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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.

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.

Selenium deficiency in humans has been associated with various diseases, the risks of which can be reduced through dietary supplementation. Selenium accumulating plants may provide a beneficial nutrient for avoiding such illnesses. Thus, leafy vegetables such asAmaranthus hybridus,Amaranthussp.,Cucurbita maxima,Ipomoea batatas,Solanum villosum,Solanum scabrum, andVigna unguiculatawere explored for their capabilities to accumulate selenium when grown on selenium enriched soil and for use as a potential source of selenium enriched functional foods. Their selenium contents were determined by spectrophotometry using the complex of 3,3′-diaminobenzidine hydrochloride (DABH) as a chromogen. The mean concentrations in the leaves were found to range from7.90±0.40to1.95±0.12 μg/g dry weight (DW), withC. maximaaccumulating the most selenium. In stems, the accumulated selenium content ranged from1.12±0.10 μg/g inAmaranthussp. to5.35±0.78 μg/g DW inC. maximaand was hence significantly different (P<0.01). The cancer cell line MDA-MB-231 was used in cytotoxicity assays to determine the anticancer potential of these extracts. With exception ofS. scabrumandS. villosum, no cytotoxicity was detected for the selenium enriched vegetable extracts up to 100 μg/mL concentration. Hence, following careful evaluation the studied vegetables may be considered as selenium enriched functional foods.

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.

Extracellular vesicles (EVs), e.g., exosomes and microvesicles, are one of the main networks of intercellular communication. In myeloproliferative neoplasms, such as polycythemia vera (PV), excess of EVs originating from overabundant blood cells can directly contribute to thrombosis through their procoagulant activity. However, the proteomic composition of these vesicles in PV patients has not been investigated before. In this work, we examined the proteomic composition of serum EVs of PV patients in comparison to healthy controls. We processed EV-enriched serum samples using the Multiple Enzyme Filter Aided Sample Preparation approach (MED-FASP), conducted LC-MS/MS measurements on a Q-Exactive HF-X mass spectrometer, and quantitatively analyzed the absolute concentrations of identified proteins by the Total Protein Approach (TPA). Thirty-eight proteins were present at statistically significant different concentrations between PV patients&rsquo, study group and healthy controls&rsquo, group. The main protein components deregulated in PV were primarily related to excessive amounts of cells, increased platelet activation, elevated immune and inflammatory response, and high concentrations of procoagulant and angiogenic agents. Our study provides the first quantitative analysis of the serum EVs&rsquo, proteome in PV patients. This new knowledge may contribute to a better understanding of the secondary systemic effects of PV disease and further development of diagnostic or therapeutic procedures.

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.

Background: Postmarketing surveillance studies (PMS) are an important tool for evaluating a drug’s effectiveness and safety in clinical practice. To our knowledge, no PMS on memantine monotherapy for moderately-severe-to-severe Alzheimer’s disease (AD) according to National Institute of Neurological and Communicative Disorders and Stroke — Alzheimer’s Disease and Related Disorders Association criteria has been conducted to date. Objective: The Lombardy Health Office, Italy, promoted this PMS to evaluate the effectiveness and safety of memantine in the treatment of moderately-severe-to-severe AD in clinical practice. Methods: A total of 451 patients with moderately-severe-to-severe AD (mean age 77 ± 7 years; 72% female), free of cholinergic medication, received memantine (standard titration to 10 mg twice daily). After 6 months of therapy, treatment effectiveness was evaluated according to two definitions of response (‘no deterioration’ and ‘improvement’), as measured by changes in baseline scores on the Clinical Global Impression of Change, Mini-Mental State Examination, Neuropsychiatric Inventory and Activities of Daily Living scales. The safety measure was the frequency of adverse events (AEs). Results: At 6-month assessment, 26.8% of subjects showed no deterioration and 3.8% showed improvement. In those showing no deterioration, response to treatment at the 3-month assessment was associated with a greater probability of a response at 6 months (adjusted odds ratio = 8.54; 95% CI 4.54, 16.05). Seventy patients (15.5%) experienced at least one AE and 39 (8.6%) discontinued treatment prematurely because of an AE. Of those who experienced an AE, 27 (38.6%) manifested behavioural and psychological symptoms of dementia. Conclusion: The proportion of responders to memantine treatment in this PMS was similar to that reported in a previous randomized clinical trial (26.8% vs 29%, respectively). The proportion of patients who discontinued treatment prematurely because of an AE (8.6%) was similar to that reported in two previous randomized clinical trials (10% and 12.4%). This PMS provides additional evidence that both the effectiveness and the tolerability of memantine may be transferred into real world medicine, where AD patients receiving treatment are not selected according to strict criteria.

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.

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 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

To further characterize JAK2 exon 12 mutations, we performed molecular screening in 409 patients with polycythemia vera or unclear erythrocytosis with unmutated JAK2V617. The frequency of JAK2exon12 mutations was 10/63 (15.9%) in PV but only 5/346 (1.4%) in the erythrocytosis cases. Nine different mutations including four new types (D544-L545del, H538DK539LI540S, H538-K539del, V536-F547dup) were detected. In 2 cases we found evidence for the presence of cells homozygous for mutated JAK2exon12. As this was the case in only 2/15 cases with JAK2exon12 mutations (13%) homozygosity seemed to be less frequent than in V617F-mutated polycythemia vera (69%) (p

HL-60 cells produce an autostimulatory growth factor. Since the stimulatory effect of HL-60 conditioned medium is only observed in the absence of exogenous transferrin we have assayed HL-60 cells for the production of transferrin and found that they produce polypeptides which react with transferrin antibodies. 35S-methionine labelling, immunoprecipitation and subsequent separation by SDS-gel electrophoresis reveals the presence of a major transferrin related 41 +/- 2 kDa species released by HL-60 cells. Physiological levels of iron salts completely abolish the requirement of exogenous transferrin which indicates that the endogenous transferrin related polypeptides in the presence of exogenous inorganic iron salts are sufficient for the proliferation of HL-60 cells provided insulin or related growth factors are present. The addition of transferrin receptor antibodies inhibits the stimulatory action of the endogenous transferrin related activity.

Recently, mutations of MPL , the gene coding for the thrombopoetin receptor, were demonstrated in ~5% of cases of primary myelofibrosis (PMF) and in ~1% of all cases of essential thrombocytosis (ET).[1][1],[2][2] They represent gain-of-function mutations that confer constitutive activation of the

At the Olympic Winter Games in 2006, Evi Sachenbacher-Stehle had an elevated hemoglobin reading of 16.4 g/dL in a doping sample and was suspended for five days. In the summer of 2006, more than 50 cyclists were involved in the scandal surrounding the team physician Fuentes in the run-up to the Tour de France (1). Investigators found stored blood units intended to artificially increase hematocrit and evidence of orders for erythropoietin. Professional cyclist Jorg Raschke recently confirmed that erythropoietin doping is normal practice in the cycling world (1). Hematocrit can be regarded as a symbol of manipulation in endurance sports: it can be increased legally by high altitude training or illegally with erythropoietin (EPO), androgens, and autologous blood transfusions. But not every elevated hematocrit is attributable to high altitude training or doping. The Finnish cross-country skier Eero Mantyranta was known since youth to have high hemoglobin levels of above 20 g dL (hematocrit above 60%). Examinations performed on the several times Olympic champion revealed increased sensitivity of erythropoietin precursors in the bone marrow to erythropoietin. Causally responsible is a hereditary point mutation in the erythropoietin receptor gene which leads to permanent activation of the EPO receptor system and thus to erythrocytosis. Unlike congenital forms of erythrocytosis, the commonest form – polycythemia vera (PV) – is acquired. Although the term polycythemia originally denoted "too many" cells in the peripheral blood, it is now used as a synonym for erythrocytosis. In polycythemia vera (PV) not the erythropoietin receptor, but the signal cascade associated with it is changed. A basic distinction can be made between relatively rare congenital and the more common acquired polycythemias. The following overview of polycythemias is based on a selective literature review and the authors’ own clinical experiences.

Under normal circumstances only mature granulocytes and monocytes cross the bone marrow sinus wall, a trilaminar structure consisting of endothelial cells, a discontinuous basal membrane and an adventitial cell layer in order to get access to the blood circulation. In leukemia, however, immature white blood cells are able to traverse the barrier and to appear in the blood stream. Very little is known about the regulatory processes which govern the egress of white blood cells in healthy individuals and their malignant counterparts in patients with leukemia. The results of the few studies performed to address this question in animal and human leukemias all agree that the extent to which adventitial cells (fibroblasts) cover the endothelium in bone marrow is drastically reduced. This implies altered interactions between the leukemic and adventitial cells and their extracellular matrix. We propose here a model to explain the egress of normal cells and their leukemic counterparts. It is based upon our own experimental data and the general at present limited knowledge of the subject. It is hoped that this model will stimulate further research into this important aspect of leukemogenesis.

We have characterized and analyzed IGF-I- and insulin-stimulated cell growth, receptor binding, and autophosphorylation in the human leukemic cell line HL-60. IGF-I-stimulated cell growth occurred at low (5 ng/ml) and insulin stimulated only at high (500 ng/ml) concentrations. Binding of 125I-IGF-I to partially purified plasma membrane proteins followed the characteristics of IGF-I receptor binding. 125I-IGF-I binding, as determined by chemical cross-linking, occurred to a 145-kDa protein. IGF-I, as well as insulin, stimulated the autophosphorylation of a 105-kDa band (pp105), but we could not detect a 95-kDa band corresponding to the known molecular mass of the IGF-I and insulin receptor beta-subunits. Phosphorylation of pp105 followed the dose-response characteristics of the IGF-I receptor. The phosphorylation of pp105 occurred at tyrosine and threonine, and the pattern of HPLC tryptic peptide maps showed marked differences when compared with that of a phosphorylated insulin receptor beta-subunit. Enzymatic deglycosylation of pp105 resulted only in a slight reduction of the molecular weight. These data suggest that pp105 is the beta-subunit of an IGF-I receptor variant with a higher molecular weight, similar to that found in fetal tissue. The HL-60 cell may acquire, at least in part, malignant growth characteristics through reexpression of the fetal version of the IGF-I receptor.

Anagrelide is an established platelet-reducing drug. Although there are gaps in the understanding of its mechanism of action, two randomized comparisons with other drugs used for therapy of patients with essential thrombocythemia (ET) have been performed. Recent progress has been made in this field with the development of better determination techniques, with the characterization of metabolites, and with studies of their mechanism of action on megakaryocytes and platelets. More data are now available from various noncomparative clinical trials on its clinical efficacy and safety. Only few investigations are available that document its long-term effects. Although the drug should not be used during pregnancy, there are a few studies that report that pregnant women have taken this drug without harm to the newborn. Studies have also investigated the effects of anagrelide on platelets, indicating that platelet function is as important as platelet counts in ET. Preliminary analyses of the mechanism of action of anagrelide have revealed that the drug interferes with the signal transduction of the thrombopoietin receptor. Results of the first phase III trial (PT1) that compared anagrelide/aspirin with hydroxyurea/aspirin have sparked an intense discussion, given that the combination of anagrelide and aspirin causes more bleeding complications in the gastrointestinal tract. It has been speculated that the higher number of transient ischemic attacks in this study arm is not caused by thrombotic events but by small bleedings that would be responsible for transient hemorrhagic attacks. More insights are expected from the recently completed ANAHYDRET trial that compared monotherapy with hydroxyurea and anagrelide.

Hemostatic complications which can occur in the arterial or venous vasculature or in the microcirculation are the major causes of morbidity and mortality in patients with ET. In order to prevent these complications, often platelet reductive drugs are used. These agents are by themselves potentially toxic, i.e. may cause leukemia or cardiac side effects. In order to avoid these adverse effects, a better understanding of the mechanism of thrombus formation which is causative in ET is mandatory. Unfortunately, until now, no biomarkers have been identified which allow the estimation of the risk of thrombotic complications. Platelet number is not a good predictor per se since thrombotic complications can occur in some patients at low platelet numbers whereas others do not encounter a thrombosis even at very high platelet levels. On the other hand, lowering of the platelet count usually results in symptomatic improvement. In ET, morphological alterations of the megakaryocyte in the bone marrow and the circulating platelets are observed: megakaryocyte nuclei show a staghorn appearance, circulating platelets are characterized by anisocytosis and giant size. Functional studies indicate that these anatomically altered platelets function abnormally. When platelets are analyzed with a platelet function analyzer (PFA-100, which uses cartridges that measure how well a patient's platelets adhere and aggregate to form a platelet plug in the first phase of thrombus formation), in many patients with ET, closure time using collagen/ADP and collagen/epinephrine cartridges is prolonged. This seems paradoxical since these patients do not show an increased bleeding time. These results indicate that either receptors and/or consecutive signaling events are abnormal in ET platelets. Proteomic analysis of platelets of ET patients has revealed individual differences but not yet led to the identification of disease-specific proteins. Moreover, the search for alternative risk factors (factor V Leiden, prothrombin gene polymorphism, etc.) has not provided evidence for the contribution of these factors to the generation of the thrombotic risk in ET patients. In summary, despite intensive research over several decades, relatively little is known about the pathogenesis and risk factors for thrombosis in ET. I expect that this conference will contribute to the development of new strategies to identify patients at risk for hemostatic complications.

Chronic myeloid leukemia (CML) was the first hematological malignancy in which a chromosomal alteration (the Philadelphia chromosome) was identified. This alteration results from a translocation with the formation of a fusion gene. The elucidation of the underlying molecular process has not only allowed a precise diagnosis and the monitoring of residual disease but has also paved the way for the development of imatinib, a therapeutic agent which directly targets the molecular alteration [1].

Essential thrombocythemia (ET) is associated with both an increased risk of thromboembolic and bleeding complications [1]. The efficacy of aspirin and cytoreductive therapy supports the concept that platelets are functionally altered in individuals with ET and contribute to the complications. However, there is no linear relationship between platelet count and risk of thromboses since the complications can occur even at low platelet counts [2] and do not necessarily occur at high platelet counts. This maybe due to a patient-specific potential to aggregate and/or the individual modulation of the activity of platelets by nonthrombocytic factors such as plasma proteins or other cellular elements.

My interest in myeloperoxidase (MPO) deficiency arose in the early 1990s when I began caring for a young female (RT) who had been in good health until the age of 11 years when she had developed osteomyelitis in her right tibia in 1979. Despite surgical intervention she had relapsed at the same site 2 years later. In 1988 she underwent partial resection of the right fourth rib because of the osteomyelitis. A specific microbiological etiology was not recovered from any of her operative specimens, blood or wound cultures. Antibiotic therapy was empiric and included agents with a broad range of activity. At no time were fungi isolated, serologic tests for Candida positive or empiric antifungal therapy used. Since her last surgical procedure she had been stable clinically requiring episodic courses of antibiotic treatment because of recurrent pain at the site of her previous infections. There was no evidence of diabetes mellitus.

Myeloperoxidase (MPO) belongs to a family of related proteins which also includes eosinophil, thyroid, and lactoperoxidase. The MPO gene is a 14-kb gene located on the long arm of chromosome 17. Thus far four mutations (R569W, Y173C, M251T and a 14-base deletion in exon 9) have been identified in patients with MPO deficiency. As in other genetically determined diseases, many more mutations will eventually be revealed that cause this disease. Present evidence shows that most patients are compound heterozygotes, i.e., they have inherited different mutations on their paternal and maternal MPO alleles. Understanding why some patients with this genetic deficiency develop clinical symptoms while others do not requires mutation analyses of a large number of patients. This includes the analysis of genotype-phenotype relationships. Genotyping has also been started in patients with EPO-deficiency.

A 58-yr-old man developed severe weight loss, arthralgias, and diarrhea. Endoscopic examination of the stomach and duodenum revealed thickened folds of duodenal mucosa. Biopsy of the gastric mucosa was negative, whereas duodenal biopsy revealed blunted epithelial villi and PAS-positive foamy macrophages within the lamina propria. Bacilli typical of those associated with Whipple's disease were found by electron microscopy. The diagnosis was confirmed by polymerase chain reaction (PCR) assay, which detected a portion of the 16S ribosomal RNA gene sequence corresponding to the Whipple bacillus (Tropheryma whippelii) in duodenum, stomach, and liver biopsies before therapy. T. whippelii DNA was eliminated from all tissues tested within 3 months of starting antibiotic treatment, but the histological improvement lagged behind the clinical and molecular evidence of improvement.

We report on our treatment experience in Germany with anagrelide, a novel platelet lowering agent, in 48 patients (27 females, 21 males) with essential thrombocythaemia. Their age was between 19 and 79 yr when anagrelide therapy was initiated. Sixteen patients were previously untreated, 15 pretreated with hydroxyurea and 17 had multiple pretreatments. Forty-one of the 48 patients had either microvascular, thromboembolic or bleeding complications. About 50% received low dose acetylsalicylic acid as an adjunct. Their platelet count prior to therapy ranged from 850,000/microl to 3,100,000/microl. Eighty-seven per cent of the patients treated with anagrelide were complete hematological responders, while 13% responded only partially or failed to respond. Twelve of our patients (25%) developed short-term (from a few days to a maximum of 4 wk) side effects including headache (most frequent), palpitations, tachycardia or nausea. Eight patients reported long-term (more than 1 month) adverse effects. However, in only 5 of all 48 patients (10%) were these side effects not acceptable so that treatment had to be discontinued. We have now treated patients for up to 7 yr (median maintenance dose 2.5 mg/d). Preliminary evidence suggests that anagrelide mediated platelet count reduction also prevents recurrence of thromboembolic complications. Hence, anagrelide has the potential to become the first-line platelet-lowering treatment in myeloproliferative disorders with high platelet counts.

MK-886, a specific inhibitor of 5-lipoxygenase inhibited DNA replication in leukemic HL-60 cells in a dose-dependent manner. Addition of exogenous leukotriene B4 reversed this effect, whereas addition of leukotriene B4 failed to modulate a prostaglandin D2-induced inhibition of DNA replication. The reversal of MK-886-induced inhibition was not observed with leukotriene C4. These results suggest that the effect of MK-886 is mediated by inhibition of leukotriene B4 biosynthesis. Moreover, MK-886 not only impaired DNA replication in HL-60 cells but also decreased cell proliferation and induced apoptotic cell death. Our results suggest a crucial role of leukotriene B4 in the regulation of cell proliferation and cell survival in HL-60 cells.