Search

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis and thrombosis, and bone marrow failure. Affected cells harbor somatic mutation in X-linkedPIGAgene, essential for the initial step in glycosylphosphatidylinositol (GPI) biosynthesis. Loss of GPI biosynthesis results in defective cell-surface expression of GPI-anchored complement regulators CD59 and DAF. The affected stem cells generate many abnormal blood cells after clonal expansion, which occurs under bone marrow failure. Here, we report the mechanistic basis of a disease entity, autoinflammation-paroxysmal nocturnal hemoglobinuria (AIF-PNH), caused by germline mutation plus somatic loss ofPIGTon chromosome 20q. A region containing maternally imprinted genes implicated in clonal expansion in 20q-myeloproliferative syndromes was lost together with normalPIGTfrom paternal chromosome 20. Taking these findings together with a lack of bone marrow failure, the mechanisms of clonal expansion in AIF-PNH appear to differ from those in PNH. AIF-PNH is characterized by intravascular hemolysis and recurrent autoinflammation, such as urticaria, arthralgia, fever and aseptic meningitis. Consistent with PIGT’s essential role in synthesized GPI’s attachment to precursor proteins, non-protein-linked free GPIs appeared on the surface of PIGT-defective cells. PIGT-defective THP-1 cells accumulated higher levels of C3 fragments and C5b-9 complexes, and secreted more IL-1β than PIGA-defective cells after activation of the complement alternative pathway. IL-1β secretion was dependent upon C5b-9 complexes, accounting for the effectiveness of the anti-C5 drug eculizumab for both intravascular hemolysis and autoinflammation. These results suggest that free GPIs enhance complement activation and inflammasome-mediated IL-1β secretion.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a clonal population of blood cells deficient in glycosylphosphatidylinositol-anchored (GPI-anchored) proteins, resulting from a mutation in the X-linked gene PIGA. Here we report on a set of patients in whom PNH results instead from biallelic mutation of PIGT on chromosome 20. These PIGT-PNH patients have clinically typical PNH, but they have in addition prominent autoinflammatory features, including recurrent attacks of aseptic meningitis. In all these patients we find a germ-line point mutation in one PIGT allele, whereas the other PIGT allele is removed by somatic deletion of a 20q region comprising maternally imprinted genes implicated in myeloproliferative syndromes. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. From studies of patients' leukocytes and of PIGT-KO THP-1 cells we show that, through increased IL-1β secretion, activation of the lectin pathway of complement and generation of C5b-9 complexes, free GPI is the agent of autoinflammation. Eculizumab treatment abrogates not only intravascular hemolysis, but also autoinflammation. Thus, PIGT-PNH differs from PIGA-PNH both in the mechanism of clonal expansion and in clinical manifestations.

(Introduction) Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare but life-threatening stem cell disease caused by expansion of PIGA mutated clone(s). PIGA mutation abolishes the expression of GPI-anchored proteins on the cell surface including CD55 and CD59 which protect red blood cells from complement attack, resulting in complement-mediated destruction of PNH erythrocytes. Eculizumab (Ecu) effectively ameliorates the intravascular hemolysis in PNH patients by blocking complement C5 in the terminal pathway. We previously reported that 3-4 % of the Japanese patients did not respond to Ecu due to C5 polymorphism c.2654G>A (predicting p.Arg885His) and one Argentina patient with similar but different polymorphism c.2653C>T (predicting p.Arg885Cys) (N Engl J Med 370:632, 2014). Since then, we have received various consults regarding poor response cases and requests for analysis from around the world. Here, we summarize the latest series of analyses in C5 polymorphisms and propose optimal management based on these findings. (Method) Once poor response to Ecu, defined as sustained high serum LDH, was suspected with sustained high serum LDH level, peripheral blood samples with clinical data were sent to our institute after obtaining patients' informed consent. DNA was extracted from the samples, and the hot spot of C5 polymorphisms at exon 21 was sequenced by Sanger method. If no polymorphism was identified, all 41 exons of C5 were sequenced. (Results) At the initial publication (2014), 11 cases of c.2654G>A were identified out of 345 PNH patients (3.2%). As of July, 2018, a total of 22 patients were identified among roughly 600 patients treated with Ecu in Japan (3.7%). To determine the distribution of the polymorphism, a DNA panel containing 120 Han Chinese persons were previously screened, and one had the polymorphism. The same C5 polymorphism was newly identified in one among 89 patients treated with Ecu in Korea. A similar polymorphism c.2653C>T (predicting p.Arg885Cys) was also previously identified in an Argentina patient. Another polymorphism c.2653C>A (predicting p.Arg885Ser) was identified in a Dutch patient. Furthermore, a novel mutation c.2422 G>A (predicting p.Val 808 Ile) was found in a poor responder in Israel, and currently under functional analyses. (Discussion) In PNH patients treated with Ecu, serum LDH level usually drops importantly after the first loading dose, and mostly comes to upper limit of normal range after second or third loading dose. If the serum LDH level remains high, poor response should be considered. CH50 is usually not detected in Ecu responsive patients, so monitoring of CH50 level is critical to evaluate the responsiveness to Ecu. Once poor response is suspected, hot spot of exon 21 should initially be sequenced, and then the whole 41 exons of C5 may need to be sequenced. c.2654G>A was found among Han Chinese in our previous study, so it is reasonable that a Korean case had the same polymorphism considering the geography. Surprisingly, a Caucasian case with the same polymorphism has been reported (Blood Advances 1:1254, 2017) in addition to the Dutch case, underlining the importance of poor responsiveness due to polymorphisms (p.Arg885) even outside of Asia. We previously reported that Coversin, a C5 inhibitor derived from tick saliva protein, blocked hemolysis in vitro using the serum from the patients with c.2654G>A, and it was reported that post-transplant thrombotic microangiopathy was successfully treated with Coversin for the patient with the polymorphism (Blood Advances 1:1254, 2017). C5 inhibitors targeting a different epitope or having different mechanisms from Ecu as well as other upcoming complement inhibitors targeting Factor D or C3 are expected to benefit patients with C5 polymorphism and resistance to Ecu. Further analyses and clinical trials may pave the way to a second generation anti-complement drug to treat PNH patients. Figure. Figure. Disclosures Ueda: Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Muus:Akari Therapeutics: Consultancy. Lee:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Nishimura:Chugai Pharmaceuticals: Consultancy, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired GPI-anchor deficiency caused by the somatic mutation of PIGA gene in a hematopoietic stem cell (PIGA-PNH). Loss of function of PIGA causes loss of GPI anchored proteins including complement regulatory proteins. Main symptoms are hemolytic anemia and venous thrombosis caused by the uncontrolled complement activation, and bone marrow failure. For X-linked PIGA, one hit of somatic mutation causes GPI-deficiency. All other genes involved in GPI-anchor biosynthetic pathway are autosomal and hits to two alleles are required to cause GPI-deficiency. In 2013, a PNH patient caused by the PIGT mutations was reported. This patient had a splice site mutation in one germ line allele and a somatic 8Mb deletion including entire PIGT. Recently, we found a Japanese patient who has a nonsense mutation in a germ line allele and a somatic 18Mb deletion in chromosome 20q including PIGT. Here we compare two patients and report that these cases of PNH are atypical in two points. One is that PIGT-PNH cases had unusual symptoms similar to auto inflammatory syndrome. Both had long lasting chronic urticaria and joint pain. Additionally, the Japanese case had recurrent aseptic meningitis and the German patient had ulcerative colitis. It should be noted that these symptoms had lasted over ten years before hemolytic attacks occurred and diagnosis as PNH was made. Urticaria, joint pain and recurrent meningitis were controllable by eculizumab, anti-C5 antibody, showing involvement of complement activation. These inflammatory symptoms are specific to PIGT-PNH and are not seen in PIGA-PNH. PIGT is involved in transfer of GPI to proteins and its defect causes accumulation of free GPI, non-protein linked GPI. In fact, FACS analysis using the antibody which recognizes free GPI revealed that GPI negative granulocytes, monocytes, and B cells from the PIGT-PNH patient, but not from PIGA-PNH patients, highly expressed free GPI. We speculate that this free GPI together with complement activation cause inflammasome activation and are establishing assay system. The other atypical nature is a mechanism of clonal expansion. In typical PNH, autoimmunity against hematopoietic stem cells causing bone marrow failure is thought to positively select GPI-deficient cells. In contrast, the somatically deleted 18Mb included a region commonly deleted in some patients with myeloproliferative noeplasms. This region contains several maternally imprinted genes, defective expression of which is likely causally related to clonal expansion in atypical PNH cases. Thus, the symptoms and mechanism of clonal expansion of PIGT-PNH are quite different from those of PIGA-PNH, so that PIGT-PNH should be categorized as an atypical PNH. Screening of PIGT-PNH among the PNH patients by detecting free GPI using FACS analysis should be feasible. Disclosures Ueda: Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharma: Honoraria, Research Funding. Kanakura:Bristol Myers: Research Funding; Toyama Chemical: Research Funding; Kyowa Hakko Kirin: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Fujimotoseiyaku: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Alexionpharma: Research Funding; Astellas: Research Funding. Kinoshita:Alexion Pharma: Honoraria.

Background: Paroxysmal Nocturnal Hemoglobinurea (PNH) is a rare stem cell disease caused by the expansion of PIGA mutated clone(s). PNH-type cells are deficient in the expression of GPI-anchored proteins including DAF and CD59, which protect red blood cells (RBC) from complement-mediated intravascular hemolysis. Eculizumab (Soliris®, Alexion Pharmaceuticals) is a humanized monoclonal antibody against C5 which efficiently inhibits hemolysis by blocking the terminal complement cascade. Eculizumab dramatically ameliorates several clinical symptoms, and improves the prognosis in PNH patients. However, among 345 Japanese PNH patients who were treated with eculizumab, 11 patients showed poor response. All the poor responders had a single missense C5 heterozygous mutation, c.2654G>A, which predicts the polymorphism p.Arg885His (Nishimura et al, NEJM. 2014 13;370(7):632-9). Two of those patients have already passed away due to severe complications related to PNH, and the rest of them are still suffered from various clinical symptoms including hemolytic episodes and RBC transfusion. In these circumstances, multiple new anti-complement drugs are under development in Japan. Coversin (Volution Immuno Pharmaceuticals) is a recombinant protein (16,740 Da) derived from a secreted protein in the saliva of the Ornithodoros moubata tick, and it blocks complement-mediated hemolysis at C5 level. In this study, we examined this new anti-complement agent to a PNH patient with C5 polymorphism c.2654G>A, as well as those without the polymorphism. Materials: Peripheral blood samples were collected from a poor responder to eculizumab and hemolytic PNH patients with written informed consent as approved by the Institutional Review Board of Osaka University Hospital. In vitro hemolytic assay: RBC from ABO-matched PNH patients off eculizumab treatment were washed 3 times in saline, and subsequently incubated with Mg2+ supplemented serum of the poor responder in the presence or absence of an anti-complement agent. Alternative pathway was activated by adding HCl (22:1 of 0.4M HCl) to the serum. Heat-inactivated (56°C for 30min) serum was used as a negative control. After a 24-hour incubation at 37°C, hemolysis was quantified by measuring the optical density at 405nm (OD405). The hemolytic activity was normalized against maximum hemolysis as induced by HCl (100%) and minimum hemolysis with inactivated acidified serum (0%). Results: A 41-year-old male with fatigue was diagnosed as aplastic anemia with PNH in 2008, and cyclosporine (CyA) was initiated at the dose of 150mg/day. The PNH clone expanded from 30.6% to 70.2% in granulocytes from 2008 to 2011 with elevated LDH (700 U/L) and the patient was referred to our hospital to undergo eculizumab treatment. CyA was reduced to 100mg/day and eculizumab was initiated in May 2012. Eculizumab treatment did not change the serum LDH level without any improvement of the symptoms: fatigue, abdominal pain, and periodical hemoglobinurea. A heterozygous mutation c.2654G>A was identified as the cause of the failure to eculizumab treatment, and he is still suffered from continuous intravascular hemolysis (LDH > 1400 U/L) with periodical acute hemolytic episodes, requiring frequent RBC transfusion. In the hemolytic assay, Coversin completely blocked hemolysis at the concentration of 10ug/ml, similar to the effective inhibition with hemolytic PNH patients without the polymorphism. Discussion: Eculizumab has dramatically improved the quality-of-life in the majority of the PNH patients by blocking intravascular hemolysis, but there are still some concerns; poor response due to C5 polymorphism, C3b deposition on the RBC, high cost and burden for scheduled infusion. Blocking the complement cascade at C5 level has shown to be relatively safe if meningococcal vaccination is properly performed, but still an extravascular hemolysis remains problematic at least in some cases. Inhibiting C3 amplification would resolve both intra and extravascular hemolysis, but susceptibility to infections remains a major concern. Our study showed that Coversin efficiently blocked in vitro hemolysis in the eculizumab resistant patient with C5 heterozygous mutation, c.2654G>A. Coversin might be a therapeutic option for the population of C5 polymorphism c.2654G>A in PNH patients. Our results warrant further investigation to explore new anti-complement agents for hemolytic PNH patients. Disclosures Ueda: Alexion Pharma: Research Funding. Osato:Alexion Pharma: Research Funding. Weston-Davies:Volution Immuno Pharmaceuticals (UK) Ltd: Employment, Equity Ownership. Nunn:Volution Immuno Pharmaceuticals: Employment, Equity Ownership. Hayashi:Alexion Pharma: Research Funding. Nishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Background Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder, characterized by the complement-mediated intravascular hemolysis. Eculizumab is a humanized monoclonal antibody that binds to the human C5 protein, thereby inhibiting terminal complement-mediated hemolysis. PNH patients treated with eculizumab responded with a significant reduction in hemolysis and improved in quality of life (QOL). Recent report shows that PNH-type cells were under the oxidative stress due to elevated levels of intracellular reactive oxygen species (ROS) and/or decreased antioxidant status. Fermented papaya preparation (FPP), a dietary nutriceutical derived from carica papaya, has been shown to modulate oxidative stress damage parameters in vitro and in vivo. To clarify the pathogenetic link between oxidative stress and hemolysis in PNH, the effects of each inhibitor, eculizumab and FPP, in Japanese patients with PNH were investigated independently. Methods Peripheral blood and sera were obtained from patients with PNH and healthy individuals after the obtaining informed consent. The study protocol was approved by the IRB at Osaka University Graduate School of Medicine. For ROS assay, RBCs were measured using 2’-7’-dichlorofluorescin-diacetate. The oxidative stress index (OSI) was derived from the percent ratio of the Reactive Oxygen Metabolites-derived compounds test (Diacron) and the Biological Antioxidant Potential test (Diacron) using a JCA-BM1650 analyzer (JEOL). LDH, biomarker of hemolysis, was measured by routine tests. Eculizumab was administrated according to standard schedule. FPP was taken orally 18g/day for 3 month. Health-related fatigue and QOL were also assessed using the FACIT-fatigue (Version 4A) and EORTC QLQ-C30 (Version 3) instruments. Results PNH patients without eculizumab treatment (pts w/o ecu) showed significantly higher levels in ROS compered to healthy individuals ([mean±SD] 1.13±0.18 vs. 0.84±0.10, p=0.004, n=8). Among these patients, CD59-negative RBCs showed a significant increase in ROS compared to GPI-positive RBCs in corresponding patients (1.23±0.26 vs. 1.13±0.27, p=0.03, n=6). Similarly, the OSI level was significantly higher in pts w/o ecu than healthy individuals (1.32±0.37 vs. 0.97±0.10, p=0.022, n=9) and oxidation activity in sera was significantly higher in pts w/o ecu than healthy volunteers (361.6±91.5 vs. 285.0±31.8, p=0.039, n=9), while antioxidant activity in sera was not significantly different between these 2 groups ([2440.6±191.5 vs. 2601.5±186.6, p=0.09, n=9). Interestingly, there was no significant difference in OSI level between PNH patients on eculizumab (n=8) and healthy individuals (0.84±0.10 vs. 1.07±0.45, p=0.55). Two patients who newly started eculizumab treatment showed improvement in LDH along with ROS generation ([LDH(IU/l)] Pt.I:6082-906, Pt.II:618-211; [ROS] Pt.I:1.57-1.06, Pt.II:1.50-1.40). FPP treatment (n=2) showed little effect on LDH, while it decreased ROS (Pt.I:1.72-1.47, Pt.II:1.16-1.04) and improved the scores in FACIT-fatigue and EORTC QLQ-C30. In addition, those 2 patients showed increase in ROS after discontinuation of FPP (Pt.I:1.47-2.04, Pt.II:1.04-1.19). Conclusion The RBCs and sera derived from Japanese patients with PNH were highly susceptible to oxidative stress compared to healthy individuals. Eculizumab was effective in controlling the oxidative stress, in addition to the hemolysis, suggesting that elevated oxidative stress in PNH was mainly due to the complement-mediated hemolysis. Since FPP showed little effect on hemolysis but had a potential to ameliorate the oxidative stress and improved QOL, FPP could alleviate symptoms associated with oxidative stress and may contribute to the therapeutic option for supportive therapy in PNH. Disclosures Osato: Alexion Pharma: Research Funding. Nishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hayashi:Alexion Pharma: Research Funding. Ueda:Alexion Pharma: Research Funding. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.