Multilineage glycosylphosphatidylinositol anchor-deficient ...

British Journal of Haematology, 2001, 115, 476±482

Multilineage glycosylphosphatidylinositol anchor-deficient haematopoiesis in untreated aplastic anaemia Galina L. Mukhina, 1 J. Thomas Buckley 2 James P. Barber, 1 Richard J. Jones 1 and Robert A. Brodsky 1 Department of Oncology Johns Hopkins University, Baltimore, Maryland USA and 2 The Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada 1

Received 9 January 2001; accepted for publication 2 July 2001

Summary. Aplastic anaemia and paroxysmal nocturnal haemoglobinuria (PNH) are closely related disorders. In PNH, haematopoietic stem cells that harbour PIGA mutations give rise to blood elements that are unable to synthesize glycosylphosphatidylinositol (GPI) anchors. Because the GPI anchor is the receptor for the channelforming protein aerolysin, PNH cells do not bind the toxin and are unaffected by concentrations that lyse normal cells. Exploiting these biological differences, we have developed two novel aerolysin-based assays to detect small populations of PNH cells. CD59 populations as small as 0´004% of total red cells could be detected when cells were pretreated with aerolysin to enrich the PNH population. All PNH patients displayed CD59-deficient erythrocytes, but no myelodysplastic syndrome (MDS) patient or control had detectable PNH cells before or after enrichment in aerolysin. Only one aplastic anaemia patient had detectable PNH red cells before

exposure to aerolysin. However, 14 (61%) had detectable PNH cells after enrichment in aerolysin. The inactive fluorescent proaerolysin variant (FLAER) that binds the GPI anchors of a number of proteins on normal cells was used to detect a global GPI anchor deficit on granulocytes. Flow cytometry with FLAER showed that 12 out of 18 (67%) aplastic anaemia patients had FLAER-negative granulocytes, but none of the MDS patients or normal control subjects had GPI anchor-deficient cells. These studies demonstrate that aerolysin-based assays can reveal previously undetectable multilineage PNH cells in patients with untreated aplastic anaemia. Thus, clonality appears to be an early feature of aplastic anaemia.

Aplastic anaemia and paroxysmal nocturnal haemoglobinuria (PNH) are closely related haematopoietic stem cell disorders (Dameshek, 1967; Young, 1992; GriscelliBennaceur et al, 1995; Nagarajan et al, 1995; Schrezenmeier et al, 1995; Brodsky, 1998). Aplastic anaemia is caused by bone marrow failure resulting from an immunemediated attack on haematopoietic stem cells (Zoumbos et al, 1985; Nakao et al, 1997; Young & Maciejewsi, 1997), whereas PNH is a clonal haematopoietic stem cell disorder caused by somatic mutations in the X-linked gene, PIGA (Miyata et al, 1993; Takeda et al, 1993; Miyata et al, 1994; Nafa et al, 1995; Nagarajan et al, 1995). PNH may arise de novo or evolve from aplastic anaemia (Hillmen et al, 1995; Nagarajan et al, 1995; Socie et al, 1996). After immunosuppressive therapy for aplastic anaemia, 10±50% of patients will develop clonal disorders, with PNH being the most common (Tichelli et al, 1988; de Planque et al, 1989;

Ohara et al, 1997). The incidence of clonal disorders, including PNH, appears to be less frequent after allogeneic bone marrow transplantation (Socie et al, 1993) or highdose cyclophosphamide (Brodsky et al, 1996; Brodsky et al, 2001). The phenotypic hallmark of PNH blood cells is a deficiency of cell surface glycosylphatidylinositol (GPI)anchored proteins (Hall & Rosse, 1996; Rosse, 1997), owing to mutation of the PIGA gene that encodes the enzyme required for the first step in the biosynthesis of the GPI anchor itself. Using monoclonal antibodies directed against individual GPI-anchored proteins, PNH cells have been detected in . 20% of patients with aplastic anaemia; (Griscelli-Bennaceur et al, 1995; Schrezenmeier et al, 1995; Iwanaga et al, 1998; Dunn et al, 1999) and in a small percentage of patients with myelodysplastic syndrome (MDS) (Iwanaga et al, 1998; Dunn et al, 1999). However, monoclonal antibodies cannot reliably detect PNH populations , 1%, so that the number of patients with aplastic anaemia or MDS who have PNH cells may be underestimated. Moreover, there is no single monoclonal

Correspondence: Robert A. Brodsky, MD, Johns Hopkins Oncology Center, Cancer Research Building, Room 242, 1650 Orleans Street, Baltimore MD, USA. E-mail: [email protected]

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Keywords: aplastic anaemia, paroxysmal nocturnal haemoglobinuria, aerolysin, GPI-anchored proteins, FLAER.

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GPI-deficient Haematopoisis in Aplastic Anaemia antibody-based flow cytometric assay that can be used to establish the presence of PNH cells unconditionally; different haematopoietic lineages display different arrays of GPIanchored proteins, and certain proteins, such as CD58 and CD16, can be displayed in both a GPI-anchored and transmembrane form. Thus, it is recommended that at least two different monoclonal antibodies directed against two different GPI-anchored proteins on at least two different cell lineages be used to definitively detect PNH cells (Vanderschoot et al, 1990; Schubert et al, 1991; Hall & Rosse, 1996; Hillmen & Richards, 2000). The limitations of PNH detection with individual monoclonal antibodies are confounded by the fact that many patients with aplastic anaemia and MDS have received prior erythrocyte transfusions, further impeding the ability of standard flow cytometric techniques to detect small PNH erythrocyte populations. Aerolysin, and its inactive precursor proaerolysin, have the unusual ability to bind tightly and selectively to a common determinant in the anchor of GPI-anchored proteins (Diep et al, 1998; Brodsky et al, 1999) After binding, aerolysin oligomerizes to form membrane channels that destroy the permeability barrier of the cell (Parker et al, 1996). PNH cells are uniquely resistant to aerolysin because they lack GPI anchors. Thus, the toxin can enrich for PNH cells mixed with normal cells (Brodsky et al, 1999). We have also established a fluorescent-labelled variant of proaerolysin (FLAER) that binds the GPI anchor but does not form channels (Brodsky et al, 2000). A fluorescent labelled variant of FLAER, that binds GPI-anchored proteins but does not form channels (Brodsky et al, 2000), can be used to detect more accurately GPI anchor deficiencies than antiCD59 in a variety of cell types, including granulocytes and monocytes (Brodsky et al, 2000). Here, we use enrichment of PNH cells with aerolysin and flow cytometry with FLAER to determine how frequently PNH cells can be detected in patients with newly diagnosed aplastic anaemia and MDS. PATIENTS AND METHODS Patients. Venous peripheral blood from PNH patients, MDS patients or control subjects was drawn into heparincontaining tubes after informed consent as approved by the Joint Committee on Clinical Investigation of The Johns Hopkins Hospital. Severe aplastic anaemia was defined as (i) , 25% bone marrow cellularity and (ii) at least two of the following three peripheral blood counts: neutrophils , 0´5 109/l; platelets , 20 109/l; and anaemia with corrected reticulocytes , 1% (Camitta et al, 1979). Patients who met the above criteria who had a neutrophil count , 0´2 109/l were categorized as super-severe disease. Moderate aplastic anaemia was defined as marrow hypoplasia with pancytopenia not severe enough to meet the above criteria. Myelodysplasia was defined using the French±American±British classification (Bennett et al, 1982) Patients were categorized as `classic' PNH if they had overt haemolysis and/or thrombosis, a demonstrable PNH phenotype in at least two lineages and absence of criteria for severe aplastic anaemia.

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Red cell enrichment assay. Whole blood was washed two times in phosphate-buffered saline (PBS) and resuspended in PBS to a concentration of 0´8% (v:v). To enrich for GPI anchor-deficient erythrocytes, aerolysin (1 1028 mol/l), produced by trypsin activation of proaerolysin as previously described (Garland & Buckley, 1988), was added to 0´25 ml of erythrocytes and incubated for 15 min at 378C. After incubation, the cells were washed in cold PBS. To measure CD59 expression, 3 106 erythrocytes were stained with phycoerythrin (PE)-conjugated antiglycophorin (Immunotech, Marseille, France) and fluorescein isothiocyanate (FITC)conjugated anti-CD59 (Research Diagnostics, Flanders, NJ, USA) for 30 min at 48C before and after exposure to aerolysin and analysed using flow cytometry (FACscan; Becton Dickinson, San Jose, CA, USA). FLAER assay for the detection of GPI anchor protein deficiency. FLAER has fluorescent properties similar to the FITC proteins and is available from Protox Biotech (web.uvic.ca/idc/protox/protox.html). After lysing erythrocytes with ammonium chloride, neutrophils were identified on the basis of cell size and granularity and by staining with a specific anti-granulocyte antibody (PE-conjugated antiCD15, Immunotech). Detection of GPI anchor expression was assessed using FLAER. Gates used to define FLAERnegative granulocytes were based on normal control granulocytes analysed on the same day (Brodsky et al, 2000). RESULTS Previously, we demonstrated that aerolysin can be used in conjunction with anti-CD59 to detect minute PNH populations that cannot be detected using conventional flow cytometric assays (Brodsky et al, 1999). In order to determine the sensitivity of enrichment using aerolysin, we performed 1:10 serial dilutions of washed erythrocytes from a PNH patient with erythrocytes from a normal control subject; the mixtures were assayed for CD59 deficiency before and after exposure to aerolysin (1 1028 mol/l). Conventional flow cytometry using anti-CD59 was able to reliably detect 1% or greater PNH erythrocytes. However, pretreatment of red cells with aerolysin allowed us to detect as few as one PNH cell among 25 000 erythrocytes, improving the sensitivity of the assay by more than 2 logs (Fig 1). Next, we screened erythrocytes from six normal control subjects, 23 patients with untreated aplastic anaemia, 10 patients with PNH, 11 patients with MDS and nine disease control subjects for a PNH phenotype before and after aerolysin treatment to enrich for GPI anchor-deficient cells (Fig 2). All PNH patients showed substantial enrichment of their PNH population after aerolysin; the mean percentage of CD592 erythrocytes before and after exposure to aerolysin was 37´0% and 95´5% respectively. None of the patients with MDS, normal subjects or disease control subjects displayed a PNH population before or after aerolysin. Although PNH red cells could be detected in one (4%) of the aplastic anaemia patients before enrichment with aerolysin, they were detected in 14 (61%) of the

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Fig 2. Detection of GPI anchor-negative erythrocytes before and after enrichment with aerolysin. Erythrocytes from six normal control subjects (N), 23 patients with aplastic anaemia (AA), 10 patients with paroxysmal nocturnal haemoglobinuria (PNH), 11 patients with myelodysplastic syndrome (MDS), and nine disease control subjects (DC) were assayed using flow cytometry for lack of CD59 before (closed symbols) and after (open symbols) enrichment in aerolysin. The dashed line represents the lower level of detection of flow cytometry (5%) after enrichment with aerolysin. Median values for each group are designated by the solid black lines. The disease control group consisted of patients with various autoimmune disorders including, systemic lupus erythematosus, pemphigus vulgaris and large granular lymphocyte disease.

Fig 1. Enrichment of PNH erythrocytes with aerolysin. PNH erythrocytes were diluted with normal control erythrocytes to make a 4% PNH population (A). Serial 10-fold dilutions were made with normal control erythrocytes to generate PNH populations of 0´4% (B), 0´04% (C) and 0´004% (D). Enrichment of the PNH populations was accomplished by adding aerolysin 1´0 1028 mol/l for 15 min at 378C. After staining with PE-conjugated antiglycophorin and FITC-conjugated anti-CD59, PNH erythrocytes were assessed using two-colour flow cytometry before (A±D) and after (E±H) exposure to aerolysin.

patients after enrichment (Fig 2). Thus, aerolysin pretreatment uncovered previously undetectable PNH cells in the majority of patients with untreated aplastic anaemia. To document a global deficiency of GPI anchors in granulocytes we used FLAER, a fluorescent-labelled variant of aerolysin that binds directly to the anchor of a number of GPI-anchored proteins and, thus, serves as a much more comprehensive assay for PNH cells than any individual monoclonal antibody (Brodsky et al, 2000). Five normal subjects, 18 patients with aplastic anaemia, eight patients with PNH, nine patients with MDS and nine disease control subjects were assessed for the presence of GPI anchordeficient granulocytes using FLAER. A representative example is shown in Fig 3. The median percentage of FLAER-negative granulocytes in the PNH patients was 95% (range 68±98%) (Fig 4). None of the normal subjects, MDS

patients or disease control subjects had detectable FLAERnegative granulocytes. However, 12 out of 18 (67%) aplastic anaemia patients displayed FLAER-negative granulocytes (Fig 4). The median number of FLAER-negative granulocytes from the 18 patients with aplastic anaemia was 0´8% (range 0±30%). A high level of concordance for detection of PNH cells was observed between the two aerolysin-based assays. All normal control subjects, disease control subjects and patients with MDS were negative in both assays. Similarly, there were eight patients with PNH who were studied using both the red cell enrichment assay and the granulocyte assay with FLAER. All patients who were positive in one assay were positive in the other. Characteristics of the patients and concordance between the aerolysin-based assays in patients with aplastic anaemia are shown in Table I. Both the FLAER assay on granulocytes and the red cell enrichment assay were performed on 16 of the 23 patients with aplastic anaemia reported in this study. All but one (10 out of 11), of the patients who were positive in the FLAER assay were also positive in the red cell enrichment assay, and all five of the patients whose granulocytes were negative in the FLAER assay were negative in the red cell enrichment assay.

DISCUSSION A close relationship between aplastic anaemia and PNH has long been established (Dameshek, 1967). However, less sensitive assays, such as the Ham test and sucrose


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Table I. Characteristics of aplastic anaemia patients studied in both aerolysin assays. Patient number

Red cell assay

Granulocyte assay

Disease severity

Disease duration (months)

Aetiology

Age(years) /sex

3 4 5 7 8 9 11 14 15 16 18 19 20 21 22 23

Positive Positive Positive Positive Negative Positive Negative Positive Negative Negative Negative Positive Positive Positive Positive Negative

Positive Positive Positive Positive Negative Positive Negative Positive Negative Positive Negative Positive Positive Positive Positive Negative

maa maa saa saa saa maa ssaa maa ssaa ssaa saa ssaa ssaa saa saa saa

18 5 1 6 4 3 3 6 0´5 1 1 4 6 2 6 2

Idiopathic Drug Idiopathic Idiopathic Idiopathic Idiopathic Idiopathic Idiopathic Idiopathic Drug Hepatitis Idiopathic Hepatitis Idiopathic Idiopathic Idiopathic

41/M 35/M 24/M 45/F 21/F 45/M 19/F 47/F 42/F 68/F 6/F 53/F 19/F 20/M 70/F 28/F

maa, moderate aplastic anaemia; saa, severe aplastic anaemia; ssaa, super severe aplastic anaemia.

haemolysis test, greatly underestimated the frequency of PNH cells in patients with aplastic anaemia when compared with assays which use monoclonal antibodies and flow cytometry (Griscelli-Bennaceur et al, 1995; Schrezenmeier et al, 1995; Iwanaga et al, 1998; Dunn et al, 1999). We found that aerolysin-based assays detect an even higher frequency of small PNH populations in aplastic anaemia than individual monoclonal antibodies such as anti-CD59. A major advantage of the aerolysin assays is that they directly assess the biochemical consequence of PIGA mutations, global loss of GPI anchors, rather than the absence of surrogate proteins. Previous studies using monoclonal antibodies have demonstrated the presence of PNH cells in 20±52% of patients with aplastic anaemia (Fores et al, 1995; Schrezenmeier et al, 1995; De Lord et al, 1998; Dunn et al, 1999). Most of the patients in these studies were evaluated after treatment with immunosuppressive therapy; very few patients with untreated aplastic anaemia have been studied. Furthermore, in most cases the presence of PNH cells was found in a single lineage. Schrezenmeier et al (1995) studied multiple lineages, but the concordance between the various lineages was low (, 15% of aplastic anaemia patients had detectable red cell involvement). PNH is a haematopoietic stem cell disorder. Therefore, it is recommended that a deficiency of two or more GPI anchor proteins be demonstrated in two or more lineages. Our study is the first to demonstrate multilineage GPI anchor deficiency in the majority of untreated patients with aplastic anaemia, suggesting that PIGA mutations are an early event in the development of aplastic anaemia. Compared with monoclonal antibodies, FLAER leads to a greater difference in fluorescence intensity between the normal granulocytes and the PNH granulocytes (Fig 3). This is particularly helpful in detecting small PNH populations. Unfortunately, FLAER cannot distinguish between normal and PNH red cells as

effectively as anti-CD59. This appears to be as a result of the fact that both normal and PNH erythrocytes contain large amounts of glycophorin, a protein shown to weakly bind aerolysin(Mackenzie et al, 1999). However, activated aerolysin used in conjunction with anti-CD59 improves the sensitivity of this assay. Although the red cell enrichment assay can detect a smaller percentage of PNH cells (0´004%), its ability for uncovering minute PNH populations in AA probably parallels that of FLAER for the following reasons. First, PNH red cells, in contrast to PNH granulocytes (Brubaker et al, 1977; Brodsky et al, 1997), have a shortened survival in the peripheral blood than their normal (GPI anchor replete) counterparts (Brubaker et al, 1977; Brodsky et al, 1997). Second, patients with aplastic anaemia are frequently transfused with packed red blood cells, which dilute out the small number of PNH erythrocytes. In fact, the only discordance between the two assays occurred in patient 16, and may have been a consequence of her heavy transfusional requirements (more than four units of red cells) immediately before the assay. Thus, although pretreating erythrocytes with aerolysin greatly improves the ability to detect PNH erythrocytes in patients with aplastic anaemia, it is not a quantitative assay. The assay was designed to preferentially detect cells completely lacking GPI anchors (type III erythrocytes) because this is usually the predominant population; , 50% of PNH patients harbour type II cells (cells with partial deficiency) with only 13% of patients having . 20% type II cells (Rosse, 1997). Nevertheless, the dose and length of exposure to aerolysin can be adjusted to detect type II cells (Brodsky et al, 1999). In the 11 patients studied with MDS, we were unable to corroborate previous findings of GPI anchor proteinnegative cells in this group of patients (Iwanaga et al, 1998; Dunn et al, 1999). This may be a consequence of the relatively small number of MDS patients we studied or be a


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Fig 4. Detection of GPI anchor-negative granulocytes in untreated aplastic anaemia. Shown are the percentage of FLAER-negative granulocytes detected by flow cytometry from five normal control subjects (N), 18 patients with aplastic anaemia (AA), eight patients with PNH, nine patients with MDS, and nine disease control subjects (DC). The dashed line represents the lower limit of sensitivity of the assay (0´5%). The solid black lines represent the median value for each group of patients.

Fig 3. Representative example demonstrating GPI anchor-negative granulocytes in patients with PNH and aplastic anaemia. Dual colour display of peripheral blood granulocytes from a normal control (A and E), a patient with aplastic anaemia (B and F), a patient with PNH (C and G) and a patient with MDS (D and H) after staining with anti-CD15 PE and anti-CD59 FITC (A±D) or antiCD15 PE and FLAER (E±H).

function of the difficulty in reliably distinguishing moderate aplastic anaemia from hypoplastic MDS. All four patients with moderate aplastic anaemia were positive in both assays (Table I). The improved sensitivity and specificity of the aerolysinbased assays will be useful in studying the biology of PNH, it's close relationship to aplastic anaemia and for monitoring small clones after therapy for aplastic anaemia. PNH progenitor cells have a proliferative disadvantage compared with normal progenitors (Maciejewski et al, 1997; Rosti et al, 1997). Thus, it is unclear as to how a PNH clone can dominate haematopoiesis. The leading hypothesis to explain this enigma is known as the theory of immune escape (Rotoli & Luzzatto, 1989; Young, 1992; Luzzatto et al, 1997). This postulates that PIGA mutations are a relatively common benign event, and that cells harbouring PIGA mutations can dominate only in the setting of an immune attack (such as that in aplastic anaemia) that kills normal progenitor cells, but not progenitors lacking GPI anchors. The demonstration of a global loss of GPI anchors in most patients with untreated aplastic anaemia shows that clonality is an early event in the evolution of this disease. Thus, aplastic anaemia (AA) and PNH may represent different responses to stem cell insults such as viruses,

radiation or chemicals. Damage to stem cells may in certain instances lead to an autoimmune attack and in other instances produce a stem cell mutation (e.g. PIGA) that confers a survival advantage to that cell. Over time, the slowly proliferating clone could become dominant (Brodsky et al, 1997). In certain instances, stem cell injury may result in the simultaneous appearance of autoimmune marrow suppression and clonality (AA/PNH). PNH cells may be favoured even more in the setting of immune attack if they are spared or if they are intrinsically resistant to the immune attack (Chen et al, 2000; Karadimitris et al, 2000). Tracking the fate of these small PNH populations in aplastic anaemia with aerolysin-based assays after immunoablative therapy should give further insight into the mechanism of clonal dominance in PNH. ACKNOWLEDGMENTS This work is supported in part by National Institutes of Health grant no. CA74990 to R.A.B., and by a Medical Research Council of Canada grant to J.T.B. R.A.B. is a Leukaemia and Lymphoma Society Scholar. REFERENCES Bennett, J.M., Catovsky, D., Daniel, M.T., Flandrin, G., Galton, D.A.G., Gralnick, H.R. & Sultan, C. and the French±American± British (FAB) Co-operative Group (1982) Proposals for the classification of the myelodysplastic syndromes. British Journal of Haematology, 51, 189±199. Brodsky, R.A. (1998) Biology and Management of Acquired Severe Aplastic Anaemia. Current Opinion in Oncology, 10, 95±99. Brodsky, R.A., Sensenbrenner, L.L. & Jones, R.J. (1996) Complete remission in acquired severe aplastic anemia following high-dose cyclophosphamide. Blood, 87, 491±494. Brodsky, R.A., Vala, M.S., Barber, J.P., Medof, M.E. & Jones, R.J. (1997) Resistance to Apoptosis Caused by PIG-A Gene Mutations


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