European Journal of Haematology 95 (190–198)

REVIEW ARTICLE

Pathophysiology, diagnosis, and treatment of paroxysmal nocturnal hemoglobinuria: a review range re Devalet1, Francßois Mullier2,3, Bernard Chatelain2, Jean-Michel Dogne 3, Christian Chatelain1 Be 1

Department of Hematology, Namur Thrombosis and Hemostasis Center (NTHC), CHU Dinant-Godinne UCL Namur, Yvoir; 2Hematology Laboratory, Namur Thrombosis and Hemostasis Center (NTHC), CHU Dinant-Godinne UCL Namur, Yvoir; 3Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), University of Namur, Namur, Belgium

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder of the hematopoietic stem cell that makes blood cells more sensitive to the action of complement. Patients experience intravascular hemolysis, smooth muscle dystonia, renal failure, arterial and pulmonary hypertension, recurrent infectious diseases and an increased risk of notably dreadful thrombotic complications. The diagnosis is made by flow cytometry. Efforts have been recently performed to improve the sensitivity and the standardization of this technique. PNH is frequently associated with aplastic anemia or low-risk myelodysplasia and may be asymptomatic. Management of the classical form of PNH has been dramatically revolutionized by the development of eculizumab, which brings benefits in terms of hemolysis, quality of life, renal function, thrombotic risk, and life expectancy. Prophylaxis and treatment of arterial and venous thrombosis currently remain a challenge in PNH. Key words hemoglobinuria; hemolysis; thrombosis; eculizumab; anticoagulation range re Devalet, MD, PhD Student, Department of Hematology – CHU Dinant Godinne – UCL Namur, Avenue Correspondence Be Dr. G. Therasse, 1 a 5530 Yvoir, Belgium. Tel: +0032(0)81 42 47 95; Fax: +0032(0)81 42 38 32; e-mail: [email protected] Accepted for publication 4 March 2015

Definition and physiopathology

Paroxysmal nocturnal hemoglobinuria is a rare acquired disorder of the pluripotent hematopoietic stem cell and therefore can affect erythrocytes, leukocytes, thrombocytes (1) and probably some endothelial cells (2). Its incidence is not really known but estimated at 0.1–0.2/100 000 persons/yr. A study showed a 0.13/100 000/yr incidence in Yorkshire (3). These hematopoietic stem cells have acquired a somatic mutation in an X-linked gene: the phosphatidylinositol glycan class A (PIG-A). This gene is required for the synthesis of the glycosyl phosphatidylinositol (GPI) anchor, which is necessary to attach some proteins to the cell membrane. The lack of synthesis of the GPI anchor leads to the underexpression of a variety of proteins on the hematopoietic stem cell surface and on all cell lines that are generated by it. By this mechanism, a lack of two important complement regulatory proteins is observed on the cell surface: ‘decay-accelerating factor’ (DAF), also called ‘CD55’ and

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‘membrane inhibitor of reactive lysis’ (MIRL), also called ‘CD59’. Thus, red blood cells are more vulnerable to the action of complement. This leads to a complement-mediated intravascular hemolysis (see Fig. 1) (4). As a result, a high concentration of free hemoglobin is found in the plasma, responsible for nitric oxide (NO) scavenging. NO depletion causes the majority of symptoms experienced by paroxysmal nocturnal hemoglobinuria (PNH) patients. Smooth muscle dystonia is responsible for dysphagia and abdominal pain. Erectile dysfunction is frequent. NO depletion may also contribute to the development of arterial constriction, leading to reduced blood flow to the kidneys (with renal failure), arterial hypertension and pulmonary hypertension (associated with frequent but underdiagnosed pulmonary embolism) (5). In PNH, a non-malignant clonal expansion of the mutated hematopoietic cell line is observed. Different mechanisms have been proposed to explain this evolution. Despite the lack of studies that demonstrate it, the most widely accepted

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Diagnosis and management of PNH

A

B

Figure 1 Action of complement in healthy subjects (A) and paroxysmal nocturnal hemoglobinuria (PNH) patients (B). (A) Due to the presence of membrane proteins MIRL and DAF, a normal RBC is protected from complement activation. (B) MIRL and DAF deficiency makes the RBC sensitive to complement attack, resulting in hemolysis. RBC, red blood cells, MAC, membrane attack complex, DAF, decay-accelerating factor.

explanation is a two steps hypothesis (6). The first step is the occurrence of the PIG-A mutation. At this time, there is no clonal expansion and the patient is asymptomatic. An injury to the normal bone marrow (second step) is then needed, which leads to an activation of T cells and NK cells. The non-PIG-A mutated but injured hematopoietic cells are destroyed by this immune attack but PIG-A mutated cells escape. This is probably due to the absence of GPI-anchored targeted peptides or accessory molecules required for T cells attack on the PIG-A mutated cells (7). This makes the PNH cells resistant to the T-cell-mediated cytotoxicity (8). The result is a bone marrow failure and a clonal expansion of the PIG-A-mutated cells.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Diagnosis approach

The diagnosis of PNH was formerly comforted by in vitro complement activation by either acidity (Ham test) or osmolarity (sucrose test). These tests are obsolete as the diagnosis of PNH by flow cytometry (FCM) refers to the detection of the pathognomonic anomaly. As a matter of fact, CD55 and CD59 are detected by their specific antibodies. These monoclonal antibodies that bind specifically to GPI-anchored proteins and their absence can be used to detect small populations of PNH cells. Determining the size of the clone needs to use at least two different monoclonal antibodies, directed against two different GPI-anchored proteins, as

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Diagnosis and management of PNH

there are rare congenital deficiencies of CD55 or CD59 that could be responsible for false positive results. Moreover, tests must be made on at least two different blood cell lineages due to the risk of false negative tests if only red cells are screened. Indeed, PNH red blood cells can hardly be detected in the case of recent hemolytic crisis or transfusion. Blood cells of PNH patients can be regarded as a genetic mosaic because of the simultaneous presence of type I cells (that express GPI-anchored proteins at normal density), type II cells (partly deficient in GPI-anchored proteins), and type III cells (completely deficient in GPI-anchored proteins). FCM can accurately distinguish these different populations (9). A reagent called ‘FLAER’ (fluorescent aerolysin) is increasingly used to diagnose PNH in FCM. Aerolysin, which is the principal virulence factor of the bacterium Aeromonas hydrophila, binds selectively and with high affinity to the GPI anchor. This allows a more accurate assessment of the GPI anchor defect in PNH. Indeed, PNH clones lower that 1% can be detected (10–12). Sutherland et al. (13) recently demonstrated the interest of a CD157-based 5color assay to simultaneously detect PNH clones in granulocytes and monocytes. However, PNH testing by FCM is limited by several problems. Because PNH is a rare disorder, screening is only performed infrequently in many laboratories. Methods used, as the choice of antibodies to detect GPI-linked antigens, greatly varied among laboratories until a few years ago. False-positive and false-negative results were common (14). Efforts were then made to make the procedure more sensitive and standardized. In 2010, the International Clinical Cytometry Society (ICCS) published the first guidelines for the diagnosis and monitoring of PNH. Sutherland et al. (15). completed them by proposing a selection of optimal cocktails for both GPI-specific and lineage-gating monoclonal antibodies. In addition, they provided concise practical protocols and detailed analytic strategies for the detection of PNH red blood cells and white blood cells (granulocytes and monocytes) across a wide range of cytometers. Following these guidelines, very good intra- and interlaboratory performance characteristics were later demonstrated. Coefficients of variation for precision and reproducibility ranged from 0.01%/0.02% to 0.48%/0.45% (big clone) and from 1.48%/3.91% to 15.01%/17.83% (minor clone) for PNH white blood cells and from 0.24%/0.48% to 1.76%/1.83% (big clone) and from 1.09%/3.36% to 10.54%/10.23% (minor clone) for PNH red blood cells, respectively (16). These guidelines for flow cytometry screening of PNH allow a high sensitivity detection of small PNH clones (up to 0.01%) in asymptomatic, non-hemolytic patients (15). This sensitivity may be useful in myelodysplastic syndromes or aplastic anemia, where very small clones are found (17). The screening of subclinical PNH clones in patients

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suffering from such bone marrow failure syndromes may be of clinical relevance, given the relation between the presence of PNH clone and the response to immunosuppressive therapy or prognosis (18, 19). In the case of unexplained thrombosis, it is recommended to screen a patient for PNH by flow cytometry if he presents with one of these four criteria: (i) being young (under 50 yr old), (ii) having a thrombosis in an unusual site (intraabdominal veins, cerebral veins, dermal veins,. . .), (iii) having evidence of hemolysis, or (iv) having any cytopenia (20). In addition to FCM, the diagnosis approach of PNH may require other tests such as complete blood count, reticulocyte count, lactate dehydrogenase (LDH), bilirubin, haptoglobin and hemosiderinuria levels, iron stores evaluation, bone marrow aspirate, and biopsy, cytogenetics. Leukocyte alkaline phosphatase is frequently decreased in PNH but is not routinely used for PNH diagnosis (12). The PIG-A mutation can be confirmed by a mutation-specific PCR-based analysis (6), but this is not usually necessary. Clinical aspects

A clinical polymorphism of PNH has been described. Three different forms are identified: classical PNH, PNH associated with aplastic anemia (AA), and subclinical PNH. However, it is important to understand that these entities are three clinical manifestations of a single disease. The classical form

The classical form of PNH affects preferentially young people. They suffer from chronic intravascular hemolytic anemia, due to a continuous state of complement activation, but brisk periods of hemolysis may result from complement activation due to infection, surgery, strenuous activity, and alcohol intake (21). Physicians may note anemia (hemoglobin level 1500/lL, platelets >120 000/ lL). Patients may complain of dysphagia, abdominal pain, or erectile dysfunction (35%). Thrombotic events are common in PNH. Renal failure is a dreaded complication of PNH. It may be due to NO depletion leading to arterial constriction but also chronic hemosiderosis and microvascular thrombosis (22). Forty per cent of patients suffer from recurrent infectious diseases (upper respiratory tract or lung infections), which is the second cause of death in PNH patients. Their risk of developing a myelodysplastic syndrome (MDS) or an acute myeloid leukemia (AML) is 5.0% and 2.5%,

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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respectively, which is a hundred times higher than for the general population (11, 23). PNH associated with aplastic anemia

When PNH is associated with AA, the majority of patients express only a small PNH clone (

Pathophysiology, diagnosis, and treatment of paroxysmal nocturnal hemoglobinuria: a review.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder of the hematopoietic stem cell that makes blood cells more sensitive to the action o...
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