scholarly journals Complement- and inflammasome-mediated autoinflammation-paroxysmal nocturnal hemoglobinuria

2019 ◽  
Author(s):  
Britta Hoechsmann ◽  
Yoshiko Murakami ◽  
Makiko Osato ◽  
Alexej Knaus ◽  
Michi Kawamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Alternative Pathway ◽  
Initial Step ◽  
Clonal Expansion ◽  
Cell Surface Expression ◽  
Intravascular Hemolysis ◽  
Complement Alternative Pathway ◽  
Hematopoietic Stem

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis and thrombosis, and bone marrow failure. Affected cells harbor somatic mutation in X-linkedPIGAgene, essential for the initial step in glycosylphosphatidylinositol (GPI) biosynthesis. Loss of GPI biosynthesis results in defective cell-surface expression of GPI-anchored complement regulators CD59 and DAF. The affected stem cells generate many abnormal blood cells after clonal expansion, which occurs under bone marrow failure. Here, we report the mechanistic basis of a disease entity, autoinflammation-paroxysmal nocturnal hemoglobinuria (AIF-PNH), caused by germline mutation plus somatic loss ofPIGTon chromosome 20q. A region containing maternally imprinted genes implicated in clonal expansion in 20q-myeloproliferative syndromes was lost together with normalPIGTfrom paternal chromosome 20. Taking these findings together with a lack of bone marrow failure, the mechanisms of clonal expansion in AIF-PNH appear to differ from those in PNH. AIF-PNH is characterized by intravascular hemolysis and recurrent autoinflammation, such as urticaria, arthralgia, fever and aseptic meningitis. Consistent with PIGT’s essential role in synthesized GPI’s attachment to precursor proteins, non-protein-linked free GPIs appeared on the surface of PIGT-defective cells. PIGT-defective THP-1 cells accumulated higher levels of C3 fragments and C5b-9 complexes, and secreted more IL-1β than PIGA-defective cells after activation of the complement alternative pathway. IL-1β secretion was dependent upon C5b-9 complexes, accounting for the effectiveness of the anti-C5 drug eculizumab for both intravascular hemolysis and autoinflammation. These results suggest that free GPIs enhance complement activation and inflammasome-mediated IL-1β secretion.

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.

The Role of Telomerase Activity and Telomere Length in Cellular Sensitivity to DNA Damaging Agents.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4192-4192
Author(s):  
Greg T. Rice ◽  
Michael A. Beasley ◽  
Ike I. Akabogu ◽  
Erik R. Westin ◽  
Dale A. Winnike ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Bone Marrow Failure ◽  
Skin Pigmentation ◽  
Cytotoxic Agents ◽  
Cell Surface Expression ◽  
Cell Transplant ◽  
Telomere Shortening ◽  
Hematopoietic Stem ◽  
Increased Sensitivity

Abstract Dyskeratosis congenita (DC) is a premature aging syndrome characterized by progressive bone marrow failure, abnormal skin pigmentation and nail dystrophy. We have described an autosomal dominant form of DC (AD DC) in a large three-generation kindred that is due to a mutation in the gene encoding human telomerase RNA (hTR). While telomere shortening is a normal consequence of the aging process, DC patients display extremely short telomeres in many somatic cell types, including hematopoietic cells, and they often suffer from bone marrow failure. Allogeneic hematopoietic stem cell transplant (HSCT) remains the only curative therapy for marrow failure in DC. However, HSCT in DC is generally poorly tolerated and associated with significant morbidity, perhaps as a consequence of increased sensitivity of dividing cells to cytotoxic agents during myeloablative therapy. To test this hypothesis, we characterized lymphocytes from various AD DC patients and age matched controls that had been placed in long term culture following in vitro exposure to irradiation (137Cs) and varying doses of Taxol, Adriamycin, and Etoposide. Cell proliferation and viability were quantified by direct visual counting on a hemocytometer, and flow cytometry was employed to assess apoptosis and cell surface expression of senescent markers. In addition to DC lymphocytes having a decreased proliferative capacity and higher basal apoptotic levels, an increased sensitivity to irradiation, Taxol, Adriamycin, and Etoposide was noted. These results suggest that telomere shortening may be an important factor in determining cellular tolerance to cytotoxic therapy and support the concept of reduced intensity HSCT regimens in both aged individuals and DC patients. Further studies have been initiated to determine whether reconstitution of telomere length in DC cells alters response to cytotoxic agents.

scholarly journals Different clinical courses with the same findings: two cases of paroxysmal nocturnal hemoglobinuria presenting with thrombocytopenia

2021 ◽  
Vol 15 (3) ◽  
Author(s):  
Volkan Karakuş ◽  
Egemen Kaya ◽  
Yelda Dere ◽  
Fahri Şahin
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Clinical Findings ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Laboratory Findings

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disease that manifests with chronic intravascular hemolysis, thrombosis, and bone marrow failure. Various degrees of cytopenias accompany the disease. Although laboratory and clinical findings are similar, the disease may show different courses and require different treatments. Herein, we report two different courses of PNH with similar clinical and laboratory findings.

scholarly journals Paroxysmal Nocturnal Hemoglobinuria with a Distinct Molecular Signature Diagnosed Ten Years after Allogenic Bone Marrow Transplantation for Acute Myeloid Leukemia

2019 ◽  
Vol 2019 ◽  
pp. 1-3
Author(s):  
Alberto Santagostino ◽  
Laura Lombardi ◽  
Gerard Dine ◽  
Pierre Hirsch ◽  
Srimanta Chandra Misra
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Somatic Mutation ◽  
Myeloid Leukemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Clonal Selection ◽  
Bone Marrow Failure ◽  
Clinical Manifestations ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Paroxysmal nocturnal hemoglobinurea (PNH) is a rare disorder of complement regulation due to somatic mutation of PIGA (phosphatidylinositol glycan anchor) gene. We herewith report a case who developed a symptomatic PNH long after an allogenic marrow transplant. Some reasonable arguments concerning the origin of PNH clone have been discussed. The molecular studies revealed presence of JAK2 and TET2 mutations without a BCOR mutation. The literature review has been performed to probe into the complex interplay of autoimmunity and clonal selection and expansion of PNH cells, which occurs early in hematopoietic differentiation. The consequent events such as hypoplastic and/or hemato-oncologic features could further be explained on the basis of next-generation sequencing (NGS) studies. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disorder of hematopoietic stem cells, characterized by a somatic mutation of the phosphatidylinositol glycan-class A (PIGA). The PIGA gene products are crucial for biosynthesis of glycosylphosphatidylinositol (GPI) anchors, which attaches a number of proteins to the plasma membrane of the cell. Amongst these proteins, the CD55 and CD59 are complement regulatory proteins. The CD55 inhibits C3 convertase whereas the CD59 blocks the membrane attack complex (MAC) by inhibiting the incorporation of C9 to MAC. The loss of complement regulatory protein renders the red cell susceptible to complement-mediated lysis leading to intravascular and extravascular hemolysis. The intravascular hemolysis explains most of the morbid clinical manifestations of the disease. The clinical features of syndrome of PNH are recurrent hemolytic episodes, thrombosis, smooth muscle dystonia, and bone marrow failure; other important complications include renal failure, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The most used therapies were blood transfusions, immunosuppressive, and steroid. Allogeneic stem cell transplantation was also practiced. At present, the therapy of choice is eculizumab (Soliris, Alexion Pharmaceuticals), a humanized monoclonal antibody that blocks activation of the terminal complement at C5. The limiting factor for this therapy is breakthrough hemolysis and the frequent dosing schedule. Ravulizumab (ALXN1210) is the second generation terminal compliment inhibitor which seems to provide a sustained control of hemolysis without breakthrough hemolysis and with a longer dosing interval.

DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1048-1048
Author(s):  
Kazuhiko Ikeda ◽  
Tsutomu Shichishima ◽  
Yoshihiro Yamashita ◽  
Yukio Maruyama ◽  
Hiroyuki Mano
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Data Set ◽  
Hematopoietic Stem ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms’ tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of >22,000 probe sets (corresponding to >14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P <0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.

A Spontaneous Reduction of Clone Size in Paroxysmal Nocturnal Hemoglobinuria Patients Treated with Eculizumab for Greater Than 12 Months.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1992-1992 ◽  
Author(s):  
Richard Kelly ◽  
Stephen Richards ◽  
Louise Arnold ◽  
Gemma Valters ◽  
Matthew Cullen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Maintenance Dose ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Hemopoietic Stem Cells ◽  
Intravascular Hemolysis ◽  
Complement Protein ◽  
Clone Size ◽  
Pnh Clone

Abstract Abstract 1992 Poster Board I-1014 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal disorder of hemopoietic stem cells that is characterized by bone marrow failure, intravascular hemolysis and venous thrombosis. Eculizumab is a humanized monoclonal antibody that specifically binds to the complement protein C5 preventing its cleavage thereby inhibiting the formation of the terminal components of the complement cascade. Eculizumab was approved by the FDA in 2007 after clinical trials showed it was efficacious in treating patients with hemolytic PNH. Prior to eculizumab therapy treatment options were mainly supportive in nature. Historical data shows that a third of patients who survive greater than 10 years undergo spontaneous recovery. We present data on 38 patients with hemolytic PNH treated at a single centre with eculizumab for 12 months or longer. Thirty six of these patients were treated with a loading dose of 600mg every week for 4 doses followed by 900mg the following week and then a maintenance dose of 900mg dose every 14 day. The other 2 patients required a higher maintenance dose of eculizumab, 1200mg every 14 days, due to symptomatic intravascular hemolysis on the standard regime. All our patients had a high PNH granulocyte clone size at the initiation of eculizumab treatment from 52.90% to 99.95% with a median of 96.38%. The duration of eculizumab therapy varied from 12 to 84 months with a median treatment duration of 50 months. Granulocyte clone size was used as it is not subject to as much variation as the erythrocyte clone size which changes both due to blood transfusions and to the extent of intravascular hemolysis present. The proportion of PNH granulocytes probably most accurately reflects the true size of the PNH clone. Seven out of these 38 patients (18.4%) have had a 10% or greater reduction in their granulocyte clone size during the course of their eculizumab treatment. These patients have had a steady and continued decline in their granulocyte clone size throughout their treatment with eculizumab. This may actually be due to an increase in the residual normal cells in some patients (see Table). Two of these patients (U.P.N. 5 and 7) have had such a dramatic reduction in their clone size that they have been able to stop their eculizumab treatment without any observed detriment to their health.TableChange in PNH clones in patients on eculizumabU.P.N.Months on eculizumabNeutrophils PNH clone size (%)Normal neutrophils (%)Pre-treatmentMost recent on treatmentPre-treatmentMost recent on treatment15097.242.82.85724878.063.222.036.335596.484.13.615.941592.577.07.523.051261.732.438.367.664788.362.511.737.578552.98.547.191.5 Two of these 7 patients were treated with ciclosporin for underlying aplasia as compared to 3 of the 31 of those who haven't had a decrease in their clone size. There was no difference in the white cell or platelet count in these 7 patients from when they started eculizumab treatment to the present day indicating that the degree of bone marrow failure present has not changed dramatically during this time course. 5 of the 7 patients had neutrophil clone sizes of less than the median perhaps indicating that recovery requires a certain number of residual normal stem cells to be present. There were no other observed differences to distinguish between patients whose clone size fell and those that did not. It is unlikely that eculizumab has a direct effect on clone size in hemolytic PNH. A more probable hypothesis is that the immune selection in favour of the PNH clone expires over time allowing normal hemopoietic stem cells to repopulate the bone marrow. Whether eculizumab has any influence on this rather than just allowing patients to survive and remain well until recovery occurs is not clear. Our data suggests that there needs to be some normal hematopoietic activity in order for the normal marrow cells to expand and clone size under 95% predicts for recovery. In conclusion, a significant minority of patients with PNH on eculizumab have a progressive reduction in the size of their PNH clone during therapy and in some of these patients the clone falls to a level at which eculizumab can safely be stopped. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria. Richards:Alexion Pharmaceuticals: Honoraria. Hill:Alexion: Honoraria. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Expression of HMGA2 in Blood Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3439-3439
Author(s):  
Yoshiko Murakami ◽  
Rieko Ohta ◽  
Norimitsu Inoue ◽  
Hideyoshi Noji ◽  
Tsutomu Shichishima ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Somatic Mutation ◽  
Untranslated Region ◽  
Selection Pressure ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Ectopic Expression ◽  
Clonal Expansion ◽  
Hematopoietic Stem ◽  
Immune Mediated

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation of PIG-A gene in one or few hematopoietic stem cells and subsequent clonal expansion of mutant stem cells that leads to development of symptoms. It is known that PIG-A mutation is insufficient to account for the clonal expansion required for clinical manifestation of PNH. We are proposing a 3-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Based on the close association of PNH with aplastic anemia, it has been suggested that the selection pressure is immune mediated. However, in spite that over 60% of patients with aplastic anemia have subclinical population of GPI-deficient hematopoietic cells at diagnosis, only 10% develop clinical PNH, suggesting that step-1 and 2 are insufficient to cause PNH. Under immune mediated selection pressure, GPI-deficient cells not only survive, but also proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate increases the chance that additional genetic mutations are acquired, in turn leads to Step 3. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative phenotype. According to this hypothesis, we searched for the candidate gene for Step 3. We reported 2 patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome12, making the break within the 3’ untranslated region in HMGA2. This gene encodes an architectural transcription factor which is deregulated in many benign mesenchymal tumors (Blood. 2006 vol.108 no.13, p4232). Recently, many reports show that truncation of 3’ untranslated region of HMGA2 disrupts binding of miRNA, let-7, which regulates both transcription and translation of HMGA2. In fact, these two PNH patients with chromosomal abnormalities had ectopic expression of HMGA2 in the bone marrow. Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation. We have established the method for stable isolation of mRNA and miRNA from blood and bone marrow cells from PNH patients and analyzed the expression of HMGA2 and let-7 by quantitative RT-PCR. We have analyzed the peripheral blood from 8 healthy volunteers and 12 PNH patients. The samples from patients had significantly higher expression of HMGA2 than those from normal volunteers (relative mRNA expression, 4.8±2.4 vs 1.3±0.3, p<0.05). We analyzed the genomic sequence of three patients including one who has highest HMGA2 expression and found no mutation in 3’ untranslated region. We also analyzed the expression of miRNA and found significantly lower expression of let7b and c in patients. Surprisingly, truncated form without 3’ untranslated region is predominantly expressed in patients. There maybe deregulation of alternative splicing of HMGA2 gene in patients, which needs further investigations. We are now analyzing more PNH samples including bone marrow, where proliferation of stem cells takes place, to investigate whether high expression of HMGA2 contributes to the pathogenesis of PNH. In addition we are going to analyze whether high expression of HMGA2 causes the clonal expansion of PNH cells using PNH mouse model.

Ham Test For Therapeutic Monitoring Of Eculizumab In Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4876-4876
Author(s):  
Miriam Arcavi ◽  
Fernanda Ceballo ◽  
Andres L. Brodsky ◽  
Nora Silvia Halperin ◽  
Norma Cantenys ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Alternative Pathway ◽  
Specific Treatment ◽  
Chimeric Antibody ◽  
Intravascular Hemolysis ◽  
Hematopoietic Stem ◽  
Gold Standard Method

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal disease, caused by an inactivating mutation in the PIG-A gene in a hematopoietic stem cell. The PIG-A gene encodes an enzyme required for glycosylphosphatidylinositol (GPI) anchor synthesis. Its inactivation results in a deficiency of many plasma membrane GPI-anchored proteins -including CD55 and CD59, natural inhibitors of the complement cascade- in the involved stem cell and all its progeny (the PNH clone). Intravascular hemolysis, anemia, thrombosis, acute and chronic renal damage, pulmonary hypertension, abdominal pain, esophagic spasm, erectile dysfunction -among others manifestations- are consequences of complement mediated damage of the sensitive PNH blood cells. In 2007 both the FDA and the EMA approved eculizumab, a monoclonal chimeric antibody targeted against C5 fraction of complement, as the first specific treatment of complement mediated PNH manifestations. Flow cytometry (FC) is the gold standard method for diagnosis. The former diagnostic test -the Ham test- is based on the susceptibility of PHN red blood cells (RBC), when they are incubated with both normal and patient sera to lysis mediated by the alternative pathway of complement (APC). APC is activated, in the Ham test, through sera acidification. Despite its physiopathological value, Ham test has been replaced with flow cytometry to diagnose PNH due to a much higher sensitivity and reproducibility. Aims To evaluate the Ham test in PNH treated patients, to monitor the eculizumab-mediated blockade of APC. Patients and methods Ham test was used to monitor APC blockade in the patient serum, testing the ability of the acidified patient serum to lyse his or her own PNH-RBC. Eight patients were diagnosed as PNH by FC and were treated with eculizumab. Six had a good therapeutic response, with decreased levels of both, LDH and the serum total complement hemolytic capacity (CH50). Ham test, in these six patients, showed hemolysis when PNH-RBC were mixed with normal acidified serum but absence of hemolysis when the acidified serum of eculizumab treated patient was added to the PNH-RBC. This result was called “blockade profile” and shows the “ex vivo” APC blockade, confirming thus the eculizumab success. The remaining two patients showed a persistent positivity of the Ham test at day 14 of eculizumab administration (as PNH-RBC lysis continued taking place with both normal and patient acidified sera). One patient demonstrated break through hemolysis occurring near the end of eculizumab dosing period as indicated by increase in LDH. As LDH may increase due to other possible factors (ie hepatic lesions) the positive Ham test confirmed that intravascular hemolysis was taking place, possibly due to a shorter eculizumab half life. An increase of the eculizumab dose to 1,200 mg/14 days reinstated lower LDH levels and the blockade profile in the Ham test (Table). There has been a single patient treated with eculizumab where LDH did not reduce. There was a persistently positive Ham test, elevated LDH and free hemoglobin levels and normal CH50 values despite a dose of 1,200 mg of eculizumab every 14 days (Table). A genetic study found in this case a C5 mutation, which seems responsible of the lack of response to eculizumab. Conclusions In our experience, the Ham test has proved to be a useful and economic method to monitor the effectiveness of eculizumab treatment in cases with high LDH levels due to either a) other causes than intravascular hemolysis, or b) no responsive patients due to pharmacokinetic (inadequate eculizumab concentration) or pharmacodynamic causes. Disclosures: Brodsky: Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Colin:Alexion Pharmaceuticals: Consultancy.

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.

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