Ann Janssens, MD, PhD

Department of Hematology, UZ LeuvenBHS course

8 november 2014

The history of platelets, megakaryocytes and thrombopoietin

Platelet Production

Platelet Pool

eTPOProduction

Normal Splenic Destruction

Endogenous thrombopoietin (eTPO)Megakaryocyte precursorMegakaryocytePlatelet

1Kuter et al PNAS 91:11104, 1994; 2Stoffel et al Blood 87:567, 1996; 3Gurney et al Science 265:1445, 1994; 4de Sauvage et al JEM 183:651, 1996

Platelet homeostasis

Platelet count: 150 -400000/µl

Normal platelet life span 9-10d

Plateletproduction:

1011/d

1000 -3000Platelets/megakaryocyte

Peripheral BloodBone Marrow

Pluripotent Progenitor

Megakaryocyte Progenitor

Mitotic Expansion

JAK2JAK2STAT5

PPPNuclear Maturation

(End mitosis)

Cytoplasmic Maturation(Platelet Specific Granules)

Thrombopoietin signaling and megakaryocytematuration

TPO-R (Mpl)

Platelets

Proplatelet Formations

Cell membrane

Thrombopoietin

Thrombopoietin production: constitutive or regulated?

Izak et al. F1000Prime reports, 2014

Peripheraldestruction

•Autoimmune•Primary ITP• Secondary ITP

•Drug induced: HIT,…

•Alloimmune: posttransfusion, neonatal

•Pregnancy induced

•TTP-HUS

•DIC

•Hemangiomas•…

Insufficientproduction

•AA

•MDS

•Myelofibrosis

•Bone marrowinvasion

•Bone marrowtoxicity

•Megaloblasticanemia

•Hereditarydisorders

•…

others

•Plateletsequestration dueto hypersplenism• Portal hypertension

(cardiac, cirrhosis, V. Porta or V. Cavathrombosis

• Gaucher• Myelofibrosis• Viral infections• …

•Dilution due to massivetransfusion

• In vitro agglutination of platelets when blood is collected in EDTA tubes ( 2% of all thrombocytopenias detected on EDTA blood)

• Measure platelets on blood collected in citrate or heparine tubes

• Look for agregates on the peripheral blood smear

• Personal and familial history• Recent infections• Vaccinations ( >MMR, < H. Influenzae, pneumococci, Hep B,..)• Malignancies• Pregnancy• Recent travels• Recent transfusions• Alcohol abuse• Dietary habits, beverages, herbal preparations• Risk factors for HIV and viral hepatitis• Medication: especially those started 1 to 2 weeks before the onset

of thrombocytopenia, recent exposure to heparine

• Clinical examination with special attention to:

– Bleeding symptoms: pecheciae, purpura, ecchymoses

– Lymphadenopathy– Spleno- , hepatomegaly

– Skeletal abnormalities– Dysmorphy– Skin abnormalities

• Complete blood count– Isolated vs pancytopenia– With neutrophilia or lymphocytosis

• Blood smear– True vs pseudo– Platelet morphology: giant platelets, vs microthrombocytes– Toxic granulation in the neutrophils– Pelger Huet, blasts– Atypical lymphocytes– Fragmentocytes– Tear drops, nucleated red blood cells

• Additional investigations– LDH– Coombs, hapto, bilirubin– Renal function– Coagulation– Liver function– Virus serology,– Bone marrow examination

Lacey et al, Semin Thromb Hemost, 1977

Cohen et al, Arch Intern Med, 2000

fatal haemorrhage if platelets < 30000/Pl persistentlyage < 40y: 0,4%/yage 40-60y: 1,2%/yage > 60y: 13% /y

PetechiaePurpuraBruises

Mucous membrane bleedingEpistaxisGingival

UG-GI tractIntracerebral bleeding

• Dentistry: ≥  10 à 20 x 109/L• Extractions: ≥  30 x 109/L• Regional dental block: ≥  30 x 109/L• Lumbar puncture: ≥  50 x 109/L• GI endoscopy with biopsy: ≥  20 x 109/L• Bronchoscopy: ≥  20 x 109/L

( 50 if also biopsy)

• Organ biopsy: ≥  50 x 109/L (lower for bone biopsy)

• Minor surgery: ≥  50 x 109/L• Major surgery: ≥  80 x 109/L• Epidural: ≥  80 x 109/L

British Committee for Standards in Haematology General Haematology Task Force. Br J Haematol. 2003;120:574-596.Webert KE, et al. Blood. 2003;102:4306-4311.

A. Janssens, C. Lambert, G. Bries, A. Bosly, D. Selleslag, Y. Beguin

Belgian Journal of Hematology. 2013;4(1) (March 2013)

• Isolated thrombocytopenia• Threshold platelets for ITP-diagnosis

− ≤  100000/µl  instead of 150000/µl

• Normal complete blood count and peripheral smear• Avoid Purpura:

– bleeding symptoms frequently absent or minimal at the onset of disease

Rodeghiero et al, Blood 2009;113:2386-2393

Basic evaluation

• Personal and family history

• Clinical examination

• Full blood count with reticulocytes and Coombs

• Peripheral blood film!!!

• Immunoglobulins

• Blood group ?• HIV, hep C, H pylori ?

• Bone marrow in selected patients

Potential utility

• Antiplatelet antibodies

• Antiphospholipid antibodies

• Thyroid function and antithyroid antibodies

• pregnancy test

• Antinuclear antibodies

• PCR for CMV and parvovirus

• Hep B• Chest radiograph• Abdominal ultrasound• Biological fitness

Unproven benefit

• Thrombopoitin

• Reticulated platelets

• Bleeding time

• Platelet survival time

• Serum complement

MANDATORY!!!

• Primary: no obvious initiating and/or underlying cause Avoid idiopathic

SLE 5%

APS 2%

CVID 1%

CLL 2%

Evan’s  2%ALPS, post-tx 1%HIV 1%

Hep C 2%H. pylori 1%

Postvaccine 1%Misc systemic infection 2%

Primary80%

Cines DB, et al. Blood. 2009;113:6511-6521.

• 25% develop thrombocytopenia• 15-25% of ITP pat are pos for ANF• Bleeding risk? Activity of SLE? Vasculitis?• Severe thr-penia with active SLE: treat SLE• Severe thr-cytopenia without active SLE:

• treat as primary ITP; rituximab; splenectomy if refractory

• thrombopenia with thrombotic events and poor outcome of pregnancy• Lupus anticoagulans and anticardiolipin AB pos• +/- 40 (10-70)% of pat with ITP has APLAs• treat as primary ITP, also good outcome with rituximab• Aspirin ?

(hypothyroidism,thyrotoxicosis)• 25-50% ITP patients has antithyroid AB• Control of the underlying thyroid disease

• 10% develop ITP with or without AIHA• Treat as primary ITP

– Avoid immunosuppressive agents– rituximab

– thrombocytopenia 1 to 4 weeks after an acute infection with mumps, rubella,  EBV,  CMV,…  

– appears sudden and can be severe– remits mostly in 2 to 4w

– HIV, hep C, H. Pylori– insidious onset, no tendency to remit

spontaneouslyStasi, Sem Hematol, 2009

• US: prevalence of anti-HCV AB: 2%• Platelets <150000/μl: 41% chronic Hep C (19% in chronic hep B)• Platelets <50000/μl: +/- 9% chronic hep C

• Associated with cryoglobulins and anticardiolipin AB• Bleeding symptoms at higher platelet counts

• Treatment– Interferon-α with antiviral treatment– Corticosteroids ( try to avoid)

• Cavé: increase in viral load, elevation in transaminases– IGIV– Splenectomy– TPO-R agonists Nagamine et al. J Hepatol 1996

Rajan et al. Br J Haematol 2005Stasi, Sem Hematol, 2009

• Before HAART: 5 to 30% of HIV + patients developthrombocytopenia (<150000/μl)

• Incidence of thrombocytopenia higher with an increase in immunosuppression

• If diagnosed before the stage of AIDS: thrombocytopenia mostlymild

• (can have additional bleeding problems: hemophilia, hep C, liverdisease in  drug  addicts,…)

• Treatment– Antiretroviral therapy ( can take weeks)– Corticosteroids, IGIV– Splenectomy– TPO-R agonists

Stasi, Sem Hematol, 2009

• Prevelance depends on socio-economic conditions• Prevalence in adult ITP: 20-80% (Japan 70%, Italy 50%, US 22%)

• Diagnostic methods: – urea breath test and stool Ag test: highest sensitivity and specificity

• Association with dyspepsia????• Older than ITP without H. Pylori

• Eradication therapy: ORR 50 (14-100) % ( higher ORR in Japan), persistent response 70%

• Higher ORR in ITP with a short duration and a higher platelet count>30000

• Platelet responses after 3d to 24 w (2 weeks in Italian trial)• No responses to eradication therapy in pat H. Pylori neg

Stasi, Sem Hematol, 2009

• AITP can occur in all lymphoproliferative disorders

• However more frequent with(1-5%) (can occur at any time in the course of CLL),

(0,2-1%) (can occur at any time in the course of Ho, also in remission)

1(severe)-20%(mild))

Liebman, Sem Hematol,2009

• Corticosteroids, IGIV, splenectomy• Rituximab monotherapy or in combination with cyclophosphamide-dexamethasone• Alemtuzumab• Cyclosporine, Cellcept• TPO-R agonists

• Ho treatment if active disease• Corticosteroids, IGIV, splenectomy• azathioprine

• Cytotoxic treatment against the LGL-clone• Cyclosporine• Alemtuzumab

• Corticosteroids, IGIV• Rituximab,cellcept, SCT

Liebman, Sem Hematol,2009

• Eliminate the trombocytopenia inducing drug– Heparin– Quinine, tonic water– Valproic acid– Non steroidal inflammatory agents– …

• http://w3.ouhsc.edu/platelets/ditp.html

• HAT: 10-30%: binding of heparin to platelets withplatelet aggregation (non-immune) ( first days of treatment, > 80000/Pl, no bleeding or thrombosis, resolves even with continuingheparin)

• HIT: 5% if IV, <1% with LMWH, thrombocytopeniamoderate to severe, 30-75% has a thromboticevent, venous predominance (2.5/1), late onsettill 20d after stop heparin

• Discontinue all heparin products• Alternative non heparin anticoagulants

• Direct thrombin inhibitors– Argatroban– Lepirudin– Bivalirudin

• Factor X inhibitors (Arixtra)• Vit K antagonists: NO• Novel anticoagulants (Pradaxa, Xarelto, Eliquis)• Heparinoids

– Danaparoid ( antiX and anti-II activity) (Orgaran)

• Non immune: directly toxic to megakaryocytes– Linezolid ( >10D 32%)

• Immune: after 1 to 2 weeks , severe bleeding– 5 different mechanisms

Mechanisms:

1 auto-antibodies

2 neo-epitope

3 hapteen

4 drug specific

5 quinine type

6 Immune complex

1

23

4

5

6

:immuunglobuline :(glyco)proteïne :drug(metaboliet) :PF-4 :neo-epitope

FAB

FC

Aster et al., NEJM 2007

Drug-induced ITP

heparin

Kinine, NSAIM, AB, anticonvulsiva

penicillins

abciximabgold

• Autologeous• Allogeneic• Post liver transplantation• …

Active bleeding OR platelets <10000/Pl• treatment is obligatory

No or mild bleeding AND platelets 10-30000/Pl• treatment is a potential option• ( evaluation of pat characteristics)

No bleeding AND platelets >30000/Pl• no need for treatment unless special circumstances

Stasi, Eur J Haematol, 2009

• Platelet count• Previous major bleeding• Age• Life style: sedendary (office workers) vs active ( physical jobs),

contact sports• Additional risk factors for bleeding

– Platelet dysfunction or hemostatic defect (clopidogrel, ASA, po anticoagulantia)– Uremia– Untreated or poorly controled hypertension, Aneurysms– Fever or Infections– Chronic liver disease or alcoholism– History of peptic ulcer

• Tolerance of expected adverse events of treatment• Medical interventions that may cause bleeding• Accessibily of care• Patient’s preferences ( anxiety,…)

Minimize bleeding symptoms or risk of

bleeding

Minimize exposure to

potentially toxic therapy

Decrease activityrestrictions and

improve QOL

Newly diagnosed ITP (<3m)(retrospective diagnosis)

Persistent ITP (3 à12m) ( time in which spontaneous remission can occur)

Chronic ITP (>12m)

Rodeghiero et al, Blood 2009:113;2386-2393

Table 2: Corticosteroids

♀ ° 25-09-1984

• 09-09-2010: vomiting 3x/d since 1week; blood exam solitarythrombocytopenia: 12000/µl; not pregnant

• Some petechiae legs, 1 bruise , last period more explicit; no mucosalbleeding

• Corticosteroids IV because of the vomiting with a rapid rise of platelets , exacerbation of migraine ( gastroscopy neg, CT brain neg)

♂° 01-12-1955

• 07-03-2010: petechiae arms, legs and face and also epistaxis; solitarythrombocytopenia 2000/µl

• Nasal cautery, corticosteroids IV and platelet transfusion• Hospital discharge at d 4 with platelets of 3000/µl, no bleeding symptoms

anymore and with Medrol 64mg/d

d18

♀ °25-06-1960

• Iron deficiency anemia, depression• 10-2007: diagnosis of ITP, Medrol

64mg/d with rapid platelet increase• Tapering corticosteroids very slowly

( stop 01-2009)• No sustained response but safe

platelet count

Table 3: Immunoglobulins (IGIV)

♀ °22-06-1934• 11-2006: diagnosis of ITP• 01-2007: platelets 16000/µl : Medrol 64 mg/d (corticorefractory)• 02-2007: IGIV monthly (4x) with tapering of corticosteroids

• Relapse after a long treatment-free interval can be managed by restartingfirst line treatment.

• Long term steroid use (> 5mg prednisone or equivalent) must be avoided.

– Azathioprine– Cyclosporine– Cyclophosphamide– Danazol– dapsone– Mycophenolate mofetil– Rituximab– Splenectomy– TPO-mimetica– Vinca alkaloiden

Splenectomy (IB)TPO-R agonists after splenectomyTPO-R agonists if a contraindication for splenectomy (IB)

TPO-R agonists after failure to corticosteroids or IgIV (2C)Rituximab after failure to corticosteroids ,IgIV orsplenectomy (2C)

• For adult ITP patients who are intolerant or unresponsive to or relapseafter initial corticosteroid treatment and have a risk of bleeding (platelets< 30.000/µl OR bleeding symptoms), the BHS guideline panel on adult ITP :

BHS recommendations• Splenectomy as it is the only treatment with a curative

potential and has an acceptable safety profile. If possible,splenectomy should be delayed to at least twelve months afterdiagnosis as spontaneous remission can occur

• TPO-R agonists for patients who are refractory to or relapseafter splenectomy or who have a contra-indication tosplenectomy irrespective of the duration of ITP

BHS suggestion in TPO-R agonist refractory patients• Rituximab, azathioprine, cyclophosphamide, cyclosporine A,

danazol, dapsone, mycophenolate mofetil andvincristine/vinblastine are potential treatment options

Vaccination strategy 11/2013:*Prevenar 13 with/or without Pneumo 23/8w; boost with Pneumo 23/5y (rationale ?)*Nimenrix

*

• For adult ITP patients who are intolerant or unresponsive to or relapseafter initial corticosteroid treatment and have a risk of bleeding (platelets< 30.000/µl OR bleeding symptoms), the BHS guideline panel on adult ITP :

BHS recommendations• Splenectomy as it is the only treatment with a curative

potential and has an acceptable safety profile. If possible,splenectomy should be delayed to at least twelve months afterdiagnosis as spontaneous remission can occur

• TPO-R agonists for patients who are refractory to or relapseafter splenectomy or who have a contra-indication tosplenectomy irrespective of the duration of ITP

BHS suggestion in TPO-R agonist refractory patients• Rituximab, azathioprine, cyclophosphamide, cyclosporine A,

danazol, dapsone, mycophenolate mofetil andvincristine/vinblastine are potential treatment options

• Characteristics of treatment with TPO-R agonists : general

• TPO-R agonists are very well tolerated: 5% of patients discontinue therapy becauseof side effects

• Experience in the clinic does not exceed seven years, suspicion for possiblerisks of long-term use is warranted

• Characteristics of treatment with TPO-R agonists : dosing

Response as long as therapy is maintained. Cavé compliance and adherence!!!

+/- 15% can stop TPO-R agonists with long-term remission

• For adult ITP patients who are intolerant or unresponsive to or relapseafter initial corticosteroid treatment and have a risk of bleeding (platelets< 30.000/µl OR bleeding symptoms), the BHS guideline panel on adult ITP :

BHS recommendations• Splenectomy as it is the only treatment with a curative

potential and has an acceptable safety profile. If possible,splenectomy should be delayed to at least twelve months afterdiagnosis as spontaneous remission can occur

• TPO-R agonists for patients who are refractory to or relapseafter splenectomy or who have a contra-indication tosplenectomy irrespective of the duration of ITP

BHS suggestion in TPO-R agonist refractory patients• Rituximab, azathioprine, cyclophosphamide, cyclosporine A,

danazol, dapsone, mycophenolate mofetil andvincristine/vinblastine are potential treatment options

• Most patients: concomitant therapy• Dose: most frequently used 375 mg/m²/w x4• ORR 62,5%, CR 46,3% (all)• ORR 57%, CR 41% (non-splenectomized: n=368))• ORR 68%, CR 39% (children: n= 323 pat)• Time to response 1 to 6,34 w• Duration of response : PR <6m, CR ≈12m• Long-term response: 5y ( 21% of adults, 26% of children)

Arnold et al, Ann Intern Med, 2007;146:25-33 Auger et al, B J Hematol 2012 epub

Liang et al, PLoS ONE 2012;5:Patel et al, Blood, 2012;119:5989-5995

Cavé:Late onset neutropeniaHypogammaglobulinemiaHep B reactivationPML

• Azathioprine, cyclophosphamide, cyclosporine A, danazol, dapsone, mycophenolate mofetiland vincristine have been used after treatment failure for decades.– Variable individual responses– Long-term side effects such as immune suppression

• Antifibrinolytic agents (tranexamic acid : 3 g/day per os)• Oral iron supplements if iron deficient• Local application of adrenalin soaked nose pads• Nasal cautery • Hormone substitutes to prevent menorrhagia• Control of blood pressure• Stop ASA, antiplatelet agents, anticoagulation if appropriate : except case

of recent stent, ischemic heart disease, several peripheral arterialocclusive disease

• Avoid nonsteroidal anti-inflammatory drugs or ASA

1. ITP patients with platelet counts higher than 30000/µl and absence of bleeding signs do not need treatment.

2. Corticosteroids with or without intravenous IVIg are the preferred treatment options for patients with ITP newly diagnosed or relapsing after a long-term treatment-free period.

3. Splenectomy is recommended as second-line treatment as it is the treatment with the highest curative potential and an acceptable safety profile. If possible, splenectomy should be delayed to at least twelve months after diagnosis as spontaneous remission can occur.

4. TPO-R agonists are recommended for patients who are refractory to or relapse after splenectomy or who are unfit for splenectomy, irrespective of the duration of ITP.

5. Rituximab, azathioprine, cyclophosphamide, cyclosporine A, danazol, dapsone, mycophenolate mofetil and vincristine/vinblastine are potential treatment options, especially for patients refractory to TPO-R agonists.

Megakaryocyte differentiationCongenital amegakaryocytic TT with absent radiiT with radio-ulnar synostosis

Megakaryocyte maturationFamilial platelet disorder /AML

Paris-Trousseau & Jacobsen syndromeGATA-1 related TGFI 1B- related T

ANKRD26- related TGray platelet syndrome

Proplatelet formation andplatelet release

MYH9-related diseaseACTN1-related T

FLNA-related TWiskott-Aldrich syndrome

& X-linked TBernard Soulier S

…Pecci et al, Br J Hematol 2014