Paroxysmal Nocturnal Hemoglobinuria Granulocytes Research Articles

Paroxysmal nocturnal hemoglobinuria–related thrombosis in the era of novel therapies: a 2043–patient-year analysis

AbstractThrombophilia is one of the principal features of paroxysmal nocturnal hemoglobinuria (PNH) and constitutes the main cause of disease morbidity/mortality. Anticomplement treatment has revolutionized the natural history of PNH, with control of the hemolytic process and abolition of thrombotic events (TEs). However, no guidelines exist for the management of thromboembolic complications in this setting, with type and duration of anticoagulation depending on individual practices. Besides, a scarcity of data is present on the efficacy of direct oral anticoagulants (DOACs). Herein, we accrued a large real-world cohort of patients with PNH from 4 US centers to explore features, predictors of TE, and anticoagulation strategies. Among 267 patients followed up for a total of 2043 patient-years, 56 (21%) developed TEs. These occurred at disease onset in 43% of cases, involving more frequently the venous system, typically as Budd-Chiari syndrome. Rate of TEs was halved in patients receiving complement inhibitors (21 vs 40 TEs per 1000 patient-years in untreated cases, with a 2-year cumulative incidence of thrombosis of 3.9% vs 18.3%, respectively), and varied according to PNH granulocytes and erythrocytes clone size, type, disease activity parameters, as well as number (≥2 mutations, or less) and variant allelic frequency of PIGA mutations. Anticoagulation with warfarin (39%), DOACs (37%), and low-molecular weight heparin (16%) was administered for a median of 29 months (interquartile range [IQR], 9-61.8). No thrombotic recurrence was observed in 19 patients treated with DOACs at a median observation of 17.1 months (IQR, 8.9-45) whereas 14 cases discontinued anticoagulation without TE recurrence at a median time of 51.4 months (IQR, 29.9-86.8).

Ahemolytic PNH: Clinical Features of a Distinct Phenotype of Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem/progenitor cell disorder caused by PIGA mutations. All hematopoietic lineages derived from the affected clone exhibit the PNH phenotype [deficiency of glycosyl phosphatidylinositol linked membrane proteins (GPI-APs)]. Diagnosis is made by flow cytometric analysis of red blood cells (RBC), granulocytes, and monocytes. The percentage of granulocytes and monocytes with absent expression of GPI-APs is a measure of the size of the PNH clone. Typically, the percentage of GPI-AP deficient RBCs is somewhat less than that of granulocytes and monocytes due to selective destruction by complement-mediated intravascular hemolysis, manifested by high LDH, bilirubin, reticulocyte count and low haptoglobin. Herein, we report five patients with large PNH clones based on percentage of GPI-AP deficient neutrophils and monocytes, but with absent or near absent PNH RBCs and no biochemical evidence of hemolysis. We call this unique disease subtype, ahemolytic PNH. Methods: The medical records of PNH patients (174 patients with PNH granulocytes >20%) at the University of Texas Southwestern, Cleveland Clinic Foundation, and National Institutes of Health from 2000 to 2022 were examined. Information pertaining to their clinical, laboratory, and molecular attributes (NGS sequencing) was collected. Five patients with granulocyte clones >50% and RBC clones <5% were identified (Table). Results: Patient 1 is a 63-year-old male with a remote history of non-severe aplastic anemia (AA) diagnosed at 11 years old. The first PNH flow cytometry, obtained at age 61 years, showed 0.02% type II PNH RBC, 0.5% type III PNH RBC, 96% PNH monocyte and 97% PNH granulocytes (Table). There was no biochemical evidence of hemolysis at the time of diagnosis or in the preceding 5 years. Patient 2 is a 50-year-old male who presented with pancytopenia and was diagnosed with acute myeloid leukemia with myelodysplastic-related mutations. Flow cytometry showed 85% PNH monocytes and 59% PNH with no detectable PNH RBCs and no biochemical evidence of hemolysis at diagnosis or in 3 years of follow-up (Table). Patient 3 is a 73-year-old female who presented with pancytopenia and marrow aplasia with unremarkable morphology, and a PNH granulocyte clone of 93.6% with 1.3% type III PNH RBCs and 1.3% and 0.5% type II PNH RBCs, with no evidence of hemolysis (Table). Patient 4 is a 21-year-old female who presented with Budd-Chiari syndrome. Flow cytometry showed 73% PNH granulocytes with 3.6% type III PNH RBCs and 1.8% type II PNH RBCs. There was no biochemical evidence of hemolysis, but because of thrombosis, she was treated complement inhibitor therapy (Table). Patient 5 is a 61-year-old female with history of severe aplastic anemia treated with immunosuppression (IST) and eltrombopag. PNH flow cytometry showed PNH granulocytes of 92.4% and 2.1% PNH type II RBCs 2.1% post IST treatment with no biochemical evidence of hemolysis (Table). Conclusion: Herein, we present five patients with a distinct PNH phenotype that we call ahemolytic PNH. Except for the absence or near absence of PNH erythrocytes, this entity shares clinical and pathophysiological features with classic PNH, including thrombophilia (Budd-Chiari Syndrome, Table). The basis of this phenotype is speculative but may be determined by the genotype of the affected HSPC in which the somatic mutation of PIGA first occurred. According to this hypothesis, somatic mutations other than PIGA skew differentiation toward granulocyte/monocyte lineages. The International PNH Interest Group recognizes the following three categories of PNH: classic PNH, PNH in the setting of another define bone marrow abnormality, and subclinical PNH. Like ahemolytic PNH, subclinical PNH has no biochemical evidence of hemolysis. In this case, however, the absence of hemolysis is due to the small size of the PNH clone (median clone size <1%) as determined by flow cytometry analysis of peripheral blood granulocytes and monocytes. We propose that ahemolytic PNH, defined as patients having less than <5% PNH red cells and >50% PNH granulocytes/monocytes, be recognized as a distinct category of PNH.

Treatment Outcomes of Complement Protein C5 Inhibition in 509 Patients with Paroxysmal Nocturnal Hemoglobinuria in the United Kingdom

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is an ultra-rare hematological disorder. Affected individuals experience chronic intravascular hemolysis and an increased tendency to thrombosis. Clinical symptoms can include hemoglobinuria, dysphagia, abdominal pain, breathlessness, erectile dysfunction, fatigue and lethargy. Inhibition of terminal complement by targeting the complement protein 5 (C5) is currently the standard care used to treat PNH in the UK. Eculizumab and ravulizumab, bind to C5, stopping its cleavage to C5a and C5b and thereby preventing terminal complement activation and its consequences. Method: A retrospective analysis of case records of all patients with PNH treated with eculizumab or ravulizumab in the UK since May 2002 was performed. Clinical features at presentation and diagnosis, prior history of bone marrow failure (BMF) as well as outcomes whilst on C5 inhibition (C5i) were collected. Results: 509 patients with PNH (237 male and 272 female) have been treated with eculizumab or ravulizumab between May 2002 and July 2022. Mean age at diagnosis was 43 years and 11 months. A history of BMF was documented in 246 patients (48.3%) of which 215 had aplastic anemia (42.2%) and 31 (6.1%) had myelodysplastic syndrome. One hundred and thirty five patients (26.5%) had experienced thrombosis prior to commencing C5 inhibition. There were 192 documented thromboses in these 135 patients with 73 (38.0%) being intra-abdominal and 57 (29.7%) being arterial (mainly myocardial infarction and stroke). Symptoms experienced by patients at diagnosis include anemia: 358 (70.3%), fatigue: 316 (62.1%), hemoglobinuria: 225 (44.2%), breathlessness: 114 (22.4%), abdominal pain: 92 (18.0%), dysphagia: 36 (7.1%) and erectile dysfunction: 32 of the 237 male patients (13.5%). The median time from developing symptoms to diagnosis of PNH was 5 months (interquartile range 0-18, available in 454 cases) with 86 (16.9%) patients being originally referred to a different specialty, mainly urology, for investigation. Sixty-four (12.6%) patients were misdiagnosed with an alternative condition before the correct diagnosis of PNH was made. Four hundred and seventy-four, (93.1%) patients have been treated with eculizumab with 77 (16.2%) requiring higher doses than is recommended in the summary of product characteristics due to pharmacokinetic breakthrough hemolysis. In 2021 ravulizumab became available for use on the National Health Service and 237 patients treated with eculizumab have switched to ravulizumab. The majority of patients have had no adverse clinical issues but 3 patients have reverted back to eculizumab due to side effects. Thirty-five patients (6.9%) presented after 2021 and have been treated with ravulizumab alone. The median PNH granulocyte clone size at initiation of C5i was 85.5% (interquartile range 67-95%, available in 494 cases). Indications for complement inhibition: hemolytic disease: 325 patients (63.8%), thrombosis: 93 patients (18.3%), other complication due to PNH: 27 patients (5.3%), pregnancy: 26 patients (5.1%), pre-allogeneic bone marrow transplantation: 14 patients (2.8%), exceptional cases after discussion at a joint service meeting: 17 patients (3.3%), indication unknown: 7 patients (1.4%). Mean hemoglobin levels 12 and 24 months after initiating C5i were 107.7g/l (in 421 patients) and 108.6g/l (in 374 patients) respectively. In the most recent 12 months on C5i 123 out of 446 (27.6%) patients needed transfusions with 94 of the 123 (76.4%) requiring 3 or more transfusions. There were 23 thrombotic events on C5i (4.5%). Over the 20-year period, 91 patients have died equating to a 79.0% survival rate (Figure 1). Infection was implicated in the cause of death in 44 individuals (48.4%). There have been 11 cases of Neisseria Meningitidis septicemia in 10 individuals over 3,130 years of treatment with C5i and 1 death from this, equating to a meningococcal infection rate of 1 event per 284 years and 6 months. Clonal evolution to acute leukemia occurred in 7 cases (1.4%) and to myelodysplastic syndrome in 10 cases (2.0%). Conclusions: We report 20 years of C5i experience from the UK, for patients with PNH. Our experience of treatment with C5i confirms the continued long-term safety and efficacy of eculizumab and ravulizumab. Overall survival remains high on C5i and complications such as thromboses, meningococcal infection and clonal evolution are uncommon events. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lactate dehydrogenase versus haemoglobin: which one is the better marker in paroxysmal nocturnal haemoglobinuria?

Lactate dehydrogenase versus haemoglobin: which one is the better marker in paroxysmal nocturnal haemoglobinuria?

Long-Term Survival Benefit of Eculizumab Treatment in Patients with Paroxysmal Nocturnal Hemoglobinuria: Data from the International PNH Registry

Long-Term Survival Benefit of Eculizumab Treatment in Patients with Paroxysmal Nocturnal Hemoglobinuria: Data from the International PNH Registry

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.

Is Nature Truly Healing Itself? Spontaneous Remissions and Clonal Replacement in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is considered to be curable only through the means of allogeneic HSCT. One of the many fascinating and scientifically instructive aspects of the pathogenesis of this disease is the rare possibility of its spontaneous remission with disappearance of PNH clone and abatement of clinical symptoms, which has always captivated the research community. Due to the orphan nature of the condition, no clinical predictors have been identified so far as harbingers of this phenomenon. In a classical scenario, exhaustion of PNH clone may be associated with reappearance of aplastic anemia (AA), in which PNH clone reflects a semi-maladaptive attempt of recovery. Consequently, one could stipulate that the retraction of PNH clone(s) would have to be associated with a compensatory re-expansion of normal hematopoiesis should normal counts be maintained. The recent insights into the AA/PNH pathobiology shed light onto molecular underpinnings of polyclonal vs oligoclonal hematopoiesis and their dynamics. Here, through application of NGS we attempted to better discern the mechanism of PNH spontaneous remission taking advantage of our internal cohort of PNH patients.Among 92 patients with a diagnosis of hemolytic PNH (M:F ratio 0.88, median age 38 years, range 9-84) 41% were primary PNH (pPNH) while 59% were secondary to AA (sPNH). Overall, patients were clinically followed-up for a median time of 68 months (2-339). Median granulocyte clone size was 73% (22-99) with the majority of cases being classified as having a type III dominant red blood cells (RBCs) clone (80%) while 20% type II. Within this cohort, a total of 3 patients underwent spontaneous remission.UPN1 was a 69-year-old male diagnosed with pPNH at the age of 46 after an episode of deep vein thrombosis. He had been managed with prednisone, transfusions and anti-coagulation because of recurrent thrombotic episodes. Once available, he was started on eculizumab and later continued on ravulizumab. His initial flow cytometry study revealed the presence of a type III RBCs clone of 40% and a granulocyte clone of 89%. After 9 years of anti-complement therapy, the patient's clone started a slow decrease and the most recent study revealed a granulocyte clone of 0.02%. Molecular analysis performed at the time of eculizumab start showed a co-dominant mutational configuration by variant allelic frequency (VAF) with PIGA deletion (p.94_95del; VAF 29%) and a BCOR nonsense (p.Y1446X; VAF 27%). No HLA class I/II mutations were found in two longitudinal samples collected 1 year before and after eculizumab start. However, at the last sequencing performed after the complete disappearance of the PNH clone, the patient developed ASXL1 (p.E635Rfs*, VAF 26%) and ZRSR2 (p.E120Gfs*, VAF 42%) mutations along with retraction of the previous PIGA lesion. No decrease in blood counts was noted.UPN2 was a 58-year-old male initially diagnosed with severe AA at the age of 48 and treated with ATG + CsA. At that time, he had a co-existing PNH granulocyte clone of 28%. After 1 year from IST his PNH clone dropped to 1% and since then has been consistently below 1%. Patient has never received anti-complement therapy. At the time of PNH clone retraction, no HLA class I/II or myeloid driver mutations were found and PIGA mutations were not detectable. However, longitudinal molecular studies performed after disappearance of PNH clone revealed the acquisition of ASXL1 p.Q512X mutation at an initial VAF of 23%, which doubled (45%) at last follow-up 5 years later while normal counts persisted.UPN3 was instead a 59-year-old lady diagnosed with pPNH at the age of 30. She had a granulocyte clone as high as 43% with a type II RBCs clone of 17% and a typical PIGA splice site c.981+1G>A mutation (VAF 15%). She was initially treated with transfusions and steroids and her course was complicated by a cerebral venous sinus thrombosis. Patient was eventually given eculizumab and her PNH clone started decreasing until it vanished (last 0.04%) after 8 years. Analysis of samples prior to and after PNH disappearance did not show any HLA class I/II nor myeloid driver gene mutations with absence of PIGA alterations at last sequencing.PNH spontaneous remissions are rare events. In addition to be replaced by polyclonal hematopoiesis, PIGA clones may be swept by CHIP lesions in myeloid genes (e.g. ASXL1) characterized by improved fitness advantage in a process of Darwinian selection. [Display omitted] DisclosuresMaciejewski: Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy.

ANALYSIS OF BASELINE CHARACTERISTICS, DISEASE BURDEN AND LONG-TERM FOLLOW-UP OF 167 PATIENTS WITH BRAZILIAN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA - ANOTHER NATURAL HISTORY

Paroxysmal nocturnal hemoglobinuria (PNH) may occur as classical hemolytic disease or a small PNH clone found in a patient with bone marrow failure. To describe clinical features and long-term follow-up of 167 patients with PNH and demonstrate differences between disease categories. Multiparametric flow cytometry performed on 1025 patients referred from Jan/2000 to Dec/2019 found 167 (16.3%) confirmed PNH clone, 87M/70F. Clinical characteristics at first visit, and laboratory results, incidence of thrombosis, treatment and outcome during follow-up visits were considered for statistical analysis. Most patients (89.2%) had hypocellular bone marrow at diagnosis; 55(32.9%) developed hemoglobinuria, and 22(13.2%) developed thrombosis during monitoring. Mean age at aplasia diagnosis 28.4 years (range 5.6 - 71.2), and mean age at PNH clone detection 31.0 years (7.2 - 71.3 years). Clonal evolution occurred in 77 (46.1%) patients at a median of 4 years (range 6-281 months) after the onset of cytopenia. The cohort was divided into 15 classic PNH, 55 hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 definitive bone marrow disease with minor PNH clones - subclinical PNH (sc-PNH). Subclinical patients had lower erythrocyte (2.0% vs 24.0% vs 57.8%) and granulocyte PNH clones (11.7% vs 58.8% vs 81.2%) than PNH/AA and classic PNH, respectively. LDH, reticulocytes, absolute neutrophils and bone marrow cellularity were all lower in sc-PNH than in the hemolytic group. Pearson's analysis showed a statistically significant correlation (p < 0.001) between PNH granulocyte clone and RBC clone (0.697), LDH (0.602), absolute neutrophils (0.310), platelet count (0.430), absolute reticulocytes (0.546), total bilirubin (0.330), and bone marrow biopsy (0.435). PNH granulocyte clone greater than 50% was found in 16/22 thrombotic patients. Mean granulocyte clone was 89.1% (3.4%-99.2%) versus 12.3%(0.1%-34.0%), p < 0.001, and erythrocytes 28.4% (3.4-99.8) versus 0.7% (0.1-14.0) in thrombotic versus the remaining 145 patients. Treatment was highly heterogeneous during this 20-year follow-up: immunosuppressive therapy with CSA and/or thymoglobulin was given to 72 patients in the subclinical group (73.1%) and 45 (64.3%) in the hemolytic group; 52 patients received HSCT (31.1%) of which 40 in sc-PNH and 11 in PNH/AA; a total of 19 patients received Eculizumab after 2008 and 3 patients never received treatment. Incidentally, there were 17 thrombotic events in hemolytic patients (38.6%) diagnosed by 2007, compared to only five events (19.2%) in patients diagnosed after 2008. The average follow-up period was 74 months (1 - 330 months), with 31 deaths in total (80.4% OS). Sepsis was the leading cause of sc-PNH mortality (16/18, 88.8%), and thrombosis in both hemolytic groups (11/13, 84.6%). Log rank test shows similar survival when comparing patients with diagnosis until and after 2007 (p = 0.04). this study suggests clonal evolution in the long-term follow-up of aplastic patients and confirms the observation that high levels of PNH and LDH clones are associated with life-threatening hemolysis and thrombosis, while small PNH clones and young age are associated with the subclinical form of the disease. Although without statistical significance, the cumulative incidence of thrombosis in the group diagnosed after 2008 appears to be lower than the previous period, perhaps due to the routine use of Eculizumab.

Analysis of baseline characteristics, disease burden and long-term follow-up of 167 patients with Paroxysmal Nocturnal Hemoglobinuria at a single center in Brazil

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure. MethodsDescribe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up. Results167 patients, mean age at diagnosis 28.4 (7.1–71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%). ConclusionLarge PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

Chronic Budd–Chiari syndrome in paroxysmal nocturnal haemoglobinuria

A 42-year-old woman with a sudden onset of abdominal pain requested to see us urgently. 5 years earlier she had been diagnosed with paroxysmal nocturnal haemoglobinuria (PNH) and had missed multiple follow-up appointments. Despite having a large PNH clone—greater than 50% of PNH granulocytes—the patient had been asymptomatic with only mildly elevated haemolytic parameters and treatment with eculizumab had been deferred.

Platelet counts on peripheral blood and Mean Platelet Volume as markers of clinical severity in Sickle Cell Disease

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure.Describe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up.167 patients, mean age at diagnosis 28.4 (7.1–71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%).Large PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

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.

Fibrinogen and Clot Structure Is Affected By Eculizumab in Patients with Paroxysmal Nocturnal Haemoglobinuria

Background Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic stem cell disorder in which an acquired somatic mutation of the PIGA gene in a haematopoietic stem cell results in the deficiency of key complement regulatory molecules, namely CD59 and CD55, from the surface of blood cells leading to an increased susceptibility of blood cells to complement-mediated activation +/- intravascular haemolysis and thrombosis. Thrombosis has been the leading cause of death in PNH. Multiple mechanisms of thrombosis in PNH have been proposed, including cross talk between the coagulation and complement system. This may lead to denser clots which are more resistant to fibrinolysis. Treatment with eculizumab, which inhibits complement factor C5 and thereby prevents terminal complement activation, reduces thromboembolic events in PNH, indicating that complement activation plays a significant role in the pathophysiology of thrombosis in this disease. Patients with other thrombotic disorders are known to form denser fibrin clots that incur greater resistance to fibrinolysis. It has not previously been explored whether changes to clot architecture contribute to thrombosis in PNH. Furthermore, the role of eculizumab in clot architecture is undocumented.Aims Characterise fibrin clot structure in patients with PNH and determine the effect of eculizumab on clot structure.Methods Informed consent was obtained from all participants. 53 patients from the PNH National Service (Leeds) were recruited. They were grouped according to the proportion of PNH granulocytes, <40% (n=18), 40> - < 90 %(n=18) and >90% (n=17) and reanalysed according to treatment with (n=19) or without (n=32) eculizumab. Plasma samples were obtained and ex vivo fibrin clot structure and function was analysed by permeation analysis of clot pore structure, turbidity analysis of clot formation and fibrinolysis, and fibrin ultrastructure by laser scanning confocal microscopy and scanning electron microscopy, according to methodology and protocols well established in our laboratory. The proportion of PNH granulocytes was measured by flow cytometry and fibrinogen levels by Clauss. P values of <0.05 were taken to indicate significance.Results The proportion of PNH granulocytes correlated with fibrinogen (r= -0.35, p=0.048), maximum absorbency (r= -0.34, p=0.046) and clot density (r= -0.36, p=0.04). Seventy per cent of patients with large proportions of PNH granulocytes were on eculizumab. Analysis of patients not on eculizumab showed a trend towards proportion of PNH granulocytes correlating positively with fibrinogen levels (r=0.42, p=ns). Patients on eculizumab had lower fibrinogen levels (3.1±0.2 v 3.6±0.1g/L, P=0.02), maximum absorbance (0.31±0.1 v 0.40±0.1, p=0.028) and fibre density/100µm (18.5±4.4 v 23.9±4.2, p=0.006), indicative of a less dense clot.Summary/Conclusions This is the first study to demonstrate that patients on eculizumab showed reduced fibrinogen, fibre thickness and fibre density. Our data indicate that the beneficial effects of eculizumab in PNH is partly due to reduced complement activation and attenuated inflammatory response, leading to lower fibrinogen levels and a less thrombotic fibrin clot structure. These novel data help to explain the impact of complement inhibition in this disease. [Display omitted] DisclosuresArnold:Alexion Pharmaceuticals, Inc.: Honoraria. Griffin:Alexion Pharmaceuticals, Inc.: Honoraria. Munir:Janssen: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Roche: Honoraria; AbbVie: Honoraria; Gilled: Honoraria. Hillmen:Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Celgene: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding. Richards:Alexion Pharmaceuticals, Inc.: Honoraria. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Frequency and perturbations of various peripheral blood cell populations before and after eculizumab treatment in paroxysmal nocturnal hemoglobinuria

While red blood cells (RBCs) and granulocytes have been more studied, platelets and reticulocytes are not commonly used in paroxysmal nocturnal hemoglobinuria (PNH) flow-cytometry and less is known about susceptibility to complement-mediated destruction and effects of anti-complement therapy on these populations.We performed flow-cytometry of RBCs and granulocytes in 90 PNH patients and of platelets and reticulocytes in a subgroup (N = 36), to unveil perturbations of these populations during PNH disease course before and after anti-complement treatment.We found that platelets and reticulocytes were less sensitive to complement-mediated lysis than RBCs but not as resistant as granulocytes, as shown by mean sensitive fraction (difference in a given PNH population vs. PNH granulocyte clone size). In treated patients, reticulocytes, platelets, RBCs (with differences between type II and III) and granulocytes significantly increased post-treatment, confirming the role of PNH hematopoiesis within the context of anti-complement therapy. Moreover, we found that PNH platelet clone size reflects PNH granulocyte clone size. Finally, we established correlations between sensitive fraction of PNH cell-types and thrombosis.In sum, we applied a flow-cytometry panel for investigation of PNH peripheral blood populations' perturbations before and after eculizumab treatment to explore complement-sensitivity and kinetics of these cells during the disease course.

Thrombotic Events with Neisseria Meningitidis Vaccination in Patients with Paroxysmal Nocturnal Hemoglobinuria, UK Experience

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic and thrombotic condition. Patients can experience severe anemia due to intravascular hemolysis, thrombotic events, renal impairment and pulmonary hypertension. Symptomatic patients are treated with complement inhibitors either in clinical trials or with eculizumab the only licensed treatment in the UK. Due to the mechanism of action of eculizumab, it increases susceptibility to Neisseria infection, including Neisseria meningitidis. To reduce this risk of infection, worldwide practice is for patients to be vaccinated at least 2 weeks prior to receiving eculizumab (serogroups A, C, Y, W 135 and B). It was noted within the PNH National Service at Leeds (UK) that a small number of patients deteriorated with enhanced intravascular hemolysis and thrombosis during the period between vaccination and eculizumab, leading to a review of practice. We report five of 121 patients with events in the intervening 2 weeks between vaccination and commencement of eculizumab from 2002-2012:A 44 year female presented with hemolysis and hemoglobinuria, with a granulocyte PNH clone of 99.4%. She was transfusion dependent and on anticoagulation. She consented to the PNH pilot eculizumab study, undergoing meningococcal vaccination as per protocol. Twenty two days later, she suffered an ischemic stroke with left hemiplegia and permanent weakness, resulting in exclusion from the study. Two years later she received eculizumab in the TRIUMPH study.A 37 year male presented with hemoglobinuria and fatigue with a granulocyte PNH clone of 99.58%. He had significant hemolysis, managed initially with warfarin and blood transfusions. He consented to start eculizumab and received meningococcal vaccination. 4 days later he presented with a symptomatic right hepatic vein thrombosis, promptly commenced eculizumab.A 29 year male, with abdominal pain and hemoglobinuria for 3 years developed a stroke and portal vein thrombosis leading to a diagnosis of PNH, with a granulocyte PNH clone of 84.99%. He commenced anticoagulation. Four months after the stroke he received meningococcal vaccination in preparation for scheduled commencement with eculizumab. He experienced a left central retinal vein thrombosis 15 days after its administration prior to starting eculizumab.A 47 year old male, was diagnosed with haemolytic PNH but only had mild symptoms and anaemia. Twenty four years later, he developed increasing hemolysis and symptoms; granulocyte PNH clone of 96.45%. Eculizumab was planned and he received meningococcal vaccination, but presented ten days later with acute renal failure secondary to massive intravascular haemolysis, necessitating emergency eculizumab therapy.A 35 year female, with a granulocyte PNH clone of 99.87%. Although she had active intravascular hemolysis, eculizumab was declined, and anticoagulation commenced. Four years later she consented to start eculizumab, receiving meningococcal vaccination. She was admitted 24 hours later with a stroke and commenced eculizumab the same day, but has persistent neurological impairment to date. See Table 1 Discussion: The close time proximity of these serious events to the patients' vaccinations raised concern that the complement system was being activated by administration of the vaccine, precipitating complications of PNH. It is also concerning that 4 of the 5 patients experienced thrombotic events despite therapeutic anticoagulation, confirming that anticoagulation only partially mitigates the risk of thrombosis in patients with PNH. The decision was taken to administer the vaccination immediately after the first dose of eculizumab, with therapeutic doses of antibiotic (ciprofloxacin 500mg bd) for the first 14 days post vaccination, followed by long term meningococcal prophylaxis (penicillin V or erythromycin 500mg bd) whilst receiving a complement inhibitor. Since this change in practice in 2012 we have commenced eculizumab therapy in 211 patients with no similar complications as described. Thus the change in practice appears to reduce the occurrence of these severe complications associated with vaccinations prior to initiating anti-complement therapy. Whilst it is possible these events could have been caused by the underlying condition of PNH, we would advise colleagues to also adopt a change in practice to reduce potentially significant complications. Disclosures Arnold: Alexion Pharmaceuticals: Honoraria. Kelly:Alexion: Honoraria. Munir:F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland; Alexion: Honoraria. Pike:Apellis: Research Funding. Riley:Alexion: Honoraria. Griffin:Alexion Pharmaceuticals: Honoraria, Other: Conference Support; Biocryst: Membership on an entity's Board of Directors or advisory committees. Hillmen:Acerta: Other: Financial or material support; Roche: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; Pharmacyclics: Other: Financial or material support, Research Funding; Janssen: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; Apellis: Consultancy, Research Funding, Speakers Bureau; Alexion: Consultancy, Research Funding, Speakers Bureau; Gilead: Other: Financial or material support, Research Funding.

Real-World Efficacy of Eculizumab for Paroxysmal Nocturnal Hemoglobinuria in South Korea: Paradox of Eculizumab

Purpose Eculizumab is reported to be highly effective in managing patients with paroxysmal nocturnal hemoglobinuria (PNH). However, eculizumab therapy is associated with high cost and thus is inaccessible to all patients with PNH. Additionally, there are some restrictions in the coverage of PNH therapy from the health insurance, which varies depending on each country. In South Korea, majority of the cost involved in eculizumab therapy is supported by the National Health Insurance Service (NHIS) with very limited indication. The patients with PNH must satisfy all of the following criteria to be eligible for financial support from NIHS: (1) PNH granulocyte count ≥ 10%; (2) lactate dehydrogenase ≥ 1.5 times the upper limit of normal; (3) at least 4 units of red blood cells must have be transfused in the past 12 months (transfusion-dependent anemia (TDA)); and (4) exhibit at least more than one of the PNH-related complications (except TDA), which include thrombosis, renal insufficiency, pulmonary insufficiency, or recurrent smooth muscle spasm. These criteria are the narrowest indication for eculizumab therapy compared with those used by other countries. Hence, we performed a nation-wide study including all the patients with PNH in South Korea using the NHIS database to investigate the real-world efficacy of eculizumab. Methods The National Health Insurance Database of the NHIS in South Korea was used to collect the data of patients diagnosed with PNH between January 1, 2002 and December 31, 2016. Propensity score (PS) method was used to evaluate the real-world effects of eculizumab on patient survival. For PS method, the patients with PNH were divided into the following two groups: (1) patients treated with eculizumab; and (2) patients not treated with eculizumab. Additionally, the patients used for the analysis were limited to those who are surviving from the time of introduction of eculizumab in South Korea (October 1, 2012). In total, 11 variables were selected for PS matching according to the indications for using eculizumab in South Korea: age, sex, TDA, venous thrombosis, arterial thrombosis, AA, MDS, acute renal failure (ARF), chronic renal failure (CRF), prescription of opioid analgesic drug (≥ 2 times), and pulmonary hypertension. The patients diagnosed with acute leukemia were excluded. The time point of the matching was designated as the date on which the eculizumab was first administered. All survival or complication analyses were performed using the Cox proportional hazard regression model. The duration of PNH was used as the explanatory variable to estimate the proper treatment effect. Results Eculizumab-treated patients exhibited significantly higher survival rate than the eculizumab-untreated patients (4-year survival after propensity score matching, 98.31% vs. 79.67%, p = 0.0489). The mean RBC transfusion units per 12 months after eculizumab therapy was significantly lower than that before eculizumab therapy (5.75 units vs. 12.28 units, p &lt; 0.0001). The median time for the first transfusion in the eculizumab-treated group was significantly longer than that in the eculizumab-untreated group (p = 0.0078). The 4-year transfusion-independence rate for the eculizumab-treated group was significantly higher than that for the eculizumab-untreated group (20.81%; 95% CI, 10.71-33.20%, vs. 10.24%; 95% CI, 4.17-19.50%). There was no significant difference in the incidence of new documented complications related to PNH between the two groups. These complications include venous thrombosis (p = 0.886), arterial thrombosis (p = 0.112), acute renal failure (p = 0.745), chronic renal failure (p = 0.827), use of opioid analgesics (p = 0.142), and pulmonary hypertension (p = 0.396). Conclusions Eculizumab therapy for high-risk PNH patients may effectively improve the survival rate and reduce the transfusion requirement. Paradoxically, the eculizumab-treated patients with severe PNH exhibit higher survival rate than the eculizumab-untreated patients with less severe PNH. Disclosures No relevant conflicts of interest to declare.

Analysis of platelets by flow cytometry in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

The marked pro-thrombotic tendency in PNH is likely to be at least partly due to the population of platelets derived from the abnormal stem cell clone. However, identification of GPI (−) platelets by flow cytometry can be technically difficult. Here we describe a technique that involves the addition of aspirin immediately after the separation of platelet rich plasma and the use of gel filtration to isolate platelets away from plasma proteins and other blood cells. In a study of 92 analyses of samples from 50 patients, we have demonstrated that the percentage of PNH platelets correlates well with the percentage of PNH granulocytes. We also provide data on several cases where there was an extreme discrepancy between the proportion of PNH granulocytes and red cells; in these cases, the demonstration of abnormal platelets suggests that the patient is likely to be at risk of thrombosis. We believe this test will be potentially useful in the evaluation of samples from such patients and may serve as a tool to investigate the causes of hypercoagulability in PNH.

S865 CHANGE IN CLONE SIZE DOES NOT PREDICT RISK OF MAJOR ADVERSE VASCULAR EVENTS: RESULTS FROM THE INTERNATIONAL PNH REGISTRY

Background:In paroxysmal nocturnal hemoglobinuria (PNH), the granulocyte clone size is often used as an indicator of disease severity. However, the prognostic value of changing clone size on clinical outcomes in PNH is not well understood.Aims:To assess the prognostic value of change in PNH granulocyte clone size on the risk of developing major adverse vascular events (MAVE) or high disease activity (HDA: elevated hemolysis [lactate dehydrogenase {LDH} ≥1.5 x upper limit of normal] and one or more PNH‐related symptom[s]) in patients with PNH.Methods:The International PNH Registry is the largest worldwide observational study of patients with a diagnosis of PNH or a detected PNH clone as low as 0.01%. This study assessed patients with a baseline value for PNH clone size (defined as the closest value to disease onset) and at least one additional clone size value, who were untreated with complement inhibitor therapy at enrollment, and had ≥12 months of untreated follow‐up. Patients were stratified by clone size at baseline and change in clone size was estimated using regression models of clone size vs duration from disease start, adjusted for baseline clone size. Patients were categorized as having stable, increasing, or decreasing clone size depending on the slope, where a change in clone size &gt;10% per year was considered increasing/decreasing. Prognostic value of change in clone size was assessed using Poisson regression to estimate rate ratios of MAVE and logistic regression to estimate odds ratios of HDA during follow‐up, with stable clone size as the reference. Adjusted models included baseline LDH ratio category, history of bone marrow disease at baseline, baseline clone size, and duration.Results:As of April 3, 2018, a total of 1894 patients were available for analysis: cohort (COH) 1 (clone size &gt;0.01% to ≤1%, n = 381); COH 2 (clone size &gt;1% to ≤5%, n = 318); COH 3 (clone size &gt;5% to ≤10%, n = 159); COH 4 (clone size &gt;10% to ≤50%, n = 480); COH 5 (clone size &gt;50% to ≤100%, n = 556). Median duration was 3.5 years. Clone size was stable in only 16.2% of patients, as compared with 45.3% increasing and 38.5% decreasing; stable clone size was primarily seen in patients with a clone size ≤1% at baseline (58% of patients in COH 1 with a stable clone), with less than 10% of patients with a stable clone in the other cohorts. Clone size change over time was highly prognostic of HDA; patients with increasing and decreasing clone size had an increased risk of HDA, compared to patients with stable clone size, although this may be due to patients with a stable clone largely in COH 1 with a low risk of HDA; after adjusting, we see no increased risk of HDA in patients with decreasing clone size, but the increased risk of HDA in patients with increasing clone size is maintained (Table). Due to limited data on covariates at baseline, only 156 patients were available for this adjusted analysis. There was no difference in the MAVE rate in patients with increasing or decreasing clone size as compared with those with a stable clone size. The lack of effect was maintained even after adjusting for covariates (Table).Summary/Conclusion:This is the first analysis of the prognostic value of PNH clone size changes on clinical outcomes over time in patients with PNH. Increasing clone size over time was predictive of patients experiencing HDA during their disease course. Interestingly, no difference in the MAVE rate was seen in patients with increasing or decreasing clone size, suggesting that change in clone size does not change the increased risk of MAVE in patients with PNH.image

PS1119 PNH CLONE SIZE BY FLOW CYTOMETRY AND ITS CORRELATION TO PIG GENE MUTATION

Background:Routine diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) is based on flow cytometric measurement of PNH clone in RBC and granulocyte. However there is no well‐established consensus on the clinically significant size of PNH clone.Aims:So we investigated whether quantitative results of PNH clone size measured by flow cytometry (FCM) correlate with mutant burden of PIG gene.Methods:A total of 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM was enrolled. We performed ultra‐deep sequencing for PIGA, PIGM and PIGX genes on these 89 consecutive specimens. Also, we followed up 6 patients who were treated with Eculizumab and analyzed their laboratory changes including hemoglobin, lactate dehydrogenase level, PNH flow cytometry and PIG gene mutant burden. Treatment response was evaluated every 6 months.Results:Sixteen out of 63 patients had &gt;10% granulocyte PNH clone and they all had PIG gene mutation. Fifteen (93.8%) had PIGA mutation and one (6.3%) PIGM mutation. Patients with &gt;15% RBC PNH clone (n = 12) all had PNH‐related symptoms (p &lt;0.001). Granulocyte clone size showed better correlation with VAF than RBC clone size (Granulocyte r = 0.61, p = 0.01, RBC r = 0.39, p = 0.14). On the other hand, RBC clone size was better reflective of PNH‐related symptoms with a cut‐off of 15% (p &lt;0.001). We found no significant laboratory parameter for prediction of Eculizumab treatment response.Summary/Conclusion:Patients with &gt;10% granulocyte PNH clone by FCM were always accompanied by PIG gene mutation. Those with &gt;15% RBC PNH clone by FCM all had PNH‐related symptoms. Weak correlation between granulocyte clone size and VAF of PIG gene mutation implies that granulocyte clone reflects the real PNH clonal burden.image

Prognostic Value of Clone Size in Paroxysmal Nocturnal Hemoglobinuria (PNH) for Thrombotic Events in Untreated Patients in the International PNH Registry

Background/Objective: The association between paroxysmal nocturnal hemoglobinuria (PNH) granulocyte clone size at disease onset and outcomes in patients with PNH remains unclear. Most but not all reports examining the relationship have shown a positive correlation between clone size and thrombotic events [TEs] in patients with PNH, but without a clear temporal association showing the prognostic value of clone size on the risk of TEs. The ongoing International PNH Registry (NCT01374360) is the largest prospective, observational study of patients with PNH conducted to date. The objective of this analysis was to examine the relationship between PNH granulocyte clone size at disease onset and risk of thrombosis after disease onset while untreated with a complement inhibitor in patients enrolled in the Registry.Methods: The current analysis included patients enrolled in the Registry as of April 2018 who had known demographics, were untreated with complement inhibitor therapy at enrollment, and had ≥12 months of untreated follow-up after disease onset. Baseline was defined as disease onset (earliest of a reported PNH clone, date of PNH diagnosis, or reported PNH symptom), and patients were stratified into 5 cohorts based on clone size at baseline (using the earliest reported clone prior to enrollment): cohort 1, clone size: 0.01-1%; cohort 2, clone size: >1-5%; cohort 3, clone size: >5-10%; cohort 4, clone size: >10-50%; cohort 5, clone size: >50%. Event rates for TEs and all major adverse vascular events (MAVEs; including TEs) were calculated for the time period from baseline to last follow-up. Other outcomes of interest included LDH ratio (LDH/LDH upper limit of normal [ULN]), hemoglobin levels, platelet counts, absolute neutrophil counts, and absolute reticulocyte counts at last follow-up.Results: A total of 2489 patients were eligible for the analysis. The majority of patients in the overall study population were female (54.0% [1343/2489]) and white (79.2% [1967/2484]). Mean (standard deviation [SD]) age at PNH start ranged from 36.4 (16.53) in cohort 5 to 44.2 (20.70) in cohort 1. Median time from baseline to last follow-up was 3.7 years in cohort 1, 4.3 years in cohort 2, 4.7 years in cohort 3, 5.6 years in cohort 4, and 6.8 years in cohort 5. Results for the outcomes of interest are summarized in the Table. All cohorts showed a risk of MAVE and TE during follow-up. Although estimated rates of MAVE and TE were highest in the >50% clone size cohort, there was no difference in the rate of MAVE or TE during follow-up across the 4 cohorts with clone size <50% at disease onset. Mean LDH ratio (LDH/LDH ULN) at last follow-up showed a statistically significant difference by clone size at baseline among the cohorts, ranging from a mean (SD) of 1.1 (1.34) in the patients with clone size <1% and increasing to 5.1 (3.81) in the patients with clone size >50% (P<0.0001). No difference in hemoglobin level at last follow-up was observed in the smaller clone size cohorts, although mean hemoglobin level was lower in patients with clone size >50% (P<0.0001). Similar trends were seen in mean platelet and absolute neutrophil counts across the smaller clone size cohorts, while patients with clone size >50% showed higher values (P<0.0001). Mean absolute reticulocyte count at last follow-up was lowest in patients with clone size 0.01-1%, and were incrementally higher in each successive clone-size cohort (P<0.0001).Conclusions: In this study, all patients with a PNH clone at disease onset were at risk for TEs and other MAVEs. Patients in the highest baseline PNH clone size strata (clone size >50%) had an approximately 2-times higher risk of TEs than patients with smaller clone sizes; Patients with small clone sizes (0.01-1%) were older and showed a higher prevalence of BMD. There was no difference in the prognostic value of clone size at disease onset on the risk of TEs and other MAVEs in patients with small (0.01-1% and 1-5%) versus medium-sized (5-10% or 10-50%) clones. [Display omitted] DisclosuresPeffault De Latour:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy. Kulasekararaj:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support . Larratt:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Dingli:Millennium Takeda: Research Funding; Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.; Millennium Takeda: Research Funding; Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.. Wilson:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Gustovic:Alexion Pharma GmbH: Employment, Equity Ownership. Kulagin:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.