Classical Paroxysmal Nocturnal Hemoglobinuria Research Articles

The immunologic abnormalities in patients with paroxysmal nocturnal hemoglobinuria are associated with disease progression.

To suggest the presence of a hyperimmune state in patients, and indicate that immune system attack on glycosylphosphatidylinositol (+) (GPI+) cells while escaping GPI- cell immunity. We retrospective the immune cell subtypes in peripheral blood from 25 patients visiting Tianjin Medical University General Hospital, Tianjin, China, with classical paroxysmal nocturnal hemoglobinuria (PNH) and 50 healthy controls. The total CD3+ and CD3+CD8+ cell levels were higher in patients with PNH. The CD3+ cells are positively, correlated with lactate dehydrogenase (LDH; r=0.5453, p=0.0040), indirect bilirubin (r=0.4260, p=0.0379) and Flear- cells in monocytes (r=0.4099, p=0.0303). However, a negative correlation was observed between CD3+ cells and hemoglobin (r= -0.4530, p=0.0105). The total CD19+ cells decreased in patients, and CD19+ cells were negatively correlated with LDH (r= -0.5640, p=0.0077) and Flear- cells in monocytes (r= -0.4432, p=0.0341). Patients showed an increased proportion of total dendritic cells (DCs), with a higher proportion of myeloid DCs (mDCs) within the DC population. Moreover, the proportion of mDC/DC was positively correlated with CD59- cells (II + III types) in red cells (r=0.7941, p=0.0004), Flear- cells in granulocytes (r=0.5357, p=0.0396), and monocytes (r=0.6445, p=0.0095). Our results demonstrated that immune abnormalities are associated with PNH development.

Transplant Outcomes for Acute Myeloid Leukemia or Myelodysplastic Syndromes Secondary to Acquired Aplastic Anemia or Paroxysmal Nocturnal Hemoglobinuria: A Report from the EBMT Severe Aplastic Anemia Working Party

Introduction Clonal evolution into myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) is the most concerning late event among non-transplanted acquired aplastic anemia (AA) or paroxysmal nocturnal hemoglobinuria (PNH) patients, occurring in nearly 10% of cases. MDS/AML secondary to AA differ from primary MDS/AML, presenting with higher risk cytogenetics and poorer prognosis, usually involving abnormalities in chromosome 7 (Gurnari C. et al., JCO 2023). The survival is poor, and the only curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT). We present the HSCT outcomes of secondary MDS/AML following AA/PNH. Methods We have extracted data from the EBMT database for patients with AA or PNH diagnosed after 2010, who later presented a clonal evolution into AML, MDS, or isolated cytogenetic abnormality insufficient for MDS diagnosis according to WHO 2016 classification, who have undergone HSCT after the clonal evolution diagnosis. We have excluded AA/PNH patients with HSCT before clonal evolution, inherited bone marrow failure syndromes, and non-transplanted patients. Our primary objective was to describe the overall survival (OS). The secondary aims were transplant outcomes according to clonal evolution category and conditioning regimen intensity. Results: We have studied 278 patients from 114 different EBMT centers, 250 patients diagnosed with AA, and 28 with classical PNH. The median age at AA/PNH diagnosis was 34.7 (20.5-51.7) years. At first evolution, 153 (56.5%) patients had a diagnosis of MDS, 45 (16.6%) AML, and 73 (26.9%) isolated abnormal cytogenetics insufficient for a diagnosis of MDS (missing data for 7 patients). The median interval from AA/PNH diagnosis to clonal evolution was 30.1 (8.1-67) and 39.8 (21.5-82.6) months for MDS and AML, respectively. The interval from clonal evolution to HSCT was 5 (3-10.2) and 3.4 (2.3-5.1) months for MDS and AML, respectively. We present transplant characteristics in Table 1. With a median follow-up of 5 (4.3 - 5.9) years, the 5-year overall survival (OS) was 65% for the entire cohort. A subgroup analysis did not find a significant 5-year OS difference among groups: 73, 62, and 60% for isolated cytogenetic abnormality, MDS, and AML, respectively (p=0.3, Figure 1). The main causes of death were transplant-related toxicities (66.7% and 75% for isolated cytogenetic abnormality and MDS, respectively) and relapse or progression for AML (43.8%). AML patients relapsed more frequently than MDS patients by 5 years [cumulative incidences of 22 (9-35) vs. 10% (4-17) for AML and MDS (p=0.046), respectively]. The 5-year non-relapse mortality (NRM) was 19 (7-31) and 28% (19-36) for AML and MDS patients, respectively (p=0.3). When stratifying by conditioning regimen intensity, the 5-year NRM was 34% (19-40) for myeloablative (MAC) vs. 18% (9-26) for reduced-intensity (RIC, p=0.025), with similar incidence of relapse in both groups (4 vs 10% for MAC and RIC, respectively, p=0.2). AML patients were more prone to have presented acute graft-versus-host disease (GvHD) by day+100 than MDS or isolated cytogenetic abnormality patients (cumulative incidences of 36, 26, and 14% respectively, p=0.022). Likewise, AML patients had more chronic GvHD (43, 28, and 12% respectively, p=0.008). Graft-failure, GvHD, relapse-free survival (GGRFS) was 71 (61-82), 60 (51-68), and 45% (29-60) for isolated abnormality, MDS, and AML, respectively (p=0.026). Discussion This study is the largest cohort of transplanted patients with secondary MDS/AML following AA/PNH. The major limitations of our study are its retrospective nature, non-adjusted analyses, and missing data on complete cytogenetics. We found a 5-year OS higher than 60% for MDS/AML compared to 45% in the previously published EBMT study (Hussein A. et al., BBMT 2014) and did not find a survival difference between AML and MDS. Interestingly, the main cause of death for MDS patients was HSCT complications. Furthermore, MAC was also associated with a higher NRM. Clinicians should indicate HSCT with discretion for post-AA/PNH MDS or isolated cytogenetic abnormality, considering the transplant toxicity, especially with MAC, and the risk of primary disease progression. For patients with AML and a high risk of relapse, early tapering of immunosuppressive agents and post-transplant maintenance, such as hypomethylating agents and donor lymphocyte infusion, should be considered.

Allogeneic Hematopoietic Stem Cell Transplantation for Classical Paroxysmal Nocturnal Hemoglobinuria

Classical paroxysmal nocturnal hemoglobinuria (cPNH) is a rare acquired clonal hematopoietic cell disease. Eculizumab is effective in PNH patients, though, it must be administered indefinitely. As the technology of allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves in recent years, what is the position of HSCT in treatment of cPNH? This study discusses the efficacy and safety of allo-HSCT in cPNH. The clinical data of 5 cPNH patients who underwent allo-HSCT from 2019 to 2022 were collected. All patients were diagnosed cPNH according to guidelines from International PNH Interest Group. Indications for HSCT were transfusion-dependent and nonavailability of eculizumab. The cohort was all male, with a median age of 26 years (range, 26 to 46 years) at the time of transplantation. All patients had documented hemolysis and no thrombosis during monitoring. The median time from diagnosis to allo-HSCT was 5.5 years (range, 3.6 to 18 years). The median PNH granulocyte clone size was 96.3% (range, 90% to 99.7%), and the median lactate dehydrogenase (LDH) level was 2224IU/L (8.896-fold of the upper limit of normal). All patients were detected bone marrow hyperplasia by trephine biopsy. The stem cell source came from 4 haploidentical donors and 1 HLA-identical sibling donor. Among the 5 patients, 3 patients underwent myeloablative conditioning (MAC) and 2 patients underwent reduced-intensity conditioning (RIC) treatment. None of the patients experienced primary implantation failure. Neutrophil implantation time was 15 (13-21) days, and platelet implantation time was 24 (13-60) days. 3 patients developed grade II acute graft-versus-host disease (aGVHD). No patients developed grade Ⅲ-Ⅳ aGVHD. 2 patients developed localized chronic graft-versus-host disease (cGVHD) involving lung and skin respectively, and no patients developed extensive cGVHD. PNH clones turned negative in all patients after 2 months (range, 1 to 3 months) of transplantation. At a median follow-up of 16 (6-34) months, 1 patient died of relapse and infection. The remaining 4 patients survived, of which 2 patients had discontinued all drugs. cPNH has its own unique clinical features. Allo-HSCT could cure cPNH patients who can withdrawal from treatment of all medication. Haploidentical donor HSCT is an acceptable choice for cPNH, and RIC constitutes a valid option.

Efficacy and Safety of Allogenic Hematopoietic Stem Cell Transplant in Paroxysmal Nocturnal Hemoglobinuria: A Single Centre Study from Pakistan

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disorder of clonal hematopoiesis1 .The resulting abnormalities in complement regulatory proteins can manifest as hemolysis, thrombosis, and marrow failure2. The management and clinical outcomes of patients with PNH significantly changed with the development of complement inhibitor therapies3. Allogenic Hematopoietic stem cell transplant (HSCT) despite being the sole curative option for PNH is not recommended as first line therapy owing to its toxicities 4,5. Current indications for HSCT include a suboptimal response to eculizumab, severe AA/PNH, clonal evolution to myelodysplastic syndrome (MDS), or acute myelogenous leukemia2. However, in low and middle income countries (LMIC) where complement inhibitor therapies are not available, allogenic HSCT still remains the only treatment option for classical PNH as well as for PNH in the setting of another specified bone marrow disorder. Objective: To analyze the efficacy and safety of Allogenic HSCT in patients with paroxysmal nocturnal hemoglobinuria (PNH). Study design: Observational, retrospective study. Place and duration of study: Armed Forces Bone Marrow Transplant Centre/ National Institute of Blood and Marrow Transplant (AFBMTC/NIBMT) Rawalpindi, Pakistan, from June 2008 till February 2022. Methodology: From June 2008 till February 2022, a total of 65 patients of PNH were registered at AFBMTC/NIBMT. Out of these16 patients underwent allogenic HSCT and were included in the final retrospective analysis. PNH diagnosis was confirmed by flow cytometry by the demonstration of GPI-negative cells in at least two cell lineages i.e neutrophils, red blood cells (RBCs) in 62.5% (n=10) patients and by fluorescent aerolysin (FLAER)-based assay in 37.5% (n=6) patients. Median time from diagnosis to transplant was 380 days. All patients received HLA identical sibling donor HSCT. Stem cell source was Bone marrow harvest (BMH), BMH plus peripheral blood stem cell (PBSC) and PBSC alone in 81% (n=13), 12.5% (n=2) and 6% (n=1) patients respectively. Graft versus host disease (GVHD) prophylaxis was with Antithymocyte globulin (ATG) + Cyclosporin (CSA) in patients receiving NMA conditioning or ATG + CSA + short term Methotrexate (MTX) in those receiving MAC. Results: Median age of the study cohort was 24 years (range, 18 to 42 years) and all were male. 68.7% (n=11) patients received immunosuppressive therapy before HSCT with partial or no response. History of thrombosis prior to HSCT was present in only one patient (cPNH). Allogenic HSCT done for classical PNH (cPNH) in 75% (n=12) patients and for PNH in the setting of another specified bone marrow disorder (PNH/AA) in 25% (n=4) patients. Patients with classical PNH received Myeloablative conditioning (MAC) i.e Flu150Bu12.8IV (75%, n=9), Bu16Cy120 (8.3%, n=1), or Flu150Cy200 (8.3 %, n=1) except for one patient who received Flu150Cy160. Patients with PNH/AA, received non myeloablative conditioning (NMA) i.e Flu150Cy120, except for one who received Flu150Bu12.8IV. Out of 16 patients, 94% (n=15) patients achieved successful engraftment with full donor chimerism. One patient (cPNH) had Primary graft failure. Median time to neutrophil and platelet engraftment was day +12 and day +22 respectively. Disease relapse was monitored by donor chimerism till Day +180 post BMT and thereafter by peripheral blood counts. PNH clone post BMT was not tested. Incidence of acute GVHD grade I/II involving skin and gut, was 25% (n=4). No incidence of chronic GVHD was noted. One patient (PNH/AA) had secondary graft failure on Day +46. With a median post-transplant follow-up of 15 months (range, 4 to 157) overall survival (OS) was 93.7% and disease-free survival (DFS) was 87.5%. Conclusion: In resource constrained, LMIC, where complement inhibitor therapies are currently unavailable, allogenic HSCT remains the valid option for treatment of PNH, with good overall survival rate and acceptable treatment related toxicites. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinico-Molecular Risk Factors and Management of Thrombosis in a Large Real-Life Cohort of Patients with Paroxysmal Nocturnal Hemoglobinuria in the New Era of Anti-Complement Inhibitors and Doacs

The increased propensity for thrombotic events (TE) is well established in patients with paroxysmal nocturnal hemoglobinuria (PNH) and represents the main cause of disease morbidity/mortality. While the precise pathogenesis of TE remains ill-defined and likely multifactorial, cases with fully blown hemolytic PNH clones show the highest risk for such complications, often occurring in atypical localizations including the hepatic circulation (e.g., Budd-Chiari syndrome, BCS). The introduction of anti-complement therapy greatly abated hemolysis and thrombosis, demonstrating the crucial involvement of the underlying hemolytic process in triggering TE. Despite the current availability of PNH-directed treatment, a scarcity of data exist on anticoagulation and specifically on the efficacy of direct oral anticoagulants (DOACs) in this setting. Here, we took advantage of a large, real-life cohort of patients with classical hemolytic PNH to unravel determinants of thrombosis and decipher anticoagulation strategies in cases developing TE. Among 106 PNH cases (M:F ratio 0.86, median age 39 years, IQR 26-58) followed-up for a median of 68 months (95% CI 50-92), 42% were primary and 58% had antecedent aplastic anemia. Median granulocyte clone size at diagnosis was 71% (46-90) with the majority of cases classified as type III dominant red blood cells (RBCs) clone (81%) by flow cytometric analysis.1In total, 77% of cases harbored PIGA mutations by multi-amplicon deep NGS at a median ploidy-adjusted VAF of 20% (9-33), of whom 58% presenting multiple hits (clonal mosaicism).1 A total of 33 patients (31%) experienced TE, of which 84% occurred as the initial disease manifestation prompting to seek medical attention, 13% during a temporary discontinuation of anti-complement treatment (e.g., preparation to allogeneic bone marrow transplant), and only 3% (N=1) during treatment. Overall, the majority of patients had TE originating in the venous system (91%), arterial cerebrovascular district (6%) or both (3%). TE occurred typically in unusual sites with BCS as the most frequent (34%) followed by deep vein thrombosis (DVT, 20%), DVT complicated by pulmonary embolism (14%), splanchnic (14%), cerebral sinuses and superficial veins (9% each). No differences in disease ontogenesis (primary vs secondary to AA), and baseline variables such as age, PNH clone size, gender, PIGA mutations (presence, type and clonal burden), blood counts, reticulocytes, LDH, creatinine and transaminases were observed. Interestingly, patients experiencing TE were more commonly presenting with type II RBC phenotype (36% vs 13%; p=.027), had median lower albumin (4 vs 4.5 g/dL; p<.001) and elevated D-dimers (1520 vs 325 ng/mL; p=.003) as compared to non-TE cases. Patients with superficial TE were promptly started on anti-complement treatment, while the others improved with additional anticoagulation. One patient with portal vein thrombosis had severe thrombocytopenia and eventually died due to progressive BCS. Anticoagulation strategy consisted of warfarin (50%), DOACs (29%) and low molecular weight heparin (LMWH, 21%) with a median treatment duration of 44 months (17-73). LMWH was preferred in patients with thrombocytopenia (median platelets 45 vs 102x109/L in cases receiving other therapeutics; p=.03). In 1 patient, warfarin was switched to DOACs for prolonged treatment, 2 BCS cases underwent TIPS, and in 1 case with DVT an inferior vena cava filter was positioned because of the concomitant diagnosis of myeloproliferative neoplasm with MPLW515L. Recurrence of TE under anticoagulants was noted in only 2 cases receiving warfarin, whereas 7 (N=5 warfarin/ 2 DOACs) experienced bleeding. Two patients on DOACs had acute rectal hemorrhage with additional bleeding risk factors (clopidogel, ulcerative colitis). No thrombotic recurrence was observed in all 9 patients treated with DOACs at a median observation time of 24 months (12-95). While previously TE prompted lifelong anticoagulation, the control of the underlying disease with anti-complement drugs enabled its successful discontinuation in 42% of patients without any TE recurrence at a median time of 50 months (15-90). This is the largest hemolytic PNH series ever reported detailing clinico-molecular risk factors, characteristics of TE and their management in the era of anti-complement therapy and DOACs, whose use in such a setting warrants attention and further studies.

Pregnancy in Paroxysmal Nocturnal Hemoglobinuria - a Systematic Review

Background Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal disorder of hematopoietic stem cells, characterized by complement-mediated intravascular hemolysis, thrombosis, and bone marrow failure. Pregnancy exacerbates the clinical features of PNH, increasing maternal and fetal morbidity and mortality. Pregnancy in patients with PNH has traditionally been discouraged. Terminal complement inhibition with eculizumab gained regulatory approval in 2007 and has revolutionized the management of PNH. Limited data suggest that eculizumab is also safe and efficacious during pregnancy. Owing to the rarity of PNH and the inability to conduct randomized controlled trials on this population, most reports are small case series, with even the largest retrospective studies including well below 100 patients. We therefore present the first comprehensive systematic review and meta-analysis of outcomes in 190 pregnancies from 135 women with PNH. We compare pregnancy complications between those receiving eculizumab and those not, since complement blockade is not universally available. For the first time we demonstrate that eculizumab is associated with a lower risk of fetal loss. Methods The PubMed (Medline) database was searched for studies reporting primary data on pregnancy outcomes in the context of classical hemolytic PNH, diagnosed through flow cytometry, acid Ham's or sucrose lysis (Figure 1). Women with evidence of recurrent hemolytic episodes including serum lactate dehydrogenase >1.5 times the upper limit of normal, and typical PNH symptoms were included. We excluded papers where there was insufficient information for meaningful downstream analysis, or where cases were duplicated elsewhere in the literature. The search was restricted to English language papers. Two reviewers independently screened articles for inclusion with a third opinion sought where there was disagreement. References of included papers and review articles were screened to identify further papers. Thirty-six papers published between 1968 to 2021 were selected for inclusion. The majority were small case series or single case studies. There were two larger retrospective studies reporting on 45 and 75 pregnancies respectively. There was one prospective study (n=15 pregnancies). Results Eculizumab was used in 130 pregnancies. No maternal deaths were reported in patients on eculizumab. One woman who had never received eculizumab died at 12 weeks postpartum from intra-abdominal sepsis following a severe episode of acalculous cholecystitis. A higher rate of fetal survival was observed in pregnancies where eculizumab was used (82%) than in pregnancies when it was not (69%) (Table 1). Thrombosis occurred at a higher rate than in the healthy pregnant population but did not significantly differ in incidence between treatment groups (Table 1). The incidence of pre-eclampsia was high in this population. There was a trend towards a reduced risk of pre-eclampsia and infection in eculizumab pregnancies, but this did not reach statistical significance (Table 1). No Neisserial infections were observed in patients treated with eculizumab. The rate of spontaneous miscarriage (<24 weeks') was nearly twice as high in women who had not received eculizumab (18.6%) compared to those who received it (9.7%). The rate of intrauterine death did not significantly differ between treatment groups (Table 1). Fetal structural anomalies occurred in three cases, none of which had been exposed to eculizumab, supporting the notion that the drug does not have a teratogenic effect. Intrauterine growth restriction (IUGR) occurred in just over 8% of pregnancies that progressed beyond 20 weeks and did not differ between treatment groups (Table 1). IUGR was associated with pre-eclampsia in eight cases (61%). 148 pregnancies progressed to delivery, the majority by vaginal delivery. Premature birth (<37 weeks' gestation) was observed in 32% of eculizumab pregnancies and 44% of non-eculizumab pregnancies. Premature delivery was usually iatrogenic due to maternal or fetal complications, with spontaneous premature labor in only eight cases (15%). Conclusion Eculizumab appears to be safe and provide a benefit in pregnancy in PNH. There were less premature births and spontaneous miscarriages and greater fetal survival when eculizumab was given. A tendency to fewer cases of pre-eclampsia and maternal infections was also seen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Aplastic anemia and paroxysmal nocturnal hemoglobinuria in children and adults in two centers of Northern Greece.

Bone marrow failure (BMF) syndromes are a group of various hematological diseases with cytopenia as a main common characteristic. Given their rarity and continuous progress in the field, we aim to provide data considering the efficiency and safety of the therapeutic methods, focusing on the treatment of aplastic anemia(AA) and paroxysmal nocturnal hemoglobinuria (PNH). We enrolled consecutive patients diagnosed with BMF in two referral centers of Northern Greece from 2008 to 2020. We studied 43 patients with AA (37 adults and 6 children/adolescents) and 6 with classical PNH. Regarding classical PNH, 4 patients have received eculizumab treatment with 1/4 presenting extravascular hemolysis. Among 43 patients with aplastic anemia, PNH clones were detected in 11. Regarding patients that did not receive alloHCT (n=15), 14/15 were treated with ATG and cyclosporine as first line, with the addition of eltrombopag in patients treated after its approval (n=9). With a median follow-up of 16.7 (1.8-56.2) months from diagnosis, 12/14 (85.7%) are alive (4-year OS: 85.1%). AlloHCT was performed in 28 patients. Five patients developed TA-TMA which did not resolve in 3/5 (all with a pre-transplant PNH clone). With the follow-up among survivors reaching 86.3 (6.3-262.4) months, 10-year OS was 56.9%, independently associated with PNH clones after adjusting for age (p=0.024). In conclusion, our real-world experience confirms that novel treatments are changing the field of BMF syndromes. Nevertheless, there is still an unmet need to personalize algorithms in this field.

Insights Into the Emergence of Paroxysmal Nocturnal Hemoglobinuria.

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

Hematopoietic Stem Cell Transplantation for Patients with Paroxysmal Nocturnal Hemoglobinuria with or without Aplastic Anemia: A Multicenter Turkish Experience

Objective:Although inhibition of the complement system at different steps is a promising therapy modality in patients with paroxysmal nocturnal hemoglobinuria (PNH), allogeneic hematopoietic stem cell transplantation (HCT) is still the only curative therapy, especially for patients with intractable hemolysis or bone marrow failure. The aim of this study is to evaluate the outcomes of allogeneic HCT in PNH patients with aplastic anemia (PNH-AA) or without.Materials and Methods:Thirty-five PNH/PNH-AA patients who were treated with allogeneic HCT in 10 transplantation centers in Turkey were retrospectively analyzed.Results:Sixteen (45.7%) and 19 (54.3%) patients were diagnosed with classical PNH and PNH-AA, respectively. The median age of the patients was 32 (18-51) years. The 2-year overall survival (OS) rate and rate of graft-versus-host disease-free, failure-free survival (GFFS) was 81.2% and 78.1%, respectively. The 2-year OS in cases of classical PNH and PNH-AA was 81.3% and 79.9%, respectively (p=0.87), and 2-year GFFS in cases of PNH and PNH-AA was 79% and 76% (p=0.977), without statistical significance. The OS and GFFS rates also did not differ between transplantations with matched sibling donors (MSDs) and matched unrelated donors (MUDs).Conclusion:Allogeneic HCT with MSDs or MUDs is a good option for selected patients with classical PNH and PNH-AA. In particular, patients with debilitating and refractory hemolysis and patients with bone marrow failure might form an excellent group of candidates for allogeneic HCT.

Pregnancy in Classical Paroxysmal Nocturnal Hemoglobinuria and Aplastic Anemia-Paroxysmal Nocturnal Hemoglobinuria: A High-Risk Constellation.

Pregnancies in paroxysmal nocturnal hemoglobinuria (PNH) are associated with increased morbidity and mortality. Retrospective studies suggest that outcome has improved with the advent of the complement inhibitor eculizumab. To substantiate this assumption we analyzed the data from patients treated in our department since 2009. All patients were included in the International PNH registry and followed prospectively. We identified 16 pregnancies in 9 patients with classical PNH, and two pregnancies in two patients with aplastic anemia (AA)-PNH. In classical PNH, 13 pregnancies were supported by eculizumab. Breakthrough hemolysis occurred in six pregnancies, necessitating an increase in the biweekly eculizumab dose from 900 mg to 1,200–1,800 mg. Red blood cell transfusions were given in six and platelet transfusions in two pregnancies. A Budd-Chiari syndrome and cholecystitis complicated the course of two pregnancies. Four of 13 pregnancies supported by eculizumab ended in spontaneous abortion or stillbirth, and one was prematurely terminated because of fetal trisomy 21. None of the three pregnancies not supported by eculizumab had a successful outcome. Half the deliveries were preterm. None of the patients died, and, in all but one patient, the post-partum period was uneventful. Both pregnancies in patients with AA-PNH took a favorable course. Our results confirm low maternal mortality and frequent breakthrough hemolysis in pregnant PNH patients receiving eculizumab. Fetal mortality and the rate of preterm delivery were higher than reported previously, possibly related to the use of registry data that are likely to reduce the risk of publication and recall biases.

Prognostic factors and follow-up parameters in patients with paroxysmal nocturnal hemoglobinuria (PNH): experience of the Austrian PNH network.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease characterized by a deregulated complement system, chronic Coombs-negative, intravascular hemolysis, and a variable clinical course with substantial risk to develop thromboembolic events. We analyzed diagnostic and prognostic parameters as well as clinical endpoints in 59 adult patients suffering from PNH in 5 hematology centers in Austria (observation period: 1978-2015). Median follow-up time was 5.6 years. The median clone size at diagnosis amounted to 55% and was higher in patients with classical PNH (81%) compared to patients with PNH associated with aplastic anemia (AA) or myelodysplastic syndromes (MDS) (50%). The clone size also correlated with lactate dehydrogenase (LDH) levels. In one patient, anemia improved spontaneously and disappeared with complete normalization of LDH after 16 years. Seventeen patients received therapy with eculizumab. The rate of thromboembolic events was higher in the pre-eculizumab era compared with eculizumab-treated patients but did not correlate with the presence of age-related clonal hematopoiesis or any other clinical or laboratory parameters. Peripheral blood colony-forming progenitor cell counts were lower in PNH patients compared with healthy controls. Only two patients with classical PNH developed MDS. Overall, 7/59 patients died after 0.5-32 years. Causes of death were acute pulmonary hypertension, Budd-Chiari syndrome, and septicemia. Overall survival (OS) was mainly influenced by age and was similar to OS measured in an age-matched healthy Austrian control cohort. Together, compared with previous times, the clinical course and OS in PNH are favorable, which may be due to better diagnosis, early recognition, and eculizumab therapy.

Clinical Characteristics of Brazilian Patients with Paroxysmal Nocturnal Hemoglobinuria and Changing Prognosis with Eculizumab

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic cells due to acquired mutations in the phosphatidylinositol glycan class A (PIG-A) gene, which is required for glycosylphosphatidylinositol (GPI) anchor biosynthesis. This leads to partial or complete absence of all GPI-linked proteins, who are complement regulatory proteins, resulting in an increased sensitivity of the red blood cells to the action of complement. PNH is characterized by signs and symptoms related to intravascular hemolysis, hypercoagulability state, and varying degrees of medullary insufficiency. The anti-complement therapy radically changed the PNH patients outcomes. However, there are little data on the clinical characteristics of PNH in Latin American countries. Methods: We performed a retrospective analysis of 109 patients with PNH clone followed from January 1987 until July 2019 in two Brazilian centers: Universidade Federal de Sao Paulo and Hospital Sirio Libanes (Sao Paulo-Brazil). Most patients (88%) were evaluated while the others had lost follow up or died and data was obtained from their medical reports. Patients were separated into 3 groups: classical PNH (n=44) PNH associated with other bone marrow disorder(n=12), and subclinical PNH, defined as PNH clone (at least 0.01% of cells with PNH clone) associated with another bone marrow disorder (n=53, aplastic anemia in 95% of cases). Median follow up was 60 months (range: 3-394). Results: Median age at diagnosis was 41 years (range: 18-81), and 51% were male. Among the 56 patients with hemolytic PNH, 86% had fatigue, 66% hemoglobinuria, 45% abdominal pain and 16% dysphagia. Venous thromboembolism was observed in 14 cases (25%), with abdominal thrombosis in 7 cases (50%). Seven patients (13%) had arterial thrombosis (stroke or transient ischemic attack). Only 5 patients (10%) in the hemolytic group had acute renal failure and needed dialysis therapy due to a hemolytic crisis, but progressed to recovery of renal function after the event. No patient in this series had moderate or severe chronic kidney disease. Most hemolytic patients (73%) were treated with eculizumab, with a median time from diagnosis to the start of eculizumab of 25 months (range: 2-275). All eculizumab-treated patients had significant reduction in intravascular hemolysis with lactate dehydrogenase (LDH) normalization. Most had significant improvement in anemia, with increase in the median hemoglobin from 9.1 g/dL before treatment to 11.7 g/dL after eculizumab. The vast majority (94%) became transfusion-independent. Overall survival (OS) at 5 years was 100% at 5 years for classical PNH (n=44), 89% for subclinical PNH (n=53) and 71% for PNH associated with another bone marrow disease (n=12). Conclusion: The clinical data and the distribution of the three subtypes of PNH in this study in this large series of Brazilian PNH patients were similar to other published series, except for a lower frequency of venous or arterial thrombosis in hemolytic patients before eculizumab treatment and a lower frequency of chronic kidney disease in our series. We also confirmed in our series the efficacy of eculizumab in controlling hemolysis and PNH-related complications and death risk. Disclosures No relevant conflicts of interest to declare.

PIG-a Mutations in a Brazilian Cohort of Patients with Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a disorder due to an acquired loss-of-function mutation in the phosphatidylinositol glycan class A (PIG-A) gene. A large spectrum of acquired PIG-A mutations has been described, like insertions or deletions involving a single base or several bases, and single base substitution that are the most common. Usually there are more than one PIGA mutations and one clone is predominant. The clinical manifestations of PNH are intrinsically related to clonal expansion of hematopoietic stem cell deficient in GPI-anchored proteins. Some hypothesis failed to explain alone this clonal expansion. Here we try to identify and to correlate PIG-A gene mutations with clinical manifestations in a series of patients with PNH. Methods: We analyzed 31 patients with classical PNH (n=23) or aplastic anemia and PNH clone (n=8). The sequencing of the PIG-A gene was performed using the Sanger technique. The electropherograms were aligned against the reference sequence of the PIG-A gene deposited in GenBank (Accession number NG_009786), and analyzed using the Geneious R10 software (Biomatters). After analysis, a search was performed in the Clinvar, dbSNP and HGMD databases to verify the pathogenicity of the mutations. For variants without description in the literature, a pathogenicity prediction analysis was performed using Mutation Taster, Polyphen 2 and Human Splicing Finder software. Results: We found 29 different variants of the PIG-A gene in 27 patients: 23 were new mutations, with no previous description in the literature, 3 were previously described mutations, and 3 were single nucleotide polymorphism (SNP). There was great variation in the type and location of somatic mutations. Mutations were predominantly small deletions and simple base changes; 42% of the mutations were described as frameshift mutations and 31% missense mutations. We did not find any specific correlation between the clinical characteristics of hemolytic PNH patients and their mutations, due to the wide variety of mutations. According the pathogenicity prediction programs, the majority (22 of 29) of the variants found were classified as probably pathogenic. Among the 23 patients with hemolytic PNH, 19 patients had at least one mutation classified as pathogenic. In patients with subclinical PNH, only SNPs were found. Fifteen patients with hemolytic PNH had more than one concomitant mutation, most of which were probably pathogenic mutations associated with a polymorphism. Conclusion: We described PIG-A mutations in a series of PNH patients in Brazil and observed no correlation exists between mutation types and clinical features in hemolytic patients. Among subclinical PNH patients, only SNPs were observed, probably because of small clone sizes. Disclosures No relevant conflicts of interest to declare.

Expression and influencing factors of hepcidin in classical paroxysmal nocturnal hemoglobinuria

目的研究铁调素在经典型阵发性睡眠性血红蛋白尿症（PNH）患者血清中的表达及其影响因素。方法以2016年3月至2017年3月36例经典型PNH患者为研究对象，采用ELISA方法检测血清铁调素水平，并结合患者铁稳态状况和临床资料，分析经典型PNH患者血清铁调素水平与血清铁蛋白（SF）、可溶性转铁蛋白受体、C反应蛋白、EPO、网织红细胞绝对值、网织红细胞血红蛋白等血液学参数的相关性。结果36例经典型PNH患者血清铁调素水平为32.03（23.11，118.48）µg/L，明显低于正常对照组的181.42（106.80，250.53）µg/L（z=−5.107，P<0.001）。其中，铁蛋白正常经典型PNH患者血清铁调素水平为56.41（44.60，95.06）µg/L，经典型PNH伴缺铁组血清铁调素水平为23.75（21.77，30.35）µg/L，均低于正常对照组；而经典型PNH伴铁蛋白升高组血清铁调素水平为336.19（304.19，375.08）µg/L，高于正常对照组、铁蛋白正常经典型PNH组及经典型PNH伴缺铁组。多重线性回归分析显示SF、转铁蛋白饱和度和血清白蛋白水平是经典型PNH患者血清铁调素水平的独立影响因素。结论经典型PNH患者血清铁调素水平减低，其表达主要受铁代谢因素影响。

PB1857 THE ROLE OF WHOLE‐BODY MAGNETIC RESONANCE IMAGING IN THE DETECTION OF VASCULAR EVENTS IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA.

Background:Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder characterized by intravascular hemolysis, cytopenias, and recurrent thrombosis. Thrombotic events are just the main cause of morbidity and mortality. Therefore, it is essential to detect them early to treat them and avoid sequels.Aims:To analyze the diagnostic value of the whole‐body magnetic resonance imaging (WB‐MRI) in the detection of symptomatic and silent vascular events.Methods:Retrospective study in which a 1.5 Tesla whole‐body magnetic resonance (Philips Achieva, Philips Medical Systems, Best, the Netherlands) with Gadolinium contrast was performed along 2017 and 2018 to patients diagnosed of classical PNH in Autonomous Community of the Region of Murcia, Spain. The image findings were analyzed.Four patients were included (3 men and 1 woman) with median age of 54 years, diagnosed of either PNH (n = 2) and AA/PNH (n = 2). At the time of investigation, mean size clon in granulocytes measured by FLAER was high (90.7 %, range 75.6‐99.3 %) and mean LDH was 341.7 U/L (range 204‐465 U/L; normal range 135‐214 U/L). Only one patient had prior history of thrombosis (Budd‐Chiari syndrome). All patients were treated according to Spanish PNH guidelines, including the use of complement inhibitor Eculizumab.Results:WB‐MRI findings showed unknown bone infarctions in two (50%) patients (patient 1 and patient 3). In patient 1, symptomatic bone infarctions was diagnosed (both femoral condyle and proximal tibias). Patient 3 showed an asymptomatic area of bone edema in distal left radial bone.Summary/Conclusion:Although the sample from our study is very small, it seems that WB‐MRI could be useful to evaluate vascular events, like bone infarctions, even subclinical ones. Our findings are consistent with the only article published to date (Alaskar et al. Sci Rep. 2018; 8: 13458). Further studies are needed to test the effectiveness of WB‐MRI in the detection of vascular events and their relationship as a thrombotic event in PNH.imageimage

Outcomes of paroxysmal nocturnal hemoglobinuria in the pediatric age group in a resource‐constrained setting

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal stem cell disorder. Eculizumab and bone marrow transplantation are disease-modifying treatments for PNH but may not be readily available in resource-constrained settings. Of 52 pediatric patients with PNH, 20 had classical PNH and 32 had PNH/aplastic anemia (PNH/AA). Median time to diagnosis was 30 months in classical PNH patients. Renal failure was present in four patients (20%). Six (30%) achieved complete response, 10 (50%) achieved partial response with androgens in classical PNH. Two underwent allogenic stem cell transplantation. In the PNH/AA group, 16 (50%) were in CR and seven (21%) were in PR with anti-thymocyte globulin ± cyclosporine.

Paroxysmal Nocturnal Hemoglobinuria in Patients with Aplastic Anemia: Challenges, Characteristics, and Analysis of Clinical Experience

Background &amp; Aims. Paroxysmal nocturnal hemoglobinuria (PNH) is a disease caused by an acquired clonal disorder of hematopoietic stem cells with clone cell membrane hypersensitivity to the complement. PNH can exist as an independent disease and can also be associated with other pathological conditions characterized by bone marrow deficiency, first of all with aplastic anemia (AA). In PNH-associated AA (AA/PNH) pathological clones may be initially of different size. In some patients a gradual growth of PNH clone is observed together with occurring signs of intravascular hemolysis and transformation into classical hemolytic PNH. In this case it is important to assess the clinical situation and determine eligibility for complement inhibitor therapy. During targeted therapy it is necessary to assess the efficacy of treatment based on monitoring of complement-mediated hemolysis and to identify probable reasons for insufficient effect. Materials &amp; Methods. The paper deals with 1 clinical case. A female patient born in 1964, with initial diagnosis of AA was followed-up from 1989 till present at the Russian Research Institute of Hematology and Transfusiology. Her treatment included blood-component therapy, the use of antilymphocyte immunoglobulin, cyclosporine, plasmapheresis, eculizumab, and symptom-relieving drugs. Results. The study deals with the case of transformation of non-severe AA with remission after immune-suppressive therapy into classical hemolytic PNH. The case report describes the characteristic features, AA/PNH diagnosis and treatment issues at different stages of the disease, and the reasons for incomplete effect of targeted therapy. Conclusion. The case under discussion confirms the relevance of current methods of detecting PNH clone at early stages of AA diagnosis and dynamic follow-up with respect to a probable growth of clone with PNH phenotype, especially at the stage of hematopoietic recovery. Determination of PNH clone size and lactate dehydrogenase serum level is required for timely amendment of treatment strategy with a switch to long-term targeted monitoring of hemolysis which allows to prevent irreversible visceral changes and severe complications. In case of insufficient effect of targeted therapy with ongoing anemia Coombs test is recommended because of probability of C3-mediated extravascular hemolysis.

A female with classical paroxysmal nocturnal hemoglobinuria misdiagnosed as megaloblastic anemia

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder of the hematopoietic stem cells, which manifests as bone marrow failure, hemolytic anemia, and thrombosis. PNH affects both sexes equally and may present at any age, although it is most often diagnosed in young adulthood. Here, we report a case of a middle-aged woman, presented with one and a half year history of anemia, significant fatigue, backache, and persistent high erythrocyte sedimentation rate. The patient has no organomegaly and did not give a history of thrombotic events previously. She was considered as a case of megaloblastic anemia at another center and treated accordingly. Her full blood count showed mild macrocytic anemia with slight polychromasia. The hemolytic anemia workup revealed slightly raised serum bilirubin (unconjugated) and undetectable haptoglobin with hemosiderin in urine. Ham's test was positive, and the diagnosis of PNH was confirmed by flow cytometry. The patient was treated with short-course steroids and folic acid, and her general condition has become stable.

PNH Clone Size By Flow Cytometry and Its Correlation with PIG Gene Mutation

Background: Routine diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) is based on flow cytometric measurement of PNH clone in RBC and granulocyte with sensitivity of 1%, and recent introduction of high-sensitive flow cytometry (FCM) makes it possible to detect 0.01% clone. However, the clinical significance of small PNH clone has not been elucidated, so even the necessity to treat or not is controversial. We investigated whether quantitative results of PNH clone size measured by FCM correlate with mutant burden of PIG gene.Methods: A total of 44 specimens from 20 patients whose PNH clone size was >1% in either RBC or granulocyte by routine FCM were enrolled (classical PNH n=10, AA/PNH n=8, MDS/PNH n=2). To detect small quantity of cells with PIG gene mutation, we performed ultra-deep sequencing (average depth 3000X) for PIGA, PIGM, PIGT, and PIGX genes on these 44 consecutive specimens.Results: Sixteen patients (80.0%) were found to harbor PIG gene mutations: 15 patients had PIGA mutation and 1 patient had PIGM mutation. Granulocyte PNH clone size and variant allele frequency (VAF) of PIG gene mutation showed higher correlation (Spearman's r=0.73, p=0.0002) than that of RBC (Spearman's r=0.61, p=0.0073). All patients harboring PIG gene mutation showed more than 10% PNH clone by FCM. In contrast, 4 patients who did not have PIG gene mutation showed less than 10% PNH clone by FCM.Conclusion: Considering the mechanism of PNH development, the presence of PIG gene mutation is a definitive evidence supporting PNH diagnosis. PNH granulocyte clone size more than 10% by FCM seems to be clinically significant in relevance to PIG gene mutation. We suggest that neither high sensitivity FCM nor the detection of PIG gene mutation is a requisite for routine clinical diagnosis of PNH. DisclosuresNo relevant conflicts of interest to declare.

Clinical and flow cytometric analysis of paroxysmal nocturnal hemoglobinuria in Indian patients

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon disease. Many cases go undiagnosed as high index of clinical suspicion is required for its detection. This study was performed to detect the presence of PNH defect by flow cytometric immunophenotyping (FCMI) in patients with suspected PNH disease and evaluate their clinical and laboratory profile. MATERIALS AND METHODS: In this retrospective study, a total of 136 patients with suspected PNH who fulfilled the inclusion criteria for the study were evaluated for PNH defect by FCMI using monoclonal antibodies against CD55 and CD59 on red blood cell, granulocytes, and monocytes. RESULTS: Forty-eight (35.3%) of 136 patients evaluated had a PNH defect. Nineteen (39.5%) of these 48 patients had classical PNH (hemolytic type). The remaining 29 patients had PNH Clone in association with aplastic anemia. The clinical and laboratory data of these 19 patients with classical PNH were analyzed in this retrospective study. The median age was 34 years (range: 19–65 years). Thrombotic events were observed in 3 (16%) of the 19 cases (one each with Budd–Chiari syndrome, cerebral venous thrombosis, and abdominal vein thrombosis). The flow cytometric data of these patients were further analyzed for the presence of and size of PNH clone on erythrocytes, granulocytes, and monocytes. PNH clone was detected in 84% of erythrocytes, 76.9 % of monocytes and in 100% granulocytes. CONCLUSION: Classical PNH is not rare in India as previously thought. A high index of clinical suspicions and evaluation by FCMI is necessary for its detection. CD59 is a better marker for identification of PNH clone than CD55 in all three cell types.