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Everything You Ever Wanted to Know About PNH(but didn’t know to ask!)

Lawrence Rice, MDChief, Division of HematologyHouston Methodist HospitalProfessor of MedicineWeill Cornell Medical CollegeHouston, Texas

25 yo woman referred for anemia

3 year hx episodic red urine, every 2 months, lasting hours to days, usually beginning on awakening in the morning

Associated Sx: substernal tightness and difficulty swallowing with episodes; chronic profound fatigue

Prior w/u (all negative): urine cultures, IVP, cystoscopies

Rxs: antibiotics; oral iron

Hgb 9.5, WBC 4.1, plate 225K, MCV110

Further w/u:Reticulocytes 7%; ferritin 38

Urine hgb and hemosiderin + (no RBC)

Ham’s Acidified Serum and Sucrose

Hemolysis tests strongly +

Dx: PNH

The year was 1976!

I can’t even pronounce what you’ve got!

Machiafava – Micheli Disease

PNH for 39 years Subsequent flow cytometries: clone size 80% Rx over the years: few days prednisone 3X/yr

often aborts hemolytic episodes

Hemoglobin levels 7.5 – 10.3 2004: Pulmonary Embolus

Coumadin ever since; also got first RBC Tx

2006: Most severe hemolytic episode; Tx 4 units; creatinine to 6.5, rapidly improved

2009: Last RBC Tx 2011: Rising creatinine (2.4)

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What is PNH? An acquired intracorpuscular RBC defect

arising from a stem cell PIG-A mutation(s)

1,500 – 5,000 affected in US (≥ 5 per

million); more people study it than have it

Illustrates important scientific insights can emerge from dissecting rare disorders

Median age of diagnosis is early 30’s

Diminished quality of life and shortened survival Chronic Intravascular Hemolysis Thrombosis occurs in about 40% of patients

Pathophysiology of PNH

GPI Anchor Defect in PNH

2 2CH CH CH

C=O

O

O

O-P-O

O

O

(18:0,1)

(22:4,5)

C=O

Asp

C=O2 2

O

O=P=O

O

O

CH CH CH

C=O

O

CH2

CH2

NH

CH2

CH2

NH

O2

O

O-P=O

O-P-O

O

O

(18:0,1)

(22:4,5)

(16:0)

N

( 1-2)

( 1-6)

( 1-4)

PROTEIN

NORMAL PNH

Stem Cells Carrying a Hereditary Gene Mutation

Embryonic Stem Cell

Somatic Stem Cells

Blood Muscle Nerve Etc.Egg or Sperm

Egg

Egg Sperm

The Cells of Each Specific Organ

Acquired “Clonal” Mutation in

Blood Stem Cell

Embryonic Stem Cell

Somatic Stem Cells

Blood Muscle Nerve Etc.Egg or Sperm

Egg

Egg Sperm

The Cells of Each Specific Organ

The Beginning of PNH

The mutation in the PIG-A gene in PNH stops the production of an anchor that ties many protein molecules to the outside of the cell (sometimes the stop is only partial and PNH II cells occur)

About 25 molecules are not attached and lost, but only one plays a major role in causing PNH This is CD59 (MIRL), which protects the cells

from complement

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Evolution of PNH in MarrowStem Cell Produces All Blood Cell Lines

ABNORMAL CLONE

NORMAL CLONES

How Do These Abnormal Cells Take Over The Bone Marrow?

Many normal people have blood stem cells with the abnormality characteristic of PNH in very small numbers (6 per million marrow cells)

In PNH, something allows the abnormal cells to become a major population in the marrow and blood (anywhere from 1% to over 90%) This “something” may be “sick” normal stem

cells, such as in aplastic anemia or MDS

What Does PNH Have To Do With Aplastic Anemia?

Many PNH patients have aplastic anemia, MDS, or a history of aplastic anemia

Many PNH patients show signs of inadequate blood cell formation, such as low white cell and platelet counts

Therefore, whatever causes aplastic anemia (immune factors?) may allow PNH to develop

Some recommend all AA and MDS patients be tested for PNH – even few such cells predict response to immunosuppression

Bone Marrow Dysfunction in PNH

PNH

Bone marrowinsult

• In the environment of bone marrow insult, PNH clones expand

• PNH is common in patients with bone marrow failure

• Up to 50% of patients with aplastic anemia

• Up to 25% of patients with MDS

1Araten, et al. Proc Natl Acad Sci. 1999;96:5209-5214.2Johnson & Hillmen. JClinPath:MolPathol. 2002;55:145-152.3Wang, et al. Blood. 2002;100:3897-3902.4Iwanga, et al. Brit J Haem. 1998;102:465-474.5Maciejewski, et al. Brit J Haem. 2001;115:1015-1022.

PIG-Amutation1,2

The Complement SystemProteins that help fight infection

Contribute to disease when things go awry

Lectin Classical Alternative

C3 C3a

C3b

C5 C5a

C5b

Weak anaphylatoxin

Microbial opsonizationImmune complex clearance

Strong anaphylatoxin

Terminal Complement Complex (TCC)Cause of Hemolysis in PNHLysis of Neisseria

Pro

xim

alTe

rmin

al

Microorganisms Antigen-AntibodyConstitutive/

Microorganisms

C5b-9

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August_20_2010US

The Defect in PNH

CD55

The Somatic Mutation of the PIG A gene in the hematopoietic stem cell prevents all GPI anchored proteins from binding to cell surface

19

1. Adapted from: Johnson RJ et al. J Clin Pathol: Mol Pathol. 2002;55:145-152. 2. Brodsky R. Paroxysmal Nocturnal Hemoglobinuria. In: Hematology - Basic Principles and Practices. 4th ed. R Hoffman; EJ Benz; S Shattil et al, eds. Philadelphia, PA: Elsevier Churchill Livingstone; 2005; p. 419-427.

CD59 GPI-anchor

CD55 Prevents formation and augments

instability of the C3 convertases, attenuating the complement cascade

CD59 Forms a defensive shield for RBCs

from complement-mediated lysis

Inhibits the assembly of the membrane attack complex

Absence of CD59 Allows Terminal Complement Complex

Formation

C5b-8 C5b-8

CD55

CD59

C9

C9 C

9

CD59

Walport MJ, et al. N Engl J Med 2001;344:1058-1066 +

What Happens in PNH When Complement Is Activated?

Complement successfully attacks the red cells and they break up (hemolysis) RBCs are destroyed, resulting in anemia Hemoglobin released from RBC into the

plasma

Free hemoglobin binds nitric oxide: esophageal spasm, abdominal pain, erectile dysfxn, fatigue

Hemoglobin is cleared by the kidney, often resulting in red urine

Hemoglobinuria

Thrombosis

Fatigue

Renal Failure

Significant Impact on Quality of

Life

Significant Impact on Survival

Smooth Muscle Dystonias including

Dysphagia,Abdominal Pain,

and Male ED

Pulmonary Hypertension

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

ComplementActivation

Reduced Red Cell Mass

Anemia

Chronic Hemolysis is Central to the Morbidities and Mortality of PNH

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Proposed Classification of PNH Classic PNH Clinical evidence of intravascular hemolysis; no evidence of

another bone marrow disorder.

PNH in the setting of another specified bone marrow disorder Same as classical PNH but concurrent bone marrow disorder such as aplastic anemia, refractory anemia-myelodysplastic syndrome, myelodysplastic or myelofibrotic disease.

PNH subclinical (PNH-sc) in the setting of another specified bone marrow disorder

No clinical evidence of intravascular hemolysis; small populations of PNH cells; concurrent bone marrow disorder.

Diagnosis of PNH

Anemias Decrease in Red Cell Number Common symptoms: fatigue, exertional

intolerance, worsening of other diseases Three mechanisms can underly anemia:

+ Decreased RBC production – such as iron, B12 deficiencies; aplasia

+ Bleeding+ Hemolytic Anemias

Initial Evaluation of Anemia+ History and Physical+ Blood smear and Reticulocyte Count

Hemolytic Anemias

Can be Hereditary (such as sickle cell anemia) or Acquired

Can be Intravascular; usually Extravascular Can be Intracorpuscular or Extracorpuscular

PNH is an Acquired, Intracorpuscular defect leading to Intravascular Hemolysis

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Screening Tests for Hemolysis

Blood Smear

Reticulocyte Count: 99% sensitive outpts,> 90% sensitive inpatients,confounders usually obvious (eg. B12

shot in B12 deficient patient)

Bilirubin, Haptoglobin, LDH are NOT screening tests for hemolysiseach has substantial issues with

sensitivity and specificity

INTRAVASCULAR HEMOLYSIS Free Hemoglobin Binds NO

Smooth MuscleContraction

Smooth MuscleRelaxation

NONO

NOFree Hemoglobin

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Which Patients Should Be Screened For PNH?

Hemoglobinuria Hemolytic anemia Bone marrow dysfunction

Aplastic anemia (AA) or MDS screened annually Coombs-negative intravascular hemolysis

Elevated serum LDH Unusual or unexplained venous thrombosis

Budd-Chiari syndrome Mesenteric, portal, cerebral, or dermal veins

Unexplained arterial thrombosis Episodic dysphagia or abdominal pain with

evidence of chronic hemolysis.

Red Urine?Me Do Cystoscopy

Who Is Smarter Here?

Joe Urologist

Engelbert Internist

Red Urine can be Hematuria,Hemoglobinuria,

Myoglobinuria, Porphyrins,Dipyrroles, Drugs,

Eating too many beets…

Who Is Smarter Here? Or, Why Don’t More Doctors

Go Into Primary Care?

Internal Med H & P (Intermed) $280

Urology H & P $280-545Cystoscopy, Urologist Fee $1,081

Chronic Hemolysis is Central to the Symptoms and Complications of PNH

1Parker, et al. Blood. 2005;106:3699-3709. 2Brodksy. Paroxysmal Nocturnal Hemoglobinuria. In: Hematology - Basic Principles and Practices. 4th ed. R Hoffman; EJ Benz; S Shattil et al, eds. Philadelphia, PA: Elsevier Churchill Livingstone; 2005; p. 419-427. 3Hillmen, et al. N Engl J Med. 1995;333:1253-1258. 4Rosse, et al. Hematology (Am Soc Hematol Educ Program). 2004:48-62. 5Rother, et al. JAMA. 2005;293:1653-1662. 6Socie, et al. Lancet. 1996;348:573-577. 7SOLIRIS® (eculizumab) [package insert]. Alexion Pharmaceuticals; 2007.

SMOOTH MUSCLEDYSTONIA

Abdominal pain Dysphagia Erectile dysfunction

THROMBOSISVenous:

MI

Cerebral Mesenteric

CVA/TIAArterial:

DVT Liver Dermal

IMPAIRED QoL Disabling fatigue Poor physical functioning Pain Dyspnea

ANEMIA Transfusions Fatigue Dyspnea Angina

END ORGAN DAMAGE Kidney GI Liver Brain

1Hill, et al. Blood. 2006;108:972. 2Moyo, et al. Br J Haematol. 2004;126:133-138.3Socie, et al. Lancet. 1996;348:573-577. 4Nishimura, et al. Medicine. 2004;83:193–

207.5Brodsky. Paroxysmal Nocturnal Hemoglobinuria. In: Hoffman, R et al., eds. Hematology - Basic Principles and Practices. 4th ed. 2005; p. 419-427.

PNH Can Be Disabling and Life-Threatening

An estimated 5,000 affected in US

Median age of diagnosis is early 30’s

Diminished quality of life Anemia, dyspnea, pain, and disabling fatigue

Life-threatening Thrombosis occurs in about 40% of patients

Significant Mortality in PNHHillmen, NEJM, 1995

5 year mortality: 35%

Median time from Dx to death: 10 yrs

100

80

60

40

20

00 5 10 15 20 25

Years After Diagnosis

Pat

ien

ts S

urv

ivin

g (

%)

Age- and Sex-Matched Controls

Patients With PNH

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Hillmen et al., NEJM, 1995;333:1253-8. De Latour RP et al., Blood, 2008;112:3099-3106.

Mortality rates in patients with PNH

80 patients with PNH treated between 1940 and 1970

454 French patients with PNH treated between 1950 and 2005

PNH in Francede Latour, Blood, Oct 2008

Survey 58 Hematology Centers 1950-2005 460 PNH patients:

-- 113 Classic PNH-- 93 Intermediate-- 224 AA-PNH Syndrome

Median survival 23 yrs; 5 yr survival 75%-- better survival Dx after 1986-- classic PNH slightly better prognosis

Thrombosis major cause of death in all groups

Thrombosis in PNHThrombosis occurs in 40% of patients

Can be the presenting symptom in PNH Contributes to end organ damage Leading cause of death (40–67% of deaths)

Multiple postulated mechanisms 25-33% of thrombotic events are DVT/PE15-16% of thrombotic events are CVA/MI27-29% unusual venous thromboses,

especially hepatic veins (Budd-Chiari), splanchnic and cerebral veins

34 Thrombotic events in 21 patients prior to eculizumab treatment

12

43

3

4

3

3 2 Budd-Chiari

Mesenteric

Cerebral

Pulmonary embolus

Deep veins

Other

Stroke

Myocardial infarction

Arterial

Incidence of VTE and Relative Risk in PNH vs Inherited Hypercoagulable States

PNH is a less common disease than inherited hypercoagulable states PNH patients have a higher risk for VTE than inherited hypercoagulable state patients

PNH4 ATDeficiency2

PSDeficiency2

PCDeficiency2

PTMutation2

Factor V2

% GeneralPopulation

Expected Patientswith VTE per year*

Rel

ativ

e R

isk

0.00153 0.021 0.031 0.201 2.01 4.81

280 1020 630 4200 6600 14400

62

8 8 82

4

*Based on US population1. De Stefano V et al. Semin Thromb Hemost. 2006;32(8):767-80. 2. De Stefano V et al. Haematologica. 2002;87(10):1095-108. 3. Hill A, et al. Blood. 2006;108(11): 290a. Abstract 985. 4. Relative risk calculated based on 1/1,00 in general population as reported in De Stefano V et al. Haematologica. 2002;87(10):1095-108.

First Ever Ischemic Stroke Incidence in PNH vs General

Population

1. Hillmen P et al. Blood. 2007;110:4123-81. 2. Data on file. 3. Gostynski M et al. J Neurol. 2006;253:86-91.

FEIS risk is elevated in patients with PNH

Age of FEIS in PNH patients is markedly less than in the general population

Thrombophilic Condition

First Ever Ischemic Stroke

(FEIS) (per 100 patient years)

FEIS Risk Relative to General Population Median Age at FEIS

PNH 0.421,2 6 462

General Population 0.083 723

PNH <54 year old 0.241 8

General Population35-54 year old

0.033

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Mechanisms of Thrombosis

Platelet hyperreactivity

Diminished NO availability Thromboplastic RBC membranes

Endothelial perturbations

Impaired fibrinolysis

Risk Factors for Thrombosis

Clinical SymptomsAbdominal pains; dysphagiaOvert hemoglobinuria

Lab Indices LDH (an index of hemolysis)D-dimer (an index of clotting)

Clone size

PNH Clone Size and Thrombosis(excluding warfarin prophylaxis patients)

Hall C et al. Blood 2003;102(10):3587-3591.

0 5 10 15 200

10

20

30

40

50

60

Inci

den

ce o

f th

rom

bo

sis,

%

Granulocyte clone size >50% (n=67)

Granulocyte clone size<50% (n=55)

P=0.0001

Follow-up (years)

3.7 thromboses/100 patient years

Incidence of Thrombosis is Highest in Patients With a Large PNH Clone

Every PNH Patient is UniqueAverage diagnosis delay > 3 yrs; may be > 10 yrs

Clinical signs or symptoms Incidence (%)

Thrombosis 40%

Anemia 89%

Bone marrow failure 10-45%

Fatigue, impaired QOL 96%

Hemoglobinuria 26%7

Abdominal Pain 57%6

Dysphagia 41%6

Erectile Dysfunction 47%6

Chronic Renal Insufficiency (GFR<60/ml/min)

30%8

53

Pain is a Common Symptom in PNH Patients

Almost 3 out of 5 (58%) patients reported significant pain

47% of patients with pain required medical intervention

Lee JW et al. Hematologica 2010. 95 (s2): Abstract #506.

No pain

Pain, No treatment

Pain, Treatment

NSAID

OtherOpioids

Medical Intervention

(N=286)

South Korean National Registry

Flow Cytometry: Diagnostic Test for PNH

Perform on peripheral blood

Test both granulocytes and erythrocytes Erythrocytes alone are not sufficient due to hemolysis

and the dilution effect of transfusions

Use monoclonal antibodies against GPI-anchored proteins, such as CD59 or CD55

Clone size = percent of cells missing GPI-anchored proteins

.

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Historical Management of PNH

Transfusions

AnticoagulantsSupplements

Folic acid; ironSteroids or androgen hormones Allogeneic bone marrow transplant

Curative in 50%, but high Rx-related mortality

56% 2 yr survival with HLA-matched sib donor

Acute GVHD in 34%; chronic GVHD in 33%

Generally conservative and supportive

SOLIRIS® (Eculizumab) Blocks Terminal Complement

C3 C3a

C3b

C5

Pro

xim

alTe

rmin

al

Figueroa, et al. Clin Microbiol Rev. 1991;4:359-395.Walport. N Engl J Med. 2001;344:1058.SOLIRIS® (eculizumab) [package insert]. Alexion Pharmaceuticals; 2007.

C5b-9Cause of Hemolysis

in PNH

C5a

C5b

SOLIRIS

• Proximal functions of complement remain intact

• Weak anaphylatoxin

• Immune complex clearance

• Microbial opsonization

• Terminal complement activity is blocked

• SOLIRIS is a monoclonal AB binding tightly to C5

Complement Cascade

Dosing Schedule Used Throughout Clinical Development

• All patients were vaccinated against Neisseria meningitidis• Concomitant medications allowed:

• Steroids, immunosuppressant drugs, anti-clotting agents and hematinics

• SOLIRIS® should be administered via IV infusion over 35 minutes every 7 days during induction and every 14 days during maintenance

• SOLIRIS® dose adjustment to every 12 days may be necessary for some patients to maintain LDH reduction

Pretreatment

Induction Phase Maintenance Phase

2 weeks before

induction Week

→1 2 3 4 5 6 7 8

9and

every2 weeks thereafte

r

Neisseria meningitidisvaccination

SOLIRIS ® dose,

mg

→

600 600 600 600 900 X 900 X 900

SOLIRIS® (eculizumab) [package insert]. Alexion Pharmaceuticals; 2007.

Long-Term Extension Trial Evaluated long-term safety,

efficacy and effect on thrombosis; Placebo patients

switched to SOLIRIS®N = 187

Pilot Study – NEJM 2004N = 11

TRIUMPH – NEJM 2006 Pivotal Phase III, Double-Blind, Placebo-

Controlled Trial, N = 87

SHEPHERD – Blood 2008Broader patient population, includingthose receiving minimal transfusions

or with thrombocytopenia, N = 97

Eculizumab Studies in PNH

TRIUMPH: Results

ParameterPlacebo(n = 44)

Soliris(n = 43)

LDH levels at end of study, median (U/L)

2,167 239*

Packed RBC units Txed per pt,median (range) †

10(2 - 21)

0*(0 - 16)

Transfusion avoidance, % 0 51*

Patients with stabilized hemoglobin 0 49*

Free hemoglobin at end, median 62 5*

*P < 0.001; † denotes co-primary endpoints

Reduction in LDH During Eculizumab Treatment in TRIUMPH and SHEPHERD

Time, Weeks

Lac

tate

Deh

ydro

gen

ase

(U/L

)

0

500

1000

1500

2000

2500

3000

0 10 20 30 40 50

TRIUMPH – Placebo/extensionTRIUMPH – SOLIRIS/extensionSHEPHERD – SOLIRIS

PI: All patients sustained a reduction in intravascular hemolysis over a total SOLIRIS exposure time ranging from 10 to 54 months.

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Effect of Soliris® on Transfusion

P<0.0000001

Tran

sfu

sed

Un

its/

Pat

ien

t(m

edia

n)

Soliris®Placebo

0

2

4

6

8

10

Clots in Patients With and Without Eculizumab

92% Fewer thrombotic events with SOLIRIS treatment 7.37 clots/100 pt yrs vs 1.07 clots/100 pt yrs Most patients (63%) received concomitant anticoagulants The effect of anticoagulant withdrawal was not studied

Hillmen P, et al. Blood. 2007;110: 4123-4128

39

3

05

1015202530354045

Pre-Soliris® Treatment SOLIRIS Treatment

Th

rom

bo

tic

Eve

nts

(#)

P=0.0001

Thrombotic eventsPatients (n) 79

Pre-treatment

Thrombotic events (n) 34

Proportion occurring on anticoagulation (%)

47

Patient years (n) 608

Thrombotic event rate (n per 100 patient years)

5.60

Eculizumab treatment

Thrombotic events (n) 2

Patient years (n) 260

Thrombotic event rate (n per 100 patient years)

0.8(p<0.001)

1Comparison of eculizumab treatment versus pre-treatment; signed rank test.

21 patients have stopped primary anticoagulation and 1 patient secondary anticoagulation without thromboses occurring (mean duration 10.8 months)

1 2 3 4 5 6 7 8 9

20

40

60

80

100

TIME (YEARS)

N= 79

AGE & SEX MATCHED NORMAL POPULATION

Mortality in 79 PNH patients on eculizumab

Patient survival was compared with age and sex matched control averages obtained using 2001 UK census data from the UK Office of National StatisticsSurvival curves were derived using the Kaplan-Meier method using the log-rank test to analyze the difference between the groups

P=0.46

1 2 3 4 5 6 7 8 9

20

40

60

80

100

TIME (YEARS)

Under 50 N= 4550-69 N= 23

70 and over N= 11

2

1 = 7.49

P = .0062

Mortality in different age groups Adverse Reactions Reported in ≥ 5% of SOLIRIS® -Treated Patients

Reaction SOLIRIS® (43) Placebo (n = 44)

Headache 19 (44) 12 (27)

Nasopharyngitis 10 (23) 8 (18)

Back pain 8 (19) 4 (9)

Nausea 7 (16) 5 (11)

Fatigue 5 (12) 1 (2)

Cough 5 (12) 4 (9)

Herpes simplex virus 3 (7) 0

Sinusitis 3 (7) 0

Respiratory tract infection 3 (7) 1 (2)

Constipation 3 (7) 2 (5)

Myalgia 3 (7) 1 (2)

Pain in extremity 3 (7) 1 (2)

Influenza-like illness 2 (5) 1 (2)

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What Does Soliris® Do?

Quickly and markedly reduces hemolysisImproves anemia (may not be normal)Markedly reduces transfusion needs

Reduces symptoms assoc. with hemolysis fatigue, esophageal spasm, abdominal pain, erectile dysfxn

Appears to reduce thrombosisMay change role of blood thinners

What is Eculizumab NOT expected to Do?

Does not improve genetic defect

Does not improve impaired hematopoiesis(bone marrow dysfunction) Low white count or low platelet count persist

Some shortcomings of Eculizumab

Does not address genetic defect Does not address underlying marrow dysfunction

Extravascular hemolysis (C3-mediated) may continue

Risk of Hemolytic Crisis with sudden withdrawal

Susceptibility to meningococcemia/ meningitis More rapid metabolism in some patients

Very rare genetic resistance (C5 mutation)

Cost Inconvenience (IV infusion every 12-14 days)

PNH Summary

A rare but fascinating blood disorder Incredibly well-understood down to genetic

and molecular levels Myriad manifestations, most importantly:

+ intravascular hemolytic anemia+ clotting tendency, including unusual

sites Scientific understanding increasing rapidly Targeted therapy now available; more

options on the horizon

Wonderful…Just Wonderful! So much

for instilling them with a sense of awe…