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

Blood ◽  
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.

Whole Exome Sequencing Reveals Spectrum of Gene Mutations in Pediatric AML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 124-124
Author(s):  
Norio Shiba ◽  
Kenichi Yoshida ◽  
Yusuke Okuno ◽  
Yuichi Shiraishi ◽  
Yasunobu Nagata ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Conflicts Of Interest ◽  
Massively Parallel Sequencing ◽  
Gene Mutations ◽  
Pediatric Leukemia ◽  
Protein Coding ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
Pediatric Aml

Abstract Abstract 124 Background Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with the current overall survival remains to be ∼60%. As for the molecular pathogenesis of pediatric AML, it has been well established that gene fusions generated by recurrent chromosomal translocations, including t(15;17), t(8;21), inv(16) and t(9;11), play critical roles in leukemogenesis. However, they are not sufficient for leukemogenesis, indicating apparent need of additional genetic hits, and approximately 20% of pediatric AML cases lack any detectable chromosomal abnormalities (normal karyotype AML). Currently, a number of gene mutations have been implicated in the pathogenesis of both adult and pediatric AML, including mutations of RAS, KIT and FLT3, and more recently, a new class of mutational targets have been reported in adult AML, including CEBPA, NPM1, DNMT3A, IDH1/2, TET2 and EZH2. However, mutations of the latter class of gene targets seem to be rare in pediatric AML cases, whereas other abnormalities such as a NUP98-NSD1 fusion are barely found in adult cases, indicating the discrete pathogenesis between both AML at least in their subsets. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be successfully applied to the genetic analysis of pediatric AML to obtain a better understanding of its pathogenesis. Methods In order to reveal a complete registry of gene mutations and other genetic lesions, we performed whole exome sequencing of paired tumor-normal specimens from 23 pediatric AML cases using Illumina HiSeq 2000. Although incapable of detecting non-coding mutations and gene rearrangements, the whole-exome approach is a well-established strategy for obtaining comprehensive spectrum of protein-coding mutations. Recurrently mutated genes were further examined for mutations in an extended cohort of 200 pediatric AML samples, using deep sequencing, in which the prevalence and relative allele frequencies of mutations were investigated. Results Whole-exome sequencing of paired tumor-normal DNA from 23 patients were analyzed with a mean coverage of more than x120, and 90 % of the target sequences were analyzed at more than x20 depth on average. A total of 237 somatic mutations or 10.3 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in adult AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1, MLL3, BCOR, BCORL1, EZH2, and major cohesin components including XXX and ZZZ. On the other hand, several genes were newly identified in the current study, including BRAF, CUL2 and COL4A5, which were validated for the clinical significance in an extended cohort of 200 pediatric cases. Discussion Whole exome sequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

Whole-Exome Sequencing Of Adult Philadephia-Negative Acute Lymphoblastic Leukemia From Diagnosis To Relapse After Allogeneic Hematopoietic Stem Cell Transplantation Reveals Clonal Evolution and Relapse-Associated Mutated Genes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 156-156
Author(s):  
Haowen Xiao ◽  
Yi Luo ◽  
Xiaoyu Lai ◽  
Jimin Shi ◽  
Yamin Tan ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Somatic Mutations ◽  
Lymphoblastic Leukemia ◽  
Gene Mutations ◽  
Genetic Alterations ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Whole Exome ◽  
Matched Samples

Abstract Introduction Although steady progress of effective chemotherapy in childhood acute lymphoblastic leukemia (ALL) carried with exceeding 80% of individuals now cured, the majority of adult patients with ALL are not cured by chemotherapy, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option. However, relapse remains the most leading cause of death after allo-HSCT. Adverse genetic alterations are generally accepted to be responsible for treatment failure and relapse. Several structural chromosomal alterations including rearrangement of the myeloid-lymphoid or mixed-lineage leukemia gene (MLL) and Philadelphia chromosome (Ph), have been mostly found in relapsed ALL. However, many Ph-negative (Ph-) ALL patients with normal karyotype , lacking known risk factors, also experienced relapse. The underlying pathologic determinants leading to relapse and prognostic markers in these cases remain poorly understood. More importantly, allo-HSCT is a distinct treatment option from tradtional chemotherapy and has 2 important forms to eliminate and select on malignant cells. The malignant cells that go on to causing relapse must initially survive ablation of chemotherapy before allo-HSCT and conditioning regimen in allo-HSCT. Then, after allo-HSCT, they must survive the effect of graft-versus- leukemia (GVL) reaction. Following this rationale, we hypothesized that there may be pivotal genetic causes confer leukemic cells a fitness advantage to undergo huge selective pressures and expand after allo-HSCT. To elucidate the genomic basis underlying relapse after allo-HSCT to aid to discover novel predictive biomarkers and identify therapeutic targets, we carried out the first whole-exome sequencing analysis in longitudinal matched samples from diagnosis to relapse after allo-HSCT in adult patients with the most common subtype of ALL, Ph- B-cell ALL (B-ALL). Methods Whole-exome sequencing was conducted for 9 genomic DNA samples from 3 relapsed cases with Ph- B-ALL (discovery cohort) at 3 specific time points including: diagnosis, complete remission (CR) after induction chemotherapy before allo-HSCT, relapse after allo-HSCT to discover candidate relapse-associated mutated genes. We identified putative somatic mutations by comparing each tumor ( diagnostic samples or relapsed samples) to normal (CR samples) from the same patient. To confirm candidate somatic gene mutations, screen relapse-associated gene mutations and define the frequency of somatic mutations identified by whole-exome sequencing analysis, we further carried out target genes whole coding regions sequencing in an ALL extended validation cohort including 58 adult Ph- B-ALL cases, where 27 patients experienced relapse at a median time of 6.5 (range 2-33) months after allo-HSCT and 31 patients did not relapse after allo-HSCT at a median follow-up for 34 (range 12–56) months. Results (1) We discovered novel associations of recurrently mutated genes (CREBBP, KRAS, PTPN21) with the pathogenesis of adult Ph- B-ALL relapse after allo-HSCT, which were mutated in at least two relapsed cases, but were not mutated in non- relapsed patients. (2) The generation of high-depth whole-exome sequencing data in longitudinal matched samples from diagnosis to relapse after allo-HSCT in initial 3 patients allowed us to directly assessed the evolution of somatic mutations. Our data suggested that in the progression of leukemia relapse after allo-HSCT, the relapse clone had a clear relationship to the diagnosis clone, either arising from a subclone already exsiting in the diagnostic tumor, or originating from a common preleukemic progenitor with the diagnosis clone. In the latter pattern, the relapse clone acquires new genetic alterations while retaining some but not all of the alterations found in the diagnostic tumor. In contrast, in some cases, leukemia recurrences afer allo-HSCT may be composed of second malignancies with completely distinct sets of mutations from the primary tumor. Conclusions Our study is the first to explore genetic basis of adult Ph- B-ALL from diagnosis to relapse after allo-HSCT over time, which will provide novel genetic biomarkers on risk “index” to improve individualized treatment intensification and intervention strategies, and potential therapeutic targets for Ph--ALL relapse after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The identification of novel gene mutations for degenerative lumbar spinal stenosis using whole-exome sequencing in a Chinese cohort

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xin Jiang ◽  
Dong Chen
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Spinal Stenosis ◽  
Lumbar Spinal Stenosis ◽  
Gene Mutations ◽  
Specific Gene ◽  
Single Nucleotide ◽  
Degenerative Lumbar Spinal Stenosis ◽  
Whole Exome ◽  
Lumbar Spinal

Abstract Background Degenerative lumbar spinal stenosis (DLSS) is a common lumbar disease that requires surgery. Previous studies have indicated that genetic mutations are implicated in DLSS. However, studies on specific gene mutations are scarce. Whole-exome sequencing (WES) is a valuable research tool that identifies disease-causing genes and could become an effective strategy to investigate DLSS pathogenesis. Methods From January 2016 to December 2017, we recruited 50 unrelated patients with symptoms consistent with DLSS and 25 unrelated healthy controls. We conducted WES and exome data analysis to identify susceptible genes. Allele mutations firstly identified potential DLSS variants in controls to the patients’ group. We conducted a site-based association analysis to identify pathogenic variants using PolyPhen2, SIFT, Mutation Taster, Combined Annotation Dependent Depletion, and Phenolyzer algorithms. Potential variants were further confirmed using manual curation and validated using Sanger sequencing. Results In this cohort, the major classification variant was missense_mutation, the major variant type was single nucleotide polymorphism (SNP), and the major single nucleotide variation was C > T. Multiple SNPs in 34 genes were identified when filtered allele mutations in controls to retain only patient mutations. Pathway enrichment analyses revealed that mutated genes were mainly enriched for immune response-related signaling pathways. Using the Novegene database, site-based associations revealed several novel variants, including HLA-DRB1, PARK2, ACTR8, AOAH, BCORL1, MKRN2, NRG4, NUP205 genes, etc., were DLSS related. Conclusions Our study revealed that deleterious mutations in several genes might contribute to DLSS etiology. By screening and confirming susceptibility genes using WES, we provided more information on disease pathogenesis. Further WES studies incorporating larger DLSS patient cohorts are required to comprehend the genetic landscape of DLSS pathophysiology fully.

scholarly journals Whole-exome sequencing associates novel CSMD1 gene mutations with familial Parkinson disease

2017 ◽  
Vol 3 (5) ◽  
pp. e177 ◽  
Author(s):  
Javier Ruiz-Martínez ◽  
Luis J. Azcona ◽  
Alberto Bergareche ◽  
Jose F. Martí-Massó ◽  
Coro Paisán-Ruiz
Keyword(s):  
Parkinson Disease ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Genome Wide Association Study ◽  
Single Gene ◽  
Gene Mutations ◽  
Complement Control Protein ◽  
Genome Wide ◽  
Whole Exome ◽  
A Genome

Objective:Despite the enormous advancements made in deciphering the genetic architecture of Parkinson disease (PD), the majority of PD is idiopathic, with single gene mutations explaining only a small proportion of the cases.Methods:In this study, we clinically evaluated 2 unrelated Spanish families diagnosed with PD, in which known PD genes were previously excluded, and performed whole-exome sequencing analyses in affected individuals for disease gene identification.Results:Patients were diagnosed with typical PD without relevant distinctive symptoms. Two different novel mutations were identified in the CSMD1 gene. The CSMD1 gene, which encodes a complement control protein that is known to participate in the complement activation and inflammation in the developing CNS, was previously shown to be associated with the risk of PD in a genome-wide association study.Conclusions:We conclude that the CSMD1 mutations identified in this study might be responsible for the PD phenotype observed in our examined patients. This, along with previous reported studies, may suggest the complement pathway as an important therapeutic target for PD and other neurodegenerative diseases.

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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5016-5016
Author(s):  
Wenrui Yang ◽  
Xin Zhao ◽  
Guangxin Peng ◽  
Li Zhang ◽  
Liping Jing ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Extrinsic Factors ◽  
Clone Size ◽  
Hematopoietic Stem ◽  
Over Time ◽  
Pnh Clone

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

Diagnostic Efficacy of Whole-Exome Sequencing in 250 Patients with Congenital Bone Marrow Failure

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4385-4385
Author(s):  
Hideki Muramatsu ◽  
Yusuke Okuno ◽  
Kenichi Yoshida ◽  
Sayoko Doisaki ◽  
Asahito Hama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Practice ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Sanger Sequencing ◽  
Bone Marrow Failure ◽  
Exome Capture ◽  
Diagnostic Efficacy ◽  
Hematopoietic Stem ◽  
Whole Exome

Abstract Introduction: Congenital bone marrow failure syndromes (CBMFSs) are a heterogeneous class of diseases with overlapping phenotypes. Therefore, a precise and comprehensive genetic diagnostic system is strongly warranted to arrive at appropriate clinical decisions to avoid ineffective therapies and/or lethal complications of allogeneic hematopoietic stem cell transplantation. However, a large panel of newly identified causative genes of CBMFSs have been identified in recent years; therefore, it is virtually impossible to establish a routine genetic diagnostic test using conventional Sanger sequencing. Whole-exome sequencing (WES) is a promising solution for the diagnosis of inherited diseases because it tests virtually all genes simultaneously. For the introduction of WES into clinical practice, it is necessary to clarify whether this technique has superior diagnostic efficacy to conventional clinical genetic tests. Methods: We performed WES in 250 patients with CBMFSs lacking genetic diagnoses. Exome capture was performed using the SureSelect® Human All Exon V3–5 kit (Agilent Technologies, Santa Clara, CA, USA), which covers all known coding exons, followed by massively parallel sequencing using the HiSeq 2000 Sequencing System (Illumina, San Diego, CA, USA). Our established pipeline for WES (genomon: http://genomon.hgc.jp/exome/) detected >20,000 candidate variants per patient. Diagnoses were based on variants of 130 genes with pathogenicities confirmed by published studies. Results: Genetic diagnoses were possible in 68 patients (27%). The best efficacy was achieved in patients with Fanconi anemia [35/73, 48%; FANCG (n = 17), FANCA (n = 14), FANCB (n = 1), FANCF (n = 1), SLX4 (n = 1), and BRCA2 (n = 1)], although Sanger sequencing was not applied because of the large sizes of its causative genes. Encouraging results were obtained in patients with Diamond–Blackfan anemia [11/ 61, 18%; RPS26 (n = 3), RPS7 (n = 2), RPS19 (n = 2), RPL5 (n = 2), RPL35A (n = 1), and RPL11 (n = 1)] and dyskeratosis congenita [7/29, 24%; TERT (n = 3), TINF2 (n = 2), and DKC1 (n = 2)]. Five genetic diagnoses (7%) were inconsistent with clinical diagnoses, possibly because of overlapping disease phenotypes. Conclusion: Relative to conventional genetic testing, WES was found to be effective for the diagnoses of CBMFSs. Furthermore, the efficacy of WES will increase as our knowledge of gene mutations expands. In conclusion, the use of WES in clinical practice is warranted. Disclosures No relevant conflicts of interest to declare.

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

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

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

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