Minor Paroxysmal Nocturnal Hemoglobinuria Research Articles

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.

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.

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.

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.