POST-ISTH: NOVITÀ DAL MEETING DI TORONTO … · POST-ISTH: NOVITÀ DAL MEETING DI TORONTO 2015 Piastrinopenie e piastrinopatie Patrizia NORIS Fondazione IRCCS Policlinico San Matteo

POST-ISTH: NOVITÀ DAL MEETING DI TORONTO 2015

Piastrinopenie e piastrinopatie

Patrizia NORIS Fondazione IRCCS Policlinico San Matteo eUniversità

Pavia

Conflitto di interessi: nessuno

THROMBOCYTOPENIAS DISORDERS OF PLATELET

FUNCTION

SLFN14-related thrombocytopenia

ETV6-related thrombocytopenia

Immune thrombocytopenia: therapy

DISORDERS OF PLATELET FUNCTION

CalDAG-GEFI deficiency


FUNCTION





AS011 Consecutive SLFN14 mutations in 3 unrelated families with an inherited bleeding disorder, thrombocytopenia and secretion defects Fletcher S1, Johnson B1, Lowe G1, Bem D1, Drake S1, Lordkipanidze M1, Guiu IS1,2, Dawood B1, Rivera J2, Simpson M3, Daly M4, Motwani J5, Collins P6, Watson S1, Morgan NV1 and on behalf of the UK Genotyping and Phenotyping of Platelets (GAPP) Study Group 1 Centre for Cardiovascular Sciences, University of Birmingham, Birmingham, UK 2 Centro Regional de Hemodonacion, Universidad de Murcia, Murcia, Spain; 3 Division of Genetics and Molecular Medicine, King’s College London, London; 4 Department of Cardiovascular Science, University of Sheffield, Sheffield; 5 Department of Haematology, Birmingham Childrens Hospital, Birmingham; 6 6Arthur Bloom Haemophilia Centre, Cardiff University, Cardiff, UK

Inherited thrombocytopenias: SLFN14-RT


J Clin Invest. 2015;125(9):3600–3605


J Clin Invest. 2015;125(9):3600–3605

Heterozygous SLFN14 mutations and platelet phenotyping in patients of the 3 families with autosomal dominant thrombocytopenia, and excessive bleeding

J Clin Invest. 2015;125(9):3600–3605


Defective platelet function by aggregometry (A), and by flow cytometry (C), Reduced ATP secretion from dense granules (B)

J Clin Invest. 2015;125(9):3600–3605


Decreased SLFN14 protein levels

Significant reduction in dense granule number (δ) by whole-mount electron microscopy


Thrombosis and Haemostasis 115.5/2016

Moderate autosomal dominant thrombocytopenia, mild or absent bleeding tendency,

and increased platelet size

Thrombosis and Haemostasis 115.5/2016


Reduced expression of SLFN14 in platelets

Reduced proportion of megakaryocytes extending proplatelets and reduced elongation and ramification of the proplatelet shafts

AUTOSOMAL DOMINANT THROMBOCYTOPENIAS DEFECTIVE PLATELET FUNCTION

MYH9-related disease (MYH9-RD)

ANKRD26-related thrombocytopenia

Monoallelic Bernard-Soulier syndrome +/-

ACTN1-related thrombocytopenia

TUBB1-related thrombocytopenia

GFI1B-related thrombocytopenia ++

CYCS-related thrombocytopenia

ITGA2B/ITGB3-related thrombocytopenia ++

Familial platelet disorder and predisposition to AML +


Platelet-type von Willebrand disease +

Paris-Trousseau and Jacobsen syndrome

Thrombocytopenia with radio-ulnar synostosis

AUTOSOMAL RECESSIVE THROMBOCYTOPENIAS

Biallelic Bernard-Soulier syndrome +++

Congenital amegakaryocytic thrombocytopenia

NBEAL2-related thrombocytopenia ++

Thrombocytopenia with absent radii

Thrombocytopenia associated with sitosterolemia

PRKACG-related thrombocytopenia

FYB-related thrombocytopenia

X-LINKED THROMBOCYTOPENIAS

Wiskott-Aldrich syndrome and X-linked thrombocytopenia +/-

FLNA-related thrombocytopenia

GATA1-related diseases +

Inherited thrombocytopenias with defective platelet function



FUNCTION





AS014 Mutation in Ets Variant Gene 6 associates with autosomal dominant thrombocytopenia and raised levels of circulating CD34+ cells

Poggi M1, Baccini V1, Favier M1, Canault M1, Mezzapesa A1, Ghalloussi D1, Chelghoum N2, Mohand-Oumoussa B2, Falaise C1, Peiretti F1, Morange PE1, Saut N1, Ghysdael J3, Nurden AT4, Guidez F5, Bernot D1, Nurden P4, Raslova H6, Tregouet D-A7 and Alessi MC1

1 Laboratory NORT, UMR1062 Inserm, Aix-Marseille Université, Marseille 2 Post-Genomic Platform of Pitié-Salpêtriere, Pierre and Marie Curie University, Paris 3 Inserm UMR 1005, Orsay 4 LIRYC, Plateforme Technologique et d’Innovation Biomedicale, Hôpital Xavier Arnozan, Pessac 5 Institut Universitaire d’Hematologié (IUH), Université Paris Diderot, Hôpital Saint-Louis, Paris 6 Inserm U1009, Gustave Roussy, Université Paris Sud, Villejuif 7 ICAN, Inserm UMR 1166, Paris

Inherited thrombocytopenias: ETV6-RT

BACKGROUND: Unraveling the defective molecular mechanisms involved in inherited thrombocytopenias is important not only to assure correct treatment but also to allow proper prognosis. AIMS: The aim of this study was to clarify the genetic cause of familial thrombocytopenia with normal platelet size with a suspected dominant mode of transmission but with no known responsible mutation documented. METHODS: We performed a whole-exome sequencing in six family members. In vitro experiments were conducted to determine the effects of the identified mutation. Peripheral CD34+ cells were differentiated in megakaryocytes. Transcriptional regulatory properties and recruitment of corepressors of the wild-type and mutant were analyzed using Luciferase reporter plasmid. RESULTS: Exome sequencing identified a heterozygous single nucleotide mutation in Ets-Variant Gene 6 (ETV6, c.641C>T; p.P214L). This mutation is located in the central region involved in the recruitment of a repression complex. Screening for ETV6 mutations in 8 unrelated patients suffering autosomal dominant thrombocytopenia without known genetic etiologies revealed another mutation in ETV6 leading to a stop codon (c.601_602insC; p.L201Pfs*15). Both mutations were associated with low repressive ETV6 activity (residual repressive activity 28% and 0% respectively). p.P214L mutation did not lead to a decreased binding of corepressors (N-CoR, SMRT, Sin3A). p.P214L carrier bone marrow displays dysmegakaryopoiesis with an increased number of megakaryoblasts and hypolobulated megakaryocytes. p.P214L associates with a large expansion of CFU-GM/G/M/MK, a high number of early megakaryocytes and decreased proplatelet formation. Carriers of p.P214L displayed a high level of circulating CD34+ cells. CONCLUSION: Defects in ETV6 activity affect the terminal maturation of megakaryocytes and is responsible of autosomal dominant thrombocytopenia.

Inherited thrombocytopenia due to ETV6 germline mutations

AS014

ETV6: the gene and the protein

HLH o PNT: Helix-loop-Helix domain o N-terminal pointed domain

ETS: E-twenty-Six domain

Receptor tyrosine kinases ETV6-PDGFRB fusion gene ETV6-PDGFRA fusion gene ETV6-NTRK3 fusion gene ETV6-FLT3 fusion gene

Non-receptor tyrosine kinases ETV6-ABL1 fusion gene ETV6-ABL2 fusion gene ETV6-JAK2 fusion gene ETV6-FGFR3 fusion gene ETV6-SYK fusion gene ETV6-FRK fusion gene ETV6-LYN fusion gene

Homeobox genes ETV6-CDX2 fusion gene PAX5-ETV6 fusion gene MNX1-ETV6 fusion gene

Transcription factors ETV6-RUNX1 fusion gene MN1-ETV6 fusion gene ETV6-ARNT fusion gene ETV6-PER1 fusion gene ETV6-MDS1/EVI1 fusion gene

Genes belonging to other families CHIC2-ETV6 fusion gene ETV6-MDS2 fusion gene TTL-ETV6 fusion gene ETV6-STL fusion gene ETV6-PTPRR fusion gene ETV6-NCOA2 fusion gene ETV6-BAZ2A fusion gene ETV6-GOT1 fusion gene ETV6-FCHO2 fusion gene ETV6-ACSL6 fusion gene ETV6-IGH co-localization

Fusion genes involving ETV6

Nature Genetics 2015;47:180-5

ETV6 somatic mutations in hematological malignancies



CMML

age 82y

MPAL

age 50y

MM

age 51y

ALL

age 7y

MDS

age 17y

Pro214Leu

Arg369Gln

Arg399Cys




2015;47:535

ALL

age 3y

ALL

age 35y

ALL

age 14y

Pro214Leu

Arg418Gly



R418G


Family/

Individual

Year of

birth/Sex

Age at

diagnosis, y

WHO bleeding

score

Platelets, x

109/L

MPV,

fL

MPD,

mm

Hgb,

g/dL

MCV,

fL

WBC,

x 109/L

Neutrophils x

109/L

A/I-1 1958/M 31 1 115 8.8 2.44 14.6 99 7.13 4.9

A/II-1 1995/F birth 2 59 8.6 2.24 10.4 68 4.98 2.3

A/II-2 1988/F birth 2 82 8.2 na 13.6 98 5.5 3.39

B/I-21 1972/F 14 0 115 10 2.82 11.1 88 5.02 1.75

B/II-11 2000/M birth 2 66 10.4 2.89 14.0 91 5.36 1.18

B/II-21 1997/F 2 0 44 10.1 3.26 13.1 97 4.04 1.42

C/I-1 1967/M 38 1 112 na 2.73 15.4 103 6.3 4

C/II-1 2002/M 3 0 87 na 2.53 14.1 86 3.84 1.81

D/I-1 1962/M 47 0 110 8.4 2.42 13.7 97 5.4 2.84

D/II-1 2008/F 1 0 109 9.2 2.28 12.6 79 6.82 1.87

E/I-1 1978/F 8 0 105 8.1 na 14.2 97 7.50 5.2

E/I-2 1973/M 5 2 765 8.91 na 14.28 938 11.1 6.29

E/I-3 1970/M 20 0 93 7.9 na 16.9 101 8.30 4.24

E/II-1 1995/M 4 1 171 na 2.73 16.2 94 8.4 5.37

E/II-2 2002/M birth 2 99 7.430 na 14.0 90 6.15 2.45

F/I-2 1966/F 7 0 105 8.9 2.55 13.4 107 7.11 4.4

F/II-1 2003/F birth 1 57 8.6 2.40 14.2 97 6.59 4

F/II-2 1998/F birth 1 70 8.7 2.36 14.4 97 8.24 5.3

G/I-21 1964/F 20 0 101 7.6 3.17 13.6 97 4.71 2.02

G/II-11 1987/M 3 1 101 7.8 2.99 15.9 97 5.3 2.39

Nature Genetics 2015;47(5):535-8 and unpublished data

ETV6-related thrombocytopenia: clinical and laboratory picture


Platelets on electron micrograph

Nature Genetics 2015;47(5):535-8

Control Patient

BOM of one affected individual without leukemia

Mild dyserythropoiesis

Immature hypolobulated megakaryocytes


Aberrant cellular localization of mutant and endogenous ETV6

Decreased transcriptional repression

Altered megakaryocyte maturation

Nature Genetics 2015;47(5):535-8


Leu349Pro

Asn385fs

Sabine Topka1,2☯, Joseph Vijai1,2☯, Michael F. Walsh3☯, Lauren Jacobs1, Ann Maria1,

Danylo Villano1, Pragna Gaddam1, Gang Wu3, Rose B. McGee3, Emily Quinn3, Hiroto

Inaba3, Christine Hartford3, Ching-hon Pui3, Alberto Pappo3, Michael Edmonson3, Michael

Y. Zhang4, Polina Stepensky5, Peter Steinherz1, Kasmintan Schrader6, Anne Lincoln1,

James Bussel7, Steve M. Lipkin7, Yehuda Goldgur8, Mira Harit5, Zsofia K. Stadler1,

Charles Mullighan3, Michael Weintraub5, Akiko Shimamura4,9, Jinghui Zhang3, James R.

Downing3, Kim E. Nichols3‡*, Kenneth Offit1,2,7‡*

preB-ALL preB-ALL

ALL + MDSAML

Few forms, all with severe bleeding tendency

Many forms, bleeding diathesis from severe to absent.

Risks other than bleeding

No treatment other than platelet transfusions

Specific treatments for specific disorders

The evolving spectrum of inherited thrombocytopenias

Semin Thromb Hemost 2013;39:161


Defects possibly associated with low platelet count in syndromic forms of inherited thrombocytopenia

haematologica | 2016; 101(1)


Predisposition to hematological malignancies

With giant or large platelets MYH9-RD bBSS TUBB1-RT GPS FLNA-RT GFI1b-RT mBSS ITGA2B/B3-RT ACTN1-RT SLFN14-RT PRKACG-RT

Normal or slightly increased platelet size ETV6-RT FDP/AML ANKRD26-RT TCPT XLTT CTRUS VWDP

Normal or slightly decreased platelet size TAR CAMT CYCS-RT XLT/WAS

Inherited thrombocytopenias based on platelet size

With giant or large platelets MYH9-RD bBSS TUBB1-RT GPS FLNA-RT GFI1b-RT mBSS ITGA2B/B3-RT ACTN1-RT SLFN14-RT PRKACG-RT

Normal or slightly increased platelet size ETV6-RT FDP/AML ANKRD26-RT TCPT XLTT CTRUS VWDP

Normal or slightly decreased platelet size TAR CAMT CYCS-RT XLT/WAS

Inherited thrombocytopenias based on platelet size


FUNCTION





OR340 Severe bleeding tendency and impaired platelet function in a patient with CalDAG-GEFI deficiency Kato H1, Nakazawa Y2, Kashiwagi H1, Tadokoro S1, Morikawa Y1, Morita D2, Kurokawa Y2, Kanakura Y1 and Tomiyama Y3

1 Hematology-Oncology, Osaka University Graduate School of Medicine, Suita; 2 Pediatrics, Shinshu University School of Medicine, Matsumoto; 3 Blood transfusion, Osaka University Hospital, Suita, Japan

Case report: the proband has been suffering from repeated severe spontaneous nasal bleeding which required transfusions since she was 1 year old. The initial laboratory assessment indicated normal platelet count, normal PT/APTT, markedly prolonged bleeding time, and reduced platelet aggregation responses to ADP and collagen.

Inherited disorders of platelet function


OR340

Results: Expression levels of her platelet surface glycoproteins were comparable to those of control. Although PMA-induced integrin aIIbb3 activation was normal, aIIbb3 activation induced by various agonists was impaired. Platelet granule release was also decreased. In addition, slower aIIbb3 activation kinetics was observed by ‘initial velocity assay’. These results, along with normal calcium mobilization and impaired Rap1 activation, suggest impaired activation process of aIIbb3. Western blotting revealed the deficiency of CalDAG-GEFI, but normal talin and Kindlin-3 expression in her platelets. Sequencing results revealed compound heterozygous mutation, R360del and K309X, in CalDAG-GEFI. Introduction of expression vector for mutant CalDAG-GEFI into 293T cells confirmed that these mutations are responsible for the deficiency of CalDAG-GEFI. Conclusion: We identified new mutations of CalDAG-GEFI causing severe bleeding problem. Our results indicate the essential role of Cal- DAG-GEFI in aIIbb3 activation as well as hemostatic function of platelets.

Disorders of platelet function from CalDAG-GEFI gene (RASGRP2) mutation

J Exp Med 2014, 211: 1349-1362

cG742T mutation in the RAS guanyl-releasing protein-2 (RASGRP2) gene

coding for calcium- and diacylglycerol-regulated guanine exchange factor-1

CalDAG-GEFI)

Severe bleeding phenotype since childhood, often requiring platelet transfusions


J Exp Med 2014, 211: 1349-1362

Impaired platelet aggregation

J Exp Med 2014, 211: 1349-1362


Expression of membrane receptors, including aIIbb3 and GPIba were normal

Platelet release reaction and clot retraction were normal

Thrombin generation was moderately affected (delayed time to peak but normal total amount)


Incomplete platelet spreading on immobilized fibrinogen

J Exp Med 2014, 211: 1349-1362


Journal of Thrombosis and Haemostasis 2015, 13 (Suppl. I): S12-S9

Pathologies of genes affecting surface constituents

Pathologies of genes affecting intracellular proteins

Journal of Thrombosis and Haemostasis 2015, 13 (Suppl. I): S12-S9


FUNCTION





PO271-TUE Diagnosis of venous thromboembolism during eltrombopag therapy for immune thrombocytopenia and successful replacement with romiplostim Amit O, Kirgner I and Avivi I Hematology and Bone Marrow Unit, Tel Aviv Medical Center, Tel Aviv, Israel Background: There has been growing evidence concerning the development of venous thromboembolism (VTE) during eltrombopag therapy in patients with refractory immune thrombocytopenic purpura (ITP). However, the feasibility of switching between thrombomimetic agents post VTE development has not been fully explored to date. Conclusion: These case reports support the feasibility of replacing eltrombopag therapy with romiplostim in conjunction with anticoagulation, for patients diagnosed with VTE during treatment with eltrombopag for refractory ITP.

Acquired thrombocytopenias: ITP and NEW DRUGS

PO311-MON Thrombopoietin receptor agonists do no not cause coagulation activation: in patients with immune thrombocytopenia Garabet L et al. Fredrikstad, Norway; New York, USA; Oslo, Norway; Los Angeles, USA. Background: Thrombopoietin receptor agonists (TPO) are effective treatment in immune thrombocytopenia (ITP). The rate of thrombotic events was reported in up to 6% of patients in TPO-agent trials raising concerns of coagulation activation. Conclusion: Coagulation is not activated by TPO-agents. Previous studies have shown that neither platelets are activated. This indicates that other mechanisms are affected by TPO to increase the risk of thromboembolism.

Acquired thrombocytopenias: ITP and NEW DRUGS

OR265 The effect of danazol and a new treatment strategy in primary immune thrombocytopenia – an analysis of 412 cases from a single center in China Liu W1, Gu X2, Li Y1, Lv M1, Sun T1, Lv C1 and Yang R1

1 Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin and 2 Kunming Medical University, Kunming, China Aims: this study aimed to assess the long-term benefit and side effects of danazol therapy, and determine the appropriate dose and duration of danazol. Results: Among the 366 cases of ITP, the overall response and complete response rate was 61.9% and 27.3% respectively. The median time to response was 1.8 ± 3 months. The median duration of remission was 18 ± 10 months, and 50.9% patients got sustained remission without danazol. The rate of relapse was 21.9% .

Acquired thrombocytopenias: ITP and OLD DRUGS

A total of 20.1% patients experienced side effects, 4 cases discontinued due to side effects, others were well-tolerated with mild side effects.

Adverse events

OR265


…age, phase of the disease, the time before danazol treatment, and the duration of danazol therapy influenced the response (p< 0,05)…

OR265


OR265 The effect of danazol and a new treatment strategy in primary immune thrombocytopenia – an analysis of 412 cases from a single center in China Liu W1, Gu X2, Li Y1, Lv M1, Sun T1, Lv C1 and Yang R1

1 Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin and 2 Kunming Medical University, Kunming, China Aims: this study aimed to assess the long-term benefit and side effects of danazol therapy, and determine the appropriate dose and duration of danazol. Conclusion: Our study shows that low to medium dosage danazol is effective and better tolerated, but long-term therapy is necessary to maintain remission. Instead of being the second line treatment, low dosage danazol can be combined with GC in the initial stage, which can reduce the dosage and duration time of GC to achieve well-tolerated and long-term remission.


Patrizia NORIS

Università di Pavia - Fondazione IRCCS Policlinico San Matteo

[email protected]

mailto:[email protected]

Documents

POST-ISTH: NOVITÀ DAL MEETING DI TORONTO … · POST-ISTH: NOVITÀ DAL MEETING DI TORONTO 2015 Piastrinopenie e piastrinopatie Patrizia NORIS Fondazione IRCCS Policlinico San Matteo