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

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.

Download Full-text

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.

Download Full-text

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

Blood ◽

10.1182/blood.v118.21.3426.3426 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3426-3426 ◽

Cited By ~ 1

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.

Download Full-text

Characteristics Of Paroxysmal Nocturnal Hemoglobinuria Clones In Colombian Patients

Blood ◽

10.1182/blood.v122.21.4869.4869 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4869-4869

Author(s):

Mónica Londoño ◽

Mario Arenas-Mantilla ◽

Alicia Maria Henao-Uribe ◽

Ricardo Novoa ◽

Andrea Naranjo ◽

...

Keyword(s):

Paroxysmal Nocturnal Hemoglobinuria ◽

Clinical Characteristics ◽

Conflicts Of Interest ◽

Moderate Correlation ◽

Unusual Site ◽

Clone Size ◽

Hematopoietic Stem ◽

Study Population ◽

Written Informed Consent ◽

Pnh Clone

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.

Download Full-text

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.

Download Full-text

Mutations in the Telomerase Complex and Expression Levels of the TERT Gene Determine Severity and Outcome of Disease in Aplastic Anemia Patients

Blood ◽

10.1182/blood.v128.22.1502.1502 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1502-1502 ◽

Cited By ~ 1

Author(s):

Arati Khanna-Gupta ◽

Durga Sarvepalli ◽

Snigdha Majumder ◽

Coral Karunakaran ◽

Malini Manoharan ◽

...

Keyword(s):

Aplastic Anemia ◽

Disease Severity ◽

Poor Prognosis ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Response To Therapy ◽

Hematopoietic Stem ◽

Expression Levels ◽

Shelterin Complex ◽

Tert Expression

Abstract Acquired Aplastic anemia (AA) is a bone marrow failure syndrome characterized by pancytopenia and marrow hypoplasia, and is mediated by immune destruction of hematopoietic stem cells. Mutations in several genes including telomerase, a ribonucleoprotein enzyme complex, consisting of a reverse transcriptase enzyme (TERT), an RNA template (TERC), and several stabilizing proteins, and the associated shelterin complexes have been found in both congenital and idiopathic AA. In particular, several TERT and TERC mutations reduce telomerase activity in vitro and accelerate telomere attrition in vivo. Shortened telomeres have been observed in a third of idiopathic AA patients, but only 10% of these patients have mutations in genes of the telomerase complex. We have recently demonstrated that in addition to keeping telomeres from shortening, telomerase directly regulates transcriptional programs of developmentally relevant genes (Ghosh et al, Nat Cell Biol, 2012, 14, 1270). We postulate that changes in expression of telomerase associated genes, specifically TERT, contribute to the etiology of aplastic anemia. In an effort to better understand the molecular and clinical correlates of this disease, 24 idiopathic AA patient samples were collected at a tertiary medical center in Bangalore, India. Following informed consent, we performed RT-PCR analysis on harvested RNA from each patient and measured levels of TERT expression compared to that of normal controls (n=6). An 8 fold reduction in TERT expression was observed in 17/24 patients, while 7/24 patients maintained normal TERT expression. In general, TERT-low patients were younger in age (mean age 29y) compared with the TERT-normal patients (mean age 40y). TERT-low patients were more likely to have severe aplastic anemia (SAA) leading to higher mortality and poorer response to therapy, with 6/17 patients dying and 4/17 not responding to ATG therapy. Targeted panel sequencing of the 24 samples on an Illumina platform revealed that while TERT-normal patients had no mutations in genes associated with the telomerase/shelterin complex, TERT-low patients carried predicted pathogenic variants in TERT, TEP1, TINF2, NBN, TPP1, HSP90A and POT1 genes, all associated with the telomerase complex. Somatic gene variants were also identified in other AA associated genes, PRF1 and CDAN1, in the TERT-low cohort. In addition, novel predicted pathogenic mutations associated with the shelterin complex were found in two TERT-low patients in the TNKS gene. We also detected mutations in TET2, BCORL1, FLT-3, MLP and BRAF genes in TERT-low patients. Mutations in these genes are associated with clonal evolution, disease progression and poor prognosis. Our observations were further illustrated in a single patient where normal TERT expression was noted at initial clinical presentation. ATG therapy led to CR, but the patient returned within a year and succumbed to E.coli related sepsis. At that stage he had low TERT expression, suggesting that TERT expression can change as the disease progresses. Taken together, our data support the hypothesis that loss of TERT expression correlates with disease severity and poor prognosis. Our observations further suggest that preliminary and periodic evaluation of TERT expression levels in AA patients is likely to serve as a predictor of disease severity and influence the choice of therapy. Disclosures No relevant conflicts of interest to declare.

Download Full-text

23 Years of Management: A Retrospective Review of Treatment for Aplastic Anemia.

Blood ◽

10.1182/blood.v114.22.1091.1091 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1091-1091

Author(s):

Connie M Piccone ◽

Marie Boorman Martin ◽

Zung Vu Tran ◽

Kim Smith-Whitley

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Retrospective Review ◽

Pediatric Patients ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Complete Response ◽

Primary Objective ◽

Hematopoietic Stem ◽

Transfusion Independence

Abstract Abstract 1091 Poster Board I-113 Introduction Aplastic anemia (AA) is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. In the past, AA was considered to be a fatal disease; however, current therapies, including bone marrow transplantation or immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CSA), are curative in the majority of patients. IST is effective at restoring hematopoietic stem cell production, but relapse and evolution to myelodysplastic syndromes remain clinical challenges. Additionally, there is no real consensus regarding optimal CSA levels, duration of CSA treatment, or the optimal use of growth factors and their relationship to the development of clonal disease. Objectives The primary objective was to review treatment management for severe AA in pediatric patients in order to elucidate treatment differences and review morbidity and mortality as they relate to treatment variation. Study Design/Methods A retrospective review of pediatric patients treated at the Children's Hospital of Philadelphia for AA (both severe and moderate) over a 23 year period was performed. Results A total of 70 patients with AA were treated at our institution from 1985 to July 2008. Exclusions included: 6 patients who received some type of initial treatment at outside institutions, 4 patients who had missing records, and 2 patients who had a diagnosis of moderate AA. Thus, a total of 58 patient records were included in the analysis. Of the total patients reviewed, 60% were male and 40% were female. 34.5% of patients were African-American, and 57% were diagnosed in 2000 or later. The mean age at diagnosis was 9.5±5.8 years. 52% fell into the category of very severe AA based on published diagnostic criteria, 45% had severe AA, and 2 patients (3%) had moderate AA. 15.5% of patients developed AA in the setting of acute hepatitis. More than half of the patients treated with IST had a complete response (CR). The average time to CR was 15±15 months. Average duration of CSA treatment was 15±13 months and 8.6±10.7 months for growth factor. Two patients (3.5%) died, one from complications unrelated to AA and one from infectious complications post-BMT after initial IST failure. Average time to transfusion independence for all patients was 8±11 months (with a range of 0-54 months). Not surprisingly, the time to transfusion independence was significantly associated with IST failure (p=0.010). Patients who failed treatment had an average time to transfusion independence of 17±16 months as compared to those who were complete responders who had an average time to transfusion independence of 3±3 months. Additionally, there was a significant association between IST failure and CSA levels (p=0.014). Patients who had nontherapeutic CSA levels overall had an increased rate of treatment failure. Of those patients who were nontherapeutic, 56% were noncompliant with CSA administration. There was no significant association between IST failure and bone marrow cellularity (p=0.251). PNH was diagnosed in 5% of patients; there were no patients with evidence of myelodysplastic syndrome (MDS). Two of the 3 patients with PNH failed initial IST. Another 2 patients had evidence of a cytogenetic abnormality (16q deletion), but never progressed to MDS. (Note: averages presented as mean±SD) Conclusions/Methods With current IST regimens, AA is curative in the majority of pediatric patients. IST failure was associated with nonadherence to CSA treatment. For patients with confirmed clonal disease, it is possible that IST failure and the ultimate development of clonal disease are related. Disclosures No relevant conflicts of interest to declare.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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

Blood ◽

10.1182/blood.v128.22.5080.5080 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5080-5080

Author(s):

Elena Shilova ◽

Tatiana Glazanova ◽

Zhanna Chubukina ◽

Lubov Stelmashenko ◽

Sergey Gritsaev ◽

...

Keyword(s):

Aplastic Anemia ◽

Combined Therapy ◽

Clonal Evolution ◽

Pregnant Patient ◽

Observation Group ◽

Intravascular Hemolysis ◽

Clone Size ◽

Dynamic Observation ◽

Long Term Observation ◽

Pnh Clone

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.

Download Full-text

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

Blood ◽

10.1182/blood.v106.11.1048.1048 ◽

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 >22,000 probe sets (corresponding to >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 <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.

Download Full-text