Paroxysmal Nocturnal Hemoglobinuria with Large Clones in Non-Hypoplastic Myelodysplastic Syndrome: Report of Two Cases

IMPLICATIONS OF RECENT INSIGHTS INTO THE PATHOPHYSIOLOGY OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

Haemolytic paroxysmal nocturnal haemoglobinuria in patients with myeloid neoplasms: A rare association with specific therapeutic implications.

Extent and Clinical Implications of Subclonal Diversity in Paroxysmal Nocturnal Hemoglobinuria

Pediatric-Specific Patterns of Clonal Evolution Arising from Acquired Aplastic Anemia

Background. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal haematopoietic stem cell disorder characterised by intravascular haemolysis, bone marrow failure and increased tendency to thrombosis. It is caused by a somatic mutation in the PIG-A gene, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIG-A mutation results in a clone of blood cells with total or partial deficiency of membrane proteins anchored to the cell surface through GPI anchor. For many years, an increased susceptibility of the PNH red cells to complement lysis in acidified serum (Ham’s test) has been essential for diagnosis of the PNH. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of the specific GPI anchor proteins on blood cells. We evaluated a three-colour flow cytometry method for detection and quantification of CD55/59 negative netrophils. Patients and methods. Nine patients (6 with PNH and 3 with aplastic anaemia) who were previously diagnosed as having PNH or aplastic anaemia were evaluated. In the period of three years the patients were serially evaluated for the extent of PNH clone by quantification of CD55/59 negative neutrophils. Three-colour flow cytometry method was used for quantification of the CD55/CD59 negative neutrophils. We used directly conjugated monoclonal antibodies anti-DC15PC5 for identification of neutrophils and anti-CD59-FITC and anti-CD55-PE for the GPI-linked antigens. Results. Patients with haemolytic PNH had > 50% CD59/CD55 negative granulocytes. The proportion of the PNH granulocytes was higher in the patient with frequent and serious haemolytic attacks. Over the period of three years slow growth of the PNH clone was seen in two cases. Two patients with PNH diagnosed 19 years ago and in remission at the time of flow cytometric analysis was devoid of the PNH clone. Three patients with aplastic anaemia (hypoplastic PNH) had the proportion of CD59/CD55 negative granulocytes < 40%. In one of them PNH clone increased. Conclusions. Three colour flow cytometry of granulocytes using combination of anti-CD15/55/59 provides the accurate technique for detection and quantification of the PNH clone. Monitoring the PNH clone size has clinical and prognostic value. The size of the PNH clone is an important determinant for thrombotic risk. Serial studies allow prediction of remission in some cases or progress from hypoplastic to hemolytic PNH in others.

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

Role of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Refractory Anemias

A retrospective analysis of presentation clinical, laboratory and immunophenotypic features of 1081 patients with paroxysmal nocturnal haemoglobinuria (PNH) clones [glycosylphosphatidylinositol (GPI)-deficient blood cells] identified at our hospital by flow cytometry over the past 25years was undertaken. Three distinct clusters of patients were identified and significant correlations between presentation disease type and PNH clone sizes were evident. Smaller PNH clones predominate in cytopenic and myelodysplastic subtypes; large PNH clones were associated with haemolytic, thrombotic and haemolytic/thrombotic subtypes. Rare cases with an associated chronic myeloproliferative disorder had either large or small PNH clones. Cytopenia was a frequent finding, highlighting bone marrow failure as the major underlying feature associated with the detection of PNH clones in the peripheral blood. Red cell PNH clones showed significant correlations between the presence of type II (partial GPI deficiency) red cells and thrombotic disease. Haemolytic PNH was associated with type III (complete GPI deficiency) red cell populations of >20%. Those with both haemolytic and thrombotic features had major type II and type III red cell populations. Distinct patterns of presentation age decade were evident for clinical subtypes with a peak incidence of haemolytic PNH in the 30-49year age group and a biphasic age distribution for the cytopenia group.

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

Morphological Spectrum of Paroxysmal Nocturnal Hemoglobinuria (PNH).

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

The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in aplastic anemia (AA) suggests immunopathogenesis, but when and how PNH clones emerge and proliferate are unclear. Hepatitis-associated aplastic anemia (HAAA) is a special variant of AA, contrarily to idiopathic AA, in HAAA the trigger for immune activation is clearer and represented by the hepatitis and thus serves as a good model for studying PNH clones. Ninety HAAA patients were enrolled, including 61 males and 29 females (median age 21years). Four hundred three of idiopathic AA have been included as controls. The median time from hepatitis to cytopenia was 50days (range 0-180days) and from cytopenia to AA diagnosis was 26days (range 2-370days). PNH clones were detected in 8 HAAA patients (8.9%) at diagnosis and in 73 patients with idiopathic AA (IAA) (18.1%). PNH cells accounted for 4.2% (1.09-12.33%) of red cells and/or granulocytes and were more likely to be detected in patients with longer disease history and less severe disease. During follow-up, the cumulative incidence of PNH clones in HAAA increased to 18.9% (17/90). Nine HAAA patients newly developed PNH clones, including six immunosuppressive therapy (IST) nonresponders. The clone size was mostly stable during follow-up, and only 2 of 14 patients showed increased clone size without proof of hemolysis. In conclusion, PNH clones were infrequent in newly diagnosed HAAA, but their frequency increased to one that was similar to the IAA frequency during follow-up. These results suggest that the PNH clone selection/expansion process is dynamic and takes time to establish, confirming that retesting for PNH clones during follow-up is crucial.

SF3B1, a Splicing Factor Gene, Is Infrequently Mutated in Rare Bone Marrow Failure Diseases but Still Associated with Ring Sideroblast Phenotype

Hypoplastic MDS with PNH Clone Treated Successfully with Eculizumab

In this large single-centre study, we report high prevalence (25%) of, small (<10%) and very small (<1%), paroxysmal nocturnal hemoglobinuria (PNH) clones by high-sensitive cytometry among 3085 patients tested. Given PNH association with bone marrow failures, we analyzed 869 myelodysplastic syndromes (MDS) and 531 aplastic anemia (AA) within the cohort. PNH clones were more frequent and larger in AA vs. MDS (p = 0.04). PNH clone, irrespective of size, was a good predictor of response to immunosuppressive therapy (IST) and to stem cell transplant (HSCT) (in MDS: 84% if PNH+ vs. 44.7% if PNH−, p = 0.01 for IST, and 71% if PNH+ vs. 56.6% if PNH− for HSCT; in AA: 78 vs. 50% for IST, p < 0.0001, and 97 vs. 77%, p = 0.01 for HSCT). PNH positivity had a favorable impact on disease progression (0.6% vs. 4.9% IPSS-progression in MDS, p < 0.005; and 2.1 vs. 6.9% progression to MDS in AA, p = 0.01), leukemic evolution (6.8 vs. 12.7%, p = 0.01 in MDS), and overall survival [73% (95% CI 68–77) vs. 51% (48–54), p < 0.0001], with a relative HR for mortality of 2.37 (95% CI 1.8–3.1; p < 0.0001) in PNH negative cases, both in univariate and multivariable analysis. Our data suggest systematic PNH testing in AA/MDS, as it might allow better prediction/prognostication and consequent clinical/laboratory follow-up timing.