Clinical Paroxysmal Nocturnal Hemoglobinuria Research Articles

Inhibition of C3 with pegcetacoplan results in normalization of hemolysis markers in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hematologic disorder characterized by complement-mediated hemolysis. C5 inhibitors (eculizumab/ravulizumab) control intravascular hemolysis but do not prevent residual extravascular hemolysis. The newly approved complement inhibitor, pegcetacoplan, inhibits C3, upstream of C5, and has the potential to improve control of complement-mediated hemolysis. The PADDOCK and PALOMINO clinical trials assessed the safety and efficacy of pegcetacoplan in complement inhibitor-naïve adults (≥ 18 years) diagnosed with PNH. Patients in PADDOCK (phase 1b open-label, pilot trial) received daily subcutaneous pegcetacoplan (cohort 1: 180 mg up to day 28 [n = 3]; cohort 2: 270–360 mg up to day 365 [n = 20]). PALOMINO (phase 2a, open-label trial) used the same dosing protocol as PADDOCK cohort 2 (n = 4). Primary endpoints in both trials were mean change from baseline in hemoglobin, lactate dehydrogenase, haptoglobin, and the number and severity of treatment-emergent adverse events. Mean baseline hemoglobin levels were below the lower limit of normal in both trials (PADDOCK: 8.38 g/dL; PALOMINO: 7.73 g/dL; normal range: 11.90–18.00 g/dL), increased to within normal range by day 85, and were sustained through day 365 (PADDOCK: 12.14 g/dL; PALOMINO: 13.00 g/dL). In PADDOCK, 3 serious adverse events (SAE) led to study drug discontinuation, 1 of which was deemed likely related to pegcetacoplan and 1 SAE, not deemed related to study drug, led to death. No SAE led to discontinuation/death in PALOMINO. Pegcetacoplan was generally well tolerated and improved hematological parameters by controlling hemolysis, while also improving other clinical PNH indicators in both trials. These trials were registered at www.clinicaltrials.gov (NCT02588833 and NCT03593200).

Clinical Significance of Small PNH-Type Cell Populations in Bone Marrow Failure Syndromes - an Interim Analysis of Japanese Multicentrer Prospective Study -

Background: Small populations of paroxysmal nocturnal hemoglobinuria (PNH)-type cells (<1.0%) are frequently found in bone marrow failure syndromes (BMF) including aplastic anemia (AA) and low-risk myelodysplastic syndromes (MDS) without evident hemolytic symptoms. The presence of small PNH-type cells has been reported to predict better response to immunosuppressive therapy (IST) both retrospectively (Blood 2006 107:1308-1314) and prospectively (Br J Haematol 2014 164:546-54), but its clinical significance is still to be elucidated. In addition, the kinetics of those small PNH-type cells have not been well studied. To better understand the clinical significance and the kinetics of PNH-type cells in BMF, we conducted a nationwide, multicenter, prospective, observational clinical study named SUPREMACY.Methods: Patients diagnosed with PNH, AA, MDS or indistinguishable BMF without malignant diseases were prospectively recruited to the study between April 1 st, 2016 and December 31 st, 2019 in Japan. Participants were excluded from the study if treated with eculizumab or ravulizumab. Peripheral blood samples were obtained with informed consent and sent to the single laboratory every 12months (mos) for 36 mos. A high-resolution flow cytometry assay known as OPTIMA method (Ann Hematol 97(12):2289-2297) was used to precisely detect a small population of GPI(-) cells, which defines ≥0.003% PNH-type granulocytes (Gran) and ≥0.005% erythrocytes as an abnormal increase. The quality of life (QOL) of the pts was assessed using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) instruments. All other lab data and clinical information were obtained at each participating institute or hospital on sample collection, and were accumulated by the Japan PNH Study Group for analysis.Results: Total 1,985 pts were registered and 1,813 pts were eligible for analysis. Median age was 67 with 50.5% male patients. PNH-type cells were positive in 50.4% (235/466) of AA, 19.7% (70/355) of MDS, 22.3% (61/273) of suspected PNH, and 33.9% (232/685) of undiagnosed BMF. PNH-type cells were increased in nearly 30% of the pts with RCUD, RCMD, MDS-U, and 5q-, but not in RARS, RAEB-1, or RAEB-2. Time-course data of the size of PNH-type cells were available in 651 pts at 12mos and in 210 pts at 36mos. Small (<1.0%) PNH-type granulocytes (sPNHg) stayed below 1.0% at 36mos in 81/86 (94.2%) pts, but increased in 5/86 (5.8%) by median 2.75% or by average 17.86% at 36mos. Clinical data for IST were available in 277 pts (12mos) and 99 pts (36mos). Pts with PNH-type cells showed a better response rate [Complete response (CR) + Partial response (PR), 159/201 (79.1%) at 12mo, 68/73 (93.2%) at 36mo] to IST compared to the pts without PNH-type cells [48/76 (63.2%) at 12mos, 17/26 (65.4%) at 36mos] (P<0.01, Chi-square test)(Figure 1). Changes of QOL data were compared between the pts with sPNHg [PNH(+)] and the pts without PNH-type cells [PNH(-)] at 36mos, and PNH(+) showed significant improvement in FACIT-Fatigue score (n=36, p=0.0348, Chi-square test) and in 6 items of EORTC QLQ-C30 (global health status, emotional functioning, social functioning, fatigue, and financial difficulties)(n=40) but no significant change of QOL was observed except in one item (constipation) in PNH(-) (n=36)(Table 1).Conclusion: PNH-type cells were detected exclusively in AA and low-risk MDS, supporting the hypothesis that the increase of PNH-type cells in BMF underpin the benign immune-mediated feature of the disease. The presence of PNH-type cells predicts a better response to IST in BMF, which is consistent with previous reports. Detection of subclinical PNH-type cells was associated with an improvement of QOL scores in multiple items at 36mos. Those small populations of PNH-type cells stayed subclinical in most of the cases, but caution should be exercised in monitoring the sizes as some may evolve into clinical PNH. [Display omitted] DisclosuresUeda: Chugai Pharmaceutical: Consultancy, Honoraria, Research Funding; Alexion Pharma: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Yonemura: Alexion Pharma: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; Novartis Pharma: Honoraria. Obara: Novartis Pharma: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Alexion Pharma: Honoraria, Research Funding. Ando: Novartis: Honoraria; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Celgene: Honoraria; Astellas Pharma: Honoraria; Takeda Pharmaceutical: Research Funding. Kawaguchi: Alexion Pharma: Honoraria. Nishimura: Alexion Pharma: Consultancy; Chugai Pharmaceutical: Consultancy; Novartis Pharma: Consultancy; Roche: Consultancy; apellis pharmaceuticals: Consultancy; Biocryst: Consultancy; Sanofi: Consultancy. Nakao: Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Symbio: Consultancy; Alexion Pharma: Research Funding.

Analysis of Baseline Clinical Characteristics and Disease Burden in Patients Enrolled in the International Paroxysmal Nocturnal Hemoglobinuria Registry

Background/Objective: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired, potentially life-threatening hematologic disorder characterized by chronic uncontrolled intravascular hemolysis, thrombosis, end organ damage, and impaired quality of life (QoL) and survival. The International PNH Registry (NCT01374360) is an ongoing, global observational study to evaluate the safety profile of eculizumab and to characterize the burden and progression of PNH including clinical outcomes, morbidity, and mortality irrespective of treatment. The objective of this analysis was to characterize the demographic and clinical characteristics, disease-associated morbidities, PNH symptoms, and health-related QoL of the largest cohort of PNH patients enrolled in the Registry.Methods: Registry patients who had data available as of May 1, 2017, were eligible for the current analysis. All patients were untreated at baseline, defined as the date of eculizumab initiation for patients ever treated with eculizumab and the date of Registry enrollment for patients never treated with eculizumab. Proportions of patients with high disease activity (HDA), history of major adverse vascular events (MAVE; including thrombotic events [TE]) and bone marrow disorders (BMD) including aplastic anemia, red blood cell (RBC) transfusions, concomitant medication use, and physician-reported symptoms were based on the number of patients with non-missing responses at baseline. Laboratory values and QoL scores assessed with the European Organisation for Research and Treatment of Cancer [EORTC] Global Health and Functional Assessment of Chronic Illness Therapy [FACIT]-Fatigue questionnaires within 6 months prior to baseline were summarized using medians and quartiles or proportions for the patients with non-missing responses. Patients with percent glycosylphosphatidylinositol (GPI)-deficient granulocyte values available prior to baseline were stratified based on the most recent value prior to baseline and statistical analysis (i.e., P values) was used to assess potential trends related to clone size.Results: A total of 4903 patients were enrolled. Of these, 4398 had non-missing data on demographics, enrollment date, and eculizumab status, and were eligible for the analysis. Median (Q1, Q3) age at disease onset was 35.3 (23.9, 53.1) years; 53% of the patients were female. Median (Q1, Q3) percent GPI-deficient granulocyte clone size was 33% (3%, 85%) in the 2654 patients with clone size reported prior to baseline. Clinical characteristics at baseline for the overall population and stratified by percent GPI-deficient clone size are summarized in the Table. Overall, patients had a high burden of disease at baseline, as reflected by the proportion with HDA (52%), history of MAVE (19%), BMD (63%), RBC transfusions (61%), and impaired renal function defined as estimated glomerular filtration rate <90 mL/min (42%). Patients frequently experienced symptoms such as fatigue (81%), dyspnea (45%), hemoglobinuria (45%), abdominal pain (35%), and impaired QoL with median (Q1, Q3) FACIT-Fatigue and EORTC Global Health scores of 34.0 (27.0, 40.0) and 58.3 (41.7, 75.0), respectively. All measures of disease burden except for history of RBC transfusions were significantly correlated with clone size (P <0.05); specifically, higher disease burden correlated with larger clone size. However, a substantial number of patients with clone size <10% had hemolysis (LDH ≥1.5 x ULN; 10%), MAVE (10%), HDA (9%), and/or experienced symptoms such as fatigue (82%), dyspnea (46%), abdominal pain (19%), or hemoglobinuria (13%). History of RBC transfusion and immunosuppressive therapy were reported in 60% and 59% of patients with clone size <10%, potentially due to patients with history of BMD. Interestingly, median LDH ratio and the proportion of patients with history of MAVE and TE were modestly higher in the cohort with clone size 10-<50% vs <10%, while the proportion of those patients was substantially higher in the cohort with clone size ≥50% vs 10-<50%.Conclusions: PNH is associated with high burden of disease, particularly in patients with larger GPI-deficient clone sizes; however, even patients with smaller clone sizes had significant disease burden and symptoms. Improved characterization of clinical measures and of relationships between these measures may help inform patient management. [Display omitted] DisclosuresSchrezenmeier:Alexion Pharmaceuticals, Inc.: Honoraria, Other: Research Funding to University of Ulm . Maciejewski:Ra Pharma: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy. Roeth:Roche: Consultancy, Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Araten:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Kanakura:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Astellas: Research Funding; Bristol Myers: Research Funding; Toyama Chemical: Research Funding; Kyowa Hakko Kirin: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Fujimotoseiyaku: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Chugai Pharmaceutical: Research Funding. Larratt:Alexion Pharmaceuticals, Inc.: Honoraria, Other: Research Funding to the laboratory at University of Alberta, for ADAMTS13 testing. Shammo:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Wilson:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Shayan:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Hillmen:Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Celgene: Research Funding; Roche: Consultancy, Honoraria, Research Funding.

Utility of FLAER and CD157 in a five-color single-tube high sensitivity assay, for diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH)-A standalone flow cytometry laboratory experience.

FLAER-based flow cytometry assay is considered the gold standard for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). CD157 is a recently reported marker for GPI-anchored protein found both on neutrophils and monocytes. This study highlights the robustness of FLAER and CD157 combination to identify PNH clones in a high sensitivity assay. Though rare, the data shown highlight the presence of CD157 negativity in few cases re-emphasizing the importance of FLAER for PNH diagnosis. A single 5-color tube-FLAER Alexa488/ CD157PE/ CD15PECy5/ CD64PE-Cy7 & CD45APCH7-was used for a high sensitivity PNH assay. Of 364 cases, 59(16.2%) cases had PNH clone in both granulocytes and monocytes. PNH clone sizes ranged from 0.02% to 96.6% in granulocytes and 0.07% to 96.3% in monocytes based on their FLAER-negative, CD157-negative phenotype. Twenty-two of the 59 PNH cases (37.3%) had WBC clone size of <1%. In addition, there were 10 cases which showed absence of CD 157 expression on both granulocytes and monocytes but on FLAER staining showed normal staining patterns. Three of these ten cases also showed a PNH clone based on absence of FLAER expression on both granulocytes and monocytes. FLAER and CD157 is a robust combination for diagnosis of clinical and subclinical PNH. Absence of CD157 expression in normal WBCs, though rare, should be kept in mind and re-emphasizes the importance of FLAER for the high sensitivity PNH assay.

Long-term outcome of a randomized controlled study in patients with newly diagnosed severe aplastic anemia treated with antithymocyte globulin and cyclosporine, with or without granulocyte colony-stimulating factor: a Severe Aplastic Anemia Working Party Trial from the European Group of Blood and Marrow Transplantation.

This follow-up study of a randomized, prospective trial included 192 patients with newly diagnosed severe aplastic anemia receiving antithymoglobulin and cyclosporine, with or without granulocyte colony-stimulating factor (G-CSF). We aimed to evaluate the long-term effect of G-CSF on overall survival, event-free survival, probability of secondary myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), clinical paroxysmal nocturnal hemoglobinuria, relapse, avascular osteonecrosis and chronic kidney disease. The median follow-up was 11.7 years (95% CI, 10.9-12.5). The overall survival rate at 15 years was 57±12% in the group given G-CSF and 63±12% in the group not given G-CSF (P=0.92); the corresponding event-free survival rates were 24±10% and 23±10%, respectively (P=0.36). In total, 9 patients developed MDS or AML, 10 only a clonal cytogenetic abnormality, 7 a solid cancer, 18 clinical paroxysmal nocturnal hemoglobinuria, 8 osteonecrosis, and 12 chronic kidney disease, without any difference between patients treated with or without G-CSF. The cumulative incidence of MDS, AML or isolated cytogenetic abnormality at 15 years was 8.5±3% for the G-CSF group and 8.2±3% for the non-G-CSF group (P=0.90). The cumulative incidence of any late event including myelodysplastic syndrome or acute myeloid leukemia, isolated cytogenetic abnormalities, solid cancer, clinical paroxysmal nocturnal hemoglobinuria, aseptic osteonecrosis, chronic kidney disease and relapse was 50±12% for the G-CSF group and 49±12% for the non-G-CSF group (P=0.65). Our results demonstrate that it is unlikely that G-CSF has an impact on the outcome of severe aplastic anemia; nevertheless, very late events are common and eventually affect the prognosis of these patients, irrespectively of their age at the time of immunosuppressive therapy (NCT01163942).

Evolution patterns of paroxysmal nocturnal hemoglobinuria clone and clinical implications in acquired bone marrow failure

The paroxysmal nocturnal hemoglobinuria (PNH) clone often presents in acquired bone marrow failure (aBMF), which is involved in more than half of aplastic anemia (AA) cases and about 10%-20% of myelodysplastic syndrome (MDS) cases. PNH clone expansion patterns and clinical implications, however, remain obscure. We conducted a large retrospective study of 457 aBMF patients with positive PNH clones to explore the wide spectrum of clone architecture, evolution patterns, and clinical implications. PNH clone size at diagnosis in AA or MDS was significantly smaller than that in clinical PNH (p < 0.001); the main clone patterns in AA and MDS were granulocyte dominant, with the remaining cases having a granulocyte-erythrocyte balance pattern in clinical PNH. In 131 AA patients at follow-up, there was no obvious difference in response rates between those with the aggressive pattern of clone evolution (73.7%) and those with the stable pattern (81.1%). A quarter of AA patients evolved into clinical hemolysis within a median interval of 11 months. AA cases progressing into clinical hemolysis after immunosuppressive therapy had significantly larger clones (granulocytes: 12.3% vs. 2.6%; erythrocytes: 5.7% vs. 1.3%) at diagnosis and presented mainly an aggressive pattern, especially the granulocyte-erythrocyte aggressive model. Clone sizes reaching 37% for erythrocytes and 28% for granulocytes were indicators of the onset of hemolysis in AA. In conclusion, aBMF patients presented significantly various PNH clone patterns at diagnosis. AA patients with either an aggressive or stable evolution pattern can achieve a response, but patients with an aggressive evolution pattern, especially the granulocyte-erythrocyte aggressive model, tend to evolve into clinical hemolysis.

Development of Paroxysmal Nocturnal Hemoglobinuria in Children with Aplastic Anemia

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells resulting from a somatic mutation in the PIGA gene. PNH frequently manifests in association with aplastic anemia (AA), in which PIGA mutations are believed to enable escape from the immune-mediated destruction by pathogenic T cells. Recent studies using next-generation sequencing have revealed that frequent somatic PIGA mutationsin AA patients are associated with a better response to IST and prognosis (Yoshizato et al N Engl J Med. 2015; 373: 35-47). However, clinical PNH is a progressive and life-threatening disease driven by chronic hemolysis that leads to thrombosis, renal impairment, poor quality of life, and death. Large studies in adults have reported that clinical PNH developed in 10%-25% of AA patients; however; the frequency of clinical PNH in children with AA has rarely been described. Here we aimed to elucidate the pathological link between PNH and AA in children.Methods: In total, 57 children (35 boys and 22 girls) diagnosed with acquired AA at our hospital between 1992 and 2010 were retrospectively studied. Patients who underwent hematopoietic stem cell transplantation as first-line treatment within 1 year after AA diagnosis and those with clinical PNH at AA diagnosis were excluded. Flow cytometry (FCM) was used to detect PNH CD13+/CD55−/CD59− granulocytes and PNH glycophorin A+/CD55−/CD59− red blood cells (RBCs). Clinical PNH was defined as the presence of intravascular hemolysis and ≥5% PNH granulocytes or PNH RBCs. Minor PNH clones were defined as those with >0.005% PNH granulocytes or >0.010% PNH RBCs. We performed targeted sequencing of bone marrow samples from patients with clinical PNH that were obtained at 2 time points: at AA diagnosis and after PNH development. The panel of 184 genes for targeted sequencing included most of the genes known to be mutated in inherited bone marrow failure syndromes and myeloid cancers, as well as PIGA.Results: The median patient age at AA diagnosis was 9.3 (1.2-17.8) years, and the median follow-up period was 123 (2-228) months. A total of 43 patients were screened for PNH clones by FCM after AA diagnosis, and 21 of these with minor PNH clones were identified. The median percentages of PNH granulocytes and PNH RBCs were 0.001% (0.000%-4.785%) and 0.000% (0.000%-3.829%), respectively. During follow-up, 5 patients developed clinical PNH after adolescence (15-22 years of age). The median time between AA diagnosis and PNH development was 4.9 (3.3-7.9) years. All clinical PNH patients were treated with IST for AA, and complete and partial response after 6 months were achieved in 1 and 4 patients, respectively. Gross hemoglobinuria was present in all clinical PNH patients, but thrombosis was not observed. The size of PNH clones varied greatly among patients: PNH granulocytes and PNH RBCs were 42.96% (10.04%-59.50%) and 48.87% (15.02%-90.80%), respectively. Oral cyclosporine A and intravenous eculizumab were administered to 3 and 1 patients, respectively; all patients showed sustained response as indicated by improvement in gross hemoglobinuria and normal blood counts after treatment. The remaining 1 patient underwent bone marrow transplantation from the HLA-identical mother and was alive without any complications. Overall, the 10-year probability of developing clinical PNH was 10.2% (95%CI, 3.6-20.7). Among 43 patients screened for PNH clones at AA diagnosis, the 10-year cumulative clinical PNH incidence was significantly higher in patients with minor PNH clones than in those without minor PNH clones at AA diagnosis [29% (95% CI, 10%-51%) vs. 0% (95% CI, 0%-0%); p = 0.015]. Among all clinical PNH patients, a total of 8 somatic PIGA mutations were detected (missense, 2; splice site, 2; and frameshift, 4). However, PIGA mutations were not detected at AA diagnosis even in patients who subsequently developed clinical PNH.Conclusion: In our cohort, the percentage of patients who eventually developed clinical PNH was comparable to that reported in adults in a previous study. Furthermore, the current study showed that the presence of minor PNH clones at AA diagnosis was a risk factor for the subsequent development of clinical PNH, although the clones were not detected by targeted sequencing. Thus, pediatric AA patients with PNH clones at AA diagnosis should undergo long-term periodic monitoring for potential clinical PNH development. DisclosuresKojima:SANOFI: Honoraria, Research Funding.

Paroxysmal nocturnal hemoglobinuria

The aim is to report on recent observations related to the natural history of paroxysmal nocturnal hemoglobinuria (PNH) and to review new therapeutic strategies for controlling the hemolysis of PNH. This review focuses on studies designed to characterize the long-term outcome of patients with PNH treated with eculizumab and to define the relationship between PNH and bone marrow failure syndromes. New therapeutic strategies aimed at controlling extravascular as well as intravascular hemolysis are also examined. Long-term safety and efficacy of eculizumab was observed in a large group of patients. Survival for the group was not different from that of a sex-matched and age-matched control group from the general population. Thrombotic complications were rare and deaths due to PNH or complications of therapy were not observed. These studies suggest that patients with clinical PNH who are treated with eculizumab have a benign clinical course. Patients with bone marrow failure who have PNH cells detected by high-sensitivity flow cytometry have aplastic anemia or low-risk myelodysplastic syndrome. For patients with a percentage of PNH cells that is below the threshold for producing laboratory evidence of hemolysis (subclinical PNH), expansion of the clone to a size sufficient to produce clinical PNH is not observed. Approximately 50% of patients with bone marrow failure who have clinical evidence of PNH at presentation will require PNH-specific therapy. Novel reagents that target the alternative pathway of complement C3 convertase are being developed with a goal of inhibiting both the extravascular and the intravascular hemolysis of PNH.

Paroxysmal nocturnal hemoglobinuria clones in severe aplastic anemia patients treated with horse anti-thymocyte globulin plus cyclosporine

Clones of glycosylphosphatidylinositol-anchor protein-deficient cells are characteristic in paroxysmal nocturnal hemoglobinuria and are present in about 40-50% of patients with severe aplastic anemia. Flow cytometry has allowed for sensitive and precise measurement of glycosylphosphatidylinositol-anchor protein-deficient red blood cells and neutrophils in severe aplastic anemia. We conducted a retrospective analysis of paroxysmal nocturnal hemoglobinuria clones measured by flow cytometry in 207 consecutive severe aplastic anemia patients who received immunosuppressive therapy with a horse anti-thymocyte globulin plus cyclosporine regimen from 2000 to 2008. The presence of a glycosylphosphatidylinositol-anchor protein-deficient clone was detected in 83 (40%) patients pre-treatment, and the median clone size was 9.7% (interquartile range 3.5-29). In patients without a detectable clone pre-treatment, the appearance of a clone after immunosuppressive therapy was infrequent, and in most with a clone pre-treatment, clone size often decreased after immunosuppressive therapy. However, in 30 patients, an increase in clone size was observed after immunosuppressive therapy. The majority of patients with a paroxysmal nocturnal hemoglobinuria clone detected after immunosuppressive therapy did not have an elevated lactate dehydrogenase, nor did they experience hemolysis or thrombosis, and they did not require specific interventions with anticoagulation and/or eculizumab. Of the 7 patients who did require therapy for clinical paroxysmal nocturnal hemoglobinuria symptoms and signs, all had an elevated lactate dehydrogenase and a clone size greater than 50%. In all, 18 (8.6%) patients had a clone greater than 50% at any given time of sampling. The presence of a paroxysmal nocturnal hemoglobinuria clone in severe aplastic anemia is associated with low morbidity and mortality, and specific measures to address clinical paroxysmal nocturnal hemoglobinuria are seldom required.

Reduced Hematopoietic Stem Cell Function in a Mouse Model with Conditional Pig-a Gene Deletion.

Abstract 2428Poster Board II-405Hematopoietic stem cells (HSCs) are capable of self-renewal as well as differentiation to produce progenitor cells of all hematopoietic lineages in order to sustain life long blood production. In normal inbred mice, HSCs survive multiple rounds of transplantation and extend function to over 100 months, 3-4 times the lifespan of a normal mouse (Harrison DE, Mech Ageing Dev, 1973). We assessed HSC functions in a murine model of paroxysmal nocturnal hemoglobinuria (PNH). PNH originates with a somatic mutation in a HSC of the X-linked phosphatydylinositol glycan class A (PIG-A) gene. We produced a mouse model with conditional Pig-a deletion (Pig-a-/-) by cross-breeding mice carrying germline insertion of two lox sites flanking exon 6 of Pig-a gene with mice carrying the transgene Cre-recombinase driven by the human c-fes promoter. The resultant B6 Fes-cre-Pig-aflox (Pig-a-/-) mice showed Pig-a gene inactivation specifically in hematopoietic cells. Mice survived without obvious abnormalities and did not show any sign of clinical PNH or bone marrow (BM) failure. In preliminary experiments, we confirmed that BM cells from Pig-a-/- donors had normal HSC functional ability to compete with BM cells from congenic B6-CD45.1 competitors in a competitive repopulation assay, as previously shown by another PNH mouse model with conditional Pig-a gene deletion (Keller P et al, J Exp Med, 2001). We then tested whether Pig-a deletion and the resultant GPI deficiency might affect HSC function long term after serial transplantation. Toward this end, we incubated BM cells from Pig-a-/- donors with aerolysin to lyse GPI-normal cells (GPI+) and then transplanted the residual GPI-deficient (GPI-) cells into lethally-irradiated normal C57BL/6 recipients. Within two months, GPI- BM cells engrafted efficiently in all recipients, and 95-100% of recipient T, B and granulocytes were GPI- cells by flow cytometry. After 11 months, we serially-transplanted the GPI- BM cells into lethally-irradiated secondary recipients. Phenotype analysis at one and three months after secondary transplantation also showed relative stable GPI- cell engraftment in T cells (29 ± 9% and 69 ± 29%), B cells (79 ± 6% and 72 ± 15%) and granulocytes (74 ± 11% and 70 ± 10%). After 8 months in the secondary recipients, we extracted BM cells from three secondary recipients with >70% GPI- cells in T, B, and granulocytes. Flow cytometry analysis showed that their BM cellularity was normal, but marrows contained three to five fold lower proportion of cells staining for the Kit+Sca1+Lin- (KSL) markers. We pooled BM cells from these three secondary recipients and transplanted BM cells into lethally-irradiated third-round recipients. Three out of five recipients did not survive transplantation and died between one and two weeks after irradiation/transplantation. The remaining two recipients showed <5% GPI- cells at two months and <2% GPI- cells at seven months following transplantation, suggesting that functional ability of GPI- BM cells had been diminished or drastically reduced. In order to test the competitive repopulating capacity, we also used pooled BM cells from secondary recipients in a competitive repopulation assay, congenic B6-CD45.1 BM cells serving as competitors. There was essentially no engraftment from GPI- donor cells in all recipients, when measured at two and seven months following transplantation, indicating that, after 19 months and two rounds of transplantation, GPI- BM cells were unable to compete with control BM cells for engraftment. In conclusion, Pig-a deletion and lack of GPI-anchored proteins may cause reduced HSC function long-term. Thus, the expansion of the mutant HSC clone seen in PNH patients is unlikely related to any intrinsic self-renewal or proliferative advantage of the HSC clone carrying Pig-a mutation. Disclosures:No relevant conflicts of interest to declare.

Expression of HMGA2 in Blood Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation of PIG-A gene in one or few hematopoietic stem cells and subsequent clonal expansion of mutant stem cells that leads to development of symptoms. It is known that PIG-A mutation is insufficient to account for the clonal expansion required for clinical manifestation of PNH. We are proposing a 3-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Based on the close association of PNH with aplastic anemia, it has been suggested that the selection pressure is immune mediated. However, in spite that over 60% of patients with aplastic anemia have subclinical population of GPI-deficient hematopoietic cells at diagnosis, only 10% develop clinical PNH, suggesting that step-1 and 2 are insufficient to cause PNH. Under immune mediated selection pressure, GPI-deficient cells not only survive, but also proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate increases the chance that additional genetic mutations are acquired, in turn leads to Step 3. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative phenotype. According to this hypothesis, we searched for the candidate gene for Step 3. We reported 2 patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome12, making the break within the 3' untranslated region in HMGA2. This gene encodes an architectural transcription factor which is deregulated in many benign mesenchymal tumors (Blood. 2006 vol.108 no.13, p4232). Recently, many reports show that truncation of 3' untranslated region of HMGA2 disrupts binding of miRNA, let-7, which regulates both transcription and translation of HMGA2. In fact, these two PNH patients with chromosomal abnormalities had ectopic expression of HMGA2 in the bone marrow. Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation. We have established the method for stable isolation of mRNA and miRNA from blood and bone marrow cells from PNH patients and analyzed the expression of HMGA2 and let-7 by quantitative RT-PCR. We have analyzed the peripheral blood from 8 healthy volunteers and 12 PNH patients. The samples from patients had significantly higher expression of HMGA2 than those from normal volunteers (relative mRNA expression, 4.8±2.4 vs 1.3±0.3, p<0.05). We analyzed the genomic sequence of three patients including one who has highest HMGA2 expression and found no mutation in 3' untranslated region. We also analyzed the expression of miRNA and found significantly lower expression of let7b and c in patients. Surprisingly, truncated form without 3' untranslated region is predominantly expressed in patients. There maybe deregulation of alternative splicing of HMGA2 gene in patients, which needs further investigations. We are now analyzing more PNH samples including bone marrow, where proliferation of stem cells takes place, to investigate whether high expression of HMGA2 contributes to the pathogenesis of PNH. In addition we are going to analyze whether high expression of HMGA2 causes the clonal expansion of PNH cells using PNH mouse model.

HLA B*1402, Cw*0802 Haplotype and DRB1*1501 Allele Are Significantly Increased in Italian Paroxysmal Nocturnal Hemoglobinuria Affected Patients.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder characterized by the emergence and expansion of a GPI-defective clonal haematopoiesis. Its clinical features are haemolytic anaemia, cytopenia and thrombosis. Several data suggests the pathogenetic relevance for auto-immune-mediated mechanisms in the expansion of the mutated clone. Since HLA genes represent a susceptibility factor for autoimmunity, we have investigated HLA genotypes in 42 Italian patients with PNH. This study analyzed the HLA class I and class II genes in a cohort of 42 Italian PNH patients. Data indicate the occurrence of a significant increase of A*33 (9.5% in PNH patients versus 2.3% in controls; p<0.05), A*0201 (52.5% versus 32.2%; p<0.05), B*1402 (23.8% versus 5.3%; p<0.0005) and Cw*0802 alleles (23.8% versus 5.9%; p<0.001) in the PNH patient cohort, as compared with a population of 301 healthy controls of the same ethnical origin. In addition, an increased frequency of DRB1*1501 (21.1% versus 5.3%; p<0.005) and DRB1*01 (31.6% versus 10.9%; p<0.005), without specific association with the DRB1*0101 or DRB1*0102 allele, has been revealed. Notably, the B*1402, Cw*0802 (23.8% versus 5.3%; p<0.0005) HLA -B,-C haplotype was significantly increased. Moreover, the A*33, B*1402, Cw*0802, DRB1*0102 HLA-A, -B, -C, -DR haplotype shows a fifteen times increased frequency in PNH patients, if compared with controls (10.5% versus 0.66%; p<0.005). HLA polymorphism represents a main autoimmune susceptibility factor. In this context, our results suggest that specific peptide/s could be relevant for the selection/dominance of PIG-A mutated clones in our cohort of Italian PNH patients. Alternatively, the linkage of the described allele/s and haplotype with not yet characterized genes and/or with gene/s likely involved in pro-inflammatory cytokine productopn might be hypothesized. The association between certain HLA types and the occurrence of PNH may provide further insight into the mechanism for selective damage to normal (GPI+) hematopoiesis, which is responsible for the clonal expansion of GPI-defective hematopoiesis that ultimately gives rise to clinical PNH.

A cohort study of the nature of paroxysmal nocturnal hemoglobinuria clones and PIG‐A mutations in patients with aplastic anemia

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the clonal expansion of blood cells, which are deficient in glycosylphosphatidylinositol anchored proteins (GPI-APs). As PNH frequently occurs during the clinical course of acquired aplastic anemia (AA), it is likely that a process inducing bone marrow failure in AA is responsible for the selection of GPI-AP deficient blood cells or PNH clone. To explore the nature and mutation of a PNH clone in AA. We performed regular repeated flow cytometric analyses of CD59 expression on peripheral blood cells from a cohort of 32 patients with AA. Mutation of phosphatidylinositol glycan class A (PIG-A) was also studied. Fifty-one episodes of occurrences of CD59 negative granulocytes out of a total cohort 167 flow cytometric analyses (31%) were observed in 22 patients (69%). CD59 negative erythrocytes were less apparent than the granulocytes. Repeated occurrences of PNH clones were observed in 16 patients. Most of the emerging PNH clones were transient in nature. They were more frequently detected during episodes of lower white blood cell and platelet counts. Persistence and expansion of the GPI-AP deficient blood cell populations to the level of clinical PNH were seen in only four patients (12.5%). Analysis of PIG-A gene demonstrated eight mutations among the four patients, with two and four independent mutations in two patients. Our study indicates that PIG-A mutations of hematopoietic stem cells with resultant PNH clones, are relatively common among AA patients. It also supports the hypothesis of selection of the PNH clone by a process or condition associated with or responsible for bone marrow failure in AA. However, there must be an additional factor favoring expansion or growth of the clone to the level of clinical or florid PNH.

Immunophenotypic Analysis of PNH Cells

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem-cell disorder in which a gene mutation results in cellular inability to synthesize the glycosylphosphatidylinositol (GPI) anchors needed to attach various proteins to the cell membrane. Traditional clinical laboratory PNH screening and diagnosis are based upon demonstration of increased erythrocyte susceptibility to lysis by activated complement. These methods are neither very specific nor very sensitive, and have largely been superseded by the rapid, sensitive, and specific flow cytometric analysis of GPI-linked antigen expression. The protocols in this unit cover immunophenotypic analysis of red blood cells, peripheral blood granulocytes, and peripheral blood monocytes, as well as a four-color modification for simultaneous analysis of peripheral blood granulocytes and monocytes.

Clinical paroxysmal nocturnal hemoglobinuria is the result of expansion of glycosyl-phosphatidyl-inositol-anchored protein-deficient clone in the condition of deficient hematopoiesis.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired, clonal hematopoietic stem cell disorder in which PIG-A, gene essential for the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor, is somatically mutated. Absence of GPI-linked proteins from the surface of blood cells is characteristic of the PIG-A mutant (PNH) clone and is also accountable fo certain manifestations, such as intravascular hemolysis. It is unclear how the PNH clone expands and comes to dominate hematopoiesis. In this study, CD34+ cells--committed progenitors (colony-forming cells) representing immature hematopoietic stem cells--and reticulocytes representing the differentiated erythroid cells were quantitated in peripheral blood of patients with PNH. Compared with normal controls (n = 29), CD34+ cell levels were significantly lower in PNH patients who did not have preexisting aplastic anemia (AA) (n = 12) (2.47+/-1.23 versus 4.68+/-1.05 x 106/L, mean +/- standard error; P = .022). PNH patients with precedent aplastic anemia (AA+/PNH) showed markedly low CD34+ cell levels compared with normal control subjects (0.6+/-0.29 versus 4.68+/-1.05 x 10(6)/L; P = .0001). In addition, colony-forming cells from PNH patients were significantly decreased compared with those from normal volunteers (erythroid burst-forming units, 2.8+/-1.2 versu 25.6+/-6.2/5 x 10(5) mononuclear cells; P = .0006; and granulocyte/macrophage colony-forming units, 1.2+/-0.5 versus 13.3+/-3.0/ 5 x 10(5) mononuclear cells; P = .0006). These findings occur in both aplastic and hemolytic types of PNH, suggesting hematopoietic failure in PNH. On the contrary, the numbers of reticulocytes and the reticulocyte production index of PNH patients were significantly higher than those of normal persons and comparable to those from patients with autoimmune hemolytic anemia, indicating accelerating erythropoiesis in PNH. The degree of reticulocytosis correlated well with the proportion of CD59- (PNH) reticulocytes. All of the findings suggest that in the condition of deficient hematopoiesis, the PNH clone arising from the mutated hematopoietic stem cell expands and maintains a substantial proportion of the patient's hematopoiesis.

High Incidence of Transiently Appearing Complement-Sensitive Bone Marrow Precursor Cells in Patients with Severe Aplastic Anemia - A Possible Role of High Endogenous IL-2 in Their Suppression

In a prospective long-term study on the incidence of paroxysmal nocturnal hemoglobinuria (PNH), 115 consecutive patients with severe aplastic anemia (SAA), 97 treated with antilymphocyte globulin (ALG) and 18 with bone marrow transplantation (BMT), were observed over a period of 4–18 years and tested for the presence of complement-sensitive hematopoietic precursor cells with the bone marrow (BM) sucrose test. Sixteen (14%) of the ALG-treated patients developed clinical signs of PNH between 0.5 and 8 years after treatment. Complement-sensitive BM precursors were found in 89% of the SAA patients at some time during their disease, but in none of 18 normal donors. At diagnosis, their proportion was significantly higher in patients who later developed PNH than in patients who later achieved disease-free complete remission (CR). After ALG, the abnormal population was found in both groups, but it was gradually replaced by normal precursors in remission patients. After BMT, the complement-sensitive population decreased to very low numbers in patients with a stable graft, but increased again in 3 patients upon graft rejection. Mimicking the PNH defect by enzymatic removal of glycosyl-phosphatidylinositol (GPI)-linked proteins from CD34+ cells resulted in their complement sensitivity, suggesting that the BM sucrose test identifies precursor cells carrying the PNH defect. In 66 patients, white blood cells (WBC) in peripheral blood (PB) were examined for GPI-deficient populations by flow cytometry (FACS). Ten patients with signs of clinical or laboratory PNH had over 25% complement-sensitive precursor cells in the BM and a GPI-deficient WBC population in the PB. Of 56 SAA patients without PNH, 8 had an abnormal population detectable with both tests, 26 only with the BM sucrose test, 4 only with PB FACS analysis, and in 18, no abnormal cells were detected with either test. In search for parameters which might explain why in some patients the abnormal population expands, while it regresses or disappears in others, we tested the release of IL-2 as a parameter of immune competence. At diagnosis, IL-2 release was approximately 50% of normal in patients who later developed PNH, while it was double the normal value in patients who later achieved CR. We conclude that the majority of SAA patients transiently harbor complement-sensitive precursor cells in the BM. Patients with more than 25% abnormal BM precursors and low endogenous IL-2 release are at risk of progression to clinical PNH.