Minor Paroxysmal Nocturnal Hemoglobinuria Clones Research Articles

ANALYSIS OF BASELINE CHARACTERISTICS, DISEASE BURDEN AND LONG-TERM FOLLOW-UP OF 167 PATIENTS WITH BRAZILIAN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA - ANOTHER NATURAL HISTORY

Paroxysmal nocturnal hemoglobinuria (PNH) may occur as classical hemolytic disease or a small PNH clone found in a patient with bone marrow failure. To describe clinical features and long-term follow-up of 167 patients with PNH and demonstrate differences between disease categories. Multiparametric flow cytometry performed on 1025 patients referred from Jan/2000 to Dec/2019 found 167 (16.3%) confirmed PNH clone, 87M/70F. Clinical characteristics at first visit, and laboratory results, incidence of thrombosis, treatment and outcome during follow-up visits were considered for statistical analysis. Most patients (89.2%) had hypocellular bone marrow at diagnosis; 55(32.9%) developed hemoglobinuria, and 22(13.2%) developed thrombosis during monitoring. Mean age at aplasia diagnosis 28.4 years (range 5.6 - 71.2), and mean age at PNH clone detection 31.0 years (7.2 - 71.3 years). Clonal evolution occurred in 77 (46.1%) patients at a median of 4 years (range 6-281 months) after the onset of cytopenia. The cohort was divided into 15 classic PNH, 55 hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 definitive bone marrow disease with minor PNH clones - subclinical PNH (sc-PNH). Subclinical patients had lower erythrocyte (2.0% vs 24.0% vs 57.8%) and granulocyte PNH clones (11.7% vs 58.8% vs 81.2%) than PNH/AA and classic PNH, respectively. LDH, reticulocytes, absolute neutrophils and bone marrow cellularity were all lower in sc-PNH than in the hemolytic group. Pearson's analysis showed a statistically significant correlation (p < 0.001) between PNH granulocyte clone and RBC clone (0.697), LDH (0.602), absolute neutrophils (0.310), platelet count (0.430), absolute reticulocytes (0.546), total bilirubin (0.330), and bone marrow biopsy (0.435). PNH granulocyte clone greater than 50% was found in 16/22 thrombotic patients. Mean granulocyte clone was 89.1% (3.4%-99.2%) versus 12.3%(0.1%-34.0%), p < 0.001, and erythrocytes 28.4% (3.4-99.8) versus 0.7% (0.1-14.0) in thrombotic versus the remaining 145 patients. Treatment was highly heterogeneous during this 20-year follow-up: immunosuppressive therapy with CSA and/or thymoglobulin was given to 72 patients in the subclinical group (73.1%) and 45 (64.3%) in the hemolytic group; 52 patients received HSCT (31.1%) of which 40 in sc-PNH and 11 in PNH/AA; a total of 19 patients received Eculizumab after 2008 and 3 patients never received treatment. Incidentally, there were 17 thrombotic events in hemolytic patients (38.6%) diagnosed by 2007, compared to only five events (19.2%) in patients diagnosed after 2008. The average follow-up period was 74 months (1 - 330 months), with 31 deaths in total (80.4% OS). Sepsis was the leading cause of sc-PNH mortality (16/18, 88.8%), and thrombosis in both hemolytic groups (11/13, 84.6%). Log rank test shows similar survival when comparing patients with diagnosis until and after 2007 (p = 0.04). this study suggests clonal evolution in the long-term follow-up of aplastic patients and confirms the observation that high levels of PNH and LDH clones are associated with life-threatening hemolysis and thrombosis, while small PNH clones and young age are associated with the subclinical form of the disease. Although without statistical significance, the cumulative incidence of thrombosis in the group diagnosed after 2008 appears to be lower than the previous period, perhaps due to the routine use of Eculizumab.

First Interim Analysis of the French National Observatory of Paroxysmal Nocturnal Hemoglobinuria Clones

Introduction: Following the published International Guidelines (Borowitz, 2010), the French-Belgian biological workshop was initiated in 2010 by the French Paroxysmal Nocturnal Hemoglobinuria (PNH) group (named HPNAFC). This effort to harmonize the diagnostic practices has been pursued through the development of an inter-laboratory comparison program, involving more than 50 French-speaking centers since 2013 (Debliquis, 2015). Thus, the French National Observatory of PNH clones opened for 5 years on the 9th International Rare Disease Day (February 29th 2016), in order to collect all PNH clone cases diagnosed by French cytometrists.Methods: The primary objective of the French National Observatory of PNH clones is to monitor all PNH clones ≥0.01%, along with two secondary objectives according to the PNH clone size: the evolution of minor clones <1% and the proportion and significance of type II and type III cells for clones ≥1%. All patients with a PNH clone ≥0.01% can be included, irrespective of age, provided that the center has validated a PNH flow cytometry quality control. For each patient, the inclusion time point always corresponds with the initial diagnosis date of the PNH clone, even if it was made before the opening of the Observatory, allowing the collection of all cases in the active French PNH file. This Observatory is based on a principle of non-opposition; inclusions are made by the referent cytometrist of each participating center by filling in the e-CRF form available on the HPNAFC website.Results: As of June 30th 2017, 39 cytometry laboratories have registered across France. Of these, 24 centers have submitted 163 inclusion points with a PNH clone above 0.01%, each corresponding to a patient. The cases were reviewed by the 2 principal investigators, who carefully re-examined all of the flow cytometry data. This method enabled the validation and inclusion of 126 cases, with 37 ongoing ones due to missing clinical or biological data. The first extraction led to the preliminary analysis of these 126 cases and showed that patient median age at diagnosis was 45 years [11-87], with 6 pediatric cases, and a M:F sex ratio of 0.83. Diagnosis was made between 1988 and 2017 and clinical information at diagnosis was available for 92% of patients. More than half of them had aplastic anemia (58.7%). Other diagnosis included: hemolytic anemia (with or without aplastic anemia) (15.9%), unexplained cytopenia(s)(7.9%), myelodysplastic syndrome (6.3%), hemoglobinuria (4.8%) and 2 cases (1.6%) of recurrent thrombosis without hemolysis. Biological data showed a median hemoglobin value of 9.3 g/dL [4.7-16.5], 1.4 x109/L [0.1-11] neutrophils and 60 x109/L [2-356] platelets. The median clone size at diagnosis was 1.9% on granulocytes with a very wide range from 0.01% to 99.4%, most of them being less than 10% and mainly composed of type III cells (median type III clone size: 1.6%). A similar distribution of PNH clones was observed on monocytes, with a good correlation between clone sizes on both lineages. RBC analysis, which was performed for less than half of the patients showed a majority of minor clones and no correlation was observed between PNH clone size on granulocytes and RBC, due to an underestimation of the clone size on RBC. Regarding the clinical context, the median PNH clone size on granulocytes in case of aplastic anemia was 1%, yet some patients had very large clones, higher than 99%. Conversely, PNH clones from patients with hemolytic anemia were very large with a median size of 88%, but some patients had smaller clones of less than 20%. The 2 patients with thrombosis displayed a much smaller clone size, below 0.05%.Conclusion: More than one year after its initiation, 126 confirmed patients were enrolled in the French National Observatory of PNH clones. Median PNH clone size was 1.9% on granulocytes and more than half of these patients had aplastic anemia. A good correlation was seen between clone sizes on granulocytes and monocytes, but not on granulocytes and RBCs. This collaborative work has established a strong network of motivated cytometrists as highlighted by the number of registered centers and the increasing inclusions since its recent opening. A comprehensive follow-up of a foreseeable larger number of PNH clones (so far 92 out of the 163 already have at least 1 follow up point) will lead to a better understanding of the physiopathology of minor PNH clones. DisclosuresDrenou:Alexion: Consultancy, Honoraria. Pruvot Debliquis:Alexion: Consultancy, Honoraria. Letestu:Alexion: Consultancy, Honoraria. Chatelain:Alexion: Consultancy, Honoraria. Socié:Alexion Pharmaceuticals, Inc.: Consultancy. Peffault De Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding. Wagner Ballon:Alexion: Consultancy, Honoraria.

Pediatric Patients with Severe Aplastic Anemia Treated with Combined Immunosuppressive Therapy, Eltrombopag and Eculizumab and Their Clinical Course to Stem Cell Transplant

Acquired aplastic anemia (AA) in children is a rare disorder characterized by pancytopenia and hypocellular bone marrow. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoetic stem cell (HCT) disorder characterized by complemented-mediated hemolysis, thrombosis and bone marrow failure secondary to deficiency of glycosylphosphatidylinositol-anchored proteins (GPI-AP) on hematopoetic stem cells. PNH and acquired AA are closely related; up to 50% of patients with AA have detectable PNH-clones at the time of diagnosis and small percentage of them can have clonal expansion throughout their disease course requiring close monitoring. Current standard therapy for severe aplastic anemia (SAA) patients without matched related donor (MRD) is immunosuppressive therapy (IST) regimen with anti-thymoglobulin (ATG), cyclosporine (CSA), and recent addition of eltrombopag. Eculizumab is a recombinant humanized monoclonal antibody that blocks complement protein C5 and prevents cell lysis. While it has been shown to be effective in children with PNH, concomitant treatment with IST for patients with SAA is unknown. To our knowledge, there has been no pediatric data on combined IST, eltrombopag and eculizumab treatment for children with AA with clinically significant PNH clones. Here we retrospectively reviewed three pediatric patients with SAA with PNH clones treated with IST, eltrombopag and eculizumab and their unique clinical courses. Two out of three patients had high PNH clone size and were started on eculizumab prior to IST with improvement in transfusion intervals. Of the two, one had decrease in PNH clone size after IST but the other patient's clone size continued to increase despite two courses of IST. Finally, the third patient had a minor PNH clone and was not started on eculizumab prior to IST. He remained asymptomatic for over a year until he developed symptomatic PNH with large clone size and aplastic bone marrow. His clone size continued to increase with no improvement in transfusion frequency despite being started on eculizumab. However, steroids were started based on anecdotal literature and his transfusion frequency decreased subsequently. All three patients are in the process of going through matched unrelated donor stem cell transplants. Several studies have reported the presence of a minor PNH clone at the time of AA diagnosis was associated a favorable response to IST. In this case series, the patient with the smallest PNH clone size at the time of diagnosis of SAA had the best response to IST compared to the other two patients who had larger PNH clone populations. These findings suggest that even though minor PNH population may lead to better response to IST compared to absence of the clone, the correlation between clone size and prognosis is unclear. Further study with larger sample size is needed to investigate this relationship. However, regardless of the population size, all three patients were able to prolong transfusion intervals using eculizumab without significant side effects. As the adoption of eltrombopag with standard IST is evolving with ongoing study, the efficacy of incorporating eculizumab to decrease transfusion frequency in patients with PNH-clone in addition to eltrombopag/IST regimen should be further investigated. Disclosures No relevant conflicts of interest to declare.

Acute kidney injury with dark urine: the case of paroxysmal nocturnal hemoglobinuria

In this case report; we describe a patient with severe attack of paroxysmal nocturnal hemoglobinuria (PNH) following Ciprofloxacin therapy. He presented with recurrent abdominal pain, repeated vomiting and dark urine. Physical examination revealed severe pallor and jaundice. Laboratory investigations showed severe intravascular hemolysis with negative Coomb’s test, progressive acute kidney injury (AKI), sterile blood cultures and negative serology for autoimmune diseases. Subsequently, he developed pancytopenia. Ham test was positive and flow cytometry, later on, confirmed PNH. He was supported with multiple transfusions of packed blood cells and hemodialysis. Ciprofloxacin was discontinued and his PNH was treated with Solumedrol 1 g daily for 3 days followed by Prednisone 60 mg/day for 2 months. Two weeks later; his hemolysis abated and his AKI improved. Up to 5 years later, he still has minor PNH clone yet without disease activity. In conclusion; our patient had acute drug-induced hemolytic crisis associated with minor PNH clone. With drug-vigilance; no further relapses were reported and his PNH clone remained stable for 5 years. The case expands the spectrum of PNH phenotypes and its triggering factors.&#x0D; Keywords: AKI, anemia, autoimmune, flow cytometry, hemolysis, PNH.

Evaluation of Clinical Usefulness of Monocyte Gating Using CD14 and CD64 for Detecting PNH Clone By Flow Cytometry

IntroductionFlow cytometry is the gold standard in diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) by detecting the absence of glycol-phosphatidyl inositol (GPI)-linked protein expression on red blood cell, granulocyte, and monocyte. The current assays are 4-color analyses of GPI-linked markers such as fluorescein-labeled proaerolysin (FLAER), CD24, CD14, CD59, and CD235a and the lineage markers for granulocyte (CD15) and monocyte (CD64) cells to detect PNH clones. We investigated the utility of CD14/CD64 monocyte gating by comparing with CD45/light scatter (LS) gating in PNH study of the patients with cytopenia and analyzed the types and cell lineages of PNH clone according to the disease groups.MethodTotal 138 cases were recruited in this study from July 2017 to February 2018 at Gachon University Gil Medical Center in Korea. Flow cytometric analysis was performed with EDTA blood by Beckman Coulter Cytomics FC500 cytometer using gating antibodies such as CD45, CD14, CD15, CD64, CD235a and GPI-linked antibodies such as CD59, CD14, CD24, FLAER. The proportion of monocyte was estimated by CD14/CD64 gating and compared with those using CD45/LS gating. The type of PNH clone was defined according to the size of PNH population. A PNH clone is defined as a PNH population exceeding 1% of the gated cells, a minor PNH clone as between 0.1 and 1%, and rare cells with GPI-deficiency defined as a PNH population less than 0.1%. The types and cell lineages of the PNH clone were analyzed according to the disease groups. Statistical analysis was done using SPSS 17.0 and MedCalc 15.2, and P<0.05 was considered statistically significant.ResultsOf the 138 cases, PNH clone was detected with 27 cases including 15 cases with a PNH clone and 12 cases with a minor PNH clone. PNH clone was observed in all 8 cases (100%) of PNH cases. Two PNH clone and 4 minor PNH clones were identified in 6 of 16 cases (38%) of acute myeloid leukemia. In 6 of 21 cases (29%) of aplastic anemia (AA) show 5 PNH clones and 1 minor PNH clone. In 5 of 78 cases (6%) of cytopenia(s) only minor PNH clone was observed. The CD45 plus LS gating in monocyte represents a sensitivity of 100%, a specificity of 40.2%, and 60% (73/89) false positive rate in detecting of PNH clone. McNemar test indicates a significant difference between CD14/CD64 and CD45/LS gating methods (P = 0.00). The Bland-Altman plot of monocyte proportion between the two gating methods revealed that CD45/LS gating method was tended to underestimate monocyte proportion and the larger the number of monocytes, the greater the difference in number of monocyte between the two gating methods. The trend of the size of PNH clone in each cell lineage was confirmed by follow-up in three patients with PNH clone. Two patients showed more abrupt changes of PNH clone in monocytes than in red blood cells or in granulocytes. However, in the other patient, a significant trend found in only PNH clone of RBC.ConclusionThe types of PNH clone observed in each disease group showed different characteristics. PNH clone was identified in 5 of 6 PNH population detected AA cases, whereas minor PNH clones were observed in all 5 PNH population detected cytopenia cases. Four minor PNH clones and two PNH clones were discovered in 6 PNH population detected AML cases. However, all observed PNH clones observed in AML cases were monocyte. Monocyte gating with CD45 and LS not only underestimated the proportion of monocyte in total WBCs but also showed a high false positive rate of 60% in detecting PNH clone. In contrast, the CD14/CD64 gating method can accurately measure the monocyte population and avoid making a false positive measurement of PNH clone. In addition, in monitoring PNH patients, the measurement of the PNH clone in monocyte tends to be more sensitive to change of PNH clone size than those measured in RBC or granulocytes. In conclusion, the gating using CD14 and CD64 is significantly valuable in flow cytometric diagnosis for detecting the PNH clone in diagnosing new patents as well as monitoring of PNH patients. DisclosuresNo relevant conflicts of interest to declare.

Updated Guidelines for the Treatment of Acquired Aplastic Anemia in Children.

This review aimed to provide updated guidelines for the management of children with acquired aplastic anemia (AA), particularly focusing on hematopoietic stem cell transplantation (HSCT). Failure-free survival for children with aplastic anemia has been shown to be better after bone marrow transplantation (BMT) from matched or one-locus mismatched related donors (MRD/1MMRD) than after immunosuppressive therapy (IST). A combination of the absence of minor paroxysmal nocturnal hemoglobinuria clones and short telomere length was identified as a strong predictor of a poor response to IST. Upfront HSCT from matched unrelated donors (MUD) and MRD was recently demonstrated to have comparable outcomes. Moreover, unrelated cord blood transplantation (UCBT) and haploidentical HSCT have shown promising outcomes, and the fludarabine/melphalan-based regimen has resulted in excellent survival without poor graft function. BMT from MRD/1MMRD is the treatment of choice. When a MRD/1MMRD is not available, upfront BMT from a MUD should be considered for patients with only a slim chance of responding to IST. UCBT and haploidentical HSCT are promising options. This updated treatment algorithm should improve overall outcomes for children with AA.

Development of Paroxysmal Nocturnal Hemoglobinuria in Children with Aplastic Anemia

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. DisclosuresKojima:SANOFI: Honoraria, Research Funding.

Biomarkers for predicting clinical response to immunosuppressive therapy in aplastic anemia.

The decision to select hematopoietic stem cell transplantation (HSCT) or immunosuppressive therapy (IST) as initial therapy in acquired aplastic anemia (AA) is currently based on patient age and the availability of a human leukocyte antigen (HLA)-matched donor. Although IST is a promising treatment option, the ability to predict its long-term outcomes remains poor due to refractoriness, relapses, and the risk of clonal evolution. Several predictive biomarkers for response to IST have been posited, including age, gender, pre-treatment blood cell counts, cytokines, gene mutations, paroxysmal nocturnal hemoglobinuria (PNH), and telomere length (TL). While previous studies have provided substantial biological insights into the utility of IST, the prognostic power of the reported biomarkers is currently insufficient to contribute to clinical decision making. Recently, a large retrospective analysis proposed the combination of minor PNH clones and TL as an efficient predictor of IST response. Identification of a reliable predictor would provide a useful tool for determining the most appropriate treatment choice for AA patients, including up-front HSCT from HLA-matched unrelated donor. The present review summarizes studies evaluating the utility of biomarkers in predicting the clinical response to IST of patients with AA, and provides a baseline for prospective studies aimed at validating previously reported biomarkers.

Evaluation of Paroxysmal Nocturnal Hemoglobinuria Screening by Flow Cytometry Through Multicentric Interlaboratory Comparison in Four Countries

Paroxysmal nocturnal hemoglobinuria (PNH) is currently diagnosed by flow cytometry; although highly sensitive, its interpretation and reporting appear as critical as its technique. Thus, we developed a quality control scheme for the French-speaking region based on the international recommendations for PNH screening. After a topical workshop, we proposed a 1-year, two-step survey program to any volunteering French-speaking clinical laboratory. The first survey consisted of sending raw data files to evaluate gating and the interpretation strategy of each center. The second stipulated sending fresh whole-blood samples to evaluate the whole process and its practice. Forty-nine participants from voluntary centers returned results for each of the two successive surveys. On virtual survey, 27% reported false-positive PNH created by immature granulocytes, whereas the minor PNH clone was not detected by 9%. On fresh survey, 63% of centers used at least the same six-color combination (CD24, CD14, CD33, CD15, CD45, and fluorescent aerolysin), and nearly 70% of participants were able to perform a sensitivity test less than 0.1% on neutrophils. All participants detected the major PNH clone, yet 16% returned false-positive results for the non-PNH clone case. We succeeded in rallying numerous French-speaking clinical laboratories for both surveys and in harmonizing the technical practice by highlighting common pitfalls.

Predicting Response to Immunosuppressive Therapy By the Combination of Minor Paroxysmal Nocturnal Hemoglobinuria Clones and Lymphocyte Telomere Length in Children with Aplastic Anemia

Introduction: Acquired aplastic anemia (AA) is an immune mediated disease characterized by severe quantitative defects in stem cell number resulting in a hypocellular marrow and peripheral blood cytopenias. Immunosuppressive therapy (IST) using anti-thymocyte globulin (ATG) and cyclosporine A (CyA) is the standard therapy for patients with AA who lack a human leukocyte antigen (HLA)-matched sibling donor. Previous studies identified minor paroxysmal nocturnal hemoglobinuria (PNH) clones and short telomere length (TL) as predictive markers of the response to IST. However, the likelihood ratio of these markers was insufficient to influence clinical decisions. Here we assessed whether the combination of these markers could more accurately predict the response to IST.Methods: Between July 2001 and November 2013, 116 patients (65 boys and 51 girls) diagnosed with AA were examined to identify a minor population of PNH-type cells and TL of peripheral blood lymphocytes. Fifty-three patients received horse ATG (Lymphoglobulin, 15mg/kg/day for 5 days) and 63 received rabbit ATG (Thymoglobulin, 2.5−3.75 mg/kg/day for 5 days). CyA (6mg/kg/day) was started on day 1 and continued to at least day 180. The dose of CyA was adjusted to maintain trough levels between 100 and 200 ng/ml. Written informed consent was obtained from all patients’ parents. The ethics committee of each participating hospital approved the study. We used a flow cytometric assay to detect PNH-type granulocytes and red blood cells (RBCs). Presence of >0.007% CD13+ CD55− CD59− granulocytes and/or >0.009% glycophorin A+ CD55− CD59−RBCs was defined as PNH positive (PNH+), based on the results from healthy individuals. The average relative TL of peripheral lymphocytes was measured by flow-fluorescence in situ hybridization (flow-FISH) using a Telomere PNA Kit (Dako Cytomation). Based on our previous study, we defined −1.0 SD of age-adjusted controls as the cut-off value.Results: The median age at IST was 9.8 years (0.9−16.0). Disease severity of patients was assessed as moderate in 39, severe in 37, and very severe in 40, respectively. The causes of AA were idiopathic in 104 patients and hepatitis-associated in 12 patients. The median follow-up period from the time of IST was 36 months (range 1−134). Thirty-nine patients (34%) carried a minor PNH clone at diagnosis. Compared with healthy individuals, their median of TL was −1.08 SD (−4.01 to +3.01 SD). Overall, 60 of 116 patients (51.7%) responded to IST at 6 months after administration of ATG. In univariate analysis, the response rate at 6 months was higher in PNH+ patients than in PNH- patients (74.4% vs. 40.3%, respectively, p = 0.001). Longer TLs (≥−1.0 SD of age-adjusted control) were also associated with a favorable response to IST at 6 months (73.2% vs. 31.7%, p<0.001). In multivariate logistic regression analysis, PNH clone presence [odds ratio (OR), 4.41; 95% CI, 1.68−11.60; p = 0.003], longer TLs (OR, 5.59; 95% CI, 2.33−13.40; p < 0.001), and male gender (OR, 2.72; 95% CI, 1.12−6.62; p = 0.028) were found to be independent favorable predictors of response to IST at 6 months. Different types of ATG (horse vs. rabbit) did not affect response rate (OR, 1.94; 95% CI, 0.92−4.10; p = 0.084). According to these findings, the cohort was stratified into two groups: possible poor responders [PPR; PNH− and shorter TLs (<−1.0 SD), 45 patients] and possible good responders (PGR; PNH+ and/or longer TLs, 71 patients). The response rate at 6 months in the PPR group was as low as 24.4%, which was significantly lower than that in the PGR group (69.0%, p < 0.001). There was no significant difference in the incidence of relapse (0.0% vs. 20.8%, p=0.190) and clonal evolution (4.5% vs. 5.7%, p=1.000) between two groups. Five-year probabilities of transplant-free survival (TFS) and failure-free survival (FFS) were significantly lower in the PPR group than in the PGR group (TFS, 49.1% vs. 71.4%, p = 0.008; FFS, 42.6% vs. 57.3%, p = 0.020). However, 5-year overall survival showed no difference between the two groups (PPR, 97.7% vs. PGR, 95.5%, p = 0.910) because of the effective salvage therapies.Conclusion: Combination of the absence of a minor PNH clone and a short TL was an efficient predictor of poor response to IST. These patients had low chances to respond to IST, which challenges the current standard of treatment. Prospective clinical trials are required to confirm these findings. DisclosuresNo relevant conflicts of interest to declare.

A New Approach of PNH Clone Detection

IntroductionThe presence of the CD157 GPI-protein on both monocytes and granulocytes only gives opportunity to use it as a target for paroxysmal nocturnal hemoglobinuria (PNH) phenotype cells detection instead of CD24 and CD14, thus improving the technique of PNH-cells evaluation. In present study we used standard technique (International Clinical Cytometry Society (ICCS) guideline proposal) together with 5 color combination of mAbs for detection of PNH-clone on the monocytes and granulocytes simultaneously in one test-tube.Objectives: improvement of PNH cells detection technique.Materials and methods: Blood samples were collected from 37 patients (pts) with bone marrow failure syndrome (aplastic anemia – 34 pts., PNH - 2 pts., myelodysplastic syndrome – 1 pt.; 23 female and 14 men; median age 24 (15 to 66). PNH clone was detected by flow cytometry in all 37 pts. Three healthy donors were in control group.The comparison of the standard 4-color technique of PNH clone size detection on monocytes (FLAER, CD14, CD64, CD45 reagents) and on granulocytes (FLAER, CD24, CD15, CD45 reagents) with the 5-color technique using CD157 GPI-protein antibodies for the both cells populations in one test-tube (FLAER, CD157, CD15, CD64, CD45) were performed.Results: Both methods showed the same high sensitivity for determining of PNH clone. The correlation coefficient was 0,9994 for granulocytes and 0,9924 for monocytes (Figure 1). [Display omitted] In group of patients with minor PNH-clone (range from 0,01% to 0,99%) the clone size was twice times bigger on monocytes compared to granulocytes using the standard method, whereas with antibodies against CD157 this difference disappeared. In patients with PNH-clone between 1% and 10% the clone was nearly three times less on granulocytes than monocytes using both methods; in the group with clone more than 10% there was no differences between cells populations.Table 1Average value of PNH clone on leukocytes in groups of patients both techniquesPNH cloneGr, % (mean)Mon, % (mean)CD24-/FLAER-CD157-/FLAER-CD14-/FLAER-CD157-/FLAER-0%-0,99%0,1780,3490,3230,3781%-9,99%3,983,769,549,98>10%71,5271,5271,8571,34Conclusion: The results of the PNH clone detection obtained with CD157 mAbs are comparable with the standard technique proposed by ISSC. However, the use of CD157 antibodies has important advantage: the PNH clone size detection in one test-tube with 5-color combination reduces time and expenses. Additionally, using of 5-color CD157 antibodies kit would be preferable for the monitoring and detection of minor PNH clone. DisclosuresNo relevant conflicts of interest to declare.

The clinical relevance of minor paroxysmal nocturnal hemoglobinuria clones in refractory cytopenia of childhood: a prospective study by EWOG-MDS

The clinical relevance of minor paroxysmal nocturnal hemoglobinuria clones in refractory cytopenia of childhood: a prospective study by EWOG-MDS

Performance characteristics of consensus approaches for small and minor paroxysmal nocturnal hemoglobinuria clone determination by flow cytometry

Evaluation of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM) is not standardized and is associated with consistent inter-laboratory variability. In order to rule out the influence of particular approach in generating final results, we analyzed the performance characteristics of individual consensus strategies for small to intermediate (1%-20%) and minor (<1%) PNH clones within the white blood cell (WBC) and red blood cell (RBC) compartments with sensitivity up to 0.1%. Coefficient of variation (CV) for precision/reproducibility analysis ranged from 0.67%/1.49% to 2.56%/3.09% for granulocytes, from 0.93%/3.09% to 7.76%/12.06% for monocytes and from 0.41%/4.73% to 6.53%/5.1% for RBCs. Coefficient of determination (r2) for linear regression analysis ranged from 0.95 to 0.99, Wilcoxon ranks test showed no statistically significant differences (p>0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from -0.18 to 1.24. Our results confirmed very good performance characteristics for precision and reproducibility analysis, excellent correlation and favorable agreement between strategies, suggesting that reported inter-laboratory variability is related mainly to incorrect performance and/or insufficient experience with PNH testing by flow cytometry, rather than to relevant limitations of any particular approach.

High-Sensitivity Flow Cytometry Testing for Paroxysmal Nocturnal Hemoglobinuria in Children with Cytopenia: A Single Center Study

Abstract 2398 Background:Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by expansion of cells with complete or partial loss of glycosyl phosphatidyl-inositol-anchored proteins. PNH usually presents with one or more of three clinical manifestations: intravascular hemolysis, thrombosis, or acquired bone marrow failure [aplastic anemia (AA) or myelodysplastic syndrome (MDS)]. Flow cytometry has become the gold standard for the diagnosis of PNH, particularly with the recent publication of guidelines for the diagnosis and monitoring of PNH and related disorders in 2010. PNH occurs rarely in children, and, consequently, the published literature regarding PNH in this pediatric population consists only of small case series, making it difficult to extrapolate the frequency of which PNH clones are identified. Moreover, no studies are available on the incidence of PNH clones in children with MDS and acquired aplastic anemia (AAA). We, therefore, sought to determine how frequently a high sensitivity FLAER-based assay, with a sensitivity of 0.01%, would detect PNH clones in children with cytopenias. Method and Results:The study period was from December 2010 to July 2011. PNH testing was performed using a high sensitivity FLAER based assay according to published guidelines using the combination of FLAER/CD64/CD15/CD33/CD24/CD14/CD45 for WBC testing and CD235a/CD59 for RBC testing. There were 31 peripheral blood samples from 29 patients (17 males/12 females) ranging in age from 4 months to 17 years (median, 10 years). All patients were tested for PNH because of cytopenia [pancytopenia (n = 14) and uni- or bicytopenia (n = 15)]. Patients had a mean Hgb of 10.7 gm/dL, mean ANC of 2.66 X103/uL and mean platelet of 115 X103/uL. Review of medical charts revealed the following clinical diagnoses: classic PNH - episodic hemolytic anemia with persistent thrombocytopenia (1), severe AA (SAA, 8), SAA with myelofibrosis (1), MDS (1), Fanconi anemia (1), chronic thrombocytopenia (2), refractory iron deficiency anemia (1), bone marrow suppression likely due to virus/medication (1), parvovirus infection (1), Copper deficiency (1), systemic lupus erythematosus (SLE, 1), and cytopenia of unknown etiology (10). Of note, all patients with AAA had SAA. PNH clones were identified in 6 out of 29 patients (20%): minor clones (<1% PNH population) in 3 patients: average clone sizes 0.12% [range 0.02–0.25] granulocytes (G), 0.51% [0.20–0.99] monocytes (M), and 0.08% [0.04–0.14] red blood cells (RBCs), and major clones (>1% PNH population) in 3 patients: average clone sizes 31.11% [3.98–67.58] G, 31.98% [6.15–71.1] M, and 14.76% [1.19–38.03] RBC, respectively, with ages ranging from 4 to 17 years. Patients who were identified to have minor PNH clones all presented with pancytopenia. Two were diagnosed with SAA; the cause of pancytopenia in the third patient is currently under investigation. None of patients with minor PNH clones had evidence of hemolysis or thrombosis. The three patients with major PNH clones had the following: Classic PNH with hemolytic anemia (1), SAA with PNH clones detected at the time of SAA diagnosis (1), and SAA with PNH clones detected 20 months after immunosuppressive therapy (1). The latter two patients did not have evidence of hemolysis or thrombosis. Of the 10 patients with a diagnosis of SAA or MDS, PNH clones were identified in 4 (40%) patients (2 with minor clones, 2 with major clones). Conclusions:This is the first study to describe the utility of using a standardized high-sensitivity FLAER-based flow cytometry assay to identify PNH clones in children. This is also the first study describing the prevalence of PNH clones in children with MDS and AAA. The identification of a PNH population in 40% of the MDS and AAA cases emphasizes the need for PNH testing in all children with these disorders using a high-sensitivity FLAER based flow cytometry assay. A low sensitivity assay would have missed 2 patients with minor PNH clones. This finding may be of significance considering SAA or MDS patients with PNH clones are more likely to respond to immunosuppressive therapy. Further studies are needed to investigate the prevalence of PNH clones in this setting and its impact on disease manifestations, course, and outcomes in children. Disclosures:No relevant conflicts of interest to declare.

The Telomere Length In Peripheral Blood Lymphocytes Is a Useful Predictor of the Response to Immunosuppressive Therapy In Patients with Acquired Aplastic Anemia

AbstractAbstract 2042 Introduction:The ability to predict the response to immunosuppressive therapy (IST) in patients with aplastic anemia (AA) will avoid unnecessary and possibly harmful treatment. To this end, several in vitro tests have been proposed as useful predictors of response in adult patients. However, we previously reported that the presence of either human leukocyte antigen (HLA)-DR15, a minor population of paroxysmal nocturnal hemoglobinuria (PNH)-type cells, or of autoantibodies to postmeiotic segregation increased 1 (PMS1) in children with AA, were not useful predictors of the response to IST, suggesting a different etiology of AA between adults and children (Yoshida, N et al Br J Haematol. 2008). Telomeres in peripheral blood cells from AA patients are shorter than those from normal individuals in 30% of patients. This shorter telomere length in AA patients is thought to result from genetic abnormalities in the genes related to telomere maintenance, stressed hematopoiesis due to immunological attack, and increased cell division. An NIH group and we reported that cryptic forms of dyskeratosis congenita (DC) are present in just under 5% of patients with AA who do not have physical abnormalities, but who harbour the gene mutation associated with telomere maintenance (Yamaguchi, H et al. N Engl J Med. 2005, Liang, J et al. Haematologica 2006). To identify such patients is important because those who have these mutations might respond poorly to IST. However, the presence of cryptic forms of DC does not explain the causes of short telomeres in the other patients. We hypothesized that patients with short telomeres may respond poorly to IST, and thus evaluated the relationship between telomere length in hematopoietic cells before IST and the response to IST in patients with AA. Patients and Methods:We enrolled 40 newly diagnosed children with AA (median age, 10 years; range, 1 to 16 years) who received IST. We obtained peripheral blood lymphocytes from the patients with AA at the time of diagnosis and prospectively measured the telomere length of lymphocytes prior to IST by flow-fluorescence in situ hybridization (flow-FISH) using Telomere PNA kits (Dako Cytomation, Glostrup, Denmark). After gating diploid cells based on propidium iodine staining, lymphocytes were isolated according to size and granularity. Relative telomere length (RTL) was calculated as the ratio between the telomere signal of each sample and the telomere signal of the control cell line. Because telomeres shorten with age, we obtained age-adjusted measurements for comparison from 71 normal age-matched individuals. Delta RTL of each patient with AA was calculated using the distance of the RTL from the line of age-adjusted normal telomere length. Results:Of the 40 patients, at diagnosis AA was very severe in 12, severe in 13, and non-severe in 15. All received IST with horse antithymocyte globulin (ATG) and cyclosporine. The causes of AA were idiopathic in 38 patients and hepatitis in 2. In total, 25 of the 40 patients (63%) responded to IST at 6 months after administration of ATG. Between the responder and non-responder groups, no significant differences in gender, age, etiology, disease severity or interval from diagnosis, and IST were observed. Univariate analysis for IST response did not find any significant differences in median WBC count, neutrophil count, Hb level, reticulocyte count, platelet count or minor PNH clone positivity between the groups. However, delta RTL was significantly lower in non-responders than in responders (p=0.035). Multivariate analysis for response to IST revealed that only delta RTL was significantly lower in non-responders than responders (p = 0.049). Conclusion:The telomere length in lymphocytes is promising not only to identify cryptic DC but also to predict the response to IST of patients with AA. A further larger analysis is warranted to evaluate the correlation between telomere length and the response to IST in AA patients. Disclosures:No relevant conflicts of interest to declare.

Sensitive Detection of Survival of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Red Blood Cells, Granulocytes and Monocytes by High Resolution Multiparameter Flow Cytometry.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by intravascular hemolysis due to GPI-deficient red blood cells sensitive to complement-mediated lysis. Accurate and sensitive detection of PNH-type cells has become important not only to diagnose PNH but also because studies have shown PNH-type cells may indicate favorable response to therapy and favorable prognosis in patients with aplastic anemia and myelodysplastic syndrome. Previous studies have suggested optimal testing for PNH-type cells by flow cytometry should be limited to within 48 hours after collection of whole blood. Our laboratory has developed a very sensitive and specific high resolution flow cytometric method for detecting PNH-type cells based on testing over 3,000 patients with known PNH, aplastic anemia, myelodysplastic syndromes and other bone marrow failure syndromes. The aim for this study was to determine the longevity of PNH clones in whole blood samples, the day-to-day variability of these clones and the rate of deterioration of the PNH clones compared to normal blood cells. We analyzed 10 whole blood samples from patients known to have PNH-type cells on seven consecutive days utilizing a two-color assay with GPA-CD59 for the red blood cells, a 5-color assay with FLAER-CD24-CD14-CD15-CD45 for the granulocytes and a 5 color assay with FLAER-CD33-CD14-CD64-CD45 for the monocytes. The results are summarized in the table below. The initial PNH clone sizes ranged from 0.02% to 90.8%. The PNH cells showed an overall similar level of deterioration to the normal blood cells with even minor PNH clones of 0.02% able to be detected at day 7. The day-to-day variability of PNH clone sizes was generally less than 10%, with smaller clone sizes showing a higher degree of variation, up to 20%, due to their smaller absolute numbers. Interestingly, Type III PNH red blood cells showed slightly better overall survival than normal red blood cells and were detected in modestly increasing percentages throughout the study. Based on this data, we propose that accurate detection of PNH type cells can be achieved up to seven days after collection of whole blood when utilizing high resolution flow cytometry. PNH Clone Size on Sequential Days as Percentage of Original PNH Clone SizeOriginal PNH Clone SizesPNH Clone Sizes as Percentage of Original PNH Clone SizeCell TypeDay 1Day 2Day 3Day 4Day 5Day 6Day 7Type III RBCs.02%–58.6%102%106%107%104%108%103%Granulocytes.29%–90.8%100%100%93%89%79%86%Monocytes.52%–89.9%96%96%92%94%97%85%

Changes of Clonality of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones during Clinical Courses in Patients with PNH.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder, in which almost all hematopoietic cells lack glycosylphosphatidylinositol (GPI)-anchored proteins due to phosphatidylinositol glycan-class A (PIG-A) gene abnormalities at the level of hematopoietic stem cells. It has been reported that several minor PNH clones in patients with aplastic anemia and myelodysplastic syndromes occur at random during the clinical course (Okamoto M et al, Leukemia, 2006), while one or more major PNH clones in patients with typical PNH continue to exist during their clinical courses (Nishimura J et al, Blood, 2002). To clarify how PNH clones in patients with PNH expand during their clinical courses, we examined sequence analyses of the PIG-A gene using peripheral blood granulocytes from PNH patients with high (>10%; n=5) and low (<10%; n=3) proportions of CD16− or CD59− granulocytes, which were determined by flow cytometry using CD16b or CD59 monoclonal antibody, respectively. A patient with over 1% of CD59− erythrocytes was judged to have PNH erythrocytes (Shichishima T et al, Br J Haematol, 1993). PNH clones were defined as GPI-negative hematopoietic cells with PIG-A mutations. Twenty to 40 bacterial clones of PCR or RT-PCR products of the PIG-A gene with GPI-negative or unsorted populations of peripheral blood granulocytes were analyzed by nucleotide sequencing. We examined sequence analyses of the PIG-A gene in peripheral blood granulocytes at least twice during the clinical course (the period of 1995–2007). Major PNH clones were decided by the Chi2 test or the Fisher's exact probability test. In all of 5 PNH patients with high proportions of PNH clones, major PNH clones were found at least once and were common during their clinical courses. However, the PNH clones in 3 of 5 PNH patients were transiently major during their clinical courses. In 2 of 3 PNH patients with low proportions of PNH clones, there were common PIG-A mutations during their clinical courses, but were not major clones. The PNH clone from one of the 2 PNH patients acquired a new PIG-A mutation, which was also minor. Another patient of the 3 patients with low proportions of PNH clones had a major PNH clone at the time of the first examination, but disappeared at the time of the next analysis. In conclusion, our findings suggest that the clonality of PNH clones in PNH patients with high and low proportions of PNH clones could change during their clinical courses and that especially, the clonality of PNH clones in PNH patients with low proportions of PNH clones is not necessarily definitive.PIG-A mutations in PNH patients with high and low proportions of PNH clonesCase No.Common mutationUncommon mutationPNH patients with high proportions of PNH clonesCase 1214 C to T, 5′ splicing site del G (Major)NDCase 2698 G to A (Major), 1152 del CNDCase 3548 G to A (Major)982 G to TCase 4ND491 C to TCase 5ND407 A to GPNH patients with low proportions of PNH clonesCase 5ND148 del G, 142 G to A (Major), 460 C to TCase 6ND11 G to C, 91 ins C, 916 del CCase 6ND67 del G, 72 del 13bp, 72 del 13bp/188 G to A, 358 G to A, 365 A to C, 952 del AAbbreviations: del , deletion; ND, not dectected; and ins, insertion

Presence of HLA-DR15, a Minor PNH Clone, or an Aplastic Anemia - Associated Autoantibody Do Not Predict a Favorable Response to Immunosuppressive Therapy in Children with Aplastic Anemia.

The pathogenenesis of aplastic anemia (AA) is thought to involve an autoimmune process. 60% to 70% of patients respond to immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CyA). The presence of HLA-DR15 and a minor paroxysmal nocturnal hemoglobinuria (PNH) clone have been shown to be closely associated with a good response to IST in adult patients with AA. Recently, several autoantibodies associated with AA have been found in sera from AA patient based on a peptide library derived from fetal liver or leukemic cell lines. Anti-PMS1 antibody was found in 10% of Japanese pediatric patients with AA. The relevance of the antibody to the pathophysiology of AA and its clinical significance is unclear.We evaluated the response rate among patients who showed the presence of HLA-DR15, a minor PNH clone, and anti-PMS1 antibody to identify potential predictive markers for response to IST.Patients and MethodsPlasma samples and peripheral red blood cells (RBCs) were obtained from 103 children with AA who were enrolled in the AA-97 study conducted by the Japan childhood AA study group. We used immunoblotting to detect anti-PMS1 autoantibodies. A minor PNH clone was defined as positive when CD55−/CD59− RBCs in peripheral blood increased more than 0.037%, as quantified by high-sensitivity flow-cytometry assay.Patients with very severe (n=53) or severe AA (n=28) received ATG (lymphoglobulin 15mg/kg/d for 5 days) and CyA (6mg/kg/d), and patients with non-severe AA (n=22) were randomized to receive only ATG or ATG plus CyA. A complete response (CR) was defined as a neutrophil count >1.5x10*8~10*9/L, a platelet count >10x10*8~10*9/L, and a hemoglobin level >11.0g/dl. A partial response (PR) was defined as a neutrophil count >0.5x10*8~10*9/L, a platelet count >20x10*8~10*9/L, and a hemoglobin level >8.0g/dl. Overall response was defined as a CR and PR 6 months after IST. Subgroup analyses were conducted and differences in overall response were tested using the Mann-Whitney U-test.ResultsA total of 103 patients (66 boys and 37 girls) were enrolled. Results of HLA-DR15 typing were available in 85 children. Median age at diagnosis was 8 years (range, 1 to 15 years). Of the 103 patients, 82 (79.6%) had idiophatic AA, 18 (17.5%) had hepatitis-associated AA, and 3 (2.9%) had AA from other causes. Fifty-eight patients (56.3%) attained CR and PR 6 months after IST.HLA-DR15 was present in 22 of 85 children (25.9%). Of the 22 patients who shared the HLA-DR15, 10 (45.5%) achieved a response. Of the 63 patients who did not share HLA-DR15, 34 (54.0%) responded (p=0.62).Minor PNH clone was positive in 20 of 103 children (19.4%). Of the 20 patients who had minor PNH clone, 14 (70.0%) responded to IST, whereas 45 of 83 patients (54.2%) without minor PNH clone responded (p=0.31).Anti-PMS1 antibody was detected in 15 of 103 children (14.6%). Of the 15 patients who had anti-PMS1 antibody, 6 (40%) responded. Of the 88 patients without anti-PMS1 antibody, 52 (59.1%) responded (p=0.26). No differences in patient demographics, including gender, age, causes, and the severity of AA were observed among subgroups.ConclusionIn our study, these 3 markers failed to identify a subset of AA patients who respond to IST. This finding may suggest a difference in pathophysiology between children and adults with AA.

High frequency of several PIG-A mutations in patients with aplastic anemia and myelodysplastic syndrome

To clarify some characteristics of phosphatidylinositol glycan-class A gene (PIG-A) mutations in aplastic anemia (AA) and myelodysplastic syndrome (MDS) patients compared with those in paroxysmal nocturnal hemoglobinuria (PNH) patients, we investigated PIG-A mutations in CD59- granulocytes and CD48- monocytes from seven AA, eight MDS, and 11 PNH Japanese patients. The most frequent base or type abnormalities of the PIG-A gene in AA and MDS patients were base substitutions or missense mutations, respectively, and deletions or frameshift mutations, respectively, in PNH patients. Several PIG-A mutations, most of which were statistically minor, were found in glycosylphosphatidylinositol-negative cells from all AA and MDS patients but not from all PNH patients. However, the common PIG-A mutations during the clinical course between CD59- granulocytes and/or CD48- monocytes from each AA or MDS patient, except for Case 5, were not found. PIG-A mutations were different between the granulocytes and monocytes from five AA and five MDS patients. Our results indicate that there were some characteristics of PIG-A mutations in AA and MDS patients compared with PNH patients and that several minor PNH clones in these patients occurred at random during the clinical course. This partly explains the transformation of AA or MDS to PNH at intervals.