Paroxysmal Nocturnal Hemoglobinuria Cells Research Articles

Abnormal expression of CUX1 influences autophagy activation in paroxysmal nocturnal hemoglobinuria.

The mechanism underlying autophagy in paroxysmal nocturnal hemoglobinuria (PNH) remains largely unknown. We previously sequenced the entire genome exon of the CD59- cells from 13 patients with PNH and found genes such as CUX1 encoding Cut-like homeobox 1. Peripheral blood samples from 9 patients with PNH and 7 healthy control subjects were obtained to measure CUX1 expression. The correlation between CUX1 messenger RNA expression and PNH clinical indicators was analyzed. To simulate CUX1 expression in patients with PNH, we generated a panel of PNH cell lines by knocking out PIGA in K562 cell lines and transfected lentivirus with CUX1. CCK-8 and EDU assay assessed cell proliferation. Western blotting was used to detect Beclin-1, LC3A, LC3B, ULK1, PI3K, AKT, p-AKT, mTOR, and p-mTOR protein levels. Autophagosomes were observed with transmission electron microscopy. Chloroquine was used to observe CUX1 expression in PNH after autophagy inhibition. Leukocytes from patients with PNH had lower levels of CUX1 messenger RNA expression and protein content than healthy control subjects. The lactose dehydrogenase level and the percentage of PNH clones were negatively correlated with CUX1 relative expression. We reduced CUX1 expression in a PIGA knockout K562 cell line, leading to increased cell proliferation. Levels of autophagy markers Beclin-1, LC3B, LC3A, and ULK1 increased, and autophagosomes increased. Furthermore, PI3K/AKT/mTOR protein phosphorylation levels were lower. CUX1 expression did not change and cell proliferation decreased in CUX1 knocked down PNH cells after inhibition of autophagy by chloroquine. In brief, CUX1 loss-of-function mutation resulted in stronger autophagy in PNH.

Neutrophil Functionalities in Patients with Paroxysmal Nocturnal Haemoglobinuria Are Not Affected By Complement C5- Inhibition

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired haematological stem cell disorder characterized by haemolysis, thrombophilia, and cytopenia. PNH is caused by a somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene at the level of pluripotent, hematopoietic stem cells leading to a lack of GPI anchored cell surface proteins, such as complement-inactivating proteins, (e.g., CD55, CD59) on stem cells, polymorphonuclear neutrophil granulocytes (PMN), erythrocytes, and platelets. Subsequently, uncontrolled complement overactivity leads to the systemic complications. Phenotypic mosaicism is a distinct characteristic of PNH. For diagnostic purposes, the absence of the GPI-anchor on granulocytes and monocytes in the flow cytometry is considered the gold standard. However, the exact consequences of GPI deficiency in PMNs and monocytes have not been studied sufficiently yet. Since the implementation of complement inhibitors, the primary cause of morbidity and mortality in PNH has been significantly reduced. Despite this, the tendency towards thrombosis, as well as the consequences of the deficiency of GPI-linked proteins on PMNs and monocytes, remain largely unclear. Therefore, the main objective of this project is to gain a deeper understanding of the impact of PNH-PMNs and monocytes on thrombo-inflammation in PNH. Specifically, the impact of complement inhibitors on these factors has been compared. Blood from PNH patients (n=14) with a median clone size of 74 % were compared with an age- and sex-matched control group (n =20). Various cellular markers indicating a state of activation (CD11b, TREM1, CD64) or showing platelet adherence (CD42), were measured using flow cytometry. Unmutated PMNs and monocytes from PNH patients (PMN) served as intrinsic control, GPI positive PMNs (WT-PMN (PNH)) and monocytes from a control group served as extrinsic control. We compared unstimulated PNH and WT cells for their basal phenotype, and then functional changes upon stimulation (phorbol 12-myristate 13-acetate (PMA)). Our data present a contrary perspective to our initial assumptions, indicating that PNH-PMNs have a direct impact on unmutated PMNs, resulting in the generation of a thrombogenic state and increased levels of activation. The unmutated GPI-positive PMNs and monocytes exhibit a significantly stronger direct aggregation with platelets compared to GPI-deficient clones. It is hypothesized that the PNH clones influence the activity and aggregation propensity of the remaining unmutated granulocytes through additional cellular mechanisms. In a subgroup analysis, the impact of complement factor 5 inhibitors (C5i) on the aforementioned factors was investigated detecting no significant differences in terms of PMN activation or platelet aggregation. Thus, it can be hypothesized that thrombosis and hyper-inflammation are not solely driven by the PNH clones directly and modulated C5 inhibition. Additional cellular mechanisms and signaling pathways appear to play an important role, and these aspects have not been adequately addressed by the current therapy.

Complement Activation Is Increased in Antiphospholipid Syndrome-Related Ischemic Stroke with Higher Risk Features

Introduction: Antiphospholipid syndrome (APS) patients with arterial thrombosis, mostly ischemic stroke, have concerningly high rates of recurrent thrombosis despite antithrombotic treatment, with over 20% thrombosis recurrence within two years. Complement activation is increased in thrombotic APS patients, although its role has not been fully investigated in APS-related stroke. This study aimed to evaluate the degree of complement activation in APS-related ischemic stroke, transient ischemic attack (TIA) or other ischemic brain injury patients and whether complement activation correlated with APS severity and platelet hyperreactivity as a potential mechanism for increased thrombotic risk. Method: Patients with confirmed APS by Sydney Criteria and imaging-proven ischemic stroke, TIA or other ischemic brain injury were recruited, subcategorized as high-risk APS (HR-APS, n=30) and lower-risk APS (LR-APS, n=41). HR-APS included triple antiphospholipid antibody (aPL) positive, anticoagulant refractory or both arterial and venous thromboembolism patients. LR-APS included patients with a single thrombotic event who were single or double aPL positive. Ischemic stroke patients without aPL (n=40) and healthy donors (n=40) were utilized as controls. Patients with active cancer, inflammation, infection or use of hydroxychloroquine, immunosuppression and heparins were excluded. Complement activation markers were measured as Bb fragment (Bb), C3a-desArg (C3a), C5a-des-Arg (C5a), and SC5b-9 using ELISA (MicroVue kits, Quidel Corp, Pathway Diagnostics Ltd). Complement-mediated cell killing was measured by the modified Ham (mHam) test, which determines the degree of cell lysis after adding patient serum to a paroxysmal nocturnal hemoglobinuria cell line (PIGA-null TF-1 cells) lacking cell surface complement regulators. A positive result was considered 20% or greater cell killing. Flow cytometry was used to evaluate whether complement activation correlated with platelet hyperreactivity. For this, plasma from mHam positive patients was added to healthy donor whole blood and procoagulant platelet response was measured as the absolute change in proportion of platelet events that were positive for annexin V and P-selectin following stimulation with thrombin 2U/mL. Results: There was no significant difference in median age between the patient cohorts, ranging from 49 to 56 years, although the healthy controls were younger, with median age 36 (range 25-73, p<0.001). Circulating complement activation markers were all significantly increased in the APS cohorts compared to healthy controls (p<0.01). The most prominent increase was observed in C3a levels: HR-APS median (interquartile range - IQR) 215 ng/mL (137.1-297.4), LR-APS 290.8 ng/mL (120.3-450.3) and healthy controls 62 ng/mL (27.9-121.1)(p=0.004 and p<0.0001 compared to healthy controls, respectively); and Bb fragment levels: HR-APS 1.2 µg/mL (IQR 1.0-1.4), LR-APS 1.3 µg/mL (IQR 1.0-1.5) and healthy controls 0.8 µg/mL (IQR 0.7-0.9)(both p<0.0001 compared to healthy controls). However, only C3a, SC5b-9 and Bb fragment levels in LR-APS remained significantly elevated when compared to stroke controls (p=0.0009, p=0.01 and p=0.03, respectively). There were no differences between HR-APS and LR-APS, nor between stroke controls and healthy controls. Complement-mediated cell killing was also significantly higher in APS patients overall, median (IQR) proportion of non-viable cells 18% (IQR 12-33), compared to stroke controls: 10% (IQR 8-15, p<0.0001) and healthy controls: 12.5% (IQR 6.3-17, p=0.0009). Notably, HR-APS had significantly higher proportion of positive mHam tests (60%), compared to LR-APS: 36.6%, stroke controls: 12.5%, and healthy controls: 17.5% (p<0.0001, Figure 1A). Importantly, HR-APS patients with positive mHam results (n=5) induced a substantially higher increase in procoagulant platelets, median 17.2% (IQR 11.2-28.6), whereas healthy controls (n=4) only resulted in median change of -0.7% (IQR -7.0-1.4, p=0.02) (Figure 1B). Conclusion: Preliminary data suggest that APS-related ischemic stroke, TIA or ischemic brain injury are associated with increased, functionally important, complement activation, more prominent in higher risk disease. Complement-mediated platelet hyperreactivity may have a contributory role that remains to be established.

Effect of Eculizumab on Iron Metabolism in Transfusion-independent Patients With Paroxysmal Nocturnal Hemoglobinuria

Effect of Eculizumab on Iron Metabolism in Transfusion-independent Patients With Paroxysmal Nocturnal Hemoglobinuria

Defected lipid rafts suppress cavin1-dependent IFN-α signaling endosome in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal disorder of haematopoietic stem cells caused by somatic PIGA mutations, resulting in a deficiency in glycosylphosphatidylinositol-anchored proteins (GPI-AP). Some researchers uncovered that PNH cells displayed a GPI-mediated defect in lipid-raft formation. However, Lipid rafts play a crucial role in signaling, the signaling underlying lipid rafts in PNH have not yet been addressed. In this study, we reported that, IFN-α was significantly increased in PNH plasma compared with normal controls. And PNH cells more resistant to the inhibitory colony[1]-forming activity of IFN-α. Here we have already established PIGA knock out K562 cell line by CRISPR/cas9, the most recognized in vitro model of PNH. PNH cells showed obviously defected endocytosis of IFNα/βRs in lipid rafts, causing suppressed STAT2 activation and the inflammatory response. We further investigated the possible mechanisms of interferon signaling endosomes mediate by cavin1. Our findings provide crucial insight into the process of reduced IFNα signal transduction in PNH cells mediated by lipid rafts and suggest that cavin1 are a potential target for suppression of IFN-α inflammatory signaling. These results might further explain the growth advantage of PNH cells in an unfavorable microenvironment.

Immune Checkpoint Molecules Regulate Paroxysmal Nocturnal Hemoglobinuria Clonal Evolution

In paroxysmal nocturnal hemoglobinuria (PNH), the acquisition of somatic PIGA mutations provides a selection advantage to hematopoietic stem cells from aplastic anemia (AA) immune attack. In addition to this immune evasion mechanism, HLA mutations, deletions, and somatic UPD involving 6p21 locus have been also described in AA, all of which implicated in such adaptive clonal escape. It is well-known that blood cells derived from a PIGA-mutant clone lack membrane-bound complement regulators (e.g., CD55, CD59) and exhibit an increased vulnerability to complement attack. Nevertheless, PIGA-mutant cells may still be susceptible targets for AA T cell-mediated immune attack. In conjunction with mutational analysis, HLA protein expression may be investigated by flow cytometry to identify a haploinsufficient (clonal) vs. possible adaptive down-modulation. Indeed, the absence of these otherwise constitutionally expressed proteins represents an excellent marker of clonality. Additionally, in an attempt to further hinder the creation of a successful immunological synapsis, other mechanisms, including modulation of immune checkpoints expression, may be also involved in such AA/PNH intertwine. Whether these immune escape mechanisms are convergent or instead represent alternative pathways is a subject of intense research. Here, we comprehensively explore the dynamics governing PNH immune ontogenesis by combining HLA/PIGA genomics and flow cytometric measurements of HLA (A, B, C, DQ, DR, DP) and the immune checkpoint B7/CD28H family (PD1, PDL1, PDL2, VISTA, B7-H6, HHLA2, CD28H), stimulatory (GITR, CD30L) and inhibitory immune checkpoints (CD47) in CD45+CD15+CD59+ and CD59- fractions. A deep targeted sequencing panel covering HLA classical loci along with an in-house developed pipeline was also applied to assess fine HLA aberrations (mutational and copy number events), whereas multi-amplicon deep sequencing was deployed to identify PIGA mutational status. A total of 27 PNH patients classified as fully blown hemolytic PNH (n=15) or PNH/AA overlap (n=12) were included in this study with additional 4 healthy controls (HC). Overall, 41% of our patients harboured concomitant myeloid clones (most common hits were in DNMT3A, TET2, ASXL1, BCOR), while 58% of cases carried somatic HLA alterations, involving allelic losses or mutations. The latter were nonsense (33%), missense (11%), or 5’ or 3’ UTR (56%) hits with a median VAF of 11% (3-95%). Somatic lesions (including losses) were found in HLA-A (10%), B (30%), DQA1 (10%), or DQB1 (50%) loci. Since HLA aberrations may constitute the extreme pole of immune evasion and a marker of enhanced immune selection, we performed sorting/sequencing experiments on CD59+vs CD59- cells. A similar distribution and clonal burden of HLA-mutant subclones was found between the 2 fractions, indicating that PNH cells do not provide absolute immune privilege.1 Next, we compared the expression of HLA and other immunomodulatory molecules across HC, CD59+ cells from PNH patients and their corresponding CD59- cells (GPI+ and GPI-, respectively). Using MFI as a readout, GPI- cells showed similar expression levels of HLA-DP, -DR, PDL2, HHLA2 and GITR as compared to HC cells, while these molecules were significantly 1.5 to 5-fold upregulated on corresponding GPI+ cells consistent with their trigger/target function. This conclusion was also supported by the higher levels of CD28H found on GPI+ cells vs. GPI-(median MFI, 13.3 vs. 6.0; p<0.0001). Of note is that GPI- cells exhibited lower levels of HLA-DQ (0 vs. 7.0, p=0.01), B7H6 (0 vs. 39, p<0.0001) and CD30L (0 vs. 41, p <0.0001) vs. HC cells and upregulation of VISTA (460 vs. 227, p=0.006), suggesting that PNH clones may exhibit additional (to PNH phenotype) immune deterrence pathways. HLA alterations resulted in downregulation of HLA-DQ (0 vs. 29, p=.03), -DR (20 vs. 136, p=.03), B7H6 (0 vs. 45, p=.02), and CD30L (7.5 vs. 52, p=.02), as well as upregulation of HLA-B (2671 vs. 1909, p=.03) and VISTA (561 vs. 291, p=.02) in GPI- vs. GPI+, while no difference was noted in HLAWT cases. In conclusion, differential expression of immunomodulatory molecules on PNH vs. residual phenotypically normal cells in PNH patients and HC highlights various stimulatory and immune escape mechanisms operating in this disease. Understanding these processes may enable the identification of therapeutically relevant immune pathways.

Dysregulation of the MiR-30a-5p-B4GALT5 Axis Leads to Cell Proliferation and Is Associated with M1 Macrophage Polarization in PNH

Background：MicroRNAs (miRNAs) play versatile roles in multiple biological processes. However, little is known about miRNA's involvement in Paroxysmal nocturnal hemoglobinuria (PNH). Methods: We constructed a PIGA knock-out K562 cell line and PIGA conditional knock-out mice model by CRISPR/cas9 technology. Furthermore, we used FACS and colony formation assays to clarify proliferative and apoptosis abilities of PNH cells. We also used FACS to clarify M1 Macrophage Polarization in mouse bone marrow-derived macrophages (BMDMs). RT-PCR, and western blot analyses were used to verify the relationship among miR-30a-5p, B4GALT5 and PI3K/AKT signaling. Results: We observed that the expression of miR-30a-5p is significantly increased in Flaer negative cells compared with Flaer positive cells from 10 PNH patients (3.31±0.4 vs 1.48±0.23, p<0.05). The miRNA-mediated gene B4GALT5 were identified by comparing RNA-seq data with gene targets predicted using TargetScan. Importantly, B4GALT5 mRNA expression is significantly decreased in Flaer negative cells compared with Flaer positive cells from 15 PNH patients(1.68±0.31 vs 2.68±0.31, p<0.05), and this decreased B4GALT5 expression is also observed at the protein levels. In PIGA knock-out cells, the transfection of miR-30a-5p and B4GalT5 knockdown significantly promoted cell proliferation by activating PI3K/AKT signaling. What's more, the mean fluorescence intensity(MFI) of CD80 and CD86 which acted as the markers of M1 macrophages were higher in PNH patients compared with normal controls(CD80:1374±156 vs 614±162, p<0.05;CD86:738±131 vs 297±78,p<0.05). Meanwhile, downregulation of B4GalT5 increased the the markers of M1 macrophages in BMDMs of PIGA CKO mice. Conclusion: Thus, miRNA-30a-5p/B4GALT5 axis plays a crucial role in modulating PNH clone proliferation and M1 macrophage Polarization. This is the first report to characterize the potential roles of miRNA-30a-5p in PNH. Keywords: PNH, miRNA-30a-5p, B4GALT5, proliferation, Macrophage Polarization Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunophenotypic assessment of PNH clones in major and minor cell lineages in the peripheral blood of patients with paroxysmal nocturnal hemoglobinuria.

Flow cytometric immunophenotyping is essential for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Most cases have easy to interpret flow cytometry profiles with red cells, neutrophils and monocytes showing complete deficiency of glycophosphatidylinositol (GPI) linked antigen expression. Some cases are more challenging to interpret due to the presence of multiple populations of PNH cells and variable levels of GPI antigen expression. We studied 46 known PNH patients, many with complex immunophenotypic profiles using a novel, single tube, multi-parameter 7-color immunophenotyping assay that allowed simultaneous detection and assessment of PNH clones within multiple lineages of peripheral blood leucocytes. Red cell PNH clones were also assessed in total and immature (CD71+) components by CD59 expression. For individual patients, total PNH clones in each cell lineage were highly correlated. Monocytes, eosinophils and basophils showed the highest proportions of PNH cells. Red cell PNH clones were typically smaller than monocyte and neutrophil PNH clones. In most cases, PNH clones were detectable in minor leucocyte populations where multiple populations of PNH cells were present, variability in the proportions of type II and type III cells was seen across different cell lineages, even though total PNH clones remained similar. This study shows that PNH patients with multiple PNH clones do not always display the same abnormality across all cell lineages routinely tested. There is no simple explanation for this but is likely due to a combination of complex molecular, genetic and biochemical dysfunction in different blood cell types.

Concomitant Immunosuppressive Therapy and Eculizumab Use in Patients with Paroxysmal Nocturnal Hemoglobinuria: An International PNH Registry Analysis

Introduction: Complement C5 inhibitor eculizumab is the first approved treatment for paroxysmal nocturnal hemoglobinuria (PNH), a rare hematologic disorder caused by uncontrolled terminal complement activation. Approximately 50% of patients with aplastic anemia (AA) have PNH cells. Limited data are available for patients with AA-PNH taking concomitant immunosuppressive therapy (IST) and eculizumab. Methods: Data from the International PNH Registry (NCT01374360) were used to evaluate the safety and effectiveness of eculizumab and IST in patients taking IST followed by concomitant eculizumab (IST + c-Ecu) or eculizumab followed by concomitant IST (Ecu + c-IST). Results: As of January 1, 2018, 181 Registry-enrolled patients were included in the eculizumab effectiveness analyses (n = 138, IST + c-Ecu; n = 43, Ecu + c-IST); 87 additional patients received IST alone. Reductions from baseline with eculizumab were observed in the least squares mean lactate dehydrogenase ratio (IST + c-Ecu, −3.4; Ecu + c-IST, −3.5); thrombotic event incidence rates were similar between groups (IST + c-Ecu, 1.3; Ecu + c-IST, 0.7). Red blood cell transfusion rate ratios decreased from baseline for IST + c-Ecu (0.7) and increased for Ecu + c-IST (1.2); there were none for IST alone. Hematological parameters generally improved for IST + c-Ecu and IST alone, and changed minimally or worsened for Ecu + c-IST. Safety signals were generally consistent with those previously described for the respective therapies. Discussion/conclusion: Although some intergroup differences were seen, concomitant eculizumab and IST were safe and effective regardless of treatment sequence.

Paroxysmal nocturnal haemoglobinuria, diagnosis and haematological findings, first report from Iran, model for developing countries.

Since paroxysmal nocturnal haemoglobinuria (PNH) was first described in 1881, the diagnosis and follow‐up patients diagnosed with the illness has remained an area of concern, with several different techniques of varying sensitivity having been described in the literature for both the diagnosis and monitoring treatment of the disease. PNH is a rare and life‐threatening disease that manifests symptoms of haemolytic anaemia. Hence, a quick and reliable technique for precise diagnosis would be crucial. PNH patients who have previously been diagnosed with aplastic anaemia or myelodysplastic syndrome carry small PNH clones and for more than a century traditional method with low sensitivity was used for such patients. In 2010, the International Clinical Cytometry Society described a highly sensitive method for detection and quantification of different types of PNH clones using multi‐colour flow cytometry. In this method, a three‐colour flow cytometer is essential to detect PNH affected cells amongst monocytes and granulocytes. This started a new era in the diagnosis of patients who carry small clones of PNH cells. Before this, flow cytometric analysis was used only for detection of PNH cells amongst erythrocytes. By using flow cytometry instruments with more light sources, the sensitivity of detection and quantification of PNH clones would be augmented. However, standardisation and crosstalk compensation would be the most concerning issue. For the first time in Iran, we set up and standardised multi‐colour flow cytometry technique to detect PNH cells in erythrocytes and leukocytes at Payvand medical laboratory.

Frequency of Paroxysmal Nocturnal Hemoglobinuria in Patients with Lymphoma

Objective: To investigate the frequency of paroxysmal nocturnal hemoglobinuria (PNH), an acquired clonal hematopoietic stem cell disease resulting in complement-mediated hemolysis, in patients with lymphoma by flow cytometry.Methods: Fifty patients with lymphoma who were admitted to the hematology clinic, newly diagnosed and not yet treated were included in this study conducted in 2014. The presence of PNH clones was checked by FLAER flow cytometry method in peripheral blood samples. FLAER is a non-hemolytic fluorescently labeled inactive toxin aerolysin method that can detect up to 0.5% of PNH cells instead of bacterial toxin aerolysin, which binds to RBCs via the GPI anchor and initiates hemolysis for PNH screening or PNH clone detection. With this technique, PNH clones in all hematopoietic cell lines can be detected in an assay.Results: PNH clone was observed over 10% in two patients, one male and the other female. However, no hemolysis was found in patients with PNH clones. The lymphoma subtypes of the patients with positive PNH clone were B-cell small-cell lymphocytic lymphoma in the male patient and primary splenic lymphoma in the female patient.Conclusion: PNH or PNH-like disorders accompanying hematological malignancies, especially lymphomas, are not very common in the literature. There is a need to elucidate the relationship between hematological malignancies and PNH with the help of more advanced molecular techniques.

Insights Into the Emergence of Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disease as simple as it is complex. PNH patients develop somatic loss-of-function mutations in phosphatidylinositol N-acetylglucosaminyltransferase subunit A gene (PIGA), required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. Ubiquitous in eukaryotes, GPI anchors are a group of conserved glycolipid molecules responsible for attaching nearly 150 distinct proteins to the surface of cell membranes. The loss of two GPI-anchored surface proteins, CD55 and CD59, from red blood cells causes unregulated complement activation and hemolysis in classical PNH disease. In PNH patients, PIGA-mutant, GPI (-) hematopoietic cells clonally expand to make up a large portion of patients’ blood production, yet mechanisms leading to clonal expansion of GPI (-) cells remain enigmatic. Historical models of PNH in mice and the more recent PNH model in rhesus macaques showed that GPI (-) cells reconstitute near-normal hematopoiesis but have no intrinsic growth advantage and do not clonally expand over time. Landmark studies identified several potential mechanisms which can promote PNH clonal expansion. However, to what extent these contribute to PNH cell selection in patients continues to be a matter of active debate. Recent advancements in disease models and immunologic technologies, together with the growing understanding of autoimmune marrow failure, offer new opportunities to evaluate the mechanisms of clonal expansion in PNH. Here, we critically review published data on PNH cell biology and clonal expansion and highlight limitations and opportunities to further our understanding of the emergence of PNH clones.

Immunodynamics of Paroxysmal Nocturnal Hemoglobinuria

In the context of immune-mediated attack in aplastic anemia (AA), PIGA mutations produce a clonally-restricted escape phenotype in an attempt to restore hematopoiesis. Along with PIGA lesions, HLA class I/II mutations may represent an alternative way to render cytotoxic T cell recognition less effective. Microdeletions of 6p21, uniparental disomy [UPD] of 6p and mutations of HLA genes have been previously identified in AA patients. One could stipulate that paroxysmal nocturnal hemoglobinuria (PNH) may be an alternative mode of clonal evolution as opposed to HLA mutation acquisition or instead be a redundant pathway of immune escape co-existing with such lesions. Here, we took advantage of a large cohort (n=92) of patients with hemolytic PNH (granulocytes clone size >20% and LDH>x2.5 ULN) to elucidate the immunodynamics of PIGA/HLA clone(s) (Fig1A).We first used a deep targeted sequencing panel covering HLA classical loci and applied an in-house newly developed pipeline on 62 patients with PNH. Overall, 35% of cases had HLA alterations, which were found at a similar rate in patients studied at onset (33%) and during the course of the disease (32%). These alterations were classified as allelic losses in 15% of cases while majority of mutations were missense (57%) followed by 5‘ or 3‘ UTR (20%), nonsense (15%) and frameshifts (8%) with a median variant allelic frequency (VAF) of 5% (range, 2-95). Of note, the canonical exon 1 HLA B14:02 mutation (c.19C>T, p.R7X) 1,2was found in 2 patients who both had de novo PNH with a granulocyte clone size of 60% at disease onset (Fig1B).To clarify whether the PNH phenotype conveys an absolute resistance from immune attack, one should determine whether in these patients HLA mutations arise indeed within non-PNH cells. Consequently, we also performed HLA sequencing on 9 patients whose samples (n=18) were previously sorted by flow cytometry to enrich for CD59+ and CD59- fractions. HLA mutations were identified in both CD59+ and CD59- fractions at similar rates (44% each) and VAF (5% vs 3%, p=0.3) while no “canonical” exon 1 mutation was found.When looking at associations with disease-specific characteristics, we did not identify any significant differences in terms of burden of HLA events and disease ontogeny (primary vs secondary to AA cases) as well as baseline demographics characteristics (gender, age at presentation), dominant erythrocytes type (II vs III), and thrombotic events. No correlations with specific types of PIGA mutations nor their burden were found in HLA-mutated vs non-mutated groups as also confirmed by the absence of relationship with PNH granulocyte clone size (62% vs 72%, p=0.5). Of note, in HLA mutants the VAFs of HLA lesions were significantly lower than those of PIGA (5% vs 18%, p<0.001) underlying their possible less powerful role in driving immune escape within the context of AA overshooting reactions. To validate these results, we have designed a highly quantitative flow cytometric method for detection of HLA expression (A, B, C for class I; DQ, DR, DP for class II) on CD59+ vs CD59- fractions of CD34+ and CD15+ cells obtained from PNH patients. Preliminary results in 7 patients showed a general downregulation of HLA expression within PNH clones (delta HLA MFI of CD59+ vs CD59- fractions), consistent with our ability to detect HLA mutations also in PNH cells (Fig1C). This preferential down-modulation on PNH cells suggests a possible selection mechanism operating agonistically and in a non-mutually exclusive fashion with the PNH-intrinsic privilege. Moreover, these findings may also represent a byproduct of antigen processing machinery impairment due to proteasomal accumulation of proteins, which would have been GPI-linked and exported to the membrane.In sum, our study indicates that PNH phenotype conveyed by PIGA mutations operates independently from HLA mutations as means of immune escape. In addition, based on these results we stipulate that HLA down-modulation via various mechanisms is operative in the context of the PNH phenotype and the immune privilege of PIGA clones is not absolutely protective. Patients with PNH may have elements of immune suppression. In fact, their responses in the context of reduced-intensity transplant do eliminate hosts PNH clones following transplantation. 3 [Display omitted] DisclosuresPatel: Alexion: Consultancy, Other: educational talks, Speakers Bureau; Apellis: Consultancy, Other: educational talks, Speakers Bureau. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Regeneron: Consultancy.

SUZ12 Participates in PNH Cloning Proliferation By Regulating Histone H3K27me3 Methylation Level

Paroxysmal nocturnal hemoglobinuria (PNH) is a disease of hematopoietic stem cell membrane defects due to acquired PIG-Amutation. Our previous study found some secondary gene mutations in PNH patients by WES. However, it is not clear exactly which mutations are associated with the disease. So, 97 target genes were selected as a target gene panel and tested in 23 PNH patients by DNA sequencing of specific target regions. We found that all PNH patients had other gene mutations except PIG-Amutations, including TTN, NCOR2, CPS1, MUC4, SUZ12, LFNG, CELSR2, JAK2, SETBP1 and KMT2D (Figure1A). Through harmful analysis, KEGG enrichment, GO enrichment analysis and protein interaction analysis, we screened out the secondary mutant gene SUZ12 that may be involved in the cloning proliferation of PNH. We detected the mRNA and protein expression levels of SUZ12 and H3K27me3 methylation in PNH patients and health volunteers, the results showed that the mRNA and protein expression levels of SUZ12 and H3K27me3 methylation in peripheral blood CD59 -cells of PNH patients were higher than those in CD59 + cells of PNH patients and healthy controls (Figure1B). The relative expression level of SUZ12 in peripheral blood CD59 -cells of PNH patients was correlated with (r=0.4162, p=0.0385), CD59 -erythrocyte ratio (r=0.4636, p=0.0196), CD59 -monocyte ratio (r=0.4052, p=0.0495), Flaer -monocyte ratio (r=0.6769, p=0.0004) and Flaer -granulocytic ratio (r=0.6146, p=0.0018), indicating that SUZ12 may be involved in abnormal PNH cloning and proliferation by regulating H3K27me3. To verify the role of SUZ12 in the proliferation of PNH cloning, we used CRISPR/Cas9 to knockdown PIG-A expression in THP-1 cells to construct A PNH cell model, the expression level of PIG-A protein in the cell model was significantly decreased, and the proportion of CD59 - cells accounted was stable at 95%. Then lentivirus transfection was used to knockdown the expression of SUZ12 in PNH cell model. The results showed when the SUZ12 expression was knockdown, the methylation level of histone H3K27me3 was decreased, the cell proliferation activity was decreased, apoptosis was increased, and the cell cycle was arrested at G0/G1 phase. The proportion of CD59 + cells increased gradually from 3 weeks after transfection, and significantly increased at 4 weeks after transfection, while no changes were observed in the empty virus group and control group (Figure1C). Four weeks after lentivirus transfection, the expression of PIG-A protein recovered in SUZ12 knockdown group compared with empty virus group and control group (Figure1D). In conclusion, SUZ12 mutation leads to the overexpression of SUZ12, which can affect cell proliferation, apoptosis and cell cycle by regulating the methylation level of histone H3K27me3, thereby promoting the proliferation of PNH abnormal cloning and participating in the pathogenesis of PNH. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Time and residual hematopoiesis are crucial for PNH clones escape in hepatitis-associated aplastic anemia.

The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in aplastic anemia (AA) suggests immunopathogenesis, but when and how PNH clones emerge and proliferate are unclear. Hepatitis-associated aplastic anemia (HAAA) is a special variant of AA, contrarily to idiopathic AA, in HAAA the trigger for immune activation is clearer and represented by the hepatitis and thus serves as a good model for studying PNH clones. Ninety HAAA patients were enrolled, including 61 males and 29 females (median age 21years). Four hundred three of idiopathic AA have been included as controls. The median time from hepatitis to cytopenia was 50days (range 0-180days) and from cytopenia to AA diagnosis was 26days (range 2-370days). PNH clones were detected in 8 HAAA patients (8.9%) at diagnosis and in 73 patients with idiopathic AA (IAA) (18.1%). PNH cells accounted for 4.2% (1.09-12.33%) of red cells and/or granulocytes and were more likely to be detected in patients with longer disease history and less severe disease. During follow-up, the cumulative incidence of PNH clones in HAAA increased to 18.9% (17/90). Nine HAAA patients newly developed PNH clones, including six immunosuppressive therapy (IST) nonresponders. The clone size was mostly stable during follow-up, and only 2 of 14 patients showed increased clone size without proof of hemolysis. In conclusion, PNH clones were infrequent in newly diagnosed HAAA, but their frequency increased to one that was similar to the IAA frequency during follow-up. These results suggest that the PNH clone selection/expansion process is dynamic and takes time to establish, confirming that retesting for PNH clones during follow-up is crucial.

Thrombosis in Non-Hemolytic Paroxysmal Nocturnal Haemogloinuria

▪Paroxysmal Nocturnal Hemoglobinuria (PNH), a rare hematological condition, presents in patients with a variety of symptoms. The risk of thrombosis is high, with a cumulative thrombosis incidence over an eight to ten year period of between 23% and 30% (in the pre-eculizumab era). 20% of patients have multi-site thrombosis and 10% of patients present with thrombosis. Thrombosis in PNH is a clinical emergency, due to risk of death, thrombosis extension and long term morbidity.The complement and coagulation systems are intimately linked, and explain some of the mechanisms for thrombosis in PNH. Intravascular haemolysis is associated with thrombosis risk however, thrombosis has been observed in patients without hemolysis, most likely related to white cell and platelet factors.The proportion of granulocyte PNH cells correlates with thrombosis risk; those with cell proportions over 50% have a cumulative ten-year incidence of thrombosis of 34.5% compared to 5.3-7% in those with a clone of <50%. Thus in the UK, those with PNH cellular proportions over 50% commence prophylactic anticoagulation.The Leeds (UK) PNH service manages 394 patients with PNH, and has observed PNH patients with minimal hemolysis suffer thrombosis. Predicting those at highest risk is difficult. A retrospective review to identify rates of thrombosis in patients with no/minimal hemolysis was undertakenMethodsThe Leeds PNH database was interrogated, and identified patients with a PNH granulocyte or monocyte clone of over 10% and an LDH less than twice the upper limit of normal for the laboratory in which the sample was tested. Anonymised clinical data was then analysed to determine risk factors for thrombosis. Comparisons between those with and without thrombosis were assessed.All patients who fulfilled the above requirements were then subdivided into groups as below:Group 1: White cell clone >30%, red cell clone <10%, LDH <2xULNGroup 2: White cell clone >30%, red cell clone >10% with higher proportion of type II red cells than type III red cells, LDH <2xULNGroup 3: White cell clone: 10-30%, red cell clone <10%, LDH <2xULNResults29 patients were identified who fit the criteria. Median age of the patient cohort was 62 years (range 22-80 years). 12 patients fit the criteria for group 1, of whom 6 (50%) had thrombosis, 12 patients fit the criteria for group 2 of whom 2 (16.6%) had thrombosis and 5 patients fit the criteria for group 3, with no thrombosis. All 8 patients with thrombosis are detailed in the Table7 patients were not on anticoagulation at the time of the thrombosis, with on patient extending their thrombosis whilst on warfarin (patient 1).Discussion and conclusionsWe report the first case series of patients with non-hemolytic PNH who experience thrombosis. Patients at higher risk of thrombosis in this cohort were those with non-hemolytic PNH, high white cell clones and low red cell clones (group I). However those with a greater proportion of type II red cell clones compared to type III red cells and LDH <2x ULN were also at risk of thrombosis. This is an under-recognised risk for patients with PNH cells.Whilst the risk of arterial thrombosis is around 20%, interestingly and surprisingly, arterial thrombosis in this patient cohort was disproportionately high, with 62.5% of patients with either arterial or mixed venous and arterial thrombosis. It should also be noted that only one patient was anticoagulated at the time of their CVA extension. This was due to a combination of patients not being diagnosed with PNH at the time of thrombosis, or thought to be unsuitable for anticoagulation due to thrombocytopenia.This study supports the theory that while hemolysis may be associated with a thrombotic event, white cell and platelet factors have a more pivotal role than previously thought in the mechanisms of thrombosis in PNH. As per the National recommendations in England, thrombosis thought to be due to PNH regardless of LDH level, is an indication to commence treatment [Display omitted] DisclosuresGriffin:Alexion Pharmaceuticals, Inc.: Honoraria. Munir:Gilled: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Janssen: Honoraria; Roche: Honoraria; AbbVie: Honoraria. Hillmen:Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Celgene: Research Funding; GSK: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Arnold:Alexion Pharmaceuticals, Inc.: Honoraria. Richards:Alexion Pharmaceuticals, Inc.: Honoraria. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

In Vitro Combination Studies of ACH-4471 with Eculizumab to Assess a Potential “Switch” Treatment Approach for Paroxysmal Nocturnal Hemoglobinuria

Introduction: ACH-4471 is an oral complement factor D (fD) inhibitor that has demonstrated clinical efficacy in an ongoing phase 2 clinical study for naïve patients suffering from paroxysmal nocturnal hemoglobinuria (PNH). PNH is a rare clonal blood disorder in which erythrocytes lacking the GPI-anchored complement regulators CD55 and CD59 are susceptible to destruction by the terminal complement complex (also known as membrane attack complex, MAC) following the normal low-level activation of the complement alternative pathway (AP) in the fluid phase. The therapeutic agent eculizumab, a monoclonal antibody directed against the terminal complement component C5, blocks MAC assembly on erythrocyte membranes and so prevents most intravascular hemolytic destruction. Eculizumab treatment however leads to increased deposition of complement C3 fragments on PNH membranes, which can result in extravascular phagocytic elimination of opsonized erythrocytes (A. M. Risitano et al, Blood, 2009, 113:4094) and incomplete inhibition of intravascular hemolysis (M. J. Harder et al, Blood, 2017, 129:970), and consequently to the continued anemia and transfusion dependence observed in a significant subset of patients. As an AP inhibitor, in contrast to eculizumab, ACH-4471 prevents both MAC assembly and C3 fragment deposition on PNH cell surfaces and therefore is expected to address the medical needs of these suboptimal responders to eculizumab. To assess this notion directly and also to support a future switch treatment approach, a further clinical study has been planned to evaluate ACH-4471 co-administration in PNH patients who respond suboptimally to eculizumab. In the present study, we conducted in vitro combination experiments to explore the potential pharmacological consequences when ACH-4471 is added on to eculizumab or another anti-C5 monoclonal antibody, using functional AP assays that include the hemolysis of erythrocytes from a PNH patient.Methods: Concentration series of ACH-4471 were assessed in pairwise combinations with eculizumab and a second monoclonal antibody inhibitor of complement C5. Functional assays for human serum AP activity included a single-plate ELISA-based functional assay (Wieslab) and the hemolysis of red blood cells from a PNH patient under AP-restricted conditions but at neutral pH. Analyses of interactions were performed using a three-dimensional surface graphing method and, where applicable, a median-effect plot method.Results: We assessed pairwise interactions using two assays for AP function. In the AP Wieslab assay, interactions between ACH-4471 and an anti-C5 monoclonal antibody ranged from additive to moderately synergistic. Next, to better approximate the physiological setting, we assessed inhibitor interactions against the AP-mediated hemolysis of red blood cells obtained from a PNH patient. ACH-4471 in this assay showed strongly synergistic interactions when tested with eculizumab. In light of a recent report on incomplete inhibition by eculizumab following strong complement activation, we propose a possible mechanism for synergy between ACH-4471 and eculizumab wherein ACH-4471 reduces the density of deposited C3b clusters, to which C5 binding is thereby more readily inhibited by eculizumab.Conclusions: ACH-4471 showed strong synergy with eculizumab. These results suggest the potential clinical utility of ACH-4471 as a PNH therapeutic agent that may be used in suboptimal eculizumab responders in support of a “switch” treatment strategy. DisclosuresPatel:Achillion: Employment. Thanassi:Achillion: Employment. Yang:Achillion: Employment. Brodsky:Alexion Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Grant Funding. Huang:Achillion: Employment. Podos:Achillion: Employment.

The incidence and prevalence of patients with paroxysmal nocturnal haemoglobinuria and aplastic anaemia PNH syndrome: A retrospective analysis of the UK's population-based haematological malignancy research network 2004-2018.

A retrospective population-based study to determine the incidence and prevalence of patients with the rare blood disease paroxysmal nocturnal haemoglobinuria (PNH). All patients were identified by flow cytometric detection of blood cells deficient in glycosylphosphatidylinositol (GPI) linked proteins at a single diagnostic reference laboratory that serves the Yorkshire based, Haematological Malignancy Research Network (HMRN) with a population of 3.8million. One hundred and ninety-seven patients with detectable PNH clones at a level of >0.01% in at least two lineages of cells (neutrophils, monocytes and/or red cells) were identified over a 15-year period (2004-2018). Of these, 88% had aplastic anaemia (AA), 8% classical PNH and 3% myelodysplastic syndrome. The overall incidence rate was estimated at 0.35 cases per 100000 people per year. This equates to 220 cases newly diagnosed in the United Kingdom each year. The overall prevalence rate was 3.81 per 100000, this equates to an estimated 2400 prevalent cases in the UK. The overall and relative 5-year survival rates were 72% and 82.7%, respectively. This study showed that classical haemolytic PNH is a rare disease and represents only a small proportion overall of patients with detectable PNH cells, the majority of which have aplastic anaemia.

Preclinical Characterization of BCX9930, a Potent Oral Complement Factor D Inhibitor, Targeting Alternative Pathway-Mediated Diseases Including Paroxysmal Nocturnal Hemoglobinuria (PNH)

INTRODUCTION: Complement factor D is a serine protease that specifically catalyzes the cleavage of factor B [bound to C3(H2O) or C3b], its only natural substrate, to Ba and Bb. Factor D has the lowest concentration in plasma among all the complement proteins and is the rate-limiting enzyme of the alternative pathway (AP) of complement. Factor D plays a key role in both AP-initiated C3 convertase formation and the amplification loop of the complement cascade. Therefore, targeting factor D is a promising therapeutic strategy to inhibit AP activation for treatment of AP-mediated diseases such as PNH, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Inhibition of terminal complement C5 by eculizumab or ravulizumab results in sustained inhibition of intravascular hemolysis in patients with PNH by blocking formation of the membrane attack complex (C5b-9). However, many PNH patients treated with eculizumab continue to have symptomatic anemia, with up to half of the patients requiring blood transfusions due to extravascular hemolysis as a result of ongoing AP activation and C3 opsonization on erythrocytes. BioCryst Pharmaceuticals has developed a small-molecule, orally bioavailable human factor D inhibitor that is currently under evaluation as oral monotherapy for the treatment of patients with PNH in a Phase 1 study (BCX9930-101; NCT04330534). Here we demonstrate that BCX9930 is a potent and highly selective factor D inhibitor, and completely blocked AP activation on PNH erythrocytes in vitro. METHODS: Factor D enzymatic activity was analyzed in vitro in an esterolytic assay with a synthetic substrate and in a proteolytic assay using its natural substrate factor B bound to C3b. The selectivity was assessed in substrate-specific assays using other human serine proteases. The inhibition of AP-mediated hemolysis was assessed in vitro with rabbit erythrocytes incubated with 10% normal human serum (NHS). The Ham test was used to evaluate the inhibition by BCX9930 of AP-mediated hemolysis of erythrocytes from PNH patients with 20% acidified NHS. Inhibition of AP-mediated C3 fragment deposition was evaluated by flow cytometry using PNH erythrocytes incubated with acidified C5-depleted NHS. Activity of the classical pathway (CP) of complement l was evaluated by measuring 50% Hemolytic Complement (CH50) activity of serum and by assay of CP-mediated hemolysis of antibody-sensitized sheep erythrocytes. Pharmacodynamic effects in rhesus monkeys dosed orally with BCX9930 (100 and 200 mg twice-a-day) were assessed ex vivo with the AP Wieslab assay for the determination of C5b-9 generation after AP activation. RESULTS: BCX9930 potently inhibited esterolytic activity of purified human factor D with a mean 50% inhibitory concentration (IC50) of 14.3 nM. BCX9930 also inhibited its proteolytic activity against factor B bound to C3b with a mean IC50 of 28.1 nM. BCX9930 inhibited AP-mediated hemolysis of rabbit erythrocytes with a mean IC50 of 29.5 nM. Using the Ham test with erythrocytes from three PNH patients, BCX9930 completely suppressed AP-mediated hemolysis with a mean IC50 of 35.4 nM. Furthermore, BCX9930 suppressed C3 fragment deposition on PNH erythrocytes with a mean IC50 value of 39.3nM. BCX9930 did not inhibit enzyme activity of the human serine proteases thrombin, activated protein C, tissue plasminogen activator and trypsin up to 28 μM, and activated factor X and activated factor XII up to 50 μM. The mean IC50 values of BCX9930 inhibition of C1s and plasmin were 936 and 2850 nM, respectively. BCX9930 at a concentration up to 3 μM had no effect on CP activity in in vitro assays. Oral dosing of BCX9930 in monkeys completely suppressed AP activity of serum measured by AP-dependent generation of C5b-9. CONCLUSIONS: BCX9930 inhibitory activity on Factor D in vitro was potent and highly specific. BCX9930 completely blocked hemolysis of PNH cells in vitro and suppressed the accumulation of C3 fragments on PNH erythrocytes, indicating that BCX9930 has the potential to inhibit both intravascular and extravascular hemolysis. After oral dosing of BCX9930 in rhesus monkeys, AP activity was completely suppressed in ex vivo assays. These data demonstrate that BCX9930, an orally administered investigational drug, is a potent and selective factor D inhibitor, and that targeting factor D with BCX9930 has potential for the treatment of patients with PNH and other alternative pathway mediated diseases. Disclosures Chen: BioCryst Pharmaceuticals, Inc.: Current Employment. Cheng:BioCryst Pharmaceuticals, Inc.: Current Employment. Liu:BioCryst Pharmaceuticals, Inc.: Current Employment. Muppa:BioCryst Pharmaceuticals, Inc.: Current Employment. Parker:BioCryst Pharmaceuticals, Inc.: Current Employment. Zhu:BioCryst Pharmaceuticals, Inc.: Current Employment. Raman:BioCryst Pharmaceuticals, Inc.: Current Employment. Kotian:BioCryst Pharmaceuticals, Inc.: Current Employment. Babu:BioCryst Pharmaceuticals, Inc.: Current Employment.

Risk Factors for Thrombotic Events in Patients with PNH: A Nested Case-Control Study in the International PNH Registry

Risk Factors for Thrombotic Events in Patients with PNH: A Nested Case-Control Study in the International PNH Registry