Long Non-Coding RNA FAM157C Contributed to Clonal Proliferation in Paroxysmal Nocturnal Hemoglobinuria

Abstract Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease of hematopoietic stem cells. However, the mechanism of proliferative advantage of PNH clone is unclear. Long noncoding RNAs (LncRNAs) have a wide range of biological functions, including regulation of gene expression, cell differentiation, and proliferation, while its role in PNH remains unclear. Methods: In our study, CD59－and CD59+ granulocytes and monocytes from 5 PNH patients were sorted, and LncRNAs and mRNAs were detected by RNA sequencing. The proliferation-related NF-κB pathway was focused on. A total of 8 mRNAs and 5 LncRNAs were verified by qRT-PCR, and analyzed the correlation with clinical data. Meanwhile, the function of LncRNA was studied.Results: LncRNA FAM157C were verified to be upregulated in PNH clone cells, which were positively correlated with LDH level and CD59- granulated and monocytes cells ratio. After knockdown of FAM157C gene in PIGA-KO-THP-1 cell line, we found that the cells were blocked in G0/G1 phase and S phase, and the apoptosis rate increased, while the proliferation ability decreased. Conclusions: LncRNA FAM157C was proved to promote PNH clone proliferation, which is the first time to explore the role of LncRNAs in PNH.

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The Study of Proliferation Relative Long Non-Coding RNA in CD59- Cell from Paroxysmalnocturnal Hemoglobinuria Patients

Blood ◽

10.1182/blood-2019-128038 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5023-5023

Author(s):

Rong Fu ◽

Honglei Wang ◽

Hui Liu ◽

Zonghong Shao ◽

Liyan Li

Keyword(s):

Conflicts Of Interest ◽

Regulation Of Gene Expression ◽

Biological Functions ◽

Transcription Analysis ◽

Hematopoietic Stem ◽

Non Coding Rna ◽

Lncrna Malat1 ◽

Wide Range ◽

Long Non Coding Rna ◽

Pnh Clone

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonogenic disease of hematopoietic stem cells. LncRNA has a wide range of biological functions, including regulation of gene expression, cell differentiation, cell proliferation and substance metabolism.LncRNA maybe contribute to the proliferation of PNH clone. Methods: PNH clone (CD59-cells) and (CD59+cells) from PNH patients were sorted and analyzed by RNA sequencing in 5 PNH patients. The results were analyzed by KEGG, we focus on the proliferation relative pathway-NF-kB pathway. The mRNA(TAB2, TLR4, LYN,CFLAR, TNFAIP3, PTGS2, TRIM25, CXCL8) which FPKM>10 and over 3 pathients were chosen to search out the upstream regulation LncRNA. MALAT1 and LINC01002 were screened by Co-expression. Then the expression of MALAT1 and LINC01002 in 30 PNH patients were detected by qRT-PCR to vertify the LncRNA sequencing results. Results: Transcription analysis revealed that 742 upregulation LncRNA and 3276 upregulation mRNA were identified in CD59- cells. The highly expressed NF-kB pathway mRNA (TAB2, TLR4, LYN,CFLAR, TNFAIP3, PTGS2, TRIM25, CXCL8) were analysed by LncRNA co-expression, after that MALAT1and LINC01002 were concerned with the 8 mRNA. The results of PNH primary cells (CD59-cells) showed that the level of MALAT1and LINC01002 expression was significantly higher than that of the CD59+ cell in 30 PNH patients (p<0.05). Conclusion: MALAT1and LINC01002 seems to be involved in the proliferation of PNH clones. Its mechanism of action in patients with PNH needs further study. 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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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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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.

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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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Allo-HSCT from MUD/MRD As a Curative Treatment in Paroxysmal Nocturnal Hemoglobinuria

Blood ◽

10.1182/blood.v124.21.1251.1251 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1251-1251

Author(s):

Miroslaw Markiewicz ◽

Anna Koclega ◽

Monika Dzierzak-Mietla ◽

Patrycja Zielinska ◽

Ewa Mendek-Czajkowska ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cyclosporine A ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Hemorrhagic Cystitis ◽

Curative Treatment ◽

Hematopoietic Stem ◽

Donor Chimerism ◽

Pnh Clone

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal abnormality of the hematopoietic stem cell caused by somatic mutation in the phosphatidylinositol glycan-class A (PIG-A) gene located on the short arm of the X chromosome. Cells with lack phosphatidylinositol glycoproteins are more sensitive to complement-mediated lysis. Despite the efficient symptomatic treatment of hemolytic PNH with eculizumab, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment of the disease, although outcomes presented in the past were controversial. Material and methods: We report 32 allo-HSCTs: 31 from MUD and 1 from MRD performed for PNH in 2004-2014. Median age of recipients was 28 years (range 20-55) and donors 33(19-53), median time from diagnosis to allo-HSCT was 18(2-307) months. Median size of PNH clone was 80% granulocytes (0.41%-98%). Indication for allo-HSCT was aplastic/hypoplastic bone marrow (15 pts), overlapping MDS (2 pts), severe course of PNH with hemolytic crises and transfusion-dependency without access to eculizumab (15 pts). Additional risk factors were Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (2 pts) and chronic hepatitis B (1 pt). The preparative regimen consisted of treosulfan 3x14 g/m2 plus fludarabine 5x30 mg/m2 (25 pts) or treosulfan 2x10 g/m2 plus cyclophosphamide 4x40 mg/kg (7 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG or thymoglobulin in MUD-HSCT. 2 pts instead cyclosporine-A received mycophenolate mofetil and tacrolimus. Source of cells was bone marrow (12 pts) or peripheral blood (20 pts) with median 7.7x10(8)NC/kg, 5.3x10(6)CD34+cells/kg, 24.2x10(6)CD3+cells/kg. Myeloablation was complete in all pts with median 9 days (6-13) of absolute agranulocytosis <0.1 G/l. Median number of transfused RBC and platelets units was 8.5(1-16) and 8(3-18). 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(13-33), 17.5(11-39) and 19.5(11-34). Acute GVHD grade I,II and III was present in 14, 6 and 1 pt, limited chronic GVHD in 11 pts. LDH decreased by 77%(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 83% what was treated with donor lymphocytes infusion (DLI). 2 patients died, 1 previously hemodialysed pt died on day +102 in a consequence of nephrotoxicity complicating adenoviral/CMV hemorrhagic cystitis and second on day +56 because of severe pulmonary infection. Complications in survivors were FUO (7 pts), CMV reactivation (8), VOD (1), neurotoxicity (1), venal thrombosis (1), hemorrhagic cystitis (1) and mucositis (8). 30 pts (93.7%) are alive 42 months (1-85) post-transplant and are doing well without treatment. Complete disappearance of PNH clone was confirmed by flow cytometry in all surviving pts. Conclusions: Our results indicate, that allo-HSCT with treosulfan-based conditioning is effective and well tolerated curative therapy in PNH. Disclosures No relevant conflicts of interest to declare.

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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.

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Long-term support of hematopoiesis by a single stem cell clone in patients with paroxysmal nocturnal hemoglobinuria

Blood ◽

10.1182/blood.v99.8.2748 ◽

2002 ◽

Vol 99 (8) ◽

pp. 2748-2751 ◽

Cited By ~ 27

Author(s):

Jun-ichi Nishimura ◽

Toshiyuki Hirota ◽

Yuzuru Kanakura ◽

Takashi Machii ◽

Takashi Kageyama ◽

...

Keyword(s):

Stem Cell ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Somatic Mutations ◽

Cell Clone ◽

Gene Mutations ◽

Polymorphonuclear Cells ◽

Hematopoietic Stem ◽

Cell Disorder ◽

Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by clonal blood cells that are deficient in glycosylphosphatidylinositol-anchored proteins because of somatic mutations of the PIG-A gene. Many patients with PNH have more than one PNH clone, but it is unclear whether a single PNH clone remains dominant or minor clones eventually become dominant. Furthermore, it is unknown how many hematopoietic stem cells (HSCs) sustain hematopoiesis and how long a single HSC can support hematopoiesis in humans. To understand dynamics of HSCs, we reanalyzed the PIG-A gene mutations in 9 patients 6 to 10 years after the previous analyses. The proportion of affected peripheral blood polymorphonuclear cells (PMNs) in each patient was highly variable; it increased in 2 (from 50% and 65% to 98% and 97%, respectively), was stable in 4 (changed less than 20%), and diminished in 3 (94%, 99%, and 98% to 33%, 57%, and 43%, respectively) patients. The complexity of these results reflects the high variability of the clinical course of PNH. In all patients, the previously predominant clone was still present and dominant. Therefore, one stem cell clone can sustain hematopoiesis for 6 to 10 years in patients with PNH. Two patients whose affected PMNs decreased because of a decline of the predominant PNH clone and who have been followed up for 24 and 31 years now have an aplastic condition, suggesting that aplasia is a terminal feature of PNH.

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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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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.bloodjournal6661247 ◽

1985 ◽

Vol 66 (6) ◽

pp. 1247-1250

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

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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