Paroxysmal Nocturnal Hemoglobinuria: An Atypical Clinical Case Report and Proof of the Disease in Blood and BONE Marrow with FLOW Cytometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5166-5166
Author(s):  
Fabienne Pineau-Vincent ◽  
Pierre Lemaire ◽  
Habib Ghnaya ◽  
Guillaume Direz ◽  
Mohamed Kaabar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Case Report ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Blood Smear ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Chromatin Condensation ◽  
Clinical Signs ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disease, associated with hemolytic anemia and bone marrow failure. The cellular abnormality is a mutation in the phosphatidylinositol glycan class (PIG A) resulting in a deficiency of glycosylphosphadityl-inositol (GPI)-anchored complement regulatory proteins, including CD 55 and CD59, on the surface of blood cells. Case report We report the case of a French, 81 year-old-man, who was admitted to our institution with an unusual clinical presentation. He had a rheumatologic monitoring in the context of polyarthritis associated with anemia (98g/L). No hemolytic events were noticed and there was no notion of either transfusion. Biological results showed hemolytic regenerative anemia (98g/L) with 136G/L of reticulocytes, neutrophil polynuclears (4.2G/L) without degranulation and nevertheless rare degranulation cells, no blasts, normal level of platelets (258G/l), increase of LDH (Nx3), low haptoglobin (0.07g/L), negative direct Coombs test. The cytology aspect of medullar cells associated dysgranulopoiesis with degranulation of myeloid lineage and abnormal chromatin condensation, dyserythropoiesis, dysmegacaryopoiesis, in favor of a multilineage dysplasia without blasts. The marrow karyotype was normal. Due to the morphological results observed on the blood smear and their dissociation with the medullary cytology, flow cytometry (FC500) for GPI‘s expression study was performed. The used antisera were: CD55, CD59, CD14, CD16, CD24, CD66b, CD157, no FLEAR was tested. Results TableBloodBone marrowMononuclear cells CD14 FL378% intermediar cells70% negative cellsNeutrophil cells CD16 PE56% intermediar cells56% negative cellsNeutrophil cells CD66b FITC57% negative cells70% negative cellsGranular cells CD24 PE49% negative cells62% negative cellsRed cells CD55 FITC10% negative cells11% negative cellsRed cells CD59 FITC12% negative cells12% negative cells Figure 1 Blood Figure 1. Blood Figure 2 Bone Marrow Figure 2. Bone Marrow The confirmation was obtained by using CD157PE antisera on bone marrow with 70% negative mononuclear and granular cells. The results confirmed the PNH clone’s presence in the blood and also in bone marrow, and the results of flow cytometry could explain the cytological aspect of neutrophil polynuclear cells. It is rare to explore the expression of GPI molecules in bone marrow and there is no publication about the PNH clone whose identification required bone marrow cells for the confirmation of abnormalities in blood. Thus, the apoptosis in the bone marrow of the defective myeloid cells would explain the difference of granularity of polynuclear cells between bone marrow and blood smear. Conclusion The significance of this observation is related to the search of a PNH clone when cytological dissociation is observed between the peripheral blood and bone marrow, associated with biological hemolysis arguments (increased LDH and decreased haptoglobin). It is well known that 6 at 8% of myelodysplasia had PNH clone; the originality of this case report is the initial clinical signs and the laboratory proof of PNH in the blood and the bone marrow. This observation was submitted at the national reference center of PNH in France (St Louis Hospital - Hematology Department - Professor SOCIE) and the treatment by eculizumab was introduced. Disclosures No relevant conflicts of interest to declare.

Paroxysmal Nocturnal Haemoglobinuria Clone In Aplastic Anaemia Patients At The Beginning Of Disease and In Remission

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4868-4868
Author(s):  
Zalina Fidarova ◽  
Elena Mikhailova ◽  
Svetlana Lugovskaia ◽  
Elena Naumova ◽  
Vera Troitskaia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Aplastic Anaemia ◽  
Partial Remission ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Paroxysmal Nocturnal Haemoglobinuria ◽  
Clone Size ◽  
Pnh Clone

Abstract Introduction Aplastic Anaemia (AA) and Paroxysmal Nocturnal Haemoglobinuria (PNH) are severe hematological diseases accompanied by bone marrow failure syndromes. The high-sensitivity flow cytometry standartised methods helped us to detect the PNH clone incedence at AA patients at the different stages of disease and of treatement and to reveal its’ influence on the immunosuppressive therapy (IST) effectiveness. Objective to detect the PNH clone at AA patients at different stages of disease and to reveal its’ influence on the IST effectiveness. Methods 63 patients with severe AA (SAA) who received combined IST with antithymocytic globulin (hATG) and cyclosporin A (CsA) have been included into the study. Mediane age – 26 years (16-65). All 63 patients were divided into 2 groups. The 1st one included de novo AA patients (n=28); the 2nd group – AA patients in complete remission (CR) after IST (n=35). The median remission duration was 3 years (2-6 y). The results of the de novo AA treatment (1stgroup) were evaluated at 3, 6 and 12 months from the start of IST. We used the flow cytometry (Becton Dickinson (BD) FACS Canto II and Beckman Coulter (BC) FC 500) to evaluate the PNH clone. Peripheral blood samples were analyzed with antibodies CD45(BD), CD15(BD), CD64(BD), CD235a(BC), GPI-tying antibodies CD59 (Invitrogen), CD14(BC), CD24(BC) and FLAER (Cedarlane). Minor PNH clone was detected when the count of GPI- deficient cells did not exceed 1%. Results The PNH clone was found in 18 patients among 28 (64%) from the 1st group. The minor clone was found in 4 patients, in 3 patients the clone size exceeded 50%. Median (Me) clone size on the Red Blood Cells (RBC: type II + type III) was 0,25% (0,03-25,3%), Granulocytes (GR) – 1,7% (0,02- 93,92%), Monocytes (Mon)- 23,2% (0,05-95,66%). 7/18 SAA patients (38,9%) with the PNH clone, showed a haematological response at 3 months from the treatment start, including 3 patients with the minor PNH clone. 2/18 patients (11.1%) underwent allogenic bone marrow transplantation (alloBMT). 9/18 did not get the remission by the 3d month. 4/18 patients (22,2%), without response at 6 months, received the second course of IST; 5/18 patients (27.7%) are being followed up and can‘t be analyzed at 6 months. It worth to note, that PNH clone disappeared after allo-BMT (n=2) and in 1 patient with CR after IST. None of 10 patients without PNH clone attained response at 3 months (p=0,02). 4 out of 10 (40%) achieved only partial remission at 6 months. In these cases minor PNH clone appeared after hematological response and persisting from 6 till 18 months. 6 other patients are still on the treatment (ATG). In the 2nd group the PNH clone was detected in 26 of 35 cases (74,3%), only 11 of them had a minor clone. The Me of PNH clone size on RBC - 1,4% (0,02 to 3,76%), Gr – 25,2% (0,01-93,73%), Mon - 23,52% (0,01-54,32%) Conclusion The PNH clone has been detected in more than 60 % of de novo SAA patients. The disease was characterized by pancytopenia and aplasia of the bone marrow without clinical signs of intravascular hemolysis. In our study we observed the quick IST response at 3 months in SAA patients with the PNH clone (38,9%), in patients without PNH clone at time of diagnosis achievement of partial remission at 6 months was followed by PNH clone appearance and persistence. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

Bone Marrow Histology in Patients with Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽  
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.

Screening Patients with Myelodysplastic Syndrome and Aplastic Anemia for Paroxysmal Nocturnal Hemoglobinuria Clones: A Retrospective Study,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3426-3426 ◽  
Author(s):  
Andrew Shih ◽  
Ian H. Chin-Yee ◽  
Ben Hedley ◽  
Mike Keeney ◽  
Richard A. Wells ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Retrospective Study ◽  
Aplastic Anemia ◽  
Board Of Directors ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Cell Surface Expression ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Pnh Clone

Abstract Abstract 3426 Introduction: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare disorder due to a somatic mutation in the hematopoietic stem cell. The introduction of highly sensitive flow cytometric and aerolysin testing have shown the presence of PNH clones in patients with a variety of other hematological disorders such as aplastic anemia (AA) and myelodysplasic syndrome (MDS). It is hypothesized that patients with these disorders and PNH clones may share an immunologic basis for marrow failure with relative protection of the PNH clone, due to their lack of cell surface expression of immune accessory proteins. This is supported by the literature showing responsiveness in AA and MDS to immunosuppressive treatments. Preliminary results from a recent multicenter trial, EXPLORE, notes that PNH clones can be seen in 70% of AA and 55% of MDS patients, and therefore there may be utility in the general screening of all patients with bone marrow failure (BMF) syndromes. Furthermore, it has been suggested that the presence of PNH cells in MDS is a predictive biomarker that is clinically important for response to immunosuppressive therapy. Methods: Our retrospective cohort study in a tertiary care center used a high sensitivity RBC and FLAER assay to detect PNH clones as small as 0.01%. Of all patients screened with this method, those with bone marrow biopsy and aspirate proven MDS, AA, or other BMF syndromes (defined as unexplained cytopenias) were analysed. Results from PNH assays were compared to other clinical and laboratory parameters such as LDH. Results: Overall, 102 patients were initially screened over a 12 month period at our center. 30 patients were excluded as they did not have biopsy or aspirate proven MDS, AA, or other BMF syndromes. Of the remaining 72 patients, four patients were found to have PNH clones, where 2/51 had MDS (both RCMD, IPSS 0) [3.92%] and 2/4 had AA [50%]. The PNH clone sizes of these four patients were 0.01%, 0.01%, 0.02%, and 1.7%. None of the MDS patients with known recurrent karyotypic abnormalities had PNH clones present. Only one of the four patients had a markedly increased serum LDH level. Conclusions: Our retrospective study indicates much lower incidence of PNH clones in MDS patients or any patients with BMF syndromes when compared to the preliminary data from the EXPLORE trial. There is also significant disagreement in other smaller cohorts in regards to the incidence of PNH in AA and MDS. Screening for PNH clones in patients with bone marrow failure needs further study before adoption of widespread use. Disclosures: Keeney: Alexion Pharmaceuticals Canada Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wells:Alexion Pharmaceuticals Canada Inc: Honoraria. Sutherland:Alexion Pharmaceuticals Canada Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Allo-HCT from MUD/MRD for Paroxysmal Nocturnal Hemoglobinuria - 12 Years of Experience

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5886-5886 ◽  
Author(s):  
Miroslaw Markiewicz ◽  
Malwina Rybicka-Ramos ◽  
Monika Dzierzak-Mietla ◽  
Anna Koclega ◽  
Krzysztof Bialas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cyclosporine A ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Conflicts Of Interest ◽  
Hemorrhagic Cystitis ◽  
Chronic Gvhd ◽  
Complete Disappearance ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Pnh Clone

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal abnormality of hematopoietic stem cell leading to lack of phosphatidylinositol glycoproteins, sensitizing cells to complement-mediated lysis. Despite the efficient symptomatic treatment of hemolytic PNH with eculizumab, allo-HCT is the only curative treatment for the disease, although outcomes presented in the past were controversial. Material and methods: We report 41 allo-HCTs: 37 from MUD and 4 from MRD performed for PNH in 2004-2016. Median age of recipients was 29(20-62) years and donors 30(19-53), median time from diagnosis to allo-HCT was 16(2-307) months. Median size of PNH clone was 80% granulocytes (0.5%-100%). Indication for allo-HCT was PNH with aplastic/hypoplastic bone marrow (19 pts), MDS (2 pts), overlapping MDS/aplasia (3 pts), severe course of PNH with hemolytic crises and transfusion-dependency without access to eculizumab (17 pts). Additional risk factors were Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (2 pts), chronic hepatitis B (1 pt) and C (1 pt). The preparative regimen consisted of treosulfan 3x14 g/m2 plus fludarabine 5x30 mg/m2 (31 pts) or treosulfan 2x10 g/m2 plus cyclophosphamide 4x40 mg/kg (10 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG in MUD-HCT. 2 pts instead of cyclosporine-A received mycophenolate mofetil and tacrolimus. Source of cells was bone marrow (13 pts) or peripheral blood (28 pts) with median 6.3x108NC/kg, 5.7x106CD34+cells/kg, 24.7x107CD3+cells/kg. Myeloablation was complete in all pts with median 9(1-20) days of absolute agranulocytosis <0.1 G/l. Median number of transfused RBC and platelets units was 9(0-16) and 8(2-18). Results: All pts engrafted, median counts of granulocytes 0.5 G/l, platelets 50 G/l and Hb 10 g/dl were achieved on days 17.5(10-33), 16(9-39) and 19.5(11-34). Acute GVHD grade I,II and III was present in 16, 7 and 3 pt, limited and extensive chronic GVHD respectively in 11 and 3 pts. LDH decreased by 73%(5%-91%) in first 30 days indicating disappearance of hemolysis. 100% donor chimerism was achieved in all pts. In 1 patient donor chimerism decreased to 81% what was treated with donor lymphocytes infusion (DLI). 3 patients died, 1 previously hemodialysed pt died on day +102 due to nephrotoxicity complicating adenoviral/CMV hemorrhagic cystitis, two other SAA patients with PNH clone<10% died on days +56 due to severe pulmonary infection and +114 due to aGvHD-III and multi organ failure. Complications in survivors were FUO (10 pts), CMV reactivation (13), VOD (1), neurotoxicity (1), venal thrombosis (1), hemorrhagic cystitis (4) and mucositis (8). 38 pts (92.7%) are alive 4.2 (0.4-12) years post-transplant and are doing well without treatment. Complete disappearance of PNH clone was confirmed by flow cytometry in all surviving pts. Conclusions: Allo-HCT with treosulfan-based conditioning is effective and well tolerated curative therapy for PNH. Disclosures No relevant conflicts of interest to declare.

Establishment of a flow cytometry assay for detecting paroxysmal nocturnal hemoglobinuria-type cells specific to patients with bone marrow failure

2018 ◽  
Vol 97 (12) ◽  
pp. 2289-2297
Author(s):  
Kohei Hosokawa ◽  
Chiharu Sugimori ◽  
Ken Ishiyama ◽  
Hiroyuki Takamatsu ◽  
Hideyoshi Noji ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Flow Cytometry Assay

Extent and Clinical Implications of Subclonal Diversity in Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 779-779
Author(s):  
Mohammad Fahad B Asad ◽  
Abhinav Goyal ◽  
Hassan Awada ◽  
Mai Aly ◽  
Cassandra Hirsch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Features ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Positive Patient ◽  
Monogenic Disease ◽  
Control Group ◽  
History Of ◽  
Serial Samples ◽  
Pnh Clone

Abstract Paroxysmal Nocturnal hemoglobinuria (PNH) has been traditionally considered a monogenic disease due to somatic mutations in PIGA gene. While selective immune pressure was implicated in PNH evolution via expansion of a PIGA mutant clone in a privileged environment, history of aplastic anemia (AA) is not always present in a manifest hemolytic PNH. Similarly, expansion of PNH clone after AA therapy does not seem to correlate with the strength of immunosuppression and the quality of response. Our initial studies (J.Clin.Invest.2014; 124 :4529-4538) indicated that additional mutational events may be present in PNH and act as intrinsic factors modifying clinical features and leading to a differential expansion. We hypothesize that subclonal hits may be present in some patients with PNH to explain their expansion dynamics and clinical features. To that end we have collected 319 patients with bone marrow failure of whom 202 had PNH clone including 70 hemolytic PNH cases. For a subgroup of these patients (n=197), deep multi-targeted NGS has been performed to identify mutations in 78 myeloid genes of which 38 genes were found to be mutated (≥1) in this PNH spectrum disease. 116 serial samples for 33 patients were also sequenced. As a control group, we have included AA stably negative for PNH clone (n=113). For analytic purposes, on clinical grounds we have sub grouped patients with PNH clone into AA/PNH (31%) defined as patients with presence of bone marrow failure but no clinical or laboratory evidence of hemolysis and WBC PNH clone &lt;20%, and PNH group (69%). The latter (69%) consisted of patients with primary hemolytic PNH (pPNH; 60%) or secondary PNH (sPNH;40%). sPNH is defined as a group showing hemolysis with an antecedent history of AA and clone size &gt;20%. Our analysis focused on driver mutations. In cross-sectional analysis, in addition to PIGA we have identified somatic hits in 10/26 (38%) of AA/PNH pts. (On average 2.1 hits/per positive patient) compared to 27/58(47%) of PNH patients (on average 1.22 hits per positive patients. Subdividing PNH patients, 20/35(57%) had a mutation in primary PNH compared to 7/23(30%) in the sPNH group. In contrast, 42/113(37%) patients had a mutation in the control AA group with a (2.1 mutations per positive patient). The mean VAF for AA is 29% compared to 23% in AA/PNH vs. 34% in PNH vs. 31%in sPNH. In all patients with PNH clone, 44% had a mutation vs. 37% in the AA group. AA/PNH subcohort contained hits in cohesins genes (RAD21-SMC3-STAG2 (8 %), ZRSR2 (8%), BCORs (4%) and TET2 (4%). PNH group had mutations in BCOR/BCORL1 (BCORs) (11%), cohesions, ZRSR2 and TET2 (6% each). In sPNH, a similar trend was observed with the most common hits in EZH2-SUZ12 (9%), ZRSR2 (9%), and BCORs, TET2, LUC7L2 (4% each). In contrast, AA without PNH, the 5 most frequently mutated genes were TET2, ASXL1, EZH2-SUZ12 AND BCORs (5% each) and RUNX 1 (4%). BCORs mutations were more frequent in PNH group compared to AA, (11% vs. 5%). In summary, BCOR/L and ZRSR genes showed more mutations in PNH clone cohort while AA cohort did not show any dominant hits. To determine whether somatic hits observed cross-sectionally led to establishment of stable subclones or were merely transient, we performed a longitudinal analysis. In PNH, 3/4 clones were transient which included EZH2, PHF6 and STAG2. In AA/PNH patients, 9/14 mutational hits appeared in the course of their disease (TET2, CEBPA, CUX1, STAG2, BCOR, SMC3) but 10/14 faded away in subsequent course and thus only 4 were stable clones. In AA cohort, 32 additional mutational hits were noted in 77 serial samples and 34 mutations vanished in subsequent course. In sum, our results show that in PNH, similar to AA, additional somatic hits are common and involve some of the commonly mutated myeloid genes seen in MDS. While likely, many of these subclonal events are transient, seemingly lacking the proper ancestral context, some of the hits resulted in significant expansions (sweeping mutations) and thus may modify the clinical course including speed of the PNH clone evolution. Disclosures No relevant conflicts of interest to declare.

DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1048-1048
Author(s):  
Kazuhiko Ikeda ◽  
Tsutomu Shichishima ◽  
Yoshihiro Yamashita ◽  
Yukio Maruyama ◽  
Hiroyuki Mano
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Data Set ◽  
Hematopoietic Stem ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms’ tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of &gt;22,000 probe sets (corresponding to &gt;14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P &lt;0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1992-1992 ◽  
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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