Shoichi Nagakura

Paroxysmal Nocturnal Hemoglobinuria Cells in Patients with Bone Marrow Failure Syndromes

Aplastic anemia and paroxysmal nocturnal hemoglobinuria (PNH) are rare hematologic diseases that often appear in the same patient. Patients with aplastic anemia have severe thrombocytopenia, neutropenia, and anemia accompanied by absent hematopoietic precursors in an empty bone marrow (1). In contrast, the classic evidence of PNH is the intermittent appearance of dark urine due to excretion of hemoglobin, the result of intravascular hemolysis (2). The knowledge that this peculiar form of erythrocyte destruction resulted from increased susceptibility of the PNH erythrocyte to complement led to the development of laboratory assays, such as the Ham and sugar hemolysis tests. Modern clinical studies have shown that patients with PNH experience serious morbidity and mortality, mainly from venous thromboses and, especially in younger patients, pancytopenia (3). On the basis of results of the Ham test in several patients, Lewis and Dacie (4) formalized the overlap between the two diseases as the aplastic anemia-paroxysmal nocturnal hemoglobinuria syndrome. With improved survival in aplastic anemia, many patients show laboratory and clinical evidence of PNH, often months or years after completion of successful immunosuppressive therapy (5, 6). In aplastic anemia, hematopoietic cells appear to be destroyed by the patients own immune system (7). In contrast, the basis of PNH is a somatically acquired mutation in a hematopoietic stem cell. Almost all patients with PNH have molecular lesions in the PIG-A gene, which is located on the X chromosome (8). The PIG-A gene product is required at an early step in the synthesis of a glycosylphosphatidylinositol (GPI) structure, which serves as an anchor for a group of proteins that are linked to the cell surface by this greasy foot rather than by the more typical transmembrane configuration. As a result, affected cells are globally deficient in GPI-anchored proteins. Deficiency in one of these proteins, CD59, which inhibits late-acting complement component activity on the erythrocyte surface, accounts for the hemolytic component of the disease (9). The pathophysiologic basis of the clinical relation between PNH and aplastic anemia is unknown. Lewis and Dacie (4) postulated the development of an abnormal clone of haemopoietic cells in a regenerating, previously aplastic marrow as a cause, and Rotoli and Luzzatto (10, 11) speculated that an insult leading to a hypocellular environment or some feature of marrow failure might lead to either aplasia or PNH. Observations of patients with lymphoma who received monoclonal antibody treatment directed against a GPI-anchored protein have demonstrated the appearance of PNH-like lymphocytes as an inadvertent result of therapy (12). These results confirmed the in vivo plausibility of a third hypothetical mechanism: that preexisting PNH clones might be selected as a result of their relative insusceptibility to autoimmune attack (6, 10). To test these hypotheses, we used a sensitive flow cytometric method to analyze blood cells from patients in various states of marrow failure for evidence of PNH clones. Methods Patients We developed a rigorous three-color flow cytometric protocol for identification of GPI-anchored protein-deficient granulocyte populations and applied this method over 1.5 years to patients presenting to our clinic at the National Heart, Lung, and Blood Institute with newly diagnosed or previously treated bone marrow failure syndromes. Most of the patients with previously treated bone marrow syndromes had received therapy at the National Institutes of Health up to 11 years before sampling for this study. Samples from 254 participants were analyzed from April 1997 to October 1998. Of these participants, 115 had aplastic anemia, 39 had myelodysplasia, 28 had recently undergone bone marrow transplantation, 20 were controls who had undergone renal transplantation and had received antithymocyte globulin as treatment for graft rejection, 13 had large granular lymphocytosis, 18 had undergone multiple cycles of chemotherapy for cancer, and 21 were healthy controls. All clinical research samples were obtained after informed consent was given under protocols approved by the institutional review board of the National Heart, Lung, and Blood Institute. Other specimens were collected during routine phlebotomy procedures and were studied without unique patient identifiers. Aplastic anemia was defined as bone marrow cellularity of 30% or less and two of the following three laboratory abnormalities: absolute neutrophil count less than 0.5 109 cells/L, platelet count less than 20 109 cells/L, and reticulocyte count less than 60 109 cells/L. Myelodysplasia was defined by the standard morphologic criteria of either dysplastic myeloid or megakaryocytic (but not solely erythroid) bone marrow elements in patients who required 2 or more units of red blood cell transfusions per month for 2 or more months, with or without thrombocytopenia or neutropenia. Monoclonal Antibodies We used CD55-PE (clone 143-30, mouse IgG1 [Research Diagnosis, Inc., Flanders, New Jersey]) and CD59-PE (clone MEM 43, mouse IgG2a [Research Diagnosis, Inc.]) for analysis of GPI-anchored protein expression on erythrocytes. Glycophorin-A-FITC (clone D2.10, mouse IgG1 [Immunotech, Westbrook, Maine]) was used as a nonanchored marker to positively identify erythrocytes. The isotypic control for erythrocyte phenotyping consisted of mouse IgG1-PE (clone X40 [Becton Dickinson, San Diego, California]) and mouse IgG2a-PE (clone X39 [Becton Dickinson]). For analysis of granulocyte GPI-anchored protein expression, we used CD66b-FITC (clone 80H3, mouse IgG1 [Immunotech]) and CD16-PECy5 (clone 3G8, mouse IgG1 [Caltag]), with CD15-PE (clone 80H5, mouse IgM [Immunotech]) as a non-GPI-anchored marker to positively identify granulocytes. The isotypic controls for granulocyte staining consisted of mouse IgG1-FITC (clone X40 [Becton Dickinson]) and mouse IgG1-PECy5 (clone MOPC-21 [Caltag]). Nonspecific Fc receptor-mediated binding of conjugated antibodies to granulocytes or erythrocytes was blocked by pre-incubating 1 mL of blood with 30 L of mouse IgG (Caltag). Antibody Staining and Flow Cytometry Analysis Blood was drawn by venipuncture into tubes containing EDTA. Samples were stained within 48 hours of collection; staining was usually done within 8 hours. (Pilot studies revealed no significant change in cytofluorometric results in samples stored for up to 48 hours.) Erythrocyte GPI-anchored protein expression was evaluated by incubating for 30 minutes at room temperature 50 L of a 1:20 dilution of whole blood with 20 L of Gly-A-FITC and either 10 L each of CD55-PE plus CD59-PE or the appropriate isotype controls. Samples were washed and resuspended in 1 mL of phosphate-buffered saline before flow cytometry. Granulocyte GPI-anchored protein expression was evaluated by incubating for 60 minutes at room temperature 100 L of whole blood with 10 L of CD15-PE and either 20 L of CD66b-FITC plus 5 L of CD16-PECy5 or the appropriate isotype controls. Erythrocytes in these samples were lysed by using a Q-Prep apparatus (Coulter, Fullerton, California) and were then fixed with paraformaldehyde. All of the samples were analyzed by using a Coulter XL flow cytometry machine equipped with a 488-nm argon laser and XL or XLII software. Strict criteria to distinguish cells lacking GPI-anchored proteins were applied for two reasons. First, like many cell surface transmembrane proteins, GPI-anchored protein can vary in expression according to the stage of cellular differentiationfor example, as a result of myeloid or erythroid maturation. In addition, in some hematologic diseases, differentiation may be abnormal, as reflected in abnormal structure and aberrant expression of specific proteins. Therefore, polymorphonuclear cells were identified on the basis of light-scatter properties that correlate with cell size and granularity and by staining with a specific antigranulocyte antibody (anti-CD15, conjugated to the fluorescent dye phycoerythrin). Two antibodies directed against distinct GPI-anchored protein (anti-CD16, conjugated to the fluorochrome PE-Cy5, and anti-CD66b, conjugated to fluorescein isothiocyanate) were used to determine the PNH phenotype. Erythrocytes were similarly analyzed by using appropriate antibody combinations (anti-glycophorin for identification of erythrocytes and anti-CD59 and anti-CD55 as labels for GPI-anchored protein). Interpretation of Flow Cytometry Data The gates used to define CD16 /CD66b granulocyte or CD55 /CD59 erythrocyte populations were set on the basis of the isotypic control analyses performed on the same day. The mean SD of double negative cells for 21 healthy participants was 0.129% 0.101% for granulocytes and 0.195% 0.116% for erythrocytes. Because many patients required red blood cell transfusion at the time of initial sampling, the classification of patients was based on granulocyte analysis; a population of GPI-anchored protein-negative granulocytes of 1% or more that was clearly separate from the wild-type CD16+/CD66b+ cells on a two-dimensional histogram (Figure 1) was required for categorization of a patient as harboring PNH cells. Figure 1. Protocol for identification of paroxysmal nocturnal hemoglobinuria ( PNH) phenotype granulocytes. A. B. C. D. E. Response Criteria Response to treatment in aplastic anemia was defined as failure to fulfill criteria for severity for at least 3 months (see above); this was previously shown to correlate with transfusion independence and absence of infections. Criteria for response to therapy in myelodysplasia were no need for transfusion for 8 or more weeks, hemoglobin values of 80 g/L or more, platelet counts greater than 20 109 cells/L, and absolute neutrophil counts of 0.5 109 cells/L or more. Statistical Analysis A Fisher exact test was used to determine two-tailed P values in 2 2 contingency tables. The chi-square test was used

NKG2D-mediated immunity underlying paroxysmal nocturnal haemoglobinuria and related bone marrow failure syndromes.

It is considered that a similar immune mechanism acts in the pathogenesis of bone marrow (BM) failure in paroxysmal nocturnal haemoglobinuria (PNH) and its related disorders, such as aplastic anaemia (AA) and myelodysplastic syndromes (MDS). However, the molecular events in immune‐mediated marrow injury have not been elucidated. We recently reported an abnormal expression of stress‐inducible NKG2D (natural‐killer group 2, member D) ligands, such as ULBP (UL16‐binding protein) and MICA/B (major histocompatibility complex class I chain‐related molecules A/B), on granulocytes in some PNH patients and the granulocyte killing by autologous lymphocytes in vitro. The present study found that the expression of NKG2D ligands was common to both granulocytes and BM cells of patients with PNH, AA, and MDS, indicating their exposure to some incitement to induce the ligands. The haematopoietic colony formation in vitro by the patients’ marrow cells significantly improved when their BM cells were pretreated with antibodies against NKG2D receptor, suggesting that the antibodies rescued haematopoietic cells expressing NKG2D ligands from damage by autologous lymphocytes with NKG2D. Clinical courses of patients with PNH and AA showed a close association of the expression of NKG2D ligands with BM failure and a favourable response to immunosuppressive therapy. We therefore propose that NKG2D‐mediated immunity may underlie the BM failure in PNH and its‐related marrow diseases.

Differential glycosylation of Bence Jones protein and kidney impairment in patients with plasma cell dyscrasia

Although Bence Jones protein (BJP) is generally accepted to be critically involved in the pathogenic process of kidney impairment in patients with myeloma, patients with BJP do not always have kidney dysfunction. As proteins often undergo glycosylation and alter their molecular nature, it is expected that the heterogeneity in kidney dysfunction can be explained at least partly by the differential affinity to the kidneys of BJP dependent on its glycosylation. Accordingly, we analyzed the structures of carbohydrates of urine BJP biochemically to correlate the structure with kidney function. BJP was obtained from 16 patients with myeloma, 2 patients with light chain amyloidosis, a patient with plasma cell leukemia, and a patient with Waldenstroms macroglobulinemia. All BJP had five forms of oligosaccharides: three forms of biantennary oligosaccharides and two forms of triantennaries. The three biantennaries correspond to previously reported oligosaccharides on only lambda-type BJP, whereas the triantennaries are novel oligosaccharides found on BJP. Among the five oligosaccharides, the triantennary oligosaccharide Gal(beta)1-4GlcNAc(beta)1-2Man(alpha)1-6 [Gal(beta)1-GlcNA(beta)1-4(Gal(beta)1-4GlcNAc(beta) 1-2)Man(alpha)1-3]Man(beta)1-4GlcNAc(beta)1-4GlcNAc showed a significant negative correlation with the serum creatinine level (p = 0.015 by Spearmans correlation test, R = 0.744). Thus determination of BJP glycosylation may be useful for the evaluation of kidney impairment in patients with BJP.

Establishment of a human T-cell line with deficient surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins from a patient with paroxysmal nocturnal haemoglobinuria.

A novel interleukin‐2 dependent T‐cell line, PMT‐2Y, was established from the peripheral blood of a patient with paroxysmal nocturnal haemoglobinuria (PNH) by human T lymphotropic virus type I (HTLV‐1)‐mediated transformation. PMT‐2Y cells are positive for CD2, CD3, CD4, CD25, T cell receptor αβ and HLA‐DR, but negative for CD1, CD7, CD8, CD19 and CD20, indicating that the clone belongs to a helper/inducer subset of T cells. PMT‐2Y cells have the monoclonal integration of HTLV‐I proviral DNA, suggesting that they derived from a single clone. Moreover, they lack surface expression of complement regulatory proteins such as DAF (CD55) and CD59, that are the most important glycosylphosphatidylinositol (GPI)‐anchored membrane proteins defective in haemopoietic cells of patients with PNH. Northern blot analysis, however, revealed the production of normal levels of DAF mRNAs. Thus, PMT‐2Y is derived from a PNH T cell clone and may be a useful model to study PNH.

Occupancy of whole blood cells by a single PIGA-mutant clone with HMGA2 amplification in a paroxysmal nocturnal haemoglobinuria patient having blood cells with NKG2D ligands.

Chou, T.C. & Talalay, P. (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation, 22, 27–55. Fahy, B.N., Schlieman, M.G., Virudachalam, S. & Bold, R.J. (2003) Schedule-dependent molecular effects of the proteasome inhibitor bortezomib and gemcitabine in pancreatic cancer. Journal of Surgical Research, 113, 88–95. Lonial, S., Kaufman, J., Tighiouart, M., Nooka, A., Langston, A.A., Heffner, L.T., Torre, C., McMillan, S., Renfroe, H., Harvey, R.D., Lechowicz, M.J., Khoury, H.J., Flowers, C.R. & Waller, E.K. (2010) A phase I/II trial combining high-dose melphalan and autologous transplant with bortezomib for multiple myeloma: a doseand schedule-finding study. Clinical Cancer Research, 16, 5079–5086. Mitsiades, N., Mitsiades, C.S., Richardson, P.G., Poulaki, V., Tai, Y.T., Chauhan, D., Fanourakis, G., Gu, X., Bailey, C., Joseph, M., Libermann, T. A., Schlossman, R., Munshi, N.C., Hideshima, T. & Anderson, K.C. (2003) The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. Blood, 101, 2377–2380. Nencioni, A., Hua, F., Dillon, C.P., Yokoo, R., Scheiermann, C., Cardone, M.H., Barbieri, E., Rocco, I., Garuti, A., Wesselborg, S., Belka, C., Brossart, P., Patrone, F. & Ballestrero, A. (2005) Evidence for a protective role of Mcl-1 in proteasome inhibitor-induced apoptosis. Blood, 105, 3255–3262. Popat, R., Oakervee, H., Williams, C., Cook, M., Craddock, C., Basu, S., Singer, C., Harding, S., Foot, N., Hallam, S., Odeh, L., Joel, S. & Cavenagh, J. (2009) Bortezomib, low-dose intravenous melphalan, and dexamethasone for patients with relapsed multiple myeloma. British Journal of Haematology, 144, 887–894. San Miguel, J.F., Schlag, R., Khuageva, N.K., Dimopoulos, M.A., Shpilberg, O., Kropff, M., Spicka, I., Petrucci, M.T., Palumbo, A., Samoilova, O.S., Dmoszynska, A., Abdulkadyrov, K.M., Schots, R., Jiang, B., Mateos, M.V., Anderson, K.C., Esseltine, D.L., Liu, K., Cakana, A., van de Velde, H. & Richardson, P.G. (2008) Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma. New England Journal of Medicine, 359, 906–917. Spanswick, V.J., Craddock, C., Sekhar, M., Mahendra, P., Shankaranarayana, P., Hughes, R.G., Hochhauser, D. & Hartley, J.A. (2002) Repair of DNA interstrand crosslinks as a mechanism of clinical resistance to melphalan in multiple myeloma. Blood, 100, 224–229. Weigert, O., Pastore, A., Rieken, M., Lang, N., Hiddemann, W. & Dreyling, M. (2007) Sequence-dependent synergy of the proteasome inhibitor bortezomib and cytarabine in mantle cell lymphoma. Leukemia, 21, 524– 528. Yarde, D.N., Oliveira, V., Mathews, L., Wang, X., Villagra, A., Boulware, D., Shain, K.H., Hazlehurst, L.A., Alsina, M., Chen, D.T., Beg, A.A. & Dalton, W.S. (2009) Targeting the Fanconi anemia/BRCA pathway circumvents drug resistance in multiple myeloma. Cancer Research, 69, 9367 –9937.

Persistently high quality of life conferred by coexisting congenital deficiency of terminal complement C9 in a paroxysmal nocturnal hemoglobinuria patient

To the editor: Paroxysmal nocturnal hemoglobinuria (PNH) clone bears a PIGA mutation and fails to express glycosylphosphatidylinositol-linked membrane proteins such as complement-regulatory CD55 and CD59, leading to complement-mediated intravascular hemolysis and thrombosis. The advent of

3H9, a monoclonal antibody capable of discriminating neutrophilic from basophilic and eosinophilic granulocytes.

To the Editor: The discovery of markers and generation of antibodies discriminative of granulocyte subtypes has been shown to promote characterization of their morphological and functional aspects (1, 2). The monoclonal antibody (mAb 3H9), which was developed by screening for the inhibition of leukocyte adherence to plastic plates, reacts distinctly with granulocytes, barely with monocytes, but not with other peripheral blood cells (3). A leukocyte antigen recognized by 3H9 was recently identi®ed as an 80 kDa glycosylphosphatidyl inositol (GPI)-linked protein (4). We report here the selective reactivity of 3H9 with neutrophils among granulocytes and its maturation-associated reactivity. Peripheral granulocytes were isolated from 12 healthy volunteers. By centrifugation with Percoll of the lymphocyte-depleted granulocyte fraction, basophils, eosinophils, and neutrophils were enriched (5) and analyzed by ̄ow cytometry (6). In brief, leukocytes (1r10) were incubated with 3H9 mAb or anti-CD59 mAb, and then labeled with ̄uorescein isothiocyanate (FITC)-conjugated antimouse IgG. For two-color ̄ow cytometry, cells were incubated with 3H9, labeled with phycoerythrin (PE)-conjugated antimouse IgG, and ®nally labeled with FITC-conjugated mouse antihuman CD59 mAb. Further, 3H9 reactivity was assessed by immunostaining and subsequent May±Giemsa counterstaining (7). In brief, cells (1r10/100 ml phosphate-buffered saline, PBS) were spun down to a microslide glass, ®xed with 4% paraformaldehyde/ PBS (7), incubated with 3H9 mAb for 60 min, labeled with alkaline phosphatase-conjugated goat antimouse IgG, and visualized with a kit containing dye substrates (Zymed Laboratories). Subsequently, cells were counterstained with May±Giemsa reagent and photographed under 400r original magni®cation. Using basophil-rich fraction (more than 70%), we con®rmed that preceding immunostaining abolished May±Giemsa counterstaining of basophilic granules; however, basophils were morphologically discriminated from other leukocytes in the fraction (data not shown). Blood cells on bone marrow (BM) smear slides were also similarly stained. Because 3H9 recognizes a GPI-anchored membrane protein, paroxysmal nocturnal haemoglobinuria (PNH) granulocytes that lack a series of GPI-linked proteins were also analyzed by ̄ow cytometry. In summary, ̄ow cytometry showed selective reactivity of 3H9 with leukocyte subtypes. Most granulocytes were reactive with 3H9, whereas a small population (ranging from 3% to 10%) of granulocytes obtained from healthy volunteers showed no reactivity with 3H9 (Fig. 1, left panel). This granulocyte population was positive for another GPI-anchored membrane protein CD59 (Fig. 1, left panel), indicating that the PNH cells were excluded since PNH cells lack both GPI-linked proteins (Fig. 1, right panel) (8). Regarding 3H9nonreactive granulocytes, their population appeared to be the same as the sum population of basophilic and eosinophilic granulocytes. Indeed, indirect immunochemical staining revealed the exclusive reactivity of 3H9 with neutrophils (Fig. 2a). In contrast, neither eosinophils nor basophils were positive (Fig. 2a). To our knowlFig. 1. Two-color ̄ow cytometry of lymphocyte-depleted granulocyte fraction. Cells were isolated from a healthy representative (left panel) and a patient with PNH (right panel). An arrow indicates a population (9.9%) of granulocytes negative for 3H9 staining but positive for CD59. Eur J Haematol 2000: 64: 275±276 Printed in UK. All rights reserved Copyright # Munksgaard 2000

Elderly ATL patients in ageing society of Japan

Results More than 70% of newly diagnosed ATL patients were elderly persons aged 65 or older. Twenty-three of 66 patients at age from 41 to 61-year-old underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) in our hospital. On the other hand, 38 patients were not suitable for allo-HSCT therapy. Their ages were from 51 to 89-year-old and elderly aged 65-yearold and over occupied 73.3% of them (22 of 30, who we confirmed their outcome). Seven patients of them died before treatment or during the primary therapy. Then, we focused patients cause acute transformation from chronic type to figure out the timing of primary intervention and the efficiency of the therapy. Elevating sCD30 was observed earlier than other markers (sIL-2R, LDH) due to acute transformation and reduced by the treatment regimen changed even during treatment of relapse (recurrence).