Classic Paroxysmal Nocturnal Hemoglobinuria Research Articles

PIG-a Gene Expression Deficiency Association with Reduced DNA Damage Checkpoint Response and Activation

Introduction:Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disease caused by mutation in the PIG-A gene. When expressed, it encodes a protein that is crucial in glycophosphatidylinositol (GPI) anchor biosynthesis. The mutation of this gene causes plasma membrane GPI anchor biosynthesis deficiency, cell membrane structure alteration, signal transduction pathway blockage and vulnerability to complement complex attack. Despite these deficiencies, PNH cells have a clonal advantage and this dominant expansion is the hallmark of PNH progression. The mechanism of this growth, however, is still unknown. Recently, multiple studies demonstrated that malignant cells harboring the PIG-A mutation appear to have a higher degree of genomic instability and disease progression. This led to the proposal of using the PIG-A gene mutation frequency as the biomarker of mutagenesis to evaluate the transgenic animal mutation or mutagenicity of test compounds. The PIG-A gene mutation assay is even considered as a valuable tool for quantifying mutational events in vivo and in vitro despite the mechanism remaining unknown. This study investigated the impacts PIG-A mutation in genomic stability, DNA damage response, and DNA damage checkpoint activity, which as a measure of cellular stability as well as a possible mechanism for clonal expansion.Methods:To investigate the relationship between PIG-A gene mutation status and DNA damage checkpoint activity we looked at 45 high-risk MDS/AML patients, 3 classic PNH patients and 12 healthy controls. Additionally, the TF-1 leukemia cell line was used to evaluate PIG-A wild-type vs PIG-A CRISPR knockout vs PIG-A siRNA transient suppression. H2AX/ɣH2AX, ⍺-pChk1, ⍺-pChk2, ⍺-ubPCNA, and ⍺-pRPA proteins and RNA levels were used as a quantitative indicator of DNA damage repair activity via qTR-PCR and western blot. The comet assay and fiber combing assay were further applied to explore the DNA damage and cellular replication events. RNAseq analysis was used to explore the impact of PIG-A mutation in global gene expression.Results:The in vitro results from the leukemia cell lines with various PIG-A mutation statuses showed that the PIG-A mutation decreases ɣH2AX expression. PIG-A mutation was also associated with down-regulated expression of ⍺-pChk1, ⍺-pChk2, ⍺-ubPCNA, and ⍺-pRPA proteins. The decreased levels of those gene expressions indicate that there is increased genomic stability and less repair activity at the DNA damage response checkpoint. The results of the comet assay support this data with the PIG-A mutated cells exhibiting significantly (p<0.01) shorter tail lengths than the wild-type counterparts. The fiber-combing assay showed a reduced helicase activity for DNA replication in PIG-A mutated cells.In patient samples, subjects with PNH showed significantly lower levels of ɣH2AX expression when compared to both healthy controls and AML/MDS patients. The comet assay revealed that PNH patients had decreased DNA double strand breaks and fiber combing assay showed a reduced DNA repair activity when compared to normal controls and AML patients with intact PIG-A gene expression (p<0.01).Additionally, RNAseq analysis reveals that the expression of 10/10 genes involved in the DNA repair mechanism were down-regulated in PIG-A mutated cells when compared to wild-type control in both cell lines and patient samples.Conclusion:The above-mentioned results showed that there were decreased expressions of both DNA damage response checkpoint genes and repair genes in both the PIG-A CRISPR knockout leukemia cell lines as well as in the PNH patients. PIG-A mutation is globally associated with reduced DNA damage response capability and increased cellular stability. Our finding explains, at least partially, why PIG-A gene mutation status could be seen as a biomarker of mutagenesis and how PNH cells dominantly expend via clonal escape. DisclosuresNo relevant conflicts of interest to declare.

Fluorescent Aerolysin (FLAER)-based paroxysmal nocturnal hemoglobinuria (PNH) screening: a single center experience from India.

Fluorescent aerolysin (FLAER) has been recommended as an important part of antibody panel used for flow cytometric detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study was aimed to observe the frequency of PNH-positive clones and their sizes in patients screened for various indications. A retrospective analysis of 624 patients screened over a period of 30 months. Frequency and size of clone sizes noted, and laboratory parameters were compared among different groups of patient being screened. There were 445 adults and 179 pediatric patients. Indications for screening included AA (n = 433), myelodysplastic syndrome (MDS) (n = 34), hemolytic anemia (n = 84), and thrombophilia workup group (n = 63). PNH clones were found in 39.03%, 5.88%, 26.19%, and 1.59% cases, respectively. No significant difference among adult or pediatric population was noted. The bone marrow failure (BMF) group [AA and MDS] with PNH clone had a significantly lower clone size (Median- 2.7%) as compared to classic PNH group (Median-77.2%). Most of the classic PNH cases (78.26%) and a small proportion of AA (9.9%) showed a large clone size (>50%). In spite of having large clone size, there was a significant difference between the median LDH values of these two groups (2511.5 vs 593 U/L). FLAER-based screening detects the presence of PNH clone in a high proportion of AA patients and some MDS patients. These patients usually have a small clone size. Even if they have a large clone, it does not get translated into a high LDH or severe clinical symptoms.

A retrospective study of paroxysmal nocturnal hemoglobinuria in pediatric and adolescent patients

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease, especially in children, characterized by intravascular hemolysis, thrombotic events, serious infections and bone marrow failure. We describe 16 patients who were diagnosed with PNH in childhood or adolescence. The time interval between the onset of symptoms and the PNH diagnosis and its treatment were compared in patients with classic PNH versus PNH associated with bone marrow disorder (PNH/BMD). A greater delay in diagnosis was observed in classic PNH compared to PNH/BMD patients. The first group of patients had higher levels of LDH, total bilirubin and absolute reticulocyte count and a bigger PNH clone size compared to PNH/BMD patients; also thrombotic events were observed only in the classic form of PNH. Conversely, PNH/BMD patients showed lower median levels of platelets. Apart from standard supportive measures, four patients with classic PNH received eculizumab whereas four patients with PNH/BMD underwent hematopoietic stem cell transplantation. Our series confirm that the most frequent presentation of PNH in the pediatric-adolescent age is PNH/BMD. The delay between the onset of symptoms and PNH diagnosis is relevant principally in the classic form. Moreover, our study showed that any case of unexpected thrombosis represents a criterium to perform a PNH screening.

Efficacy of eculizumab in paroxysmal nocturnal hemoglobinuria patients with or without aplastic anemia: prospective study of a Korean PNH cohort.

BackgroundPatients with paroxysmal nocturnal hemoglobinuria (PNH) often have concurrent aplastic anemia (AA). This study aimed to determine whether eculizumab-treated patients show clinical benefit regardless of concurrent AA.MethodsWe analyzed 46 PNH patients ≥18 years of age who were diagnosed by flow cytometry and treated with eculizumab for more than 6 months in the prospective Korean PNH registry. Patients were categorized into two groups: PNH patients with concurrent AA (PNH/AA, N=27) and without AA (classic PNH, N=19). Biochemical indicators of intravascular hemolysis, hematological laboratory values, transfusion requirement, and PNH-associated complications were assessed at baseline and every 6 months after initiation of eculizumab treatment.ResultsThe median patient age was 46 years and median duration of eculizumab treatment was 34 months. Treatment with eculizumab induced rapid inhibition of hemolysis. At 6-month follow-up, LDH decreased to near normal levels in all patients; this effect was maintained until the 36-month follow-up regardless of concurrent AA. Transfusion independence was achieved by 53.3% of patients within the first 6 months of treatment and by 90.9% after 36 months of treatment. The mean number of RBC units transfused was significantly reduced, from 8.5 units during the 6 months prior to initiation of eculizumab to 1.6 units in the first 6 months of treatment, for the total study population; this effect was similar in both PNH/AA and classic PNH.ConclusionThis study demonstrated that eculizumab is beneficial in the management of patients with PNH/AA, similar to classic PNH.

Long-Term Outcome of Allogeneic Stem Cell Transplantation in Patients with Paroxysmal Nocturnal Hemoglobinuria with or without Aplastic Anemia

Background: Although recently, Eculizumab, humanized monoclonal antibody directed against complement component C5, has used increasingly for the patients with hemolytic paroxysmal nocturnal hemoglobinuria (PNH), allogeneic stem cell transplantation (allo-SCT) can be curative treatment option especially for PNH patients with combined aplastic anemia (AA). The aim of the present study was to evaluate long-term outcome of allo-SCT in patients with AA/PNH. In addition, patients with classic PNH who underwent allo-SCT in the pre-eculizumab era were also evaluated.Methods: Total of 33 patients with PNH clones underwent allogeneic SCT at our institution between Jan 1998 and Jan 2016. Among them, seven patients had classic PNH and 26 patients with cytopenia had AA/PNH (with bone marrow evidence of a concomitant AA).Results: There were 21 male and 12 female patients with a median age of 34 years (range, 13-56 years). Pre-transplant GPI-AP deficient neutrophils and erythrocytes were 5.6% (0-92) and 21% (0-98.5), respectively. Median white blood cell, absolute neutrophil count, hemoglobin, and platelet at transplant were 2.4×109/L, 0.8×109/L, 7.7 g/dL, and 27×109/L, respectively. Median LDH level was 727 U/L (232-7721 U/L) and 19 (58%) patients had LDH ≥1.5x upper limit of normal. Classic PNH (n=7) and AA/PNH [SAA (n=15), VSAA (n=9), or non-SAA (n=2)] received SCT from HLA-matched sibling (MSD, n=24), unrelated (URD, n=7), or haplo-identical donor (Haplo-SCT, n=2). Since 2003, the conditioning regimen for MSD-SCT was changed from Busulfex (12.8 mg/kg) + cyclophosphamide (CY, 120 mg/kg) to fludarabine (180 mg/m2) + CY (100 mg/kg) + rATG (10 mg/kg). The conditioning regimen for URD-SCT and Haplo-SCT were TBI (800 cGy) + CY (100-120 mg/kg) ± rATG (2.5 mg/kg) and TBI 600cGy + Fludarabine (150 mg/m2) + rATG (5 mg/kg), respectively. After a median follow-up of 57 months (range 6.0-151.3), the 5-year estimated OS rates were 87.9 ± 5.7%. Four patients died of treatment-related mortality (TRM), including acute GVHD (n=1), pneumonia (n = 2), and cerebral hemorrhage (n=1), respectively. Except one patient with early TRM, 32 patients engrafted. Two patients who experienced delayed graft-failure received second transplant and recovered. The cumulative incidence of acute GVHD (≥grade II) and chronic GVHD was 27.3 ± 7.9% and 18.7 ± 7.0%, respectively. Among 25 patients with available follow-up data, PNH clone disappeared at median 3.0 months (range 0.7-45.5) after SCT and reemerging of PNH clones was observed in two patients; one patient showed re-appearance of 2.6% GPI-negative neutrophils at 12 months without PNH symptoms, but disappeared again at 21 months. Another patient suffered from labile graft and received a booster with peripheral blood stem cells.Conclusion: This study showed that long-term transplant outcome in patients with AA/PNH were comparable to that of allogeneic SCT in SAA (the 3-year estimated OS rates were 92.7 and 89 % for MSD-SCT and URD-SCT, respectively) at our institution (ASH Annual Meeting Abstracts 2012;120:4151). Reduced-intensity conditioning regimen was sufficient for the eradication of PNH clone in allogeneic SCT. Therefore, application of allogeneic SCT should be considered in PNH patients with AA in case of availability of well matched donor. DisclosuresNo relevant conflicts of interest to declare.

Aplastic Anemia Associated with PNH-Clone - a Single Centre Experience

Introduction: Pathogenetic relationship of aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) was registered a long time ago. Approximately in 50-60% of patients with AA is detected PNH-clone, and during long-term observation the transformation of AA into classic PNH is likely. At the time of diagnosis PNH-clone sizes range from small proportion of PNH-cells, requiring only monitoring, up to combination of clinically manifested AA and PNH requring combined therapy. Nevertheless, the peculiar characteristics of the disease in cases of different AA-PNH-combinations still remain unclear.Aim: to evaluate the relationship between size and dynamics of PNH-clone with clinical features of AA/PNH.Materials and methods: We have evaluated blood samples of 81 patients with AA (whole group) for the presence of PNH-clone using flow cytometry according to ICCS standards. 23 patients aged 23 to 54 years (median 46 years) were under constant observation for 3 years or more, control examination was performed once every 6 months (dynamic observation group). Of them 17 had severe AA (SAA) and 6 - nonsevere AA (NAA). Twelve patients received standard immunosupressive therapy (IST) for the treatment of AA, other patients were in remission and did not require IST.Results: In the whole group of patients (n=81) 47 patients (58%) were AA/PNH-positive (PNHpos) with clone size ranging from 0.01% to 97.9%. PNH-clone less than 1% was identified in 10% of subjects; PNH-clone more than 10% was observed in 50% of subjects. In dynamic observation group 18 patients were AA/PNHpos with initial clone size from 0.1% to 95.9%, and 5 patients - AA/PNH negative (AA/PNHneg). In AA/PNHneg patients pathological clone still has not been detected in repeated studies for the observed period. Among patients with AA/PNHpos stable clone was observed in 11 subjects (61%), it increased to 10% and more in 5 subjects (28%) and decreased in 2 patients (11%) with small PNH-clone. Clinical and laboratory signs of chronic intravascular hemolysis were usually observed in patients with pathological clone over 25%. PNH-clone less than 10% was not accompanied by significant deviations in clinico-laboratory indices. Most of patients in whom PNH-clone size increased, had remission of AA. Four patients had clonal evolution to manifest classical PNH, of them in 2 patients with SAA аplasia of hematopoiesis persisted. In one patient, with complete remission of SAA lasting more than 3 years and minor PNH-clone, was observed transformation into myeloid leukemia leading to death. Ekulizumab was prescribed to 4 patients: 2 with clinically manifest hemolysis and clone size > 90% (1 - in conjunction with IST for AA), and 2 with clone size 24% and 70% and LDH > 1.5 ULN due to unplanned pregnancies (both on Cyclosporin therapy). All of them achieved the target level of LDH <1.5 ULN in the first 2 weeks of therapy. Of 2 transfusion-dependent patients, one still does not require transfusion support after the Ekulizumab initiation, and in other patient transfusion dependency decreased, despite refractory TAA. In one patient with PNH-clone 70% Ekulizumab therapy was initiated in III trimester of pregnancy, delivery was without complications, full-term baby, 8-9 points Apgar. In the 2nd pregnant patient with PNH-clone 24% Ekulizumab was prescribed because of pre-eclampsia and LDH>4 ULN at 21-22 weeks with antenatal fetal death, and it allowed for accelerating cure of pathological symptoms.Conclusion: PNH-clone was detected in 58% of patients with AA. Our data confirm the need for monitoring of PNH clone in patients with AA and for timely adequate therapy, including inhibitors of intravascular hemolysis, according to specific indications in each case. DisclosuresFominykh:Novartis Pharma: Honoraria; BMS: Honoraria.

The First Follow-up Data Analysis of Patients with Acquired Bone Marrow Failure Harboring a Small Population of PNH-Type Cells in the Japanese, Multicenter, Prospective Study Optima

▪Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem cell disorder with the expansion of PIGA mutant clone(s), which is deficient in GPI-anchored proteins including CD55 and CD59. The lack of CD55 and CD59 renders PNH-type red blood cells (RBC) susceptible to complement attacks, resulting in intravascular hemolysis. Classic PNH manifests 3 major symptoms: anemia, bone marrow failure, and thrombosis. Small populations of PNH-type cells (<1.0%) have been found in approximately 60% of aplastic anemia (AA) and in 20% of low-risk myelodysplastic syndrome (MDS) patients with no clinical or biochemical evidence of hemolysis. The presence of increased PNH-type cells in bone marrow failure (BMF) patients was shown to predict better response to immunosuppressive therapy by retrospective (Blood 2006 107:1308-1314) and prospective (Br J Haematol 2014 164:546-54.) studies. To ascertain the clinical significance of PNH-type cells, we conducted a nationwide, multicenter, prospective, observational clinical study named OPTIMA in Japan.Methods: Patients diagnosed with PNH, AA, MDS or indistinguishable BMF were prospectively recruited to the study since 2011 in Japan. A high-resolution FCM assay was established to precisely detect a small population of PNH-type granulocytes (with FLAER) and RBCs (with anti-CD55 and CD59 antibodies) ≤ 0.01% of the total granulocyte or RBC population based on the Kanazawa method (Blood 2006 107:1308-1314). Six university laboratories across Japan were designated as regional analyzing centers and measured the percentages of PNH-type cells in the study population, as well as collected clinical and laboratory data. Periodic blind cross validation tests using a positive control sample containing 0.01% PNH-type cells and a negative control sample were performed to minimize inter-laboratory variations.Results: As of July 2016, 2,849 patients were enrolled in the study and 2,734 patients were analyzed. Nine hundred twelve patients (33.4%) were positive for PNH-type cells (≥ 0.005% PNH-type erythrocytes and/or ≥ 0.003% PNH-type granulocytes) and 238 (8.7%) patients had more than 1% of PNH-type cells. PNH-type cells were positive in 90/90 PNH (100%), 512/982 AA (52.1%), 132/822 MDS (16.1%), and 141/512 indistinguishable BMF (27.5%) patients. Among the MDS patients, PNH-type cells were positive in approximately 20% of patients with RCUD, RCMD, MDS-U, or 5q- syndrome, but not in any patients with RARS, RAEB-1, and RAEB-2 (Fig. 1). The serum LDH level increased in proportion to the PNH clone size of RBCs, and 63.3% of the patients possessing ≥1.0% RBCs showed LDH levels more than 1.5 times the upper limit of normal. Of 171 patients who completed submission of 3-year follow-up data, BMF patients with PNH-type cells showed a better response rate [CR+PR, 99/107 (92.5%)] to IST compared to those without PNH-type cells [44/64 (68.8%)] (P<0.001, Chi-square test) (Fig. 2).Conclusions: These interim analyses of the OPTIMA study demonstrate that our high-resolution FCM is reliable in detecting small populations of PNH-type cells and produces consistent results among different laboratories. The presence of PNH-type cells exclusively in patients with AA and low-risk MDS suggests a link between benign pathophysiology of BMF and an increase in the number of PNH-type cells. The better response of PNH-type cell-positive BMF to IST compared to BMF without PNH-type cells was consistent with previous reports. Our data, for the first time, prospectively confirms the significance of small populations of PNH cells in BMF patients in Japan and warrants further worldwide, prospective studies on non-Japanese patients with BMF. [Display omitted] [Display omitted] DisclosuresUeda:Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Yonemura:Alexion Pharmaceuticals: Research Funding. Obara:Alexion Pharmaceuticals: Honoraria, Research Funding. Shichishima:Alexion Pharmaceuticals, Inc. Japan: Honoraria. Ninomiya:Alexion Pharmaceuticals: Honoraria. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Kanakura:Fujimotoseiyaku: Research Funding; Toyama Chemical: Research Funding; Bristol - Myers: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Pfizer: Research Funding; Chugai Pharmaceutical: Research Funding; Shionogi: Research Funding; Kyowa Hakko Kirin: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

Impact of eculizumab treatment on paroxysmal nocturnal hemoglobinuria: a treatment versus no-treatment study.

Intravascular hemolysis in Paroxysmal nocturnal hemoglobinuria (PNH) can effectively be controlled with eculizumab, a humanized monoclonal antibody that binds complement protein C5. We report here a retrospective comparison study between 123 patients treated with eculizumab in the recent period (>2005) and 191 historical controls (from the French registry). Overall survival (OS) at 6 years was 92% (95%CI, 87 to 98) in the eculizumab cohort versus 80% (95%CI 70 to 91) in historical controls diagnosed after 1985 (HR 0.38 [0.15 to 0.94], P = 0.037). There were significantly fewer thrombotic events (TEs) in the group of patients treated with eculizumab (4% [1-10]) as compared to the historical cohort (27% [20-34]). However, we found that TEs may still occur after the initiation of eculizumab treatment and that previous TEs still have a negative impact on survival. Evolutions to myelodysplastic syndrome or acute leukemia were similar in both cohorts. There was less evolution to aplastic anemia in the treatment group. In multivariate analysis, absence of a previous TE and treatment with eculizumab were associated with a better OS. Treatment with eculizumab improves overall survival in classic PNH patients without increasing the risk of clonal evolution.

Clinical Features of Chinese Patients with Paroxysmal Nocturnal Hemoglobinuria Diagnosed By FLAER

Objective:To summary the clinical features of Chinese patients with paroxysmal nocturnal hemoglobinuria (PNH) diagnosed by FLAER.Methods:The clinical data of 98 cases diagnosed by FLAER from September 2011 to March 2014 were analyzed retrospectively, including clinical features, laboratory examination results and complications. Patients were divided into three clinical groups according to the standard proposed by the International PNH Interest Group. This classification has been applied to each patient considering the clinical characteristics of the disease, bone marrow failure and PNH clone size. Statistical analysis: We presented continuous data as mean and standard deviation or median and interquartile range (IQR), with extreme values. The distributions of the presentation characteristics were compared among the three subcategories and between classic PNH and PNH-sc/AA by chi-square test or Fisher exact test when necessary for qualitative characteristics, and by Kruskal-Wallis (three subcategories) or Mann-Whitney (two groups) test for continuous characteristics. Spearman's rank correlation coefficient was used to measure correlations. Overall survival (OS) estimated by the Kaplan-Meier method was compared using the log-rank test. The Cox proportional hazards model was used to assess the risk factors for survival in both univariate and multivariate analyses. All of the analyses were performedusing statistical package SPSS 17.0. P<0.05 was considered as statistically significant.Results:There were 43 cases of classic PNH, 45 of PNH combined with other specific bone marrow disorders, 10 cases of subclinical PNH. 11 patients with PNH less than 18 years of age belong to adolescents, and other 87 PNH were adults. 70 patients were with fatigue, 41 patients with PNH manifested hemoglobinuria. Thrombosis in 6 cases, mostly formed in rare sites. 17 cases and 19 cases concurrented with renal and liver impairment respective. Only 2 cases of PNH were suffered with pulmonary hypertension simultaneously. Classic PNH was more susceptible to hemolysis, the bone marrow failure features of PNH combined with other specific bone marrow disorder was markedly obvious. The value of lactate dehydrogenase (LDH) level over normal range was related with the size of clone PNH-the GPI negative granulocyte, linearly (R=0.710, P<0.001). Patients with high PNH clone were specially prone to thrombosis. The incidence of hemoglobinuria in adolescent group was significantly lower than the adult group, but the rate of bleeding was higher than that of adult group.Conclusion:In Chinese patients with PNH, bone marrow failure, renal and liver impairment and thrombosis in rare sites was relatively common. But pulmonary arterial hypertension happened seldomly. Adult patients were more susceptible to hemolysis, but the megakaryocyte hematopoietic failure of adolescent patients combined with other specific bone marrow disorder was more obvious. DisclosuresNo relevant conflicts of interest to declare.

Clinical Features and Survival of Asian Pediatric Patients with Paroxysmal Nocturnal Hemoglobinuria: Results from a Single Center in China

Objective: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease, especially in children. To characterize the clinical presentations and survival, we performed a retrospective analysis of pediatric patients. Methods: We reviewed 55 pediatric patients with PNH referred to our hospital from January 1990 through June 2012 to assess clinical presentations, survival, and differences among subcategories. Results: The overall survival 10 years after diagnosis estimated via the Kaplan-Meier method was 77.6%. The cohort of patients was divided into subcategories of classic PNH, PNH/aplastic anemia (AA), and subclinical PNH (PNH-sc)/AA based on the recently proposed PNH working clinical classification. We found that patients with classic PNH and PNH/AA had larger PNH clones and many more parameters of hemolysis, but patients with PNH-sc/AA had smaller PNH clones, fewer parameters of hemolysis, and a higher rate of bone marrow failure. Our results revealed a high rate of bone marrow failure and a low rate of hemoglobinuria at presentation. Furthermore, thrombotic events were not observed in our patients, which is significantly different from the rate seen in Caucasian patients. Additionally, pediatric patients with PNH may develop bone marrow cytogenetic abnormalities. Conclusion: This study provides insight into Chinese pediatric PNH patients and may aid in setting up individualized therapeutic regimens.

Paroxysmal nocturnal hemoglobinuria clone in 103 Brazilian patients: diagnosis and classification.

BackgroundParoxysmal nocturnal hemoglobinuria is an acquired chronic hemolytic anemia, which often manifests as peripheral blood cytopenias and thrombosis.ObjectiveThe aim of this study is to describe a Brazilian population of paroxysmal nocturnal hemoglobinuria patients.MethodsOne hundred and three paroxysmal nocturnal hemoglobinuria cases were retrospectively reviewed and the clinical presentation, thrombosis, survival, and clone size were assessed. Diagnosis was established by flow cytometry.ResultsFifty-two male and 51 female patients with a median age of 24.1 years (5.5–62 years) were studied. Clinical symptoms included hemoglobinuria (18.4%), infection (46.6%) and thrombosis (16.5%), and 80.6% had pancytopenia. Patients were classified as classic paroxysmal nocturnal hemoglobinuria (10), paroxysmal nocturnal hemoglobinuria with aplastic anemia (39), and paroxysmal nocturnal hemoglobinuria with subclinical features and aplastic anemia (54). There were significant differences in terms of median age, size of clone, clinical symptoms, and peripheral blood cell counts between the three subcategories. The clone size in erythrocytes and granulocytes were respectively 0.04% (range: 0–18%) and 7.3% (range: 0.3–68.7%) in patients with subclinical features and aplastic anemia, 15.8% (range: 0–99.7%) and 63.0% (range: 1.7–99.8%) in patients with aplastic anemia alone, and 82.2% (range: 0–99.85%) and 98.0% (81.3–100.0%) in Classic disease. Statistical differences were identified for platelets (p-value = 0.001), lactate dehydrogenase (p-value = 0.002) and the clone size (p-value < 0.001) in patients who suffered thrombotic events compared to those who did not. Overall survival was 81.7%, with patients with subclinical features and aplastic anemia having lower overall survival (76.5%).ConclusionThis retrospective review of 103 patients over an 11-year period represents the largest collection of paroxysmal nocturnal hemoglobinuria cases from a single center in Brazil. Flow cytometry showed that a larger clone was associated with classical symptoms and increased risk of thrombosis, even in patients with bone marrow failure, whereas a smaller clone was associated with bone marrow aplasia.

Long-Term Outcome of Allogeneic Stem Cell Transplantation in Patients with Combined Paroxysmal Nocturnal Hemoglobinuria and Aplastic Anemia (AA/PNH syndrome)

Background:Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disorder of hematopoietic stem cells characterized by a somatic mutation in the PIG-A gene, encoding the glycosyl phosphatidylinositol (GPI) moiety. PNH clones lack GPI-anchored proteins (GPI-AP) which inhibit the activation and cytolytic functions of complement. Recently, Eculizumab, humanized monoclonal antibody directed against complement component C5, has used increasingly for the patients with hemolytic PNH. However, the patients with PNH clone and bone marrow failure syndrome (i.e. aplastic anemia) should be treated as their predominant clinical manifestation. Allogeneic stem cell transplantation (SCT) can be curative treatment option especially for PNH patients with combined aplastic anemia (AA). The aim of the present study was to evaluate long-term outcome of allogeneic SCT in patients with AA/PNH.Methods: Total of 27 patients with PNH clones underwent allogeneic SCT at our institution between Jan 1998 and Mar 2014. Among them, seven patients had classic PNH and 20 patients with cytopenia had AA/PNH (with bone marrow evidence of a concomitant AA). We analyzed long-term transplant outcomes in 20 patients with AA/PNH.Results: There were 12 male and 8 female patients with a median age of 34 years (range, 13-51 years). The median interval from the diagnosis to transplantation was 8 months (range; 1-201 months). The median transfusions prior to SCT were 33 units (range; 8-208 units). Pre-transplant GPI-AP deficient neutrophils and erythrocytes were 46% (0-99) and 15.6% (0-88), respectively. Median white blood cell, absolute neutrophil count, hemoglobin, and platelet at transplant were 2.3×109/L, 0.7×109/L, 7.9 g/dL, and 21×109/L, respectively. Median LDH level was 714 U/L (range; 273-6499 U/L) and 11 (55%) patients had LDH ≥1.5x upper normal limit. PNH patients with SAA (n=14), VSAA (n=4), or non-SAA (n=2) received SCT from sibling (s) donor (n=15) or unrelated (u) donor (n=5). The conditioning regimen for s-SCT consisted of fludarabine (180 mg/m2) + cyclophosphamide (CY, 100 mg/kg) + ATG (10 mg/kg) (n=11), or busulfex (12.8 mg/kg) + CY (120mg/kg) (n=4). The conditioning regimen for u-SCT was TBI (fractionated, 800 cGy) + CY (100-120 mg/kg) ± ATG (2.5 mg/kg). GVHD prophylaxis consisted of CsA + MTX in s-SCT and FK506 + mini-MTX in u-SCT, respectively. After a median follow-up of 57 months (range 4.7-122.1), the 5-year estimated OS rates were 90.0 ± 6.7%. Two patients died of treatment-related mortality (TRM), including acute GVHD (n=1) and cerebral hemorrhage (n=1), respectively. Except one patient with early TRM, 19 patients engrafted with no secondary graft-failure. The cumulative incidence of acute GVHD (≥grade II) and chronic GVHD was 25.0 ± 1.0% and 26.3 ± 10.4%, respectively. PNH clones disappeared at median 1.8 months (range 0.9-11.9) after SCT and reemerging of PNH clone was not observed in all patients.Conclusion: This study showed that long-term transplant outcome in patients with AA/PNH were comparable to that of allogeneic SCT in SAA (the 3-year estimated OS rates were 92.7 and 89 % for s-SCT and u-SCT, respectively) at our institution (ASH Annual Meeting Abstracts 2012;120:4151). Therefore, application of allogeneic SCT should be considered in PNH patients with AA in case of availability of well matched donor. DisclosuresNo relevant conflicts of interest to declare.

Paroxysmal nocturnal haemoglobinuria. Experience over a 10 years period

Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic, clonal and acquired disorder of the hematopoietic stem cell with a deficiency of all glycophosphatidyl-inositol (GPI) linked proteins. The aim of this retrospective study was to analyse haematological and biochemical data from 152 patients referred to our laboratory for diagnosis of PNH by flow cytometry (FC). Patients and healthy donor (152 and 99 respectively) were studied. Ham, sucrose, lactate dehydrogenase (LDH), Iron, haptoglobin (Hp), blood cell morphology and Kaplow cytochemical stain for leukocyte alkaline phosphatase (LAP) were carried out. GPI-proteins anti-CD55 and CD59 in erythrocytes and the former, plus anti CD16b and CD66b on neutrophils were evaluated by FC. Anemia and/or leukopenia and/or thrombocytopenia, increased reticulocyte count and LDH were observed in patients with PNH clone. Some of them had dacriocytes, schistocytes. LAP was low. On average, we detected 50% CD59 (-) erythrocytes and 29, 83, 78% CD55/59 (-), CD16b (-), CD66b (-) neutrophils, respectively. Paroxysmal nocturnal hemoglobinuria clone was detected in 20/152 patients. Negative population's percentages were high in patients with classic PNH, Hematimetry, LAP and adequate use of CF contribute to PNH clone detection in the laboratory.

Perisurgical induction of eculizumab in a patient with paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement (C')-induced lysis of PNH red blood cells (RBCs), which are deficient in the expression of CD55 and CD59. Surgery is one of the major clinical situations that trigger hemolytic attack and thrombosis in PNH. We describe here a case of 64-year-old man with classic PNH complicated by early-stage gastric cancer requiring distal gastrectomy under general anesthesia. We administered humanized monoclonal anti-C5 antibody (eculizumab; Soliris) for a limited period (600 mg, once a week × four times) perisurgically. Eculizumab effectively inhibited the C' system and the patient underwent a curative distal gastrectomy without significant surgery-triggered hemolytic attack. Although discontinuation of eculizumab induced mild hemolysis 2 weeks after the last administration, it was treated conservatively without thrombotic complication. Limited-term induction of eculizumab could be an option for PNH patients with transient and anticipated high risks, with careful preparation for the discontinuation-related risks afterwards.

Paroxysmal nocturnal hemoglobinuria and concurrent JAK2V617F mutation

Paroxysmal nocturnal hemoglobinuria and concurrent JAK2V617F mutation

Clinical features and prognostic factors of Asian patients with paroxysmal nocturnal hemoglobinuria: results from a single center in China

Although all patients with paroxysmal nocturnal hemoglobinuria (PNH) have acquired mutations in the phosphatidylinositol glycan class-A(PIG-A)gene, their clinical courses are highly variable. We reviewed 280 PNH cases referred to our hospital from January 1990 through June 2010 to assess clinical presentations, prognostic factors influencing survival, difference among subcategories, and clinical significance of PNH clone size. The overall survival at 10years after diagnosis estimated by Kaplan-Meier was 77.6%. Both univariate and multivariate analyses identified risk factors affecting survival, including age >40years, absolute neutrophil count<0.5 × 10(9) cells/L, development of thrombotic events, evolution to myelodysplastic syndrome or acute myelogenous leukemia, and recurrent infections. The cohort of patients were divided into subcategories of classic PNH, PNH/AA, and PNH-sc/AA based on the recent proposed PNH working clinical classification, hemoglobinuria as the initial symptomatic manifestation was high up to 82.0% in classic PNH subcategory, whereas only as low as 1.4% in PNH-sc/AA subcategory; the frequencies of infectious (26.0%) and bleeding symptoms (14.0%) in classic PNH subcategory were significantly less than those in PNH/AA (25.3% and 51.7%, respectively) and PNH-sc/AA (48.3% and 83.2%, respectively) subcategories. Our results revealed that large PNH clone was associated with increased risks for hemoglobinuria and thrombosis, whereas small PNH clone was associated with bone marrow failure. Thus, this study may shed insights into Chinese PNH patients to set up individually therapeutic regimens.

Successful Management of Obstructive Jaundice due to Gallstones with Eculizumab in a Patient with Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) makes patients susceptible to intravascular hemolysis and thrombosis, and it can be life-threatening in stressful situations. Eculizumab, a humanized monoclonal antibody that inhibits the complement protein C5, has been evaluated as a novel therapy for PNH. We herein describe the case of a 59-year-old Japanese woman with classic PNH, who had been successfully treated with eculizumab, but who later developed acute cholecystitis/cholangitis from gallstones. Although the severe obstructive jaundice requiring endoscopic therapy following cholecystectomy was complicated, critical intravascular hemolysis and thrombosis were not observed. Therefore, utilizing eculizumab during the peri-operative management of PNH patients should be carefully taken into consideration.

Management of Paroxysmal Nocturnal Hemoglobinuria in the Era of Complement Inhibitory Therapy

Despite the availability of safe, effective targeted therapy that controls intravascular hemolysis, the management of paroxysmal nocturnal hemoglobinuria (PNH) remains complicated because of disease heterogeneity and close association with BM failure syndromes. The purpose of this review is to provide a framework for individualizing treatment based on disease classification. According to the recommendations of the International PNH Interest Group, patients can be placed into one of the following 3 categories: (1) classic PNH, (2) PNH in the setting of another BM failure syndrome, or (3) subclinical PNH. The PNH clone in patients with subclinical disease is insufficiently large to produce even biochemical evidence of hemolysis, and consequently, patients who fit into this category require no PNH-specific therapy. Patients with PNH in the setting of another BM failure syndrome (usually aplastic anemia or low-risk myelodysplastic syndrome) have at least biochemical evidence of hemolysis, but typically the PNH clone is small (< 10%) so that hemolysis does not contribute significantly to the underlying anemia. In these cases, the focus of treatment is on the BM failure component of the disease. Intravascular hemolysis is the dominant feature of classic PNH, and this process is blocked by the complement inhibitor eculizumab. The thrombophilia of PNH also appears to be ameliorated by eculizumab, but the drug has no effect on the BM failure component of the disease. Low-grade extravascular hemolysis due to complement C3 opsonization develops in most patients treated with eculizumab, and in some cases is a cause for suboptimal response to treatment. Allogeneic BM transplantation can cure classic PNH, but treatment-related toxicity suggests caution for this approach to management.

High-Sensitivity Flow Cytometry Testing for Paroxysmal Nocturnal Hemoglobinuria in Children with Cytopenia: A Single Center Study

Abstract 2398 Background:Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by expansion of cells with complete or partial loss of glycosyl phosphatidyl-inositol-anchored proteins. PNH usually presents with one or more of three clinical manifestations: intravascular hemolysis, thrombosis, or acquired bone marrow failure [aplastic anemia (AA) or myelodysplastic syndrome (MDS)]. Flow cytometry has become the gold standard for the diagnosis of PNH, particularly with the recent publication of guidelines for the diagnosis and monitoring of PNH and related disorders in 2010. PNH occurs rarely in children, and, consequently, the published literature regarding PNH in this pediatric population consists only of small case series, making it difficult to extrapolate the frequency of which PNH clones are identified. Moreover, no studies are available on the incidence of PNH clones in children with MDS and acquired aplastic anemia (AAA). We, therefore, sought to determine how frequently a high sensitivity FLAER-based assay, with a sensitivity of 0.01%, would detect PNH clones in children with cytopenias. Method and Results:The study period was from December 2010 to July 2011. PNH testing was performed using a high sensitivity FLAER based assay according to published guidelines using the combination of FLAER/CD64/CD15/CD33/CD24/CD14/CD45 for WBC testing and CD235a/CD59 for RBC testing. There were 31 peripheral blood samples from 29 patients (17 males/12 females) ranging in age from 4 months to 17 years (median, 10 years). All patients were tested for PNH because of cytopenia [pancytopenia (n = 14) and uni- or bicytopenia (n = 15)]. Patients had a mean Hgb of 10.7 gm/dL, mean ANC of 2.66 X103/uL and mean platelet of 115 X103/uL. Review of medical charts revealed the following clinical diagnoses: classic PNH - episodic hemolytic anemia with persistent thrombocytopenia (1), severe AA (SAA, 8), SAA with myelofibrosis (1), MDS (1), Fanconi anemia (1), chronic thrombocytopenia (2), refractory iron deficiency anemia (1), bone marrow suppression likely due to virus/medication (1), parvovirus infection (1), Copper deficiency (1), systemic lupus erythematosus (SLE, 1), and cytopenia of unknown etiology (10). Of note, all patients with AAA had SAA. PNH clones were identified in 6 out of 29 patients (20%): minor clones (<1% PNH population) in 3 patients: average clone sizes 0.12% [range 0.02–0.25] granulocytes (G), 0.51% [0.20–0.99] monocytes (M), and 0.08% [0.04–0.14] red blood cells (RBCs), and major clones (>1% PNH population) in 3 patients: average clone sizes 31.11% [3.98–67.58] G, 31.98% [6.15–71.1] M, and 14.76% [1.19–38.03] RBC, respectively, with ages ranging from 4 to 17 years. Patients who were identified to have minor PNH clones all presented with pancytopenia. Two were diagnosed with SAA; the cause of pancytopenia in the third patient is currently under investigation. None of patients with minor PNH clones had evidence of hemolysis or thrombosis. The three patients with major PNH clones had the following: Classic PNH with hemolytic anemia (1), SAA with PNH clones detected at the time of SAA diagnosis (1), and SAA with PNH clones detected 20 months after immunosuppressive therapy (1). The latter two patients did not have evidence of hemolysis or thrombosis. Of the 10 patients with a diagnosis of SAA or MDS, PNH clones were identified in 4 (40%) patients (2 with minor clones, 2 with major clones). Conclusions:This is the first study to describe the utility of using a standardized high-sensitivity FLAER-based flow cytometry assay to identify PNH clones in children. This is also the first study describing the prevalence of PNH clones in children with MDS and AAA. The identification of a PNH population in 40% of the MDS and AAA cases emphasizes the need for PNH testing in all children with these disorders using a high-sensitivity FLAER based flow cytometry assay. A low sensitivity assay would have missed 2 patients with minor PNH clones. This finding may be of significance considering SAA or MDS patients with PNH clones are more likely to respond to immunosuppressive therapy. Further studies are needed to investigate the prevalence of PNH clones in this setting and its impact on disease manifestations, course, and outcomes in children. Disclosures:No relevant conflicts of interest to declare.

Clinical Characteristics of Classic Paroxysmal Nocturnal Hemoglobinuria (PNH) in Pediatric Patients: A Comparison with Classic PNH in Adults. An International PNH Registry Study

Abstract 2102 Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a chronic, life-threatening hematopoietic stem cell disorder characterized by deficiency of the GPI-anchored complement inhibitory proteins CD55 and CD59. Uncontrolled complement activation is responsible for chronic hemolysis that leads to the serious clinical morbidities including thromboembolism (TE) and chronic kidney disease, which increase risk of mortality. PNH is infrequent in children and as such, there is a paucity of data in the literature examining their burden of disease. This is the largest series of pediatric patients with PNH, and the first comparison with adult PNH patients. AIM: To assess the clinical differences between pediatric and adult PNH patients with classic/hemolytic PNH. Methods: Data from 1143 PNH patients from 204 clinical sites in 21 countries on 5 continents (as of 6/30/11) were analyzed to evaluate clinical characteristics of PNH. Patients registered as having classic/hemolytic PNH were initially included (n=559). Of these, 62 additional patients were excluded due to either a Results: The median age at disease start for pediatric patients was 15 years (5–17), and for adults was 33 years (18–87). Hemoglobin, leucocytes, neutrophils, reticulocytes, LDH, and number of transfusions were similar between both pediatric and adult patients. There were no differences in GPI-deficient granulocyte or RBC clone sizes between pediatric and adult patients. Follow-up was longer for children (mean + SD, in years: 14.5 + 13.6 vs 9.6 + 9.1, P 18 years at disease onset and kidney disease were predictors of TE (odds ratio [OR] 3.01, 95% CI 1.15–7.72; P=0.02, and OR 1.87, 95% CI 1.03–3.4; P=0.04, respectively), while clone size was not a significant predictor of TE (OR 1.010, 95% CI 0.995–1.025; P=0.18). Conclusions: Pediatric PNH patients experience a significant burden of disease similar to adult PNH patients. There were no differences in LDH levels, GPI-deficient clone size, or in the incidence of bone marrow disorders, renal impairment, hemoglobinuria, abdominal pain, shortness of breath and dysphagia between pediatric and adult patients. TE event rate was lower in pediatric PNH patients when compared to adults, especially for those with concomitant kidney disease However, TE still remains an issue in this population and pediatric PNH patients are at significant risk for TE. Disclosures: Urbano-Ispizua:Alexion: Membership on an entity9s Board of Directors or advisory committees. Schrezenmeier:Alexion Pharmaceuticals, Inc.: Membership on an entity9s Board of Directors or advisory committees. Muus:Celgene: Membership on an entity9s Board of Directors or advisory committees; Alexion: Membership on an entity9s Board of Directors or advisory committees; Amgen: Membership on an entity9s Board of Directors or advisory committees; Novarts: Membership on an entity9s Board of Directors or advisory committees. Maciejewski:Celgene: Research Funding; Eisai: Research Funding; Alexion: Consultancy. Socie:Alexion: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Szer:Alexion Pharmaceuticals, Inc.: Membership on an entity9s Board of Directors or advisory committees. Brodsky:Alexion Pharmaceuticals, Inc.: Membership on an entity9s Board of Directors or advisory committees. Bessler:Alexion Pharmaceuticals, Inc.: Consultancy; Novartis: Membership on an entity9s Board of Directors or advisory committees; Taligen: Consultancy. Rosse:Alexion: Consultancy, Membership on an entity9s Board of Directors or advisory committees. Kanakura:Alexion: Consultancy, Membership on an entity9s Board of Directors or advisory committees. Khursigara:Alexion: Employment, Equity Ownership. Bedrosian:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Hillmen:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding.