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

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

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Paroxysmal Nocturnal Hemoglobinuria Clones Are Infrequent in Hepatitis-Associated Aplastic Anemia at the Time of Diagnosis

Blood ◽

10.1182/blood-2019-125620 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5016-5016

Author(s):

Wenrui Yang ◽

Xin Zhao ◽

Guangxin Peng ◽

Li Zhang ◽

Liping Jing ◽

...

Keyword(s):

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Clonal Evolution ◽

Extrinsic Factors ◽

Clone Size ◽

Hematopoietic Stem ◽

Over Time ◽

Pnh Clone

Aplastic anemia (AA) is an immune-mediated bone marrow failure, resulting in reduced number of hematopoietic stem and progenitor cells and pancytopenia. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clone in AA usually suggests an immunopathogenesis in patients. However, when and how PNH clone emerge in AA is still unclear. Hepatitis associated aplastic anemia (HAAA) is a special variant of AA with a clear disease course and relatively explicit immune pathogenesis, thus serves as a good model to explore the emergence and expansion of PNH clone. To evaluate the frequency and clonal evolution of PNH clones in AA, we retrospectively analyzed the clinical data of 90 HAAA patients that were consecutively diagnosed between August 2006 and March 2018 in Blood Diseases Hospital, and we included 403 idiopathic AA (IAA) patients as control. PNH clones were detected in 8 HAAA patients (8.9%,8/90) at the time of diagnosis, compared to 18.1% (73/403) in IAA. Eight HAAA patients had PNH clone in granulocytes with a median clone size of 3.90% (1.09-12.33%), and 3 patients had PNH clone in erythrocytes (median 4.29%, range 2.99-10.8%). Only one HAAA patients (1/8, 12.5%) had a PNH clone larger than 10%, while 24 out of 73 IAA patients (32.9%) had larger PNH clones. Taken together, we observed a less frequent PNH clone with smaller clone size in HAAA patients, compared to that in IAAs. We next attempted to find out factors that associated with PNH clones. We first split patients with HAAA into two groups based on the length of disease history (≥3 mo and < 3mo). There were more patients carried PNH clone in HAAA with longer history (21.4%, 3/14) than patients with shorter history (6.6%, 5/76), in line with higher incidence of PNH clone in IAA patients who had longer disease history. Then we compared the PNH clone incidence between HAAA patients with higher absolute neutrophil counts (ANC, ≥0.2*109/L) and lower ANC (< 0.2*109/L). Interestingly, very few VSAA patients developed PNH clone (5%, 3/60), while 16.7% (5/30) of non-VSAA patients had PNH clone at diagnosis. We monitored the evolution of PNH clones after immunosuppressive therapy, and found increased incidence of PNH clone over time. The overall frequency of PNH clone in HAAA was 20.8% (15/72), which was comparable to that in IAA (27.8%, 112/403). Two thirds of those new PNH clones occurred in non-responders in HAAA. In conclusion, PNH clones are infrequent in HAAA compared to IAA at the time of diagnosis, but the overall frequency over time are comparable between the two groups of patients. In SAA/VSAA patients who are under the activated abnormal immunity, longer clinical course and relatively adequate residual hematopoietic cells serve as two important extrinsic factors for HSCs with PIGA-mutation to escape from immune attack and to expand. Disclosures No relevant conflicts of interest to declare.

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Bone Marrow Histology in Patients with Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽

10.1182/blood.v114.22.4215.4215 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4215-4215

Author(s):

Sandra van Bijnen ◽

Konnie Hebeda ◽

Petra Muus

Keyword(s):

Bone Marrow ◽

Mast Cells ◽

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Plasma Cells ◽

Bone Marrow Failure ◽

Clone Size ◽

Immune Mediated ◽

Lymphoid Nodules ◽

Pnh Clone

Abstract Abstract 4215 Introduction Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disease of the hematopoietic stem cell (HSC) resulting in a clone of hematopoietic cells deficient in glycosyl phosphatidyl inositol anchored proteins. The clinical spectrum of PNH is highly variable with classical hemolytic PNH at one end, and PNH in association with aplastic anemia (AA/PNH) or other bone marrow failure states at the other end. It is still largely unknown what is causing these highly variable clinical presentations. Immune-mediated marrow failure has been suggested to contribute to the development of a PNH clone by selective damage to normal HSC. However, in classic PNH patients with no or only mild cytopenias, a role for immune mediated marrow failure is less obvious. No series of trephine biopsies has been previously documented of patients with PNH and AA/PNH to investigate the similarities and differences in these patients. Methods We have reviewed a series of trephine biopsies of 41 PNH patients at the time the PNH clone was first detected. The histology was compared of 27 patients with aplastic anemia and a PNH clone was compared to that of 14 patients with classic PNH. Age related cellularity, the ratio between myeloid and erythroid cells (ME ratio), and the presence of inflammatory cells (mast cells, lymphoid nodules and plasma cells) were evaluated. The relation with clinical and other laboratory parameters of PNH was established. Results Classic PNH patients showed a normal or hypercellular marrow in 79% of patients, whereas all AA/PNH patients showed a hypocellular marrow. Interestingly, a decreased myelopoiesis was observed not only in AA/PNH patients but also in 93% of classic PNH patients, despite normal absolute neutrophil counts (ANC ≥ 1,5 × 109/l) in 79% of these patients. The number of megakaryocytes was decreased in 29% of classic PNH patients although thrombocytopenia (< 150 × 109/l) was only present in 14% of the patients. Median PNH granulocyte clone size was 70% (range 8-95%) in classic PNH patients, whereas in AA/PNH patients this was only 10% (range 0.5-90%). PNH clones below 5% were exclusively detected in the AA/PNH group. Clinical or laboratory evidence of hemolysis was present in all classical PNH patients and in 52% of AA/PNH patients and correlated with PNH granulocyte clone size. Bone marrow iron stores were decreased in 71% of classic PNH patients. In contrast, increased iron stores were present in 63% of AA/PNH patients, probably reflecting their transfusion history. AA/PNH patients showed increased plasma cells in 15% of patients and lymphoid nodules in 37%, versus 0% and 11% in classic PNH. Increased mast cells (>2/high power field) were three times more frequent in AA/PNH (67%) than in PNH (21%). Conclusion Classic PNH patients were characterized by a more cellular bone marrow, increased erythropoiesis, larger PNH clones and clinically by less pronounced or absent peripheral cytopenias and more overt hemolysis. Decreased myelopoiesis and/or megakaryopoiesis was observed in both AA/PNH and classic PNH patients, even in the presence of normal peripheral blood counts, suggesting a role for bone marrow failure in classic PNH as well. More prominent inflammatory infiltrates were observed in AA/PNH patients compared to classical PNH patients. Disclosures: No relevant conflicts of interest to declare.

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Clonal hematopoiesis in acquired aplastic anemia

Blood ◽

10.1182/blood-2016-01-636381 ◽

2016 ◽

Vol 128 (3) ◽

pp. 337-347 ◽

Cited By ~ 81

Author(s):

Seishi Ogawa

Keyword(s):

Aplastic Anemia ◽

High Throughput Sequencing ◽

Somatic Mutations ◽

Clonal Selection ◽

Clonal Evolution ◽

Uniparental Disomy ◽

Close Monitoring ◽

Clone Size ◽

Close Link ◽

Clonal Hematopoiesis

AbstractClonal hematopoiesis (CH) in aplastic anemia (AA) has been closely linked to the evolution of late clonal disorders, including paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML), which are common complications after successful immunosuppressive therapy (IST). With the advent of high-throughput sequencing of recent years, the molecular aspect of CH in AA has been clarified by comprehensive detection of somatic mutations that drive clonal evolution. Genetic abnormalities are found in ∼50% of patients with AA and, except for PIGA mutations and copy-neutral loss-of-heterozygosity, or uniparental disomy (UPD) in 6p (6pUPD), are most frequently represented by mutations involving genes commonly mutated in myeloid malignancies, including DNMT3A, ASXL1, and BCOR/BCORL1. Mutations exhibit distinct chronological profiles and clinical impacts. BCOR/BCORL1 and PIGA mutations tend to disappear or show stable clone size and predict a better response to IST and a significantly better clinical outcome compared with mutations in DNMT3A, ASXL1, and other genes, which are likely to increase their clone size, are associated with a faster progression to MDS/AML, and predict an unfavorable survival. High frequency of 6pUPD and overrepresentation of PIGA and BCOR/BCORL1 mutations are unique to AA, suggesting the role of autoimmunity in clonal selection. By contrast, DNMT3A and ASXL1 mutations, also commonly seen in CH in the general population, indicate a close link to CH in the aged bone marrow, in terms of the mechanism for selection. Detection and close monitoring of somatic mutations/evolution may help with prediction and diagnosis of clonal evolution of MDS/AML and better management of patients with AA.

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Evolution of GPI-Deficient Clones Predicts Clinical Course in Paroxysmal Nocturnal Haemoglobinuria.

Blood ◽

10.1182/blood.v104.11.172.172 ◽

2004 ◽

Vol 104 (11) ◽

pp. 172-172 ◽

Cited By ~ 5

Author(s):

Stephen J. Richards ◽

Matthew J. Cullen ◽

Anita J. Dickinson ◽

Claire Hall ◽

Anita Hill ◽

...

Keyword(s):

Aplastic Anemia ◽

Clinical Course ◽

Spontaneous Remission ◽

Red Cells ◽

Red Cell ◽

Clone Size ◽

Clinical Behavior ◽

The Mean ◽

Pnh Clone

Abstract Flow cytometric analysis of GPI-linked antigens has had a major impact on the diagnosis of PNH. Significant numbers of patients with aplastic anemia have small PNH clones, and due to the precision in clone size measurement, reliable serial monitoring can now be undertaken although the clinical value of this is not proven. From our series of 234 PNH patients, we analysed clinical correlates between disease type and red cell and granulocyte peripheral blood clone sizes as determined by flow cytometry at presentation. For hemolytic patients (n = 99) the mean PNH clone sizes were: granulocytes 84.8%; red cells 45.3% (type III cells 33.6%). For aplastic patients (no macroscopic hemolysis) the mean clone sizes were: granulocytes 18.5%; red cells 6.4% (type III cells 4.5%). The two groups were statistically different (Mann Whitney U; P<0.001). Monitoring of PNH clones in 86 of these patients who had at least 3 samples over a minimum of 12 months (mean 55 months; range 15–174) not only showed distinct groups of patients with highly characteristic patterns of disease but also provided insights into the incidence of spontaneous remission, progression from aplastic to hemolytic disease, and development of leukemia. Firstly, hemolytic patients that present with >90% granulocyte clones (n = 30; mean follow up 48 months) with virtually all their hematopoiesis maintained from PNH stem cells have clone sizes that remain stable and their clinical behavior suggests that their PNH can persist for up to 40 years. The second group of patients (n = 16) were those with hemolytic PNH with granulocyte clones of <90%. Mean granulocyte clone size at presentation was 68.4% (range 34.7– 90%) with a mean follow-up of 66 months (range 24–164). Of these, 6 showed stable clone sizes, 2 increasing clone size, and 8 showing reductions in granulocyte clone size. The third group were those presenting with aplastic anemia (n = 34). This group showed the most significant variation in clone size and clinical behavior. Of the 12 patients with persistent aplastic anemia, the majority had slowly increasing clone sizes with 5 patients progressing to hemolytic PNH after a variable time period ranging from 26 to 79 months. Only 3 patients developed MDS or AML. Two of these were from the >90% granulocyte clone group (2/30) and developed as a terminal event, one with GPI-MDS, and the second with a rapid emergence of GPI+MDS. One patient in the aplastic group showed progression to AML (1/34). 27% of patients had an improvement in cytopenias with concurrent decrease in PNH clone size. For hemolytic patients with granulocyte clones of <90%, the 8 patients with falling clone sizes had improving blood counts. The PNH granulocyte clone halved in a mean of 74 months. Of the patients with aplastic anemia, 15 showed resolution of anemia with normalization of counts and all but one had an associated fall in granulocyte PNH clone sizes. Eleven patients have been treated in clinical trials of the anti-complement antibody, eculizumab, for a period of up to 2 years and over this period the proportion of PNH granulocytes has remained stable. This data demonstrates that the size and type of granulocyte and red cell PNH clones at presentation predicts the clinical course for individual patients assisting long term clinical management planning. Moreover, regular clone size monitoring predicts the likelihood of spontaneous reduction in the PNH clone and potentially for spontaneous remission.

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A Spontaneous Reduction of Clone Size in Paroxysmal Nocturnal Hemoglobinuria Patients Treated with Eculizumab for Greater Than 12 Months.

Blood ◽

10.1182/blood.v114.22.1992.1992 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1992-1992 ◽

Cited By ~ 4

Author(s):

Richard Kelly ◽

Stephen Richards ◽

Louise Arnold ◽

Gemma Valters ◽

Matthew Cullen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Maintenance Dose ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Failure ◽

Hemopoietic Stem Cells ◽

Intravascular Hemolysis ◽

Complement Protein ◽

Clone Size ◽

Pnh Clone

Abstract Abstract 1992 Poster Board I-1014 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal disorder of hemopoietic stem cells that is characterized by bone marrow failure, intravascular hemolysis and venous thrombosis. Eculizumab is a humanized monoclonal antibody that specifically binds to the complement protein C5 preventing its cleavage thereby inhibiting the formation of the terminal components of the complement cascade. Eculizumab was approved by the FDA in 2007 after clinical trials showed it was efficacious in treating patients with hemolytic PNH. Prior to eculizumab therapy treatment options were mainly supportive in nature. Historical data shows that a third of patients who survive greater than 10 years undergo spontaneous recovery. We present data on 38 patients with hemolytic PNH treated at a single centre with eculizumab for 12 months or longer. Thirty six of these patients were treated with a loading dose of 600mg every week for 4 doses followed by 900mg the following week and then a maintenance dose of 900mg dose every 14 day. The other 2 patients required a higher maintenance dose of eculizumab, 1200mg every 14 days, due to symptomatic intravascular hemolysis on the standard regime. All our patients had a high PNH granulocyte clone size at the initiation of eculizumab treatment from 52.90% to 99.95% with a median of 96.38%. The duration of eculizumab therapy varied from 12 to 84 months with a median treatment duration of 50 months. Granulocyte clone size was used as it is not subject to as much variation as the erythrocyte clone size which changes both due to blood transfusions and to the extent of intravascular hemolysis present. The proportion of PNH granulocytes probably most accurately reflects the true size of the PNH clone. Seven out of these 38 patients (18.4%) have had a 10% or greater reduction in their granulocyte clone size during the course of their eculizumab treatment. These patients have had a steady and continued decline in their granulocyte clone size throughout their treatment with eculizumab. This may actually be due to an increase in the residual normal cells in some patients (see Table). Two of these patients (U.P.N. 5 and 7) have had such a dramatic reduction in their clone size that they have been able to stop their eculizumab treatment without any observed detriment to their health.TableChange in PNH clones in patients on eculizumabU.P.N.Months on eculizumabNeutrophils PNH clone size (%)Normal neutrophils (%)Pre-treatmentMost recent on treatmentPre-treatmentMost recent on treatment15097.242.82.85724878.063.222.036.335596.484.13.615.941592.577.07.523.051261.732.438.367.664788.362.511.737.578552.98.547.191.5 Two of these 7 patients were treated with ciclosporin for underlying aplasia as compared to 3 of the 31 of those who haven't had a decrease in their clone size. There was no difference in the white cell or platelet count in these 7 patients from when they started eculizumab treatment to the present day indicating that the degree of bone marrow failure present has not changed dramatically during this time course. 5 of the 7 patients had neutrophil clone sizes of less than the median perhaps indicating that recovery requires a certain number of residual normal stem cells to be present. There were no other observed differences to distinguish between patients whose clone size fell and those that did not. It is unlikely that eculizumab has a direct effect on clone size in hemolytic PNH. A more probable hypothesis is that the immune selection in favour of the PNH clone expires over time allowing normal hemopoietic stem cells to repopulate the bone marrow. Whether eculizumab has any influence on this rather than just allowing patients to survive and remain well until recovery occurs is not clear. Our data suggests that there needs to be some normal hematopoietic activity in order for the normal marrow cells to expand and clone size under 95% predicts for recovery. In conclusion, a significant minority of patients with PNH on eculizumab have a progressive reduction in the size of their PNH clone during therapy and in some of these patients the clone falls to a level at which eculizumab can safely be stopped. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria. Richards:Alexion Pharmaceuticals: Honoraria. Hill:Alexion: Honoraria. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Prognostic and Predictive Impact of Small PNH Clones in a Large Cohort of Patients with Myelodysplastic Syndromes and Aplastic Anemia: A Single-Center Experience

Blood ◽

10.1182/blood-2018-99-116772 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3870-3870 ◽

Cited By ~ 1

Author(s):

Bruno Fattizzo ◽

Alan Dunlop ◽

Robin M. Ireland ◽

Shireen Kassam ◽

Dario Consonni ◽

...

Keyword(s):

Aplastic Anemia ◽

Myelodysplastic Syndromes ◽

Response Rates ◽

Male Gender ◽

Response To Therapy ◽

Older Age ◽

Size Increase ◽

Clone Size ◽

Tertiary Center ◽

Pnh Clone

Abstract Background: the use of high sensitive FLAER in the last 10 years improved the detection of very small PNH clones (<1%) in various hematologic conditions, including aplastic anemia (AA)/myelodysplastic syndromes (MDS). Although generally considered unremarkable, the recent observation of asymptomatic end-organ damage due to undiagnosed thrombosis even in patients without overt hemolysis questions the assertion that small PNH clones are of subclinical value. Aim: To evaluate the prevalence of PNH clones ≥0.01% in AA/MDS patients tested at a single tertiary center, and to assess their impact on disease prognosis (leukemic evolution, death), occurrence of thrombosis, and response to current therapies. Methods: We retrospectively collected clinical (diagnosis, stage, therapy, complications and outcome) and laboratory features (complete blood counts, LDH, PNH clone size) of 869 MDS and 531 AA patients tested from March 1998 till October 2017. Results: Figure 1 shows clinical and laboratory characteristics of MDS and AA patients, divided according to the presence/absence of PNH clones. A PNH clone was less frequently found in MDS cases versus AA (20.3% vs 61%). Focusing on MDS, PNH+ cases were significantly more hypoplastic, mainly displayed IPSS low/int-1 score, and showed deeper cytopenias (significantly for Hb and PLT) and higher LDH levels. Dividing patients according to clone size (negative, 0.01-1%, 1.01-10%,10.01-50%, and >50% on granulocytes), we observed a significant worsening of cytopenia and raise of LDH along with clone size increase. Likewise, lower IPSS risk patients more frequently displayed a greater clone size. As regards therapy, PNH+MDS showed significantly higher response rates to immunosuppressive therapies (ATG and CyA, 84% vs 44.7%, p=0.01) and to HSCT (71% vs 56.6%, p=0.09) compared to PNH-, and the cumulative probability of response to any treatment significantly improved along with clone size increase (from 52 to 100%, p=0.03). In addition, PNH+MDS showed lower rate of progression and AML evolution, and a longer OS [mean 11.9+0.7 years (10.5-13.3) vs 7.3+0.3 (6.6-7.9), p<0.0001] compared to PNH- ones. Interestingly, we observed a significant reduction of death frequency along with clone size increase (56% in PNH- vs 24% in PNH+ with clone size 0.01-1%, p<0.0001). However, PNH+MDS had a higher incidence of thrombotic events, with greater frequency along with clone size increase (from 5% in PNH- to 50% in PNH+ with clone size >50%, p<0.0001). As expected, worse OS also significantly correlated with older age, male gender, transfusion dependence, MDS progression/AML evolution, higher IPSS score, and non-response to therapy. Regarding AA, together with a higher frequency of PNH clones, we also found a higher frequency of large clones (>10%) compared to MDS (p=0.04, bars chart). PNH+AA showed deeper thrombocytopenia, higher reticulocyte counts and LDH values. As observed for MDS, we found a significant worsening of cytopenia and raise of LDH along with clone size increase. In addition, PNH+ AA showed higher response rates compared to PNH- (97 vs 77% for HSCT, p=0.01; 78 vs 50% for IST, p<0.0001; and 88% vs 65% considering any treatment, p<0.0001). PNH+AA also showed lower rate of MDS progression and death (p=0.01 and p<0.0001), and longer OS [mean 15.8+0.43 years (14.9-16.7) vs 6.5+0.35 (5.8-7.21), p<0.0001]. Also in AA, we observed a significant reduction of death frequency along with clone size increase (32.1% in PNH- vs 9.2% in PNH+ with clone size 0.01-1%, p<0.0001). Worse OS also correlated with older age, male gender, presence of cytopenia, and non-response to therapy. Conclusions: Prevalence of PNH clones of any size is high in patients with MDS and AA. We firstly show a positive impact of PNH clone positivity on response to IST and HSCT in MDS. The presence of a PNH clone also correlated with lower disease progression and better OS. Furthermore, we confirm the known favourable prognostic and predictive value of PNH clones in a large AA cohort. Clone size analysis suggests that even small clones (0.01-1%) have a clinical and prognostic significance. Figure 1. Figure 1. Disclosures Kassam: AbbVie: Equity Ownership. Yallop:Pfizer: Consultancy; Servier: Other: Travel funding. Mufti:Celgene: Research Funding. Kulasekararaj:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support .

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Incidence, Clinical Characteristics and Outcome of Symptomatic Thromboembolic Events (TE) in 276 Patients with Paroxysmal Nocturnal Hemoglobinurea (PNH)

Blood ◽

10.1182/blood.v128.22.5072.5072 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5072-5072

Author(s):

Alexander Kulagin ◽

Olesya Klimova ◽

Alexey Dobronravov ◽

Elena Pavlyuchenko ◽

Elena Babenko ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Aplastic Anemia ◽

Median Time ◽

Cumulative Incidence ◽

Clinical Characteristics ◽

Cerebral Veins ◽

Intravascular Hemolysis ◽

Clone Size ◽

Hematopoietic Stem ◽

History Of

Abstract Introduction: PNH is an orphan clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, cytopenias and TE. TE is the leading cause of death in the natural course of PNH. The data on epidemiology, clinical characteristics and outcomes of TE are limited to a few studies and not available in the Russian population of patients with PNH. Methods: We conducted a retrospective and prospective analysis of TE in a large cohort of patients with classic PNH and hemolytic PNH associated with aplastic anemia (AA/PNH) in one reference center in Russia. Hemolytic PNH was defined as a granulocyte clone size ≥ 10% and lactate dehydrogenase (LDH) level ≥ 1.5 of upper limit of normal (ULN). The analysis included only the documented episodes of symptomatic TE. Cases of subclinical thrombosis detected using sensitive methods were not considered. Cumulative incidence of TE was calculated using unrelated to TE death and allogeneic HSCT as competing risks. Patients without TE were censored at the last follow-up or date of first treatment with eculizumab. Results: A total of 276 patients with classic PNH (n=141) and hPNH associated with aplastic anemia (n=135) were included in analysis between 1996 and 2016 (Table 1). Sixty seven patients (24.3%) developed a total of 118 TE (median, 1; range, 1-7) with the median time between PNH onset and first TE of 2.7 years (range, 0-29). Cumulative incidence of first TE reached 29.7% and 45.5% at 10 and 20 years respectively (Fig. 1). Recurrent TEs were documented in 30 of 67 patients (45%) with the cumulative incidence of second TE of 27.7% and 46.7% at 1 and 5 years respectively (Fig. 2). The median time between the initial TE and the first recurrence was 0.74 years (range, 0.05-10). Routine secondary anticoagulant prophylaxis carried out in 34 patients had no significant impact on 5-year cumulative incidence of recurrent TE (43% vs 50%, p=0.919). The proportion of TE(+) patients was significantly higher in the classical PNH compared to AA / PNH, but taking into account the duration of the disease, these differences were not found in the cumulative incidence analysis. TE occurred in venous (64% of patients), arterial (22%) and both venous and arterial sites (14%). The most common sites of venous TE were intra-abdominal veins (n=26, 22%), deep veins (n=22, 18.6%), superficial veins (n=16, 13.5%), pulmonary embolism (n=15, 12.7%) and cerebral veins (n=5, 4.2%). Arterial TE included TIA/ischemic stroke (n=23, 19.5%) and myocardial infarction (n=7, 5.9%). Patients with TE were also characterized by 2-fold increase in the incidence of acute kidney injury (AKIN) episodes, the presence of chronic kidney disease (CKD) ≥ 2 stage and pulmonary hypertension. Seventeen of 67 (25%) TE(+) patients died which resulted in 10-year overall survival of 66% (CI 95%, 51-82). The mortality rate was highest in eculizumab naïve cohort: 11 of 24 (46%) patients died and all deaths were directly caused by TEs. In contrast, among the 43 patients with a history of TE treated with eculizumab there was only one death related to the consequences of TE, but 5 patients died due to neutropenic infections (2), solid tumor (1), MDS/AML (1) and complications after HSCT (1). Conclusions: These data confirm the high incidence and poor prognosis of symptomatic TE in patients with natural history of PNH. TE are often associated with organ dysfunction (AKIN, CKD, pulmonary hypertension), and characterize the highest burden of disease. Early targeted control of intravascular hemolysis is required in all cases at high risk or history of TE in PNH. Disclosures Kulagin: Alexion: Speakers Bureau.

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Distribution of PNH Clone Sizes within High Risk Diagnostic Categories Among 481 PNH Positive Patients Identified by High Sensitivity Flow Cytometry

Blood ◽

10.1182/blood.v120.21.1271.1271 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1271-1271 ◽

Cited By ~ 4

Author(s):

Mayur K Movalia ◽

Andrea Illingworth

Keyword(s):

Flow Cytometry ◽

High Risk ◽

Aplastic Anemia ◽

High Risk Patient ◽

High Sensitivity ◽

Risk Patient ◽

Clone Size ◽

Diagnostic Categories ◽

Patient Groups ◽

Pnh Clone

Abstract Abstract 1271 Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder associated with patients (pts) with aplastic anemia (AA), myelodysplastic syndrome (MDS), unexplained cytopenias, unexplained thrombosis, and hemolysis. High sensitivity flow cytometry (HSFC) is recommended by The International Clinical Cytometry Society (ICCS) as the method of choice for diagnosing PNH. The incidence of PNH clones in these patient groups has been previously reported, but the distribution of clinically significant PNH clones not been previously explored. The purpose of this study is to analyze the distribution of PNH clone sizes using HSFC with sensitivity up to 0.01% among 7,699 pts who were screened for PNH clones utilizing CD235a/CD59 for RBCs, FLAER/CD24/CD15/CD45 for neutrophils and FLAER/CD14/CD64/CD45 for monocytes. We evaluated the distribution of PNH clones sizes against the provided ICD-9 diagnostic (DX) codes and evaluated the significance of hemolysis on PNH clone size. Based on a sensitivity of at least 0.01%, 6.2% of all pts (481/7,699) were found to be PNH positive. Of those pts, 3.8% (293/7,699) were found to have a PNH clone size of >1%, while 2.4% (188/7,699) were found to have a PNH clone size of <1%. Of the 481 PNH positive patients, the distribution of PNH clone sizes among high risk diagnostic categories is shown in Table 1. Aplastic anemia (AA) and hemolysis were more commonly associated with PNH clone sizes >20%. In 32 patients reported to have both aplastic anemia and hemolysis, 20 pts (63%) had PNH clone sizes >20%, while 30 pts (94%) had PNH clone sizes >1%. Pts with MDS, cytopenias and thrombosis more frequently showed PNH clones sizes of <1%. However, in 18 pts reported to have either MDS or cytopenias and hemolysis, 4 pts (22%) showed PNH clone sizes >20%, while 10 pts (56%) showed PNH clone sizes >1%. In this single-laboratory experience, we evaluated the distribution of PNH clone sizes among high risk patient groups based on ICD-9 diagnostic code. Pts with large PNH clone sizes are more likely to have clinical symptoms, particularly those associated with hemolysis, and thus most likely to benefit from therapy. In this study, pts with hemolysis showed a higher incidence of PNH clone sizes >20%, underscoring the need to test for hemolysis in these high risk patient groups. In addition, this study confirmed the need to continue actively testing high risk patient populations, including aplastic anemia, myelodysplastic syndrome, unexplained cytopenias, unexplained thrombosis and hemolysis for PNH based on the ICCS recommendations to ensure accurate diagnosis and appropriate therapy. Table 1. Distribution of PNH Clone Sizes within high risk diagnostic categories among 481 PNH+ Patients at Dahl-Chase Diagnostic Services PNH Clone Sizes Total <1% 1–20% >20% Aplastic Anemia 32 39 40 25.8% (111) MDS 20 9 7 8.4% (36) Cytopenias (including pancytopenia, leukopenia and non-hemolytic anemia) 78 27 10 26.7% (115) Hemolysis (including hemolytic anemia and hemoglobinuria) 24 29 129 42.3% (182) Thrombosis 12 4 5 4.9% (21) Miscellaneous 15 2 5 5.1% (22) Not provided 17 6 28 11.9% (51) Note: Table reflects patients who had more than one associated ICD-9 code. Disclosures: Illingworth: Alexion: Consultancy, Honoraria.

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Evaluation of Patients with PNH Treated By Eculizumab: Real World Data from Turkey

Blood ◽

10.1182/blood-2019-128743 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4805-4805

Author(s):

Fatma Keklik Karadag ◽

Mustafa Nuri Yenerel ◽

Yılmaz Mehmet ◽

Hava uskudar Teke ◽

Vildan Ozkocaman ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Retrospective Analysis ◽

Bone Marrow Failure ◽

Thrombotic Event ◽

Specific Information ◽

Intravascular Hemolysis ◽

Clone Size ◽

Cell Counts ◽

Pnh Clone

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease which is characterized with complement mediated intravascular hemolysis, bone marrow failure and thrombosis. The prevelance of PNH is estimated 1 to 16 cases per million in USA. X-linked somatic mutation of phosphatidylinositol glycan‐A (PIGA) gene in hematopoetic stem cells causes an impairment of glycosylphosphatidylinositol (GPI) anchored proteins. The absence of GPI‐dependent molecules that are CD55 (decay accelerating factor; DAF) and CD59 (membrane inhibitor of reactive lysis; MIRL), normally protect the cell from complement‐mediated hemolysis by preventing the formation of the membrane attack complex. The diagnosis is based on flow cytometry which can detect the deficiency of these two complement regulary proteins. We report a retrospective analysis of demographic and clinical characteristics of PNH patients from different centers. Material and methods: We conducted a retrospective analysis of the patients' recorded data. Patients' demographics, medical and treatment history, comorbid conditions, PNH clone size, disease characteristics and outcomes, symptoms, PNH-specific treatments, PNH-related events, morbidity (including myeloproliferative disease, other malignancies, and infections), mortality. Clinical data captured include lactate dehydrogenase (LDH) levels, PNH clone size, hemoglobin levels, thrombotic events, renal functional tests at the time of diagnosis, and other laboratory data. Specific information collected for eculizumab-treated patients includes dosage and dose adjustments and blood cell counts, reticulocyte count and LDH level after eculizumab treatment. Results: 138 patients were included from 28 different centers. All patients were diagnosed by flow cytometry for GPI-linked antigens on red cells and neutrophils. The number of male (69/138) and female (69/138) patient was equal and the median age was 41 years. Median hemoglobin (hb) level was 8.75±2.13 gr/dL; Platelet (plt) level was 131× 109/L at the time of diagnosis. Overall, 49(35,5%) of the patients had been diagnosed with bone marrow failure, including aplastic anemia or hypoplastic anemia (n=31; 22,5%), myelodysplastic syndromes (n=18; 13%). A history of any prior thrombotic event was reported in 45 patients (32,6%). At the time of analysis, 12 (8,7%) patients had pulmonary hypertension. The median granulocyte and monocyte clone size was 63,6% (±32.26) and 66.76 ±28.75 respectively. Fatigue (58%) is the most commonly reported symptom and abdominal pain was seen in 8% of patients. After the eculizumab therapy, the median time for normalization of Hb and LDH level were 7 and 14,6 months, respectively. There was no correlation between thrombosis and clone size, hb, plt, LDH level at the time of diagnosis. LDH level was higher in fatigue patients compared with the patients who were not fatigue (p=0.021). Discussion: PNH is a clonal but non malignant disease that is very rare and knowledge on large case series is really limited. The clinical findings and symptoms could be variable and unfortunately it takes very long time to diagnose because of unawareness of physicians. Eculizumab is a good option in treatment of PNH for improving symptoms of intravascular hemolysis but we still need better understanding of thrombosis mechanism of PNH to better management. In the future, novel inhibitors of the alternative pathway of complement will be used to improve survival and quality of life for PNH patients. Disclosures No relevant conflicts of interest to declare.

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Pediatric Patients with Severe Aplastic Anemia Treated with Combined Immunosuppressive Therapy, Eltrombopag and Eculizumab and Their Clinical Course to Stem Cell Transplant

Blood ◽

10.1182/blood-2020-134553 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 13-13

Author(s):

Kazuhiro Sabet ◽

Arun Ranjan Panigrahi

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Aplastic Anemia ◽

Immunosuppressive Therapy ◽

Pediatric Patients ◽

The Other ◽

Severe Aplastic Anemia ◽

Clone Size ◽

A Minor ◽

Pnh Clone

Acquired aplastic anemia (AA) in children is a rare disorder characterized by pancytopenia and hypocellular bone marrow. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoetic stem cell (HCT) disorder characterized by complemented-mediated hemolysis, thrombosis and bone marrow failure secondary to deficiency of glycosylphosphatidylinositol-anchored proteins (GPI-AP) on hematopoetic stem cells. PNH and acquired AA are closely related; up to 50% of patients with AA have detectable PNH-clones at the time of diagnosis and small percentage of them can have clonal expansion throughout their disease course requiring close monitoring. Current standard therapy for severe aplastic anemia (SAA) patients without matched related donor (MRD) is immunosuppressive therapy (IST) regimen with anti-thymoglobulin (ATG), cyclosporine (CSA), and recent addition of eltrombopag. Eculizumab is a recombinant humanized monoclonal antibody that blocks complement protein C5 and prevents cell lysis. While it has been shown to be effective in children with PNH, concomitant treatment with IST for patients with SAA is unknown. To our knowledge, there has been no pediatric data on combined IST, eltrombopag and eculizumab treatment for children with AA with clinically significant PNH clones. Here we retrospectively reviewed three pediatric patients with SAA with PNH clones treated with IST, eltrombopag and eculizumab and their unique clinical courses. Two out of three patients had high PNH clone size and were started on eculizumab prior to IST with improvement in transfusion intervals. Of the two, one had decrease in PNH clone size after IST but the other patient's clone size continued to increase despite two courses of IST. Finally, the third patient had a minor PNH clone and was not started on eculizumab prior to IST. He remained asymptomatic for over a year until he developed symptomatic PNH with large clone size and aplastic bone marrow. His clone size continued to increase with no improvement in transfusion frequency despite being started on eculizumab. However, steroids were started based on anecdotal literature and his transfusion frequency decreased subsequently. All three patients are in the process of going through matched unrelated donor stem cell transplants. Several studies have reported the presence of a minor PNH clone at the time of AA diagnosis was associated a favorable response to IST. In this case series, the patient with the smallest PNH clone size at the time of diagnosis of SAA had the best response to IST compared to the other two patients who had larger PNH clone populations. These findings suggest that even though minor PNH population may lead to better response to IST compared to absence of the clone, the correlation between clone size and prognosis is unclear. Further study with larger sample size is needed to investigate this relationship. However, regardless of the population size, all three patients were able to prolong transfusion intervals using eculizumab without significant side effects. As the adoption of eltrombopag with standard IST is evolving with ongoing study, the efficacy of incorporating eculizumab to decrease transfusion frequency in patients with PNH-clone in addition to eltrombopag/IST regimen should be further investigated. Disclosures No relevant conflicts of interest to declare.

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