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Acute intermittent porphyria (AIP) is a very rare (orphan) metabolic disorder of porphyrin biosynthesis which is characterized by elevated plasma and urine levels of 5-aminolevulinic acid (5-ALA) and porphobilinogen (PBG). Patients with this disorder which is caused by a germline mutation of the hydroxymethylbilan-synthase (HMBS)-gene have a high risk of primary liver cancer which may be determined by disease activity. The exact mechanism of carcinogenesis of this rare tumor is unknown, however.We analyzed paraffin-embedded formalin-fixed liver tumor and normal liver specimens of two female AIP patients treated at the Munich EPNET center. One patient had developed hepatocellular carcinoma (HCC), the other intrahepatic cholangiocarcinoma (CCA). Since biallelic inactivation of HMBS had been observed in one study, we used Sanger and next-generation sequencing with a 8 gene porphyria panel plus 6 potential modifier loci to search for mutations in DNA extractions.In the patient with the HCC, we found a second inactivating mutation in the HMBS gene in the tumor but not in the adjacent normal liver tissue. No mutation could be found in the liver tissues of the patient with CCA, however.Biallelic inactivation of HMBS or protoporphyrinogen-oxidase (PPOX), another enzyme of porphyrin biosynthesis, has been observed in patients with acute porphyrias and liver tumors. We could confirm this in our patient with HCC with a mutation in HMBS but not in the one with CCA. Since 5-ALA can be converted into carcinogenic substances such as 4,5-dioxovaleric acid (DOVA) or 3,6-dihydropyrazine-2,5-dipropanoic acid (= cyclic dimerization product of 5-ALA), local production of these metabolites in hepatic areas with complete loss of HMBS activity may contribute to liver carcinogenesis.

Givosiran is a novel approach to treat patients with acute intermittent porphyrias (AIP) by silencing of ∂-ALA-synthase 1, the first enzyme of heme biosynthesis in the liver. We included two patients in the Envision study who responded clinically well to this treatment. However, in both patients, therapy had to be discontinued because of severe adverse effects: One patient (A) developed local injection reactions which continued to spread all over her body with increasing number of injections and eventually caused a severe systemic allergic reaction. Patient B was hospitalized because of a fulminant pancreatitis. Searching for possible causes, we also measured the patients plasma homocysteine (Hcy) levels in fluoride-containing collection tubes: by LC–MS/MS unexpectedly, plasma Hcy levels were 100 and 200 in patient A and between 100 and 400 μmol/l in patient B. Searching for germline mutations in 10 genes that are relevant for homocysteine metabolism only revealed hetero- and homozygous polymorphisms in the MTHFR gene. Alternatively, an acquired inhibition of cystathionine-beta-synthase which is important for homocysteine metabolism could explain the plasma homocysteine increase. This enzyme is heme-dependent: when we gave heme arginate to our patients, Hcy levels rapidly dropped. Hence, we conclude that inhibition of ∂-ALA-synthase 1 by givosiran causes a drop of free heme in the hepatocyte and therefore the excessive increase of plasma homocysteine. Hyperhomocysteinemia may contribute to the adverse effects seen in givosiran-treated patients which may be due to protein-N-homocysteinylation.

Homocysteine is a sulfur-containing amino acid formed in the intermediary metabolism of methionine. Amino acid metabolism and heme biosynthesis pathways are complexly intertwined. Plasma homocysteine elevation,This article summarizes published case series in which givosiran, a subcutaneously administered small interfering RNA approved for AHP treatment, appeared to exacerbate dysregulated homocysteine metabolism in patients with AHP. A comprehensive exploratory analysis of ENVISION trial data demonstrated that on a population level, givosiran increased homocysteine but with wide interpatient variations, and there is no proof of correlations between HHcy and changes in efficacy or safety of givosiran.The strong correlation and co-increase of homocysteine and methionine suggest that HHcy associated with givosiran is likely attributable to the impaired trans-sulfuration pathway catalyzed by cystathionine β-synthase, which uses vitamin B6 as a cofactor. Data-based consensus supports monitoring total plasma homocysteine and vitamin B6, B12, and folate levels before and during givosiran treatment; supplementing with pyridoxine/vitamin B6 in patients with homocysteine levels100 μmol/L; and involving patients with homocysteine levels30 μmol/L in decisions to supplement.

Im vorangegangenen Kapitel wurde die Zusammensetzung des Blutplasmas erlautert. Hier geht es nun um die zellularen Elemente des Blutes. Erythrocyten, Thrombocyten und Leukocyten ist gemeinsam, dass sie durch den Vorgang der Hamatopoese aus pluripotenten hamatopoetischen Stammzellen entstehen.

Als flussiges und stromendes Organ ubernimmt das Blut eine Vielzahl von Transportfunktionen innerhalb des Organismus. An erster Stelle ist der Gastransport zu nennen. Sauerstoff wird von der Lunge zu den verbrauchenden Geweben, Kohlendioxid als Abfallprodukt des oxidativen Stoffwechsels in die entgegengesetzte Richtung transportiert.

Mit Hilfe des Gefassystems wird das Blut uber den gesamten Organismus verteilt. Mechanismen zur Abdichtung von Blutgefasen im Fall von Verletzungen sind daher von groser Wichtigkeit. Sie beruhen auf der Fahigkeit zur reversiblen Bildung eines Fibrinpfropfes. Dieser wird durch die Thrombocyten als zellulare Bestandteile sowie die plasmatischen Komponenten des Blutgerinnungssystems gebildet.

Objective: To present the Central European Myeloproliferative Neoplasm Organisation (CEMPO) treatment recommendations for polycythaemia vera (PV). Methods: During meetings held from 2015 through 2017, CEMPO discussed PV and its treatment and recent data. Results: PV is associated with increased risks of thrombosis/thrombo-haemorrhagic complications, fibrotic progression and leukaemic transformation. Presence of Janus kinase (JAK)-2 gene mutations is a diagnostic marker and standard diagnostic criterion. World Health Organization 2016 diagnostic criteria for PV, focusing on haemoglobin levels and bone marrow morphology, are mandatory. PV therapy aims at managing long-term risks of vascular complications and progression towards transformation to acute myeloid leukaemia and myelodysplastic syndrome. Risk stratification for thrombotic complications guides therapeutic decisions. Low-risk patients are treated first line with low-dose aspirin and phlebotomy. Cytoreduction is considered for low-risk (phlebotomy intolerance, severe/progressive symptoms, cardiovascular risk factors) and high-risk patients. Hydroxyurea is suspected of leukaemogenic potential. IFN- has demonstrated efficacy in many clinical trials;its pegylated form is best tolerated, enabling less frequent administration than standard interferon. Ropeginterferon alfa-2b has been shown to be more efficacious than hydroxyurea. JAK1/JAK2 inhibitor ruxolitinib is approved for hydroxyurea resistant/intolerant patients. Conclusion: sGreater understanding of PV is serving as a platform for new therapy development and treatment response predictors.

Anagrelide is an established therapy for essential thrombocythemia. Common adverse effects have been linked to peak plasma concentrations of anagrelide and its 3OH metabolite. Our study was performed to investigate the pharmacokinetics (PK) of a novel anagrelide extended‐release (AER) formulation and its active metabolites. Thirty healthy volunteers were randomized to receive either 2 mg AER (under fasting and fed conditions) or 2 mg commercially available reference product (CARP) in an open‐label, 3‐way crossover trial with washout periods of 6 days. Plasma concentrations of anagrelide and its active metabolites were assessed by tandem mass spectrometry. The PK differed significantly between all treatment periods. Bioavailability of AER was 55% of the CARP under fasting conditions and 60% under fed conditions. Cmax, AUCt, and AUC∞ were significantly higher and Tmax and T1/2 were significantly shorter after the CARP compared with AER. Food had a significant impact on the PK of AER, increasing the Cmax and AUCt while reducing the T1/2, plateau, and mean residence time. Both formulations were well tolerated, with a trend toward more frequently occurring adverse events after the CARP. The PK of AER and the CARP differed significantly in all parameters. Food enhanced the bioavailability of AER.

In Germany, analyses of clinical and laboratory features of patients with acute porphyrias are only available for hereditary coproporphyria (HCP) but not with other acute porphyrias, acute intermittent porphyria (AIP) and variegate porphyria (VP). The aim of the study was to analyze a large cohort of patients with particular focus upon quality of life aspects. Sixty-two individuals from separate families with acute porphyrias (57 AIP, 5 VP) were included into an observational study collecting biochemical, genetic, and clinical data. A questionnaire was designed to complete anamnestic information and to assess the influence on quality of life. Most frequent signs and symptoms or laboratory abnormalities were abdominal colicky pain, red coloration of urine, and hyponatremia. Depression or anxiety was reported by 61% or 52% individuals, respectively. Fatigue was mentioned as the most quality of life-limiting symptom. In 59/61 patients, mutations could be identified. 44% (20/45) had to be admitted to an intensive care unit. Heme arginate was used in 64% (29/45) of patients for treatment of acute attacks at least once and in 33% for long-term treatment with high frequency of administration. Serum creatinine values increased in 47% (7/17) of the patients with recurrent attacks. Our analysis confirms a substantial influence of the diseases on the quality of life on patients. Percentages of urine discoloration and intensive care unit admissions were much higher than in other reports. Long-term treatment with heme arginate requires careful monitoring of iron status and renal values.

Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPN) comprise a heterogeneous group of chronic hematologic malignancies. The quality of life, morbidity, and mortality of patients with MPN are primarily affected by disease-related symptoms, thromboembolic and hemorrhagic complications, and progression to myelofibrosis and acute leukemia. Major bleeding represents a common and important complication in MPN, and the incidence of such bleeding events will become even more relevant in the future due to the increasing disease prevalence and survival of MPN patients. This review discusses the causes, differential diagnoses, prevention, and management of bleeding episodes in patients with MPN, aiming at defining updated standards of care in these often challenging situations.

Cancer can trigger thromboembolism. There is a 4-10% chance of finding an asymptomatic occult cancer in patients with idiopathic venous thromboembolism (VTE). Current guidelines recommend limited cancer screening with history, physical examination, and screening examinations according to age after idiopathic VTE. Recent studies found that a more extensive screening program, including endoscopy and computed tomography, may increase the cancer detection rate. The Hemostasis Working Group of the German Society of Hematology and Oncology recommends a more extensive screening program after idiopathic VTE.

Patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) like polycythemia vera and essential thrombocythemia are at increased risk of arterial and venous thrombosis. Strategies of prevention may consist of platelet aggregation inhibitors and/or cytoreductive agents depending on the underlying disease and the individual risk. Clinical evidence for management of acute venous thromboembolic events in MPN patients is limited. Modality and duration of therapeutic anticoagulation after venous thrombosis has to be evaluated critically with special regard to the increased risk for spontaneous bleeding events associated with the underlying diseases. Both for therapy of the acute event and for secondary prophylaxis, low-molecular-weight heparins should preferentially be used. A prolongation of the therapeutic anticoagulation beyond the usual 3 to 6 months can only be recommended in high-risk settings and after careful evaluation of potential risks and benefits for the individual patient. New direct oral anticoagulants (NOAC) should not preferentially be used due to lack of clinical experience in patients with MPN and potential drug interactions (e.g. with JAK inhibitors). Consequent treatment of the underlying myeloproliferative disease and periodical evaluation of the response to therapy is crucial for optimal secondary prophylaxis of thromboembolic events in those patients.

Aquagenic pruritus (AP) is a symptom typical for polycythemia vera, but very little is known about its exact frequency, characteristics, influence on quality of life, and proper treatment. Therefore, we investigated these aspects in a large cohort of German patients with polycythemia vera using a patient directed questionnaire. Our analysis revealed that 301 of 441 analyzed patients suffered from AP. In 64.8%, AP occurred on average 2.9 years prior to diagnosis of polycythemia vera. Only in 15.4% did this lead to a hematological investigation. AP occurs primarily on the trunk and proximal parts of the extremities. Most patients complain about itching (71.8%), the remainder about tickling, stinging, or burning sensations. Forty-four patients (14.6%) classified the pruritus as "unbearable." Patients with AP reported reduced global health status and higher fatigue, pain, and dyspnea. Only 24% of patients received pruritus specific treatment for pruritus consisting mostly of histamine antagonists, which ameliorated symptoms in about half of the patients. In 5.6% of patients, polycythemia vera directed therapy (phlebotomy/cytoreduction) resolved the symptoms. In summary, AP is a serious symptom in patients with polycythemia vera, which until recently was difficult to treat. The advent of the novel JAK2 inhibitors, however, may open new ways for therapy.

High platelet counts in essential thrombocythemia (ET) can be effectively lowered by treatment with either anagrelide or hydroxyurea. In 259 previously untreated, high-risk patients with ET, diagnosed according to the World Health Organization classification system, the efficacy and tolerability of anagrelide compared with hydroxyurea were investigated in a prospective randomized noninferiority phase 3 study in an a priori-ordered hypothesis. Confirmatory proof of the noninferiority of anagrelide was achieved after 6 months using the primary end point criteria and was further confirmed after an observation time of 12 and 36 months for platelet counts, hemoglobin levels, leukocyte counts (P < .001), and ET-related events (HR, 1.19 [95% CI, 0.61-2.30], 1.03 [95% CI, 0.57-1.81], and 0.92 [95% CI, 0.57-1.46], respectively). During the total observation time of 730 patient-years, there was no significant difference between the anagrelide and hydroxyurea group regarding incidences of major arterial (7 vs 8) and venous (2 vs 6) thrombosis, severe bleeding events (5 vs 2), minor arterial (24 vs 20) and venous (3 vs 3) thrombosis and minor bleeding events (18 vs 15), or rates of discontinuation (adverse events 12 vs 15 or lack of response 5 vs 2). Disease transformation into myelofibrosis or secondary leukemia was not reported. Anagrelide as a selective platelet-lowering agent is not inferior compared with hydroxyurea in the prevention of thrombotic complications in patients with ET diagnosed according to the World Health Organization system. This trial was registered at http://www.clinicaltrials.gov as #NCT01065038.

Background Severe hereditary coagulation factor XIII deficiency is a rare homozygous bleeding disorder affecting one person in every two million individuals. In contrast, heterozygous factor XIII deficiency is more common, but usually not associated with severe hemorrhage such as intracranial bleeding or hemarthrosis. In most cases, the disease is caused by F13A gene mutations. Causative mutations associated with the F13B gene are rarer. Design and Methods We analyzed ten index patients and three relatives for factor XIII activity using a photometric assay and sequenced their F13A and F13B genes. Additionally, structural analysis of the wild-type protein structure from a previously reported X-ray crystallographic model identified potential structural and functional effects of the missense mutations. Results All individuals except one were heterozygous for factor XIIIA mutations (average factor XIII activity 51%), while the remaining homozygous individual was found to have severe factor XIII deficiency (

Anagrelide hydrochloride is an anti-thrombotic agent indicated for the treatment of essential thrombocythemia (ET). In various previously published clinical trials of 2 branded formulations of anagrelide in patients with ET at high risk for thrombohemorrhagic events, the rates of adverse events and discontinuation were strikingly divergent between brands. Because the formulations and manufacturers differed, the differences in tolerability, as well as platelet counts, might have been related to differences in pharmacokinetic properties between the 2 formulations.The present series of investigations (1) determined the pharmacokinetic profile of anagrelide and its metabolites; (2) compared the pharmacokinetic profiles of the test and reference formulations of anagrelide; (3) investigated the in vitro release of anagrelide as a marker of intragastric anagrelide release of the test and reference formulations; and (4) compared the platelet-reducing effects of the test and reference formulations in patients with thrombocythemia in 2 longitudinal studies over 4 weeks.A series of 4 in vivo studies and 1 in vitro study were conducted. In a pilot, prospective, singledose study in healthy volunteers, the pharmacokinetic properties (C(max), T(max), and AUC(0-infinity)) of a test formulation of anagrelide were assessed using high-performance liquid chromatography analysis of plasma samples. Based on the results from that study, a single-dose, randomized, double-blind, 2-period crossover study in healthy volunteers was conducted to determine bioequivalence of 2 formulations of anagrelide 2 mg/d (taken as 4 capsules). In vitro dissolution properties of the test or reference formulation containing 0.5 mg anagrelide as the active ingredient were studied in an assay mimicking gastrointestinal release. To test for effects on platelet counts of switching from the reference formulation (previous treatment on stable dose for 3 months) to the test formulation, two 4-week longitudinal trials were conducted: one in patients with ET (in Germany), and one in patients with thrombocythemia associated with chronic myeloproliferative disorders (CMPDs) (in Austria).The pilot pharmacokinetic study of the test formulation in 16 volunteers (10 women, 6 men; mean [SD] age, 20.5 [1.5] years; weight, 69.0 [10.0 kg) suggested that anagrelide was metabolized to 3-hydroxyanagrelide (AUC(0-infinity) 50% compared with anagrelide) and the inactive metabolite 2-amino-5,6-dichloro-,4-dihydroquinazolone. The subsequent bioequivalence study in 24 volunteers (14 women, 10 men; mean [SD] age, 23 [4] years; white, 100%; weight, 67.5 [10.2] kg) found that the test formulation was associated with a significantly lower C(max) (point estimation [PE], 66%; 90% CI, 58%-76%; P0.001) and AUC(0-infinity) (PE, 77%; 90% CI, 68%-86%; P = 0.001). T(max) values for anagrelide and 3-hydroxyanagrelide were 1 hour longer with the test formulation compared with the reference formulation. The total number of adverse events with the reference formulation was 46; the test formulation, 29 (P = 0.05). In vitro, anagrelide from the reference formulation was immediately released (89.1% at 5 minutes), whereas there was a delayed release (93.6% at 30 minutes) from the test formulation (P0.05). In the last 2 studies, 2 cohorts of white patients (cohort 1, 15 patients with ET; 10 women, 5 men; mean [SD] age, 49.0 [10.7] years [range, 31-66 years]; weight, 73.2 [12.6] kg; cohort 2, 19 patients with thrombocythemia associated with CMPD; 12 women, 7 men; age, 62.6 [12.4] years [range, 38-80 years]; weight, 66.1 [13.3] kg) who had received treatment foror =3 months with the reference formulation were switched to the same dose of the test formulation and maintained on this dose for 4 weeks. Platelet counts did not change significantly from baseline over 4 weeks and stayed within a predefined margin of 150 x 10(3) cells/microL.The pharmacokinetic properties, adverse event rates, and in vitro dissolution profile differed between the test and reference anagrelide formulations in these healthy volunteers. In patients with ET or thrombocythemia associated with CMPD, platelet counts did not differ significantly from baseline at 4 weeks when subjects were switched from the reference to the test anagrelide formulation.

Die primare Thrombozythamie (PT) ist eine seltene, erworbene chronische Erkrankung des Knochenmarks, die in jedem Lebensalter auftreten kann. Die Diagnose beruht auf erhohten Thrombozytenwerten, morphologisch und funktionell veranderten Thrombozyten und charakteristischen Knochenmarkveranderungen sowie dem Ausschluss verwandter myeloproliferativer Erkrankungen, die ebenfalls mit erhohten Thrombozytenzahlen einhergehen konnen. Mogliche Komplikationen der Erkrankung sind Thromboembolien und Hamorrhagien sowie der Ubergang in eine Fibrose des Knochenmarks (Myelofibrose) oder eine akute Leukamie. Die Hemmung der Thrombozytenaggregation mit Aspirin zur Pravention thromboembolischer Komplikationen stellt die Basistherapie dar; eine Senkung der erhohten Thrombozytenwerte (zytoreduktive Therapie mit Hydroxyharnstoff oder Interferon-α bzw. thromboreduktive Therapie mit Anagrelid) ist bei bereits bei Diagnose eingetretenen thromboembolischen Komplikationen (Sekundarpravention) bzw. bei steigenden Thrombozytenwerten und dem gleichzeitigen Vorliegen verschiedener Risikofaktoren angezeigt (Primarpravention). Aufgrund der potentiellen Nebenwirkungen der verschiedenen Therapien bei Langzeitanwendung ist eine strenge Nutzen-Risiko-Analyse vor ihrer Einleitung erforderlich. Bei bis zu 50% der Patienten ist die kurzlich entdeckte V617F-Mutation im Gen der Januskinase 2 (JAK2) nachweisbar, die an der Pathogenese der PT beteiligt ist. Wegen der Seltenheit der Erkrankung existieren bisher nur zwei prospektiv randomisierte klinische Studien zum Vergleich zyto- und thromboreduktiver Therapien. In der britischen PT1-Studie wurde die obligate Kombination von Anagrelid und Aspirin mit der von Hydroxyharnstoff und Aspirin verglichen, in der europaischen ANAHYDRET-Studie die Monotherapie mit Hydroxyharnstoff und Anagrelid. Dieses unterschiedliche Design ist der Grund fur eine kontrovers gefuhrte Diskussion uber die bisherigen Studienergebnisse. Die Durchfuhrung klinischer Therapiestudien lasst fur die Zukunft erwarten, dass Expertenmeinungen zunehmend durch evidenzbasierte Konzepte ersetzt werden. Die verbesserte Kenntnis der molekularen Grundlagen der Erkrankung durch die Entdeckung der V617F-Mutation im JAK2-Gen hat die molekulare Diagnostik verbessert und den Weg zu molekular orientierten Therapien eroffnet.

Primary thrombocythaemia (PT) is the most frequent among the rare chronic myeloproliferative disorders. Life expectancy is determined by thromboembolic and haemorrhagic complications, which can be prevented by cytoreductive therapy. For a long time, hydroxyurea has been considered as the therapeutic gold standard. However, hydroxyurea treatment is not lineage-specific, may not be tolerated because of adverse effects (skin, gastrointestinal tract) and is leukaemogenic when sequentially used with other DNA-targeting drugs. Hence, anagrelide was welcomed in 1988 when it was first described as being efficient at normalising elevated platelet counts, specific for megakaryocytes and non-mutagenic. Since then, anagrelide has been approved in the US and Canada (Agrylin), Shire Pharmaceuticals) as well as in Austria and other countries of the EU (Thromboreductin), AOP Orphan Pharmaceuticals). Clinical Phase III trials (PT1 and ANAHYDRET) are underway to directly compare the efficacy and safety of anagrelide and hydroxyurea.

Acute intermittent porphyria (AIP) is a very rare autosomal dominant disorder with low penetrance. Mutations in the gene of the porphobilinogen deaminase (PBG-D), also called hydroxymethylbilane synthase (HMBS), cause a partial deficiency of this enzyme of the heme biosynthetic pathway. Overstimulation of heme biosynthesis causes clinical symptoms. Because of the variability of the symptoms, diagnosis is often delayed. Using two approaches for genetic analysis, first in a stepwise manner, then sequencing extensive parts of the gene, the screening of the DNA of 20 unrelated individuals revealed 20 different mutations, 11 of which had not been reported previously. The novel mutations affected intron 1 (33 + 2 T→C), exon 5 (181 G→C), intron 6 (267–61 del 8 bp), intron 7 (345–1 G→C), intron 9 (498 + 15 G→T and 499–13 Δ-14 bp indel TGA), intron 13 (825 + 1 G→C and 825 + 2 T→C), exon 15 (962 G-A, 1067 del A and 1067–1068 ins 5 bp). The other nine mutations detected affected intron 14, exons 6, 7, 8, 9, 10 (3×) and 12. In the majority of AIP patients, the genotype does not predict phenotypic expression. Since the sudden manifestation of the disease maybe prevented by early diagnosis, identification of AIP gene carriers is the best preventive measure. This was performed in five families, revealing 10 additional AIP gene carriers.

Porphyrine bestehen aus vier jeweils unterschiedlich substituierten Pyrrolringen, die durch Methinbrucken verknupft sind. Das wichtigste Porphyrin fur den tierischen Organismus ist das eisenhaltige Ham, da es Bestandteil des Hamoglobins und vieler anderer Ham-Proteine ist. Die Ham-Synthese erfolgt in allen Zellen aus Glycin und Succinyl-CoA. Sie wird in erythroiden und nicht-erythroiden Geweben unterschiedlich reguliert.

Tumorzellen sind strukturiert und hochspezialisiert, was auch beinhaltet, dass sie von den normalen Organzellen und der organspezifischen extrazellularen Matrix Signale empfangen und fur ihr Wachstum nutzen.

Die morphologische Definition eines malignen Tumors als Ansammlung von Zellen, die keine Kontaktinhibition zeigen, invasiv uber Basalmembranen hinweg wachsen und in entfernten Organen Tochtergeschwulste (Metastasen) bilden konnen, ist seit mehr als 100 Jahren diagnostisch relevant.

Background : Anagrelide hydrochloride (ana), is a well-known compound used to selectively normalize platelet counts (plc) by inhibiting megakaryocyte development and maturation. Common side-effects of licensed formulations may be largely due to peak plasma concentrations of ana and its 3OH-metabolite, whereas efficacy is proportional to the Area under the curve (AUC). A novel extended-release formulation (ana retard, AR) has superior pharmacokinetics (Petrides 2015) and has been demonstrated to be equally effective as the licensed formulation (Gisslinger 2016). Here, we investigate a "treat-early" concept in non-high risk ET patients. Study design:AR was compared to placebo in a phase 3, randomized, parallel group, multicenter, subject- and sponsor-blinded trial in patients diagnosed with ET according WHO 2008 and a defined risk status including JAK2 mutation, protein C/S or antithrombin III deficiency, factor V Leiden or prothrombin mutation, cardiovascular risk factors. The primary endpoint was time to first ET-related event adjudicated by a blinded expert panel, or progressive thrombocytosis (plc≥1000G/L or plc increase >300G/L within 3 months) requiring medical intervention. Secondary endpoints included plc response and change of risk status. Results : 146 patients were randomized and dosed (all Caucasian, mean age 43 years, 74% females), and 112 completed the first year with drop-out rates of 22% in AR and 25% in placebo. Only 49% of placebo patients vs. 74% of AR patients consented to enter the extension phase. Consequently, over the entire study duration, median exposure times differed significantly with 123 weeks for AR and 62 weeks for placebo. The primary endpoint was met in all analysis sets, with p Conclusions: Long term treatment of ET patients with defined risk status, using this novel modified-release anagrelide formulation proofed well tolerable, normalized plc in a vast majority, significantly reduced progression to high-risk status and most importantly was associated with significantly less clinical ET related events. These data have important implications for the optimal management of ET and support a "treat-early" approach. References Results of a phase I, single dose, randomized, 3-way crossover study, to assess the bioavailability of a novel anagrelide extended release (ER) formulation in comparison to a commercially available anagrelide reference product (CARP) in healthy volunteers, Petrides P, Zagrijtschuk O, Klade C, DGHO, 2015 Phase 3 trial TEAM-ET in 106 high-risk Essential Thrombocythemia patients, demonstrating non-inferiority of Anagrelide Retard, a novel, extended-release anagrelide formulation, to the licensed comparator Gisslinger H, Radinoff A, Karyagina E,Kyrcz-Krzemień S, Abdulkadyrov K, Gerbutavicius R,Melikyan A, Burgstaller S, Hus M, Kłoczko J, Yablokova V, Tzvetkov N, Całbecka M, Shneyder T, Warzocha K, Jurgutis M, Kaplanov K, Hodisch J, Klade C, Buxhofer-Ausch V, European School of Hematology EHA 21st Congress, 2016 Disclosures Gisslinger: AOP Orphan Pharmaceuticals AG, Novartis, Celgene, Baxalta: Consultancy, Honoraria, Research Funding. Klade:AOP Orphan: Employment. Kyrcz-Krzemien:AOP Orphan Pharmaceuticals AG, Novartis, BMS, Medac: Honoraria. Warzocha:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Grohmann-Izay:AOP Orphan Pharmaceuticals AG: Employment. Hodisch:AOP Orphan Pharmaceuticals: Employment. Widmann:AOP Orphan Pharmaceuticals: Employment. Kralovics:Qiagen: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Research Funding. Schwarz:AOP Orphan Pharmaceuticals: Consultancy. Kiladjian:Novartis: Research Funding; AOP Orphan: Research Funding.

We report the case of a 46-year-old female with coexisting type I Gaucher's disease and chronic myeloid leukemia (CML). The diagnosis of Gaucher's disease was made in early childhood by bone marrow biopsy and was recently confirmed by biochemical demonstration of reduced leukocyte beta-glucocerebrosidase activity and the presence of Gaucher cells in a bone marrow aspirate. We analyzed the patient's genomic DNA for the underlying glucocerebrosidase mutations and have found homozygosity for a C-->T transition in cDNA nucleotide 593 (159 Pro-->Leu), presently an undescribed mutation. After initiation of replacement therapy with alglucerase we observed a significant increase of the platelet count in our patient. The diagnosis of CML was based on standard hematological parameters and the detection of the Philadelphia chromosome (Ph). With intermittent treatment with busulfan the patient has remained in chronic phase for nine years. The patient suffered from hepatosplenomegaly and thrombocytopenia, both of which can be caused by Gaucher's disease and CML. The aggravation of skeletal manifestations of Gaucher's disease, which occurred at the time of diagnosis of CML, could be due to increased production of leukocyte-derived glucocerebrosides that were not appropriately degraded because of the genetic beta-glucocerebrosidase deficiency.

Matrix metalloproteinases have been reported to be involved in tumor cell invasion and metastasis. Dissemination of malignant cells in acute myeloid leukemia (AML) may be mediated by similar mechanisms. Here, we report, that the t(15/17)+ acute promyelocytic leukemia (APL) cell line NB4 constitutively expresses and releases the proenzyme form of matrix metalloproteinase-9 (MMP-9, 92 kDa type IV collagenase/gelatinase, gelatinase B), as well as tissue inhibitor of metalloproteinases-1 (TIMP-1). Both proteins were identified by N-terminal amino acid sequence analysis after purification using gelatin Sepharose affinity chromatography. Whereas 12-O-tetradecanoylphorbol-13 acetate (TPA) increased both MMP-9 and TIMP-1 mRNA levels, tumor necrosis factor-alpha (TNF-alpha) stimulated only MMP-9 gene expression in a dose- and time-dependent manner. Neutralizing monoclonal antibodies (MoABs) to TNF-alpha (anti-TNF-alpha) decreased the constitutive and TPA-dependent expression of MMP-9 but did not influence TIMP-1 expression, either in unstimulated or in TPA-treated NB4 cells. FACS analyses showed that NB4 cells express both TNF receptor 1 (TNF-R1) and TNF-R2 to a similar extent. Blocking MoABs against TNF-R 1 (anti-TNF-R1) decreased the constitutive expression of MMP-9, whereas anti-TNF-R2 had almost no effect. Our results show, that in NB4 cells the expression of MMP-9 but not of TIMP-1 is maintained by autocrine stimulation with TNF-alpha. Thus, leukemic cells may be enabled to leave the bone marrow and infiltrate peripheral tissues by a dysfunction in the regulation of the MMP-9:TIMP-1 equilibrium, possibly triggered through autostimulation by TNF-alpha.

Chronic myelogenous leukemia (CML) is usually treated with hydroxyurea or interferon-alpha. In some patients high platelet counts develop although leukocyte counts are well controlled with these drugs. If in such a situation cytoreductive therapy has to be intensified by a increase of the dosage, anemia and leukocytopenia as well as adverse effects of the drugs are likely to occur. In twelve CML patients we have therefore combined the basic CML treatment with anagrelide. This drug which selectively reduces platelet counts has been shown to be efficacious in the control of thrombocytosis in essential thrombocythemia. The diagnosis had been confirmed in all CML patients by cytogenetic and/or molecular biological analysis. The median age of our group was 58 years. Five were women and seven men. All patients were on treatment with hydroxyurea, some of them had previously received treatment with interferon-alpha (alone or in combination with hydroxyurea), busulfan or melphalan. Prior to the initiation of anagrelide treatment the platelet count was between 970,000 and 3,600, 000/microl (median about 2,000,000/microl). Seven patients had thrombohemorrhagic complications. All twelve patients, experienced hematologic responses, since their platelet counts decreased to less than 600,000/microl. The median platelet count after reduction was 343,000/microl. The median dosage required to achieve these responses and to maintain them for a period of at least four weeks was 1.9 mg/day. Thrombohemorrhagic complications disappeared or did not recur in all symptomatic patients. Adverse effects were seen in 3/12 patients: headache (1), tachycardia (1), palpitation (1) and fluid retention (1). Whereas these symptoms were mild and transitory they caused one patient to request discontinuation of treatment. Currently five patients are still on treatment with anagrelide (median duration of treatment 11 months) while therapy had to be discontinued in the seven others because of bone marrow transplantation, development of osteomyelofibrosis, blast crisis or on patient request. In our experience anagrelide is a useful therapeutic adjunct when thrombocytosis in patients with CML cannot properly controlled alone with traditional drugs.

Acute intermittent porphyria (AIP) is the most frequent acute porphyria. Symptomatic patients and asymptomatic gene carriers are characterized by a reduction of their porphobilinogen-deaminase (PBG-D) activity to 50%, which is sufficient for porphyrin biosynthesis. PBG-D is encoded by two different mRNAs which are expressed in a tissue-specific manner. In classical AIP, the enzyme activity is reduced in erythroblasts and all other heme-forming body cells, whereas in the variant form of AIP, the PBG-D activity in erythroid tissues remains normal. Acute porphyria attacks can occur in gene carriers when the biosynthesis of heme is increased by drugs, low calory intake, alcohol consumption or infections. Under these conditions, PBG-D cannot convert the precursors adequately so that PBG and δ-aminolevulinate accumulate. This may lead to neurovisceral symptoms and other neurological complications which are potentially life threatening. In patients with AIP, mutation analysis by PCR-DGGE (denaturing gradient gel electrophoresis) is becoming increasingly important since it also permits rapid identification of their presymptomatic relatives. Using this technique, more than 120 mutations have been identified in the PBG-D gene. When identified, the family members are informed about their genetic predisposition and are taught how to prevent porphyric attacks. Here, I illustrate this preventive strategy by describing a German kindred of an affected patient with the variant form of AIP with 17 family members.

Gaucher disease is the most common lysosomal storage disease with a high prevalence in the Ashkenazi Jewish population but it is also present in other populations. The presence of eight mutations (1226G, 1448C, IVS2+1. 84GG, 1504T, 1604T, 1342C and 1297T) and the complete deletion of the beta-glucocerebrosidase gene was investigated in 25 unrelated non-Jewish patients with Gaucher's disease in Germany. In the Jewish population, three of these mutations account for more than 90% of all mutated alleles. In addition, relatives of two patients were included in our study. Restriction fragment length polymorphism analysis and sequencing of PCR products obtained from DNA of peripheral blood leukocytes was performed for mutation analysis. Gene deletion was detected by comparison of radioactively labelled PCR fragments of both the functional beta-glucocerebrosidase gene and the pseudogene. Among the unrelated patients, 50 alleles were investigated and the mutations identified in 35 alleles (70%), whereas 15 alleles (30%) remained unidentified. The most prevalent mutation in our group of patients was the 1226G (370Asn--Ser) mutation, accounting for 18 alleles (36%), followed by the 1448C (444Lcu--Pro) mutation, that was found in 12 alleles (24%). A complete gene deletion was present in two alleles (4%). The IVS1+2 (splicing mutation), the 1504T (463Arg--Cys) as well as the 1342C (409Asp--His) mutations were each present in one allele (2%). None of the alleles carried the 84GG (frame-shift), 1604A (496Arg--His) or the 1297T (394Val--Leu) mutation. This distribution is different from the Ashkenazi Jewish population but is similar to other Caucasian groups like the Spanish and Portuguese populations. Our results confirm the variability of mutation patterns in Gaucher patients of different ethnic origin. All patients were divided into nine groups according to their genotype and their clinical status was related to the individual genotype. Genotype/phenotype characteristics of the 1226G, 1448C, and 1342C mutations of previous studies were confirmed by our results.

We studied expression and functional characteristics of the insulin- and insulin-like-growth-factor-I (IGF-I) receptors in human renal carcinoma. Ligand-binding properties and tyrosine-kinase activity of both receptors, as well as the expression of the 2 isoforms of the human insulin receptor (HIR-A and -B) were analyzed in renal carcinoma and normal adjacent kidney tissue of 8 adult patients. Partially purified insulin- and IGF-I receptors from normal and renal cell carcinoma tissue possessed identical affinities for their ligands. Renal cell carcinoma, however, contained 3- to 4-fold more specific insulin-binding sites and 2-fold more IGF-I binding sites than adjacent normal kidney tissue. In addition, we determined the relative content of insulin/IGF-I receptor hybrids in both tissues. Renal cell carcinoma and adjacent normal tissue revealed similar amounts of insulin/IGF-I receptor hybrids, i.e., 44 +/- 8.2% of tracer IGF-I binding in normal tissue and 46 +/- 12.0% in renal cell carcinoma. When equal amounts of insulin- and IGF-I receptor protein were studied, we found significantly increased receptor autophosphorylation and elevated substrate phosphorylation in carcinoma tissue. To assess whether the differences in insulin-receptor tyrosine-kinase activity were caused by an altered pattern of insulin receptor isoform expression, we determined mRNA levels for HIR-A and -B. The 2 insulin receptor isoforms were, however, expressed in highly variable ratios in both normal and tumor tissue. Our experiments show that renal carcinoma expresses an elevated amount of insulin- and IGF-I receptor protein with increased specific autophosphorylation and tyrosine-kinase activity each. The increase of insulin-receptor tyrosine-kinase activity in renal carcinoma cannot be explained by an altered expression pattern of insulin receptor isoforms.

Abnormal fibrogenesis is a process which in practically every organ of our body can contribute to disease. In contrast to its paramount importance, too little is known about the pathophysiology of this process and therapeutical ways to interfere with this process. In order to spark progress in the field our idea was to bring together a small group of leading experts of tissue fibrogenesis to focus on this topic by investigating different tissues and diseases. Cross fertilization through intense discussion should allow us to work out general principles and develop new strategies for targeted therapies. For the venue we chose the beautiful island of Frauenchiemsee in Upper Bavaria where international meetings on various topics had been held over the last decade. Leading experts had met to discuss the role of peroxidases (1998), chronic myeloproliferative disorders (2000), iron metabolism (2003) and thrombus formation (2005). For details the interested reader is referred to www.innova-med.de. On this secluded island in beautiful surroundings a group of 30 experts (see Figure ​Figure1)1) in the field came together in fall 2010 for a few days to catalyze interpersonal communication, to stimulate the fruitful exchange of ideas and foster future collaborations. Figure 1 Group photo. The meeting started with basic concepts, continued with fibrosis in various organs, and went on to the reversibility of this process and the development of antifibrotic therapies. Although each organ is unique, all fibrotic diseases share core pathogenic pathways. For this publication in Fibrogenesis & Tissue Repair all authors have updated their presentations. The meeting was financially made possible as in the past through the support of a peer reviewed grant from the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany and additional funds from AOP Orphan Pharmaceuticals, Vienna, Austria as well as private contributions. We hope that the reader will enjoy these proceedings and gain some inspiration for the research in his/her own area of interest.

Matrix metalloproteinase 9 (MMP-9), also known as 92-kD type IV collagenase/gelatinase, is believed to play a critical role in tumor invasion and metastasis. Here, we report that MMP-9 was constitutively released from the human promyelocytic cell line HL-60 as determined by zymographic analysis. Tumor necrosis factor-alpha (TNF-alpha) enhanced the enzyme release threefold to fourfold and the protein kinase C (PKC) activator and differentiation inducer 12-O-tetradecanoylphorbol-13- acetate (TPA) eightfold to ninefold. Gelatinase induction by TNF-alpha and TPA was inhibited by actinomycin D or cycloheximide, indicating that de novo protein synthesis was required. Neutralizing monoclonal antibodies to TNF-alpha (anti-TNF-alpha) decreased the basal MMP-9 release of these cells. In addition, these antibodies also significantly interfered with the TPA-induced enzyme release. Agents that inhibit TNF-alpha expression in HL-60 cells, such as pentoxifylline and dexamethasone, completely abrogated both the constitutive and TPA-evoked MMP-9 release. Diethyldithiocarbamate, which is known to stimulate TNF-alpha production in HL-60 cells, exerted a positive effect on MMP-9 release in untreated cells but was inhibitory in TPA-treated HL-60 cells. The PKC inhibitor staurosporine at low concentrations (100 ng/mL) caused a significant augmentation of MMP-9 release in untreated cultures that was blocked by the addition of anti-TNF-alpha. High concentrations (2 mumol/L) of staurosporine completely abolished the extracellular enzyme activity both in untreated and TPA-stimulated cells. These results suggest, that TNF- alpha is required for basal and PKC-mediated MMP-9 release in HL-60 leukemia cells. Thus, MMP-9 secretion may be regulated by TNF-alpha not only in a paracrine but also in an autocrine fashion. This may potentiate the matrix degradative capacity of immature leukemic cells in the processes of bone marrow egress and the evasion of these cells into peripheral tissue.

Hydroxyurea is used in polycythemia vera for cytoreductive treatment. Some patients show marked fluctuations of platelet and leukocyte counts while taking hydroxyurea. We identified 12 patients in our patient cohort and performed a mathematical analysis which revealed oscillatory behavior with a