Molecular pathogenesis of large granular lymphocytic leukemia

Large granular lymphocytic (LGL) leukemia is a chronic incurable disorder characterized by expansion of cytotoxic T- or natural killer (NK)-lymphocytes infiltrating blood and bone marrow. The diagnostic criteria include persistent lymphocytosis in peripheral blood, and in the case of T-LGL leukemia, detection of a clonal rearrangement of the T cell receptor. Typical clinical and hematological characteristics, including anemia, neutropenia, and autoimmune disorders, further support the diagnosis. Current immunosuppressive treatment options, such as methotrexate, induce remission in only 50% of cases and no targeted therapies exist. The aim of this project was to characterize the molecular pathogenesis of LGL leukemia and find molecular markers that could be used for diagnosis and novel therapeutic approaches. In the first study, the molecular background of LGL leukemia was analyzed by exome sequencing of a T-LGL leukemia patient. The index patient carried a novel D661V mutation in the Signal transducer and activator of transcription 3 (STAT3) gene. STAT3 is a known oncoprotein and a transcription factor. In the subsequent screening, the prevalence of somatic STAT3 mutations in T-LGL cases was 40%. Based on functional studies, the mutations located in the SRC-like homology 2 (SH2) domain increased phosphorylation and transcriptional activity of STAT3. A larger LGL-leukemia patient cohort, including both T-LGL leukemia and chronic lymphoproliferative disorder of NK cells (CLPD-NK) patients, was analyzed in the second study. Activating STAT3 mutations were seen in 27% and 30% of cases, respectively. The analysis of clinical characteristics showed that patients with STAT3 mutations were more likely to have neutropenia and rheumatoid arthritis and also required more frequent treatment when compared with unmutated patients. The third study concentrated on the analysis of STAT3 mutation-negative patients by exome sequencing. Two T-LGL leukemia patients carried Y665F mutation in STAT5B gene. In the screening of over 200 T-LGL leukemia and CLPD-NK cases, two patients presented with additional N642H mutation in STAT5B. The mutations were situated in the SH2 domain and led to increased phosphorylation and transcriptional activity of STAT5B. The in vitro effects of N642H mutation were more prominent, and both patients harboring N642H mutation had an untypically aggressive clinical presentation. In the fourth study, the clonal hierarchy and STAT3 mutation spectrum during immunosuppressive treatment was analyzed by deep amplicon-sequencing of STAT3 and simultaneous deep sequencing analysis of a T cell receptor beta chain (TCRB) repertoire. A total of 22% of STAT3-mutated patients harbored multiple STAT3 mutations mostly in separate lymphocyte clones. Mutated clones did not share common T cell receptor beta chain (TCRB) sequences by deep sequencing method in three T-LGL leukemia cases analyzed. Complete remission after immunosuppressive therapy resulted in the eradication of the STAT3-mutated clone in most patients, whereas partial responses reflected only modest changes in the leukemic clone burden. In conclusion, the activating STAT3 and STAT5B mutations provide a basis for understanding the molecular pathogenesis of leukemic LGL expansion. LGL leukemia can result from chronic antigen exposure in combination with immunogenetic factors such as STAT mutations. The detection of STAT3 and STAT5B mutations can be included in the diagnostic criteria of LGL leukemia, and facilitate development of novel therapeutics.Suurten granulaaristen lymfosyyttien (large granular lymphocyte, LGL) leukemia on harvinainen, parantumaton krooninen verisairaus, joka johtuu klonaalisten sytotoksisten T- tai NK-lymfosyyttien lisääntymisestä veressa sekä luuytimessä. Taudin tyypillisiin oireisiin kuuluvat anemia, neutropenia sekä erilaiset autoimmuunisairaudet kuten nivelreuma. LGL-leukemian syntymekanismi on tuntematon. Koska LGL-solut ovat fenotyypiltään jo antigeeninsä kohdanneita kypsiä lymfosyyttejä, on mahdollista että jokin tuntematon, autoantigeenien kanssa ristiin reagoiva epitooppi on toiminut lymfoproliferaation käynnistäjänä. Ohjelmoitu solukuolema eli apoptoosi, joka rajoittaa aktivoituneiden lymfosyyttien määrää, toimii huonosti LGL-leukemiassa. Sairauden hoitona käytetään immuunijärjestelmää lamaavia lääkeaineita, mutta vain noin puolet potilaista saa niille hoitovasteen. Tutkimuksen tavoitteena oli selvittää LGL-leukemian syntymekanismi ja löytää taudille ominaisia molekyylitason tunnusmerkkejä ja tyyppimutaatioita, joita voitaisiin käyttää diagnostiikassa ja täsmähoitojen kohteena. Väitöskirjan osatöissä analysoitiin näytteitä yli 200:lta LGL-leukemia-potilaalta. Virtaussytometrisen valkosoluanalyysin ja erottelun lisäksi menetelminä käytettiin uusimpia, herkkiä sekvenointimenetelmiä, kuten koko eksomin sekvenointia sekä yksittäisiin geeneihin kohdistettua syväsekvenointia. Todettujen mutaatioiden toiminnallista vaikutusta analysoitiin käyttämällä potilasnäytteitä ja solulinjoja. Väitöskirjan tärkeimpänä tuloksena merkittävällä osalla, noin 40%:lla, T- ja NK-LGL-leukemiaa sairastavilla potilailla todettiin mutaatio STAT3-geenin aktiivista dimeerirakennetta säätelevässä SH2-osassa. STAT3 on perimän luentaa ohjaava proteiini, jolla on merkittävä rooli esimerkiksi puolustusjärjestelmän toiminnassa osana JAK-STAT-akselia. LGL-leukemiassa todetut hankinnaiset STAT3-mutaatiot aktivoivat STAT3-geeniä ja sen säätelelemiä signalointireittejä. Mutaatiot sijaitsivat taudille ominaisissa sytotoksisissa lymfosyyteissä, ja osalla potilaista todettiin jopa useita STAT3-mutatoituneita lymfosyyttiklooneja. Nivelreuma ja neutropenia olivat selvästi yleisempiä potilailla, joilla todettiin STAT3-mutaatio, ja mutaatiot olivat yhteydessä suurempaan leukeemisen populaation kokoon. Kvantitatiivisen STAT3-syväsekvenoinnin käytettävyyttä testattiin lisäksi hoitovasteen arvioinnissa: täydellisen hoitovasteen yhteydessä myös STAT3-mutaatiota kantava klooni katosi. Seuraavassa vaiheessa LGL-leukemian syntymekanismia selvitettiin koko eksomin sekvenoinnilla niillä potilailla, joilla ei oltu todettu STAT3-mutaatiota. Eksomisekvenoinnissa ja sitä seuranneessa LGL-leukemia-kohortin analyysissa 2%:lla (4/211) potilaista todettiin hankittu mutaatio STAT5B-geenin SH2-osassa. Funktionaaliset kokeet osoittivat, että todetut STAT5B-mutaatiot olivat luonteeltaan STAT5B:n toimintaa aktivoivia. Aineistossa oli kaksi potilasta, jotka sairastivat epätyypillistä agressiivista LGL-leukemiaa, ja molemmat olivat STAT5B N642H-mutaation kantajia. On mahdollista, että kyseinen mutaatio liittyy poikkeavaan taudinkuvaan. Loppupäätelmänä voidaan esittää, että STAT3- ja STAT5B-mutaatiot ovat merkittävässä roolissa LGL-leukemian synnyssä. On todennäköistä, että näiden mutaatioiden lisäksi mahdolliset muut hankitut mutaatiot, perinnöllinen alttius, erilaiset kohdatut antigeenit sekä immunosuppressiivinen hoito vaikuttavat lymfosyyttikloonien syntyyn ja taudinkulkuun. STAT3- ja STAT5B-mutaatioiden analyysi voidaan ottaa yhdeksi kriteeriksi LGL-leukemiaa diagnosoidessa, ja nämä proteiinit sekä niihin liittyvät signalointireitit ovat uusia kiinnostavia kohteita täsmähoitoja suunniteltaessa

LGL-leukemia ja autoimmuniteetti : autoimmuunisairauden ja syövän rajapinta hämärtyy

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OSMO - a collaborative network testing knowledge and tools for resource-efficient soil health management

Implication Soil and knowledge are the most important resources for agriculture. In order to create new knowhow on managing soils, collaboration between research, advisory services, product development and farmers is necessary. OSMO –project has provided these opportunities in farmer learning groups, workshops and field trials. Based on the first year of results it is clear that soil health is a complex system, where different aspects interact. Many of the problems observed as nutrient deficiencies can be sourced to compaction from machinery or lack of drainage. On the other hand problems in soil nutritional status may be behind compaction and weed problems. The current hypothesis is that each field has its own set of problems. Soil health can be improved only by identifying the crop yield reducing factors, determining their causes and planning for effective ways to remedy them. Simplified decision support tools are needed to couple the complexity of soil systems into the needs of farm management. Identifying and remedying soil problems have a large potential for increasing the productivity of organic and conventional agriculture. Background and objectives Agricultural soils are under increasing pressure. During the last decades crop rotations have simplified, annual crop areas have increased and machinery has gotten heavier. At the same time the progress in crop yields has stagnated. Compared to the yield potential which is theoretically possible from sunlight and water availability, crop yields are very low, especially in organic agriculture. At the same time, variability between farms, fields and field parts is high. It is unclear why some fields have low yields while other adjacent fields have very good yields. The concept of soil health is an emerging paradigm for looking at soil as a system. It appreciates the interconnections between components of soils and different views on problems (i.e. chemical, physical and biological). The objective of the project is to apply new knowledge on soil quality and health and to test their applicability in practice on farms. This is done through improving knowledge on soil testing, farmer know-how on soil health management and developing tools and study materials. Methods for analyzing and repairing soil problems are tested on 8 experimental problem fields, with adjacent good fields serving as the reference. The approach has been problem oriented, analysis methods and tested techniques have been tailored to each problem field. The aim is to identify and fix all barriers to better productivity and soil health and to test how (and if) the approach works. Key results and discussion The project has been running since 2016 and most of the work is still ongoing. Final results for the test farms will be available in 2018. For now, five regional learning groups are running, each with ca. 20 farms. These have included a six month intensive period of soil management education and application of skills to on-farm work. The separate tools for soil management have been assembled into a soil management planning toolbox, which is being refined based on user experience. Several problems have been identified in the test farms and trials have been run to test for potential solutions. Soil structure has been improved through vetch based cover crop mixes and subsoiling. Gypsum applications have been targeted to remedy Ca:Mg ratio problems and manganese, potassium and boron fertilizers have been tested to remedy common nutrient deficiencies. In 2017, new methods for identifying and solving drainage and compaction issues are being tested. The interaction between research and farmers has been valuable. In the intensive six month courses, researchers have applied new scientific information and converted it into calculators for farm use. Receiving rapid feedback on their applicability and being able to redevelop them into tools has provided useful tools for soil management. How work was carried out? The core of the project was five learning groups with ca. 20 farms in each. Each group held regular meetings and online lectures on the main aspects of soil health (chemical, physical and biological). The participants also had access and guidance on using tools for managing soil fertility, compaction, drainage and crop rotations. The participants filled an online soil management plan for their farm during the course and reported on their own trials and tested solutions for managing soil health. In addition several indepth workshops were arranged on special topics. The scientific work focused on the 8 test fields. Their status was quantified through different soil tests (ammonium acetate extraction, Mehlich 3, Soil Health Tool), physical soil evaluation (visual evaluation of soil structure, soil cover, earthworm counts, water infiltration) and plant nutrient analysis

A novel class of somatic mutations in blood detected preferentially in CD8+cells

Somatic mutations have a central role in cancer but their role in other diseases such as autoimmune disorders is poorly understood. Earlier work has provided indirect evidence of rare somatic mutations in autoreactive T-lymphocytes in multiple sclerosis (MS) patients but such mutations have not been identified thus far. We analysed somatic mutations in blood in 16 patients with relapsing MS and 4 with other neurological autoimmune disease. To facilitate the detection of somatic mutations CD4 +, CD8 +, CD19 + and CD4-/CD8-/CD19- cell subpopulations were separated. We performed next-generation DNA sequencing targeting 986 immune related genes. Somatic mutations were called by comparing the sequence data of each cell subpopulation to other subpopulations of the same patient and validated by amplicon sequencing. We found non-synonymous somatic mutations in 12 (60%) patients (10 MS, 1 myasthenia gravis, 1 narcolepsy). There were 27 mutations, all different and mostly novel (67%). They were discovered at subpopulation-wise allelic fractions of 0.2%-4.6% (median 0.95%). Multiple mutations were found in 8 patients. The mutations were enriched in CD8 + cells (85% of mutations). In follow-up after a median time of 2.3 years, 96% of the mutations were still detectable. These results unravel a novel class of persistent somatic mutations, many of which were in genes that may play a role in autoimmunity (ATM, BTK, CD46, CD180, CLIP2, HMMR, IKEF3, ITGB3, KIR3DL2, MAPK10, CD56/NCAM1, RBM6, RORA, RPM and STAT3). Whether some of this class of mutations plays a role in disease is currently unclear, but these results define an interesting hitherto unknown research target for future studies. (C) 2016 The Authors. Published by Elsevier Inc.Peer reviewe

Gemtuzumab-Ozogamicin-Related Impaired Hemoglobin-Haptoglobin Scavenging as On-Target/Off-Tumor Toxicity of Anti-CD33 AML Therapy : A Report of Two Cases

Gemtuzumab-ozogamicin (GO) is a humanized anti-CD33 antibody, which is conjugated to a cytotoxic calicheamicin. It is used to treat acute myeloid leukemia (AML) in combination with chemotherapy. We describe here two GO-treated acute myeloid leukemia (AML) cases: both patients suffered from a toxic syndrome, which manifested as impaired hemoglobin-haptoglobin scavenging and accumulation of hemolysis-related products. Our observations and earlier reports indicated that the reaction was caused by GO-targeted destruction of CD33 + CD163+ monocytes/macrophages, which are responsible for the clearance of hemoglobin-haptoglobin complexes. The rise of plasma lactate dehydrogenase was an early sign of the reaction, and both patients had high levels of free plasma hemoglobin, but plasma haptoglobin and bilirubin levels were paradoxically normal. Symptoms included septic fever and abnormalities in cardiac tests and in the case of the first patient, severe neurological symptoms which required intensive care unit admittance. Therapeutic plasma exchanges supported the patients until the recovery of normal hematopoiesis. The symptoms may be easily confounded with infectious complications-related organ damage. Regarding the increasing use of gemtuzumab-ozogamicin and other emerging CD33-targeted cell therapies, we want to highlight this mostly unknown and probably underdiagnosed toxicity.Peer reviewe

Aikuisten immunologinen trombosytopenia ja sen uudet hoidot

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STAT3 activation in large granular lymphocyte leukemia is associated with cytokine signaling and DNA hypermethylation

Large granular lymphocyte leukemia (LGLL) is characterized by somatic gain-of-function STAT3 mutations. However, the functional effects of STAT3 mutations on primary LGLL cells have not been studied in detail. In this study, we show that CD8+ T cells isolated from STAT3 mutated LGLL patients have high protein levels of epigenetic regulators, such as DNMT1, and are characterized by global hypermethylation. Correspondingly, treatment of healthy CD8+ T cells with IL-6, IL-15, and/or MCP-1 cytokines resulted in STAT3 activation, increased DNMT1, EZH2, c-MYC, l-MYC, MAX, and NF kappa B levels, increased DNA methylation, and increased oxidative stress. Similar results were discovered in KAI3 NK cells overexpressing gain-of-function STAT3(Y640F) and STAT3(G618R) mutants compared to KAI3 NK cells overexpressing STAT3(WT). Our results also confirm that STAT3 forms a direct complex with DNMT1, EZH2, and HDAC1. In STAT3 mutated LGLL cells, DNA methyltransferase (DNMT) inhibitor azacitidine abrogated the activation of STAT3 via restored SHP1 expression. In conclusion, STAT3 mutations cause DNA hypermethylation resulting in sensitivity to DNMT inhibitors, which could be considered as a novel treatment option for LGLL patients with resistance to standard treatments.Peer reviewe