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Summary: Background: Erythrocytosis is a hematological disorder usually related to hematopoietic stem cell somatic mutations. However, unexplained erythrocytosis remains frequent. In this study, we evaluated the involvement of IgA1, a regulator of erythropoiesis also implicated in IgA nephropathy (IgAN) pathophysiology, in unexplained polycythemia/erythrocytosis (PE) of IgAN patients. Methods: IgAN-PE patients’ serum was collected, analyzed and used to study IgA1 effect on proliferation and differentiation of erythroid progenitors. Hematological parameters of transgenic mice for human alpha1 heavy chain were studied. Multicentric observational cohorts of chronic kidney disease (CKD) patients, including both native kidney diseases and renal transplants, were studied to analyze patient hemoglobin levels. Findings: We retrospectively identified 6 patients with IgAN and unexplained PE. In large CKD cohorts, IgAN was associated with PE in 3.5% of patients (p

Classical BCR-ABL–negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell malignancies associated with an erythroid maturation defect, resulting in anemia. Treatments for MDS include erythropoiesis-stimulating agents (ESAs). The identification of prognostic markers is important to help predict response and improve outcomes. Various scoring systems have been developed to help predict response to ESAs. Despite limitations in its assessment, serum erythropoietin (sEPO) level is an important predictor of hematologic response to ESAs in patients with lower-risk MDS. Numerous studies have reported significantly lower sEPO levels among responders versus non-responders. Furthermore, treatment response is significantly more likely among those with sEPO levels below versus those above various cutoffs. Other prognostic indicators for response to ESAs include lower transfusion requirement, fewer bone marrow blasts, higher hemoglobin, lower serum ferritin, lower-risk MDS, and more normal cytogenetics. Studies of other MDS therapies (e.g., lenalidomide and luspatercept) have also reported that lower sEPO levels are indicative of hematologic response. In addition, lower sEPO levels (up to 500 IU/L) have been included in treatment algorithms for patients with lower-risk MDS to define whether ESAs are indicated. Lower sEPO levels are predictive of hematologic response—particularly to ESAs. Further, clinical trials should use sEPO thresholds to ensure more homogeneous cohorts.

Mutations of calreticulin (CALRm) define a subtype of myeloproliferative neoplasms (MPN). We studied the biological and genetic features of CALR-mutated essential thrombocythemia and myelofibrosis patients. In most cases, CALRm were found in granulocytes, monocytes, B and NK cells, but also in T cells. However, the type 1 CALRm spreads more easily than the type 2 CALRm in lymphoid cells. The CALRm were also associated with an early clonal dominance at the level of hematopoietic stem and progenitor cells (HSPC) with no significant increase during granulo/monocytic differentiation in most cases. Moreover, we found that half of type 2 CALRm patients harbors some homozygous progenitors. Those patients were associated with a higher clonal dominance during granulo/monocytic differentiation than patients with only heterozygous type 2 CALRm progenitors. When associated mutations were present, CALRm were the first genetic event suggesting that they are both the initiating and phenotypic event. In blood, type 1 CALRm led to a greater increased number of all types of progenitors compared with the type 2 CALRm. However, both types of CALRm induced an increase in megakaryocytic progenitors associated with a ruxolitinib-sensitive independent growth and with a mild constitutive signaling in megakaryocytes. At the transcriptional level, type 1 CALRm seems to deregulate more pathways than the type 2 CALRm in megakaryocytes. Altogether, our results show that CALRm modify both the HSPC and megakaryocyte biology with a stronger effect for type 1 than for type 2 CALRm.

Background and Aims Myeloproliferative neoplasms (MPN) are the leading cause of splanchnic vein thrombosis (SVT). Janus kinase 2 gene ( JAK2 ) V617F mutations are found in 80 to 90% of patients with SVT and MPN. Mutations of the calreticulin ( CALR ) gene have also been reported. However, as their prevalence ranges from 0 to 2%, the utility of routine testing is questionable. This study aimed to identify a group of patients with SVT at high risk of harboring CALR mutations and thus requiring this genetic testing. Methods CALR , JAK2 V617F and thrombopoietin receptor gene ( MPL ) mutations were analysed in a test cohort that included 312 patients with SVT. Criteria to identify patients at high risk of CALR mutations in this test cohort was used and evaluated in a validation cohort that included 209 patients with SVT. Results In the test cohort, 59 patients had JAK2 V617F , five had CALR and none had MPL mutations. Patients with CALR mutations had higher spleen height and platelet count than patients without these mutations. All patients with CALR mutations had a spleen height ⩾16cm and platelet count >200×10 9 /L. These criteria had a positive predictive value of 56% (5/9) and a negative predictive value of 100% (0/233) for the identification of CALR mutations. In the validation cohort, these criteria had a positive predictive value of 33% (2/6) and a negative predictive value of 99% (1/96). Conclusion CALR mutations should be tested in patients with SVT, a spleen height ⩾16cm, platelet count >200×10 9 /L, and no JAK2 V617F . This strategy avoids 96% of unnecessary CALR mutations testing. Lay summary: Mutations of the CALR gene are detected in 0 to 2% of patients with SVT, thus the utility of systematic CALR mutation testing to diagnose MPN is questionable. This study demonstrates that CALR mutations testing can be restricted to patients with SVT, a spleen height ⩾16cm, a platelet count >200×10 9 /L, and no JAK2 V617F . This strategy avoids 96% of unnecessary CALR mutations testing.

Aim To assess the safety and immunogenicity of subcutaneous (SC) HX575 (epoetin-α) in dialysis- and nondialysis-dependent adult patients with chronic kidney disease (CKD). Methods Open-label, single-arm, multicenter study in patients (n = 416) from Germany, Italy, Poland, Romania, Russia, Turkey, and Ukraine. Results Mean (standard deviation (SD)) age was 52.3 (15.8) years, all patients were Caucasian, and similar proportions were male/female. 250 patients (60.1%) were erythropoiesis-stimulating agent (ESA)-naive, and 166 (39.9%) were receiving ESA maintenance therapy at study start; mean (SD) on-study treatment duration with HX575 was 43.4 (15.8) weeks and 45.3 (13.7) weeks, respectively. Binding antierythropoietin (EPO) antibodies were detected by radioimmunoprecipitation (RIP) assay in 7 patients (1.7%; incidence 0.019); 5 of these were ESA-naive at study entry. No patient developed neutralizing antibodies as determined in a cell-based epoetin neutralizing assay. Of the 7 patients with a positive binding anti-EPO RIP assay, 4 tested negative at later time points while continuing HX575 treatment. Three patients had low titers of anti-EPO antibodies at the last study assessment. There were no clinical signs of immunogenicity or hypersensitivity. Conclusions SC HX575 was effective for correcting and maintaining correction of anemia, and the mean weekly dose remained stable over time. .

Resume Le medicament biosimilaire (BS) correspond a un medicament biologique declare comme similaire a un medicament biologique de reference et dont le plan de developpement inclut des etudes de comparaison par rapport a la reference, afin de confirmer la similarite du produit au niveau qualite, securite preclinique, et profil d’efficacite et de securite cliniques dont l’immunogenicite. Les biosimilaires (BS) sont differents des generiques, tant par leur complexite moleculaire que par leur reglementation. Ils representent des enjeux economiques considerables avec l’expiration dans les 5 prochaines annees des brevets de nombreux biomedicaments couvrant des aires therapeutiques majeures comme la cancerologie, la rhumatologie ou la gastroenterologie, qui representent des depenses importantes pour l’Assurance maladie. La table ronde (TR) a emis des recommandations pour que l’arrivee des BS soit un succes, en s’appuyant sur le medecin prescripteur comme acteur de l’information et de la confiance, en mettant en place un suivi des patients traites par biomedicaments, et en accelerant l’acces au marche de leurs biosimilaires.

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.

Changes to the manufacturing of biological agents can lead to drugs with different components from the original medicine tested in clinical trials, challenging assumptions about safety, say David Hunt and colleagues

Approval of the first biosimilar in the USA may occur by the end of 2014, yet a naming approach for biosimilars has not been determined. Biosimilars are highly similar to their biologic reference product but are not identical to it, because of their structural complexity and variations in manufacturing processes among companies. There is a need for a naming approach that can distinguish a biosimilar from its reference product and other biosimilars and ensure accurate tracing of adverse events (AEs) to the administered product. In contrast, generic small-molecule drugs are identical to their reference product and, therefore, share the same nonproprietary name. Clinical trials required to demonstrate biosimilarity for approval may not detect rare AEs or those occurring after prolonged use, and the incidence of such events may differ between a biosimilar and its reference product. The need for precise biologic identification is further underscored by the possibility of biosimilar interchangeability, a US designation that will allow substitution without prescriber intervention. For several biologics, the US Food and Drug Administration (FDA) has used a naming approach that adds a prefix to a common root nonproprietary name, enabling healthcare providers to distinguish between products, avoid medication errors, and facilitate pharmacovigilance. We recommend that the FDA implement a biosimilars naming policy that likewise would add a distinguishable prefix or suffix to the root nonproprietary name of the reference product. This approach would ensure that a biosimilar could be distinguished from its reference product and other biosimilars in patient records and pharmacovigilance databases/reports, facilitating accurate attribution of AEs.

This article was originally published without an Open Access but due to the authors final decision to opt for Open Choice this correction was created.

Introduction Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). These are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. MPN are caused by three main recurrent mutations: JAK2V617F, mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL) genes. Interferon alpha (IFNα) treatment induces not only a hematological response in around 70% of ET, PV and early myelofibrosis, but also a significant molecular response on both JAK2V617F- and CALR-mutated cells. However, a complete molecular response is only achieved in around 20% of patients. Our aim is to predict the long-term efficacy of IFNα in JAK2V617F- and CALR-mutated patients by monitoring the fate of the disease-initiating mutated HSC in order to better stratify the molecular responders. Methods A longitudinal observational study (3-5 years) was performed in 46 IFNα-treated patients. The MPN disease distribution was 42% ET, 47% PV and 11% PMF. We detected 33 patients with JAK2V617F mutation, 11 with CALR mutations (7 type 1/type 1-like and 4 type 2/type 2-like), 1 with both JAK2V617F and CALR mutation and 1 with JAK2V617F, CALR mutation and MPLS505N. At 4-month intervals, the JAK2V617For CALR mutation variant allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was determined by studying the presence of the mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38-HSC-enriched, CD90-CD34+CD38- immature and CD34+CD38+committed progenitors). We used a combination of mathematical modeling (Michor et al., Nature, 2005) and Bayesian analysis to infer the long-term behavior of mutated HSC. Results After a median follow-up of 40 months, IFNα targeted more efficiently and more rapidly the HSPC, particularly the HSC-enriched progenitors, than the mature blood cells in JAK2V617Fpatients (p Since it is very difficult to purify true HSC from patients, we used a combination of mathematical and statistical modeling to infer the behavior and the kinetics of IFNα-targeted mutated HSC. The model gave a good fit to the data and indicated that mutated HSC are exhausted slowly (> 1 year) with concomitant increase in mutated HSPC and granulocytes in well-responding patients. We calculated the rate of HSC decrease for each patient. Rates of decrease are very low for heterozygous JAK2V617F and CALR-mutated HSC and greater for homozygous JAK2V617FHSC, but all increase with high IFNα dose (>100 µg/week). Moreover, very low proportion of heterozygous mutated HSC compared to high proportion can be targeted more easily in patients. The associated mutations at diagnosis and at the last timepoint were also investigated using an NGS-targeted myeloid panel. Results indicate that IFNα does not induce any further mutations on additional genes and the mathematical approach predicts that associated mutations have no major impact on the ratio of HSC decrease. Conclusion Altogether, using a rigorous method of statistical inference, our results show that IFNα exhaust the human mutated HSC by differentiation in HSPC and mature cells. This is likely due to IFNα inducing a stronger proliferation of mutated compared to wild-type HSC, as previously shown in a mouse model (Mullally et al., Blood, 2013). Our study predicts that IFNα can slowly eradicate the mutated HSC, but this beneficial effect would be more efficient: i) in patients with homozygous JAK2V617F versus those with heterozygous JAK2V617F or CALR-mutated, ii) with high IFNα dose, iii) in patients with very low proportion of heterozygous JAK2V617F and CALR-mutated HSC. Thus, this study will help to stratify patients for IFNα treatment.These results might also explain the different outcomes in current IFNα clinical trials. Disclosures Constantinescu: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AlsaTech: Other: Co-Founde; AgenDix GmbH: Other: Co-Founder, MyeloPro Research and Diagnostics; Wiley & Sons: Other: Editor in Chief, Journal of Cellular and Molecular Medicine. Kiladjian:AOP Orphan: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Consultancy.

Antibody-mediated pure red cell aplasia is a very rare but devastating condition affecting patients receiving treatment with erythropoiesis-stimulating agents. New cases continue to emerge, generally in clusters, consistent with an ‘environmental’ trigger to its pathogenesis. Defining the causes of antibody-mediated pure red cell aplasia is clearly of importance for patients with chronic kidney disease, but any developments in this area may also have relevance to other disease areas as therapeutic delivery of endogenous proteins rapidly increases. This review focuses on the current knowledge regarding the etiology of antibody-mediated pure red cell aplasia and the current approach to therapy.

Purpose Following two cases of neutralizing antibodies to epoetin alfa in an investigational clinical study, a small number of individual syringes of two drug product batches were found to contain unusually high levels of aggregation at the end of the clinical trial. Methods We undertook an extensive analytical approach to determine the root-cause of the increased aggregation in the affected batches. Results Soluble tungsten was found in the syringes, most likely derived from the pins used to manufacture the syringes. Spiking of epoetin alfa with sodium polytungstate or an extract of tungsten pins used to manufacture the syringes induced the formation of aggregates, both dimers that appeared to be covalently linked by disulphide bonds as well as higher-order aggregates. Sodium polytungstate had also a strong denaturing effect on the protein. Conclusions We propose tungsten-mediated unfolding and aggregation of epoetin alfa in pre-filled syringes as a potential root cause for increased immunogenicity. This finding may be more broadly applicable to this and other classes of therapeutic proteins.

International audience; BACKGROUND: Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma.DESIGN AND METHODS: Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively.RESULTS: This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category.CONCLUSIONS: As observed with germline von Hippel-Lindau mutations, there are functional differences between the PHD2 mutants with regards to hypoxia inducible factor regulation. PHD2 mutation carriers do, therefore, need careful medical follow-up, since some mutations must be considered as potential candidates for tumor predisposition.

TET2 converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and is frequently mutated in myeloid malignancies, including myeloproliferative neoplasms. Here we show that the level of 5-hmC is decreased in granulocyte DNA from myeloproliferative neoplasm patients with TET2 mutations compared with granulocyte DNA from healthy patients. Inhibition of TET2 by RNA interference decreases 5-hmC levels in both human leukemia cell lines and cord blood CD34+ cells. These results confirm the enzymatic function of TET2 in human hematopoietic cells. Knockdown of TET2 in cord blood CD34+ cells skews progenitor differentiation toward the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. In addition, by monitoring in vitro granulomonocytic development we found a decreased granulocytic differentiation and an increase in monocytic cells. Our results indicate that TET2 disruption affects 5-hmC levels in human myeloid cells and participates in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation.

Introduction: Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. They are due to three main recurrent mutations affecting the JAK/STAT signaling pathway: JAK2V617F and mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL). Interferon alpha (IFNα) is the only drug that not only induces a hematological response in ET, PV and early MF, but also a significant molecular response on both JAK2V617F or CALR-mutated cells. Our broad aim was to understand the mechanism of action of IFNα. Previously, our group and others have shown that IFNα specifically targets JAK2V617F HSC in a chimeric JAK2V617F knock-in mouse model. In this study, we wanted to know how and how fast IFNα impacts the different mutated human hematopoietic compartments. Methods: A prospective study was performed with a cohort of 47 patients treated by IFNα for 3-5 years. The MPN disease distribution was 40% ET, 49% PV and 11% MF. This cohort included 33 JAK2V617F-mutated patients, 11 CALR-mutated patients (7 type 1/type 1-like and 4 type 2/type 2-like), 2 both JAK2V617F- and CALR-mutated patients and 1 MPLW515K-mutated patient. At 4-month intervals, the JAK2V617F or/and CALR mutation allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was also determined by studying the presence of the JAK2V617F or CALR mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38- HSC-enriched progenitors, CD90-CD34+CD38- immature progenitors and CD90- CD34+CD38+ committed progenitors). Results: After a median follow-up of 33 months, IFNα targets more efficiently and rapidly the HSPC particularly in HSC-enriched progenitors, than the mature blood cells in JAK2V617F patients (p Using Ba/F3-MPL cellular models and primary cells, we observed that JAK2V617F was more prone to sensitize to IFNα signaling (increased Phospho-STAT1 and IFN-stimulating genes (ISGs)) compared to controls or CALRdel52 mutated cells. Conclusion: Altogether, our results show that IFNα targets more efficiently the human JAK2V617F-HSPCthan the mature cells. Moreover, IFNα has a greater efficacy on JAK2V617F HSPC thanCALR-mutated HSPC. This former result was associated with a greater priming of the IFNα signaling by JAK2V617F than by CALRdel52. The molecular response was dependent not only on mutational status, but also on the presence of other associated mutations for the CALR-mutated HSPC. Patient data are currently incorporated into a mathematical model taking into account clonal architecture and associated mutations to develop an algorythm able to predict patient response. Figure. Figure. Disclosures Kiladjian: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.

During these past 5 years several studies have provided major genetic insights into the pathogenesis of the so-called classical myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The discovery of the JAK2V617F mutation first, then of the JAK2 exon 12 and MPLW515 mutations, have modified the understanding of these diseases, their diagnosis, and management. Now it is established that almost 100% of PV patients present a JAK2 mutation. Nearly 60% of ET patients and 50% of patients with PMF have the JAK2V617F mutation. The MPLW515 mutations are also present in a small proportion of ET and PMF patients. These mutations are oncogenic events that cause these disorders; however, they do not explain the heterogeneity of the entities in which they occur. Genetic defects have not been yet identified in around 40% of ET and PMF. There are likely additional somatic genetic factors important for the MPN phenotype like the recently described TET2, ASXL1, and CBL mutations. Moreover, polymorphisms in the JAK2 gene have been recently described as associated with MPN. Additional studies of large cohorts are required to dissect the genetic events in MPNs and the mechanisms of these oncogenic cooperations.

We investigated whether a novel, synthetic, peptide-based erythropoietin-receptor agonist (Hematide, Affymax) can stimulate erythropoiesis in patients with anemia that is caused by antierythropoietin antibodies.In this open-label, single-group trial, we enrolled patients with chronic kidney disease who had pure red-cell aplasia or hypoplasia due to antierythropoietin antibodies and treated them with a synthetic peptide-based erythropoietin-receptor agonist. The agonist was administered by subcutaneous injection at an initial dose of 0.05 mg per kilogram of body weight every 4 weeks. The primary end point was a hemoglobin concentration above 11 g per deciliter without the need for transfusions.We treated 14 patients with the peptide agonist for a median of 28 months. The median hemoglobin concentration increased from 9.0 g per deciliter (with transfusion support in the case of 12 patients) before treatment to 11.4 g per deciliter at the time of the last administration of the agonist; transfusion requirements diminished within 12 weeks after the first dose, after which 13 of the 14 patients no longer required regular transfusions. Peak reticulocyte counts increased from a median of 10x10(9) per liter before treatment to peak counts of greater than 100x10(9) per liter. The level of antierythropoietin antibodies declined over the course of the study and became undetectable in six patients. One patient who initially responded to treatment had a diminished hematologic response a few months later despite increased doses of the agonist and required transfusions again; this patient was found to have antibodies against the agonist. One patient died 4 months after the last dose of the agonist, and a grade 3 or 4 adverse event occurred in seven other patients during the study period.This novel agonist of the erythropoietin receptor can correct anemia in patients with pure red-cell aplasia caused by antierythropoietin antibodies. (ClinicalTrials.gov number, NCT00314795.).

The JAKV617F mutation is responsible for the majority of breakpoint cluster region (BCR)/Abelson (ABL)–negative myeloproliferative disorders. Ongoing clinical trials of Janus kinase 2 (JAK2) inhibitors in myeloproliferative disorder patients use small molecules targeting both wild-type and mutated JAK2. To selectively target malignant cells, we developed JAK2V617F-specific small interfering RNAs or short hairpin RNAs. Expression of these RNAs in cell lines or CD34+ cells from patients reduced JAK2V617F-driven autonomous cell proliferation. Mechanisms of inhibition involved selective JAK2V617F protein down-regulation, and consequently, decrease in signal transducer and activator of transcription 5 phosphorylation, cell-cycle progression, and cell survival. However, the addition of high concentrations of cytokines to cell lines or erythropoietin to patient cells greatly reduced growth inhibition. Similarly, the efficacy of a JAK2 small molecule inhibitor on cell line and patient cell proliferation dose dependently decreased with the addition of cytokines. Our results demonstrate that it is possible to specifically target JAK2V617F by RNA interference (RNAi) strategies. In addition, cytokines partially reverse the inhibition induced by both RNAi and small molecule approaches. This strongly suggests that patient cytokine levels in current JAK2 inhibitor clinical trials modulate the outcome of these therapies.

Myeloproliferative diseases (MPDs) represent the commonest cause of splanchnic vein thrombosis (SVT), including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT), but their diagnosis is hampered by changes secondary to portal hypertension, while their influence in the outcome of SVT remains unclear. We assessed the diagnostic and prognostic value of JAK2 and MPL515 mutations in 241 SVT patients (104 BCS, 137 PVT). JAK2V617F was found in 45% of BCS and 34% of PVT, while JAK2 exon 12 and MPL515 mutations were not detected. JAK2V617F was found in 96.5% of patients with bone marrow (BM) changes specific for MPD and endogenous erythoid colonies, but also in 58% of those with only one feature and in 7% of those with neither feature. Stratifying MPD diagnosis first on JAK2V617F detection would have avoided BM investigations in 40% of the patients. In BCS, presence of MPD carried significantly poorer baseline prognostic features, required hepatic decompression procedures earlier, but had no impact on 5-year survival. Our results suggest that JAK2V617F testing should replace BM investigations as initial test for MPD in patients with SVT. Underlying MPD is associated with severe forms of BCS, but current therapy appears to offset deleterious effects of MPD on the medium-term outcome.

Contexte. - Un groupe de travail d'experts mis en place par la Federation nationale des centres de lutte contre le cancer FNCLCC) a publie en 1998 puis a mis a jour regulierement (1999 et 2003) des Recommandations pour l'indication des agents stimulant l'erythropoiese (ASE: epoetine alpha, epoetine beta, darbepoetine) en cancerologie. Cet article presente les resultats de la mise a jour realisee en 2007. Methode. - La methode d'actualisation des SOR repose sur l'analyse critique des meilleures donnees scientifiques disponibles et sur le jugement argumente des experts au sein d'un groupe de travail pluridisciplinaire representatif des modes de pratique et des disciplines concernees par la prise en charge des patients atteints de cancer. Sur la base de l'analyse de la litterature, les conclusions et leur niveau de preuve sont etablies. Ensuite, les conclusions accompagnees du jugement argumente des experts conduisent a l'elaboration des Recommandations. Une Recommandation est une proposition d'une ou de plusieurs attitudes cliniques pour l'aide a la decision du professionnel de sante, destinee a ameliorer la prise en charge du patient atteint de cancer. Avant publication, les RPC-SOR sont revues par des experts independants selectionnes selon le meme principe que le groupe d'experts redacteurs. Resultats. - Les nouvelles donnees de la litterature concernant l'indication de l'ASE dans la prise en charge de l'anemie induite par la radiotherapie chez les patients atteints de cancer n'ont pas engendre de modifications dans les recommandations initiales qui restent donc valides. En revanche, au vu des nouvelles donnees, les recommandations relatives a la prevention de l'anemie en cancerologie et a l'indication du Fer en association avec l'ASE n'etaient plus valides et ont donc ete actualisees. Conclusions. - La validite de ces Recommandations sera verifiee dans le cadre d'un processus de veille systematique qui aura lieu dans 2 ans.

Thirty-six unrelated cases with erythrocytosis of unknown origin were investigated. Exons 5-8 of the erythropoietin receptor gene (EPOR), the von Hippel-Lindau gene, and the prolyl hydroxylase domain protein 2 gene (PHD2) were screened by direct DNA sequencing. The Janus kinase 2 mutation, JAK2 (Val617Phe), was screened by allele specific PCR. In this study, three new mutations of EPOR causing deletions in exon 8 were found: the first led directly to a stop codon [g.5957_5958delTT (p.Phe424X)], the second to a stop codon after one residue [g.5828_5829delCC (p.Pro381GlnfsX1)] and the third to a stop codon following a frameshift sequence of 23 residues [g.5971delC (p.Leu429TrpfsX23)]. One patient had a previously reported EPOR mutation [g.6146A>G (p.Asn487Ser)] and another, a silent one (g.5799G>A). All were heterozygotes. In addition, 2 patients were positive for JAK2 (Val617Phe), and 2 reported elsewhere, were mutated in the PHD2 gene [c.606delG (p.Met202IlefsX71).

Pure red cell aplasia in patients who are treated for anemia of chronic kidney disease with erythropoiesis-stimulating agents such as epoetin was first reported in 1998. Although the incidence of pure red cell aplasia peaked in 2002, it remains important for nephrologists to know how to investigate a suspected case of pure red cell aplasia and how to identify other causes of hyporesponsiveness to erythropoiesis-stimulating agents, which account for the vast majority of such cases. The authors reviewed the current status of information in the literature and drew on their personal experiences with patients regarding the diagnosis and management of epoetin-induced pure red cell aplasia. The mechanism for development of epoetin-induced pure red cell aplasia remains unconfirmed. It generally occurs after the production of neutralizing anti-erythropoietin antibodies. Elucidation of a suspected pure red cell aplasia case requires a systematic approach, beginning with simple measurements such as blood cell counts, because most cases of erythropoiesis-stimulating agent hyporesponsiveness are attributable to other causes. If these criteria indicate that the patient's response to erythropoiesis-stimulating agent therapy is very poor, then bone marrow examination and measurement of anti-erythropoietin antibodies is justified. If pure red cell aplasia is confirmed, then cessation of erythropoiesis-stimulating agent therapy and initiation of immunosuppressive therapy are recommended. Continued study of epoetin-induced pure red cell aplasia is needed to help nephrologists prevent or manage future cases and will have implications for the use of other protein-based therapeutic agents.

Purpose The pathophysiology and causes of retinal vein occlusion (RVO) remain largely unknown. Latent forms of myeloproliferative disorders, which are diagnosed by the presence of in vitro endogenous erythroid colony (EEC) formation, are a well-known cause of intraabdominal vein thrombosis. The suspected diagnosis of a latent myeloproliferative disorder in a patient with RVO, based on the presence of EEC formation, led us to evaluate the association between latent myeloproliferative disorders and RVO. Design Observational case series in a national eye center. Participants Forty-four patients, with a mean age of 46 years (range, 21–62) and central (n = 38) or peripheral (n = 6) RVO responsible for visual acuity decreased to 6/12 or less. Methods In vitro bone marrow culture. Main Outcome Measure Endogenous erythroid colony formation in cytokine-free culture medium. Conventional diagnostic criteria for myeloproliferative disorders and the JAK2 V617F mutation (which is strongly associated with myeloproliferative disorders) were assessed in RVO patients showing EECs. Results Endogenous erythroid colony formation was observed in 12 of 44 (27%) patients with RVO, 13 of 35 (37%) patients with Budd–Chiari syndrome, and 52 of 53 (98%) patients with primary polycythemia (positive control groups) but not in 22 healthy bone marrow donors (negative controls) evaluated at the same time and by the same hematology laboratory. Neither conventional nor genetic diagnostic criteria for myeloproliferative disorders were observed in any patient with both RVO and an EEC at the time of diagnosis or during follow-up. Conclusions Endogenous erythroid colony formation is frequently observed in patients with RVO independently of any detectable myeloproliferative disorder. This opens a new aspect of research on the pathophysiology of this sight-threatening disease.

Immunogenicity profiles of recombinant therapeutic proteins are important to understand because antibodies raised against these molecules may have important clinical sequelae. The purpose of the present study was to demonstrate that a flow cytometric bead array could be used to detect clinically relevant antibodies with specificity to such therapeutics. We chose to evaluate well-characterized specimens from persons treated with epoetin alfa that developed antibody-mediated pure red blood cell aplasia as a means to demonstrate the utility of this platform. Our data show that this assay is capable of detecting anti-epoetin alfa antibodies with a relative antibody concentration of 50 ng/ml, where 25 of 25 sera spiked with antibodies at this concentration scored positive. Moreover, the assay was designed to include positive and negative control beads for each specimen that is processed to ensure the specificity of the signal when detected. Measurement of interassay precision supports quantitative estimates of relative antibody concentrations in the range of 313 to 5,000 ng/ml, where the percent coefficient of variation did not exceed 20%. With respect to clinical specimens, antibodies with specificity for epoetin alfa could be easily detected in a set of specimens from persons with pure red blood cell aplasia that had prior exposure to the EPREX brand of recombinant epoetin alfa. Further development and validation of this approach may facilitate successful widespread application of the method for detection of anti-epoetin alfa antibodies, as well as antibodies directed against other recombinant therapeutic proteins.

Objective To evaluate the potential of Hematide, a PEGylated, synthetic peptide–based erythropoiesis-stimulating agent that is in clinical development for the treatment of anemia associated with chronic kidney disease and cancer, to correct antierythropoietin antibody–associated pure red cell aplasia (PRCA). Materials and Methods The binding of anti-Hematide antibodies (mouse, rabbit, and monkey) to recombinant human erythropoietin (rHuEPO) and of anti-rHuEPO antibodies (mouse, goat, rat, and human) to Hematide were evaluated. An anti-EPO antibody–mediated anemia rat model was developed by subcutaneously administering rHuEPO to rats three times weekly for 4 weeks. Sixty percent of the animals developed PRCA as characterized by severe anemia, reduced reticulocytes, anti-EPO antibodies, and limited bone marrow erythroid precursors. The effect of Hematide administration on the PRCA rats was evaluated. Results Antibodies to EPO do not cross react with Hematide and, conversely, antibodies to Hematide do not cross react with EPO. Hematide corrected antibody-induced anemia in a rat PRCA model. Conclusions The data support the potential of Hematide to correct anti-EPO antibody–associated PRCA in humans. In addition, the data suggest a negligible risk for development of anti-EPO antibody–induced PRCA secondary to Hematide administration.

Rituximab is used in the treatment of lymphoma and autoimmune diseases, for which late-onset neutropenia (LON) were reported. LON-related mechanisms remain unclear. To obtain insights into the mechanisms, we assessed serum, peripheral blood and bone marrow (BM) samples of a patient with LON. Factors classically associated with neutropenia such as anti-neutrophil antibodies, T-LGL, soluble Fas Ligand were not detectable. We then evaluated the kinetics of various cytokines involved in B-cell and granulocyte homeostasis. We found that LON is related to a lack of granulopoiesis in the BM that coincides with a very high level of BAFF, a strong stimulator of B-cell recovery, and hypothesized a hematopoietic lineage competition due to an excessive B-cell recovery in the BM by promotion of B-cell lymphopoiesis over granulopoiesis within common developmental niches. Assessment of serum BAFF levels following rituximab could detect patients at risk of developing LON.

International audience; Mutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia Vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in Essential Thrombocythemia (ET) with mutations in JAK2, the thrombopoietin receptor (MPL) and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L and CALR mutations, using Whole Exome Sequencing and Next Generation Sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, one homozygous SH2B3 mutation, one MPLS505N, one MPLW515R and two MPLS204P mutations. In the remaining patients, four presented a clonal and seven a polyclonal hematopoiesis, suggesting that certain triple negative ETs are not MPNs. NGS on 26 additional triple negative ETs detected only one MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN.

A biosimilar is a biological medicinal product claimed to be similar to a reference biological medicinal product. Its development plan includes studies comparing it with the reference product in order to confirm its similarity in terms of quality, preclinical safety, clinical efficacy, and clinical safety, including immunogenicity. Biosimilars differ from generics both in their molecular complexity and in the specific requirements that apply to them. Since patents on many biological medicinal products will expire within the next 5 years in major therapeutic areas such as oncology, rheumatology and gastroenterology and as those products are so costly to the French national health insurance system, the availability of biosimilars would have a considerable economic impact. The round table has issued a number of recommendations intended to ensure that the upcoming arrival of biosimilars on the market is a success, in which prescribing physicians would have a central role in informing and reassuring patients, an efficient monitoring of the patients treated with biologicals would be set up and time to market for biosimilars would be speeded up.

A biosimilar is a biological medicinal product claimed to be similar to a reference biological medicinal product. Its development plan includes studies comparing it with the reference product in order to confirm its similarity in terms of quality, preclinical safety, clinical efficacy, and clinical safety, including immunogenicity. Biosimilars differ from generics both in their molecular complexity and in the specific requirements that apply to them. Since patents on many biological medicinal products will expire within the next 5 years in major therapeutic areas such as oncology, rheumatology and gastroenterology and as those products are so costly to the French national health insurance system, the availability of biosimilars would have a considerable economic impact. The round table has issued a number of recommendations intended to ensure that the upcoming arrival of biosimilars on the market is a success, in which prescribing physicians would have a central role in informing and reassuring patients, an efficient monitoring of the patients treated with biologicals would be set up and time to market for biosimilars would be speeded up.

Background: Subcutaneous administration of Eprex(®) (epoetin alfa) in patients with chronic kidney disease (CKD) was contraindicated in the European Union between 2002 and 2006 after increased reports of anti-erythropoietin antibody-mediated pure red cell aplasia (PRCA). The Prospective Immunogenicity Surveillance Registry (PRIMS) was conducted to estimate the incidence of antibody-mediated PRCA with subcutaneous administration of a new coated-stopper syringe presentation of Eprex(®) and to compare this with the PRCA incidence with subcutaneous NeoRecormon(®) (epoetin beta) and Aranesp(®) (darbepoetin alfa). Methods: PRIMS was a multicentre, multinational, non-interventional, parallel-group, immunogenicity surveillance registry. Adults with CKD receiving or about to initiate subcutaneous Eprex(®), NeoRecormon(®) or Aranesp(®) for anaemia were enrolled and followed for up to 3 years. Unexplained loss or lack of effect (LOE), including suspected PRCA, was reported, with antibody testing for confirmation of PRCA. Results: Of the 15 333 patients enrolled, 5948 received Eprex(®) (8377 patient-years) and 9356 received NeoRecormon(®)/Aranesp(®) (14 286 patient-years). No treatment data were available for 29 patients. Among 23 patients with LOE, five cases of PRCA were confirmed (Eprex(®), n = 3; NeoRecormon(®), n = 1; Aranesp(®), n = 1). Based on exposed time, PRCA incidence was 35.8/100 000 patient-years (95% CI 7.4-104.7) for Eprex(®) versus 14.0/100 000 patient-years (95% CI 1.7-50.6) for NeoRecormon(®)/Aranesp(®). The incidence of PRCA with Eprex(®) was not significantly different versus comparator ESAs (rate ratio: 2.56; 95% CI 0.43-15.31). An analysis based on observed time produced similar findings. Conclusion: This large, prospective registry demonstrates that PRCA is rare with subcutaneous administration of either the new coated-stopper syringe presentation of Eprex(®), or NeoRecormon(®) or Aranesp(®). This study was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.

International audience; No major predisposition gene for familial myeloproliferative neoplasms (MPN) has been identified. Here we demonstrate that the autosomal dominant transmission of a 700-kb duplication in four genetically related families predisposes to myeloid malignancies, including MPN, frequently progressing to leukemia. Using induced pluripotent stem cells and primary cells, we demonstrate that overexpression of ATG2B and GSKIP enhances hematopoietic progenitor differentiation, including of megakaryocytes, by increasing progenitor sensitivity to thrombopoietin (TPO). ATG2B and GSKIP cooperate with acquired JAK2, MPL and CALR mutations during MPN development. Thus, the germline duplication may change the fitness of cells harboring signaling pathway mutations and increases the probability of disease development.

Purpose: Patients with adult acute myeloid leukemia (AML) with intermediate cytogenetics remain a heterogeneous group with highly variable individual prognoses. New molecular markers could help to refine cytogenetic stratification. Experimental Design: We assessed P-glycoprotein (Pgp) activity and Flt3 internal tandem duplication (ITD+) because of their known prognostic value and because they might lead to targeted therapy. We did a multivariate analysis on 171 patients with adult AML treated in the European Organization for Research and Treatment of Cancer protocols. Results: ITD+ and high Pgp activity (Pgp+) were found in 26 of 171 (15%) and 55 of 171 (32%) of all patients, respectively. ITD and Pgp activities were negative in 94 of 171 (55%, Pgp−ITD− group), mutually exclusive in 73 of 171 (43%, Pgp−ITD+ and Pgp+ITD− groups), and only 4 of 171 (2%, Pgp+ITD+ group) patients were positive for both. In multivariate analyses, Pgp+ITD+ (P < 0.0001) and age (P = 0.0022) were independent prognostic factors for the achievement of complete remission (CR). Overall survival (OS), CR achievement (P < 0.0001), WHO performance status (P = 0.0007), and Pgp+ITD+ status (P = 0.0014) were also independent prognostic factors. In 95 patients with intermediate cytogenetics, the CR rates of ITD+ patients were 40% versus 62% for ITD− (P = 0.099) and 41% versus 67% (P = 0.014) for Pgp+ versus Pgp− patients. In the Pgp−ITD− group (41 of 95), CR rates were 70% versus 44% for others (P = 0.012), OS achieved 48% versus 16% (P < 0.0001) and disease-free survival was 56% versus 27% (P = 0.024), respectively. Furthermore, the OS curves of the intermediate cytogenetics-Pgp−ITD− group were not significantly different from the favorable cytogenetic group. Conclusion: Flt3/ITD and Pgp activity are independent and additive prognostic factors which provide a powerful risk classification that can be routinely used to stratify the treatment of patients with intermediate cytogenetic AML. ITD+ and Pgp+ patients should be considered for targeted therapy.

To study the prevalence of the Val617Phe JAK2 mutation in familial cases of myeloproliferative disorder (MPD) and its possible implication as a predisposing genetic factor, we analyzed 72 families including 174 patients (81 polycythemia vera [PV], 68 essential thrombocythemia [ET], 11 myelofibrosis with myeloid metaplasia [MMM], 12 chronic myeloid leukemia), 1 systemic mastocytosis, and 1 chronic myelomonocytic leukemia (CMML). The JAK2 mutation was found in three quarters of patients with PV and MMM and in half of patients with ET. Among 46 families with at least 2 cases of PV, ET, or MMM, the JAK2 mutation was absent in 6 families, heterogeneously distributed in 18, and present in all MPD patients in 22. Among these 22 families, the absence of the JAK2 mutation both in purified T and B cells in 13 unrelated patients and the observation of variable ratios of the JAK2 mutant allele in patient leucocytes indicated that the Val617Phe JAK2 mutation was acquired in familial MPDs. The JAK2 mutation was present in natural killer cells in two thirds of tested patients (27 of 40), suggesting its occurrence in a multipotent hematopoietic progenitor cell. The analysis of the hematologic profile showed that the homozygous JAK2 mutation confers a proliferative advantage and is associated with the progression of the hematologic disease. (Blood. 2006;108:346-352)

The intimate mechanisms of early onset anemia observed in critically ill patients with septic shock remain unclear.We investigated erythropoiesis abnormalities in this setting by studying morphologic, functional, and biochemical patterns of erythroid lineage.Erythroid lineage in the bone marrow from patients with septic shock who developed early-onset anemia was compared with that of healthy control subjects. Survival and proliferation capacities were quantified in both groups. Biochemical and flow cytometry patterns of apoptosis were dissected by exploring antiapoptotic (erythropoietin [Epo] receptor-dependent) and proapoptotic (death receptor-dependent) pathways.Erythroid lineage was morphologically similar in both groups. Apoptosis of glycophorin-A-positive erythroid precursors was increased in patients versus control subjects as assessed by labeling with annexin V (26.1 +/- 8.8 vs. 3.1 +/- 2.9%, p0.05) or 3-3'-dihexyloxacarbocyanine iodide (55.9 +/- 10.5 vs. 19.1 +/- 5.4%, p0.05), respectively. This was associated with significant overexpression of Fas on erythroid precursors and higher tumor necrosis factor-alpha plasma levels in patients with septic shock vs. control subjects. Moreover, growth capacities of late erythroid progenitors of burst-forming unit erythroids (BFU-Es) at Day 10 were impaired in the presence of serum from patients with septic shock as compared with the effect of serum from control subjects (27 +/- 12 vs. 109 +/- 27 per 10(5) seeded cells, respectively; p0.001). Saturating concentrations of recombinant human Epo (rHuEpo) restored growth capacity of patients' BFU-Es (72 +/- 14 per 10(5) seeded cells) in autologous conditions of serum.Early-onset anemia that may be observed in patients with septic shock is associated with defective erythropoiesis related to an excess of apoptosis that can be counterbalanced in vitro by rHuEpo.

The diagnosis of polycythemia vera (PV) is currently based on clinical and biological criteria defined either by the World Health Organization (WHO) or the Polycythemia Vera Study Group (PVSG).1, 2 Both of these classifications use clinical and biological markers organized in major and minor criteria, allowing to diagnose a PV when a define combination of major and minor criteria is present. Recently, a somatic point mutation of the tyrosine kinase JAK2 (JAK2 V617F) has been described in about 80% of PV patients as well as 30% of essential thrombocythemia (ET) and 50% of idiopathic myelofibrosis (IMF)3, 4, 5, 6 and several in vitro and in vivo experiments demonstrated that this mutation of JAK2 was the molecular event at the origin of PV.3 It was therefore tempting to use the detection of JAK2V617F as a diagnostic test for erythrocytosis. Such an attitude supposes the development of other techniques than sequencing, as this latter is time consuming and not always feasible in hematology laboratories. We thus compared sequencing with two techniques of real-time PCR-based mutation detection (one using the LightCycler® instrument, the other the Taqman® ABI Prism 7500), for the efficiency to detect the JAK2 V617F mutation in 119 samples from patients with suspicion of myeloproliferative disorder (MPD). The three techniques were equivalent in all samples but one, where sequencing failed to detect the mutation revealed by both LightCycler® and Taqman® technologies. To evaluate the sensitivity of these techniques, we tested serial dilutions of the homozygously mutated HEL cell line DNA in the nonmutated TF-1 cell line DNA, and serial dilutions of the genomic DNA from a patient homozygous for the JAK2 V617F mutation in normal DNA. Sequencing failed to detect the mutated allele under 5% of HEL cell line DNA diluted in TF-1 cell line DNA, and under 10% of the homozygously mutated patient's DNA diluted in normal DNA. The sensitivity of LightCycler® and Taqman® techniques were equivalent, slightly higher than sequencing, reaching 0.5–1% of HEL cell line DNA diluted in TF-1 cell line DNA (Figure 1), and 2–4% of a homozygously mutated patient's DNA diluted in normal DNA.

Myeloproliferative disorders include several pathologies sharing the common feature of being clonal hematopoietic stem cell diseases. The molecular basis of chronic myeloid leukemia was characterized many years ago with the discovery of the t(9;22) translocation and its product the BCR-ABL oncoprotein. The recent finding of a recurrent mutation in the Janus 2 tyrosine kinase gene is a major advance in our understanding of the pathogenesis of several other myeloproliferative disorders, including polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Although this work clearly identifies a frequent ( approximately 50%) subgroup of myeloproliferative disorders and explains most biological abnormalities described so far, it also raises the major question of how a single mutation can explain disease heterogeneity. Such a recurrent and unique mutation leading to a tyrosine kinase deregulation would make a suitable target for the development of specific therapies.

Protein tyrosine kinases (PTKs) and phosphatases (PTPs) play a crucial role in normal cell development, and dysfunction of these enzymes has been implicated in human cancers. Polycythemia vera (PV) is a clonal hematologic disease characterized by hypersensitivity of hematopoietic progenitor cells to growth factors and cytokines. Recently, a unique and clonal mutation in the JAK homology 2 (JH2) domain of JAK2 that results in a valine to phenylalanine substitution at position 617 (V617F) was found in the majority of PV patients. This mutation leads to constitutive JAK2 activation and abnormal signaling and induces erythrocytosis in an animal model. The mutation is also found in a significant percentage of patients with idiopathic myelofibrosis (50%) and essential thrombocythemia (30%). Thus, it seems probable that this mutation associates with other molecular genetic events to cause different myeloproliferative disorders (MPDs). One of these secondary events is the transition to homozygosity of the mutated gene in 30% of the PV patients. Other events may include defects in PTPs, but these remain to be characterized. Recent studies represent a great step forward in the molecular pathogenesis in PV and the development of targeted new drugs to treat the disease.

The increased incidence of pure red cell aplasia with an Eprex formulation in uncoated rubber stopper syringes.BackgroundThe incidence of pure red cell aplasia (PRCA) in chronic kidney disease patients treated with epoetins increased substantially in 1998, was shown to be antibody mediated, and was associated predominantly with subcutaneous administration of Eprex®. A technical investigation identified organic compounds leached from uncoated rubber stoppers in prefilled syringes containing polysorbate 80 as the most probable cause of the increased immunogenicity.MethodsThis study investigated whether the incidence of PRCA was higher for exposure to the product form containing leachates than for leachate-free product forms. Antibody-mediated PRCA cases were classified according to indication, product form, and route of administration. Exposure estimates were obtained by country, indication, route of administration, and product form.ResultsFor 2001 to 2003, the PRCA incidence rate for patients with subcutaneous exposure to Eprex in prefilled syringes with polysorbate 80 and uncoated rubber stoppers (leachates present) was 4.61/10,000 patient years (95% CI 3.88–5.43) versus 0.26/10,000 patient years (95% CI 0.007–1.44) for syringes with coated stoppers (leachates absent). The rate difference was 4.35/10,000 patient years (95% CI 3.44–5.26; P < 0.0001); the rate ratio was 17 (95% CI 3.14–707). A substantial rate difference remained in sensitivity analyses that adjusted for exposure to multiple product forms.ConclusionThe epidemiologic data, together with the chemical and immunologic data, support the hypothesis that leachates from uncoated rubber syringe stoppers caused the increased incidence of PRCA associated with Eprex. Currently, all Eprex prefilled syringes contain fluoro-resin coated stoppers, which has contributed to decreased incidence of PRCA with continued surveillance.

We determined bone marrow karyotype at diagnosis in four female acute myeloid leukemia (AML) or myelodysplasia patients, aged between 52 and 56 years. In each case, we observed chromosome rearrangement involving the same 4q24 band. Three patients had a balanced reciprocal translocation as the sole abnormality - t(3;4)(q26;q24), t(4;5)(q24;p16) and t(4;7)(q24;q21) - and the fourth had del(4)(q23q24), +4. We used a set of 4q BAC probes for fluorescent in situ hybridization (FISH) in these four cases. We found a 4q24 submicroscopic deletion in all three translocations, with a common deletion of approximately 0.5 Mb. In three cases, we concluded that rearrangement occurred in an early hematopoietic stem cell, as it was detected, in mosaic with a normal karyotype, in a fraction of remission bone marrow cells, peripheral T and B lymphocytes, malignant lymph node T-lymphoma cells in one case and B-lymphoblastoid cell lines established in two cases. Moreover, one of 10 additional AML patients tested by FISH had a normal karyotype and deletion of one of the commonly deleted probe sequences. A tumor suppressor gene may therefore be involved, especially as two patients developed malignant lymphoma at the same time as myeloid proliferation.

In the vast majority of patients with antibody (Ab)-mediated pure red cell aplasia (PRCA), simple withdrawal of the erythropoiesis-stimulating agent (ESA) does not effectively reverse PRCA. In contrast, immunosuppressive treatments can induce the disappearance of anti-erythropoietin Abs and a reversal of PRCA. Consensus opinion on the optimal therapy has not been established, but individual case reports or case series suggest that kidney transplantation or treatment with corticosteroids plus cyclophosphamide are the most effective therapies. However, treatment with cyclosporine is an interesting alternative, since it appears to be effective in at least two-thirds of patients and with minimal side effects. Due to the key role of ESAs in the management of patients with chronic kidney disease (CKD), some patients have been re-treated with an ESA following resolution of Ab-mediated PRCA. In all reported cases, this treatment increased haemoglobin levels, alleviated the need for transfusions and did not have side effects. However, one should be extremely cautious when deciding to re-treat a patient with ESA, due to the small number of reported cases and the possibility of publication bias.

Summary Myeloproliferative disorders (MPD) are reported in 25‐65% of patients with splanchnic vein thrombosis (SVT). Diagnostic criteria for MPD have not been fully established in this context. Using clusters of abnormal megakaryocytes in bone marrow (BM) biopsy as a reference standard for Philadelphia negative MPD, we assessed the relevance of other criteria currently recommended for the diagnosis of MPD in SVT (128 consecutive SVT patients). First, usual criteria were compared with BM results: endogenous erythroid colony formation (EEC) was strongly correlated with BM results; splenomegaly, blood cell count, total red cell volume, erythropoietin level and cytogenetic were much less accurate. Then, patients were assigned to three groups according to the combination of BM and EEC findings (group I: both present; group II: both absent; group III: other patients); clinical presentation and outcome were compared in each group. At a mean follow-up of 6AE09 ± 6AE6 years, progression to a severe form of MPD occurred in 7 of 31 group I patients (23%), in 1 of 34 group III patients (3%) and 0 of 63 group II patients. The combination of marked splenomegaly and platelet count >200 · 10 9 /l was restricted to groups I and III. In conclusion, in patients with SVT, BM findings and EEC allowed the diagnosis of MPD at risk of aggravation. Marked splenomegaly in association with platelet counts >200 · 10 9 /l constitute a simple index with high specificity but low sensitivity.

Myeloproliferative disorders are clonal haematopoietic stem cell malignancies characterized by independency or hypersensitivity of haematopoietic progenitors to numerous cytokines(1,2). The molecular basis of most myeloproliferative disorders is unknown. On the basis of the model of chronic myeloid leukaemia, it is expected that a constitutive tyrosine kinase activity could be at the origin of these diseases. Polycythaemia vera is an acquired myeloproliferative disorder, characterized by the presence of polycythaemia diversely associated with thrombocytosis, leukocytosis and splenomegaly(3). Polycythaemia vera progenitors are hypersensitive to erythropoietin and other cytokines(4,5). Here, we describe a clonal and recurrent mutation in the JH2 pseudo-kinase domain of the Janus kinase 2 (JAK2) gene in most (>80%) polycythaemia vera patients. The mutation, a valine-to-phenylalanine substitution at amino acid position 617, leads to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity and induces erythrocytosis in a mouse model. As this mutation is also found in other myeloproliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches.

Pure red cell aplasia (PRCA) is a rare condition characterised by an arrest in red blood cell production, which may be congenital or acquired. Recombinant human erythropoietin (epoetin) was introduced in 1989 for the treatment of anaemia of chronic kidney disease patients and has maintained an excellent therapeutic and safety record while treating hundreds of thousands of patients. A very rare, but serious adverse event associated with epoetin administration is a condition in which patients develop neutralising anti-erythropoietin antibodies and, consequently, PRCA. This condition is referred to as epoetin-induced PRCA (epo-PRCA). Since it is a rare condition, many haematologists and nephrologists around the world see the condition infrequently and may be uncertain about the diagnosis. For this reason, an ad hoc international working group of expert haematologists and nephrologists met together to derive new recommendations for the haematological diagnosis of epo-PRCA. These recommendations, which represent the consensus opinions of the working group, address haematological approaches to monitor and investigate suspected epo-PRCA and should help physicians differentiate between PRCA and other bone marrow diseases, as well as, between PRCA and epo-PRCA.

Most, if not all, nephrologists are aware of the explosion of epoetin-associated cases of pure red cell aplasia, particularly in Europe, from 1998 onwards [1–4]. The dramatic increase in reported cases was largely associated with a particular brand of epoetin alfa (Eprex; Erypo) [5], but a few cases have also been reported with epoetin beta [3]. Epoetin-associated pure red cell aplasia is characterized by a sudden loss of response to the drug, with the haemoglobin falling to 5 or 6 g/dl, and requiring regular blood transfusions to maintain a more respectable haemoglobin. The white cell and platelet counts are characteristically normal, but there is usually a profound decrease in circulating reticulocytes (to

Context "The Standards, Options and Recommendations" (SOR) project, started in 1993, is a collaboration between the Federation of French Cancer Centres (FNCLCC), the twenty French cancer centres, and specialists from French public universities, general hospitals and private clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and the outcome of cancer patients. The methodology is based on a literature review and critical appraisal by a multidisciplinary group of experts, with feedback from specialists in cancer care delivery. Objectives To update the Standards, Options and Recommendations clinical practice guidelines for the use of recombinant erythropoietin (epoietin alpha and beta darbepoietin-alpha, EPO) in the management of anaemia in oncology for patient undergoing radiotherapy. Methods The working group identified the questions requiring up-dating from the previous guideline. Medline and Embase were searched using specific search strategies from January 1999 to October 2002. Literature monitoring was performed to identify randomised clinical trials published between October 2002 to November 2003. In addition several Internet sites were searched in October 2002. Results There is no standard attitude for use of rHuEPO in patients undergoing radiotherapy. There is no evidence to support use of rHuEPO in patients with ENT cancer receiving radiotherapy alone. In patients undergoing curative radiotherapy, it is recommended to correct anaemia under I Og/dL using transfusion rather than rHuEPO. When the haemoglobin concentration is between 12g/dL and 14g/dL initial use of rHuEPO can be an option under certain conditions for radiochemotherapy if the risk of anaemia is high with the chemotherapy regimen used. Anaemic patients should be included in clinical trials to clarify the impact of rHuEPO in terms of local control of the tumour and survival.

Summary Background Recombinant human erythropoietin is the standard treatment for anaemia related to chronic kidney disease, and its widespread use has been favoured by a very high therapeutic index. However, since 1998, more than 200 patients worldwide with chronic kidney disease treated in this way have developed neutralising antibodies to erythropoietin, causing pure red cell aplasia. We aimed to collate clinical and pathological features in patients unequivocally shown to have erythropoietin-induced pure red cell aplasia. Methods We retrospectively obtained data from the files of 47 patients with pure red cell aplasia. We assessed treatment and outcome of patients and defined recovery from pure red cell aplasia as an increase in reticulocyte counts to more than 20 000 per μL in patients who were no longer transfusion-dependent. Findings When patients developed pure red cell aplasia, all were receiving erythropoietin subcutaneously, and the product most typically prescribed was epoetin alfa (Eprex, Ortho Biotech). The median delay between start of erythropoietin treatment and occurrence of pure red cell aplasia was 11 months (IQR 7·5–14). Nine patients received no immunosuppressive treatment, and none of these recovered. Of 37 patients who received immunosuppressive therapy, 29 (78%) recovered. All six patients who received a kidney transplant recovered within 1 month, and recovery rates were between 56% and 88% in patients treated with corticosteroids, corticosteroids plus cyclophosphamide, or ciclosporin. No relapse of pure red cell aplasia happened after stopping immunosuppressive treatment, but no patient was rechallenged with erythropoietin. Interpretation Immunosuppressive treatment accelerates recovery from erythropoietin-induced pure red cell aplasia.

The use of recombinant human erythropoietin (rHuEPO) is a major advance in the treatment of patients with anemia caused by chronic renal failure (CRF). The development of antierythropoietin (anti-EPO) antibodies following treatment with rHuEPO has been observed in an increasing number of patients. This causes pure red cell aplasia (PRCA) and requires the definitive withdrawal of rHuEPO. Many patients require immunosuppressive therapy before anti-EPO antibodies disappear completely. We report a case of PRCA owing to anti-EPO in a 20-year-old hemodialyzed man who was receiving immunosuppressive therapy for a liver transplantation carried out in childhood. He required repeated red cell transfusions until a kidney transplantation was performed. He received an induction therapy with antithymocyte globulins and a maintenance regimen consisting of steroids, tacrolimus and mycophenolate mofetil. This new immunosuppressive treatment led to the complete disappearance of anti-EPO antibodies within a few weeks after the kidney transplantation. Erythropoiesis and endogenous erythropoietin synthesis were restored following transplantation, without leading to an increase in the titer of anti-EPO antibodies.