Paroxysmal Nocturnal Hemoglobinuria

1
Paroxysmal Nocturnal Hemoglobinuria

• March 8, 2005

2
Case

• 43 y old Hispanic man who presented to his PCP
  for headaches. Labs revealed a pancytopenia.
  Referred to hematologist.
• Bone marrow revealed hypocellular marrow (5-10).
  
• Bone marrow repeated 6 months later showed
  minimally hypocellular marrow (30).
• Two years later, he developed dark urine and
  hemolytic anemia.
• Bone marrow showed hypercellularity (80) with
  normoblastic erythroid hyperplasia. A significant
  population of myeloid cells (85) demonstrated
  atypically diminished expression of CD16, as well
  as an aberrant lack of CD55 and CD59 expression.
  A significant population of monocytic cells (83)
  lacked expression of CD14, CD55, and CD59.
• Findings consistent with PNH.

3
History

• Investigator Year
  Contribution
• Gull 1866 Described nocturnal and
  paroxysmal nature of intermittent
  haematinuria in a young man.
• Strubing 1882 Distinguished PNH from
  paroxysmal cold haemoglobinuria and march
  haemoglobinuria. Attributed the problem to
  the red cells.
• van den Burgh 1911 Red cells lysed in
  acidified serum. Suggested a role for
  complement.
• Enneking 1928 Coined the name
  paroxysmal nocturnal haemoglobinuria.
• Marchiafava 1928- Described perpetual
  hemosiderinemia.
• and Micheli 1931 Their names became eponymous
  for PNH in Europe.
• Ham 1937- Identified the role
  of complement in lysis of PNH red 1939 cells.
  Developed the acidified serum test, also called
  the Ham test, which is still used to diagnose
  PNH. Demonstrated that only a portion of PNH
  red cells are abnormally sensitive to
  complement.
• Davitz 1986 Suggests defect in membrane protein
  anchoring system responsible
• Hall Rosse 1996 Flow cytometry for the
  diagnosis of PNH

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Paroxysmal Nocturnal Hemoglobinuria

• Described as a clinical entity in 1882.
• Acquired disorder of hematopoiesis.
• Triad intravascular hemolysis, thrombosis, and
  decreased hematopoiesis.
• Nocturnal refers to belief that hemolysis is
  triggered by acidosis during sleep and activation
  of complement to hemolyze abnormal RBCs.
• However, hemolysis is shown to occur throughout
  the day and is not paroxysmal. Urine concentrated
  overnight may cause dramatic change in color.

5
Paroxysmal Nocturnal Hemoglobinuria

• Due to an acquired hematopoietic stem cell
  mutation defect.
• Somatic mutation (from deletions to point
  mutations identified) of the PIGA
  (phosphotidyl-inositol glycan class A) gene on
  the X-chromosome. Namely the transfer of
  N-acetylglucosamine to phosphatidylinositol.

Hillmen and Richards, Br J Haematol, 2000
6
Paroxysmal Nocturnal Hemoglobinuria

• Cells derived from the abnormal clone deficient
  in surface proteins normally attached to the cell
  membrane by a glycosylphoshpatidylinositol (GPI)
  anchor.
• Essential group of membrane proteins lacking are
  called complement regulating surface proteins
  decay accelerating factor (DAF) or CD55,
  homologous restriction factor (HRF) or C8 binding
  protein, and membrane inhibitor of reactive lysis
  (MIRL) or CD59.
• These proteins interact with complement proteins
  and interfere with the assembly of complement's
  membrane-attack complex.
• Deficiency CD59 is largely responsible for the
  hemolysis and implicated in the thrombotic
  tendency (induces platelet activation).

7
GPI Linked Proteins
Rosti, Haematologica, 2000
8
GPI anchored Proteins
Johnson and Hillmen,Mol Pathol, 2002
9
Paroxysmal Nocturnal Hemoglobinuria

• Frequency rate determined to be 5-10x less than
  aplastic anemia (2/million). Perhaps more
  frequent in Southeast Asia and Far East.
• Men and women affected equally.
• At any age, but frequently found among young
  adults.
• Mortality/Morbidity median survival of 10.3 yrs.
  Morbidity depends on variable expression of
  hemolysis, bone marrow failure, and
  thrombophilia.
• Main cause of death is venous thrombosis followed
  by complications of bone marrow failure.

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Hemolysis

• Hemoglobinuria/hemosiderinuria.
• Intravascular hemolysis - elevated retic count
  and LDH with low haptoglobin in the absence of
  hepatosplenomegaly.
• Hemolytic anemia of variable severity proportion
  of abnormal cells, degree of abnormality of the
  cells, degree of complement activation (viral or
  bacterial infections).
• Bone marrow usually markedly erythroid with
  decreased iron stores.
• Can be precipitated by administration of Fe to an
  Fe deficient patient due to large number of
  complement sensitive cells delivered to
  circulation at once.

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Thrombosis

• Hepatic vein (Budd-Chiari syndrome) jaundice,
  abdominal pain, hepatomegaly, ascites.
• Abdominal vein thrombosis can lead to bowel
  infarction.
• Cerebral vein thrombosis if sagittal vein
  affected can lead to papilledema and pseudotumor
  cerebri.
• Dermal vein thrombosis raised, painful, and red
  nodules in skin affecting large areas.
• Increased platelet aggregation, enhanced
  expression of tissue factor, and impaired
  fibrinolysis.
• In two series, almost all patients developing
  thrombosis had more than 50 and 61 PNH
  granulocytes.

12
Cytopenias

• Deficient hematopoiesis
• Usually presents with anemia despite the presence
  of an erythroid marrow with suboptimal
  reticulocytosis.
• Neutropenia and thrombcytopenia can occur in a
  hypoplastic bone marrow.

13
Other

• Esophageal spasms can occur concurrently with
  episodes of hemoglobinuria. Manometry shows
  generation of peristaltic waves of great
  intensity.
• Males can have impotence.
• Absence of nitric oxide (taken up by hemoglobin
  in plasma)?

14
Abnormal Cells

• PNH I cells normal in sensitivity to complement.
• PNH II cells moderately more sensitive than
  normal cells (partial absence).
• PNH III cells markedly sensitive, requiring one
  fifteenth to one twentieth of complement for an
  equal degree of lysis (complete absence). This
  group is increased in patients with more severe
  PNH and is associated with a mean life span of
  10-15 days.

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Diagnostic Test

• Acid hemolysis (Ham test) PNH red cells
  incubated in separate tubes to fresh acidified
  serum (0.5mL), unacidified serum, and heated
  acidified serum. Lysis determined by optical
  density of the supernatant fluid after 1hr
  incubation and addition of 4mL of 0.15 M NaCl.
• Positive test gt1 lysis in acidified serum.
• May be positive in congenitial dyserythro-poietic
  anemia
• Specific but not very sensitive.

16
Diagnostic Test

• The sugar water or sucrose lysis test uses the
  ionic strength of serum that is reduced by adding
  an iso-osmotic solution of sucrose, which then
  activates the classic pathway sucrose molecules
  enter red cells through defects and produce
  osmotic lysis.
• PNH diagnosed by gt5 lysis.
• Less specific but more sensitive.

17
Diagnostic Test

• Low-tech test. The patient is asked to collect a
  sample of urine each hour for 48 hours. The
  physician lines them up and eyes their colors,
  and the diagnosis is plain.

18
Diagnostic Test

• Flow Cytometry
• Expression of GPI-anchored proteins CD55 and CD59
  analyzed on hematopoietic cells using monoclonal
  antibodies and flow cytometry.
• Highly specific. No other condition in which red
  cells are a mosaic of normal and GPI linked
  protein deficient cells.

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Flow Cytometric Analysis
Hillmen et al, NEJM, 1995
20
Pathogenesis

• Relative/absolute bone marrow failure
• present to some degree in all patients
• relative granulocytopenia/thrombocytopenia
• decreased capacity to form myeloid colonies
• Two stage model
• somatic mutation in PIG-A gene (understood)
• some cause for bone marrow failure (not
  understood)
• Is damage directed at a GPI linked molecule?
• Mutant clone may expand as a result of an
  immune-escape from antigen-driven lymphocyte
  attack on hematopoietic progenitors.

21
Dual Pathogenesis Hypothesis
Hillmen and Richards, Br J Haematol, 2000
22
Aplastic Anemia and PNH

• The association between PNH and aplastic anemia
  goes both ways.
• Although current emphasis is on patients with
  marrow failure who are found to have PNH clones,
  the older literature describes PNH patients with
  apparent progression to aplastic anemia. In such
  instances, the endstage has been called "spent
  PNH.
• All four of the following clinical scenarios are
  based on the same pathophysiologic mechanisms.
  The only difference is the temporal relationship
  between bone-marrow failure and somatic PIG-A
  mutation.

23
Aplastic Anemia and PNH
24
Natural History of PNH
Hillmen et al, NEJM, 1995

• Long term study of 80 patients with PNH seen at
  one institution between 1940 and 1970
• Results
• median age at diagnosis 42 (16-75)
• median survival 10 years
• 28 survived more than 25 years
• 39 had one or more episodes of venous thrombosis
• 12 experienced spontaneous clinical recovery
• leukemia did not develop in any of the patients

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Sites of Thrombosis in PNH
Hillmen et al, NEJM, 1995
26
Natural History of PNH
Hillmen et al, NEJM, 1995
27
Prognostic Factors

• Thrombosis
• Evolution to pancytopenia, myelodysplastic
  syndrome, or acute leukemia (1 10-100x more
  than normal)
• Age gt55
• Evidence of deficient hematopoiesis at disease
  onset, such as aplastic anemia or
  thrombocytopenia

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Treatment

• Supportive, prevent complement activation
• Prednisone beneficial. Moderate doses (15-30 mg)
  administered on alternate days. Higher doses for
  acute episodes.
• Fe supp given urinary loss (10-20x normal).
  Suppress hemoglobinuric episode with prednisone.
• Folic Acid supp given increased need of
  hyperplastic marrow for cofactor.
• pRBC transfusion as needed.
• Androgenic hormones effective but mechanism
  unclear.
• Eculizumab a monoclonal Ab that binds to C5
  component of complement and inhibits terminal
  complement activation being studied.

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Treatment

• Therapy for/prevention of thrombosis
• Thrombolytics acutely
• Anticoagulation
• First episode - managed as other patients with
  similar event
• Recurrent episodes lifetime
• Prophylaxis
• Retrospective studies only, suggest warfarin
  prophylaxis effective in patients with PNH if the
  granulocyte clone size is gt50, platelet count
  gt100K, no contraindications to ac.
• Heparin or LMWH should be used in any
  perioperative period, during immobilization, or
  with use of indwelling intravenous catheter.
  Also start in 1st trimester of pregnancy until
  4-6 weeks post-partum.
• Efficacy of anti-platelet agents not clear.

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Treatment

• Stimulate hematopoiesis
• G-CSF
• Immunosuppression - hypothesis that
  immune-mediated bone marrow damage in PNH is
  primarily directed against the normal
  GPI-positive cells, producing growth advantage
  for PNH cells. Improved impaired hematopoiesis,
  but hemolysis and PNH clone not affected.
• ATG
• Cyclosporine

31
Treatment

• Protein transfer Transfer of GPI-linked
  proteins feasible using either high density
  lipoproteins or washed RBC microvesicles. PNH
  cells show increased cell-associated CD55 and
  CD59 levels and decreased hemolysis.
• Gene Therapy - PIG-A gene cloned. But PIG-A
  inactivation alone does not confer a
  proliferative advantage to hematopoietic stem
  cell. Correcting the PNH defect may allow
  exposure to the insult causing bone marrow
  failure.

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Treatment

• Stem Cell Transplantation
• Indications severe bone marrow hypoplasia,
  severe thrombotic events (hepatic vein), children
• Syngeneic (identical twin) shown to be
  successful
• Autologous - not very successful due to inability
  to obtain sufficient numbers of normal cells
• Allogeneic
• Allogeneic hematopoietic cell transplantation
  (HCT) after high-dose conditioning is the only
  curative treatment however, it is associated
  with high treatment-related mortality.

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Circulating Stem Cells in PNH
Johnson et al, Blood, 1998
34
Stem Cell Transplantation in PNH

• Summary of single institution trials
• Approximately 12 reported
• Number of patients ranges from 1-16
• Survival rates typically higher (58-100)
• Likely high degree of reporting bias, small
  studies

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Stem Cell Transplantation in PNH
IBMTR Data Saso et al, Br J Haematol, 1999
36
Results

• Sustained engraftment 77
• Graft failure 17
• Grade 2-4 acute GVHD 34
• Chronic GVHD 33
• Causes of death
• graft failure (7), int. pneumonitis (4), GVHD
  (3), infection (3), ARDS (2), hemorrhage (1)

IBMTR Data Saso et al, Br J Haematol, 1999
37
Stem Cell Transplantation in PNH
Matched siblings
IBMTR Data Saso et al, Br J Haematol, 1999
38
Stem Cell Transplantation in PNH

• Conclusions from reported series
• BMT may cure 50-60 of selected patients with
  HLA-identical siblings
• Most patients transplanted have been lt 30 years
  of age
• Regimen related toxicity and GVHD remain
  significant hurdles
• Role of alternative donor transplants unclear,
  though initial reports are not encouraging except
  in pediatric population

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Alternative Treatment

• http//www.herbchina2000.com/therapies/HPH.shtml
•  

40
Prognosis Based on Management
Luzzatto, Haematologica, 2000
41
PNH Management Guidelines
Luzzato, ASH, 2001
42
Case

• Started on prednisone, Fe, and Folate
  supplementation by Hematologist.
• One year after diagnosed with PNH, patient
  admitted to PHD for pneumonia. WBC 2900
  (ANC1800), H/H 11.7/33.6, plts 81,000 (labs one
  week prior normal CBC). LDH 2147, Tbili 2, Retic
  3.
• Obtained records (bone marrow results) revealing
  diagnosis.
• Prophylactic lovenox and higher dose prednisone
  given. Blood counts improved.
• Did well. Discharged to f/u with his
  Hematologist.

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References

• Firkin, F, Goldberg, H, Firkin, BG.
  Glucocorticoid management of paroxysmal nocturnal
  hemoglobinuria. Australia Ann Med 1968 17127.
• Rosse, WF. Treatment of paroxysmal nocturnal
  hemoglobinuria. Blood 1982 6020.
• Hartmann, RC, Jenkins, DE Jr, Mckee, LC, heyssel,
  RM. Paroxysmal nocturnal hemoglobinuria Clinical
  and laboratory studies relating to iron
  metabolism and therapy with androgen and iron.
  Medicine 1966 45331.
• Saso, R, et al. Bone marrow transplants for
  paroxysmal nocturnal haemoglobinuria. Br J
  Haematol. 1999 Feb104(2)392-6
• Risitano AM, et al. Large granular lymphocyte
  (LGL)-like clonal expansions in paroxysmal
  nocturnal hemoglobinuria (PNH) patients Leukemia.
  2005 Feb19(2)217-22
• Raiola, AM, et al Bone marrow transplantation for
  paroxysmal nocturnal hemoglobinuria.Haematologica
  . 2000 Jan85(1)59-62

44
References

• Moyo, VM, Mukhina, GL, Garrett ES, Brodsky, RA.
  Natural history of paroxysmal nocturnal
  hemoglobinuria using modern diagnostic assays. Br
  J Haematol 2004 126133.
• Nishimura, J, Kanakura, Y, Ware, RE, et al.
  Clinical course and flow cytometric analysis of
  paroxysmal nocturnal hemoglobinuria in the United
  States and Japan. Medicine (Baltimore) 2004
  83193.
• Karadimitris, A, Luzzatto L. The cellular
  pathogenesis of paroxysmal nocturnal
  haemoglobinuria. Leukemia. 2001 Aug15(8)1148-52
  
• Hillmen, P, et al. Natural History of Paroxysmal
  Hemoglobinuria. NEJM. 1995 333 19.
• Diagnosis and Treatment of paroxysmal nocturnal
  hemoglobinuria. UTD.
• Clinical Manifestations of paroxysmal nocturnal
  hemoglobinuria. UTD.
• http//hematology.im.wustl.edu/conferences/present
  ations/devine011703.
• http//www.path.sunysb.edu/labs/pnh/PNH_files/fram
  e.

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References

• Luzzatto L, Araten DJ. Allogeneic bone marrow
  transplantation for paroxysmal nocturnal
  hemoglobinuria.Haematologica. 2000 Jan85(1)1-2

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Research