Anemias part II

Semmelweis University

Faculty of Medicine

Department of Internal of Medicine and Oncology

Prof. Judit DemeterSemmelweis University

Faculty of Medicine

Department of Medicine and Oncology

Anemias part II

4 February 2020

AnaemiasHead of the Department

Professor István Takács

MD,PhD, DSc

Aplastic anaemia

Hemolytic anaemiasExtravascular destruction of red blood cells

inherited

congenital sphaerocytosis

thalassamias

sickle cell anaemia

Intravascular destruction of red blood cells

PNH

OUTLINE AND EXAMPLES



MD,PhD, DSc

Aplastic anaemia

AA



MD,PhD, DSc

Definition, disease severity & clinical presentation ofAA

• AA is a rare & heterogeneous disorder.

• pancytopenia witha hypocellular bone marrow in the absence of an abnormal

infiltrate or marrow fibrosis.

• To diagnose AA there must be at least two of the following

- haemoglobin concentration (Hb) <100 g/l,

- platelet count <50 × 109/l,

- neutrophil count <1·5 × 109/l.

The majority (70‐80%) of cases are idiopathic

The incidence is 2‐3 per million per year in Europe, higher in East Asia)

There is a biphasic distribution, with peaks at10‐25 years & over 60years



MD,PhD, DSc

Overlap of AA with PNH, MDS, and constitutional marrow

failure syndromes, as well as to other immune-mediated

diseases in which a single organ is targeted.

Aplastic anemia in relationship to other diseases

Aplastic Anemia NS Young N Engl J Med. 2018 Oct 25; 379(17): 1643–1656



MD,PhD, DSc

Aplastic anaemia: assessment of severity

• Severe AA (SAA); Marrow cellularity <25% (or 25–50% with

<30% residual haematopoietic cells),

• plus at least 2 of:

• (i) neutrophils <0·5 ×109/l,

(ii) platelets <20 × 109/l

(iii) reticulocyte count <20 × 109/l (

• Very Severe AA (VSAA); As for SAA but neutrophils <0·2×

109/l

• Non‐severe AA (NSAA); AA not fulfilling the criteria for SAA or

VSAA

AA:Classification• Inherited

Fanconi’s anemia, dyskeratosis congenita, Shwachman-Diamond Syndrome, Reticular dysgenesis, Amegakaryocytic thrombocytopenia, familial aplastic anemia, preleukemia (monosomy 7)

• Acquired

Irradiation

drugs and chemicals: cytotoxic agents, benzene, idiosyncratic reaction,

chloramphenicol, NSAIDS, antiepileptics, Gold

viruses: EBV, Hepatitis virus (non-A,non-B, non-C, non-G),

Parvovirus (transient aplastic crisis or pure red cell aplasia), HIV

Immune diseases: eosinophilic fasciitis, hyperimmunoglobulinemia,

thymoma and thymic carcinoma, GvHD in immunodeficiency

PNH

Pregnancy

Idiopathic

Drugsusedin thetreatment of AA

• Antithymocyte globulin (ATG):• Appears to be immunomodulatory as well as lymphocytotoxic- producing a state of

tolerance by preferential depletion of activated T cells.

• Source: Horse or Rabbit

• Cyclosporine:• its selective effects on T-cell function is due to direct inhibition on the expression

of nuclear regulatory proteins, resulting in decreased T-cell proliferation and activation.

• Steroids: side effects, toxicity

• eltrombopag



MD,PhD, DSc

Aplastic anaemia. NS Young. N Engl J Med. 2018 Oct 25; 379(17): 1643–1656

Treatment algorithms for adults with immune aplastic anemia.



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Aplastic anemia (AA): Clinical Endpoints

• Response defined as transfusion independence.

About 50% response rate with horseATG.

• Relapse defined as requirement of additional immunosuppresants.

Happens in 30-40% of patients.

• Clonal evolution occurs in 15% of cases.

Into MDS, AML, PNH

AA: Differential Diagnosis

• Pancytopenia with hypocellular bone marrow

Acquired aplastic anaemia - Inherited aplasticanaemia

Hypoplastic MDS - HypoplasticAML

• Pancytopenia with cellular bone marrow

Primary bone marrow diseases -MDS

PNH

Myelophthisis

Hairy cell leukemia

Hypersplenism

Overwhelming infection

Brucellosis

Sarcoidosis

- Myelofibrosis

- Bone marrow lymphoma

- SLE, Sjogren’s disease

- Vitamin B12 and folate deficiency

- Alcoholism

- Ehrlichiosis

- tuberculosis

• Hypocellular bone marrow with or without cytopenia

Q fever

Mycobacteria

Hypothyroidism

- Legionaires disease

- Tuberculosis

- Anorexia nervosa

Improving on ATG & cyclosporine for first line management of AA?

• Addition of high dose steroids did not improve outcomes and just added to toxicity.

• Addition of G-CSF and GM-CSF did not improve outcomes

• Addition of mycophenolate did not improve response rates or outcomes.

• Sirolimus was equally ineffective.

• Cyclophosphamide was associated with a higher death rate due to prolonged neutropenia.

Guidelines for the diagnosis and management of adult aplastic anaemia



MD,PhD, DSc

• Born: 1967.o5.o9.

• Symptoms from May 2000 – Hair loss

– Skin bleeding

Hb: 60 g/l, PLT: 1, 5 G/l, WBC 3,1 absz. gran. 1, 0 G/l

dg (BM biopsy) aplastikus anemia.

kezelés:– No HLA identical donor – no SCT possibility

– szubstitution (RBC and platelet transzfusions)

– Immunsuppressive treatment was started

Case presentation– V. A.



MD,PhD, DSc


treatment 1:

– Oct. 2000

ATG Fresenius (rabbit origin), cyclosporin and steroids• Elevation of liver transzaminases, icterus.

• Hepatoprotective drugs, reduction of Cyclosporin dose, improvement (with fluctuations)

– Substitution: 22 units of RBC., és 264 units of platelets

• Sepsis (repeatedly) due to granulocytopenia,

severe immunodeficiency

(Enterobacter cloacea, Klebsiella pneumoniae stb.)



MD,PhD, DSc


• Further treatment (IST2):– Nov 2001 antilymphocyte globulin (ATG)

(horse origin), cyclosporin and steroids

– Deterioration of liver function, cyclosporin had to be omitted

– Substitution: 8 Units RBC, és 7o Units platelet suspension

– hypertension transitorily.

– REMISSION LASTING SINCE 2002 !!!



MD,PhD, DSc


• Control yearly , last time:31 jan 2020 .

– WBC: 5,1 G/L

– Neutrophils: 54 %

– Absolute numer of neutrofils : 2,8 G/L

– Haemoglobin: 139 g/L

– Hematokrit: 0,41

– Platelets:164 G/L

– Liver function tests: norm.

– Ferritin: 3237 mg/ml norm.: 16-300)



MD,PhD, DSc

AA quiz

A 24-year-old man undergoes evaluation for treatment of AA

Two of his siblings are HLA-identical matches.

Hemoglobin

8.3 g/dL (83 g/L) (following

transfusion of 1 unit of

irradiated packed erythrocytes

last week)

Leukocyte count

500/µL (0.5 × 109/L) with 23%

neutrophils, 3% band forms,

and 71% lymphocytes

Platelet count 26,000/µL (26 × 109/L)

Reticulocyte count 0.2%

• Review of the bone marrow biopsy done confirms the diagnosis of AA, demonstrating an aplastic bone marrow with normal cytogenetics.

• Which of the following is the most appropriate treatment?

A: Allogeneic hematopoietic stemcell transplantation

B: Antithymocyte globulin (ATG), corticosteroids,and cyclosporine

C: Autologous hematopoietic stemcell transplantation

D: Corticosteroids

E: Granulocyte colony-stimulating factor



MD,PhD, DSc

Bone marrow smear in hemolytic anaemia



MD,PhD, DSc

Symptoms of hemolytic anemia

AnaemiaIcterus (jaundice)SplenomegalyAbdominal painCholelithiasisWeaknessLack of appetite

In children growth retardation



MD,PhD, DSc

Common causes of intravascular and extravascular hemolysis in adultsExtravascular destruction of red blood cells

Intrinsic red blood cell defectsEnzyme deficiencies (eg, deficiencies of G6PD, pyruvate kinase)Hemoglobinopathies (eg, sickle cell disease, thalassemias, unstable Hbs)Membrane defects (eg, hereditary spherocytosis, hereditary elliptocytosis)

Extrinsic red blood cell defectsLiver diseaseHypersplenismInfections (eg. Babesia, malaria)Oxidant agents (eg, dapsone, nitrites, aniline dyes)

Other agents (eg, lead, copper, snake and spider bites)Autoimmune hemolytic anemia (warm- or cold-reacting, drugs) !!!!

Intravascular destruction of red blood cellsMicroangiopathic hemolyt. anaemia (eg TTP, HUS, aortic stenosis, prosthetic valve leak)Transfusion reactions (eg, ABO incompatibility)Infection (eg, clostridial sepsis, severe malaria)Paroxysmal cold hemoglobinuria; cold agglutinin disease (on occasion)Paroxysmal nocturnal hemoglobinuriaFollowing intravenous infusion with hypotonic solutionsSnake bites

G6PD: glucose-6-phosphate dehydrogenase; TTP: thrombotic thrombocytopenic purpura; HUS: hemolytic uremic syndrome.



MD,PhD, DSc

Copyrights apply



MD,PhD, DSc



MD,PhD, DSc

The integrity of the cytoskeleton bound to the RBC membrane is lost

pathological, sphaeric form of RBC+

decrease of membrane stability

a sequestration of pathologic RBCs in the spleen(their life time decreases from ) days to a few days

Hereditary spherocytosis (HS)



MD,PhD, DSc


is a heterogeneous group of disorderscaused by variants in certain genes that encode proteins of the red blood cell (RBC) membrane and cytoskeleton,

most commonly spectrin (SPTA1 and SPTB genes), ankyrin (ANK1 gene), and band 3 (SLC4A1 gene).

These abnormalities decrease the levels of proteins that link the RBC inner membrane skeleton to the outer lipid bilayer, which in turn leads to membrane vesiculation, progressive spherocyteformation, and hemolysis.

Inheritance: three-fourths of these variants act in an AD manner, with the remainder AR.



MD,PhD, DSc

HS affects as many as 1 in 2000 to 1 in 5000 (prevalence, approximately 0.02 to 0.05 percent)


HS can present at any age and with any severity. The majority of affected individuals have mild or moderate hemolytic anemia



MD,PhD, DSc

Dg suspected based

on the history,

examination, and

results of initial laboratory testing

confirming the diagnosis of HS EMA (eosin-5-maleimide) binding is the preferred test because of its

high sensitivity and specificity.

Other options: osmotic fragility, glycerol lysis and cryohemolysis.




MD,PhD, DSc

●CBC and RBC indices –The mean corpuscular hemoglobin concentration (MCHC) is often the most useful parameter for assessing spherocytosis; an MCHC ≥36 g/dL isconsistent with spherocytes

●Blood smear review – abundance of spherocytes,the degree of polychromatophilia, which reflectsreticulocytosis.

●Hemolysis - increase in LDH, - increase in indirect bilirubin, - decreased or absent haptoglobin- an elevated reticulocyte count.

●Coombs testing – to eliminate the possibility of immune-mediated hemolysis,




MD,PhD, DSc



MD,PhD, DSc

• In the case of intercurrent infections anaemia

might become severe,

• In the case of parvovirus infection the hemolytic

crisis might become aplastic




MD,PhD, DSc

5% severe

chronic severe anaemia

tranfusion dependent

• frequent crisis when infections occur

• choleithiasis (in the first decade already)

95%:medium or light forms

• slight anaemia if any

• Crisis very rare

• Gallstones only late and rarely

Diffferent degrees of severity in congenital sphaerocytosis



MD,PhD, DSc

EMA (eosin-5-maleimide) teszt

EMA „stain” + flowcytometry

Specificity: 99,1%,

Sensitivity :92,7%,

quick (a few hours and has results)

UH (splenomegaly, gallstones)

BM aspiration

Genetics

Diagnosis of congenital sphaerocytosis



MD,PhD, DSc

A sequestration of RBCs and thus haemolysisstops definitely

Reduction/end of anaemia

Redution of serum-bilirubin

Reduction of reticulocyte count

Reduction of transfusion requirement

End of abdominal pain/discomfort

Cholelithiasis develops later if at all

Hereditary spherocytosis (HS): arguments for splenectomy



MD,PhD, DSc

Risk of OPSI (Overwhelming PostsplenectomyInfection Syndrome) sepsis (1%-2,4%)

Earlier the life-time risk following SP was huge:

33%-75%

Long antbiotic prophylaxis (?)

Partial splenectomy??

Preventive vaccination!!!!!

(Pneumococcus

N. meningitidis

H. influenzae)

Hereditary spherocytosis (HS): arguments against splenectomy



MD,PhD, DSc



MD,PhD, DSc

normal Hb-molecule• Hemoglobin = heme + globin

• Tetramer structure, with central haemcomponent

• 2 alpha, 2 non-alpha chains

• (Adult) Hb (HbA): haem + α2β 2 (95 %)

• (Adult) Hb 2 (HbA 2): haem + α2δ2 (2.5-3%)

• Foetal Hb (HbF): haem + α 2 γ 2

• Alpha globin: Ch16

• Beta globin: Ch 11

. 39

www.sagan.blog.cz



MD,PhD, DSc

Minőségi Hb-pathiák: thalassemiák• Discovery

• von Jaksch (D) Cooley (US)• Pathogenesis

• Imbalance of the globin chains (alpha, beta, gamma, delta)

• Hb-deficiency• → less Hb in the RBCs→ hypochromasia• → smaller RBCs → microcytosis• Excessive alpha chains (in beta thalassaemia) buld

chains with delta chains (elevated HbA2), partlyprecipatate the cells as „foreign” bodies –haemolysis

• Thalassemic haemoglobinopathies: exon 1 beta chain is pathologic, both qunat and qualit. Defect

• Names, forms

According to the name of the deficient chain• From light (β+)thalassaemia to zero (β0)thalassaemia

• HbBart: 4γ, HbH: 4β



MD,PhD, DSc

Thalassemiák: aniso-poikilocytosis,

“target cells”

∝-

thalassaemia

β-

thalassaemia



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The mechanism of clinical complications in non-transfusion dependent thalassaemia

42



MD,PhD, DSc

Distribution of α-thalassaemia

(the most frequent mutations are shown)



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Beta thalassaemias

• Silent carrier β thalassemia

• β-thalassaemia trait

• β-thalassaemia intermedia

• β-lánc variáns β-thalassaemia (pl. HbE β)

• β-thalassemia maior (Cooley-anaemia)



MD,PhD, DSc

Thalassaemias: treatment I

• Genetic counseling

• Aim: elevation of Hb to physiologial levels

Prevention of haemosiderosis

• Transfusions 2-3 weekly – 1-2 units

• Iron-chelating agents

• iv: desferroxamin

• Oral iron-chelators (deferiprone)

• Both

• Vitamin C, E, folic acid

45



MD,PhD, DSc

Thalassaemiák: treatment II

• Splenectomy? (only if transfusion requirement is huge)

• HbF production preference• histon deacetylase inhibition, e.g.. hydroxyurea

• Allogenic SCT

• Genetherapy?

46



MD,PhD, DSc

New therapeutic targets in β-thalassemias: (A,D) impaired α:β-globin ratio, (B) ineffective erythropoiesis, and (C) iron metabolism and hemolysis.

TMPRSS6: transmembraneprotease serine 6

Hematology Am Soc Hematol Educ Program. 2017 Dec 8; 2017(1): 278–283.



MD,PhD, DSc

FDA approval in 2019: LUSPATERCEPT



MD,PhD, DSc

Fenaux P et al, Blood, 2019

Luspatercept for the treatment of anemia in myelodysplastic syndromes and primary myelofibrosis, Blood, 2019,

Luspatercept is a soluble fusion protein with an adjusted extracellular domain of the activin receptor type IIB (ActRIIB) linked to the Fc domain of human immunoglobulin G1.



MD,PhD, DSc

Luspatercept improves hemoglobin levels and blood transfusion requirements in a study of patients with β-thalassemia

Piga et al, Blood 2019

Transfusion burden reduction vs baseline for patients with β-thalassemia treated with luspatercept (n = 32). (A) Percentage change in RBC transfusion burden over a continuous 12-week period post baseline vs the 12-week baseline period. Each bar represents 1 patient(B) Absolute change in RBC units on study vs baseline. Each circle represents 1 patient’s baseline RBC transfusion burden, For both panels, only patients with a baseline transfusion burden of ≥2 RBC units and 12-week postbaseline transfusion data are shown.



MD,PhD, DSc

A new medical therapy for anemia in thalassemia

Emanuele Angelucci, A new medical therapy for anemia in thalassemia, Blood, 2019,

Copyright © 2020 American Society of Hematology



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MD,PhD, DSc

Hb-pathies: geography• Geographics• HbS: Equatorial-Africa, South-Turkey,

Palestin, Saudi Arabia, Mediterraneancoast

mutation Africa?HbC: West-Africa (North-Ghana)HbE: South-East- Asia (frequent in

Thailand)HbD Punjab: Sics, India (Punjab)county

Heterozygotic state: „balanced polymorphism”

• Pl. malaria hipothesis: P. falciparum malaria and HbS, less proneness forinfections, ill. less severe clinicalcourse (RBC fall apart more easilybecause of the sickling)

Hijmans RJ et al Int J Climatol, 2005, 25, 1965



MD,PhD, DSc

Sickle cell disease



MD,PhD, DSc

• First desciption :Herrick JB (2001). "Peculiar elongated and sickle-shaped red blood corpuscles in a case of severe anemia. 1910". The Yale Journal of

Biology and Medicine. 74 (3): 179–84. PMC 2588723.

• Haemoglobinopathy, AR inheritance

• Genetics: Normál Hb összetétel:HbA(2 alpha és 2 beta lánc ) HbF(2 alpha és 2

gamma lánc),HbA2(2 alpha és 2 delta lánc)

HbS: in the DNA coding for the beta chain in the 6th position adenin instead

of timin van, thus Glu is produced instead of Valin

J.E.Maakaron:Sickle Cell Anemia Medscape (Drugs&Diseas.-Hematology 2019)

Sickle cell disease

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2588723

https://en.wikipedia.org/wiki/PubMed_Central

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2588723



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Sickle cell disease



MD,PhD, DSc

Sickle cell disease

S. L- Thein and J.Howard:

:How I treat the older adult with sickle cell disease

(Blood. 2018;132(17):1750-1760)



MD,PhD, DSc

Sickle cell disease• Hb electrophoresis: in Hb SS (Homozygotic SS disease)

HBA NOT detectable,

HBF variable amount (in our new pt HBF: 4,7%, HBA2: 3,7%)

diagnostics

• Chest roentgen

• CT and MRI

• Transcranial Doppler

• Abdominal ultrasound

• Cardiac ultrasound

Pulmonary

hypertension

Splenic infarcation Bone necrosisDactylitis

Sickle cell anaemia



MD,PhD, DSc

Sickle cell disease: treatmentHydroxiurea: antimetabolit, elevation of Hb F level

less veno-occlusiv crisislessens transzfusion requirement

Pain killers: opiatesTrombosis profilaxis: LMWH, aspirinTransfusions if necessaryFolic acidTreatment of infections:: Streptococcus pneumoniae, Salmonella typhiVaccination:autoasplenia : Pneumococcal,meningococcal prophylaxis

influenza vaccinationParvovírus B19 infection:immunglobulinsPulmonary hypertension : endothelin 1receptorantagonist (bosentan),

foszfodiészterase inhibitors(sildenafil)Iron-chelating agenst



MD,PhD, DSc

Sickle cell disease: treatment

Voxelotor : increases the affinity of Hb to the O2 molecule and inhibits thepolymerization of HbS

Gene therapy

Allogenic SCT



MD,PhD, DSc

Change in Hemoglobin Level from Baseline to Week 24.

Vichinsky E. A Phase 3 Randomized Trial of Voxelotor in Sickle Cell Disease.N Engl J Med. 2019 Aug 8;381(6):509-519

Persons with SS disease have abnormally elevated blood viscosity and are generally recognized to have an increased risk of vaso-occlusive crisis with excessive increases in the hemoglobin level (e.g., after simple transfusion).

The absence of an increased incidence rate of vaso-occlusive crisis with voxelotor despite significant increases in the hemoglobin level suggests that voxelotor raises hemoglobin levels without negatively affecting blood viscosity.

This may be due to the upstream mechanism of action of voxelotor(inhibition of HbS polymerization), which results in improved red-cell deformability and reduced blood viscosity with voxelotor in vitro.

Conclusion: Voxelotor provided a significant, sustained increase in hemoglobin level and reduced the incidence of worsening anemia and hemolysis in persons with sickle cell disease

Vichinsky E. A Phase 3 Randomized Trial of Voxelotor in Sickle Cell Disease. N Engl J Med. 2019 Aug 8;381(6):509-519

Metcalf et al, 2017

GTB 440=voxelotor

Change in Hemoglobin Level from Baseline to Week 24.


VOXELOTOR STUDY: Change in Hemoglobin Level from Baseline to Week 24.


FDA approval in 2019



MD,PhD, DSc

Common causes of intravascular and extravascular hemolysis in adultsExtravascular destruction of red blood cells

Intrinsic red blood cell defectsEnzyme deficiencies (eg, deficiencies of G6PD, pyruvate kinase)Hemoglobinopathies (eg, sickle cell disease, thalassemias, unstable Hbs)Membrane defects (eg, hereditary spherocytosis, hereditary elliptocytosis)

Extrinsic red blood cell defectsLiver diseaseHypersplenismInfections (eg. Babesia, malaria)Oxidant agents (eg, dapsone, nitrites, aniline dyes)Other agents (eg, lead, copper, snake and spider bites)Autoimmune hemolytic anemia (warm- or cold-reacting, drugs) !!!!

Intravascular destruction of red blood cellsMicroangiopathic hemolyt. anaemia (eg TTP, HUS, aortic stenosis, prosthetic valve leak)Transfusion reactions (eg, ABO incompatibility)Infection (eg, clostridial sepsis, severe malaria)Paroxysmal cold hemoglobinuria; cold agglutinin disease (on occasion)Paroxysmal nocturnal hemoglobinuriaFollowing intravenous infusion with hypotonic solutionsSnake bites

G6PD: glucose-6-phosphate dehydrogenase; TTP: thrombotic thrombocytopenic purpura; HUS: hemolytic uremic syndrome.



MD,PhD, DSc

PNH



MD,PhD, DSc

• Rare disease

• Prevalence: 1-2/1000000

• Prognosis poor if untreated

• Cause:

mutation of PIGA-gene

deficiency/absence of

GPI anchor

deficency of GPI anchored

proteins (e.g. CD55, CD59)

Increased complement activation, haemolysis

100

80

60

40

20

0

0 5 10 15 20 25Years After Diagnosis

Pati

en

ts S

urv

ivin

g (

%)

Actuarial Survival From the Time of

Diagnosis in 80 Patients With PNH2

Age- and sex-

matched controls

Patients with PNH

Peffault de Latour R et al. Blood 2008;112(8):3099-106

PNH

PNH - haemoglobinuria

Toronto Medical HospitalParoxysmalis nocturnalis hemoglobinuria (PNH)



MD,PhD, DSc

• Cytopenia:

• Complement mediated haemolysis and aplastic

component

• Dominating hemolysis in the classic type

• Thrombosis:

• Main cause of mortality

• Mainly venous, often visceral veins

• Pathomechanism multifaktorial: NO depletion,

platelet activation, lack of other GPI-anchoed

proteins

• Best treatment: treatment of PNH

PNH-clinical symptoms



MD,PhD, DSc

• 3 groups:

• Classic PNH:

• Dominant haemolysis, thrombosis frequent

• PNH with other primary BM disorders

• Aplastic anemia, myelodysplastic syndrome (MDS), myelofibrosis (MF)

• Subclinical PNH:

• Small PNH clone present, but no active haemolysis or thrombosis

• Problem:

• Not always clearly distinguishable

• Cytopenia, BM insufficiency might be present also in the classic form

Clinical grouping of PNH

Normal circumstances and PIG-A gene

RBCCD55 CD59

GPI-anchor

C3 konvertáz

• No complementactivation

• Nohaemolysis

INHIBITION

PNH, mutated PIG-A gene

RBC

AbsentGPI-anchor

C3 konvertáz

C5

C5b C6 C7 C8 C9

Membrane-attack complex

• Complementmediatedintravascularhaemolysis

• Lower pH (atnight) helpscomplementactivation

HAEMOLYSIS

Effect of eculizumab in PNH

VVT

AbsentGPI-anchor

C3 convertase

C5

C5b C6 C7 C8 C9

Membrane attack complex (MAC)

Eculizumab:Anti C5 monoclonalantibody

RBC

• No complementaktivation

• No haemolysis

• Thrombotic is also reducedwhile treated

Humanized monoclonal anti –C5 antibodyWeekly once for 5 weeks., than beweekly once .Counteracts CD59 deficiency, but not totally CD55 deficiency

77

93% of Patients with PNH Have Concomitant Cytopenias1

Anemia and

thrombocytopenia

Anemia and

neutropenia

Other combinations

Unknown

Unexplained VTE/ATEUnexplained CytopeniasRA-MDSAAHemoglobinuriaCoombs-negative Hem A

1. Socie G et al. Lancet 1996;348:573-7.

33%

32%

17%

7%

4%7%

Anemia

Pancytopenia

PN

HE

U11002

78

High-risk Patients to Test for PNH1,2


Intravascular hemolysis as evidenced by

hemoglobinuria or elevated plasma hemoglobin

Other acquired Coombs-negative, non-schistocytic,

non-infectious hemolytic anemia

1. Borowitz MJ et al. for the International Clinical Cytometry Society. Cytometry B Clin Cytom 2010;78B: 211-30. 2 . Parker C et al. for the International PNH Interest

Group. Blood 2005;106:3699-709.

Rule PNH in or out

using flow cytometry

and clinical assessment

PN

HE

U11002



MD,PhD, DSc

Laboratory Investigation of PNH

• Flow cytometry immunophenotyping is the method of choice for

PNH testing

• Diagnosis or identification of PNH cells by demonstrating deficiency of GPI-linked proteins from granulocytes/monocytes/red cells

Preferred granulocyte reagents

are CD24, CD66b, CD16,

FLAER

Identification of Patients at High Risk for PNH1-3

80

Unexplained

thrombosis

(venous or arterial)

Unexplained

cytopeniasRA-MDSAAHemoglobinuria

Coombs-negative

hemolytic anemia


1. Richards SJ et al. Cytometry B Clin Cytom 2009;76B:47-55. 2. Borowitz MJ et al. for the International Clinical Cytometry Society. Cytometry B Clin Cytom 2010;

78B: 211-30. 3. Parker C et al. for the International PNH Interest Group Blood 2005;106:3699-709.

Rule PNH in or out

using flow cytometry

and clinical assessment

AA – aplastic anemia; RA-MDS – Refractory anemia – myelodysplastic syndrome PN

HE

U11002

81

Thrombosis

Fatigue

Renal Failure

Abdominal Pain

Dyspnea

Dysphagia

Hemoglobinuria

Erectile Dysfunction

Normal red blood cells

are protected from

complement attack by a

shield of terminal

complement inhibitors

Without this protective

complement inhibitor

shield, PNH red blood

cells are destroyed

Intact RBC

Complement

Activation

PNH is a Progressive Disease of Chronic Hemolysis1

Significant

Impact on

Survival

Significant

Impact on

Morbidity

Free Hemoglobin

Anemia

Pulmonary Hypertension

1. Lee JW et al. EHA 2010, abstract 506 2. Figure adapted from Rachidi S et al. Eur J Int ern Med2010;21:260-7.

PN

HE

U11002

82

Chronic Kidney Disease

Renal insufficiency

Dialysis

Hypertension

End Organ Damage

Brain

Liver

GI

Anemia Transfusions

Hemosiderosis

Fatigue / ImpairedQuality of Life

Abdominal pain

Dysphagia

Poor physical functioning

Erectile dysfunction

Pulmonary Hypertension

Dyspnea

Cardiac Dysfunction

ThrombosisVenous

PE/DVT

Cerebral

Dermal

Hepatic/Portal

Abdominal ischemia

Arterial

Stroke/TIA

MI

Pulmonary HypertensionThrombosis Fatigue / Impaired QOLChronic Kidney Disease End Organ Damage Anemia

Chronic Hemolysis is the Underlying Cause of Progressive Morbidities and Mortality of PNH1,2

1. Lee JW et al. EHA 2010, abstract 506. Figure adapted from 2. Rachidi S et al. Eur J Intern Med 2010; 21:260-7.

PM

HE

U11001



MD,PhD, DSc

• curative treatment: hemopoetic stem cell transplantation:

• in cases when eculizumab not available

• Heterozygotic c.2654G-A mutáció (non-responsive to eculizumab)

• PNH with severe AA

• Symptomatic treatment:

• Corticosteroids (?):

• Iron and folic acid replacement

• Thrombosis prophylaxis – individual decision

• Inhibition of complement activation: eculizumab

Treatment of PNH

84

Eculizumab: Humanized

First in Class Anti - C5 Antibody1

Hinge

CH

3C

H2

Human IgG4 Heavy Chain

Constant Regions 2 and 3

(Eliminates complement activation)

Complementarity Determining Regions

(murine origin)

Human Framework Regions

• No mutations

• Germline

Human IgG2 Heavy Chain

Constant Region 1 and Hinge

(Eliminates Fc receptor binding)

1. Rother R et al. Nat Biotech 2007;25:1256

SO

LE

U11002

Eculizumab® Blocks Terminal Complement1

C3 C3a

C3b

C5

Pro

xim

al

Te

rmin

al

1. Rother RP et al. Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria. Nature

Biotech. 2007;25(11):1256-64. 2..SOLIRIS® SmPC: SOLIRIS® (eculizumab) summary of product characteristics. Alexion Europe SAS 2007.

C5b-9Cause of Hemolysis

in PNH

C5a

C5b

Eculizumab

• Proximal functions of

complement remain intact1

• Weak anaphylatoxin

• Immune complex clearance

• Microbial opsonization

• Terminal complement

activity is blocked1

• Eculizumab® binds with high

affinity to C51,2

Complement Cascade1

SO

LE

U11002

Eculizumab in PNH – reduction of LDH

P Hillmen et al: The Complement Inhibitor Eculizumab in Paroxysmal Nocturnal Hemoglobinuria, N Engl J Med 2006; 355:1233-1243.

P Hillmen et al: Effect of Eculizumab on Hemolysis and Transfusion Requirements in Patients with Paroxysmal Nocturnal

Hemoglobinuria, N Engl J Med 2004; 350:552-559

Eculizumab – reduction of transfusion need

P Hillmen et al: The Complement Inhibitor Eculizumab in Paroxysmal Nocturnal Hemoglobinuria, N Engl J Med 2006; 355:1233-1243.

*

**

*

(n=87) (n=30) (n=35) (n=22)0

2

4

6

8

10

12

14

16

Overall 4-14 15-25 >25

Pre-treatment Transfusion Requirement (RBC units)◘

Me

dia

n U

nits T

ran

sfu

se

d

18

0

0,5

1

1,5

2

2,5

3

2012.0

7.0

5

2012.0

8.0

5

2012.0

9.0

5

2012.1

0.0

5

2012.1

1.0

5

2012.1

2.0

5

2013.0

1.0

5

2013.0

2.0

5

2013.0

3.0

5

2013.0

4.0

5

2013.0

5.0

5

2013.0

6.0

5

2013.0

7.0

5

2013.0

8.0

5

2013.0

9.0

5

2013.1

0.0

5

2013.1

1.0

5

rel. LDH (1=460 U/L) rel. Haptogl. (1=2g/L) rel. Thrombocyta (1=150 G/L)

rel. HGB (1=125g/L) rel. RET

2E vvt cc.

4E thr cc.

2E vvt cc.

4E thr cc.

2E vvt cc.

4E thr cc.

1E vvt cc.

4E thr cc.

2E vvt cc.

4E thr cc.

4E thr cc.4E vvt cc.

4E thr cc.

2E vvt cc.

8E thr cc.

6E vvt cc.

20E thr cc.

4E thr cc.4E vvt cc.

4E thr cc.

2E vvt cc.

8E thr cc.

Eculizumab

600 mg

09.16.

eculizumab

előtt:

09.20.

eculizumab

kezdése után

09.23.

eculizumab

kezdése

után

09.27.

eculizumab

kezdése

után

09.30.

eculizumab

kezdése után

Össz-komplement

(haemolyticus

teszt):

(ref.: 48-103

CH50/ml)

76

H50/ml

6

CH50/ml

0

CH50/ml

0

CH50/ml

0

CH50/ml

Alternatív út

összkomplement

(WIELISA-ALT):

(ref.: 70-105%)

112 % 15 % 3 % 7 % 3 %

Komplement C3:

(ref.: 0,9-1,8 g/l)1,33 g/l 1,54 g/l 1,39 g/l 1,16 g/l 1,17 g/l

Komplement C4:

(ref.: 0,15-0,55

g/l)

0,45 g/l 0,51 g/l 0,46 g/l 0,49 g/l

H A eculizumab treatment

Eculizumab in PNH -survival

Kelly R J et al. Long-term treatment with eculizumab in paroxysmal nocturnal hemoglobinuria: sustained efficacy and improved

survival Blood 2011;117:6786-6792.



MD,PhD, DSc

Diagnosis of PNH : QUIZ

1.Cytogenetics

2.sucrose test

3.flow cytometry

4. BM biopsy



MD,PhD, DSc

Diagnosis of PNH

1.Cytogenetics

2.sucrose test

3.flow cytometry- FLAER

4. BM biopsy



MD,PhD, DSc

Most frequent cause of death in PNH1. Infection2. Transformation into acute leukemia3. Bleeding4. Thrombosis



MD,PhD, DSc

Most frequent cause of death in PNH1. Infection2. Transformation into acute leukemia3. Bleeding4. Thrombosis



MD,PhD, DSc

Optimal treatment of PNH outside clinical studies

1. allogeneic stem cell transplantation

2. intravenous immunoglobulins

4. monoclonal anti-CD20 antibody –Rituximab

5. complement-inhibitor - Eculizumab



MD,PhD, DSc

Optimal treatment of PNH outside clinical studies

1. allogeneic stem cell transplantation

2. intravenous immunoglobulins

4. monoclonal anti-CD20 antibody –Rituximab

5. complement-inhibitor - Eculizumab



MD,PhD, DSc

Aplastic anaemia

Hemolytic anaemiasExtravascular destruction of red blood cells

inherited

congenital sphaerocytosis

thalassamias

sickle cell anaemia

Intravascular destruction of red blood cells

PNH



MD,PhD, DSc

Documents

Anemias part II