clinical diagnosis of itp · t cell b cell megakaryocyte treatment who: - platelet count, symptoms,...
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ISTH Advanced Training Course
Dubai, UAE
ISTH Advanced Training Course
Clinical Diagnosis of ITP
Nichola Cooper
September 2016
ISTH Advanced Training Course
Dubai, UAE
Disclosures for
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In compliance with COI policy, ISTH requires the following
disclosures to the session audience:
Research Support/P.I. Pfizer
Employee No relevant conflicts of interest to declare
Consultant Novartis, Amgen
Major Stockholder No relevant conflicts of interest to declare
Speakers Bureau No relevant conflicts of interest to declare
Honoraria Amgen, Novartis
Scientific Advisory
BoardAmgen, Novartis
Presentation includes discussion of the following off-label use of a drug or medical device:
<N/A>
ISTH Advanced Training Course
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Immune Thrombocytopenia (ITP)
Isolated thrombocytopenia (platelets <100)
Exclusion of other causes:
Inherited platelet disorders
Increased destruction: (other autoimmunity, viruses)
Decreased production: (viruses, infiltration, marrow failure)
1 in 40,000 F>M, approximately 1-2% familial
Definitions:
Newly diagnosed (0-3 months)
Persistent (3-12 months)
Chronic (>12 months)
Refractory
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ITP terminology: 3 ITP phases
0–3 months 3–12 months >12 months
Newly diagnosed ITP
Persistent ITP Chronic ITP
Possibility of spontaneous remission:
0 to 12 months
Rodeghiero F, et al. Blood 2009; 113: 2386–93.
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Bone marrow
APAs
T cell
B cell
Megakaryocyte
Treatment
Who:
- Platelet count, symptoms,
psychology
When:
- Newly diagnosed,
persistent, chronic
What:
- Steroids, IVIg, rituximab,
TPO-RAs, MMF, other…
Challenges in managing ITP
Diagnosis
Pathogenesis
Treatment
APA, against phospholipid antigens; IVIg, intravenous immunoglobulin;
TPO-RA, thrombopoietin receptor agonist; MMF, mycophenolate mofetil
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Overview
There is no diagnostic test for ITP, it is a
diagnosis of exclusion
The pathology of ITP is unclear and may
have multiple causes
This is relevant when considering
treatment, which should be tailored to
individual patient details
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Pathogenesis (known and presumed)
Genetic susceptibility vs environment
Serum derived effects: antiplatelet antibodies
Unregulated T helper cells and activated
cytotoxic T cells
Inhibition of megakaryopoiesis (?)
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Genetic susceptibility vs infection
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Cines DB et al. Blood 2009;113:6511-6521
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Platelet count after infusion with patient plasma
Antiplatelet antibodies: 19511 Inhibition of megakaryocytes byplasma from ITP patients: 20042
What is the evidence for immune
pathology in ITP? B-cell disease
1. Harrington WJ et al. J Lab Clin Med 1951;38:1–10;
2. McMillan R et al. Blood 2004;103:1364–1369
Time
Pla
tele
ts (
x10
9/L
)
0
400
600
800
1000
200
1 2 3 1 2 3 4 5 6 7 8 9Hours Days
Co
ntr
ol m
egak
aryo
cyte
s(%
)
100
75
50
25
0
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T-cell proliferation in response to own platelets and Th1-mediated disease: 1990s1
Cytotoxic T-cell attack against platelets: 20032
What is the evidence for immune
pathology in ITP? T-cell-mediated
1. Semple JW & Provan D. Curr Opin Hematol 2012;19:357–362;
2. Olsson B et al. Nat Med 2003;9:1123–1124
200
24
20
16
12
3H
-th
ymid
ine
inco
rpo
rati
on
(x
10–3
)
8
4
00 50 100 150
Platelets (x109/L)
40
30
20
10
0
Control
Active ITP
ITP in remission
Pla
tele
t ly
sis
CD4
IL-2IFN-γIL-15
IL-4IL-10TGF-β
Active disease(Th1)
Remission(Th2/3)
CD, cluster of differentiation; IFN-γ, interferon-γ; IL, interleukin;
TGF-β, transforming growth factor-β; Th, helper T cell
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Activated T cells,
Th1,
Th17 profile1–4
Pro-inflammatory
cytokines1–6
Impaired Treg
function7
Immunological profile in ITP
Figure reproduced with permission from Chong BH. Blood 2010;116:4388–4390 (professional
illustration by Marie Dauenheimer); 1. Gernsheimer T. Oncologist 2009;14:12–21; 2. Semple JW &
Freedman J. Blood 1991;78:2619–2625; 3. Semple JW et al. Blood 1996;87:4245–4254; 4. Wang T et
al. Haematologica 2005;90:914–923; 5. Ogawara H et al. Eur J Hematol 2003;71:283–288;
6. Olsson B et al. Nat Med 2003;9:1123–1124; 7. Yu J et al. Blood 2008;112:1325–1328 Treg, regulatory T cell
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Complex ITP pathophysiology underlies
platelet destruction
MHC, major histocompatibility complex;
Tc, cytotoxic T cell; TCR, T-cell receptor
Th cell
IL-2IFN-γ
B cell
Impaired megakaryocyte
maturation
Reduced platelet production
Megakaryocyte
Tc cell
Tc-cell-mediated
platelet
destruction
Platelets
Epitope
spreading
Macrophage
Platelet
phagocytosis
CD80
CD80
CD28
CD28
CD40
CD40
MHC IITCR
CD154
CD154
Platelet autoantibody production
Adapted from Stasi R et al. Thromb Haemost 2008;99:4–13
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MK development:GATA1, RUNX1, FLI1,
ANKRD26, NBEAL2, GFI1N
TPO
Stem cell niche Vascular nicheMigration: SD1a
Pro-platelet development: Cytoskeleton: Rock, myosin
(inhibition Inc)
Collagen, VWF, fibrinogen (GpIb-IX-V, aIIbB3)
Sheerstress
Blood vessel
Bo
ne
Many factors are involved in making
platelets
Adapted from Pecci A & Balduini CL. Br J Haematol 2014;165:179–192 MK, megakaryocyte; TPO, thrombopoietin
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Ashwell–Morell receptor in hepatocytes:
A possible method of platelet regulation
Kile BT. Nat Med 2015;21:11–12
Old platelets are recognized by the Ashwell–Morell receptor (AMR) in hepatocytes causing thrombopoietin production
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Thrombopoietin levels are lower than
expected in ITP
1. Kosugi S et al. Br J Haematol 2003;93:704–706;
2. Aledort LM et al. Am J Hematol 2004;76:205–213
14
Thro
mb
op
oie
tin
leve
l
Normal
12
10
8
6
4
2
0ITP Aplastic
anaemia
No correlation between platelet count and TPO levels in patients with ITP1
No significant difference in TPO levels between patients with ITP and controls2
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Thymus
Bone Marrow
Liver
Spleen
Lymph node
Blood vessel
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Causes of Thrombocytopenia
Platelet production and regulation is not
fully understood
Thrombocytopenia can be the end result of
many events
Gene defects in megakaryocyte development
Bone marrow infiltration
Infection
Increased destruction (APAs and T cells)
? Abnormalities of thrombopoietin regulation
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Clinical diagnosis of ITP: medical history
• Presenting Complaint
– Extent of bleeding (inherited platelet disorders)
• Past Medical History
– Infections (immunodeficiency)
– Previous bleeding (tonsils, teeth)
• Review Of Systems
– Joints, rashes, hair loss, mouth ulcers, night sweats, weight loss (lupus, lymphoproliferative disorders)
• Family History
– Bleeding disorders, thrombocytopenia, autoimmune diseases
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Examination
Extent of bleeding, area of bleeding
Bleeding scores
Lymphadenopathy (not consistent with ITP)
Splenomegaly (not consistent with ITP)
Hepatomegaly (not consistent with ITP)
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pseudothrombocythaemia
ErythrocytesFragmentation: TTP/HUS/DIC/HELLPMacrocytes: megaloblastosis, haemolysisSpherocytes: Evans
PlateletsLarge: hereditary thrombocytopeniasSmall: Wiscott-Aldrich syndromeLarge and small: ITP
LeukocytesToxic granulesPMN InclusionsAtypical lymphocytes (infection, ITP)Blasts: leukaemia
? Bone marrow examination
Blood film examination
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.
Drachman J G Blood 2004;103:390-398
©2004 by American Society of Hematology
Other causes of thrombocytopenia
based on platelet size
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Small platelets MPV <7fL Normal platelets MPV 7-11 fL
Large/Giant platelets, MPV >11 fL
Wiskott-Aldrich syndrome Familial platelet disorder/AML
MHY9-related diseasesMay-Hegglin anomalySebastian syndromeFechtner syndromeEpstein syndrome
X-linked thrombocytopenia Chromosome 10/THC2 Bernard-Soulier syndrome
CongenitalAmegakaryocyticthrombocytopenia
Paris-Trousseauthromboctopenia/Jacobsen syndrome
Thrombocytopenia and absent radii
Velcardiofacial/DiGeorgesyndrome
GATA1 mutation
Grey platelet syndrome
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Platelet size for distinguishing between inherited
thrombocytopenias and immune thrombocytopenia: a
multicentric, real life study
British Journal of HaematologyVolume 162, Issue 1, pages 112-119, 25 APR 2013 DOI: 10.1111/bjh.12349http://onlinelibrary.wiley.com/doi/10.1111/bjh.12349/full#bjh12349-fig-0001
ITPControl
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Investigations in persistent ITP
Immunodeficiency:
Immunoglobulins
Lymphocyte subsets
Platelet disorders:
vWAg (children)
Autoimmunity:
DRVVT
ACL
Autoimmune profile
TFTs
Infections:
Hep B, Hep C, HIV,
CMV
H pylori
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Controversial Investigations in
persistent ITP
Bone marrow examination: Guidelines recommend only in those with abnormal features,
those over 60 and pre-splenectomy
Anti-platelet antibodies: not recommended (not sensitive or
specific enough to direct treatment)
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Bone marrow in ITP
Useful to confirm no other features causing
thrombocytopenia
Useful to describe megakaryocyte features
Useful for a baseline test pre long term
treatment (3 of our cohort have developed
other bone marrow conditions – OS, MF, MDS)
May be useful in the future to direct treatment
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We analysed the interaction of CD4 cells
CD8 cells with megakaryocytes in bone
marrow
29Thomas Mayo Unpublished work Thomas Mayo and Anwar Sayed
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ITP patients have higher MGK density in
the bone marrow but no difference in size
Mann Whitney U
Thomas Mayo
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ITP patients have similar T-cell numbers
to disease controls
Mann Whitney U
Thomas Mayo
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But more CD4/MK interactions and
clustering (more than 1 CD4 per MK)
Mann Whitney U
Thomas Mayo
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Increased CD4/MK interactions in
patients with chronic disease
Mann Whitney U
Thomas Mayo
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No difference in CD8/MK interactions or
CD8/MK clustering
Mann Whitney U
Thomas Mayo
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Co
ntro
l
I TP
0
2 0
4 0
6 0
8 0
1 0 0
C D 8 I F N y+
CD
8%
Co
ntro
l
I TP
0
1 0
2 0
3 0
4 0
C D 8 I L - 2+
CD
8%
Peripheral blood inflammatory CD8
is significantly higher in ITP patients
** **
** P value <0.01
CD8 INF g CD8 IL2
Control Patient Control Patient
Anwar Sayed
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Expression of proinflammatory markers
is not increased in CD4 cells of ITP
patients
Co
ntro
l
I TP
0
2 0
4 0
6 0
8 0
C D 4 T N F a+
CD
4%
Co
ntro
l
I TP
0
2 0
4 0
6 0
8 0
C D 4 I L - 2+
CD
4%
*
* P value <0.05
CD8 TNF a CD8 IL2
Control Patient Control Patient
Anwar Sayed
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C o n t r o l I T P
0
2
4
6
8
1 0
R e g u l a t o r y T c e l l
%
of
R
eg
ul
at
or
y
T
ce
ll
s i
n
CD
4+
po
pu
la
ti
on
C o n t r o l
I T P
Regulatory T cells are significantly
reduced in the periphery in ITP patients
*
* P value <0.05
Control Patient
Anwar Sayed
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Bone marrow changes
May reflect disease activity
Correlation with clinical findings and
peripheral blood abnormalities is in
progress
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Antiplatelet antibodies are difficult to
measure
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Published in: Linlin Shao; Yang Wu; Hai Zhou; Ping Qin; Heyu Ni; Jun Peng; Ming Hou; Platelets 2015, 26, 495-497.DOI: 10.3109/09537104.2014.948838Copyright © 2014 Informa UK Ltd.
Successful treatment with oseltamivir
phosphate (tamiflu) in a patient with
chronic immune thrombocytopenia
positive for anti-GPIb/IX autoantibody
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Potential use for APAs
Identification of specific APAs may allow:
Identification of types of ITP
May be able to predict bleeding and
May predict treatment responses
New methods are needed
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Thrombopoietin levels may distinguish
between consumptive or hypoproliferative
causes of thrombocytopenia
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TPO level
☐Consumptive Hypoproliferative
L
Maker et al Haematology 2013: Thrombopoietin levels in patients with disorders of platelet production: Diagnostic potential and utility in predicting response to TPO Receptor agonists
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Reduced absolute immature platelet
fraction (IPF), but increased % IPF in
ITP
Barsam, et al Blood: Platelet production and platelet destruction: assessing mechanisms of treatment effect in immune thrombocytopenia
IPF %
Absolute IPF
ITP patients
Controls
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IPF may allow a better understanding of
treatment effects
Barsam, et al Blood: Platelet production and platelet destruction: assessing mechanisms of treatment effect in immune thrombocytopenia
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Relevance to ITP patients
Variable findings in the pathology
Only 60% have identifiable antibodies
T cell and bone marrow changes variable
Variable responses to many treatments
with no biomarkers
60% respond to splenectomy
50% respond to rituximab
30-50% respond to immunosuppression
Wide variability in responses shows a
diverse disease
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Treatment recommendations based on
platelet count
Images courtesy of N Cooper
Treat adults
Only treat if bleeding, requiring surgery or requiring anticoagulation or antiplatelet agents
Treatment depends on individual patient factors: Age, other
comorbidity, success of treatment
0 10x109/L 30x109/L 150x109/L
Platelet count
Only treat children with symptoms
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Risk of fatal/severe bleeding in ITP
Presence of other conditions such as
hypertension or cerebrovascular disease
Increased rates of fatal haemorrhage rates
with age
0.004 per patient-year <40 yrs
0.012 per patient-year 40 to 60 yrs
0.130 per patient-year >60 yrs
Increased risk of VTE
George JN. J Thromb Haemost. 2006;4(8):1664-72.; Cohen YC, et al. Arch Intern Med. 2000;160:1630–38; Sarpatwari A, et al. Haematologica. 2010 Jul;95(7):1167-75.
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Management of immune thrombocytopenia (ITP)
Newly diagnosed ITP (0-3 months)Steroids or IVIG
Persistent ITP (3 to 12 months)MMF or thrombopoietin receptor
agonists or Rituximab
Chronic ITP (>12 months)Continuous TPO-RAs
Repeated rituximab (+/- dexamthasone)Continuous MMF
Other: (danazol, dapsone, hydroxychloroquine)
Splenectomy
ThrombocytopeniaDiagnosis: infection, drugs, inherited, acquired
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What factors are involved in treatment
decision making?
Emergency therapy
HRQoL Efficacy
CostPatient
type Ease of use
SafetyClinic
attendance
Rituximab: Increased response in women within first 2 years of diagnosis
MMF: Less effective in patients with virus-associated ITP
TPO-RAs: Potential risk of thrombosis
Consider other factors
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Refractory ITP (approx 5%)
Consider the diagnoses, is it ITP?
Role at this stage for bone marrow
examination
Role for antiplatelet antibody testing
? Role for NGS
There is a real need for better
understanding of ITP and better diagnostic
markers
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Summary
ITP is a diagnosis of exclusion of other
causes of thrombocytopenia
Better understanding of the underlying
pathology is needed
Anti-platelet antibodies may be helpful
TPO levels, bone marrow biopsies and
NGS have potential
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Plan
Establish better antiplatelet antibody testing
Establish a B and T cell
immunophenotyping panel to differentiate
patients in to subtypes
Analyse more bone marrow samples
Correlate TPO levels with outcome
Prospective studies to establish biomarkers
Better phenotyping will help to guide
genomic studies…….- 53 -