scholarly journals PAROKSIZMALNA NOČNA HEMOGLOBINURIJA - PRIPOROČILA ZA ODKRIVANJE BOLEZNI TER PREGLED POPULACIJE BOLNIKOV V SLOVENIJI

2016 ◽  
Vol 85 (7-8) ◽  
Author(s):  
Jasmina Hauptman ◽  
Darja Žontar ◽  
Irena Preložnik Zupan
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Hemolytic Anemia ◽  
Bone Marrow Failure ◽  
Poor Quality ◽  
Medical Centre ◽  
Specific Flow ◽  
Dark Urine ◽  
Pnh Clone

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease associated with hemolytic anemia, bone marrow failure, thrombosis, and, frequently, poor quality of life. It is caused by defects in the membrane of blood cells, where there is a lack of protein on the cell surface, which inhibits complement activation. We wanted to know the recognition of the disease in Slovenia and the incidence. We prepared the recommendations for discovering of the disease. Patients and methods: We collected data of 68 patients with prospective analysis of one – year period from 1.10.2013 to 30.9.2014 whose blood was sent to the laboratory of immunology and cytology because of suspected presence of PNH clone. The analysis of peripheral blood was performed with multiparametric high specific flow cytometry in a specialized laboratory KO of Haematology University Medical Centre in Ljubljana. Results: PNH clone was positive in 13/68 (19%) patients, 55/68 (81%) patients had a negative PNH clone, most positive samples were sent from the University Medical Centre Ljubljana (7/13). 4/13 positive patients were newly discovered. In average the incidence through 10-years was 1,3 / 1,000,000 population/year. The most common cause of PNH patient referral to a specialist hematologist was unexplained cytopenia - 12/13 (92.3%), the most common symptoms were fatigue and dyspnea (100%), in 2/13 patients was present dark urine with hemoglobinuria, 2/13 patients had transient renal insufficiency. 11/13 patients with positive PNH clone had associated a bone marrow failure (aplastic anemia or myelodysplastic syndrome).  The size of PNH clone varied from patient to patient. Conclusions: Early identification of PNH is a key to effective treatment and survival of patients. We recommend determining PNH clone at Coombs negative hemolytic anemia, hemoglobinuria, an unexplained cytopenia, aplastic anemia, myelodysplastic syndrome with laboratory evidence of haemolysis and unexplained thrombosis.

Intravenous Immunoglobulin Is an Effective Treatment for LGL-Mediated Immune Cytopenias in Myelodysplastic Syndromes and Other Bone Marrow Failure Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1704-1704
Author(s):  
Francesca Schieppati ◽  
Erin P. Demakos ◽  
Odchimar Rosalie-Reissig ◽  
Shyamala C. Navada ◽  
Lewis R. Silverman
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Intravenous Immunoglobulin ◽  
Hemolytic Anemia ◽  
Bone Marrow Failure ◽  
Myeloproliferative Neoplasm ◽  
Recurrent Fever

Abstract Background: Myelodysplastic Syndrome (MDS) and Aplastic Anemia (AA) are often associated with clinical immune manifestations. An abnormal profile of the T-cell repertoire can be detected in these patients (pts) and is thought to play a role in bone marrow (BM) insufficiency. The presence of a co-existent large granular lymphocytic (LGL) clone may exacerbate cytopenias independent of the primary disease mechanism and offers another target for therapeutic intervention. Treatment for LGL proliferation is usually immunosuppressive therapy but there is no accepted standard of care. Methods: We explored the role of intravenous immunoglobulin (IVIG) as a treatment for immune-related cytopenias, i.e. Coombs negative (C-) hemolytic anemia, in a series of 12 consecutive pts with an LGL clonal proliferation documented by flow cytometry and TCR clonal rearrangements. Of the 12 cases, 9 had MDS (7 lower-risk), 1 AA with LGL liver involvement, and 1 primary myelofibrosis. One patient (pt) had suspected MDS. Overall response was assessed by MDS IWG criteria 2006. We defined a hemolysis response (HLR) as complete normalization (CR) or, a greater than 50% improvement (PR) in deviation from normal values of LDH, reticulocytes, indirect bilirubin and haptoglobin. Duration of HLR was defined as the time from onset of HLR to the time of resumption of hemolysis and loss of effect of IVIG. Results: All pts were treated with IVIG administered at a dose of 500mg/kg of IVIG once per week, in repeated cycles, with a duration ranging from 1-4 week(s) per cycle. Clinical characteristics (Table 1): M/F ratio 10/2; median age 69. Ten pts had a CD3+ T-LGL and 2 had a CD3-/CD16+/CD56+ NK-LGL circulating clone. Karyotype abnormalities were non-specific; 8 pts had 1-3+ reticulin BM fibrosis; 4 had mutations in RNA-splicing genes: SF3B1 (2); SETBP1 (1); SRSF2 (1). Ten pts were evaluable for response: 8 pts responded (ORR 80%): Hematological improvement (HI-erythroid) 8/8 (100%); a hemolysis CR (HLR-CR) occurred in 7 (87.5%) and hemolysis PR (HLR-PR) in 1 pt (12.5%). Median number of cycles, follow up, and duration of treatment were 16, 21.5 and 9.5 months (mo), respectively. The HLR-CR was durable and prolonged in 3/8 (38%) pts; 2 of these 3 pts (67%) did not require maintenance IVIG. Relapse from HLR occurred in 4, during infection or chemotherapy, but the response returned to the original level by shortening the intervals between administration of IVIG. One pt had relapsed after an initial response and then became refractory to IVIG. In follow up at month 38, 75% of pts were still responding to treatment, and 1 pt was still in remission after 46 mo. In 4 of 6 pts, corticosteroid treatment was discontinued and no longer required for chronic hemolysis, with general improvement of steroid related symptoms. Some patients had been on steroids maintenance for periods ranging from months to years. Response was more durable with continuous rather than sporadic dosing. Adverse events were not specific: 1 pt with self-limited isolated palpitations; 1 pt with hypertension not requiring intervention. Conclusions: Treatment with IVIG of immune cytopenias associated with LGL clones and BMF yields durable responses in 80% of pts. IVIG, especially at high concentrations, may enhance apoptosis, suppress proliferation of T-cells and induce immune-regulation. Given the relative rarity of LGL clones in MDS, further investigational studies will help define the role of IVIG and clarify the mechanism of action in this group of pts with MDS and BMF associated with LGL clones. Table 1. Variable Observed % Symptomatic anemia (fatigue, SOB) 9/12 75 B symptoms (recurrent fever) 2/12 16.6 Infections (bacteremia Campylobacter with migratory arthritis and dermatitis; cellulitis bacteremia S. epidermidis and osteomyelitis) 2/12 16.6 Skin lesions (leg focal ulceration and dermal fibrosis) 1/12 8.3 Splenomegaly 7/12 58.3 Hepatomegaly 2/12 16.6 Adenopathy (mediastinal) 1/12 8.3 Neuropathy 2/12 16.6 Hematologic disorders 11/12 91.6 Myelodysplastic syndrome 9/12 75 Severe aplastic anemia 1/12 8.3 Myeloproliferative neoplasm (PMF) 1/12 8.3 Lymphoproliferative neoplasm (FL+MDS) 1/12 8.3 Hemolytic anemia 11/12 91.6 Solid tumors (anal, squamous cell; breast ca) 2/12 16.6 Autoimmune disorders 7/12 58.3 ITP 3/7 42.8 Ulcerative colitis 1/7 14.3 Pernicious anemia 1/7 14.3 Systemic lupus erythematosus 1/7 14.3 Immune pancreatitis 1/7 14.3 MGUS 4/12 33.3 Disclosures Off Label Use: IVIG.

scholarly journals STAT3 mutations indicate the presence of subclinical T-cell clones in a subset of aplastic anemia and myelodysplastic syndrome patients

Blood ◽  
2013 ◽  
Vol 122 (14) ◽  
pp. 2453-2459 ◽  
Author(s):  
Andres Jerez ◽  
Michael J. Clemente ◽  
Hideki Makishima ◽  
Hanna Rajala ◽  
Ines Gómez-Seguí ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Somatic Mutation ◽  
Bone Marrow Failure ◽  
Cell Clones ◽  
Key Points ◽  
Stat3 Mutations

Key PointsSTAT3+ T cells are found not only in detected concomitant LGL-BMFs, but in cases in which an LGL expansion was not suspected. Transformation via acquisition of a somatic mutation in T cells may be a mechanism of immune, mainly hypoplastic, bone marrow failure.

Bone Marrow Histology in Patients with Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4215-4215
Author(s):  
Sandra van Bijnen ◽  
Konnie Hebeda ◽  
Petra Muus
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Plasma Cells ◽  
Bone Marrow Failure ◽  
Clone Size ◽  
Immune Mediated ◽  
Lymphoid Nodules ◽  
Pnh Clone

Abstract Abstract 4215 Introduction Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disease of the hematopoietic stem cell (HSC) resulting in a clone of hematopoietic cells deficient in glycosyl phosphatidyl inositol anchored proteins. The clinical spectrum of PNH is highly variable with classical hemolytic PNH at one end, and PNH in association with aplastic anemia (AA/PNH) or other bone marrow failure states at the other end. It is still largely unknown what is causing these highly variable clinical presentations. Immune-mediated marrow failure has been suggested to contribute to the development of a PNH clone by selective damage to normal HSC. However, in classic PNH patients with no or only mild cytopenias, a role for immune mediated marrow failure is less obvious. No series of trephine biopsies has been previously documented of patients with PNH and AA/PNH to investigate the similarities and differences in these patients. Methods We have reviewed a series of trephine biopsies of 41 PNH patients at the time the PNH clone was first detected. The histology was compared of 27 patients with aplastic anemia and a PNH clone was compared to that of 14 patients with classic PNH. Age related cellularity, the ratio between myeloid and erythroid cells (ME ratio), and the presence of inflammatory cells (mast cells, lymphoid nodules and plasma cells) were evaluated. The relation with clinical and other laboratory parameters of PNH was established. Results Classic PNH patients showed a normal or hypercellular marrow in 79% of patients, whereas all AA/PNH patients showed a hypocellular marrow. Interestingly, a decreased myelopoiesis was observed not only in AA/PNH patients but also in 93% of classic PNH patients, despite normal absolute neutrophil counts (ANC ≥ 1,5 × 109/l) in 79% of these patients. The number of megakaryocytes was decreased in 29% of classic PNH patients although thrombocytopenia (< 150 × 109/l) was only present in 14% of the patients. Median PNH granulocyte clone size was 70% (range 8-95%) in classic PNH patients, whereas in AA/PNH patients this was only 10% (range 0.5-90%). PNH clones below 5% were exclusively detected in the AA/PNH group. Clinical or laboratory evidence of hemolysis was present in all classical PNH patients and in 52% of AA/PNH patients and correlated with PNH granulocyte clone size. Bone marrow iron stores were decreased in 71% of classic PNH patients. In contrast, increased iron stores were present in 63% of AA/PNH patients, probably reflecting their transfusion history. AA/PNH patients showed increased plasma cells in 15% of patients and lymphoid nodules in 37%, versus 0% and 11% in classic PNH. Increased mast cells (>2/high power field) were three times more frequent in AA/PNH (67%) than in PNH (21%). Conclusion Classic PNH patients were characterized by a more cellular bone marrow, increased erythropoiesis, larger PNH clones and clinically by less pronounced or absent peripheral cytopenias and more overt hemolysis. Decreased myelopoiesis and/or megakaryopoiesis was observed in both AA/PNH and classic PNH patients, even in the presence of normal peripheral blood counts, suggesting a role for bone marrow failure in classic PNH as well. More prominent inflammatory infiltrates were observed in AA/PNH patients compared to classical PNH patients. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3426-3426 ◽  
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.

Mutations in the RNA Component of Telomerase, TERC, in Children with Aplastic Anemia and Myelodysplastic Syndrome: An NMDP Study.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3736-3736
Author(s):  
Joshua J. Field ◽  
Philip J. Mason ◽  
Yvonne J. Barnes ◽  
Allison A. King ◽  
Monica Bessler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Stem Cell Transplant ◽  
Bone Marrow Failure ◽  
Juvenile Myelomonocytic Leukemia ◽  
Cell Transplant ◽  
Bone Marrow Failure Syndrome ◽  
Base Pair Deletion

Abstract Mutations in TERC, the RNA component of telomerase, result in autosomal dominant dyskeratosis congenita (DC), a rare bone marrow failure syndrome. DC is clinically heterogeneous and TERC mutations have been detected in a subset of patients previously diagnosed with idiopathic aplastic anemia (AA) and myelodysplastic syndrome (MDS). Unrecognized TERC mutations are clinically relevant as patients with DC respond poorly to immunotherapy and have an increased risk of complications following conventional conditioning for stem cell transplant (SCT). We aimed to determine the frequency of TERC mutations in pediatric patients with AA and MDS who require a SCT. We obtained 315 blood or bone marrow samples from the National Donor Marrow Program Registry from children under age 18 with bone marrow failure who underwent an unrelated stem cell transplant. We screened these samples for mutations in the TERC gene using direct DNA sequencing. To exclude polymorphisms, we also screened 537 racially diverse healthy controls. The study group was composed of patients with MDS (n=151), AA (n=123), and juvenile myelomonocytic leukemia (JMML) (n=41), which may be difficult to distinguish from MDS. The mean age at the time of transplant was 9 years. We found sequence alterations in the promoter region of TERC in 2 patients. A 2 base pair deletion (-240delCT) was identified in a 4 year-old child with MDS and a 1 year-old child with JMML was found to have a point mutation (-99C→G), which was identified previously in an 18 year-old patient with paroxysmal nocturnal hemoglobinuria and is known to affect the Sp1 binding site. The pathogenicity of this mutation is unclear. In summary, our findings suggest that screening for TERC gene mutations is unlikely to diagnose occult DC in children with severe bone marrow failure who require a stem cell transplant but have no clinical features or history to suggest a familial bone marrow failure syndrome.

Comprehensive Analysis of Telomere Biology in Patients with Aplastic Anemia and Hypoplastic Myelodysplastic Syndrome: Further Evidence for a Common Mechanism

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2858-2858
Author(s):  
Anne-Sophie Bouillon ◽  
Monica S. Ferreira ◽  
Benjamin Werner ◽  
Sebastian Hummel ◽  
Jens P. Panse ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Bone Marrow Failure ◽  
Common Mechanism ◽  
Telomere Shortening ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
The Individual

Abstract Introduction: Acquired aplastic anemia (AA) is typically characterized by pancytopenia and bone marrow (BM) failure mostly due to an autoimmune attack against the hematopoietic stem cell compartment. Thus, AA patients frequently respond to immunosuppressive therapy (IST). Hypoplastic myelodysplastic syndrome (hMDS) frequently mimics clinical and morphological features of AA and proper clinical discrimination of hMDS from AA sometimes remains difficult. Interestingly, some cases of hMDS respond at least partially to IST and on the other hand, AA can clonally evolve to hMDS. Telomeres shorten with each cell division and telomere length (TL) reflects the replicative potential of somatic cells. Whereas it is proposed that TL can to some degree discriminate hereditary subtypes of bone marrow failure syndromes from classical acquired forms, the role of TL for disease pathogenesis in hMDS remains unclear. In this study, we therefore aimed to investigate accelerated TL shortening as a possible (bio-)marker to distinguish hMDS from AA. Patients and Methods: TL of BM biopsies at diagnosis of 12 patients with hMDS and 15 patients with AA treated in the University Hospital Düsseldorf were analyzed. Mean age was 45.2 years in AA patients and 65.2 years in patients with hMDS. Confocal Q-FISH protocol was used for TL measurement as published previously (Blood, 2012). TL analysis was performed in single-blinded fashion. 28 patients (range 18-80 years) with newly diagnosed M. Hodgkin without BM affection were used as controls for linear regression and calculation of age-adapted TL difference. For the analysis of the data, we made use of a recently developed mathematical model of TL distribution on the stem cell level allowing us to extrapolate mean TL shortening per year (TS/y) based on the individual TL distributions of captured BM biopsies. Results: Using the controls to adjust for age, we found that age-adapted TL was significantly shortened both in patients with AA (median: -2.96 kb, range -4.21 to 0.26, p=0.001) and patients with hMDS (median: -2.26, range -3.85 to -0.64, p=0.005). In direct comparison, telomere shortening was more accelerated in patients with AA as compared to hMDS (p=0.048). Next, we analyzed the TL shortening per year (TS/y) based on the individual telomere distribution. Calculating the extrapolated TL shortening per year (TS/y), we found significant increased TS/y in AA patients (mean±SD: 235,8 bp/y±202,9, p=0.001) and hMDS patients (120,5±41,7 bp/y, p=0.0001) compared to controls (37,5±18,9 bp/y). Interestingly, the extrapolated rate of TS/y remained stable at different ages in hMDS patients as observed in healthy controls. In contrast, TS/y in AA patients showed a strong age-dependence with a maximum of TS/y in patients younger than 30 years (R²=0.42, p=0.008). Finally, we set to test whether TS/y can be used to identify AA or hMDS patients. Using 150 bp TS/y as a cut-off (4-fold the mean of controls), we found significantly more AA patients (10/15, 66.7%) had accelerated TL shortening compared to hMDS (1/12, 8.3% p=0.005). Conclusion: We provide first retrospective data on TL in patients with hMDS using confocal Q-FISH. Age-adapted TL is significantly shorter in patients with AA compared to hMDS. Interestingly, telomere shortening per year is both significantly increased in AA as compared to hMDS patients as well as in both groups compared to controls. The rate of telomere shortening TS/y might represent a new marker in patients with bone marrow failure syndromes that allows to discriminate AA from hMDS patients pending prospective validation. Disclosures No relevant conflicts of interest to declare.

Development of Paroxysmal Nocturnal Hemoglobinuria in Children with Aplastic Anemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1499-1499 ◽  
Author(s):  
Atsushi Narita ◽  
Hideki Muramatsu ◽  
Yusuke Okuno ◽  
Yuko Sekiya ◽  
Kyogo Suzuki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Subsequent Development ◽  
Poor Quality ◽  
Targeted Sequencing ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells resulting from a somatic mutation in the PIGA gene. PNH frequently manifests in association with aplastic anemia (AA), in which PIGA mutations are believed to enable escape from the immune-mediated destruction by pathogenic T cells. Recent studies using next-generation sequencing have revealed that frequent somatic PIGA mutationsin AA patients are associated with a better response to IST and prognosis (Yoshizato et al N Engl J Med. 2015; 373: 35-47). However, clinical PNH is a progressive and life-threatening disease driven by chronic hemolysis that leads to thrombosis, renal impairment, poor quality of life, and death. Large studies in adults have reported that clinical PNH developed in 10%-25% of AA patients; however; the frequency of clinical PNH in children with AA has rarely been described. Here we aimed to elucidate the pathological link between PNH and AA in children. Methods: In total, 57 children (35 boys and 22 girls) diagnosed with acquired AA at our hospital between 1992 and 2010 were retrospectively studied. Patients who underwent hematopoietic stem cell transplantation as first-line treatment within 1 year after AA diagnosis and those with clinical PNH at AA diagnosis were excluded. Flow cytometry (FCM) was used to detect PNH CD13+/CD55−/CD59− granulocytes and PNH glycophorin A+/CD55−/CD59− red blood cells (RBCs). Clinical PNH was defined as the presence of intravascular hemolysis and ≥5% PNH granulocytes or PNH RBCs. Minor PNH clones were defined as those with >0.005% PNH granulocytes or >0.010% PNH RBCs. We performed targeted sequencing of bone marrow samples from patients with clinical PNH that were obtained at 2 time points: at AA diagnosis and after PNH development. The panel of 184 genes for targeted sequencing included most of the genes known to be mutated in inherited bone marrow failure syndromes and myeloid cancers, as well as PIGA. Results: The median patient age at AA diagnosis was 9.3 (1.2-17.8) years, and the median follow-up period was 123 (2-228) months. A total of 43 patients were screened for PNH clones by FCM after AA diagnosis, and 21 of these with minor PNH clones were identified. The median percentages of PNH granulocytes and PNH RBCs were 0.001% (0.000%-4.785%) and 0.000% (0.000%-3.829%), respectively. During follow-up, 5 patients developed clinical PNH after adolescence (15-22 years of age). The median time between AA diagnosis and PNH development was 4.9 (3.3-7.9) years. All clinical PNH patients were treated with IST for AA, and complete and partial response after 6 months were achieved in 1 and 4 patients, respectively. Gross hemoglobinuria was present in all clinical PNH patients, but thrombosis was not observed. The size of PNH clones varied greatly among patients: PNH granulocytes and PNH RBCs were 42.96% (10.04%-59.50%) and 48.87% (15.02%-90.80%), respectively. Oral cyclosporine A and intravenous eculizumab were administered to 3 and 1 patients, respectively; all patients showed sustained response as indicated by improvement in gross hemoglobinuria and normal blood counts after treatment. The remaining 1 patient underwent bone marrow transplantation from the HLA-identical mother and was alive without any complications. Overall, the 10-year probability of developing clinical PNH was 10.2% (95%CI, 3.6-20.7). Among 43 patients screened for PNH clones at AA diagnosis, the 10-year cumulative clinical PNH incidence was significantly higher in patients with minor PNH clones than in those without minor PNH clones at AA diagnosis [29% (95% CI, 10%-51%) vs. 0% (95% CI, 0%-0%); p = 0.015]. Among all clinical PNH patients, a total of 8 somatic PIGA mutations were detected (missense, 2; splice site, 2; and frameshift, 4). However, PIGA mutations were not detected at AA diagnosis even in patients who subsequently developed clinical PNH. Conclusion: In our cohort, the percentage of patients who eventually developed clinical PNH was comparable to that reported in adults in a previous study. Furthermore, the current study showed that the presence of minor PNH clones at AA diagnosis was a risk factor for the subsequent development of clinical PNH, although the clones were not detected by targeted sequencing. Thus, pediatric AA patients with PNH clones at AA diagnosis should undergo long-term periodic monitoring for potential clinical PNH development. Disclosures Kojima: SANOFI: Honoraria, Research Funding.

Increased frequency of HLA-DR2 in patients with paroxysmal nocturnal hemoglobinuria and the PNH/aplastic anemia syndrome

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3513-3519 ◽  
Author(s):  
Jaroslaw P. Maciejewski ◽  
Dean Follmann ◽  
Ryotaro Nakamura ◽  
Yogen Saunthararajah ◽  
Candido E. Rivera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Clinical Manifestations ◽  
Normal Incidence ◽  
Primary Diagnosis ◽  
Hla Alleles ◽  
Predictors Of Response ◽  
Pnh Clone

Abstract Many autoimmune diseases are associated with HLA alleles, and such a relationship also has been reported for aplastic anemia (AA). AA and paroxysmal nocturnal hemoglobinuria (PNH) are related clinically, and glycophosphoinositol (GPI)–anchored protein (AP)–deficient cells can be found in many patients with AA. The hypothesis was considered that expansion of a PNH clone may be a marker of immune-mediated disease and its association with HLA alleles was examined. The study involved patients with a primary diagnosis of AA, patients with myelodysplastic syndrome (MDS), and patients with primary PNH. Tests of proportions were used to compare allelic frequencies. For patients with a PNH clone (defined by the presence of GPI-AP–deficient granulocytes), regardless of clinical manifestations, there was a higher than normal incidence of HLA-DR2 (58% versus 28%; z = 4.05). The increased presence of HLA-DR2 was found in all frankly hemolytic PNH and in PNH associated with bone marrow failure (AA/PNH and MDS/PNH). HLA-DR2 was more frequent in AA/PNH (56%) than in AA without a PNH clone (37%;z = 3.36). Analysis of a second cohort of patients with bone marrow failure treated with immunosuppression showed that HLA-DR2 was associated with a hematologic response (50% of responders versus 34% of nonresponders; z = 2.69). Both the presence of HLA-DR2 and the PNH clone were independent predictors of response but the size of PNH clone did not correlate with improvement in blood count. The results suggest that clonal expansion of GPI-AP–deficient cells is linked to HLA and likely related to an immune mechanism.

Incidence of PNH Clones by Diagnostic Code Utilizing High Sensitivity Flow Cytometry

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1033-1033 ◽  
Author(s):  
Mayur K Movalia ◽  
Ilene c Weitz ◽  
Seah H Lim ◽  
Andrea Illingworth
Keyword(s):  
Bone Marrow ◽  
Hemolytic Anemia ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
High Sensitivity ◽  
Diagnostic Code ◽  
Follow Up Study ◽  
Follow Up Studies ◽  
Pnh Clone

Abstract Abstract 1033 Paroxysmal nocturnal hemoglobinuria (PNH) is a chronic and life-threatening hematopoietic stem cell disorder characterized by deficiency of the GPI-anchored complement inhibitory proteins CD55/59. Chronic hemolysis from this deficiency leads to serious clinical morbidities including thromboembolism, chronic kidney disease, and increased mortality. The International Clinical Cytometry Society (ICCS) recommends multiparameter high sensitivity flow cytometry (HSFC) as the method of choice for diagnosing PNH. The ICCS also provides guidance on the clinical indications for testing for PNH, including patients (pts) with bone marrow failure (BMF), unexplained cytopenias, unexplained thrombosis, hemoglobinuria and hemolysis. The aim of this study is to use HSFC with sensitivity up to 0.01% to analyze 6,897 pts who were screened for PNH clones utilizing CD235a/CD59 for RBCs, FLAER/CD24/CD15/CD45 for neutrophils and FLAER/CD14/CD64/CD45 for monocytes. We evaluated the clinical indications for PNH testing with the provided ICD-9 diagnostic (DX) codes and examined the change in PNH clone sizes among pts who had follow-up studies in 3–12 months. Based on a sensitivity of at least 0.01%, 6.1% of all pts (421/6897) were found to be PNH positive. Of those pts, 5,545 pts (80.1%) had ICD-9 DX codes provided. The distribution of PNH clone sizes in these PNH+ pts is shown in Figure 1. Aplastic anemia (AA) and hemolytic anemia comprised the most common reasons for testing. In bone marrow failure syndromes, AA pts had the highest incidence of PNH+ clones, 26.3%, followed by pts with unexplained cytopenia, 5.7%, myelodysplastic syndrome (MDS), 5.5%, and anemia (unspecified or in chronic illness), 3.6% (Table 1). The incidence of PNH+ clones for symptoms such as hemolytic anemia was 22.7%, followed by hemoglobinuria 18.9%, and unspecified hemolysis, 7.9%, unspecified iron deficiency, 2.5%, and thrombosis, 1.4%. Of the 421 PNH positive pts, 89 pts (22%) were identified as having follow-up studies in 3–12 months. These pts were categorized into PNH clone sizes of 0.01% – 0.1% (27 pts, 30%), 0.11% – 1% (7 pts, 8%), 1.1% – 10% (18 pts, 20%) and 10.1% – 100% (37 pts, 42%). Of the 64 pts who had PNH clone sizes of 0.01% – 0.1% or 10.1 – 100%, one patient (0.02%) had a follow-up study that resulted in a change of category. Of the 25 pts with PNH clones sizes between 0.11% – 1% and 1.1% – 10%, 10 pts (40%) had a follow-up study resulting in an increase in category, 6 pts (24%) had a follow-up study resulting in a decrease in category and 9 pts (36%) had a follow-up study resulting in no change in category.Figure 1.Distribution of PNH Clone Sizes based on 421 PNH+ PatientsFigure 1. Distribution of PNH Clone Sizes based on 421 PNH+ PatientsTable 1:Incidence of PNH Clones in Patients with ICD-9 Diagnostic Code at Dahl-Chase Diagnostic ServicesICD-9 Diagnostic CodeGeneral DescriptionIncidence of PNH Clone284, 284.01, 284.8, 284.81, 284.89, 284.9Aplastic Anemia26.3% (94/357)238.7, 238.72, 238.73, 238.74, 238.75, 238.76Myelodysplastic Syndrome (MDS)5.5% (32/585)287.5Unexplained Cytopenia5.7% (13/230)284.1Pancytopenia6.0% (63/1058)285.2, 285.21, 285.29, 285.9Anemia Unspecified3.6% (40/1122)283, 283.1, 283.10, 283.11, 283.19, 283.2, 283.9Hemolytic Anemia22.7% (147/647)791, 791.2Hemoglobinuria18.9% (14/74)790.6, 790.99, 790.4Hemolysis7.9% (18/227)325, 415.1, 415.11, 434, 434.01, 444.22, 451.11, 451.19, 452, 453, 453.0, 453.2, 453.4, 453.41, 453.89, 453.9, 557, 557.1Thrombosis1.4% (14/967)280.9Unspecified Iron Deficiency2.5% (7/278)Other ICD-9 diagnostic codes2.1% (26/1232)Not Provided4.8% (51/1065)Note: Table reflects patients who had more than one ICD9 code associated with their laboratory tests. In this single-laboratory experience, we evaluated the incidence of PNH in these high risk groups. In this study, 26.3% of pts with the diagnosis of BMF had PNH+ clones detected, underscoring the need to test this group of pts. The study confirmed the utility of testing pts with unexplained hemolytic anemia, hemolysis and hemoglobinuria where the combined rate of positivity was 48%. In addition, this study highlights the need to monitor pts with small PNH clones by HSFC analysis as these pts may show significant variation over time. This examination of ICD-9 DX code association with presence of PNH+ clones confirms the need to actively test high risk populations for PNH based on the ICCS recommendations to ensure accurate diagnosis and early intervention. Disclosures: Weitz: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Illingworth:Dahl-Chase: Employment; Alexion: Consultancy, Honoraria, Research Funding.

Comparison Of Lymphocytic Immune Profiles Of Aplastic Anemia and Hypocellular Myelodysplastic Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3719-3719
Author(s):  
Jeffrey J. Pu ◽  
Guillermo Rangel Rivera ◽  
Abigail Sido ◽  
Arthur Berg ◽  
Cinda Boyer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Cell Population ◽  
Bone Marrow Failure ◽  
Expression Patterns ◽  
Lymphocyte Population ◽  
Cd19 Expression

Abstract Background Aplastic anemia (AA) and hypocellular myelodysplastic syndrome (MDS) are two common acquired bone marrow failure diseases. AA is mostly an acquired bone marrow disease caused by cellular and humoral mediated immune attack of hematopoietic stem cells (HSC) due to dysregulation of lymphocytic system, which leads to hematopoietic progenitor cell apoptosis and bone marrow failure. MDS is a group of heterogeneous acquired clonal HSC disorders with ineffective hematopoiesis. Approximately 10% to 20% of MDS manifests a reduced bone marrow cellularity, which comprises hypocellular MDS. There is increasing experimental and clinical indication that an immune-mediated damage to hematopoietic HSCs and changes in the hematopoiesis-supporting microenvironment contribute to the pathogenesis of hypocellular MDS. Because of the similarity of their bone marrow manifestation, hypocellular MDS and AA are often hard to distinguish. Mounting evidence indicates that abnormal activation of cytotoxic T cells plays a crucial role in the pathophysiology of these diseases. One study showed that AA patients have an abnormally activated subpopulation of CD4+ helper cells and a decreased number and function of T regulatory cells in the bone marrow. GVHD mouse models further demonstrated that self-reactive T cells were capable of recognizing non-polymorphic tissue or commensally-derived antigens. Recent literature suggests that immune dysregulation plays a major role in pathogenesis of acquired bone marrow failure disease. However immune profiles of these two diseases have not been thoroughly studied, specially the role of B lymphocyte population. Our study aims to find lymphocytic surface marker expression patterns of hypocellular MDS and AA in both immature cell and lymphocyte populations. Methods This retrospective study analyzed flow cytometry lymphocytic antigen expression profiles from patients diagnosed as AA and hypocellular MDS as per standard criteria. A total of 31 AA and 26 hypocellular MDS patient cases were recruited. The bone marrow aspirate/biopsy data, bone marrow aspiration flow cytometry reports, and Complete Blood Counts (CBC)s from individual patients were analyzed. Using side scatter (SSC) vs. CD45 gating flow cytometry panels, we identified immature cell population (SSClow/CD45low) and lymphocyte population (SSClow/CD45high). We then quantitatively analyzed the expression patterns of 33 cluster differentiation (CD) molecules on individual sample. Finally, we compared the CD expression patterns between AA and hypocellular MDS in both cell populations respectively. Results CD19 expression was significantly higher in AA than in hypocellular MDS in both SSClow/CD45low cell population (P=0.001) and SSClow/CD45high cell population (P=0.003). Hypocellular MDS contains significantly higher CD34high cells than AA in SSClow/CD45low populations (mean:28.5% vs 8.5%; range; 1% to 94% vs 2% to 27%; P=0.04). However, patients with both diseases similarly contains very few CD34high cells in SSClow/CD45high cell population (mean: 0.6% vs 2.6%; range: 0.0% to 2% vs 0.0% to 32%; P=0.99). Conclusion 1. In AA, B cells are highly proliferative in both immature stage and mature stage. This data indicates that B cells which may play a unique role in AA pathogenesis but not in hypocellular MDS. 2. In both AA and hypocellular MDS, the majority of lymphocyte population are mature cells. This data suggests that the pathogeneses of both diseases caused by a persistently dysregulated immune microenvironment, not by an acute insult. CD19 expression pattern may be a useful marker to distinguish AA and hypocellular MDS. Disclosures: No relevant conflicts of interest to declare.

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