Paroxysmal nocturnal hemoglobinuria

This is a rare acquired hematological disorder with three main clinical features: intravascular hemolysis, a tendency to thrombosis and BMF of variable severity. As in IAA, the precise cause of the BMF remains unclear; by contrast, the molecular and cellular events responsible for hemolysis are now explained. For this reason, the space devoted here to this condition is out of proportion to its prevalence.

Molecular pathogenesis. The initiating event in the pathogenesis of PNH consists in somatic mutation(s) in the X-linked gene PIG-A (Figure 12.4) in multipotent HSCs. The protein product of PIG-A, although not yet physically isolated, is thought to be the enzymatically active subunit of a N-acetylglycosamine transferase. This enzyme catalyzes an early step in the formation of a complex glycolipid molecule called glucosylphosphatidylinositol (GPI; Figure 12.4). The synthesis of GPI takes place initially on the cytoplasmic surface of the endoplasmic reticulum, and is completed on its luminal surface. Once formed, the GPI molecules (anchors) are attached through a transpeptidation reaction to the carboxy-terminus of a variety of proteins. The GPI-linked proteins, after post-translational modifications in the Golgi apparatus, emerge eventually on the cell surface, to which they remain attached through the GPI anchor. As a result of the impaired synthesis of GPI, blood cells are either completely (PNH III) or partially (PNH II) deficient in GPI-linked proteins. Although for most of these proteins the functional consequences of this deficiency are not known, some are directly implicated in the pathogenesis of PNH: CD55 and especially CD59, which normally protect red cells from the lytic effect of activated complement. Intravascular hemolysis in PNH (manifesting clinically as hemoglobinuria) is the direct consequence of the fact that CD55- and CD59-deficient PNH red cells are susceptible to complement-mediated lysis. The in vitro counterpart of this phenomenon is the Ham test, whereby the patient's red cells are lysed by either autologous or ABO-compatible donor acidified serum. As for thrombosis, this may result in part from complement-mediated activation of platelets deficient in CD55 and CD59. Other, as yet unidentified, genetic or

Nucleus q \

Nucleus q \

Fig. 12.4 PIG-A, GPI anchors, GPI-anchored proteins and PNH

PIG-A is a protein encoded by the X-linked gene PIG-A. PIG-A is a member of a multi-subunit enzymatic complex which catalyzes, in the endoplasmic reticulum (ER), the first step in the biosynthesis of GPI: the addition of acetylglucosamine (GlcN) to phosphatidylinosito: (inositol-P; inset). The synthesis of the GPI anchor is completed by the serial addition of a glycan moiety consisting of three mannose molecules and a molecule of phosphoethanolamine (Etn-P), to which, through a transpeptidation reaction, proteins with the appropriate carboxy-terminal amino acid motif are attached covalently. The GPI-protein complex subsequently travels to the cell surface, where the protein becomes anchored to the lipid bilayer through GPI. In PNH, PIG-A has suffered a somatic mutation in one or few HSCs. As a result, very little GPI is synthesized, or none at all, with consequent severe deficiency of GPI-anchored proteins on the surface of the mutated HSCs and their progeny. From Karadimitris A, Luzzatto L. (2001) The cellular pathogenesis of paroxysmal nocturnal hemoglobinuria. Leukemia, 15, 1148-1152.

acquired factors affecting coagulation and/or fibrinolysis may have an additive or synergistic effect in producing thrombosis, which may be devastating, especially as it tends to take place in the abdominal veins.

Cellular pathogenesis. Since PNH-HSCs lack GPI-linked proteins, in principle they would be expected to be poorly competitive in growth with respect to normal HSCs. Instead, PNH-HSCs can expand until they largely supplant normal hematopoiesis. As a first approximation, this paradox may be explained by invoking an intrinsic proliferative advantage of PNH-HSCs over normal HSCs, as is the case with leukemic cells: however, the fact that patients with PNH can live for decades with normal and PNH hemopoiesis coexisting in their bone marrow militates against this notion. Lack of a competitive growth advantage by the PNH hemopoiesis has also been demonstrated experimentally in pig-a null mouse models.

An alternative pathogenetic model—the escape model— was suggested by the long-known association between PNH and IAA. In this model, the link between PNA and IAA is the effect of HSC-specific T cells. It is predicted that in PNH, such cells would selectively target normal HSCs but not PNH-HSCs. Under these circumstances, PNH-HSCs would expand and contribute to hemopoiesis (in some cases as much as 90% of it). Clinical evidence in support of the escape model is provided by the appearance of small PNH clones in as many as 50% of patients with bona fide IAA and by the presence of minute PNH clones (~1 in 105 of granulocytes) even in normal individuals. Further evidence supporting the immune model is provided by (1) the presence of expanded T-cell clones in the blood of PNH patients at a frequency three-fold higher than in appropriate controls; and (2) the increased representation of the HLA-DR2 allele in PNH patients compared with population controls (as in IAA).

The molecular target of the postulated autoreactive T cells is not known. Potential targets are a surface GPI-linked protein, failure of PNH-HSCs to present in the context of HLA an immunogenic peptide derived from GPI-linked protein or the GPI molecule itself. Experimental evidence has ruled out the first mechanism; the last two are still to be tested. Recently, it was reported that NK cells are more effective in lysing GPI+ targets than GPI- targets. However, two previous studies had found no difference; clearly, the role of NK cells in the pathogenesis of PNH needs further exploration.

Molecular pathology. All types of mutations have been observed in the PIG-A gene in patients with PNH: a few are large deletions, the majority (~75%) are small insertions or deletions causing frameshifts, and the rest are nonsense and missense point mutations. Interestingly, the nonsense and frameshift mutations are spread throughout the coding sequence (exons 2-6), presumably because they cause complete inactivation of the gene product wherever they fall, whereas missense mutations are clustered mainly within exon 2, where it is presumed that amino acid residues critical for catalytic activity must be located.

Clinical aspects and treatment of PNH. Although hemoglobinuria is paroxysmal by definition in the prototypical PNH patient, the brisk intravascular hemolysis is in fact continuous. Additionally, patients with florid PNH (Table 12.5) often experience acute exacerbations of their hemolysis during intercurrent illnesses, such as infections (presumably because this is associated with activation of complement through either the classical or the alternative pathway), other stressful events, or for no obvious reason.

Table 12.5 PNH: clinical heterogeneity and proposed terminology.

Predominant clinical features

Blood findings

Size of PNH clone

Designation

Hemolysis ± thrombosis Hemolysis ± thrombosis

Anemia; little or no other cytopenia Anemia; mild to moderate other cytopenia(s)

Large Large

Florid PNH PNH, hypoplastic

Purpura and/or infection Purpura and/or infection Thrombosis

Moderate to severe pancytopenia

Severe pancytopenia

Normal or moderate cytopenia(s)

AA with PNH clone Mini-PNH

From Tremml G, Karadimitris A, Luzzatto L. (1998) Paroxysmal nocturnal hemoglobinuria Haema, 1, 12-20.

learning about PNH cells from patients and from mice.

Hemolytic anemia with macrocytosis (partly due to re-ticulocytosis and partly due to BMF), different degrees of thrombocytopenia and leukopenia, iron deficiency and hemosiderinuria should raise the suspicion of PNH. The diagnosis can be confirmed with the Ham test (which only detects the PNH abnormality in erythrocytes) and/or flow cytometric analysis of erythrocytes and leukocytes; for this purpose, anti-CD59 is the most reliable antibody. In about 40% of PNH patients, venous thrombosis of small or large vessels (particularly at unusual sites, such as the abdomen or the brain) is a serious and potentially life-threatening complication. If promptly diagnosed, venous thrombosis may respond to thrombolytic therapy, which must be followed by short- and long-term anticoagulants. Supportive treatment consists of blood transfusions, folic acid and iron supplements when needed. The advent of the humanized monoclonal antibody Eculizumab, an inhibitor of the C5 component of complement, has been a major therapeutic advance. In a pilot study of 12 patients hemolysis was almost completely halted and at least half of the patients became transfusion dependent; the rest had a dramatic reduction of their transfusion requirements. Side effects were mild. It seems very likely, that if these results are confirmed in a larger study, Eculizumab will replace blood transfusion as the treatment of choice for anemia.

Long-term therapeutic options for PNH include immunosuppressive agents (e.g. combination of ALG/ATG and cyclosporin A, especially for patients with moderate to severe cytopenias) and, for selected patients, HSC transplantation from an HLA-identical sibling. In principle, PNH should also be amenable to reduced intensity allografting protocols.

PNH and other clonal disorders. Patients with PNH have a small risk (<4%) of developing MDS (the reverse, i.e. patients with MDS developing PNH, is discussed above) and AML. Because a similar risk exists in IAA, it is probably the perturbed marrow environment that allows the emergence of premalignant (MDS) or malignant (AML) clones, rather than the PIG-A mutations per se predisposing to MDS and AML.

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