Idiopathic Erythrocytosis Patients Research Articles

Idiopathic erythrocytosis: a germline disease?

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis, suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2. Only 2 patients presented high mutation burden somatic variants, involving DNMT3A, TET2, ASXL1 and WT1. We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT, Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE.Graphical abstract

PB2050: EVALUATION OF NON-CLONAL ERYTHROCYTOSIS: SINGLE CENTRE EXPERIENCE

Background: Secondary erythrocytosis has a broad differential diagnosis that includes hypoxic lung disease, cyanotic congenital heart disease, kidney disease, medications and erythropoietin-producing malignant disorders. Aetiology remains unknown in some patients, they have the so-called idiopathic erythrocytosis (IE). Among the latter, patients with inherited/congenital erythrocytosis can be found, especially if they have a positive family history. Aims: The aim of our study was to analyse the characteristics of non-clonal erythrocytosis in patients referred to our Haematology department in a 5-year period (2016 – 2021) and to compare the IE group to the group with known causes. Methods: We included all individuals who were Janus kinase 2 (JAK2) mutation negative and had erythrocytosis. Data was retrospectively collected with the help of the hospital database and statistically analysed with SPSS. Results: The study included 250 patients, 176 (70%) men and 74 (30%) women, with mean age 58.8 years (21 – 94 years). Their average haemoglobin values within a 1-year time period were 182.5 g/L for men and 169 g/L for women. Average haematocrit levels were 0.53 for men and 0.50 for women. Elevated erythropoietin (EPO) levels were found in 10.5% patients, 83.4% had normal and 6.1% decreased values. Symptoms were reported by 48% of the patients, mostly itchiness (16.4%), sweating (12%) and other (30%) such as tinnitus, myalgia, joint pain and snoring. The most probable reasons for secondary erythrocytosis were smoking/chronic obstructive pulmonary disease (COPD) (32%), IE (29%), dehydration (7%), nephrological aetiology (6%), obesity (4%), sleep apnoea (4%), asthma (4%), infection (4%), heart or pulmonary disease hypoxia (4%), possible hemochromatosis (3%), medications (2%) and high altitude/training (1%). Patients with IE had an average age of 57.2 years (24 – 83 years), 57 (78%) were men and 8 (11%) had a positive family history. EPO levels in this group were elevated in 7.8%, normal in 88.2% and decreased in 3.9%. Past thrombotic events were reported by 23.3% of individuals with IE and 20.3% of patients with known secondary cause. There was no statistical difference in age, gender, EPO, haemoglobin levels and past thrombotic events between the IE group and group with known causes. Patients with IE significantly differed from the group with known causes in haematocrit levels (p=0.0219) with lower levels in IE patients. Summary/Conclusion: Among 250 patients with non-clonal erythrocytosis the majority had acquired aetiology. The most common cause was hypoxia caused by smoking or COPD. Patients with IE significantly differed from the group with known causes in haematocrit levels, while there were no significant differences between groups in age, gender, EPO levels, haemoglobin levels and past thrombotic events.

Single Centre Analysis of JAK2 V617F and Exon 12 Negative Idiopathic Erythrocytosis

▪IntroductionThe molecular categorisation of myeloproliferative neoplasms (MPNs) has changed the landscape of diagnosis and treatment. Polycythaemia vera (PV) is characterised by red blood cell proliferation and JAK2 V617F or Exon 12 mutations in up to 98% of patients 1. However, some patients without such mutations have an arduous diagnostic course with varying management and prognostic outcomes 2. We present our experience in managing this challenging cohort and aim to illuminate a potential diagnostic pathway for patients.MethodWe searched electronic records of patients attending haematology clinics over the last 20 years at University College Hospital with a prior diagnosis of PV / erythrocytosis (presenting with raised Haemoglobin (Hb) &/ Haematocrit (Hct)) with no evidence of JAK2 exon 12 or 14 mutations on bone marrow or peripheral blood molecular analysis (multiplex PCR sensitivity 0.1%). We reviewed their diagnostic workup, which included full blood count & where available, bone marrow myeloid (Illumina TruSight Myeloid) & erythroid next generation sequencing (NGS) panels.Results37 patients with JAK2 V617F & Exon 12 mutation negative erythrocytosis were identified. Patients were categorised in to 3 groups 2; idiopathic erythrocytosis (IE), secondary polycythaemia (SP) & high affinity haemoglobinopathies (HAH); patient characteristics are summarised in Table 1. The median age of IE & HAH was younger, their presenting Hb/Hct levels was higher compared to SP, with a male predominance. Constitutional symptoms were only reported in the IE cohort. Erythropoietin (EPO) was elevated in HAH & IE patients but within normal range in SP. Thrombotic events occurred in all cohorts, most frequently in IE. Splenomegaly was reported in 4/21 IE, 1/13 SP, but was not a feature in HAH patients. When performed, IE red cell mass (RCM) studies were raised but within normal range in SP patients. Table 2 details IE cohort erythroid mutations. Myeloid NGS only identified MPL and BCOR mutations of pathogenic significance and multiple single nucleotide polymorphisms of no known significance. No abnormalities were demonstrated in 15% of SP patients that underwent bone marrow myeloid mutational analysis. Venesections (VS) were instigated in 95% of the IE cohort, antiplatelets (AP) in 52%, anticoagulation (AC) in 14% and cytoreductive therapy (CT) in 19% due to intolerance/failure of VS. VS programme was instigated in 46% of SP patients, AP in 7% and AC in 47%.DiscussionThe median age of our IE cohort was 48 yrs with a 19% incidence of thrombosis. Where performed, bone marrow histology demonstrated hypercellularity but was not consistent with MPN diagnostic criteria. Myeloid NGS panel mutations such as BCOR may represent clonal haematopoieis of indeterminate potential. Heterozygous VHL C598T & C376A mutations, in keeping with Chuvash polycythaemia, were demonstrated. Mutations in EGLN1 &BPGM,were detected in our patients, however there was an absence of correlating haematological parameters or family history to support a diagnosis of congenital erythrocytosis (2). Variants of unknown significance were also detected in SH2B3, BMP6 &EGLN3 gene duplication. SP patients were older (median age 68 years) and where performed had normal RCM and no myeloid mutations identified.The initial approach adopted at our centre for diagnosing and managing JAK2 V617F & Exon 12 negative erythocytosis begins with clinical evaluation for secondary causes. This is followed by assessment of EPO level, RCM study, extended molecular mutational analysis involving screening for high affinity haemoglobins, congenital erythrocytosis and bone marrow histology. Where patients are symptomatic or considered at high risk for thrombosis, we venesect to a personalised target and patients are offered AP and/or AC. CT is instigated where VS is not tolerated or ineffective, however this approach is not corroborated in the literature. Our experience highlights the clinical heterogeneity of JAK2 negative erythrocytosis and the need to develop a robust and systematic diagnostic and treatment algorithm with further clarification of the role of molecular profiling.1. William W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017 Feb; 129(6):667-6792. McMullin MF. Idiopathic erythrocytosis: a disappearing entity. Hematology Am Soc Educ Program. 2009; 2009(1):629-635 [Display omitted] DisclosuresSekhar: Novartis: Consultancy, Research Funding.

Diagnosis and management of non-clonal erythrocytosis remains challenging: a single centre clinical experience

Erythrocytosis has a diverse background. While polycythaemia vera has well defined criteria, the diagnostic approach and management of other types of erythrocytosis are more challenging. The aim of study was to retrospectively analyse the aetiology and management of non-clonal erythrocytosis patients referred to a haematology outpatient clinic in an 8-year period using a 3-step algorithm. The first step was inclusion of patients with Hb > 185 g/L and/or Hct > 0.52 in men and Hb > 165 g/L and/or Hct > 0.48 in women on two visits ≥ two months apart, thus confirming true erythrocytosis. Secondly, polycythaemia vera was excluded and secondary causes of erythrocytosis (SE) identified. Thirdly, idiopathic erythrocytosis patients (IE) were referred to next-generation sequencing for possible genetic background evaluation. Of the 116 patients, 75 (65%) are men and 41 (35%) women, with non-clonal erythrocytosis 34/116 (29%) had SE, 15/116 (13%) IE and 67/116 (58%) stayed incompletely characterized (ICE). Patients with SE were significantly older and had significantly higher Hb and Hct compared to patients with IE. Most frequently, SE was attributed to obstructive sleep apnoea and smoking. Phlebotomies were performed in 56, 53 and 40% of patients in the SE, IE, and ICE group, respectively. Approx. 70% of patients in each group received aspirin. Thrombotic events were registered in 12, 20 and 15% of SE, IE and ICE patients, respectively. Congenital erythrocytosis type 4 (ECYT4) was diagnosed in one patient. The study demonstrates real-life management of non-clonal erythrocytosis which could be optimized using a 3-step diagnostic algorithm.

Study of the p.V617F and Exon 12 Mutations in JAK2 Gene Among Iranian Chronic Myeloproliferative Patients

Background: Chronic myeloproliferative disorders (CMPD) occur due toclonal proliferation of the single hematopoietic stem cells and result in an increased number of mature and immature cells in the peripheral blood. The mutations in JAK2 gene are identified in large numbers of CMPD patients. Objectives: The aim of this study was to investigate thep.V617F (c.1849G > T) mutation as well as exon 12 mutations in JAK2 gene in the CMPD patients. Methods: Philadelphia chromosome negative CMPD patients were recruited for this study. In order to study p.V617F and JAK2 exon 12 mutations in JAK2 gene, FRET probe real-time PCR, allele specific PCR and PCR-direct sequencing were utilized. Results: JAK2 p.V617F mutation was found in polycythemia vera, Essential thrombocytosis and idiopathic myelofibrosis (67%, 52% and 50% respectively) but not in idiopathic erythrocytosis patients. Also no mutation was found in JAK2 exon 12 of these patients. Conclusions: Our data regarding p.V617F was in concordance with the previous studies. The absence of any mutation in exon 12 of our patients may be due to extracting DNA from whole blood cells instead of granulocytes, that may impact the detection rate of cycle sequencing method.

Comprehensive Molecular Profiling of Idiopathic Erythrocytosis By Genetic Ancestry

Introduction: Idiopathic erythrocytosis (IE) is characterized by a persistently elevated hemoglobin, equivocal erythropoietin (EPO) levels, absence of janus kinase 2 (JAK2) mutations suggestive of polycythemia vera (PV) and no secondary cause. One study used a targeted 21 gene next-generation sequencing panel and identified novel variants in known erythrocytosis-related genes as well as novel genes associated with the oxygen-sensing pathway. However, expanded sequencing of blood and matched tissue samples in a large ethnically diverse group of IE patients has not been performed. Methods: All patients signed informed consent to participate in an observational study approved by the Institutional Review Board; they provide blood and buccal mucosa samples at study entry and at 24-month follow-up. Patients were enrolled if JAK2 testing and a complete work up for secondary causes was negative. They were required to have hemoglobin levels greater than 16 g/dL on two occasions or greater than 15 g/dL if undergoing phlebotomy. Our initial sequencing of 20 IE patients was performed utilizing high resolution whole-exome sequencing of circulating blood samples (disease) at a mean coverage of 390x and matched normal (buccal) samples at mean coverage of 300x. To stratify samples by genetic ancestry, we performed a population stratification principle component analysis (PCA) and STRUCTURE using Ancestry Informative Markers derived from 1K Genome Phase1_v3 Exome database. The primary in-silico analysis was performed on the baseline samples from treatment-naïve patients. The whole-exome data was generated in accordance to GATK's best practices with same filters applied as described by Exome Aggregation Consortium. The additional downstream in-silico paired analysis was performed using MutSig2.0 (Mutation Significance) algorithm to determine significant mutations and GISTIC (The Genomic Identification of Significant Targets in Cancer) to identify the significant copy number events, IPA (Ingenuity Pathway Analysis) to determine pathways along with other computational . Results: Median age at baseline was 52 years (range 35-71). Six patients (30%) were female and 14 (70%) were male. Median values and ranges for laboratory parameters at baseline were as follows: WBC 6.6 x 109/L (5-9.7), Hgb 17 g/dL (15.5-19.8), Plt 218 x 109/L (86-374), and EPO level 9.8 IU/L (2-14.3). Three patients had a personal history of malignancy, including 2 with lymphoma. Two patients had a family history of myeloid malignancy (chronic myeloid leukemia and PV). Our ancestry analysis of initial 20 patients with IE identified 6 patients with high European percent ancestry (EUR), 1 patient with high Asian percent ancestry (EAS) and 13 patients with high percent Ad Mixed ancestry (AMR). In our cohort, 60% (12/20) of patients had been also diagnosed with a liver disorder (11 with fatty liver, 1 with cirrhosis) that was not significantly different across populations. We identified, on average, 42 non-silent somatic mutations (not present in the buccal samples) in whole blood across our cohort with no statistical difference (p=0.671) in mutation burden between ancestry groups or between patients with and without liver disease. Age, gender, and ethnicity were not associated with mutation burden. Utilizing MutSig algorithm, we identified a novel candidate gene, CHAF1A, with high mutation prevalence of 30% in patients with IE. Further analysis of mutation landscape identified somatic nonsilent mutations in 25 known oncogenes which were present in at least 10% of patients. Our mutation signatures in IE identified a significant association with failure of double stranded DNA repair. Only one patient had a mutation in TET2. Further analysis of copy number indicated copy number loss in genes such as SETD3 and GSH associated with chromatin assembly which may suggest alterations in chromatin assembly and changes in the epigenome. Our analysis also identified a high number of 9p and 13q gains in patients with IE. Conclusion: In this study, we utilized high-resolution next generation sequencing in association with comprehensive clinical annotation to determine potential molecular drivers of IE in a multi-ethnic population. We identified somatic mutations in a subset of patients which may represent clonal hematopoiesis. Long term follow up of outcomes in this cohort may clarify the significance of these mutations in the pathogenesis of IE. Disclosures No relevant conflicts of interest to declare.

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.

PF665 SERUM FERRITIN LEVELS ARE INCREASED IN IDIOPATHIC ERYTHROCYTOSIS BUT REDUCED IN POLYCYTHEMIA VERA

Background:Absolute erythrocytosis is characterized by persistently raised Haemoglobin (Hb) and hematocrit (Ht) levels. The most studied form is Polycythemia Vera (PV), a clonal disease in which the proliferation of red cells is driven by mutations in JAK2. Secondary erythrocytosis are represented by rare congenital diseases, acquired forms, or idiopathic erythrocytosis (IE), a poorer studied disease in which the erythrocytosis has not a definite cause. Over‐production of red blood cells should drive a depletion of iron, and thus low serum ferritin levels. However, we often observe increased levels of ferritin at diagnosis in IE and we recently found that mutations or polymorphisms of HFE gene are commonly found in these patients.Aims:Could serum ferritin be an early help to hypothesize the cause of erythrocytosis in a patient? To answer this question, we retrospectively analysed levels of ferritin at diagnosis in PV and IE patients.Methods:We studied 72 patients with PV and 67 patients with IE, followed in our department. In each patient we recorded ferritin at diagnosis out of phlebotomy treatment. Other causes of hypo‐ and hyper‐ ferritinemia were excluded. Ferritin normal levels were considered 20–300 μg/L in men and 15–200 μg/L in women. All patients with a diagnosis of PV carried JAK2V617F or JAK 2 exon 12 mutation, while none of the patients with IE carried JAK2 or EPOR, VHL, PHD2, HIF‐1 alpha genes mutations. Presence and absence of HFE mutations in IE patients, if known, were registered. Statistical analysis was performed using the Mann‐Whitney test, Fisher's exact test and the t Student test.Results:Results are shown in the Table I.In addition, HFE mutations had been searched in 26 (39%) IE patients, and 13 (50%) of them had at least a mutated allele.Summary/Conclusion:Our results show a dramatic difference between levels of ferritin at diagnosis in PV and IE: in fact, 22% of PV patients had reduced ferritin at diagnosis out of phlebotomies, while on the contrary 28% of IE patients had increased levels of ferritin. Then, serum ferritin at diagnosis could address the physician to the origin of erythrocytosis. In addition, nearly half of IE patients have been already studied for presence of HFE mutations, and half of them carry at least one HFE mutated allele. We believe that a large part of IE patients carries a genetic impairment of the iron metabolism that could drive an enhancement of iron production: this could explain why in the presence of erythrocytosis iron is more consumed in PV patients than in IE ones. Further studies are needed about the presence of iron metabolism impairments in IE patients and, on the other hand, possible overlays of JAK2 and HFE mutations in PV patients.image

Absence of CALR Mutations in Idiopathic Erythrocytosis Patients with Low Serum Erythropoietin Levels

Absence of CALR Mutations in Idiopathic Erythrocytosis Patients with Low Serum Erythropoietin Levels

A nonsynonymous LNK polymorphism associated with idiopathic erythrocytosis

AbstractNo abtract.

The frequency of JAK2 exon 12 mutations in idiopathic erythrocytosis patients with low serum erythropoietin levels

Idiopathic erythrocytosis (IE) is characterized by erythrocytosis in the absence of megakaryocytic or granulocytic hyperplasia, and is associated with variable serum erythropoietin (Epo) levels. Most patients with IE lack the JAK2 V617F mutation that occurs in the majority of polycythemia vera patients. Four novel JAK2 mutant alleles have recently been described in patients with V617F-negative myeloproliferative disorders presenting with erythrocytosis. The aims of this study were to assess the prevalence of JAK2 exon 12 mutations in IE patients, and to determine the associated clinicopathological features. A cohort of 58 IE patients with low to normal serum Epo levels and no known causative mutation were identified from 181 individuals diagnosed with IE. Patients' DNA samples were screened for the presence of a JAK2 exon 12 mutation by allele-specific polymerase chain reaction and sequencing. Bone marrow trephines were examined for morphological abnormalities and the erythroid activity assessed immunohistochemically. Eight mutation-positive cases were identified, including one with a previously undescribed mutant JAK2 exon 12 allele and another with biallelic involvement. The hematologic features of mutation-positive and mutation-negative patients were similar, although Epo-hypersensitive erythroid progenitors occurred exclusively in patients with an exon 12 mutation (p=0.0002; n=15). Patients' bone marrows were moderately hypercellular, as the result of erythroid hyperplasia, and several had mild megakaryocyte atypia. JAK2 exon 12 mutations were detected in 27% of patients with low serum Epo levels, all of whom had Epo-independent erythroid progenitors. Consequently, IE patients presenting with either of these features should be tested for the presence of a JAK2 mutation.

JAK2 Mutations at Exon 12 and 14 in Polycythemia Vera and Idiopathic Erythrocytosis: Incidence and Correlation with Clinical Characteristics.

Introduction. Exon 12 mutations of the JAK2 gene have been described in polycythemia vera (PV) and idiopathic erythrocytosis (IE) patients. These patients display a clinical phenotype different to that observed in V617F-positive PV patients, but no information is available on their clinical outcome.Patients and methods. Twenty JAK2 V617F-negative PV or IE patients and 86 V617F-positive PV patients were assessed for exon 12 and exon 14 mutations. Analysis of cell lineage mutational status was assessed following cell sorting. Aim. To analyze the presence of JAK2 mutations at exon 12 and 14 in a cohort of V617F-negative PV and IE patients and to correlate them with the patient clinicohematological and evolutive data.Results. Exon 12 mutations were detected in 4 (20%) V617F-negative patients (K539L, two cases, N542_E543del and H538_K539delinsL, one case each). In 16 patients, no mutations were found in either exon 12 or exon 14. Additional mutations in exon 14 were found in two V617F-positive patients. Two patients with exon 12 mutations developed thrombosis after diagnosis, with the probability of thrombosis-free survival being 75% at 5 years. Another patient with exon 12 mutation developed myelofibrosis at 20 years of PV diagnosis. JAK2 mutations were present in granulocytes, platelets and monocytes, but not in lymphocytes or NK cells.Conclusions. Patients harboring exon 12 mutations represent a subset of JAK2 V617F-negative PV or IE patients. These patients have initial hematological data different from V617F-positive patients, but they do not differ with regard to thrombosis development and myelofibrotic transformation.Table 1:JAK2 exon 12 and exon 14 mutations in blood cell subpopulations.MutationGranulocytesPlateletsCD14+CD3+CD19+CD56+Patient 1*K539L (AAA →TTA)++−−−−Patient 2*K539L (AAA →CTA)+++−−−Patient 3*H538_K539delinsL++ND−−NDPatient 4*N542_E543del+++−−−Patient 5#V617F (GTC →TTT)++−−−NDC618R (TGT →CGT)++−−−NDPatient 6#V617F (GTC →TTC)+++−−NDC616C (TGT →TGC)+++−−ND* exon 12; # exon 14; + indicates the presence of the mutation; − indicates no detectable mutation ; ND not determined

Jak2 Exon 12 Mutational Status in a Cohort of Idiopathic Erithrocytosis V617F Negative Patients.

Background: V617F gain-of-function mutation in exon 14 of Janus Kinase 2 gene (JAK2) has been recognized as a major pathogenetic event of many cases of chronic myeloproliferative disorders, in particular in Polycythemia Vera (PV). The molecular basis of myeloproliferative disorders in patients without the V617F is still unclear but, recently, four new heterozygous allelic variants affecting JAK2 exon 12 have been described in V617F negative patients receiving a diagnosis of PV or Idiopathic Erythrocytosis (IE) (Scott et al, NEJM 2007). Revised WHO criteria for PV have been proposed considering these mutations as indicative for PV diagnosis (Tefferi et al, 2007).Aim: To determine the JAK2 exon 12 mutational status in patients with IE in a cohort of 40 consecutive IE V617F negative patients; 2 PV patients, V617F negative, were also included. 36 males and 6 females with a median age of 63 years (range: 25–85), median follow-up 109 months (range: 7–205) were studied. IE was diagnosed in presence of increased haemoglobin (>186 gr/L) and hematocrit levels (>51%) and normal white blood cells and platelet counts. In addition, no splenomegaly, and a normal or low serum erythropoietin (Epo) levels were present along with a bone marrow biopsy showing erythroid hyperplasia with normal megakaryopoiesis and granulopoiesis. They were treated only with phlebotomy therapy. PV was diagnosed according to the WHO criteria.Methods: Granulocytes were isolated from the lower interface of a density gradient separation of peripheral blood samples; the granulocytes were then submitted to erythrocytes lysis (mean purity of the granulocytes 97%) and DNA was extracted. Exon 12 was amplified using the couple of JAK2exon12F (5′-ctcctctttggagcaattca-3′) and JAK2exon12R (5′-caatgtcacatgaatgtaaatcaa-3′) primers. The amplicons were submitted to denaturing high performance liquid chromatography (dHPLC) analysis and runs were performed at 53° C. The amplicons showing a heteroduplex profile were sequenced directly in both strands.Results: 4 of the 40 (10%) IE patients were heterozygous positive for N542-E543del JAK2 exon 12 mutation; both PV patients were negative for JAK2 exon 12 mutations (figure 1).Conclusion: N542-E543del JAK2 was found only in 10% of patients with a diagnosis of IE, a frequency lower than that previously reported (Scott et al, NEJM 2007). This result could be due to the low levels of mutation prevalence in peripheral blood granulocytes. The sensitivity could be increase using erythroid colonies grown on colture media. In addition, different patients selection criteria could explain our results. Accordingly with the proposed revised WHO criteria for Polycythemia, a diagnosis of PV instead of IE should be made in these four patients. [Display omitted]

Low Prevalence of JAK2 Val617Phe Mutation in Patients with Idiopathic Erythrocytosis.

Idiopathic Erythrocytosis (IE) is a frequent clinical condition (estimated prevalence 1.1 cases every 1,000 normal subjects), characterized by an absolute increase of red blood cell mass, without the diagnostic features of polycythemia vera (PV), secondary polycythemias and congenital erythrocytosis. The clinical course of IE is marked by a lower rate of thrombotic complications than observed in PV and a very low, if any, spontaneous transition to acute leukemia. The differential diagnosis between IE and PV may be sometimes difficult but it is clinically relevant, also because cytotoxic drugs, frequently used in PV, should be avoided in IE. The recent description of a Val617Phe mutation in the exon 12 of JAK2 gene in 74–97% of patients with PV may represent a reliable molecular marker to differentiate PV from IE. However, the prevalence of JAK2 mutation in patients with IE is presently unknown. For this reason, we evaluated JAK2 mutation on granulocyte DNA or RNA using an allele-specific PCR in 31 patients with IE (29 males, 2 females, median age 46 years, range 19–74) and results were compared with those of 31 patients with overt PV (16 males and 15 females, median age 56 years, range 34–74). PV was diagnosed according to the WHO criteria. At variance, cases with IE were identified by increased hematocrit (median 54%, range 50–61%) and red blood cell mass (>25% above mean normal predicted value), but normal leukocyte and platelet counts, normal arterial O2 saturation and chest X-ray, no splenomegaly by abdominal ultrasound scanning and normal erythropoietin level and PRV-1 expression on peripheral blood granulocytes. Both IE and PV patients were either newly diagnosed or established cases in follow-up. Median follow-up was 5.5 years (range 0–20) in IE and 4.1 years (range 0–17) in PV patients respectively. Twenty-eight patients with PV (90%) but only 3 with IE (10%) showed the JAK2 Val617Phe mutation (p<0.001). In 3 patients with PV (10%) but none with IE, the JAK2 mutation was homozygous. The annual incidence of thrombotic complications was 3.2% in PV vs. 0.85% patient-year in IE (p<0.05). Our data confirm the hypothesis that JAK2 mutation is an useful molecular test to distinguish IE from PV in the great majority of patients with absolute erythrocytosis. Patients with “JAK2negative IE” require a different therapeutic approach than those with the classical “JAK2 positive PV”, since the incidence of thrombosis appears to be different. The few patients (about 10%) with discordant molecular and clinical diagnosis (i.e. “JAK2positive IE” or “JAK2 negative PV”) deserve particular attention. The long period of observation of our “JAK2positive IE” patients (3, 8 and 20 years, respectively) seems to exclude that these cases may represent an early phase of PV. However, whether this molecular marker confers a more aggressive or different clinical behaviour in a small subgroup of IE patients remains to be demonstrated in larger prospective studies.

Mutations in the VHL Gene Are the Major Identified Cause of Inherited Erythrocytosis.

The molecular basis of inherited erythrocytosis in most patients remains to be defined. Although all such patients have an absolute increase in red cell mass, their erythropoietin (EPO) levels differ widely, so they constitute a heterogeneous group of disorders known collectively as idiopathic erythrocytosis (IE). A proportion of individuals with IE progress to polycythemia vera (PV), a clonal disorder arising from a multipotent progenitor. Recently a gain-of-function mutation in Janus kinase 2 (JAK2), V617F, has been described in myeloproliferative disorders (MPD) and a stream of publications has confirmed its presence in the majority of patients with PV. Screening IE patients for this mutation will provide a useful additional means for delineating inherited and clonal disorders of erythrocytosis. Over the last decade we have maintained a registry of British and Irish erythrocytosis patients, consisting of clinical information and DNA samples obtained following full ethical approval. Screening the EPO receptor (EPO-R) in 120 patients identified one patient with a G6002A mutation, which leads to truncation of the receptor by 70 amino acids, increased sensitivity to EPO, and erythrocytosis. Screening the same patients for mutations in the von Hippel Lindau (VHL) gene has revealed individuals from 8 families of Asian origin who are homozygous for the Chuvash (R200W) mutation causing erythrocytosis. In addition, one Caucasian individual of English descent is compound heterozygous for R200W and the recently described G144R VHL mutation. A further individual, D1 (Percy et al, 2003 Blood 102:1097), of the same ethnicity is heterozygous for the Chuvash mutation and has been found to express the wild type allele. Both his mother and son, who are heterozygous for the Chuvash mutation, do not have IE, suggesting that the patient harbors a second unidentified genetic defect. Several such individuals have already been described (reviewed by Randi et al, 2005 Haematologica 90:689). In order to estimate the proportion of IE patients likely to progress to PV, 65 individuals from the registry with EPO levels in the low to normal range were screened by amplification refractory mutation system (ARMS) PCR for the MPD-associated V617F JAK2 mutation. Two individuals were positive, one of whom subsequently proceeded rapidly to PV, while the other has remained stable without any disease progression. In addition 9 families with VHL mutations were also screened and all were found to be negative for the JAK2 mutation, suggesting that the occurrence of these mutations tends to be mutually exclusive. Also the V617F JAK2 mutation does not constitute the second genetic defect in patient D1 who is heterozygous for the Chuvash VHL mutation. Although VHL mutations are the most frequent cause of inherited erythrocytosis in our registry they are present in only ~10% of patients, while the gain-of-function of JAK2 mutation is rare, leaving ~90% of the IE cases unexplained. Further study of this group may reveal additional regulatory mechanisms involved in red cell homeostasis.

The Natural History of Idiopathic Erythrocythosis: A Cohort Study of 74 Patients.

Patients with absolute erythrocytosis not due to a detectable cause and not fulfilling the criteria for diagnosis of polycythemia vera (PV) are descriptively classified as Idiopathic Erythrocytosis (IE). Based on scanty and retrospective data, this disease is considered to be an heterogeneous entity, including “early” PV, unrecognized secondary erythrocytosis and other miscellaneous conditions. However, appropriate prospective studies to evaluate the natural history of patients with IE are not available. We report here the results of a cohort study of 74 patients with IE (66 males, 8 females, median age 56 years, range 14–82) followed in two Italian institutions. By definition, at baseline all IE patients had increased hematocrit (median 54%, range 48–68%) and increased red blood cell mass (> 25% above mean normal predicted value), but normal leukocyte (median values 8.1 x 109/L, range 2.3–12) and platelet counts (median values 197 x 109/L, range 117–467), as well as normal erythropoietin level, arterial O2 saturation, chest X ray and abdominal ultrasound scanning (i.e. no splenomegaly). Granulocyte PRV-1 expression was also normal in 29 patients (39%) analyzed. At diagnosis, 12 patients (16%) reported a previous history of major thrombosis (7 ischemic cardiopathies, 4 cerebral ischemic events and 1 deep vein thrombosis). All IE patients were treated with phlebotomy to maintain a target hematocrit <45% and 24 patients (32%) were given aspirin, 100 mg/die, for previous thrombosis or microvascular symptoms. No cytotoxic drugs were given. The IE cohort was followed in the outpatient clinic with physical examination and full blood count at least every three months for a median period of 3.5 years (range 1–23). Twentythree patients (31%) were followed for more than 8 years. No patient was lost to follow-up. During the observation period, no disease potentially associated with secondary eryhtrocytosis emerged and no hematological transition into overt PV, myelofibrosis or acute leukemia occurred; two patients had a major thrombotic event (1 cerebral ischemia and 1 deep vein thrombosis) with an estimated incidence of thrombotic complications of 0.8% patient-year. The incidence of thrombosis was significantly lower than observed in 205 patients with overt PV followed during the same period in one of the two institutions (Bergamo, 3.49% patient-year, p<0.05). This study indicates that: a. the natural history of patients with IE, at least in the first years, is characterized by a remarkable and unexpected homogeneity without appearance of overt PV or diseases associated with secondary erythrocythosis; b. the diagnosis of IE identifies a group of absolute erythrocythoses at lower risk of thrombotic complications not requiring cytotoxic drug therapy; c. the diagnostic work-up of patients with absolute erythrocythosis should carefully distinguish IE from PV because the natural history and management of the two diseases is different.

A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1α (HIF-1α) does not impair Pro-564 hydroxylation

BackgroundThe hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the α subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1α in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).ResultsPatients with IE were screened for changes in the HIF-1α coding sequence, and a change in the ODD domain that converts Pro-582 to Ser was identified in several patients. This same change, however, was also detected at a significant frequency, 0.073, in unaffected controls compared to 0.109 in the IE patient group. In vitro hydroxylation assays examining this amino acid change failed to reveal a discernible effect on HIF hydroxylation at Pro-564.ConclusionThe Pro582Ser change represents a common polymorphism of HIF-1α that does not impair HIF-1α prolyl hydroxylation. Although the Pro582Ser polymorphism is located in the ODD domain of HIF-1α it does not diminish the association of HIF-1α with VHL. Thus, it is unlikely that this polymorphism accounts for the erythrocytosis in the group of IE patients studied.

ABNORMAL PRODUCTION OF INTERLEUKIN (IL)-11 AND IL-8 IN POLYCYTHAEMIA VERA

We studied the production of interleukin (IL)-11 and IL-8, two cytokines known to affect erythropoiesis, in polycythemia vera (PV). In vivo, IL-11 was detected more frequently in serum and bone marrow (BM) plasma of PV patients than in controls (healthy donors and patients with idiopathic erythrocytosis (IE)). In addition, serum IL-11 levels of PV patients were higher than those of controls. IL-8 was elevated in serum of both PV and IE patients (respective median levels: 38.6 and 242pg/ml, vs 4.4pg/ml for healthy donors). BM plasma IL-8 levels of PV patients (508pg/ml) were significantly higher than those of IE patients (120pg/ml). In vitro, bone marrow (BM) stromal cells (BMSC) of PV patients produced significantly more IL-11 (x6.4) and IL-8 (x8.3) than BMSC of healthy donors or IE patients. In conclusion, both IL-11 and IL-8 are overproduced in PV, apparently by BMSC; IL-8 is also overproduced in IE, by cells other than BMSC.

Serum erythropoietin values in erythrocytoses and in primary thrombocythaemia.

Serum erythropoietin (Epo) values were estimated in samples from 125 patients with erythrocytosis to examine the specificity and sensitivity of reduced and raised values in the diagnosis of polycythaemia vera (PV) and secondary erythrocytosis (SE) respectively. Additionally, Epo values were estimated in samples from 49 patients with primary thrombocythaemia (PT) to determine whether Epo values were altered. We found high specificity (92%) and moderate sensitivity (64%) of low serum Epo values (below the reference range) in the diagnosis of PV, and also poor sensitivity (47%) of raised Epo values in the diagnosis of SE. Raised Epo values were not observed in PV patients with Hb > 14.0 g/dl and were only observed in one PV patient with a relatively low Hb recovering from a gastro-intestinal haemorrhage. Raised Epo values occurred in some patients with apparent erythrocytosis (AE) and idiopathic erythrocytosis (IE), mainly at normal (rather than raised) Hb values (< 16 g/dl). Low Epo values occurred in a few AE, IE and SE patients at higher Hb values (> 16 g/dl). Low Epo values were less specific for PV when the Hb was raised, while raised Epo values were less specific for SE when the Hb was not raised. Approximately one third of patients with PT had a low (below the reference range) Epo value, this being associated with a high normal Hb (> 14 g/dl, P < 0.001) and showing a trend towards association with absence of treatment. The high normal Hb values were in turn associated with an increased incidence of thrombotic events (P < 0.05). These findings could influence the future investigation and management of PT patients.