Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease associated with a defect in the synthesis of the glycosylphosphatidylinositol (GPI) anchor and the lack of GPI-anchored protein (GPI-AP) binding in the plasma membranes of the hematopoietic cells. The most common clinical symptoms are hemolytic anemia, bone marrow failure (BMF), thrombosis, as well as renal, heart, and lung failure, and several symptoms related to smooth muscle dystonia. The deficiency of cell membrane proteins: CD55 / decay-accelerating factor (DAF) and CD59 / membrane inhibitor of reactive lysis (MIRL), which are inhibitors of the complement system, is of the greatest importance in the pathophysiology of PNH.1,2

The cause and pathobiological background of the increasing clone dominance of GPI-defective cells have not been fully elucidated.3 It is suspected that this dominance may be associated with clonal selection of hematopoietic cells, whereby cells without GPI-AP over-proliferate in comparison with normal cells.4 The researchers followed for the relationship with the immune attack of T lymphocytes and natural killer (NK) cells against the normal clone,5-7 dysfunction of immunomodulating proteins anchored by GPI in the membrane of hematopoietic cells,8 or secondary mutations causing proliferative predominance of the PNH clone. According to one theory, a clone of defective (GPI-negative) cells is characterized by increased resistance to apoptosis as compared with normal GPI-positive cells and gradually gains an advantage, leading to an exacerbation of clinical symptoms of the disease.9 The pathogenesis of PNH may be influenced by an excess of the antiapoptotic signal transducer, that is, phosphatidylinositol (3,4,5)-trisphosphate in hematopoietic stem cells (HSCs), as well as a strongly increased level of the plasma chemokine CCL3 and granulocyte colony stimulating factor (G-CSF) and decreased levels of regulated on activation of normal T cells expressed and secreted (RANTES), macrophage inflammatory protein 1β, platelet-derived growth factor-BB, and interleukin 9.10

Morbidity in PNH is estimated at approximately 1.59 to 1.86 cases per 100 000 population.11 It affects men and women equally, less frequently children, with the peak of incidence between 30 and 45 years of age.12,13 Epidemiological data show that the disease is more common in Asia (eg, in Japan, Korea, and China) than in Western countries,13-15 but patients from the United States and Europe are at a higher risk of thromboembolism (ca 30%–40%) than patients of Asian descent (ca 10%–15%).16 It is estimated that the monoclonal form of the disease is 1500 times more frequent than the biclonal one.11

Characteristics of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria classification

PNH classification proposed by the International PNH Interest Group is based on the presence of clinical symptoms and laboratory evidence of hemolysis or thrombosis, signs of BMF such as aplastic anemia (AA), myelodysplastic syndromes (MDS), and cytometric analysis of the clone size of cells affected by the GPI defect.

The following subtypes of PNH are distinguished:

  1. Classic PNH, type I: no significant signs of BMF. In the complete blood count (CBC), the number of leukocytes and platelets is usually normal. The GPI(–) cell clone typically exceeds 50%. Serum lactate dehydrogenase (LDH) activity and bilirubin concentration may be increased.
  2. Aplastic PNH, type II: approximately 10% to 50% of a GPI(–) cell clone is found, as well as laboratory features of mild hemolysis and concomitant leukopenia and / or thrombocytopenia. CBC is usually as follows: hemoglobin <⁠10 g/dl, neutrophils <⁠1 G/l, platelets <⁠80 G/l. The clinical picture is dominated by symptoms of BMF.
  3. Subclinical form of PNH, type III: in patients diagnosed with MDS or AA, with a very small clone of GPI(–) cells (1%–10%) and no clinical and laboratory evidence of hemolysis.1,17

Individuals with a GPI(–) cell clone below 1% are considered healthy or non-PNH but positives are at risk of progression to PNH.18

Characteristics of the functions of selected glycosylphosphatidylinositol-anchored proteins

Out of more than 150 type II proteins, over 20 proteins are present on hematopoietic cells (Table 1). The GPI-anchored proteins belong to different groups, such as complement inhibitors, adhesion molecules, blood group antigens, receptors, enzymes, co-receptors in the signal transduction pathways, and other functional proteins (Table 1).

Table 1. List of selected proteins that are attached to the cell membrane by glycosylphosphatidylinositol anchor and are not or are lowly expressed on paroxysmal nocturnal hemoglobinuria cells

Enzymes

Neutrophil alkaline phosphatase

Acetylcholinesterase

5`-lymphocyte ectonucleotidase

Adhesion molecules

NCAM

Neural cell adhesion molecule

CD48 / Blast-1 / LFA2

Ligand of the CD2 molecule found on T cells

CD58 / LFA3

Ligand of the CD2 molecule found on T cells

CD66b / CD67

Neutrophil activator, CEA family, adhesion to CD66c

CD66c

Neutrophil activator, CEA family, homophilic adhesion, and adhesion to CD66b

CD66e / CEA

Cancer-fetal antigen, homophilic and heterophilic adhesion to endothelial E-selectin

CD157 / BST-1

NAD+ metabolizing ectoenzyme, bone marrow stromal cell antigen 1, fibronectin receptor, expressed on mature myeloid cells

Complement regulatory proteins

CD55 / DAF

Decay accelerating factor

CD59 / MIRL

Membrane inhibitor of reactive lysis

C8-binding protein

Receptors

CD16 / FcγRIII

Low-affinity immunoglobulin gamma Fc region receptor III

CD14

Pattern recognition receptor

CD87 / u-PAR

Urokinase type plasminogen activator receptor

Blood group antigens

Cromer (CD55)

DAF, Echovirus receptor

Yt

Acetylcholinesterase, Cartwright blood group

JMH

Semaphorin 7A on erythrocytes, adhesion, migration, Plasmodium falciparum receptor

Dombrock

ADP-ribosyltransferase 4

Varia

Scrapie prion protein

CD52

Marker of mature lymphocytes; anti-CD52 humanized monoclonal antibody was called CAMPATH-1H, Alemtuzumab, Lemtrada, etc.

CD24 / HSA

Proapoptotic cell adhesion molecule, heat-stable antigen, multispecific ligand: P-, L-, E-selectins, high mobility group box 1, L1 cell adhesion molecule, neural cell adhesion molecule 1, Siglec-G

Abbreviations: CAMPATH, Pathology Department of Cambridge University; CD, cluster of differentiation; CEA, carcinoembryonic antigen; JMH, John-Milton-Hagen blood group antigen; LFA, lymphocyte function antigen; NAD, nicotinamide adenine dinucleotide

The most important proteins for PNH pathology are the CD55 (DAF) and CD59 (MIRL) proteins, which are natural inhibitors of the complement system. Their absence on red blood cells (RBCs) leads to uncontrolled activation of the complement system and the development of symptoms of chronic hemolysis, platelet activation, and thrombosis, as well as symptoms affecting the lungs, kidneys, bone marrow, and others (Table 2). The CD55 protein, a glycoprotein with a molecular weight of 68 000 Da, inhibits the activity of the membrane enzyme C3 complement convertase by classic and alternative complement activation pathways. The process inhibits the formation of a stable complement complex comprising components C3b and C4b, which prevents the opsonization and destruction of RBCs by the spleen macrophages. In the absence of CD55, the activity of C3 convertase (C4b2a) increases on the surface of erythrocytes. Opsonization and extravascular hemolysis also increase. Active C5 convertase is generated and activation of the classic complement pathway is initiated, leading to the formation of the membrane attack complex (MAC) and intravascular hemolysis.19

Table 2. Clinical symptoms of paroxysmal nocturnal hemoglobinuria

Intravascular hemolysis

  • Anemia or multilineage cytopenia, fatigue, abdominal pain, flatulence, back pain, headache, dysphagia, erectile dysfunction, gallstones, hemoglobinuria
  • Acute / chronic renal failure, recurrent urinary tract infections
  • Pulmonary hypertension

Venous thrombosis

  • Intra-abdominal venous thrombosis:
    • Hepatic veins, eg, Budd–Chiari syndrome (portal hypertension, esophageal varices, caput medusae—widening of the abdominal veins);
    • Splenic veins (splenomegaly, hypersplenism);
    • Mesenteric veins (abdominal pain, especially after eating, bloating, diarrhea, vomiting)
  • Renal venous thrombosis
  • Cerebral venous thrombosis: headache, hemorrhagic infarction
  • Retinal venous thrombosis: loss of vision
  • Cerebral venous sinus thrombosis
  • Deep vein thrombosis in extremities: pulmonary embolism
  • Rare: cutaneous vein thrombosis, purulent gangrenous dermatitis

Arterial thrombosis

  • Ischemic stroke
  • Myocardial infarction

Bone marrow failure

  • Anemia, infections, bleeding
  • Bone pain

Associated

  • Transfusion iron overload
  • Myelodysplastic syndrome
  • Transformation into acute myeloid leukemia

The CD59 protein is a 19 000-Da glycoprotein that interacts directly with MAC. Under normal conditions, CD59 acts as an inhibitor in the cell membrane by blocking the incorporation of the complement component C9 into the C5b-C8 complex. Consequently, no MAC is formed. In PNH, the absence of CD59 on erythrocytes leads to the uncontrolled formation of MAC, which triggers the complement-dependent mechanism of intravascular hemolysis.20,21

Hemolytic anemia in paroxysmal nocturnal hemoglobinuria and clinical symptoms

Among the clinical symptoms of PNH, the greatest importance is attributed to chronic hemolysis in the intravascular system caused by a deficiency of the complement inhibitory proteins CD55 and CD59 on RBCs.22 Due to the formation of MAC on erythrocytes and hemolysis, free hemoglobin is released into the plasma and excreted by the kidneys. Long-term hemoglobinuria leads to kidney damage, including an increased tendency to recurrent urinary tract infections.23,24

The binding of free hemoglobin with nitric oxide (NO) leads to a reduction of NO resources in the blood. The NO deficiency most often leads to gastrointestinal muscular dystonia manifested by abdominal pain and flatulence. Vascular muscular dystonia also causes back pain, headaches, fatigue symptoms, esophageal contractility disorders, dysphagia, and erectile dysfunction in men. Decreased NO concentration leads to narrowing of the blood vessels, may cause arterial hypertension, pulmonary hypertension, and pulmonary microembolism, as well as changes in blood flow through the parenchymal organs.25,26 Depletion of the NO pool affects the activation of platelets and causes an increase in the expression of P-selectin, a protein that is also involved in the aggregation of platelets and can activate the complement system, the thrombin-antithrombin system, and the fibrin system, increasing the tendency to thrombosis.

While intravascular hemolysis is the main cause of hemolytic anemia in PNH, the underlying anemia may be aggravated by BMF. In classic PNH, there is significant intravascular hemolysis with moderate or severe anemia. In the case of hemolysis associated with BMF, abnormal erythropoiesis is found. CBC shows a decrease in the number of peripheral blood reticulocytes, thrombocytopenia, and leukopenia.

Bone marrow failure in the course of paroxysmal nocturnal hemoglobinuria

The mechanism of BMF in PNH is poorly understood; however, autoimmunity and activation of T and NK cells directed against normal stem cells are implicated. This may be related to the increased expression of human leukocyte antigen (HLA) class II molecules on HSCs in patients with PNH.27 Also, the strong association of PNH with HLA alleles and haplotypes known to initiate an immune response may suggest autoimmune pathogenesis for PNH.28,29 In aplastic anemia, activation of immunocompetent cells leads to tissue damage due to excessive secretion of lymphokines, especially tumor necrosis factor (TNF) and interferon-gamma (IFN-γ).30 No autoimmune causes have been identified in the classic hemolytic type of PNH.31 Moreover, our recent studies of the cytokine profile did not confirm a sustained increase in TNF and IFN-γ levels in the hemolytic type of PNH, which seems to challenge the autoimmune etiology of PNH.10 At the same time, the imbalance of chemokines and cytokines in patients with PNH was strongly associated with HSC apoptosis.10 Reduced apoptosis may therefore be a major selection factor for PNH-defective hematopoietic cell clones.

BMF may occur independently or may precede the PIGA gene mutation and together with it contribute to the clonal dominance of the defective HSCs in patients with PNH.32 In the course of PNH, BMF of varying severity is found in almost all patients.33 In the classic form of PNH, approximately 30% to 40% of patients develop AA, MDS, or even acute myeloid leukemia during the 10-year follow-up.34 In AA, cytometric analysis shows a small clone of PNH cells (<⁠1% in ca 70% of patients), and approximately 50% to 60% of patients develop subclinical PNH. About 20% of patients with MDS may also develop subclinical PNH.34,35 The appearance of a defective cell clone and symptoms of PNH may also be a late complication of antithymocyte globulin or cyclosporin therapy.36

Thrombosis

Symptoms of thrombosis are frequent during the initial diagnosis of PNH patients. Thromboembolic events are diagnosed in about 29% to 44% of patients at least once in the course of the disease, and before the introduction of eculizumab treatment, they were reported as the main cause of death in about 22% to 67% of cases.37 Still, the thrombosis rate in PNH is probably underestimated. Tests with the use of sensitive imaging techniques allow the detection of abnormalities suggesting previous subclinical thrombosis in various internal organs, including the lungs.38

The risk of thromboembolism increases with the elevation of a PNH clone GPI(–) granulocytes above 50% to 60% and with increasing hemolysis. Thrombosis has also been reported in patients with a small PNH neutrophil clone.39,40

The pathophysiology of PNH-related thrombosis is complex. It is assumed that the mechanism of thrombosis is mediated by the activated complement system in association with the coagulation system, platelet activation, hemolysis caused by complement activation, free hemoglobin formation, NO depletion, increased plasma level of soluble urokinase plasminogen activator receptor, vascular endothelial activation and impaired fibrinolytic system, and activated mediators of inflammation.37

Diagnosis of paroxysmal nocturnal hemoglobinuria

Early detection of a PNH defect is the most important for the treatment and prognosis of the course of the disease; however, due to the variety of clinical symptoms, the diagnosis is frequently late. The high-risk groups of patients that should be screened for PNH are presented in Table 3.

Table 3. High-risk patients that should be screened for paroxysmal nocturnal hemoglobinuria

  1. Patients with unexplained hemolysis
  • Coombs-negative hemolytic anemia
  • Hemoglobinuria / hemosiderinuria
  • Hemolysis with renal dysfunction
  1. Patients with unexplained cytopenia
  1. Patients with evidence of bone marrow dysfunction
  • Patients with aplastic anemia
  • Patients with myelodysplastic syndromes (MDS):
    • Any type of MDS with evidence of hemolysis
    • MDS with any of the following: hypoplastic bone marrow, refractory cytopenia
  1. Patients with unexplained thrombosis and evidence of hemolysis without obvious cause
  • Venous and arterial thrombosis in unusual sites (eg, intra-abdominal veins, cerebral veins, dermal veins) or with any cytopenia
  • Nonresponsive to anticoagulants
  • Patients of young age
  1. Patients with unexplained clinical symptoms
  • Dyspnea
  • Abdominal pain, headache, low back pain
  • Dysphagia
  • Erectile dysfunction
  • Weight loss

Flow cytometry is the gold standard used in the diagnosis and monitoring of PNH. The use of the latest International Clinical Cytometry Society / European Society for Clinical Cell Analyzes recommendations enables not only to detect a clone of GPI-defective cells in each of the 3 forms of PNH but also to identify even few cells with the PNH phenotype in patients with BMF. The high sensitivity of the tests is particularly important, as 1% or less GPI-negative cells are detected in more than 40% of the patients referred for testing in accordance with the above recommendations. The ability to detect and monitor such minor clones is critically important for patients as some of them will progress to subclinical and clinical PNH.18 The diagnosis of PNH is based on the identification of a GPI-defective clone (reduced expression or no anchors at all) on at least 2 blood cell lines, confirming their origin from an HCS. The presence of GPI-defective cells in only 1 cell line may be due to the formation of a mutation at a later stage of differentiation, as seen in some MDS cases. The presence of abnormal cells may then be temporary.41

Tests are routinely performed on neutrophils, monocytes, and erythrocytes from the peripheral blood. Due to the low number of monocytes in the blood of the tested patients, it is not always feasible to evaluate the small amount of GPI-negative cells. It is not recommended to evaluate the bone marrow.42 The identification of GPI-defective cells is performed by labeling the cells with monoclonal antibodies directed against antigens that belong to GPI-APs or fluorescently labeled inactivated aerolysin (FLAER), which has an affinity directly to GPI anchors.43 In the assessment of neutrophils or monocytes, antibodies against markers of cell lines (CD45, CD15, CD64) together with antibodies against GPI-APs (CD24, CD14, CD157) or FLAER are used.44,45 Double negative cells, for example, FLAER(–)CD24(–) in the neutrophil population (CD45+CD15++) and FLAER(–)CD14(–) in the monocyte population (CD45+CD64++) are considered GPI-negative cells (type III). Some patients also have type II granulocytes or monocytes characterized by weak FLAER binding and a low expression of GPI-APs. The PNH clone is then the sum of type II and III cells.18 It has been shown that some individuals have an isolated deficiency of type II CD157 protein. In such cases, the neutrophils and monocytes are FLAER-positive, which excludes a PNH defect.42,46

If a PNH defect is detected in the patient’s neutrophil or monocyte population, it is advisable to perform an erythrocyte test. In contrast to the assessment of neutrophils or monocytes, erythrocytes need only be tested for the expression of a single GPI-AP (eg, CD59). CD235a monoclonal antibodies (glycophorin A, erythrocyte marker) are additionally used in high-sensitivity 2-color tests.44

The assessment of the size of the PNH clone in the neutrophil or monocyte population best reflects the advancement of the disease. The percentage of PNH erythrocytes (type II and III) is usually much lower for several reasons. Normal erythrocytes (CD59-positive, type I) are long-living cells (ca 120 days), while erythrocytes with reduced CD59 expression (type II) are characterized by a 3 to 5 times higher sensitivity to the complement. CD59-negative red cells (type III) are 15 to 25 times more sensitive to the complement than type I cells, and their circulating survival time is reduced to 10 to 15 days. Such RBCs are eliminated more quickly from the circulation, and their number decreases while the activation of the complement system increases (eg, during an infection). Moreover, many patients are tested for a PNH clone after transfusions of GPI-positive RBCs, which lowers the subset of PNH defective cells.47

In such a situation, the acquisition of an increased number of cells and appropriate gating strategies in the multicolor analysis allow for the lower limit of quantification to be achieved, down to 0.02% to 0.05% for neutrophils and 0.01% for erythrocytes. In fact, it is difficult to achieve such a high sensitivity for samples from patients with cytopenias.18,42

There is no need to retest if a patient with hemolytic anemia or thrombosis has no PNH-defective cells. The situation is different with patients with BMF; especially those diagnosed with AA or MDS require systematic monitoring for the occurrence and development of a PNH clone.48,49 It is recommended to perform cytometric tests every 6 months for the first 2 years after diagnosis, and then once a year if no clone is detected in the patient. If GPI-defective cells are detected, retesting should be performed initially every 3 to 6 months, and if the clone size stabilizes for 2 years, the frequency of testing may be reduced. If there is a significant improvement or worsening of hematological parameters, the time interval to the next test can be modified.18,50 Retests should also be performed in PNH patients after allogeneic HSC transplantation (allo-HSCT) and those treated with complement inhibitors.51-53

Treatment of paroxysmal nocturnal hemoglobinuria

There are 2 effective strategies for the treatment of PNH: the use of complement C5 inhibitors and allo-HSCT. However, supportive therapy plays an important role in treating the symptoms and complications of PNH.

The choice of therapy depends on the presentation of PNH. Inhibitors of the complement component C5 (C5 inhibitors) are the current standard of care for the classic hemolytic form of PNH. In PNH accompanied by BMF, treatment should focus primarily on bone marrow disease—aplastic anemia (in PNH/AA) or myelodysplastic syndrome (in PNH/MDS). In the subclinical form, no standard of care has been established.1,54

Eculizumab is the first monoclonal antibody to inactivate the C5 complement component, approved for the treatment of PNH in 2007. In 2018, another inhibitor, ravulizumab, was approved. Both inhibitors prevent the formation of terminal MAC, thus preventing lysis of the blood cells.55 C5 inhibitors changed the natural course of the disease. Before the introduction of eculizumab, the median survival of patients with PNH ranged from 10 to 22 years, and the 5-year overall survival was estimated at 65%.56 Since eculizuamb has been introduced to clinics, life expectancy has increased and it is now similar to that of the population without PNH, with a 5-year survival of 96.5% in the treated patients.57

Eculizumab prevents complications of chronic PNH hemolysis, such as kidney damage, pulmonary hypertension, and complications of thromboembolism, which have the greatest impact on the disease course and mortality.58-60 Treatment with eculizumab is safe and well tolerated. In patients with renal dysfunction or damage, it improves renal function both in individuals at the initial stages of chronic kidney disease (CKD) (stage 1 or 2; improvement by 67.1%, P <⁠0.001), and in those with stage 3 or 4 CKD (P = 0.05). Improvement occurs quickly and is sustained for at least 18 months of treatment.59

The beneficial effect on the quality of life in patients with PNH treated with eculizumab was confirmed in the study by Ueda et al,61 which also showed a relationship between the components of the quality-of-life test and the concentration of hemoglobin and LDH.

The diagnosis of PNH does not mean that treatment with C5 inhibitors should be started. Eculizumab therapy should be initiated in patients with severe hemolysis with increasing LDH concentration (≥1.5 upper limit of normal [ULN]) and the presence of symptoms or conditions such as:1,54,62,63

  • anemia with a hemoglobin level below 7 g/dl or below 10 g/dl in patients with cardiovascular symptoms;
  • PNH-related thrombosis;
  • complications of hemolysis: increasing renal failure and pulmonary hypertension manifested by dyspnea;
  • abdominal pain and / or dysphagia and / or erectile dysfunction;
  • pregnancy, especially in the case of previous miscarriages or other pregnancy complications.

The requirement for a transfusion or the size of a PNH clone are not among the indications for treatment with C5 inhibitors, although patients with a large clone (>50% of PNH granulocytes and >10% of PNH erythrocytes) in combination with a markedly elevated LDH level and a high count of reticulocytes will benefit most from the treatment with eculizumab. Patients without or with mild symptoms of hemolysis, with normal CBC, and a small clone of PNH-defective granulocytes (<⁠30%) do not require treatment with C5 inhibitors.54,63

The beneficial effect of eculizumab treatment in patients with PNH/AA or PNH/MDS without hemolysis or thrombosis has not been proven. However, patients with AA or MDS and a concomitant high percentage of PNH cells may also benefit from the use of C5 inhibitors. In individuals with predominant BMF, immunosuppressive therapy and / or allo-HSCT are used, similarly to those without PNH.54,63

Eculizumab is administered intravenously every 7 days during 4 weeks of induction, followed by a maintenance dose of 900 mg every 14 days. Shortening the time interval (up to 12 days) and increasing the dose of eculizumab should be considered if there are signs of worsening hemolysis prior to the next dose.62 Monitoring of eculizumab treatment should include the laboratory tests listed in Table 4.1,54,62,63

Table 4. Monitoring of treatment with C5 inhibitors

Laboratory tests

Time interval

Complete blood count with microscopic smear, reticulocyte count, LDH, and bilirubin

Weekly during the induction, then monthly for the first 3 months, then every 3 months

Kidney function markers: urea, electrolytes, eGFR

Every 3 months

Iron metabolism: transferrin saturation, ferritin concentration

Every 6 months

Concentration of folic acid and vitamin B12

Yearly or if symptoms of folate / vitamin B12 deficiency are present

Evaluation of PNH clones by flow cytometry

Every 6 months for the first two years then yearly for those with stable disease

Abbreviations: eGFR, estimated glomerular filtration rate; LDH, lactate dehydrogenase; PNH, paroxysmal nocturnal hemoglobinuria

Over 90% of patients respond to eculizumab, but only about 10% achieve a complete remission with normalization of hemoglobin levels; 35% to 40% of patients become independent of RBC transfusions, but still experience mild anemia; and another 30% of patients have moderate anemia and require RBC transfusions.64,65

The criteria for the response to treatment have not yet been clearly defined, especially regarding partial and suboptimal responses. In 2021, Risitano and de Latour66 proposed modified response criteria, taking into account the concentration of hemoglobin, LDH, reticulocytosis, the number of transfused RBC units, and the occurrence of hemolytic crises (Table 5).

Table 5. Response assessment criteria for patients with hemolytic paroxysmal nocturnal hemoglobinuria

Type of response

RBC transfusions

Hemoglobin

Hemolysis indicators and hemolytic crises

Complete response

No

≥13 g/dl (men); ≥12 g/dl (women)

LDH ≤1.5 × ULN and RET ≤150 G/l; no episodes of hemolytic crisis

Major response

No

≥13 g/dl (men); ≥12 g/dl (women)

LDH >1.5 × ULN and / or RET >150 G/l; only subclinical episodes of hemolytic crisis

Good response

No

≥10 and <⁠13 g/dl (men) or ≥10 and <⁠12 g/dl (women)

Any value of LDH and RET, only subclinical hemolytic crisis (excluding bone marrow failure)

Partial response

No or occasional (≤2 every 6 months)

≥8 and <⁠10 g/dl

–

Minor response

No or occasional (≤2 every 6 months)

<⁠8 g/dl

–

Regularly (3–6 every 6 months)

<⁠10 g/dl

Reduction by ≥50%

<⁠10 g/dl

No response

Regularly (>6 every 6 months)

<⁠10 g/dl

–

Abbreviations: RBC, red blood cell concentrates; RET, reticulocytes; ULN, upper limit of normal; others, see Table 4

The quality of the response depends on the size of the PNH clone, the degree of BMF, the accompanying inflammatory process, and genetic factors.54,63,65

Outcomes expected in the course of treatment with eculizumab are reduced hemolysis (as measured by LDH) and fatigue, improved quality of life, and relief of dyspnea after 1 to 3 weeks; reduced number of transfusions and stabilization of hemoglobin levels after 2 to 6 months; stable improvement in quality of life, reduction of fatigue, normalization of LDH activity, stable hemoglobin concentration, and independence from RBC transfusions after 6 months. Within 36 months to 10 years, maintenance of low rates of hemolysis (measured with LDH) and continuous improvement in thrombotic events are expected.

There are 3 main causes of an incomplete response to eculizumab treatment. First, an insufficient erythropoietic bone marrow response may be due to concomitant BMF, but folate, vitamin B12, and iron deficiencies should also be considered in such cases. Second, residual intravascular hemolysis due to suboptimal inhibition of C5 may contribute to residual anemia, but may be managed by increased doses of eculizumab. Third, persistent extravascular hemolysis may be associated with the activation of the C3 component caused by no effect of C5 inhibitors on the CD55-dependent activation of C3 convertase.67-69

A rare cause of nonresponse to eculizumab treatment is the presence of genetic polymorphisms in the complement receptor 1 (CR1) gene. Complement 1 receptors enhance the breakdown of C3 convertases, and their density on the surface of erythrocytes modulates the binding of C3 fragments to GPI-negative RBCs when C5 is inhibited.70,71

However, eculizumab does not modify the basic defect of the HSCs responsible for PNH, which is a mutation in the PIGA gene. PNH-defective blood cells are still produced in the bone marrow.63,72

Ravulizumab differs from eculizumab only in 4 amino acids and is associated with the same benefits as eculizumab, with similar safety and tolerability. Ravulizumab has a 4-fold longer half-life than eculizumab and is administered intravenously every 8 weeks.73 In phase III studies comparing ravulizumab and eculizumab, both drugs showed equal efficacy in stabilizing hemoglobin levels, reducing / normalizing LDH levels, ensuring transfusion independence, and improving quality of life.74

Novel therapeutic agents at less advanced stages of research are shown in Table 6. Among them, crovalimab is a long-acting monoclonal antibody possible to be self-administered by patients subcutaneously in small volumes.75 In phase I / II studies, the optimal dosing frequency was found to be every 4 weeks for patients with a sustained response with an LDH below 1.5 ULN. Phase III trials are ongoing, for both treatment-naïve and eculizumab-exposed patients.

Table 6. Novel therapeutic agents at less advanced stages of research

Agent

Mechanism of action

Phase of clinical trials

Safety

Treatment outcomes

Reference

Crovalimab

C5 inhibitor

III

Subcutaneous, self-administration, small volumes, every 4 weeks

Sustained LDH response

75

Nomacopan

C5 inhibitor

III

Self-administration, safe, well-tolerated for long-term treatment

Transfusion independence

76

Tesidolumab

C5 inhibitor

II

Favorable safety profile

Decreased transfusion dependency, reduction of LDH concentrations

10.3324/haematol.2020.265868

Pozelimab

C5 inhibitor

II

Well-tolerated

Normalization of LDH in normal and variant C5 carriers

10.1182/blood-2021–146178

Zilucoplan

C5 inhibitor

II

Well-tolerated

Under study

10.1038/nrneph.2017.156

Cemdisiran

siRNA targeting C5 mRNA, C5 inhibitor

II

Tolerated

Under study

10.1182/blood-2021–146205

Pegcetacoplan

Proximal complement pathway inhibitor, C3 inhibitor

approved for PNH treatment

Well-tolerated

Prevents extravascular hemolysis, may affect residual intravascular hemolysis

77

Danikopan

Proximal complement pathway inhibitor, factor D inhibitor

II/III

Oral, well-tolerated

Prevents extravascular hemolysis, may affect residual intravascular hemolysis

78,79

Iptacopan

Proximal complement pathway inhibitor, factor B inhibitor

II/III

Oral, well-tolerated

Prevents extravascular hemolysis, may affect residual intravascular hemolysis

80

Abbreviations: mRNA, messenger RNA; siRNA, small interfering RNA; others, see Table 4

There are also C5 inhibitors at phase III (nomacopan) or less advanced stages of research: tesidolumab (LFG316), pozelimab, zilucoplan, and cemdisiran. The data indicate that nomacopan is safe and well tolerated for long-term treatment of PNH by patient self-administration and offers significant therapeutic benefits to patients in terms of transfusion independence.76

Inhibitors of the proximal complement pathway are noteworthy. They primarily prevent extravascular hemolysis dependent on the activation of the complement component C3, but may also affect residual intravascular hemolysis. Drugs that target 3 different components of the complement pathway are under development: complement C3, complement factor D, and complement factor B.

The first C3 complement inhibitor approved in 2021 for PNH treatment is pegcetacoplan, which is a pegylated compstatin.77 The first oral factor D inhibitor is danikopan, an oral inhibitor of the alternative proximal complement pathway,78,79 and the first oral complement inhibitor of factor B is iptakopan.80 These drugs demonstrated significant efficacy in phase II and III studies, in combination with eculizumab or as monotherapy, in both intravascular and extravascular hemolysis. They were well tolerated.

The main risks associated with complement inhibitor use are infections. Eculizumab increases the risk of life-threatening infections with Neisseria spp., including N. meningitidis, by blocking the terminal complement activation. The risk was estimated at 0.5% per year or 5% after 10 years.63 Therefore, all patients treated with eculizumab should be vaccinated against Neisseria with a quadrivalent vaccine against ACYW135 serotypes and serogroup B at least 2 weeks prior to receiving the first dose of eculizumab.81 Although no formal studies have been conducted, all patients taking eculizumab should receive penicillin prophylaxis. Revaccination is usually recommended every 2.5 to 3 years according to current guidelines.82

As a result of intravascular hemolysis in PNH, iron is lost in the urine, and increased erythropoiesis in the bone marrow leads to increased consumption of factors necessary for the production of RBCs; therefore, patients with PNH often require iron, folic acid, and vitamin B12 supplementation.2

Individuals with PNH may also require RBC transfusions. The transfusions temporarily raise hemoglobin levels because the transfused erythrocytes have the proteins CD59 and CD55 that are resistant to complement lysis.83 Leucocyte-depleted products should be used, and irradiated blood products are recommended for patients scheduled for allo-HSCT.84,85

Glucocorticoids do not affect the complement-dependent hemolysis in PNH but can be used in the treatment of extravascular hemolysis at doses of 0.3 to 0.6 mg/kg/day of prednisone. About 70% of adult patients achieve an increase in hemoglobin levels, but long-term therapy is associated with complications.86

Some patients with reduced erythropoiesis may benefit from taking high doses of erythropoietin or darbepoetin.87 Recombinant G-CSF can be used in patients with granulocytopenia during the infection period,88 despite the fact that erythropoietin and G-CSF are already persistently increased in plasma of PNH patients, especially in PNH with bone marrow aplasia.10,89 In fact, in the majority of individuals with PNH, there is no indication for treatment with recombinant erythropoietin or G-CSF.

Anticoagulant treatment

Thromboembolic complications occur in approximately 50% of patients with PNH, and in 30% they are the cause of death.90,91 The risk of thrombosis increases with the growing size of the PNH clone.

Treatment with eculizumab significantly reduces the incidence of thrombotic events.37,58 For patients already taking eculizumab, primary anticoagulant prophylaxis with warfarin or acenocoumarol is of little benefit and may increase the risk of bleeding complications. In patients not treated with eculizumab, prophylactic anticoagulation with warfarin or acenocoumarol should be considered in the presence of a large PNH clone and in the absence of contraindications to such therapy.56,92 Low-molecular-weight heparin prophylaxis should be used during the perioperative and immobilization periods, as well as from the first trimester of pregnancy to the end of puerperium.63 Patients with acute thrombotic events should receive anticoagulant therapy, and in the cases of clinically significant thrombosis, it should be continued long-term.93,94

Transplantation of hematopoietic stem cells

Allo-HSCT is the only treatment that offers a chance to cure PNH.72 Allogeneic transplantation can eliminate the autologous PNH clone but is associated with significant morbidity and mortality. In addition, not all patients have an HLA-compatible donor available, and acute and chronic graft-versus-host disease occurs in a third of patients.1,95,96 A retrospective analysis of the Italian bone marrow transplant group (GITMO) of 26 patients transplanted between 1988 and 2006 from fully HLA-matched siblings, showed transplant-related mortality of 42%, and 10-year disease-free survival was 57%.97

These results have improved in recent years. A retrospective analysis of a group of 78 patients with PNH (27 and 51 individuals with type I and type II PNH, respectively) transplanted between 2002 and 2016 in 11 centers of the Polish Adult Leukemia Group showed a 3-year overall survival (OS) of 87% in the total cohort and of 92% in the group of patients without thrombosis.51,98 In a large retrospective study of 211 PNH transplant patients treated at 83 European Society for Blood and Marrow Transplantation centers from 1978 to 2007, the probability of 5-year OS was 86% in those with recurrent hemolytic anemia, 54% in those with thromboembolism, and 69% in those with AA. However, the outcome was worse than that of patients with classic PNH treated with eculizumab.57,99 Therefore, in the era of eculizumab treatment, allo-HSCT is not a therapeutic option for most patients and is limited to individuals with severe BMF and those who do not respond well to eculizumab treatment or do not have access to it.2,83 Efficacy of allo-HSCT for serious thromboembolic complications seems to be debatable in the era of C5 inhibitors due to unacceptably high toxicity and high mortality rate.100 Patients with PNH/AA or PNH/MDS benefit more from allo-HSCT.