Management of Paroxysmal Nocturnal Hemoglobinuriain the Era of Complement Inhibitory Therapy

Charles J. Parker1

1Hematology Division, Department of Internal Medicine, The University of Utah School of Medicine,

Salt Lake City, UT

Despite the availability of safe, effective targeted therapy that controls intravascular hemolysis, the management ofparoxysmal nocturnal hemoglobinuria (PNH) remains complicated because of disease heterogeneity and closeassociation with BM failure syndromes. The purpose of this review is to provide a framework for individualizingtreatment based on disease classification. According to the recommendations of the International PNH Interest Group,patients can be placed into one of the following 3 categories: (1) classic PNH, (2) PNH in the setting of another BMfailure syndrome, or (3) subclinical PNH. The PNH clone in patients with subclinical disease is insufficiently large toproduce even biochemical evidence of hemolysis, and consequently, patients who fit into this category require noPNH-specific therapy. Patients with PNH in the setting of another BM failure syndrome (usually aplastic anemia orlow-risk myelodysplastic syndrome) have at least biochemical evidence of hemolysis, but typically the PNH clone issmall (� 10%) so that hemolysis does not contribute significantly to the underlying anemia. In these cases, the focus oftreatment is on the BM failure component of the disease. Intravascular hemolysis is the dominant feature of classicPNH, and this process is blocked by the complement inhibitor eculizumab. The thrombophilia of PNH also appears tobe ameliorated by eculizumab, but the drug has no effect on the BM failure component of the disease. Low-gradeextravascular hemolysis due to complement C3 opsonization develops in most patients treated with eculizumab, andin some cases is a cause for suboptimal response to treatment. Allogeneic BM transplantation can cure classic PNH,but treatment-related toxicity suggests caution for this approach to management.

IntroductionParoxysmal nocturnal hemoglobinuria (PNH) has a special place inthe fields of hematology and complementology because identifica-tion of the molecular basis of the hemolytic anemia that is theclinical hallmark of this disease led to a remarkable number ofdiscoveries that helped to identify and characterize the alternativepathway and define the physiology of the complement system inhumans.1 The discoveries began with the seminal observations ofThomas Hale Ham in the late 1930s that suggested a novel,antibody-independent mechanism for complement activation. Sub-sequently, Ham’s observations contributed to elucidation of theproperdin pathway (now known as the alternative pathway) byLouis Pillemer while the two were on the faculty at Case WesternReserve University in the 1950s. Systematic investigation of theaberrant regulation of complement on PNH erythrocytes contributedto the identification and characterization of the complement regula-tory proteins decay accelerating factor (DAF, CD55) and membraneinhibitor of reactive lysis (MIRL, CD59) in the 1970s and 1980s andultimately led to the development of the first successful targetedtherapy for a complement-mediated disease when eculizumab wasapproved for treatment of PNH in 2007.2

In contrast to all other intrinsic abnormalities of the erythrocyte,PNH is an acquired disorder; and although the focus of this review ison the complement-mediated hemolytic anemia component of thedisorder, PNH is actually a disease of the hematopoietic stem cell.PNH arises as a result of the nonmalignant clonal expansion of oneor several hematopoietic stem cells that have acquired a somaticmutation of the X-chromosome gene PIGA that is required for

synthesis of the glycosyl phosphatidylinositol (GPI) moiety thatanchors some proteins to the cell surface. As a consequence ofmutant PIGA, the progeny of affected stem cells (erythrocytes,granulocytes, monocytes, platelets, and lymphocytes) are deficientin all GPI-anchored proteins (GPI-APs) that are normally expressedon hematopoietic cells (and all GPI-APs that are normally expressedon hematopoietic cells are deficient on the progeny of PIGA mutantstem cells). Among the GPI-APs that are deficient in PNH are DAF(CD55) and MIRL (CD59), the 2 primary erythrocyte membraneregulators of complement. Deficiency of CD55 and CD59 accountsfor the complement-mediated intravascular hemolysis that is thehallmark of the disease. The clinical manifestations of PNH arehemolytic anemia, thrombophilia, and BM failure, but only thehemolytic anemia is unequivocally a direct consequence of somaticmutation of PIGA.

PNH and complementThoughtful management of patients with PNH is facilitated by anunderstanding of the mechanisms involved in the activation andregulation of complement on the erythrocyte surface (Figure 1). Thechronic intravascular hemolysis of PNH is mediated by the alterna-tive pathway of complement (APC). A component of innateimmunity, this ancient system evolved to protect the host againstinvasion by pathogenic microorganisms.3 Unlike the classicalcomplement pathway that is part of the system of acquiredimmunity and requires antibody for initiation of activation, the APCis in a state of continuous activation, armed always to protect thehost.4 The APC cascade can be divided into 2 functional compo-nents, the amplification C3 and C5 convertases and the cytolyticmembrane attack complex (MAC) (Figure 1). The C3 and C5

ACQUIRED HEMATOPOIETIC DISORDERS: COMPLEMENT-MEDIATED BLOOD DISORDERS

Hematology 2011 21

convertases are enzymatic complexes that initiate and amplify theactivity of the APC and ultimately generate the MAC. The MAC isthe common pore-forming, cytolytic subunit of the classical andlectin complement pathways and the APC. Because the APC isalways primed for attack, overlapping and redundant mechanismsfor self-recognition and protection of the host against APC-mediated injury have evolved. Both fluid-phase and membrane-bound proteins are involved in these processes. Normal humanerythrocytes are protected against APC-mediated cytolysis primar-ily by DAF (CD55)5-7 and MIRL (CD59),8 and these proteins act atdifferent steps in the complement cascade. CD55 regulates theformation and stability of the C3 and C5 convertases, whereasCD59 blocks the formation of the MAC (Figure 1).9 Deficiency ofboth CD55 and CD59 is the pathophysiological basis of theCoomb-negative, intravascular hemolysis that characterizes thedisease in its untreated state.9

Phenotypic mosaicism is characteristic of PNHThe peripheral blood of patients with PNH is a mosaic of normaland abnormal cells (Figure 2). Although PNH is a clonal disease, itis not a malignant disease and, for reasons that are unclear, theextent to which the PIGA-mutant clone expands varies widelyamong patients.10 As an example, in some cases, � 90% of theperipheral blood cells may be derived from the PIGA-mutant clone,

whereas in others, � 10% of the circulating cells may be GPI-APdeficient. This peculiar feature (variability in the extent of mosa-icism) is clinically relevant because patients with small PNH cloneshave minimal or no symptoms and require no PNH-specifictreatment, whereas those with large clones are often debilitated bythe consequences of chronic complement-mediated intravascularhemolysis and respond dramatically to complement-inhibitorytherapy.

Another remarkable feature of PNH is phenotypic mosaicism basedon PIGA genotype,11 which determines the degree of GPI-APdeficiency.10 PNH III cells are completely deficient in GPI-APs,PNH II cells are partially (� 90%) deficient, and PNH I cellsexpress GPI-APs at normal density (putatively, these cells are theprogeny of residual normal stem cells) (Figure 2). Phenotype variesamong patients. Some patients have only type I and type III cells(the most common phenotype); some have type I, type II, and typeIII cells (the second most common phenotype); and some have onlytype I and type II cells (the least common phenotype). Further, thecontribution of each phenotype to the composition of the peripheralblood varies. Phenotypic mosaicism is clinically relevant becausePNH II cells are relatively resistant to spontaneous hemolysis, andpatients with a high percentage of type II cells have a relativelybenign clinical course (Figure 2).

Figure 1. Complement-mediated lysis of PNH erythrocytes. Top panel shows that the C3 convertase (left blue rectangle) of the APC consists ofactivated C3 (C3b), activated factor B (Bb, the enzymatic subunit of the complex), and factor P (a protein that stabilizes the complex, formally calledproperdin). The C5 convertase (right blue rectangle) has the same components as the C3 convertase except that 2 C3b molecules are required to bindand position C5 for cleavage by activated factor B (Bb). C3a and C5a are bioactive peptides that are generated by cleavage of C3 and C5,respectively, by their specific activation convertases. The C3 and C5 convertases greatly amplify complement activation by cleaving multiple substratemolecules. The MAC (red rectangle) consists of activated C5 (C5b), C6, C7, C8, and multiple molecules of C9 (C9n). The MAC is the cytolytic unit ofthe complement system. The GPI-anchored complement-regulatory protein CD55 restricts the formation and stability of both the C3 and the C5amplification convertases by destabilizing the interaction between activated factor B (Bb) and C3b (indicated by the blue arrows), whereas GPI-anchored CD59 blocks formation of the MAC by inhibiting the binding of C9 to the C5b-8 complex (indicated by the brown arrow). Inhibition of MACformation by the humanized anti-C5 mAb eculizumab (indicated by the red arrow) ameliorates the intravascular hemolysis of PNH. Bottom panel showsthat normal erythrocytes (left) are protected against complement-mediated lysis (lightning bolts) primarily by CD55 (blue circles) and CD59 (greencircles). Deficiency of these GPI-anchored complement-regulatory proteins results in APC activation on PNH erythrocytes (right). Consequently, MACsform pores in the red cell membrane, resulting in colloid osmotic lysis and release of hemoglobin (red circles) and other contents of the red cell,including LDH, into the intravascular space.

22 American Society of Hematology

The anemia of PNH is multifactorialThe anemia of PNH is multifactorial because an element of BMfailure is present in all patients, although the degree of dysfunctionis variable.12,13 In some patients, PNH arises in the setting of aplasticanemia. In this case, BM failure is the dominant cause of anemia. Inother patients with PNH, evidence of BM dysfunction may be subtle(eg, an inappropriately low reticulocyte count), with the degree ofanemia being determined primarily by the rate of hemolysis, whichis determined by the PNH clone size and erythrocyte phenotype(Figure 2).

Diagnosis of PNHOnce suspected, diagnosing PNH is straightforward because adeficiency of GPI-APs on peripheral blood cells can be readilydemonstrated by flow cytometry.14 Analysis of both RBCs andperipheral mononuclear cells is warranted, because clone size willbe underestimated if only RBCs are examined due to the fact thatGPI-AP–deficient red cells are selectively destroyed by comple-ment. Recent transfusion will also affect the estimate of clone size ifonly RBCs are analyzed, but delineation of PNH phenotypes (ie, thepercentage of type I, type II, and type III cells) requires flowcytometric analysis of the erythrocyte population (Figure 2).

In addition to flow cytometric analysis, the basic initial evaluationof a patient with PNH should include: complete blood count to

assess the effects of the disease on the production of leukocytes,platelets, and erythrocytes; measurement of serum concentration oflactate dehydrogenase (LDH), bilirubin (fractionated), and haptoglo-bin, which are biochemical markers of hemolysis; determination ofiron stores; BM aspirate and biopsy; and cytogenetics. Thesediagnostic studies will allow classification into 1 of 3 groups basedon the recommendation of the International PNH Interest Group(Table 1).13

In patients with classic PNH, the leukocyte and platelet counts areusually normal or nearly normal, whereas leukopenia, thrombocyto-penia, or both invariably accompany PNH in the setting of anotherBM failure syndrome. The reticulocyte count is needed to assess theongoing capacity of the BM to respond to the anemia. Although thereticulocyte count is elevated in patients with classic PNH, as notedabove, it may be inappropriately low for the degree of anemia,reflecting underlying relative insufficiency of hematopoiesis that ischaracteristic of the disease. Serum LDH is always markedlyelevated in classic PNH. The degree of serum LDH elevation isvariable in patients with PNH in the setting of another BM failuresyndrome (determined by the size of the PNH clone); however, in alarge majority of patients with PNH/BM failure, the clone sizeis � 10%, with � 10% of patients with PNH/BM failure having aclone size of � 50% (Table 1).15,16

Figure 2. Clinical manifestations of PNH are determined by clone size and erythrocyte phenotype. Mock flow cytometry histograms oferythrocytes from hypothetical patients with PNH stained with anti-CD59 are illustrated. The proportion and type of abnormal erythrocytes varies greatlyamong patients with PNH, and these characteristics are important determinants of clinical manifestations. Patients with a high percentage of type IIIerythrocytes have clinically apparent hemolysis (A). If the erythrocytes are partially deficient (� 10% of normal expression) in GPI-AP (PNH II cells),hemolysis may be modest even if the percentage of the affected cells is high (B). A patient may have a diagnosis of PNH, but if the proportion of type IIIcells is low, only biochemical evidence of hemolysis may be observed (C).

Hematology 2011 23

By definition, patients with subclinical PNH have neither clinicalnor biochemical evidence of hemolysis (Table 1). Patients withclassic PNH may be iron deficient due to chronic hemoglobinuriaand hemosiderinuria. BM aspirate and biopsy are needed todistinguish classic PNH from PNH in the setting of another BMabnormality. Nonrandom cytogenetic abnormalities are rare inclassic PNH.17

Management of PNH based on classificationCompleting the recommended diagnostic evaluation will allow thedevelopment of a systematic treatment plan (Figure 3) based ondisease classification (Table 1).

Subclinical PNHA close association exists between PNH and aplastic anemia and, toa lesser extent, between PNH and low-risk myelodysplastic syn-drome (MDS). Using high-sensitivity flow cytometry, approxi-mately 60% of patients with aplastic anemia and 20% of patientswith low-risk MDS have been found to have a detectable populationof GPI-AP–deficient erythrocytes and granulocytes.18-20 In � 80%of these cases, the proportion of GPI-AP–deficient cells is � 1.0%of the total. These patients (designated subclinical PNH patients)with very small populations of GPI-AP–deficient erythrocytes haveno clinical or biochemical evidence of hemolysis and require nospecific treatment for PNH. However, finding a population ofGPI-AP–deficient erythrocytes in patients with aplastic anemia maybe clinically relevant, because some,19,20 but not all,21 studiessuggest that these patients have a particularly high probability ofresponding to immunosuppressive therapy with a more rapid rate ofonset of response compared with patients with aplastic anemiawithout a population of GPI-AP–deficient erythrocytes.

The presence of PNH cells has also been observed in patients withMDS.19,20,22,23 The association between PNH and MDS appears tobe confined to low-risk categories of MDS, particularly the refrac-tory anemia (RA) variant.18-20,22 Using high-sensitivity flow cytom-etry in which � 0.003% of GPI-AP–deficient RBCs or peripheralmononuclear cells was classified as abnormal, Wang et al reportedthat 21 of 119 (18%) patients with RA MDS had a population ofPNH cells, whereas GPI-AP–deficient cells were not detected inpatients with RA with ringed sideroblasts, RA with excess of blasts,or RA with excess of blasts in transformation.20 Compared withpatients with RA without a population of PNH cells, RA patientswith a population of PNH cells had a distinct clinical profilecharacterized by the following features: (1) less pronounced morpho-logical abnormalities of the blood cells, (2) more severe thrombocy-topenia, (3) a lower rate of karyotypic abnormalities, (4) a higherincidence of HLA-DR15, (5) a lower rate of progression to acuteleukemia, and (6) a higher probability of response to cyclosporinetherapy. More recently, the findings by Wang et al that a populationof PNH cells was associated only with low-risk MDS variants inJapanese patients were confirmed in a North-American study of 137patients classified by World Health Organization criteria.22

When combined with evidence of polyclonal hematopoiesis (basedon the pattern of X-chromosome inactivation in female patients), thepresence of a population of PNH cells in patients with MDS predictsa relatively benign clinical course and high probability of responseto immunosuppressive therapy.18 A relatively good response toimmunosuppressive therapy for patients with MDS and aplasticanemia was also predicted by expression of HLA-DR15 in studiesof both North American and Japanese patients.24,25 These observa-tions support the hypothesis that aplastic anemia and a subgroup ofTa

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24 American Society of Hematology

low-risk MDS are immune-mediated diseases, and that the immunepathophysiological process provides the selection pressure thatfavors the outgrowth of PIGA mutant, GPI-AP–deficient stem cells.

PNH in the setting of another BM failure syndromePatients with a BM failure syndrome (aplastic anemia or MDS) anda PNH clone with clinical/biochemical evidence of hemolysis areclassified as PNH in the setting of another BM failure syndrome(Table 1). In these patients, BM failure dominates the clinicalpicture and hemolysis is primarily an incidental finding.15,16,19 Thelarge majority of patients with PNH/AA and PNH/MDS haverelatively small PNH clones (� 10%) and require no specific PNHtherapy; in these cases, treatment should focus on the underlyingBM failure syndrome (Table 1 and Figure 3).

The PNH clone will be eradicated by the conditioning regimen incombination with the GVH effect in patients undergoing allogeneictransplantation for aplastic anemia or MDS. In most cases, the sizeof the PNH clone is unaffected by treatment with immunosuppres-sive therapy, and the presence of a PNH clone should not deterimmunosuppressive therapy if that approach to treatment of theunderlying BM failure syndrome is considered appropriate.15,16 Inthe uncommon cases in which, after immunosuppressive therapy,the size of the PNH clone is sufficiently large to produce clinicalsymptoms, the patient can be managed using the same approach asfor patients with classic PNH.

Classic PNHPatients with classic PNH have a large clone (� 50%), andconsequently this disease subcategory is characterized by floridintravascular hemolysis as indicated by a markedly elevated serumLDH (Table 1). Patients may complain of episodic hemoglobinuria,and most experience ongoing constitutional symptoms dominatedby lethargy, malaise, and asthenia that can be debilitating. Thecomplement-mediated intravascular hemolysis of PNH can beinhibited by blocking formation of the MAC (Figure 1). The MACconsists of complement components C5b, C6, C7, C8, and multiplemolecules of C9. Eculizumab (Soliris; Alexion Pharmaceutics) is ahumanized mAb that binds complement C5, preventing its activa-tion to C5b by the APC C5 convertase and thereby inhibiting MACformation (Figure 1).2 In 2007, eculizumab was approved by boththe US Food and Drug Administration and the European UnionCommission for the treatment of the hemolysis of PNH. Treatmentof classic PNH patients with eculizumab reduces transfusionrequirements, ameliorates the anemia of PNH, and improves qualityof life by resolving the debilitating constitutional symptoms associ-ated with chronic complement-mediated intravascular hemolysis(Figure 3).26-28 After treatment, serum LDH concentration returns tonormal or near normal, with approximately one-half to two-thirds ofpatients achieving transfusion independence26,27,29; however, mildto moderate anemia, hyperbilirubinemia, and reticulocytosis persistin essentially all treated patients.

Figure 3. Treatment algorithm based on disease classification. Disease classification is based on the recommendations of the International PNHInterest Group.13

Hematology 2011 25

Eculizumab appears to reduce the risk of thromboembolic complica-tions.30 For patients being treated with eculizumab who have noprior history of thromboembolic complications, prophylactic antico-agulation may be unnecessary. Because PNH patients with priorthrombosis are at higher risk for recurrent thrombosis, anticoagula-tion for eculizumab-treated patients who experienced a priorthromboembolic event should be continued.29

Eculizumab is expensive (� $400 000/year in the United States)and has no effect on either the underlying stem cell abnormality oron the associated BM failure. Consequently, treatment must con-tinue indefinitely and leukopenia, thrombocytopenia, and reticulocy-topenia, if present, persist. Treatment with eculizumab appears tohave a favorable impact on survival,31 because a recent study of 79patients treated between 2002 and 2010 showed the same survivalrates as those of age- and sex-matched controls from the generalpopulation.29 The contribution of eculizumab to survival cannot bequantified accurately, however, because a control patient group wasnot included in that study.

Reasons for suboptimal response to treatment witheculizumabThe recommended maintenance dose of eculizumab is fixed (900 mgevery 2 weeks � 2 days) rather than being based on weight or bodysurface area. Some patients may show evidence of breakthroughintravascular hemolysis (ie, increases in LDH and development ofconstitutional symptoms) near the end of a treatment cycle. In these

cases, breakthrough hemolysis can be ameliorated by reducing thelength of the treatment cycle from 14 days to 13 or 12 days, and insome cases, the maintenance dose of eculizumab may also have tobe increased.

All patients with PNH have an element of BM failure, and patientstreated with eculizumab who have higher degrees of relativereticulocytopenia may remain anemic or even transfusion dependentdespite excellent control of intravascular hemolysis. Iron stores andserum erythropoietin concentration should be quantified in thesepatients, and if iron stores are adequate and serum erythropoietinconcentration is inappropriately low, a trial of recombinant erythro-poietin is warranted in patients who have symptomatic anemia orwho are transfusion dependent.

After treatment with eculizumab, serum LDH returns to normal ornear normal, but mild to moderate anemia and laboratory evidenceof hemolysis persist in essentially all treated patients.26,27 A smallsubgroup of eculizumab-treated patients experiences little or noimprovement in either anemia or constitutional symptoms. In thesepatients, hemolysis is mediated by opsonization of the PNHerythrocytes by activation and degradation products of complementC3, which, when tested, are found to be Coomb-positive for C3 butnot IgG.32-34 The known pathophysiology of the PNH predicts thatCD55 deficiency would result in ongoing extravascular hemolysisof PNH erythrocytes as a consequence of C3 opsonization (Figure4) because eculizumab does not block the activity of the APC C3

Figure 4. Generation of C3 opsonins on PNH erythrocytes in patients treated with eculizumab. Deficiency of DAF on PNH cells results inactivation of the APC on PNH erythrocytes. Eculizumab blocks MAC-mediated complement lysis, allowing accumulation of C3 opsonins on PNH cells.The opsonized erythrocytes are recognized by reticuloendothelial cells of the spleen and liver that express receptors (primarily CR2 for C3dg and CR3for iC3b), resulting in extravascular hemolysis. The figure illustrates covalent binding of activated C3 (C3b) to glycophorin A on the erythrocytemembrane surface. The bound C3 serves as the nidus for formation of the APC C3 convertase (C3b, activated factor B [Bb], and factor P) thatenzymatically activates many molecules of C3 to C3b, which then bind covalently via an exposed thioester bond to carbohydrate residues onglycophorin A. Supported by interaction with sialic acid residues on glycophorin A, the plasma protein factor H binds to C3b and serves as a cofactorfor degradation of C3b to iC3b by the plasma protein factor I. CR1 also binds to C3b and to iC3b and serves as a cofactor for the degradation of C3b toiC3b and then C3dg by factor I.

26 American Society of Hematology

convertase that is unregulated because of DAF deficiency (Figure1). Support for this hypothesis is provided by the studies of Risitanoet al, who showed that in patients treated with eculizumab, a portionof the PNH erythrocytes (ie, the CD59-deficient population) hadcomplement C3 bound.34 Those studies also confirmed the Coomb-negative designation of PNH: no C3 was found bound to PNHerythrocytes before initiation of treatment with eculizumab, imply-ing that PNH erythrocytes upon which complement has beenactivated are destroyed directly as a consequence of MAC-mediatedcytolysis. These studies provide a plausible explanation for thepersistent hemolytic anemia observed in PNH patients treated witheculizumab. By inhibiting the formation of the MAC, eculizumabprevents direct cytolysis of PNH erythrocytes, allowing themanifestations of DAF deficiency to become apparent in the formof aberrant regulation of the APC C3 convertase and the consequentdeposition of activated C3 on the cell surface (Figures 1 and 4).4

Covalently bound activation and degradation products of C3then serve as opsonins that are recognized by specific receptors onreticuloendothelial cells, resulting in extravascular hemolysis(Figure 4).

The extravascular hemolysis of patients with PNH receivingeculizumab does not require treatment in the absence of constitu-tional symptoms, symptoms of anemia, or transfusion dependence.Because the process is extravascular, splenectomy or corticosteroidsmay ameliorate the hemolysis in symptomatic or transfusion-dependent patients by removing or inhibiting the function ofphagocytic cells (Figure 3).35 Long-term use of corticosteroids isassociated with significant toxicity, however, and concerns aboutboth postoperative and late complications temper enthusiasm forsplenectomy. It is also conceivable that the primary site ofphagocytosis is hepatic rather than splenic. In such cases, responseto splenectomy would likely be inadequate. Based on experience inthe treatment of refractory autoimmune hemolytic anemia, a trial ofDanazol can be considered; however, Rituxan is not indicatedbecause the process is mediated by C3 opsonization rather thanopsonization by IgG antibody.

Hematopoietic stem cell transplantation for PNHBefore the availability of eculizumab, the primary indications fortransplantation for PNH were bone failure, recurrent life-threaten-ing thrombosis, and uncontrollable hemolysis.13 The latter processcan be eliminated by treatment with eculizumab and the thrombo-philia of PNH may also respond to inhibition of intravascularhemolysis by eculizumab.30 Nonetheless, transplantation is the onlycurative therapy for PNH, and the availability of molecularlydefined, matched unrelated donors; less toxic conditioning regi-mens; reductions in transplantation-related morbidity and mortality;and improvements in posttransplantation supportive care make thisoption a viable alternative to medical management. The decision ofwho should receive a transplantation and when it should beperformed is complex, however, and requires an understanding ofthe unique pathobiology of PNH and the input of physiciansexperienced in transplantation and medical management of PNH.36

The recent studies of Kelly et al29 showing normal survival forpatients with PNH treated with eculizumab make the decisionconcerning medical management versus transplantation even morechallenging.

Other treatments for PNHBased on anecdotal experience, a portion of patients with classicPNH responds to Danazol as first-line therapy.37,38 The basis of this

response is unknown but likely involves complement inhibitionbecause reduction in hemolysis is observed quickly (within a fewdays) after initiation of therapy and PNH WBC clone size does notchange during treatment (C.J.P., unpublished observation). Whysome patients respond dramatically to Danazol whereas others donot is unknown, but it seems plausible to suggest that respondersproduce a metabolite that inhibits complement whereas nonre-sponders do not (or they produce a subtherapeutic concentration ofthe putative inhibitory metabolite).

Although hemolysis is ameliorated in some patients by treatmentwith glucocorticoids, the harm that can accrue from long-term useof prednisone cannot be overemphasized.13 Although their use in themanagement of PNH is a matter of ongoing debate, the main valueof glucocorticoids may be in attenuating acute hemolytic exacerba-tions. Under these circumstances, brief pulses of prednisone mayreduce the severity and duration of the crisis while avoiding theuntoward consequences associated with long-term use.

Because hemolysis is a consequence of a defect intrinsic to apatient’s erythrocytes, the anemia of PNH responds to transfusion.In addition to increasing the hemoglobin concentration, transfusionmay lessen hemolysis by suppressing erythropoiesis. Concernsabout inducing a hemolytic exacerbation as a consequence ofinfusion of small amounts of donor plasma that may contaminatered cell preparations appear unwarranted.39

Patients with classic PNH frequently become iron deficient as aresult of renal loss (both hemoglobinuria and hemosiderinuria).38

Clinically important iron loss from hemosiderinuria can occur evenin the absence of gross hemoglobinuria. Concern for inducing ahemolytic exacerbation should not deter iron repletion, because irondeficiency not only limits erythropoiesis but also exacerbates thehemolysis of PNH.38 If hemolytic exacerbation occurs in the settingof iron repletion, the episode can be controlled by treatment withcorticosteroids or by suppression of erythropoiesis by transfusion.There is no concern about iron replacement therapy inducing ahemolytic exacerbation in patients being treated with eculizumabbecause the drug inhibits hemolysis. Patients treated with eculi-zumab should not become iron deficient because treatment willresolve hemoglobinuria and hemosiderinuria by blocking intravas-cular hemolysis.

Conclusions and future directionsSystematic investigation of the molecular basis of PNH hasprovided a framework for management based on an understandingof disease pathophysiology and has led to development of targetedtherapy that has improved the lives of patients and changed thenatural history of the disease. Nonetheless, continued investigationof new approaches to therapy aimed at obviating the extravascularhemolysis that limits eculizumab efficacy in some patients iswarranted.4 A better understanding of the pathobiology that under-lies the thrombophilia of PNH is needed, and defining the complexrelationship between PNH and BM failure syndromes that deter-mine clonal selection and clonal expansion40 may lead ultimately totherapy that targets the disease at the level of the hematopoietic stemcell. In particular, an understanding of the molecular basis of clonalexpansion will be facilitated by the availability of next-generationsequencing that will allow comparison between the genomes ofGPI-AP–positive and GPI-AP–negative cells from individual pa-tients with PNH.

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DisclosuresConflict-of-interest disclosure: The author declares no competingfinancial interests. Off-label drug use: None disclosed.

CorrespondenceCharles J. Parker, Hematology Division, Department of InternalMedicine, The University of Utah School of Medicine, 30 N 1900 E,Room 5C402, Salt Lake City, UT 84132; Phone: (801) 585-3229;Fax: (801) 585-0309; e-mail: charles.parker@hsc.utah.edu.

References1. Parker CJ. Paroxysmal nocturnal hemoglobinuria: an historical

overview. Hematology Am Soc Hematol Educ Program. 2008:93-103.

2. Parker C. Eculizumab for paroxysmal nocturnal haemoglobin-uria. Lancet. 2009;373:759-767.

3. Thurman JM, Holers VM. The central role of the alternativecomplement pathway in human disease. J Immunol. 2006;176:1305-1310.

4. Lindorfer MA, Pawluczkowycz AW, Peek EM, Hickman K,Taylor RP, Parker CJ. A novel approach to preventing thehemolysis of paroxysmal nocturnal hemoglobinuria: bothcomplement-mediated cytolysis and C3 deposition are blockedby a monoclonal antibody specific for the alternative pathwayof complement. Blood. 2010;115:2283-2291.

5. Nicholson-Weller A, Burge J, Fearon DT, Weller PF, AustenKF. Isolation of a human erythrocyte membrane glycoproteinwith decay-accelerating activity for C3 convertases of thecomplement system. J Immunol. 1982;129:184-189.

6. Nicholson-Weller A, March JP, Rosenfeld SI, Austen KF.Affected erythrocytes of patients with paroxysmal nocturnalhemoglobinuria are deficient in the complement regulatoryprotein, decay accelerating factor. Proc Natl Acad Sci U S A.1983;80:5066-5070.

7. Pangburn MK, Schreiber RD, Muller-Eberhard HJ. Deficiencyof an erythrocyte membrane protein with complement regula-tory activity in paroxysmal nocturnal hemoglobinuria. ProcNatl Acad Sci U S A. 1983;80:5430-5434.

8. Holguin MH, Fredrick LR, Bernshaw NJ, Wilcox LA, ParkerCJ. Isolation and characterization of a membrane protein fromnormal human erythrocytes that inhibits reactive lysis of theerythrocytes of paroxysmal nocturnal hemoglobinuria. J ClinInvest. 1989;84:7-17.

9. Parker CJ. Hemolysis in PNH. In: Young NS, Moss J, eds.Paroxysmal Nocturnal Hemoglobinuria and the Glycosylphos-phatidylinositol-Linked Proteins. San Diego, CA: AcademicPress; 2000;49-100.

10. Parker CJ. The pathophysiology of paroxysmal nocturnalhemoglobinuria. Exp Hematol. 2007;35:523-533.

11. Endo M, Ware RE, Vreeke TM, et al. Molecular basis of theheterogeneity of expression of glycosyl phosphatidylinositolanchored proteins in paroxysmal nocturnal hemoglobinuria.Blood. 1996;87:2546-2557.

12. Richards SJ, Rawstron AC, Hillmen P. Application of flowcytometry to the diagnosis of paroxysmal nocturnal hemoglobin-uria. Cytometry. 2000;42:223-233.

13. Parker C, Omine M, Richards S, et al. Diagnosis and manage-ment of paroxysmal nocturnal hemoglobinuria. Blood. 2005;106:3699-3709.

14. Borowitz MJ, Craig FE, Digiuseppe JA, et al. Guidelines for thediagnosis and monitoring of paroxysmal nocturnal hemoglobin-uria and related disorders by flow cytometry. Cytometry B ClinCytom. 2010;78:211-230.

15. Pu JJ, Mukhina G, Wang H, Savage WJ, Brodsky RA. Naturalhistory of paroxysmal nocturnal hemoglobinuria clones inpatients presenting as aplastic anemia. Eur J Haematol. 2011;87(1):37-45.

16. Scheinberg P, Marte M, Nunez O, Young NS. Paroxysmalnocturnal hemoglobinuria clones in severe aplastic anemiapatients treated with horse anti-thymocyte globulin plus cyclo-sporine. Haematologica. 2010;95:1075-1080.

17. Inoue N, Izui-Sarumaru T, Murakami Y, et al. Molecular basisof clonal expansion of hematopoiesis in 2 patients withparoxysmal nocturnal hemoglobinuria (PNH). Blood. 2006;108:4232-4236.

18. Ishiyama K, Chuhjo T, Wang H, Yachie A, Omine M, Nakao S.Polyclonal hematopoiesis maintained in patients with bonemarrow failure harboring a minor population of paroxysmalnocturnal hemoglobinuria-type cells. Blood. 2003;102:1211-1216.

19. Sugimori C, Chuhjo T, Feng X, et al. Minor population ofCD55-CD59- blood cells predicts response to immunosuppres-sive therapy and prognosis in patients with aplastic anemia.Blood. 2006;107:1308-1314.

20. Wang H, Chuhjo T, Yasue S, Omine M, Nakao S. Clinicalsignificance of a minor population of paroxysmal nocturnalhemoglobinuria-type cells in bone marrow failure syndrome.Blood. 2002;100:3897-3902.

21. Scheinberg P, Wu CO, Nunez O, Young NS. Predictingresponse to immunosuppressive therapy and survival in severeaplastic anaemia. Br J Haematol. 2009;144:206-216.

22. Wang SA, Pozdnyakova O, Jorgensen JL, et al. Detection ofparoxysmal nocturnal hemoglobinuria clones in patients withmyelodysplastic syndromes and related bone marrow diseases,with emphasis on diagnostic pitfalls and caveats. Haemato-logica. 2009;94:29-37.

23. Dunn DE, Tanawattanacharoen P, Boccuni P, et al. Paroxysmalnocturnal hemoglobinuria cells in patients with bone marrowfailure syndromes. Ann Intern Med. 1999;131:401-408.

24. Saunthararajah Y, Nakamura R, Nam JM, et al. HLA-DR15(DR2) is overrepresented in myelodysplastic syndrome andaplastic anemia and predicts a response to immunosuppressionin myelodysplastic syndrome. Blood. 2002;100:1570-1574.

25. Sugimori C, Yamazaki H, Feng X, et al. Roles of DRB1 *1501and DRB1 *1502 in the pathogenesis of aplastic anemia. ExpHematol. 2007;35:13-20.

26. Hillmen P, Hall C, Marsh JC, et al. Effect of eculizumab onhemolysis and transfusion requirements in patients with paroxys-mal nocturnal hemoglobinuria. N Engl J Med. 2004;350:552-559.

27. Hillmen P, Young NS, Schubert J, et al. The complementinhibitor eculizumab in paroxysmal nocturnal hemoglobinuria.N Engl J Med. 2006;355:1233-1243.

28. Brodsky RA, Young NS, Antonioli E, et al. Multicenter phase 3study of the complement inhibitor eculizumab for the treatmentof patients with paroxysmal nocturnal hemoglobinuria. Blood.2008;111:1840-1847.

29. Kelly RJ, Hill A, Arnold LM, et al. Long term treatment witheculizumab in paroxysmal nocturnal hemoglobinuria: sustainedefficacy and improved survival. Blood. 2011;117(25):6786-6792.

30. Hillmen P, Muus P, Duhrsen U, et al. Effect of the complementinhibitor eculizumab on thromboembolism in patients with parox-ysmal nocturnal hemoglobinuria. Blood. 2007;110:4123-4128.

31. de Latour RP, Mary JY, Salanoubat C, et al. Paroxysmalnocturnal hemoglobinuria: natural history of disease subcatego-ries. Blood. 2008;112:3099-3106.

28 American Society of Hematology

32. Berzuini A, Montanelli F, Prati D. Hemolytic anemia aftereculizumab in paroxysmal nocturnal hemoglobinuria. N EnglJ Med. 2010;363:993-994.

33. Risitano AM, Notaro R, Luzzatto L, Hill A, Kelly R, Hillmen P.Paroxysmal nocturnal hemoglobinuria–hemolysis before andafter eculizumab. N Engl J Med. 2010;363:2270-2272.

34. Risitano AM, Notaro R, Marando L, et al. Complement fraction3 binding on erythrocytes as additional mechanism of disease inparoxysmal nocturnal hemoglobinuria patients treated by eculi-zumab. Blood. 2009;113:4094-4100.

35. Risitano AM, Marando L, Seneca E, Rotoli B. Hemoglobinnormalization after splenectomy in a paroxysmal nocturnalhemoglobinuria patient treated by eculizumab. Blood. 2008;112:449-451.

36. Parker CJ. Bone marrow failure syndromes: paroxysmal noctur-

nal hemoglobinuria. Hematol Oncol Clin North Am. 2009;23:333-346.

37. Rosse WF. Treatment of paroxysmal nocturnal hemoglobin-uria. Blood. 1982;60:20-23

38. Hartmann RC, Jenkins DE, Jr., McKee LC, Heyssel RM.Paroxysmal nocturnal hemoglobinuria: clinical and laboratorystudies relating to iron metabolism and therapy with androgenand iron. Medicine (Baltimore). 1966;45:331-363.

39. Brecher ME, Taswell HF. Paroxysmal nocturnal hemoglobin-uria and the transfusio of washed red cells: A myth revisited.Transfusion. 1989;29:681-685.

40. Ikeda K, Mason PJ, Bessler M. 3�UTR-truncated Hmga2 cDNAcauses MPN-like hematopoiesis by conferring a clonal growthadvantage at the level of HSC in mice. Blood. 2011;117(22):5860-5869.

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