropoietin signaling, and prolonged Stat5 phosphorylation while the wtHEPOR fetuses are anemic. Our study demonstrates the in vivo effect of excessive EPO/EPOR signaling on developmental erythropoiesis switch and describes that fetal polycythemia in this PFCP model is followed by transient correction of polycythemia in perinatal life associated with low Epo levels and increased exposure of erythrocytes' phosphatidylserine. We suggest that neocytolysis contributes to the observed perinatal correction of polycythemia in mtHEPOR newborns as embryos leaving the hypoxic uterus are exposed to normoxia at birth. Human gain-of-function EPOR (mtHEPOR) causes fetal polycythemia in knock-in mice. Wild-type human EPOR causes fetal anemia in knock-in mouse model. mtHEPOR mice have delayed switch from primitive to definitive erythropoiesis. Polycythemia of mtHEPOR mice is transiently corrected in perinatal life. mtHEPOR newborns have low Epo and increased exposure of erythrocytes' phosphatidylserine.

Polycythemia of Mice with Human Gain-of-Function EPOR (mtHEPOR) Is Transiently Corrected in Perinatal Life in Association with Low Epo and Increased Erythrocyte Phosphatidylserine Exposure

Gain-of-function mutations of erythropoietin receptor (EPOR) resulting from truncation of the cytoplasmic domain of EPOR are associated with primary familial and congenital polycythemia (PFCP). We generated and previously reported a PFCP mouse model where the murine EpoR gene (mEpoR) was replaced with wild-type human EPOR (wtHEPOR) or mutant human EPOR (mtHEPOR) genes. We showed that mtHEPOR mice become polycythemic at 3-6 weeks of age, but not at birth, similar to the polycythemia phenotype of affected humans, while the wtHEPOR mice were anemic (Divoky et al, Proc Natl Acad Sci U S A 2001;98:986). In addition, we previously reported (Yoon et al, ASH Annual Meeting Abstracts 2005 106: Abstract 567) that mtHEPOR fetuses are polycythemic at the mid- and late-gestation stages (embryonic days, E12.4-18.5), while wtHEPOR fetuses are anemic, similar to what we observed in adult mice. The mtHEPOR fetal liver cells (FLCs) revealed augmented and sustained activation of Stat5, while wtHEPOR FLCs had significantly reduced Stat5 activation. We also previously presented (Yoon and Prchal, ASH Annual Meeting Abstracts 2007 110: Abstract 3664) that the fetal polycythemia of mtHEPOR mice is associated with a delayed switch from primitive to definitive erythropoiesis, and that polycythemia in mtHEPOR mice is transiently corrected in perinatal life and reappears at 3 weeks after birth. Here, we hypothesized that the change of oxygen tension at delivery (from the uterus to ambient atmosphere) and differences in Epo levels may account for the inexplicable perinatal absence of polycythemia in mtHEPOR mice.Epo levels in adult mice were substantially elevated in wtHEPOR (1271±240 pg/mL) and significantly reduced in mtHEPOR (98±51 pg/mL) when compared to mEpoR mice (265±62 pg/mL). We then proposed that destruction of young red blood cells (RBCs), termed neocytolysis (a mechanism that overcorrects increased RBC mass generated during chronic hypoxemia after restoration of normoxic conditions) contributes to the anemia of newborns (Song et al, J Mol Med 2015;93:857). When adult mice were challenged by rapid conversion from 10 days of hypoxia (12% of O2) to normoxia, the mtHEPOR mice had greater changes of hematocrit (drop of 16.9%) than other genotypes (drop of 14.8% for mEpoR and13.8% for wtHEPOR). Similarly, the most pronounced decrease of hematocrit after birth, at perinatal day 7, (PN7) was detected in mtHEPOR mice (drop of 56%) followed by mEpoR (drop of 48%) and only a relatively moderate decrease of hematocrit in wtHEPOR neonates (drop of 24%). In parallel, PN7 mtHEPOR newborns had the lowest levels of Epo (310±102 pg/mL), while PN7 wtHEPOR neonates had Epo levels markedly increased (2757±912 pg/mL); the Epo levels of control mEpoR at PN7 littermates were 494±102 pg/mL. This suggests that Epo protects from neocytolysis. These changes corresponded to exposure of phosphatidylserine; a stimulus for RBC removal from circulation. There was a gradual increase of phosphatidylserine-positive RBCs in mtHEPOR neonates and, to a lower extent, also in mEpoR between PN0 and PN7 with a maximum positivity at PN7 (mean fluorescence: 6.5±2.8 and 6.2±1.7 for mtHEPOR and mEpoR, respectively). This correlated with a maximal decrease of hematocrit and lowest Epo levels at PN7 in mtHEPOR. The proportion of phosphatidylserine-positive RBCs then declined with age (mean fluorescence of 0.6 in adult mtHEPOR mice). In contrast, wtHEPOR mice had the lowest numbers of phosphatidylserine-positive RBCs, which were comparable at all analyzed time-points (mean fluorescence at PN0=0.7±0.1; PN7=1.3±0.3; in adults=0.6±0.04), consistent with the lowest reduction in perinatal hematocrit and very high Epo levels in these mice.In conclusion, our study describes that transient correction of polycythemia of the mtHEPOR PFCP mouse model in the perinatal period is associated with low Epo levels and increased erythrocyte phosphatidylserine exposure. Increased phosphatidylserine exposure on the membrane of mtHEPOR RBCs is consistent with accelerated destruction of these cells by macrophages, thus contributing to the decrease of hematocrit in mtHEPOR neonatal mice.Authorship: JS and BK: equal credit as first authorsAcknowledgment: VD and MH were supported by the Czech Science Foundation (project P301/12/1503) and by the Ministry of Health Czech Republic (project NT13587). DisclosuresNo relevant conflicts of interest to declare.

Somatic mosaicism of EPAS1 mutations in the syndrome of paraganglioma and somatostatinoma associated with polycythemia.

We recently described a novel, non-inherited syndrome of tumor-specific mutations of hypoxia-inducible factor 2α, encoded by EPAS1, leading to formation of multiple paragangliomas and somatostatinomas in the setting of congenital polycythemia. Although we had suspected that somatic mosaicism of EPAS1 mutations was the underlying cause of tumorigenesis, we could not validate this theory in our initial findings. In this report, we developed a sensitive, peptide nucleic acid sequencing assay to uncover the presence of EPAS1 mutations in blood and other somatic tissues of the two patients who were described in the initial characterization of this syndrome. As such, the current study demonstrates that the underlying pathogenesis of the syndrome of multiple paraganglioma and somatostatinoma formation with congenital polycythemia is somatic mosaicism of EPAS1 mutations.

Retrospective outcomes in patients with BCR-ABL positive Acute Lymphoid Leukemia without Allogeneic stem cell transplantation at the Houston Methodist

S176 Introduction: Ph-like acute lymphoblastic leukemia (ALL) is characterized by a variety of rearrangements and other genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We recently identified recurring rearrangements of the erythropoietin receptor (EPOR) into the immunoglobulin heavy (IGH) or kappa (IGK) chain loci. The goal of this study was to examine the role of these alterations in kinase signaling and lymphoid transformation.Methods:Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of EPO. EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Luciferase-marked xenografts of human EPORrearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo. Results: Eight cases out 154 Ph-like ALL (5.2%) harbored rearrangements of EPOR into either the IGH or IGK loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR, resulting in overexpression of a C-terminal truncated receptor that retains the phosphorylation site required for STAT5 activation, but lacking multiple residues required for negative EPOR regulation. Notably, the location of the truncation sites is similar to those arising from inherited mutations in primary familial congenital polycythemia. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Xenograft leukemic cells from EPOR-rearranged cases exhibited high levels of mutant EPOR on the cell surface, constitutive pSTAT5 and noteworthy ex vivo and in vivo sensitivity to the JAK2 inhibitor ruxolitinib. Conclusions: Rearrangements of truncated EPOR into the IGH/IGK chain loci define a new subgroup of Phlike ALL and unexpectedly implicate EPOR signaling as an important factor influencing B-lymphoid malignancies. Finally, these findings suggest that aberrant EPOR signaling is amenable to JAK-STAT5 inhibition in cases harboring these rearrangements.