Tsutomu Shichishima

Clinical Course and Flow Cytometric Analysis of Paroxysmal Nocturnal Hemoglobinuria in the United States and Japan

Abstract: To determine and directly compare the clinical course of white and Asian patients with paroxysmal nocturnal hemoglobinuria (PNH), data were collected for epidemiologic analysis on 176 patients from Duke University and 209 patients from Japan. White patients were younger with significantly more classical symptoms of PNH including thrombosis, hemoglobinuria, and infection, while Asian patients were older with more marrow aplasia. The mean fraction of CD59-negative polymorphonuclear cells (PMN) at initial analysis was higher among Duke patients than Japanese patients. In both cohorts, however, a larger PNH clone was associated with classical PNH symptoms, while a smaller PNH clone was associated with marrow aplasia. Thrombosis was significantly more prevalent in white patients than Asian patients, and was associated with a significantly higher proportion of CD59-negative PMN. For individual patients, CD59-negative populations varied considerably over time, but a decreasing PNH clone portended hematopoietic failure. Survival analysis revealed a similar death rate in each group, although causes of death were different and significantly more Duke patients died from thrombosis. Japanese patients had a longer mean survival time (32.1 yr vs. 19.4 yr), although Kaplan-Meier survival curves were not significantly different. Poor survival in both groups was associated with age over 50 years, severe leukopenia/neutropenia at diagnosis, and severe infection as a complication; additionally, thrombosis at diagnosis or follow-up for Duke patients and renal failure for Japanese patients were poor prognostic factors. These data identify important differences between white and Asian patients with PNH. Identification of prognostic factors will help the design of prospective clinical trials for PNH. Abbreviations: BMT = bone marrow transplantation, GPI = glycosylphosphatidylinositol, GPI-AP = glycosylphosphatidylinositol-anchored protein, Hb = hemoglobin, PMN = polymorphonuclear cells, PNH = paroxysmal nocturnal hemoglobinuria, RBC = red blood cells, WBC = white blood cells

Genetic variants in C5 and poor response to eculizumab.

BACKGROUND Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear. METHODS We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients. RESULTS Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G → A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C → T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab. CONCLUSIONS The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).

Diagnosis of paroxysmal nocturnal haemoglobinuria by phenotypLc analysis of erythrocytes using two-colour flow cytometry with monoclonal antibodies to DAF and CD59/MACIF

Summary We investigated the relationship between the complement lysis sensitivity test and two‐colour flow cyto‐metric analysis using monoclonal antibodies to decay accelerating factor (DAF) and CD59/membrane attack complex inhibitory factor (MACIF) in patients with paroxysmal nocturnal haemoglobinuria (PNH) and other haematological diseases.

Collection of MNCs and progenitor cells by two separators for PBPC transplantation: a randomized crossover trial.

BACKGROUND: Efficient collection of progenitor cells is essential for PBPC transplantation. Two apheresis machines (Amicus, Baxter Healthcare; and Spectra, Gambro BCT, software version 4.7) were compared prospectively by a crossover trial.

Heterogenous expression of decay accelerating factor and CD59/membrane attack complex inhibition factor on paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes.

Summary In order to clarify the characterization of pheno‐types of paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes (E), we analysed the expression of decay accelerating factor (DAF) and CD59/membrane attack complex inhibition factor (MACIF) on the membrane surface of PNH‐E by means of the flow cytometric method using anti‐DAF and/or CD59/MACIF monoclonal antibodies in nine PNH‐patients. In two‐colour analysis, this expression on PNH‐E was classified into three fractions; negative, intermediate and positive according to intensity. The negative fraction was classified into two groups; one group an exclusively negative population, and the other a negative population having slightly DAF‐positive E. The intermediate fraction was recognized on PNH‐E of cases with PNH II‐E and extremely heterogenous. In the positive fraction, this expression was almost the same as on normal E except for case 8. In single‐colour analysis for DAF or CD59/MACIF, three fractions were classified as well as the definition in two‐colour analysis. In single‐colour analysis, the expression on PNH‐E was also heterogenous in each fraction and among PNH‐patients. However, the intermediate fraction for CD59/MACIF was not found on PNH‐E of cases without PNH II‐E, although intermediate fraction for DAF was recognized on PNH‐E of some cases with PNH III‐E in addition to those with PNH II‐E.

Paroxysmal nocturnal haemoglobinuria with coexisting deficiency of the ninth component of complement: lack of massive haemolytic attack

A 47‐year‐old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement‐sensitivity lysis tests, 80% of her erythrocytes were markedly complement‐sensitive (PNH‐III). Laser cytofluorimetry with a monoclonal antibody against decay‐accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF‐negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusions of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patients erythrocytes. When the patients serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.

Complement sensitivity of erythrocytes in a patient with inherited complete deficiency of CD59 or with the Inab phenotype

We investigated the complement sensitivity of erythrocytes from three patients, one with inherited complete deficiency of CD59, one with the Inab phenotype, and one with paroxysmal nocturnal haemoglobinuria (PNH). The complement lysis sensitivity units on the erythrocytes were 11.7, 4.6, and 47.6 for inherited CD59 deficiency, Inab phenotype, and PNH, respectively. Two‐colour flow cytometric analysis showed that the erythrocytes from the three patients consisted of a single population negative for CD59, negative for decay accelerating factor (DAF), and negative for both proteins, respectively. In addition, only the Inab phenotype patient had no haemolysis in vivo. These facts suggest that CD59 deficiency plays a more important role than DAF deficiency in complement‐mediated haemolysis in vitro and in vivo, and that deficiency of both proteins, but not CD59 or DAF alone, causes complement sensitivity corresponding to that of PNH III erythrocytes in vitro.

Deregulated expression of HMGA2 is implicated in clonal expansion of PIGA deficient cells in paroxysmal nocturnal haemoglobinuria.

Patients with paroxysmal nocturnal haemoglobinuria (PNH) have expanded clonal cells bearing a somatic mutation in the PIGA gene. Our previous study on two PNH patients with chromosome 12 rearrangements demonstrated the involvement of HMGA2 expression in clonal expansion. The present study investigated HMGA2 expression in PNH patients without chromosomal abnormalities. The expression of short HMGA2 with latent exon was significantly high in peripheral blood cells from 18 of 24 patients. Over‐expression of truncated HMGA2 in mouse bone marrow cells caused expansion in recipient mice. These results support the idea that deregulated expression of HMGA2 causes expansion of PNH cells.

High frequency of several PIG-A mutations in patients with aplastic anemia and myelodysplastic syndrome

To clarify some characteristics of phosphatidylinositol glycan-class A gene (PIG-A) mutations in aplastic anemia (AA) and myelodysplastic syndrome (MDS) patients compared with those in paroxysmal nocturnal hemoglobinuria (PNH) patients, we investigated PIG-A mutations in CD59− granulocytes and CD48− monocytes from seven AA, eight MDS, and 11 PNH Japanese patients. The most frequent base or type abnormalities of the PIG-A gene in AA and MDS patients were base substitutions or missense mutations, respectively, and deletions or frameshift mutations, respectively, in PNH patients. Several PIG-A mutations, most of which were statistically minor, were found in glycosylphosphatidylinositol-negative cells from all AA and MDS patients but not from all PNH patients. However, the common PIG-A mutations during the clinical course between CD59− granulocytes and/or CD48− monocytes from each AA or MDS patient, except for Case 5, were not found. PIG-A mutations were different between the granulocytes and monocytes from five AA and five MDS patients. Our results indicate that there were some characteristics of PIG-A mutations in AA and MDS patients compared with PNH patients and that several minor PNH clones in these patients occurred at random during the clinical course. This partly explains the transformation of AA or MDS to PNH at intervals.

Bernard-Soulier syndrome with a homozygous 13 base pair deletion in the signal peptide-coding region of the platelet glycoprotein Ibβ gene

We report a family with Bernard–Soulier syndrome with a homozygous mutation within the GPIbβ gene. The proband was a 24-year-old Japanese male who has suffered from life-long bleeding tendency. The patients sister also had severe bleeding episodes. The proband and the affected sister had no apparent complications including organic or skeletal anomaly, or mental disturbance. They had thrombocytopenia [(35–40) × 109/l] with giant platelets. In addition to platelet size, electron microscopic analysis revealed abnormalities in the internal structures of platelets. Ristocetin-induced platelet aggregation was defective. Flow cytometric analysis and western blot analysis showed that glycoprotein IX was nearly absent in platelets, whereas GPIbα and GPV were detectable. Genetic studies revealed a 13 base pair deletion in the signal peptide-coding sequence of GPIbβ. The deletion would cause a frame-shift, resulting in the appearance of a stop codon following an indifferent polypeptide sequence. Analysis of platelet RNA showed that the mutant GPIbβ gene was transcribed. The propositus and his affected sister were homozygous for the deletion, whereas their unaffected father and mother were heterozygotes. The molecular defects of this family would help understand the relevance of GPIbβ for complex formation of the glycoprotein Ib/IX/V receptor.