Baseline Assessment Of GPI-Anchored Protein Deficient Blood Cells In Patients With Bone Marrow Failure (The OPTIMA study)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1241-1241 ◽  
Author(s):  
Naoshi Obara ◽  
Shigeru Chiba ◽  
Kohei Hosokawa ◽  
Chiharu Sugimori ◽  
Masaki Yamamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
High Resolution ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Treatment Plan ◽  
Low Frequencies ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Patient Groups

Abstract Background Paroxysmal nocturnal hemoglobinuria (PNH) is a disease derived from an acquired mutation of the phosphatidylinositol glycan class A (PIGA) gene in the hematopoietic stem cells. In some cases with aplastic anemia (AA) or low-risk types of myelodysplastic syndromes (MDS), it is known that glycosylphosphatidylinositol-anchored protein deficient (PNH-type) cells can be often detected at low frequencies (about 0.01%) through the high-resolution flow cytometry-based methFod. Because these patient groups are reported to have a good reactivity towards immunosuppressive therapies as opposed to the other patient groups lacking PNH-type cells, detection of these cells is potentially useful in determining a treatment plan for the patients with bone marrow failure syndromes. To confirm this preliminary information, a large cohort study is needed. Method A nationwide multi-center prospective observational study (OPTIMA) was started in July 2011 to determine the prevalence of patients with bone marrow failure syndromes who carried PNH-type cells and to clarify the significance of the presence and quantitative changes of these cells with regard to the clinical features. Each of the six laboratories in different universities was assigned as a regional analyzing center. The percentage of PNH-type cells was measured by the high-resolution flow cytometry-based method, originally established in Kanazawa University. At six individual laboratories, cross validations were conducted to minimize the inter-laboratory variations in the detection sensitivities, cutoff values, etc. The liquid FLAER method (≥0.003%) and cocktail method (≥0.005%) with CD55 and CD59 antibodies were used for the detection of PNH-type granulocytes and erythrocytes, respectively. Results Quality of the assay was managed in all the laboratories by periodic blind validation tests using standard blood samples containing 0.01% PNH-type cells. Until July 2013, 1214 cases were examined; 461 (38%) were positive for PNH-type cells and 141 (11.6%) had ≥1% PNH-type cells. Out of 1214, 783 patients were diagnosed to have AA (n=386), MDS (n=341), and PNH (n=56) based on the case report forms. PNH-type cells were detected in 56.2%, 19.1% and 100% of patients with AA, MDS and PNH, respectively. In a half of patients having ≥1% PNH-type cells, lactate dehydrogenase levels exceeded the ≥1.5×upper limits of normal. Conclusion Our study has successfully established the high-resolution flow cytometry-based method that enables the detection of minimal PNH-type cells (below 0.01%). Also, by implementing a uniform protocol to six individual laboratories across the country, a system has been established for the patients to undergo the detection test with equal accuracy in all of these laboratories. Further accumulation of case studies and prolonged observations are required to determine the clinical significance of the minimal PNH-type cells, especially in terms of its relation to response to immunosuppressive therapy. Disclosures: Obara: Alexion Pharmaceuticals: Honoraria. Chiba:Alexion Pharmaceuticals, In: Research Funding. Sugimori:Alexion Pharma: Honoraria. Noji:Alexion Pharmaceuticals: Honoraria. Yonemura:Alexion Pharma: Research Funding. Ando:Alexion Pharma: Research Funding. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Shichishima:Alexion Pharmaceuticals, In: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Ninomiya:Alexion Pharma: Honoraria. Nishimura:Alexion Pharma: Research Funding, Speakers Bureau. Kanakura:Alexion Pharmaceuticals: Research Funding, Speakers Bureau. Nakao:Alexion Pharmaceuticals, In: Research Funding.

An Interim 4-Year Analysis of Prospective Multicenter Observational Study of PNH-Type Cells in Japanese Patients with Bone Marrow Failure Syndrome (OPTIMA study)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4781-4781
Author(s):  
Yukari Shirasugi ◽  
Hideyoshi Noji ◽  
Tsutomu Shichishima ◽  
Chiharu Sugimori ◽  
Naoshi Obara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
High Resolution ◽  
Clinical Significance ◽  
Interim Analysis ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
High Response Rate ◽  
Bone Marrow Failure Syndrome ◽  
Hematopoietic Stem

Abstract Background: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder caused by the clonal expansion of the phosphatidylinositol glycan class A (PIGA) mutant hematopoietic stem cells, which results in a deficiency in glycosylphosphatidylinositol-anchored proteins (GPI-APs). Through the high-resolution flow cytometry-based method, GPI-APs deficient blood cells (i.e., PNH-type cells) are often detectable in patients with bone marrow failure syndromes (BMF), such as aplastic anemia (AA) and low-risk types of myelodysplastic syndromes (MDS). Sugimori et al reported that when BMF patients possessed increased PNH-type cells, the patients had a good prognosis and showed a high response rate to immunosuppressive therapies, suggesting that detection of PNH-type cells is potentially useful in determining an optimal treatment for BMF patients. Thus, we conducted a nationwide, multi-center prospective observational investigation, the OPTIMA study. Methods: From July 2011, we start recruiting the patients with BMF that were diagnosed at various hematology clinics throughout Japan to the OPTIMA study. The primary endpoint of this study was to determine the prevalence of BMF patients with PNH-type cells and to clarify the clinical significance of the presence and quantitative changes of these cells with regard to the clinical features. Six different university laboratories were assigned as regional analyzing centers. The percentage of PNH-type cells was measured by the high-resolution flow cytometry-based method, originally established in Kanazawa University. At six individual laboratories, cross validations were conducted twice a year to minimize the inter-laboratory variations in the detection sensitivities, cutoff values, etc. The liquid FLAER method (≥0.003%) and cocktail method (≥0.005%) with CD55 and CD59 antibodies were used for the detection of PNH-type granulocytes and erythrocytes, respectively. Results Between July 2011 and May 2015, a total of 2328 patients were enrolled to this study, and we analyzed 2212 patients who were eligible for the interim analysis. Of these patients, 74 (3.3%) were diagnosed with PNH, 690 (31.2%) with AA, 592 (26.8%) with MDS, and 856 (38.7%) with undiagnosed BMF. Using high-resolution flow cytometry-based method, 755 (34.1%; 95.9% in PNH, 52.8% in AA, 18.2% in MDS, and 24.8% in undiagnosed BMT) patients had ≥0.005% PNH-type erythrocytes and ≥0.003% PNH-type granulocytes. Overall, 181(8.2%) patients had ≥1% of both PNH-type erythrocytes and granulocytes; the prevalence in each disease subset was 68/74 (91.9%) in PNH, 67/690 (9.7%) in AA, 22/592 (3.7%) in MDS, and 24/856 (2.8%) in undiagnosed BMF. Regarding FAB and WHO classifications of MDS subtype, no patients with RARS (0/22), RAEB-1 (0/37) or RAEB-2 (0/23) had PNH-type cells. In contrast, 20.4% (56/275) patients with RCMD, 18.3% (26/153) patients with RCUD and 50% (2/4) patients with del (5q) MDS possessed increased PNH-type cells. Blood samples from 75 (65 with and 10 without PNH-type cells) patients were analyzed three years after the first examination. Of 65 PNH (+) patients, PNH-type cells disappeared in 4 (6.2%), while the percentage remained stable in 61 (93.8%). All of the 10 PNH (-) at the enrollment were also negative for PNH-type cells in 3 years. Conclusions: A high-resolution flow cytometry-based method that enables the detection of minimal PNH-type cells below 0.01% was successfully transferred from Kanazawa University to other laboratories in Japan. Our interim analysis confirmed previous findings that PNH-type cells were detectable in patients with 52.8% of AA and 18.2% of MDS patients. Regarding FAB and WHO classifications of MDS subtype, PNH-type cells were not detected in any of MDS RARS, RAEB-1 or RAEB-2 patients. Further analysis are required to determine the clinical significance of the minimal level of PNH-type cells as well as chronological changes in the PNH-type cell percentage, especially in terms of their relation to response to immunosuppressive therapy. Disclosures Ninomiya: Alexion Pharmaceuticals: Honoraria. Ando:Eisai Co., Ltd.: Honoraria, Research Funding. Yonemura:1. Chugai Pharma, 2. Alexion Pharma, 3. Japan Blood Products Organization, 4. OHARA Pharma: Research Funding. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Ueda:Alexion Pharma: Research Funding. Nishimura:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

scholarly journals Disease Modeling and Phenotype Rescue Using Inducible Pluripotent Stem Cells from Patients with Diamond-Blackfan Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2496-2496
Author(s):  
Senthil Velan Bhoopalan ◽  
Min-Joon Han ◽  
Steven Ellis ◽  
Harry Lesmana ◽  
Jeremie H. Estepp ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Wild Type ◽  
Dna Breaks ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Bone Marrow Failure Syndromes

Diamond-Blackfan anemia (DBA) is a congenital ribosomopathy and bone marrow failure syndrome manifesting typically in infancy with erythroid hypoplasia. Approximately half of affected individuals also have developmental anomalies. Over time, additional cytopenias can develop, including reduced hematopoietic stem and progenitor cells (HSPC). Heterozygous loss-of-function mutations in over 20 ribosomal protein (RP) genes cause approximately 70% of DBA cases, although only 7 genes (RPS19, RPL5, RPS26, RPL11, RPL35a, RPS24 and RPS7) account for over 90% of patients with a known DBA genotype. Medical therapies including steroids, chronic transfusions are partially effective but have considerable side effects. Hematopoietic stem cell transplantation (HSCT) from matched related or unrelated donors is curative with recently reported good outcomes, although many patients lack a suitable donor and/or have serious treatment-related comorbidities that increase HSCT-related toxicities. Case reports of spontaneous genetic reversion in DBA suggest that RP gene-corrected HSPC have competitive advantage over RP-deficient cells, thus providing the rationale for gene therapy as a feasible therapeutic approach. Induced pluripotent stem cell (iPSC) technology provides a robust model of human disease and can recapitulate hematopoietic defects encountered in bone marrow failure syndromes. The goals of this study was to establish a culture system from patient-derived iPSCs that can recapitulate key aspects of DBA pathophysiology and provide a preclinical model for gene manipulation to correct the abnormal phenotype (Figure 1). We developed iPSCs from individuals with DBA who were enrolled on INSIGHT (NCT02720679), an IRB-approved, prospective study that includes biobanking of peripheral blood mononuclear cells (PBMNC) from patients with bone marrow failure syndromes. We first reprogrammed these DBA PBMNC into iPSCs using non-integrating Sendai virus to establish lines with pathogenic mutations in RPS19 (c.191>T, p.Leu64Pro), RPS19 (c.184C>T, p.Arg62Trp), RPL11 (c.61dupT, p.Cys21Leufs*13), and a variant of uncertain significance (VUS) in RPS7 (c.277_279delGTC, pVal93del). Undifferentiated iPSC lines exhibited abnormal ribosomal biogenesis revealed by polysome profiling and pre-rRNA analysis. Upon in vitrodifferentiation to hematopoietic lineages, the mutant iPSCs recapitulated DBA phenotypes with reduced CD34+ HSPCs, near absence of erythroid colonies (BFU-E and CFU-E) colonies and failure to produce erythroid cells in liquid culture. We used two methods to correct single nucleotide RP mutations in DBA iPSCs (Figure 1): i) CRISPR/Cas9-mediated homology-directed repair, and ii) base-editing, which utilizes catalytically inactive Cas9 fused to a deaminase that interconverts nucleotides directly in the absence of double-stranded DNA breaks. Corrected "isogenic" lines showed phenotype similar to wild type controls, with restored erythroid differentiation, and normal polysome maturation and pre-rRNA ratios. Because some patients carry large intragenic or whole RP gene deletions that are not amenable to gene correction, we also explored the feasibility of gene rescue by inserting a wild type copy of the defective gene (Figure 1). Using zinc-finger nuclease (ZFN), we inserted wild type RP cDNA constructs into the "safe harbor" AAVS1 locus on chromosome 19, thereby rescuing abnormal phenotypes of patient-derived iPSC lines with RPS19(p.Arg62Trp) and RPL11(p.Cys21Leufs*34) mutations. Additionally, we explored lentiviral gene delivery as an alternative method for RP gene replacement. We compared different promoters including MND, PGK and EF1a and found that the latter was most effective at rescuing RP gene expression in iPSC cells. Transduction of lentiviral vectors with wild type RPS19or RPL11fused to the EF1a promoter into three iPSC lines with RPS19or RPL11mutations resulted in stable transgene expression of RPS19or RPL11genes and phenotypic rescue. This study supports the feasibility of establishing iPSCs from DBA subjects with different genotypes. These iPSC lines provide a useful resource for numerous studies of DBA including preclinical approaches to gene therapy, evaluating the pathogenicity of RP gene variants of unknown significance and examining the pathophysiology of RP haploinsufficiency. Disclosures Estepp: Esperion: Consultancy; Forma Therapeutics: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Eli Lilly and Co: Research Funding; Daiichi Sankyo: Consultancy. Weiss:GlaxoSmithKline: Consultancy; Rubius Inc.: Consultancy; Cellarity Inc.: Consultancy; Beam Therapeutics: Consultancy; Esperion: Consultancy.

Characteristics of Laboratory Screening Suggest That Paroxysmal Nocturnal Hemoglobinuria (PNH) Is Perceived Primarily as a Bone Marrow Failure Syndrome Rather Than as a Hemolytic Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4107-4107
Author(s):  
Hwee Yong Lim ◽  
Marjorie Farley ◽  
Carl Wittwer ◽  
Charles Parker
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Small Populations ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Bone Marrow Failure Syndromes

Abstract PNH is a hematopoietic stem cell disorder in which the predominant clinical manifestations are hemolysis, bone marrow failure and thrombophilia. PNH arises as a result of somatic mutation of PIGA, an X-linked gene required for synthesis of the glycosyl phosphatidylinositol (GPI) moiety that anchors some proteins to the cell surface; and consequently, progeny of affected stem cells are deficient in all GPI-anchored proteins (GPI-APs). The hemolysis of PNH is the result of deficiency of CD55 and CD59, GPI-APs that normally inhibit complement activation on the red cell surface, but the relationship between GPI-AP deficiency and the bone marrow failure and thrombophilia of PNH are enigmatic. The peripheral blood of patients with PNH is a mosaic of normal and abnormal cells, and the degree of mosaicism varies greatly among patients. By using fluorescently labeled antibodies, GPI-AP deficient cells (GPI-AP−) can be distinguished form GPI-AP sufficient cells (GPI-AP+) cells by flow cytometric analysis, allowing quantitation of mosaicism. Flow cytometry has been used diagnostically for more than a decade, and technical modifications have improved resolution so that very small populations of GPI-AP− peripheral blood cells can be accurately detected. The purpose of these studies was to generate insights into how PNH is perceived in the community by analyzing the results of a commercially available screening assay using data from a national clinical diagnostic laboratory (ARUP Laboratories, Salt Lake City, UT). The flow cytometric method used in these studies is a modification of the high-resolution two-color assay of Sugimori and colleagues (Blood2006, 107:1308–1314). Clients are given the choice of testing for PNH by analyzing peripheral blood RBCs or PMNs (or both). The acidified serum test (Ham’s test) and the sucrose lysis test (sugar water test) are also available for screening for PNH. For flow cytometric analysis of RBCs, a value of ≥0.005% GPI-AP− cells is considered abnormal, while for PMNs ≥0.003% is abnormal. From January 1, 2008 to June 30, 2008, 1,113 RBC assays and 133 PMN assays were performed. An abnormally large population of GPI-AP− RBCs was identified in 55 cases (5%). The percentage of GPI-AP− RBC ranged from 0.009–69.603% with a median of 1.405%. Twenty-two cases (40%) had >5% GPI-AP− RBCs, while 18 cases (33%) had >10% GPI-AP− RBCs. Of the 133 PMN assays performed, 15 (11%) were abnormal. The range of GPI-AP− PMNs was 0.004–97.727% with a median of 18.327 %. Eight samples (53%) had >10% GPI-AP− PMNs. During the 1-year period from July 1, 2007-June 30, 2008 the acidified serum lysis test (Ham’s test) was performed on 212 samples while the sucrose lysis test was performed on 148 samples. These studies suggest that screening for PNH is common (~43 RBC assays/week compared to 44 assays/week for flow cytometric screening of peripheral blood for lymphoproliferative disorders and leukemia), but the vast majority of samples tested show normal expression of GPI-APs. That so many of the test samples are negative, and that the median for abnormal RBC samples is ~1.5 % GPI-AP− cells, suggest that most of the screening is done because of the association of PNH with bone marrow failure syndromes rather than because of evidence of intravascular hemolysis. These studies underscore the need to understand the pathophysiological basis and clinical implications of small populations of GPI-AP deficient cells in patients with bone marrow failure syndromes. Nonetheless, 18 cases with >10% GPI-AP− RBCs were detected during the 6 months of observation, indicating that the prevalence of classic PNH in the US is substantial. That PNH clone size is best determined by analysis of GPI-AP expression on PMNs does not appear to be widely appreciated in the community as the PMN assays is requested 12% as often as the RBC assay. Flow cytometry has largely, but not completely, replaced Ham’s test and the sucrose lysis test as screening assays for PNH.

The Interim Analysis of the Optima (observation of GPI-anchored protein-deficient [PNH-type]) Cells in Japanese Patients with Bone Marrow Failure Syndrome and in Those Suspected of Having PNH) Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1595-1595
Author(s):  
Hideyoshi Noji ◽  
Tsutomu Shichishima ◽  
Chiharu Sugimori ◽  
Naoshi Obara ◽  
Kohei Hosokawa ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Japanese Patients ◽  
Unknown Etiology ◽  
Normal Range ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Validation Tests

Abstract [Background] Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic　stem cell disorder derived from an acquired mutation of the phosphatidylinositol glycan class A (PIGA) gene in the hematopoietic stem cells which results in the expansion of glycosylphospatidylinositol-anchored protein (GPI-AP)-deficient (PNH-type) hematopoietic cells. PNH-type blood cells are also observed in patients with bone marrow failure (BMF). PNH is conventionally diagnosed when patients have >1% of GPI-AP-deficient erythrocytes and granulocytes determined by flow cytometry. Analyses with high resolution flow cytometry by several different groups have shown that patients with aplastic anemia (AA) or low-risk types of myelodysplastic syndromes (MDS) have small percentages of PNH-type erythrocytes, granulocytes, and/or other lineages of blood cells and that these patients respond better to immunosuppressive therapies compared with BMF patients lacking PNH-type cells. In order to determine the prevalence and clinical significance of PNH-type cells in BMF patients, we conducted a nationwide multi-center prospective observational investigation, the OPTIMA study. [Methods] From July 2011, Japanese patients with PNH, AA, MDS or BMF of uncertain origin have been prospectively enrolled into the study. Six laboratories in different cities in Japan were assigned as regional analyzing centers and measured the percentages of PNH-type cells in the study population as well as collecting clinical and laboratory data. The high-resolution flow cytometry assessments used a liquid fluorescein-labeled proaerolysin (FLAER) method and a cocktail method with anti-CD55 and anti-CD59 antibodies for the detection of PNH-type granulocytes and erythrocytes, respectively. Periodic blind cross validation tests using a standard blood sample containing 0.01% PNH-type cells and a normal control were conducted to minimize inter-laboratory variations. From analysis of 68 healthy individuals >0.003% of PNH-type granulocytes and >0.005% of PNH-type erythrocytes were considered to be abnormal (Sugimori et al, Blood, 2006). [Results] As of May 2014, flow cytometry data have been collected from 1685 patients and are included in this interim analysis. Of these patients, 65 (4%) were diagnosed with PNH, 523 (31%) with AA, 459 (27%) with MDS, and 638 (38%) with BMF of unknown etiology. Overall, 154 (9%) patients had ≥1% of both PNH-type erythrocytes and granulocytes: 63 (97%) patients with PNH; 57 (11%) with AA; 18 (4%) with MDS; and 16 (3%) with BMF of unknown etiology. In total, 545 (32%) patients had ≥0.005% PNH-type erythrocytes and ≥0.003% PNH-type granulocytes. These consisted of the followings; all 65 (100%) patients with PNH; 264 (51%) with AA; 76 (17%) with MDS; and 140 (22%) with BMF of unknown origin. Lactate dehydrogenase (LDH) levels ≥1.5 × upper limit of normal range were seen in 14/329 (4%) patients with 0.005-1% PNH-type erythrocytes, 23/62 (37%) patients with 1-10% PNH-type erythrocytes, and 69/71 (97%) patients with ≥10% PNH-type erythrocytes. Periodic blind validation tests revealed that inter-laboratory differences in absolute measurements of PNH-type cells were always within 0.02%. [Conclusion] A high-resolution flow cytometry-based method, based on the Kanazawa method, that enables the detection of very low percentages of PNH-type cells was successfully transferred to 6 laboratories across Japan. Our results demonstrated that the proportion of patients identified as having small percentages of PNH-type cells differed depending on diagnosis (PNH, AA, MDS, or unknown BMF) and that elevated LDH levels (>1.5 x upper limits of normal range) were more frequently associated with higher percentages of PNH-type erythrocytes. Our findings suggest that the high resolution method is helpful as a diagnostic tool in BMF syndromes, including AA, MDS, and PNH, and may prove useful in understanding the pathophysiology of these disorders. Disclosures Noji: Alexion Pharma: Honoraria. Shichishima:Alexion Pharmaceuticals, Inc; and Medical Review Company: Honoraria, Research Funding. Obara:Alexion Pharma: Research Funding. Chiba:Alexion Pharma: Research Funding. Ando:Alexion Pharma: Research Funding. Hayashi:Alexion Pharma: Research Funding. Yonemura:Alexion Pharma: Research Funding. Kawaguchi:Alexion Pharma: Honoraria. Ninomiya:Alexion Pharma: Honoraria, Research Funding. Nishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

scholarly journals Secondary CNL after SAA reveals insights in leukemic transformation of bone marrow failure syndromes

2020 ◽  
Vol 4 (21) ◽  
pp. 5540-5546
Author(s):  
Laurent Schmied ◽  
Patricia A. Olofsen ◽  
Pontus Lundberg ◽  
Alexandar Tzankov ◽  
Martina Kleber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Failure ◽  
Driver Mutations ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Proinflammatory Responses ◽  
Stem And Progenitor Cells

Abstract Acquired aplastic anemia and severe congenital neutropenia (SCN) are bone marrow (BM) failure syndromes of different origin, however, they share a common risk for secondary leukemic transformation. Here, we present a patient with severe aplastic anemia (SAA) evolving to secondary chronic neutrophilic leukemia (CNL; SAA-CNL). We show that SAA-CNL shares multiple somatic driver mutations in CSF3R, RUNX1, and EZH2/SUZ12 with cases of SCN that transformed to myelodysplastic syndrome or acute myeloid leukemia (AML). This molecular connection between SAA-CNL and SCN progressing to AML (SCN-AML) prompted us to perform a comparative transcriptome analysis on nonleukemic CD34high hematopoietic stem and progenitor cells, which showed transcriptional profiles that resemble indicative of interferon-driven proinflammatory responses. These findings provide further insights in the mechanisms underlying leukemic transformation in BM failure syndromes.

Investigation of TCR-Vβ CDR3 Spectrotypes in CD4 and CD8 Lymphocytes from Paroxysmal Nocturnal Hemoglobinuria (PNH) Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2827-2827
Author(s):  
Akiko Nakamura ◽  
Tsutomu Shichishima ◽  
Hideyoshi Noji ◽  
Kazuhiko Ikeda ◽  
Yukio Maruyama
Keyword(s):  
Bone Marrow ◽  
Healthy Volunteer ◽  
Bone Marrow Failure ◽  
Interferon Γ ◽  
Relative Increase ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Abnormal Cells ◽  
Ifn Γ ◽  
Cd4 Lymphocytes

Abstract PNH is one disorder of bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome. It is considered that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) and interferon-γ (IFN-γ) contribute to hypoplastic bone marrow of these disorders. In addition, PNH is an acquired clonal disorder of the hematopoietic stem cell. Recently, it has been reported that analysis of T cell-antigen receptor (TCR)-Vβ repertoires, especially TCR-Vβ CDR3 (complementarity- determining region 3) spectrotypes, is an effective tool to study immunologic mechanisms by CTLs in pathophysiology of PNH (Karadimitris et al, Blood, 2000; Kook et al, Blood, 2002; Risitano et al, Blood, 2002). In the present study, we investigated 21 kinds of TCR-Vβ repertoires by flow cytometry in CD4 and CD8 lymphocytes from 5 PNH patients and a healthy volunteer and the TCR-Vβ CDR3 spectrotypes using polymerase chain reaction assay in CD4 and CD8 lymphocytes from 3 of 5 PNH patients and the control. We also quantitated intracellular IFN-γ in CD4 and CD8 lymphocytes from 5 PNH patients and the control according to the method by Sloand et al (Blood, 2002). We found no specific TCR-Vβ repertoires in CD4 and CD8 lymphocytes from PNH patients compared with the control. The TCR-Vβ repertoires with relative increase of CD4 or CD8 lymphocytes (over 10 of ratio of the proportion of each TCR-Vβ repertoire in a PNH patient/the proportion of the same TCR-Vβ repertoire in a healthy volunteer) were 13.6 or 4 and 22 in Case 1, 3 and 11 or 1 in Case 2, 3 and 13.6 or 3 in Case 3, 5.3 and 7.2 or 2, 3, 7, and 18 in Case 4, and 4, 5.2, 13.6, 16, and 23 or 1 and 14 in Case 5, respectively. TCR-Vβ CDR3 spectrotyping showed that in CD4 lymphocytes most CDR3 patterns were chiefly polyclonal, except for one oligoclonal (Case 1) and one monoclonal (Case 3) patterns of TCR-Vβ25; in CD8 lymphocytes most CDR3 consisted of polyclonal, oligoclonal, and/or monoclonal patterns, suggesting the possibility that CD8 lymphocytes recognize much more antigens of abnormal cells, probably including PNH clones, than CD4 lymphocytes. Unfortunately, we found the same patterns as described above in CD8 lymphocytes from the control, although CD4 lymphocytes from the control presented only polyclonal pattern of CDR3. Quantitative analyses of IFN-γ showed that index values of IFN-γ in CD4 and CD8 lymphocytes from PNH patients were higher than those from the control. However, we did not find any significant correlations between the spectrotypes of TCR-Vβ CDR3 and the index values of IFN-γ in PNH patients, suggesting that TCR-Vβ repertoires with monoclonal and oligoclonal CDR3 patterns do not necessarily produce much IFN-γ. In conclusion, our findings suggest that TCR-Vβ CDR3 spectrotyping is more effective tool to resolve some immune mechanisms of pathophysiology in PNH, especially by auto-reactive CTLs.

Copy Number Variants Underlying Inherited Bone Marrow Failure Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2416-2416
Author(s):  
Nicolas Waespe ◽  
Santhosh Dhanraj ◽  
Manju Wahala ◽  
Tom Enbar ◽  
Bozana Zlateska ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Short Stature ◽  
Copy Number ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Organ System ◽  
Severe Phenotype ◽  
Nucleotide Level ◽  
Bone Marrow Failure Syndromes ◽  
Genome Wide

Abstract Background. Inherited bone marrow failure syndromes (IBMFSs) comprise a genetically heterogeneous group of diseases with hematopoietic failure and varying degrees of physical malformations. The diagnosis of an IBMFS and categorizing the specific syndrome critically impact on clinical care; however, these are commonly challenging and rely on genetic testing. Since over 80 genes have been associated with IBMFSs and might be affected by different types of DNA aberrations, the best strategy to establish a diagnosis in a timely and cost effective manner is unknown. The aims of this study were to evaluate the role of genome-wide copy number variant (CNV) analysis in unraveling causal genetic alterations in IBMFS patients with unknown genotype and determine whether correlation exists between large CNVs and more severe phenotype. Methods. Patients from the Canadian Inherited Marrow Failure Registry (CIMFR) who were genetically investigated were included in this analysis. Genetic and clinical data were extracted and analyzed. Mann-Whitney test and Fisher's exact test were used to assess statistical significance. Results. Among 328 patients from the CIMFR who underwent molecular investigation, a causal genotype was identified in 185 cases (56.4%). 69 patients had genome-wide CNV analysis by SNP/CGH arrays, among which ten (14.5%) had positive results. In four out of ten cases who were genotyped by SNP/CGH array, genome-wide CNV analysis was critical for establishing the diagnosis. Among 308 patients who were tested for nucleotide-level mutations by either targeted gene analysis or next generation sequencing panels, casual mutations were found in 169 (54.9%). Three patients had compound heterozygosity for a CNV and nucleotide-level mutation. To determine whether large deletions are correlated with more severe phenotype we included nine additional patients with causal CNVs whose genotype was identified by MLPA (n=1), targeted FISH (n=1), DNA-qPCR analysis (n=1), Southern blotting (n=1) or metaphase cytogenetics (n=5). The causal CNVs among patients in our cohort ranged from 0.02 to 145.5 Mb in size. The most common disease associated with causal CNVs was Diamond-Blackfan anemia (four patients). Patients with CNVs tended to have significantly more non-hematological organ system involvement (p=0.03), developmental delay (mean=56% vs. 28%, p=0.03) and short stature (mean=67% vs. 40%, p=0.04) than patients with nucleotide-level mutations. The difference remained significant when we compared all patients with mutations that are predicted to result in truncation or lack of protein from the respective allele (large CNV, nonsense, and indel/ frameshift) to patients with mutations that are predicted to be hypomorphic or affect function (splicing, indel/ inframe and missense). There was no correlation between CNVs and the severity of the hematological disease. Conclusions. Most patients with IBMFSs have nucleotide-level mutations. However, a significant proportion of patients without such mutations have large CNVs that are not efficiently detected by current nucleotide-level testing methods. Therefore, genome-wide CNV analysis should be considered in IBMFS cases, where nucleotide-level sequencing does not reveal the causal mutation. Patients with IBMFSs and large CNVs had more non-hematological organ system involvement, a higher prevalence of developmental delay and short stature. This might be related to an additional impact of the CNVs on other genes close to the affected IBMFS gene or the severe damaging effect of the CNVs. Disclosures Lipton: Teva: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

Premature Senescence in Hematopoietic Stem and Progenitor Cells in Ribosomopathy-Associated Bone Marrow Failure Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 298-298
Author(s):  
Hengjun Chao ◽  
Johnson M. Liu
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Premature Senescence ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Bone Marrow Failure Syndromes ◽  
P53 Activation ◽  
Adult Bone

Abstract Introduction: Aged hematopoietic stem cells (HSCs) are known to functionally decline and are prone to development of myeloid malignancies. Recent work has highlighted the twin roles of replication stress and decreased ribosome biogenesis as drivers for the accumulation of DNA damage and senescence. Certain bone marrow failure syndromes, including Shwachman-Diamond syndrome (SDS), Diamond-Blackfan anemia (DBA), and the acquired 5q- syndrome, are characterized by defects in ribosome biogenesis. Furthermore, recent work has suggested a role for p53 activation, through the 5S ribonucleoprotein particle (RNP), in driving cells to senescence following perturbation of ribosome biogenesis. Methods and Results: Here, we have used multiplexing flow cytometry protocols to define, enumerate, and characterize hematopoietic cells of distinct differentiation stages and lineages in 2 DBA cord bloods and 4 adult bone marrows (2 SDS, 1 DBA, and 1 patient with a diminutive somatic deletion of 5q: ages 27, 32, 40, and 30, respectively), as compared with 4 normal cord bloods and 6 normal adult bone marrows. We included a patient with bona fide MDS (diminutive somatic deletion of 5q including RPS14 in a young adult) to compare with the SDS and DBA patients, who do not meet criteria for MDS. Our preliminary results revealed significant defects in the primitive HSC and multipotent progenitor (MPP) compartments in both DBA and SDS. Specifically, we found in DBA and SDS bone marrow and cord blood samples (compared to normal controls): significantly decreased numbers of primitive HSCs (Lin-CD34+CD133+CD38-CD45RA-CD49f+CD90+) and MPPs (Lin-CD34+CD133+CD38-CD45RA-CD49f-CD90-); increased levels of apoptosis and dysregulated proliferation; and G0-1/S cell cycle arrest. We also found significant increases in senescence-associated β-galactosidase staining and G0-1/S cell cycle arrest in Lin-CD34+ and Lin-CD34+CD38-CD133+ subpopulations in all 4 adult patient bone marrows, as compared with normal adult bone marrows processed in identical fashion [see Fig. 1 for representative data from Lin-CD34+CD133+ hematopoietic progenitor cells (HPCs) from one SDS patient]. Foci of the phosphorylated form of the variant histone H2AX (γH2AX) mark DNA damage, and γH2AX staining was similarly increased in comparison to controls (Fig. 1). The mechanism whereby disturbed ribosome biogenesis induces senescence has been suggested as involving 5S RNP-mediated p53 activation. However, our experiments did not demonstrate increased levels of p53 in the SDS patient marrows, as assessed by intracellular staining. Levels of p16, a well known marker of senescence, were markedly increased in the SDS patient samples, when compared to controls. Finally, in the 2 DBA cord bloods analyzed, there was increased senescence-associated β-galactosidase staining but to a lesser degree than in the adult bone marrow samples (as might be expected with temporal progression). Discussion: Taken together, our data suggest that ribosomopathies (which often present in childhood) are disorders of premature senescence. Consequent DNA damage accumulation and decreased repair and compensation may account for the development of MDS and acute myeloid leukemia, disorders seen in young ribosomopathy patients that ordinarily are rare in the general pediatric and young adult population. Disclosures No relevant conflicts of interest to declare.

Utility of Telomere in Diagnosis of Bone Marrow Failure Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4793-4793
Author(s):  
Hasan Ahmed Abdel-ghaffar ◽  
Hosam Zaghloul ◽  
Ahmed El-Waseef ◽  
Mohamed El-Naggar ◽  
Mohamed Mabed ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Quantitative Polymerase Chain Reaction ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Relative Telomere Length ◽  
Bone Marrow Failure Syndromes ◽  
Significant Difference

Abstract Background and aim of the work: Bone marrow failure syndromes (BMFS) includes inherited and acquired conditions. Inherited bone marrow failure includes a number of syndromes; with Fanconi anemia (FA) being the most common one of them. Telomeres are eroded with cell division, but in hematopoietic stem cell, maintenance of their length is mediated by telomerase. Short telomeres can result in instability of cell function where diseases occur. Bone Marrow Failure might be developed due to low telomerase activity or short telomeres. Our study is aiming to evaluate the utility of Real Time Quantitative-Polymerase Chain Reaction (RT-qPCR) in measuring the relative telomere length and its significance in diagnosis and prognosis of patients with BMFS. Materials and methods: The study includes 3 groups: A group of congenital BMF (29 patients), a group of acquired BMF (10 patients) and a third control group (15 cases). The relative telomere length is evaluated for them using RT-qPCR. Results: We have found that there is a significant difference in relative telomere length between congenital group and controls (p=0.001), also a significant difference between acquired group and controls (p= 0.029). However, there is no significant difference between congenital and acquired groups (p= 0.479). There is no significant correlation between the telomere length and the overall survival or prognosis of the patients of BMFS. Conclusion: We conclude that the telomere length is significantly altered in patients with BMFS whether being congenital or acquired compared to the control group. Disclosures No relevant conflicts of interest to declare.

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