ALBERT

Description: Key Points The mechanism of bone marrow failure (BMF) in PNH is not known. Novel CD1d-restricted, GPI-specific T cells are present in PNH patients and might be responsible for BMF.

Description: CD157, a glycosylphosphatidylinositol (GPI)–anchored protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, is expressed on the surface of most human circulating neutrophils. This work demonstrates that CD157 is a receptor that induces reorganization of the cytoskeleton and significant changes in cell shape, and that signals mediated by CD157 act through modulation of cytosolic Ca2+ concentration. These signals are independent of the products of CD157's enzymatic activities (ie, cyclic adenosine diphosphate [ADP]–ribose and ADP-ribose). Indeed, the enzymatic activities of CD157 in circulating neutrophils as well as in dimethyl sulfoxide (DMSO)–differentiated (CD157+/CD38-) HL-60 cells, are hardly detectable. This work also shows that the receptorial activity relies on cross-talk between CD157 and β2 integrin. CD157 localizes in GM1-enriched lipid rafts and, upon activation, it migrates to the uropod, a structure specialized in motility and adhesive functions. Indeed, CD157 is involved in adhesion to extracellular matrix proteins and in chemotaxis induced in vitro by formyl-methionyl-leucyl-phenylalanine (fMLP). These findings were consistent with the results obtained in neutrophils from patients with paroxysmal nocturnal hemoglobinuria (PNH), in which CD157 is deficient. These neutrophils showed constant defects in adhesion and migration. Our data attribute specific and crucial roles to CD157 in the regulation of innate immunity during inflammation.

Description: PNH is an acquired clonal disorder of the hematopoietic stem cell (HSC) characterized by intravascular hemolysis, venous thrombosis, and variable degrees of bone marrow failure. In PNH a somatic mutation of the X-linked PIG-A gene in HSC results in complete or partial deficiency of all proteins anchored by the glycosylphosphatidylinositol (GPI) on the membrane of the mutated HSC and in its mature progeny. The close association between PNH and Idiopathic Aplastic Anemia (IAA), and other lines of evidence support the hypothesis that auto-reactive T cells might be responsible for the expansion of hematopoietic PNH clone(s), which is required to cause clinical PNH. Stemming from our observation of a unique patient with PNH and with a large granular lymphocyte (LGL) leukemia with NKT phenotype (Karadimitris et al, Br J Haematol115:1010, 2001), we have measured systematically the percentage of NKT [CD3+ CD8+(bright) CD57+] cells in the peripheral blood of PNH patients. The proportion of NKT cells was quite variable and very similar in 18 patients (6.9±5.9; range: 0.8 – 22.3%) and in 18 healthy individuals (6.5±5.2; range: 0.9 – 21.2; P〉0.5). However, when we analyzed the size distribution of the complementarity-determining region 3 (CDR3) of the TCR-beta chain genes in sorted NKT cells, there was a sharp difference. In healthy individuals we observed a normally distributed ladder of bands of different sizes. By contrast, in 14 out of 15 PNH patients we found a markedly non-random (“oligoclonal”) pattern; and in each patient some clones were predominant. In 6 out of 6 patients followed-up longitudinally over 6–12 months the “oligoclonal” pattern was consistent and persistent. In each of 10 patients in whom we carried out systematic sequencing of the TCR-beta CDR3 of sorted NKT cells we have observed an average of 25 different TCR-beta CDR3 sequences (out of an average of 80 total sequences obtained): but only one or two sequences were predominant. Interestingly, an identical or quasi-identical (single amino acid difference) sequence was found in 4 patients; and in two of these the sequence belonged to one of the predominant clones. In addition, in 5 cases a sequence found in one patient was subsequently found also in another patient. These data are reminiscent of recent findings reported in patients with IAA (Risitano et al, Lancet364:355, 2004): in these patients, however, no identity of sequence was detected. We surmise that in both groups of patients specific T cells clones may be responsible for damage to normal HSCs. However, it is possible that in IAA a number of different antigens are recognized on HSCs in individual patients; whereas in PNH the range of potential target antigens is much more restricted, because they must be present on normal HSC but not on PNH HSCs, thus enabling them to survive the auto immune attack and to expand.

Description: Background: PNH is a rare, acquired blood disorder. A somatic mutation in the PIGA gene of one or more hematopoietic stem cells generates a clone of abnormal erythrocytes (RBCs) that lack alternative pathway (AP) regulatory complement proteins CD55 and CD59. This leads to uncontrolled complement activation on affected RBCs and intravascular hemolysis (IVH). Standard of care is C5 inhibition to prevent membrane attack complex (MAC) formation. Despite preventing MAC-mediated IVH, many patients experience extravascular hemolysis (EVH). Despite C5 inhibition, ~ 70% of patients remain anemic and 〉1/3 were transfused 〉 1 time in the prior 12 months. Factor D (FD), a serine protease, catalyzes complement factor B cleavage, allowing formation of AP C3 convertase. By inhibiting FD, oral danicopan blocks C3 convertase formation, the control point for AP activation and amplification of all pathways. This leads to inhibition of C3 cleavage, C3 fragment deposition, terminal pathway activation and MAC formation. Thus, danicopan can control both IVH and EVH therefore, making FD a promising target. Aim: Demonstrate that danicopan is a potential treatment for PNH patients with an inadequate response to C5 inhibition. Methods: Data are presented for this Ph 2, dose-finding, proof of concept trial of danicopan in patients with an inadequate response to eculizumab who were transfusion dependent. Additional criteria in Table 1. In addition to the current eculizumab regimen, danicopan starting doses were 100-150 mg TID, with dose escalation up to 200 mg TID, based on clinical and biochemical response, at protocol defined time points (Figure 1). The primary endpoint was change in Hgb at Treatment Week (TW) 24. Secondary efficacy parameters included transfusion needs, effect on lactate dehydrogenase (LDH), and an exploratory an endpoint of Functional Assessment of Chronic Illness Therapy (FACIT) FATIGUE scores. General safety, tolerability, and PK/PD of danicopan were measured. After TW 24, patients entered a long-term extension. Results: Twelve patients received at least one dose of danicopan. One patient discontinued after 2 doses, due to a serious adverse event of worsening of an underlying condition (pulmonary hypertension/edema), considered unlikely related to danicopan. This patient is excluded from this analysis. Eleven patients continue to receive treatment. Dose titrations are shown in Table 2. Benefits were observed in multiple laboratory markers of PNH, shown in Table 3. Hgb improved in all patients, with a mean Hgb gain of 2.6 g/dL at 24 Weeks of treatment (n=4). Meaningful improvement in FACIT Fatigue scores were reported, with a mean increase of 8 points relative to the baseline on eculizumab. A 3-point change is clinically meaningful on this scale. Transfusion needs dramatically reduced, with one patient receiving one transfusion during the trial, as compared to 34 transfusions (58 units) in 10 patients, in the 6 months prior to screening. One patient (who does not accept blood products due to religious objections and also has hereditary elliptocytosis) had a baseline Hgb=5 g/dL with a 3.5 g/dL improvement at TW24 and significant improvement in fatigue. C3 fragment deposition was inhibited; reticulocytes, total/ direct bilirubin and LDH returned to normal range. Baseline C5 inhibition (classical pathway activity) was essentially inhibited (mean=1 [range 60-144]). Three of 11 patients received 〉 900 mg eculizumab, making PK a less likely reason for inadequate response. Danicopan has been well tolerated; 96% of treatment emergent adverse events (TEAEs) were mild to moderate in severity and no discontinuations due to TEAEs. Two patients had Grade 3 TEAEs which resolved and continued in the trial. Conclusion: Proof of concept is established with danicopan, an oral, small molecule FD inhibitor in the treatment of PNH in combination with eculizumab. Meaningful improvements in Hgb, transfusion needs, FACIT-FATIGUE and other parameters of interest were achieved. These clinically significant improvements demonstrate that further benefit can be achieved by blocking the alternative pathway at FD with danicopan, in combination with C5 inhibition. This benefit is likely due to the prevention of C3-mediated EVH, in addition to control of IVH. Danicopan targets an unmet need in PNH and will be further evaluated in a pivotal trial in combination with standard of care C5 inhibition. Disclosures Kulasekararaj: Achillion: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Akari: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Membership on an entity's Board of Directors or advisory committees. Risitano:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; Achillion: Research Funding; Amyndas: Consultancy; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Alexion: Honoraria, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Research Funding; Achillion: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ra Pharma: Research Funding; Biocryst: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Samsung: Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Notaro:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: letture fees. Browett:Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Beigene: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding. Lee:Alexion: Consultancy, Honoraria, Research Funding; Achillion: Research Funding. Huang:Achillion: Employment, Equity Ownership. Geffner:Achillion: Employment, Equity Ownership. Brodsky:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding; Achillion: Research Funding.

Description: Idiopathic aplastic anemia (IAA) is an acquired bone marrow disease probably caused by an auto-immune attack against hematopoietic stem cells (HSCs), which leads to bone marrow failure. Many abnormalities have been observed in the T cell compartment, but the putative auto-antigen(s) remain elusive. A large body of evidence links paroxysmal nocturnal hemoglobinuria (PNH) to IAA, supporting the notion that autoimmunity is a key pathogenic mechanism in both diseases. In PNH, auto-reactive T cells may be the 'noxious agent' capable of killing GPI positive HSCs while sparing GPI negative HSCs. Recently, CD1d restricted, GPI-specific T-cells have been demonstrated in PNH patients. Here, we investigate whether CD1d restricted, GPI-specific T cells are also present in IAA patients. When peripheral blood mononuclear cells (PBMNCs) from 14 newly diagnosed IAA patients [12 of whom had a small percentage (between 0.003% and 5%) of GPI-negative granulocytes] were co-cultured with antigen presenting cells (APCs) expressing CD1d and competent for the synthesis of GPI, we detected GPI specific T cells (CD8+CD1d/GPI dimer+ T cells) in 10 out of 14 patients (71%) at a significantly higher abundance than in co-culture experiments performed with PBMNCs from healthy controls (Fig. 1). In fact, the frequency of CD8+CD1d/GPI dimer+ T cells was below the cutoff value of 0.35% in all the 15 healthy controls but only in 4 out 14 IAA patients (Fisher test, P

Description: PNH is a rare acquired clonal disorder of the hematopoietic stem cell, characterized by a somatic mutation that inactivates the X-linked PIGA gene: this in turn results in deficiency on the cell surface of all proteins anchored by the glycosylphosphatidylinositol (GPI) molecule. Two of these proteins,CD55 andCD59, are complement regulators and their deficiency is responsible for the susceptibility of red cells (RBCs) from the mutant clone to lysis by activated complement. Since oxidative damage is another well-known mechanism of hemolysis (as in G6PD deficient red cells), we have investigated whether this plays a role also in PNH. To this end, we have carried out experiments on RBCs from healthy donors and on PNH-like RBCs (obtained in vitro from the same donors through the use of anti-CD55 and anti-CD59 blocking moAb). After exposure to AB0-compatible serum (in which thecomplement alternative pathway was activated by mild acidification) all PNH-like (but not normal) RBCs were lysed. In parallel experiments in which complement was blocked by eculizumab (ECU) - a moAb that binds to the complement component C5 and controls intravascular hemolysis in PNH patients - we measured the levels of reactive oxygen species (ROS) by the dichlorofluorescin diacetate assay. We found no significant difference of ROS levels between normal RBCs and PNH-like RBCs. We next tested in a similar way G6PD-deficient RBCs, because these are known to be exquisitely sensitive to oxidative damage. We found that ROS levels were significantly higher in the G6PD deficient RBCs that have been made PNH-like (Fig. 1). Thus, complement activation on the surface of PNH-like RBCs results in the production of ROS that can be demonstrated when C5-blockade prevents complement-mediated lysis of RBCs. The notion that G6PD deficiency can interact with PNH was strongly corroborated by the clinical observation of a 40yo woman from Sardinia (Italy) with a 2 years history of pancytopenia, who then developed florid hemolytic PNH: she had anemia with normal granulocyte and platelet counts, dark urine, high reticulocytosis, LDH up to 5x upper normal level, 95% GPI-negative granulocytes. When the patient was started on ECU. LDH levels promptly returned to normal, PNH RBCs rose from 20% (before ECU) to 42%, but reticulocyte count (~250x109/L) and blood transfusion requirement remained high (10 units in the last year). 39% of the GPI-negative RBCs had bound C3 fragments The peripheral blood smear revealed marked macro-anisocytosis, poikilocytosis, spherocytes, and hemighosts: a picture consistent with oxidative damage as seen in G6PD deficient patients during a hemolytic attack. The RBC G6PD activity was about one-half of normal (5 IU/g Hb), and DNA analysis revealed heterozygosity for the G6PD Mediterranean (Med) mutation. By mRNA sequence analysis we found that the GPI-negative clone expressed only the G6PD Med allele, suggesting that the PIG-A mutation took place in a stem cell in which the normal G6PD gene was on the inactive X-chromosome (G6PD, like PIG-A, is on the X chromosome); therefore, all the patients' PNH RBCs were also all G6PD deficient. We have previously shown that the clinical expression of PNH can be influenced by inherited factors: specifically, a polymorphism of the complement receptor 1 (CR1) gene correlates with the blood transfusion requirement of patients on ECU (Rondelli et al, Haematologica 2014). However, the patient here reported was homozygous for the more favorable allele ofCR1. Instead, in keeping with our experimental data, the poor response to ECU seen in this patient results probably from a unique interaction, within the same population of RBCs, between the acquired PNH abnormality and her inherited G6PD deficiency This type of interaction is novel and it seems to have pharmacogenetic implications. Indeed, on its own G6PD deficiency affects mildly the clinical expression of PNH, because complement activation causes RBC lysis regardless; however, paradoxically, when the lysis of PNH RBCs is prevented by C5 blockade, complement activation results in oxidative damage, with which PNH G6PD deficient RBCs are unable to cope. Except for one case previously reported by Oni et al (Blood 1970), this is the first detailed study of PNH associated with G6PD deficiency. Since in some parts of the world the frequency of G6PD deficiency can be as high as 30% or more, we expect that more cases of this association will be discovered in the future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: In paroxysmal nocturnal hemoglobinuria (PNH) hemolytic anemia is due mainly to deficiency of the complement regulator CD59 on the surface of red blood cells (RBCs). Eculizumab, an antibody that targets complement fraction 5 (C5), has proven highly effective in abolishing complement-mediated intravascular hemolysis in PNH; however, the hematologic benefit varies considerably among patients. In the aim to understand the basis for this variable response, we have investigated by flow cytometry the binding of complement fraction 3 (C3) on RBCs from PNH patients before and during eculizumab treatment. There was no evidence of C3 on RBCs of untreated PNH patients; by contrast, in all patients on eculizumab (n = 41) a substantial fraction of RBCs had C3 bound on their surface, and this was entirely restricted to RBCs with the PNH phenotype (CD59−). The proportion of C3+ RBCs correlated significantly with the reticulocyte count and with the hematologic response to eculizumab. In 3 patients in whom 51Cr labeling of RBCs was carried out while on eculizumab, we have demonstrated reduced RBC half-life in vivo, with excess 51Cr uptake in spleen and in liver. Binding of C3 by PNH RBCs may constitute an additional disease mechanism in PNH, strongly enhanced by eculizumab treatment and producing a variable degree of extravascular hemolysis.

Description: Abstract 4239 Paroxysmal Nocturnal Hemoglobinuria (PNH) is associated with clonal expansion of stem cells with an acquired somatic PIG-A mutation. The PIG-A gene is essential for the biosynthesis of glycosylphosphatidylinositol (GPI), and mature blood cells derived from the PNH stem cell clone exhibit a loss of all proteins that require this structure for attachment to the cell surface, notably the complement inhibitors CD55 and CD59. The loss of these proteins on the surface of red cells is responsible for hemolysis, and thrombosis may be the consequence of the loss of these proteins from the surface of platelets: both hemolysis and thrombosis can be attenuated by anti-complement therapy. Thrombosis can occur, however, despite anticomplement therapy and despite anticoagulation and has been historically the most important determinant of death in patients with PNH. Some patients will present with thrombosis and some patients will not be candidates for anticoagulation or anticomplement therapy: therefore treatment of thrombosis remains an important part of the management of PNH patients. Thrombolysis with tissue plasminogen activator (tPA) in PNH has been reported in small series or case reports, generally with encouraging outcomes. Here we report what we believe to be the largest series on the outcome of the use of tPA. Of 38 patients with PNH who had at least one thrombotic event, 13 were thought to have had a thrombus sufficiently recent to be amenable to fibrinolysis; of these, 4 patients were regarded as ineligible on account of active hemorrhage or high risk of hemorrhage. Of the 9 eligible patients who received tPA, all of whom had potentially life-threatening thromboses, 3 also required tPA on subsequent hospitalizations, and the results of a total of 15 hospitalizations during which tPA infusions were given are reported here. tPA was given in the ICU by systemic infusion through a peripheral vein at a dose of 1 mg/kg delivered over 24 hours, with anticoagulants withheld temporarily during this time. Response was monitored by follow-up imaging, and most patients required several 24 hour infusions. Platelets were given for thrombocytopenia and FFP was given to reverse oral anticoagulation or when low circulating plasminogen was documented. On all 15 occasions a radiologically documented response was obtained, including reversal of thrombosis in hepatic veins, portal veins, the IVC, cerebral dural venous sinuses, and an intrahepatic portocaval shunt. Among the 15 courses of tPA, serious hemorrhagic complications developed in 3 cases. At last follow-up visit, of the 9 patients treated, 3 have expired, one patient (who has been non-compliant with post treatment anticoagulation and anticomplement therapy) was in good clinical condition despite extensive residual occlusions, and 5 others were in good to excellent condition in terms of clinical and radiological outcome. The only patient in whom tPA may have contributed to a fatal outcome also had complications of ‘heparin induced thrombocytopenia with thrombosis’ (HITT), which we diagnosed in a milder form in 3 additional patients. The other two fatalities were associated with bowel edema (probably due to progressive small vessel thrombosis) in one case, and a progressive concurrent myeloproliferative disorder associated with a JAK2 mutation in the other case. On the other hand, we feel tPA must be credited as having been immediately life-saving in 2 patients who had been moribund with Budd-Chiari syndrome, and in one who had impending renal failure associated with an IVC thrombosis. Given the high incidence of HITT, we favor the use of direct thrombin inhibitors or fondaparinux rather than heparin products in patients with PNH. Given the high mortality and morbidity associated with thrombosis in PNH patients, and given the excellent radiographic responses, we conclude that, in spite of the risk of hemorrhage, thrombolysis is strongly indicated to reverse intra-abdominal and intracranial thromboses. Disclosures: No relevant conflicts of interest to declare.