Christa Kunert

Clinical impact of nucleophosmin mutations and Flt3 internal tandem duplications in patients older than 60 yr with acute myeloid leukaemia

Background:  Nucleophosmin (NPM1) and Flt3 internal tandem duplications (Flt3‐ITD mutations) represent the most frequent molecular aberrations in patients with acute myeloid leukemia (AML). While NPM1 mutations are associated with favourable prognosis in younger AML patients, Flt3‐ITD mutations reflect an unfavourable prognostic factor in these patients. So far, especially NPM1 mutations have not yet been evaluated exclusively in older patients.

Molecular-defined clonal evolution in patients with chronic myeloid leukemia independent of the BCR-ABL status

To study clonal evolution in chronic myeloid leukemia (CML), we searched for BCR-ABL-independent gene mutations in both Philadelphia chromosome (Ph)-negative and Ph-positive clones in 29 chronic-phase CML patients by targeted deep sequencing of 25 genes frequently mutated in myeloid disorders. Ph-negative clones were analyzed in 14 patients who developed clonal cytogenetic abnormalities in Ph-negative cells during treatment with tyrosine kinase inhibitors (TKI). Mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1 and ZRSR2. In two patients, the mutations were also found in corresponding Ph-positive diagnostic samples. To further investigate Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Somatic mutations additional to BCR-ABL were found in 5/15 patients (33%) affecting ASXL1, DNMT3A, RUNX1 and TET2. Analysis of individual hematopoietic colonies at diagnosis revealed that most mutations were part of the Ph-positive clone. In contrast, deep sequencing of subsequent samples during TKI treatment revealed one DNMT3A mutation in Ph-negative cells that was also present in Ph-positive cells at diagnosis, implying that the mutation preceded the BCR-ABL rearrangement. In summary, BCR-ABL-independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the clonal evolution of CML.

Paraneoplastic inflammation in myelodysplastic syndrome or bone marrow failure: case series with focus on 5‐azacytidine and literature review

Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal disorders of haematopoietic stem cells, characterised by dysplastic haematopoiesis and dysregulated apoptosis resulting in various degrees of cytopenia, whereas canonical cytologic, cytogenetic and histopathologic findings guiding the diagnosis MDS are widely accepted, the MDS‐phenotype can be masked by coexisting/paraneoplastic immunologic disease. Autoimmune disorders have an estimated incidence of 10% among patients suffering from MDS and are causally related to increased morbidity and mortality, younger age at diagnosis and more complex genetics. Conversely, systemic inflammatory disorders may be an early manifestation of MDS, show good response to immunosuppressive therapy and frequently disappear during the course of specific haematologic therapy.

Specific pattern of protein expression in acute myeloid leukemia harboring FLT3-ITD mutations

FLT3 activating mutations can be detected in about 35% of acute myeloid leukemia (AML). FLT3 internal tandem duplications (FLT3-ITD) represent the majority of FLT3 mutations (25 – 30%) while FLT3-TKD (tyrosine kinase domain) mutations can be found in about 7% of AML patients. In this study, we addressed the question whether especially primary AML cells carrying FLT3-ITD mutations show differences in terms of their protein expression pattern compared to FLT3 wild-type blasts. We investigated bone marrow samples that were isolated at diagnosis from 36 AML patients expressing either FLT3 wild-type (n = 16) or an activating FLT3 mutation (FLT3-ITD, n = 15; FLT3-TKD, n = 5). Proteomic analysis was performed by means of surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry which has shown its high efficiency in finding biomarkers in solid tumors. Here, we demonstrate that a large series of proteins is differently expressed in primary AML blasts harboring FLT3-ITD mutations. Furthermore, there are also significant differences of the protein expression profile between FLT3-ITD and FLT3-TKD mutations. Interestingly, further analysis of FLT3-ITD positive AML according to its response to the induction chemotherapy demonstrates putative prognostic markers for this subgroup of AML. We suggest that SELDI-TOF mass spectrometry represents a promising tool of proteomic analysis of AML that might help to establish new prognostic markers in AML.

Die hämophagozytische Lymphohistiozytose (HLH) und das Makrophagenaktivierungssyndrom (MAS): Klinisches Erscheinungsbild und Diagnostik

Zusammenfassung: Die hämophagozytische Lymphohistiozytose (HLH) ist ein Hyperinflammations-Syndrom, welchem neben genetischen Defekten insbesondere in Genen der die Immunsynapse regulierenden Proteine auch erworbene Defekte der effektiven Pathogen-Elimination zugrunde liegen. Das rasche Erkennen und zielgerichtete Diagnostizieren einer HLH ist bei weiterhin hoher Mortalitätsrate zwischen 40%–70% essentiell, um Therapieverbesserungen zu erreichen. Hierfür ist der wichtigste Schritt für den Kliniker, an eine HLH zu denken. Prolongiertes Fieber unklarer Genese, eine Hepatosplenomegalie und eine Bi- oder Panzytopenie sind die führende Symptomentrias. Bei bekannter Familienanamnese oder bekanntem Gendefekt sind rasche bestätigende Untersuchungen einzuleiten, um die häufig notwendige Stammzelltransplantation nicht zu verzögern. Insbesondere bei Erwachsenen, bei denen auch genetische Defekte mit verzögerter Manifestation vorliegen können (v.a. bei de novo EBV-Infektion), muss eine breite Diagnostik zur Ursachenforschung einer HLH angestrengt werden. Die HLH ist keine eigenständige Erkrankung. Sie ist gemeinsame Endstrecke eines Immundefekts, welcher genetisch bedingt, oder durch infektiöse, autoimmune, autoinflammatorische, maligne oder auch iatrogene Trigger (Immunsuppression, Stammzelltransplantation) erworben werden kann. Diesem breiten Spektrum der Pathogenese der HLH muss die labormedizinische Diagnostik Rechnung tragen, um dem Kliniker sehr zeitnah die klinisch zu stellende Verdachtsdiagnose zu erhärten und schnellstmöglich die Therapie einleiten zu können. Abstract: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome caused by genetic and acquired defects of the molecular machinery, which regulate the cellular immune synapse. Rapid recognition of symptoms resembling HLH, and a targeted diagnostic approach are essential in improving outcome. The condition is associated with a mortality rate between 40% and 70%. For the clinician, the most important step towards diagnosing HLH is to include it in the list of potential differential diagnoses. The leading triad of symptoms consists of prolonged fever of unknown origin, hepatosplenomegaly, and bi- or pancytopeniae. A known family history or known gene mutations require rapid confirmatory testing, which facilitates the initiation of a life saving risk-adapted treatment that includes stem cell transplantation. In adults with de-novo infection with Epstein-Barr virus, a broad diagnostic approach is required to identify potential late-onset hereditary HLH. HLH is not a diagnosis, per se, but represents a common terminal pathway of diseases with the ability to trigger HLH. Such diseases include infections, malignant disorders, autoimmune or autoinflammatory diseases, and iatrogenic triggers such as immunosuppressive treatment or stem cell transplantation itself. This broad spectrum of HLH pathogenesis must be considered in order to achieve rapid diagnosis, which is a prerequisite for the initiation of rationale treatment.

Der Einfluß einer rh-EPO-Therapie auf die In-vitro-Proliferation granulozytär-monozytär determinierter Stammzellen aus Knochenmark und peripherem Blut

Zusammenfassung□ ZielDas Ziel dervorliegenden Untersuchungen bestand in der Erfassung des Einflusses der Therapie mit rekombinantem Erythropoetin (rh-EPO) bei Hämodialysepatienten auf die Proliferationskapazität von granulozytär-monozytär (GM)-determinierten Stammzellen des Knochenmarks und des peripheren Blutes.□ Patienten und Methoden28 Hämodialysepatienten (15 weiblich, 13 männlich) im Alter zwischen 22 und 78 Jahren mit einer Dauer der Dialysebehandlung vor Beginn der Untersuchung von vier bis 99 Monaten wurden einbezogen. Die Stammzelluntersuchungen des peripheren Blutes und des Knochenmarks wurden vor Beginn der Therapie und nach Erreichen eines Zielhämatokrits von 0,30 durchgeführt und Absolutwerte der peripheren Leukozyten bestimmt.□ ErgebnisseUnter einer rh-EPO-Therapie konnten keine Veränderungen der peripheren Leukozytenzahlen (Granulozyten, Lymphozyten, Monozyten) beobachtet werden. Die Proliferationsfähigkeit von CFU-GM des Knochenmarks war bei Hämodialysepatienten im Vergleich zu Gesunden vermindert und nahm unter der rh-EPO-Therapie signifikant zu (34,3±5,8×105/ml vs. 120,5±37,8×105/ml, p<0,05). Die gegenüber Normalpersonen vermehrte Stammzellproliferation des peripheren Blutes zeigte keine Dynamik (46,7±7,1 5×105/ml vs. 37,6±12,05×105/ml).Abstract□ AimThe aim of the investigation was to determine the effect of recombinant human erythropoietin (rh-EPO) therapy on in-vitro proliferation of granulocyte/monocyte(gm)-determined stem cells in bone marrow and peripheral blood.□ Patients and MethodsThe study comprised 28 dialysis patients (15 female, 13 male, aged between 22 and 78 years, dialysis sessions 3 times weekly 4 to 5 hours, mean duration time of dialysis treatment 4 to 99 months). Stem cell parameters were estimated before and after reaching the target hematocrit and the absolute number of leukocytes in peripheral blood was measured.□ ResultsNo alteration in total leukocyte number (PMN, monocytes, lymphocytes) of peripheral blood was found. Dialysis patients had a diminished proliferation capacity of colony forming unit-granulocyte/monocyte (CFU-GM) in bone marrow and an increased one in peripheral blood. The CFU-GM proliferation capacity in bone marrow improved during rh-EPO (34,3±5,8×105/ml vs 120,5±37,8×105/ml), whereas no changes in peripheral blood could be observed (46,7±7,15×105/ml vs 37,6±12,05×105/ml).AIM The aim of the investigation was to determine the effect of recombinant human erythropoietin (rh-EPO) therapy on in-vitro proliferation of granulocyte/monocyte(gm)-determined stem cells in bone marrow and peripheral blood. PATIENTS AND METHODS The study comprised 28 dialysis patients (15 female, 13 male, aged between 22 and 78 years, dialysis sessions 3 times weekly 4 to 5 hours, mean duration time of dialysis treatment 4 to 99 months). Stem cell parameters were estimated before and after reaching the target hematocrit and the absolute number of leukocytes in peripheral blood was measured. RESULTS No alteration in total leukocyte number (PMN, monocytes, lymphocytes) of peripheral blood was found. Dialysis patients had a diminished proliferation capacity of colony forming unit-granulocyte/monocyte (CFU-GM) in bone marrow and an increased one in peripheral blood. The CFU-GM proliferation capacity in bone marrow improved during rh-EPO (34.3 +/- 5.8 x 10(5)/ml vs 120.5 +/- 37.8 x 10(5)/ml), whereas no changes in peripheral blood could be observed (46.7 +/- 7.15 x 10(5)/ml vs 37.6 +/- 12.05 x 10(5)/ml).