Characteristics Of Paroxysmal Nocturnal Hemoglobinuria Clones In Colombian Patients

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder, characterized by the clonal expansion of hematopoietic stem cells lacking glycosylphosphatidylinositol-anchored proteins. PNH clones are frequently found in patients with aplastic anemia (AA), myelodysplastic syndrome (MDS), unusual site thrombosis and non-inmume hemolysis. In Colombia, the DECF laboratory analyses blood samples from all over the country, summited by the APEC (association of patients with complement diseases) for PNH screening. Methods We reviewed the results of flow cytometry (FC) analyses from 1448 patients screened for PNH in DECF laboratory, between 2010 and 2013, and evaluated the association between clinical characteristics and distribution of PNH clone sizes. Clinical characteristics were considered as referred by treating physicians in the FC analysis request forms. All patients included gave written informed consent. Results Mean age of the study population was 44.6±18.5 years and 60.5% were female patients. The most frequent indications for screening were thrombosis (23.5%) and unexplained cytopenias (21.8%). Only 14% of the samples were PNH positive. Table 1 shows the results of FC analysis according to the indications for screening. Median clone sizes were 0.1% (interquartile range (IQR): 0-5.8%) in erythrocytes, 4.2% (IQR: 1.1-53.9%) in granulocytes and 3.8% (IQR: 0.7-64.1%) in monocytes. PNH clone size in granulocytes showed strong correlation with clone size in monocytes (r=0.86, p<0.001) and moderate correlation with clone size in erythrocytes (r=0.65, p=0.001). Figure 1 depicts the distribution of PNH granulocyte clone sizes among indications for screening. In the group of patients with PNH granulocyte clone sizes >5%, those with AA had smaller clone sizes than the rest (Figure 2), while patients with hemolysis showed larger clone sizes than the rest (Figure 3). No significant differences in clone size distribution were found within patients with PNH granulocyte clone sizes ≤5%. Conclusions Our findings are comparable to those reported in other countries. PNH granulocyte clone sizes had stronger correlation with PNH monocyte clones than with PNH erythrocyte clones. About one third of the patients with AA and one fourth of those with hemolysis were PNH positive. Over 50% of patients with AA showed PNH clones sizes <5%. Patients with hemolysis have larger clones than the rest. Further studies are needed to establish the association between PNH clone sizes and clinical outcomes of related disorders. Disclosures: No relevant conflicts of interest to declare.

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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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Periodic Evaluation Of The Clone Size Is Mandatory In PNH: Study Of The Spanish Cohort Of The International PNH Registry

Blood ◽

10.1182/blood.v122.21.3715.3715 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3715-3715

Author(s):

Ana Villegas ◽

Ana Gaya ◽

Emilio Ojeda ◽

Ataulfo Gonzalez ◽

Alvaro Urbano ◽

...

Keyword(s):

Conflicts Of Interest ◽

Small Sample Size ◽

Small Sample ◽

Additional Patient ◽

Clone Size ◽

Hematopoietic Stem ◽

Future Analysis ◽

Group I ◽

Group Ii ◽

Pnh Clone

Abstract Background Paroxysmal nocturnal hemoglobinuria (PNH) is a chronic, life threatening hematopoietic stem cell disorder with chronic hemolytic anemia, peripheral blood cytopenias and thrombosis Aims To observe the PNH clone and LDH evolution of the Spanish patients enrolled in the International PNH Registry, the thrombotic events and the role of eculizumab Methods We analyzed the 117 patients enrolled in the Registry until Dec. 31st 2012, classified in 3 groups: Classic/ hemolytic (group I, n 59), PNH with another bone marrow disorder (group II, n 42) and Subclinical (group III, n 14). The variables analyzed were PNH clone size, LDH levels, and incidence of thrombosis. Medians and percentages should be taken with caution due to the relatively small sample size. In addition to data collected in the Registry, additional patient information was obtained from local physicians. Results The median (range) age at presentation was 36.6 yrs. (16-83); 48 patients (41.0%) were women. Median (range) time from disease start to enrollment was 11.3 years in group I (0.1-41.2), 3.5 in II (0.1-33.8) and 3.4 (0.3-20.8) in III. A total of 49 patients (39 in group I) were started on eculizumab, 38 prior to enrollment (31 in group I) and 11 on or after enrollment; 3 were treated prior to enrollment but discontinued for different reasons (pregnancy, ending trial, access problems). Clone evolution (Table 1). In group I the median clone size remained stable during the follow-up period in the Registry; however, 4 patients in group II evolved to group I, with granulocyte clones > 50% and LDH levels >2000 U/L, while 3 initially in group I evolved to group II at 6, 12 and 18 months respectively. At enrollment 64 patients had a clone ≥30% and 31<30%; 7 patients in group II had a clone ≥30% despite hypoplasia, and they were treated with eculizumab. In groups I and II median clone size increased from Diagnosis to Enrollment in line with the physiopathology of the disease. LDH evolution (Table 2). Median LDH levels at diagnosis were higher in group I. In this group the decrease in LDH level between Diagnosis and Enrollment could be attributed to the start of treatment in 31 patients before the enrollment visit, but that hypothesis will need confirmation in future analysis. Thrombotic episodes (Table 3). Twenty six patients (22.6%) presented 52 TEs along the study period, 41 in group I and 11 in group II. Of the 26, fourteen presented 1 TE, six 2, one 3, four 2 and one 5. Twenty five patients presented 51 TEs since the moment of diagnosis while they were not being treated with eculizumab. Only one patient in the treated group presented a TE (CVA), of which he recovered well; after 30 months of the episode continues with the treatment and scores 90 in the Karnofsky index. Fifty eight percent of the patients presenting TEs were male, showing they may be more prone to TE than women. Conclusions These data show the dynamic features of the disease in some patients, which justifies the necessity of regularly monitoring the clone size LDH levels are higher in patients with classical PNH at diagnostic; the effect of the treatment in the whole cohort will require future analysis Thrombosis is highly prevalent in PNH; 22.6% of the patients in this sample had at least 1 episode along their time in the study. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v128.22.5886.5886 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5886-5886 ◽

Cited By ~ 1

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.

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Different Clinical Characteristics of Paroxysmal Nocturnal Hemoglobinuria in Pediatric and Adult Patients

Blood ◽

10.1182/blood.v126.23.3341.3341 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3341-3341

Author(s):

Alvaro Urbano-Ispizua ◽

Petra Muus ◽

Hubert Schrezenmeier ◽

Antonio Medina Almeida ◽

Amanda Wilson ◽

...

Keyword(s):

Research Funding ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Clinical Characteristics ◽

Pediatric Patients ◽

Adult Patients ◽

Vascular Event ◽

Clone Size ◽

History Of ◽

Pnh Clone ◽

Hypoplastic Anemia

Abstract Introduction: Studies of children with paroxysmal nocturnal hemoglobinuria (PNH) are scarce and include a very limited number of patients. The objective of this analysis was to describe characteristics of PNH at enrollment for the largest available registry of pediatric patients, and to compare demographic and clinical characteristics with those of adult patients. Methods: The International PNH Registry is a prospective, multi-center worldwide, observational study of patients with a PNH clone of 0.01-100%. Data are collected from patient medical records at the time of Registry enrollment and every six months thereafter. Adult patients were ³18 years of age at enrollment and disease start and pediatric patients were <18 years at enrollment. Demographics and clinical parameters in patients untreated with eculizumab at enrollment for the two age cohorts were compared using the Wilcoxon-Mann-Whitney test for medians and PearsonÕs chi-square for frequencies. The rate of thrombotic events (TE) between disease start (defined as the earliest reported PNH symptoms, granulocyte clone, or PNH diagnosis) and enrollment was calculated per 100 person-years. Results: As of March 2, 2015, a total of 2,184 patients were eligible for analysis: 94 (4.3%) pediatric patients and 2,090 (95.7%) adult patients. Median age (range) at enrollment was 14.0 years (3-17) in pediatrics and 45.5 years (18-100) in adults; median disease duration was 0.7 years and 2.1 years, respectively (p<0.001). More pediatric than adult patients had a PNH clone of <10% and severe cytopenia (Table). Pediatric patients had lower percent of reticulocytes compared with adults (2.1% vs. 2.6%, respectively; p=0.015). History of aplastic or hypoplastic anemia was more frequent in pediatric than adult patients (76.5% vs 54.4%, respectively; p<0.001). History of TE and any major adverse vascular event was less frequent in pediatrics (2.1% vs 8.7%; p=0.025, and 4.3% vs. 14.4%; p=0.005). The rate of TE between disease start and enrollment was lower in pediatric patients, but not statistically significant: 1.4 per 100 person-years (95%CI 0.2-5.2) compared to adult patients (2.3 per 100 person-years (95%CI 2.0-2.6). More pediatric patients than adults had abdominal pain at enrollment. Conclusions: The International PNH Registry provides the largest available pediatric cohort of patients with a PNH clone to characterize this understudied population and demonstrate an important disease burden. Pediatric patients were more likely to have smaller PNH clones and a higher component of aplastic/hypoplastic anemia. Pediatric patients had fewer vascular events. These findings may reflect the natural evolution of the disease and can be useful in the clinical management of PNH. Table 1. Clinical Characteristics at Enrollment of Pediatric and Adult Patients with PNH Pediatric(n=94) Adult(n=2,090) P-value Clone size (percent GPI-deficient granulocytes), n (%)<10% 10 to < 50% ³50% 47 (55.3) 16 (18.8) 22 (25.9) 550 (38.3) 322 (22.4) 565 (39.3) 0.006* Cytopenia status, n (%)None (neutrophils ³ 1.5 x 109/L and platelets ³100 x 109/L) Moderate (neutrophils <1.5 x 109/L or platelets <100 x 109/L) Severe (neutrophils <0.5 x 109/L or platelets <20x109/L) 22 (29.3) 28 (37.3) 25 (33.3) 735 (42.2) 784 (45.1) 221 (12.7) <0.001* Percent reticulocytesMedian (Q1, Q3) 2.1 (1.1, 3.5) 2.6 (1.6, 4.6) 0.015 Hemolytic status, n (%)Hemolytic (LDH ³1.5 x ULN and/or reticulocytes ³60 x 109/L) Not hemolytic (LDH <1.5 x ULN and reticulocytes <60 x 109/L) 33 (58.9) 23 (41.1) 1,038 (65.3) 551 (34.7) NS LDH Ratio, n (%)<1.5 x ULN³1.5 x ULN 30 (58.8) 21 (41.2) 684 (47.0) 770 (53.0) NS History of TE, n (%)Yes No 2 (2.1) 92 (97.9) 181 (8.7) 1,902 (91.3) 0.025 Rate of TENumber of TE, n Person-years (disease start to enrollment) Rate/100 person-years (95% CI) 2 139.9 1.4 (0.2-5.2) 255 11,119.8 2.3 (2.0-2.6) NS History of MAVE, n (%) 4 (4.3) 300 (14.4) 0.005 GPI, glycosylphosphatidylinositol; LDH, lactate dehydrogenase; MAVE, major adverse vascular event; TE, thrombotic event; ULN, upper limit of normal *P-values for clone size and cytopenia status represent overall comparison of categories. Disclosures Muus: Alexion Pharmaceuticals: Honoraria. Schrezenmeier:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Almeida:Celgene: Consultancy; Novartis: Consultancy; Bristol Meyer Squibb: Speakers Bureau; Shire: Speakers Bureau. Wilson:Alexion Pharmaceuticals: Employment. Ware:Bayer Pharmaceuticals: Consultancy; Biomedomics: Research Funding; Eli Lilly: Other: DSMB membership; Bristol Myers Squibb: Research Funding.

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Detection of Myeloid and Clonal Expansion Related Gene Mutations By Whole-Exome Sequencing in Patients with Paroxysmal Nocturnal Hemoglobinuria

Blood ◽

10.1182/blood-2020-135965 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-10

Author(s):

Fangfei Chen ◽

Bing Han ◽

Jian Li

Keyword(s):

Cell Proliferation ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conflicts Of Interest ◽

Gene Mutations ◽

Clonal Expansion ◽

Hematopoietic Stem ◽

Whole Exome ◽

Pnh Clone

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a disease presented with hemolysis, cytopenia and thrombosis. Apart from PIGA gene on hematopoietic stem cells which accounts for the glycosylphosphatidylinositol (GPI) anchor deficiency on the cell membrane, other mutations have also been detected in PNH through whole-exome sequencing (WES). However, the characteristics of mutations in patients with PNH and genes which may contribute to PNH clonal expansion have not been well defined. Methods: Peripheral blood samples were collected from 41 patients with PNH, among them samples from 6 patients were further separated into CD59- and CD59+ fractions by CD59 magnetic beads. Gene mutations were tested by whole-exome sequencing(WES). 178 genes commonly mutated in myeloid cancer were analyzed in the sequencing results, as well as their correlation with clinical indicators. Mutated genes correlated with cell proliferation were compared between sorted CD59+ and CD59- cells. Results: The most frequently mutated myeloid cancer-related genes were MAP3K4 and CSMD1 (12.2% respectively). Among them, RUNX1T1 mutation was found to be correlated with larger clone size, higher level of uncombined bilirubin, and lower level of hemoglobin (P<0.05). No other correlation between clinical parameters and gene mutations were found. The proportion of mutations (DNMT3A、RUNX1、JAK2、JAK3、CSMD1) which have been shown to indicate poor outcome in patients with aplastic anemia decreased as PNH clone increased (p=0.026). Mutations related to cell proliferation tended to happen more frequently in CD59- fractions compared with CD59+ fractions of the same patient (P=0.062). Conclusions: Myeloid cancer-related mutations can be detected in patients with PNH with some correlation with clinical manifestations. Larger PNH clone may "save" patients from mutation indicating poor prognosis. CD59- fractions seemed to carry more proliferation related mutations, which may contribute to PNH clonal expansion. Disclosures No relevant conflicts of interest to declare.

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Data From The Russian Cohort Of The International Disease Registry For Paroxysmal Nocturnal Hemoglobinuria (PNH)

Blood ◽

10.1182/blood.v122.21.4866.4866 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4866-4866 ◽

Cited By ~ 1

Author(s):

Igor Lisukov ◽

Alexander Kulagin ◽

Alexey Maschan ◽

Elena Shilova ◽

Kudrat Abdulkadyrov ◽

...

Keyword(s):

Paroxysmal Nocturnal Hemoglobinuria ◽

Continuous Variables ◽

Clone Size ◽

Hematopoietic Stem ◽

Multicenter Observational Study ◽

Disease Characteristics ◽

History Of ◽

Medical Histories ◽

Pnh Clone

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disease that can lead to life-threatening complications including thrombotic events (TE), chronic kidney disease (CKD) and pulmonary hypertension. An international PNH Registry was implemented in 2008 to enhance understanding of the natural history of PNH, to describe treatment outcomes, and to evaluate the long term safety of eculizumab in treated patients. Methods This Registry is a non-interventional, prospective, multicenter, observational study. All patients with a diagnosis of PNH (confirmed in accordance with international diagnostic guidelines) or a detected PNH clone are enrolled irrespective of age or therapy. Data on patient demographics, medical histories, disease characteristics and treatment are collected at enrollment, every 6 months thereafter and/or at discontinuation. Descriptive statistics are used to describe the data; n, median and range (min–max) for continuous variables and percentages for categorical parameters. Results As of May 1, 2013, the Registry has enrolled 248 patients from Russia, over 50% of whom have a history of aplastic anemia or other bone marrow disorder (BMD) (Table 1). Disease characteristics for the overall population and by clone size or LDH level are presented in table1.A total of 25 patients have received eculizumab and have available follow-up data after starting treatment; median (range) follow-up time 4.4 (0.3–8.3) months. Among the 11 patients treated with eculizumab and with available LDH levels, the median LDH ratio was 5.7 X ULN before treatment and 1.0 X ULN at last follow-up assessment. Conclusion Russian patients included in the International PNH Registry show broad ranges of age, clone size, and degrees of hemolysis. History of TE, impaired renal function, and signs of chronic hemolysis are present among these patients regardless of PNH clone size. History of TE was recorded more frequently in patients with PNH clone sizes ≥20%, and was also more frequent among patients with LDH levels ≥1.5 x ULN. Among patients treated with eculizumab there was a marked decrease in hemolysis (as measured by LDH levels). Disclosures: Lisukov: Alexion: Honoraria. Kulagin:Alexion: Honoraria. Shilova:Alexion: Honoraria. Afanasyev:Alexion: Honoraria.

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Natural History of Paroxysmal Nocturnal Hemoglobinuria Clones In Patients Presenting as Aplastic Anemia

Blood ◽

10.1182/blood.v116.21.4428.4428 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4428-4428

Author(s):

Jeffrey J Pu ◽

Galina Mukhina ◽

Hao Wang ◽

William Savage ◽

Robert A Brodsky

Keyword(s):

Bone Marrow ◽

Natural History ◽

Aplastic Anemia ◽

Median Time ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Clone Size ◽

Hematopoietic Stem ◽

History Of ◽

Pnh Clone ◽

Ldh Value

Abstract Abstract 4428 Introduction: Acquired aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are closely related bone marrow failure disorders. Most AA results from an autoimmune attack directed against hematopoietic stem/progenitor cells. PNH originates from a multipotent hematopoietic stem cell (HSC) that acquires a PIG-A mutation. The PIG-A gene mutation leads to glycosylphosphatidylinositol-anchor protein (GPI-AP) biosynthesis deficiency and subsequent hemolysis secondary to the absence of complement regulatory proteins (CD55 and CD59). Both PNH and AA can be cured by allogeneic bone marrow transplantation (alloBMT), but only a minority of patients is offered this approach due to the potential morbidity and mortality. AA can be treated with immunosuppressive therapy (IST) and PNH can be controlled by eculizumab. It has been estimated that more than 50% of AA patients harbor small, but expandable PNH populations at diagnosis. The natural history of PNH clones in AA patients following non-transplant therapy is not well studied. The purpose of this study is to determine the fate and clinical relevance of these PNH clones in patients with AA who did not receive an alloBMT. Patients and Method: Twenty-seven patients with AA and a detectable PNH clone were monitored for a median of 5.3 years (range,1.5 to 11.5 years). The PNH granulocyte clone sizes were measured using flow cytometry and analyzed via CellQuest software. PE-conjugated anti-CD15 and fluoresceinated aerolysin variant (FLAER) staining were used to define granulocytes and GPI-AP deficient cells respectively. Serum lactate dehydrogenase (LDH) value was used as a surrogate for monitoring hemolysis and 1.5× the upper limit of normal LDH value (330mg/dL) as a cut-off point to define clinically apparent hemolysis. A PNH size change <2.5% was considered as stable. Patients were treated with IST, HiCy, or both. Result: We found a linear relationship between PNH granulocyte clone size and LDH values (Pearson correlation coefficient=0.73; P<0.0001). A PNH clone size above 23% was the threshold to identify hemolysis as measured by LDH (ROC analysis with AUC=0.88). Higher LDH values over the period of follow-up were associated with larger PNH granulocyte clone size at diagnosis (P=0.03). Patients with small (≤15%) initial PNH granulocytes had lower LDH levels at 5 years after diagnosis (mean±SD: 236.9±109.9 vs 423.1±248.8; P=0.02), and were less likely to develop hemolysis (13.3% vs 55.6%, P=0.06) comparing to those with larger (>15%) initial PNH granulocytes. Of 9 patients who initially were treated with traditional IST (ATG, CsA, and prednisone), 7 did not respond to treatment and eventually received high-dose cyclophosphamide (HiCy) salvage therapy, 2 achieved a remission and did not require further treatment though one demonstrated PNH clone size expansion to 50% after 37 months. After HiCy salvage, all 7 patients became transfusion independent and 4 of them had no further PNH clone expansion. PNH clone expansion was observed in 7 of 9 patients at a median time of 3 (range: 2 to 87) months after treatment. Of 15 patients who received HiCy as initial therapy, 14 achieved remissions. Later expansion of PNH size was observed in 7 patients, of which 5 eventually required intermittent blood transfusion but only 1 developed symptomatic hemolysis necessitating eculizumab therapy. The median time to PNH granulocyte clone expansion after HiCy was 52 (range: 18 to 106) months. In 5 patients who received HiCy and then relapsed, their PNH clone size only increased (1±0.7)% in (71±31) months observation during post treatment remission; however, their PNH clone size increase accelerated to (38±14)% in (34±21) months after AA relapse (P=0.04). Two nSAA patients with an initial PNH clone size ≤15% spontaneously recovered hematopoiesis at 84 and 56 months respectively, neither had PNH clone size expansion. In this study, 25.9% patients kept a stable PNH size, 48.1% patients increased the size, and 26% patients decreased the size. The group with small initial PNH clone sizes (≤15%) was the most stable over time. Conclusion: The risk of developing clinically significant PNH over 10 years appears to be low in AA patients with PNH clones, especially for those with small initial PNH granulocyte clones (≤15%) and for those who maintain remission following therapy. Disclosures: No relevant conflicts of interest to declare.

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Flow Cytometry Screening for Paroxysmal Nocturnal Hemoglobinuria Abnormal Clones in Saudi Arabia

Blood ◽

10.1182/blood.v124.21.5162.5162 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5162-5162

Author(s):

Nahlah AlGhasham ◽

Yasmeen Abulkhair ◽

Salem Khalil

Keyword(s):

Flow Cytometry ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conflicts Of Interest ◽

Clone Detection ◽

Sample Type ◽

Type Ii ◽

Inhibitory Protein ◽

Clone Size ◽

The Past ◽

Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease with insidious process, chronic course and life-threatening condition. PNH is clinically defined by the deficiency of the endogenous glycosyl phosphatidylinositol (GPI)-anchored complement inhibitory protein. It has always aroused interest in the medical profession rendering screening and proper diagnosis by flow cytometry (FCM) technology a priority We reviewed all samples submitted for PNH/ FCM screening for the past 2 years (2012-2013) at hematology section, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center (General Organization). We collected the positive cases and reviewed them for age, gender, indication for screening, sample type, size of the PNH clone and cell type affected. Immunophenotypic analysis was performed using gating antibodies CD45, CD15, CD33, CD235a GPI-linked antibodies, CD59, CD14, and CD24 as well as fluorescent Aerolysin (FLAER). In a total of 366 peripheral blood samples submitted for PNH/ FCM screening fifteen samples (4%) were positive for PNH clones but only 12 patients were available for analysis. The median age for our patients was 34 years with approximately equal male to female distribution. 12 cases showed type II and III clones within the RBCs with clone size ranging between 0.04% and 56%. Analysis of granulocytes and monocytes revealed type III clone in 8 cases, type II and III clone in 3 cases and non in one case. The percentage of the clone varies between the granulocytes and monocytes and ranges from 1% up to 100%. Of 12 positive PNH cases, 8 (66.7%) patients were diagnosed as having aplastic anemia (AA), 1 (8.3%) patient with Budd-Chiari syndrome, 1 (8.3%) patient has chronic immune thrombocytopenia (ITP), and 2 (16.7%) patients presented with pancytopenia. This study confirms the rarity of the disease since only 4% of the submitted samples for analysis turned to be positive for PNH. The detection limit for a PNH clone by FCM in the RBC or WBC is 0.01%. Identification of small PNH clone is greater FCM sensitivity relative to old test used for the same purpose (Ham test). The use of the FLAER allowed us to detect granulocytic PNH clone, however, granulocytes PNH clone detection alone without RBCs clone detection is not recommended. This review confirms the previous percentage of positive cases (5.9%) reported from this center on a smaller number of cases during the past few years. Disclosures No relevant conflicts of interest to declare.

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Bone marrow transplantation for paroxysmal nocturnal hemoglobinuria: eradication of the PNH clone and documentation of complete lymphohematopoietic engraftment

Blood ◽

10.1182/blood.v66.6.1247.1247 ◽

1985 ◽

Vol 66 (6) ◽

pp. 1247-1250 ◽

Cited By ~ 60

Author(s):

JH Antin ◽

D Ginsburg ◽

BR Smith ◽

DG Nathan ◽

SH Orkin ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conditioning Regimen ◽

Erythroid Cell ◽

Polymorphism Analysis ◽

Hematopoietic Stem ◽

Curative Therapy ◽

Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) involves the proliferation of an abnormal and possibly premalignant hematopoietic stem cell. Successful treatment of PNH by marrow grafting requires that the PNH clone be eradicated by the pretransplant conditioning regimen. Four patients with PNH-associated marrow aplasia were transplanted with marrow from their HLA-matched, MLR-nonreactive siblings. Three patients were conditioned with cyclophosphamide, procarbazine, and antithymocyte serum (CTX/PCZ/ATS), and one was conditioned with busulfan/CTX/PCZ/ATS. Persistent complete engraftment of myeloid, lymphoid, and erythroid cell lines was demonstrated in all four patients by DNA sequence polymorphism analysis or cytogenetics, and RBC typing. There was no recurrence of the abnormal clone of cells for up to five years after transplantation despite the use of a conditioning regimen in three of them, which is not usually associated with permanent marrow aplasia. Bone marrow transplantation is a curative therapy in patients whose illness is severe enough to warrant the risk.

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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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