scholarly journals Is Nature Truly Healing Itself? Spontaneous Remissions and Clonal Replacement in Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4303-4303
Author(s):  
Carmelo Gurnari ◽  
Simona Pagliuca ◽  
Tariq Kewan ◽  
Waled Bahaj ◽  
Ishani Nautiyal ◽  
...  
Keyword(s):  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Spontaneous Remission ◽  
Hla Class I ◽  
Class I ◽  
Driver Gene ◽  
Complete Disappearance ◽  
Type Ii ◽  
Initial Flow ◽  
Type Iii ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is considered to be curable only through the means of allogeneic HSCT. One of the many fascinating and scientifically instructive aspects of the pathogenesis of this disease is the rare possibility of its spontaneous remission with disappearance of PNH clone and abatement of clinical symptoms, which has always captivated the research community. Due to the orphan nature of the condition, no clinical predictors have been identified so far as harbingers of this phenomenon. In a classical scenario, exhaustion of PNH clone may be associated with reappearance of aplastic anemia (AA), in which PNH clone reflects a semi-maladaptive attempt of recovery. Consequently, one could stipulate that the retraction of PNH clone(s) would have to be associated with a compensatory re-expansion of normal hematopoiesis should normal counts be maintained. The recent insights into the AA/PNH pathobiology shed light onto molecular underpinnings of polyclonal vs oligoclonal hematopoiesis and their dynamics. Here, through application of NGS we attempted to better discern the mechanism of PNH spontaneous remission taking advantage of our internal cohort of PNH patients. Among 92 patients with a diagnosis of hemolytic PNH (M:F ratio 0.88, median age 38 years, range 9-84) 41% were primary PNH (pPNH) while 59% were secondary to AA (sPNH). Overall, patients were clinically followed-up for a median time of 68 months (2-339). Median granulocyte clone size was 73% (22-99) with the majority of cases being classified as having a type III dominant red blood cells (RBCs) clone (80%) while 20% type II. Within this cohort, a total of 3 patients underwent spontaneous remission. UPN1 was a 69-year-old male diagnosed with pPNH at the age of 46 after an episode of deep vein thrombosis. He had been managed with prednisone, transfusions and anti-coagulation because of recurrent thrombotic episodes. Once available, he was started on eculizumab and later continued on ravulizumab. His initial flow cytometry study revealed the presence of a type III RBCs clone of 40% and a granulocyte clone of 89%. After 9 years of anti-complement therapy, the patient's clone started a slow decrease and the most recent study revealed a granulocyte clone of 0.02%. Molecular analysis performed at the time of eculizumab start showed a co-dominant mutational configuration by variant allelic frequency (VAF) with PIGA deletion (p.94_95del; VAF 29%) and a BCOR nonsense (p.Y1446X; VAF 27%). No HLA class I/II mutations were found in two longitudinal samples collected 1 year before and after eculizumab start. However, at the last sequencing performed after the complete disappearance of the PNH clone, the patient developed ASXL1 (p.E635Rfs*, VAF 26%) and ZRSR2 (p.E120Gfs*, VAF 42%) mutations along with retraction of the previous PIGA lesion. No decrease in blood counts was noted. UPN2 was a 58-year-old male initially diagnosed with severe AA at the age of 48 and treated with ATG + CsA. At that time, he had a co-existing PNH granulocyte clone of 28%. After 1 year from IST his PNH clone dropped to 1% and since then has been consistently below 1%. Patient has never received anti-complement therapy. At the time of PNH clone retraction, no HLA class I/II or myeloid driver mutations were found and PIGA mutations were not detectable. However, longitudinal molecular studies performed after disappearance of PNH clone revealed the acquisition of ASXL1 p.Q512X mutation at an initial VAF of 23%, which doubled (45%) at last follow-up 5 years later while normal counts persisted. UPN3 was instead a 59-year-old lady diagnosed with pPNH at the age of 30. She had a granulocyte clone as high as 43% with a type II RBCs clone of 17% and a typical PIGA splice site c.981+1G>A mutation (VAF 15%). She was initially treated with transfusions and steroids and her course was complicated by a cerebral venous sinus thrombosis. Patient was eventually given eculizumab and her PNH clone started decreasing until it vanished (last 0.04%) after 8 years. Analysis of samples prior to and after PNH disappearance did not show any HLA class I/II nor myeloid driver gene mutations with absence of PIGA alterations at last sequencing. PNH spontaneous remissions are rare events. In addition to be replaced by polyclonal hematopoiesis, PIGA clones may be swept by CHIP lesions in myeloid genes (e.g. ASXL1) characterized by improved fitness advantage in a process of Darwinian selection. Figure 1 Figure 1. Disclosures Maciejewski: Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy.

Allo-HCT from MUD/MRD for Paroxysmal Nocturnal Hemoglobinuria - 12 Years of Experience

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5886-5886 ◽  
Author(s):  
Miroslaw Markiewicz ◽  
Malwina Rybicka-Ramos ◽  
Monika Dzierzak-Mietla ◽  
Anna Koclega ◽  
Krzysztof Bialas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cyclosporine A ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Conflicts Of Interest ◽  
Hemorrhagic Cystitis ◽  
Chronic Gvhd ◽  
Complete Disappearance ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Pnh Clone

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal abnormality of hematopoietic stem cell leading to lack of phosphatidylinositol glycoproteins, sensitizing cells to complement-mediated lysis. Despite the efficient symptomatic treatment of hemolytic PNH with eculizumab, allo-HCT is the only curative treatment for the disease, although outcomes presented in the past were controversial. Material and methods: We report 41 allo-HCTs: 37 from MUD and 4 from MRD performed for PNH in 2004-2016. Median age of recipients was 29(20-62) years and donors 30(19-53), median time from diagnosis to allo-HCT was 16(2-307) months. Median size of PNH clone was 80% granulocytes (0.5%-100%). Indication for allo-HCT was PNH with aplastic/hypoplastic bone marrow (19 pts), MDS (2 pts), overlapping MDS/aplasia (3 pts), severe course of PNH with hemolytic crises and transfusion-dependency without access to eculizumab (17 pts). Additional risk factors were Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (2 pts), chronic hepatitis B (1 pt) and C (1 pt). The preparative regimen consisted of treosulfan 3x14 g/m2 plus fludarabine 5x30 mg/m2 (31 pts) or treosulfan 2x10 g/m2 plus cyclophosphamide 4x40 mg/kg (10 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG in MUD-HCT. 2 pts instead of cyclosporine-A received mycophenolate mofetil and tacrolimus. Source of cells was bone marrow (13 pts) or peripheral blood (28 pts) with median 6.3x108NC/kg, 5.7x106CD34+cells/kg, 24.7x107CD3+cells/kg. Myeloablation was complete in all pts with median 9(1-20) days of absolute agranulocytosis <0.1 G/l. Median number of transfused RBC and platelets units was 9(0-16) and 8(2-18). Results: All pts engrafted, median counts of granulocytes 0.5 G/l, platelets 50 G/l and Hb 10 g/dl were achieved on days 17.5(10-33), 16(9-39) and 19.5(11-34). Acute GVHD grade I,II and III was present in 16, 7 and 3 pt, limited and extensive chronic GVHD respectively in 11 and 3 pts. LDH decreased by 73%(5%-91%) in first 30 days indicating disappearance of hemolysis. 100% donor chimerism was achieved in all pts. In 1 patient donor chimerism decreased to 81% what was treated with donor lymphocytes infusion (DLI). 3 patients died, 1 previously hemodialysed pt died on day +102 due to nephrotoxicity complicating adenoviral/CMV hemorrhagic cystitis, two other SAA patients with PNH clone<10% died on days +56 due to severe pulmonary infection and +114 due to aGvHD-III and multi organ failure. Complications in survivors were FUO (10 pts), CMV reactivation (13), VOD (1), neurotoxicity (1), venal thrombosis (1), hemorrhagic cystitis (4) and mucositis (8). 38 pts (92.7%) are alive 4.2 (0.4-12) years post-transplant and are doing well without treatment. Complete disappearance of PNH clone was confirmed by flow cytometry in all surviving pts. Conclusions: Allo-HCT with treosulfan-based conditioning is effective and well tolerated curative therapy for PNH. Disclosures No relevant conflicts of interest to declare.

scholarly journals Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria

Blood ◽  
1995 ◽  
Vol 86 (4) ◽  
pp. 1586-1589 ◽  
Author(s):  
RE Ware ◽  
WF Rosse ◽  
SE Hall
Keyword(s):  
Bone Marrow ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Clinical Manifestations ◽  
Surface Expression ◽  
Surface Proteins ◽  
Type Ii ◽  
Type Iii ◽  
Hematologic Disorder ◽  
Marrow Stem Cell ◽  
Proliferative Advantage

The hematologic disorder paroxysmal nocturnal hemoglobinuria (PNH) occurs following an acquired somatic mutation in the Piga gene within a bone marrow stem cell. The progeny of this mutated cell cannot synthesize glycosylphosphatidylinositol (GPI) anchors, with a resultant deficiency in surface expression of all GPI-linked proteins. The protean clinical manifestations of PNH presumably result from the deficiency of these GPI-linked surface proteins. To explain the observation that neutrophils are affected at a significantly higher percentage than circulating erythrocytes and to analyze the proliferative rates of erythroid production in PNH, we studied 25 patients using flow cytometry. The fluorescent dye thiazole orange was used to detect reticulocytes, and CD59 monoclonal antibody was used to identify GPI-deficient cells. In contrast to the mature circulating erythrocytes, the percentage of abnormal reticulocytes was similar to the percentage of affected neutrophils. However, the vast majority of reticulocytes was completely GPI-deficient, ie, were type III cells, even in patients with only modest numbers of circulating type III erythrocytes. In addition, greater than 5% type II reticulocytes were identified in only 3 patients, although greater than 5% type II mature erythrocytes were identified in 10 of 25 patients. The results show that the erythroid and neutrophil bone marrow precursors have an equivalent proliferative advantage in PNH. The data also have important implications for the origin of type-II erythrocytes in PNH.

scholarly journals Immunogenomics of Paroxysmal Nocturnal Hemoglobinuria: A Model of Immune Escape

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-22
Author(s):  
Carmelo Gurnari ◽  
Simona Pagliuca ◽  
Cassandra M Kerr ◽  
Hassan Awada ◽  
Sunisa Kongkiatkamon ◽  
...  
Keyword(s):  
T Cell ◽  
Somatic Mutations ◽  
Immune Escape ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
Cytotoxic T Cell ◽  
Disease Evolution ◽  
Immune Pressure ◽  
Pnh Clone

Unlike leukemic driver mutations, PIGA mutations produce an escape phenotype in the context of immune-mediated bone marrow failure such as aplastic anemia (AA). Another way to create clinical advantage will be to disable HLA-mediated cytotoxic T cell recognition. Determinants of cytotoxic T cell response might include some accessories glycosylphosphoinositol (GPI)-linked moieties but the main stimulus is likely to be provided by HLA-presented antigenic peptides. Somatic hits in HLA genomic region (microdeletions, uniparental disomies [UPDs] of HLA locus on 6p and later mutations) have been previously assessed in AA patients [1-3]. Mechanistic analogy to immune-privileged GPI-anchor protein deficiency in PNH due to PIGA mutations[4] or deletion[5] of PIGA locus are obvious. We stipulate that HLA mutations may contribute to the intrinsic expansion of PNH clones under immune pressure being: i) additive to the effects of PIGA mutations in creating immune escape or ii) redundant and thus less frequent in PNH clones as in patients without PIGA mutation. Using a deep targeted-sequencing panel covering HLA classical loci, and applying an in-house newly developed pipeline for the study of the HLA region (AbstractID#142501), we detected class I/II HLA somatic mutations of 10 patients with PNH. An integrative mutational analysis of PIGA and myeloid genes was then performed in order to comprehensively evaluate the role of HLA somatic hits within the scenario of PNH clonal evolution. At the time of this submission HLA sequencing was completed for a total of 35 patients but full analysis is available for the first 10 cases. Overall, of these 10 PNH patients 20 samples were analyzed from sorted GPI(+) and GPI(-) myeloid fractions (mean purity &gt;95%). Median age at diagnosis was 36 years (11-66) while median PNH granulocyte clone size at time of sampling was 76% (5.11-99). A total of 41 PIGA mutations (Fig.1A) were detected solely in the GPI(-) fraction (mean VAF 58%), with 8 patients harboring clonal mosaicism as previously described.[6] Six somatic mutations of HLA class I (N=3, Fig.1B) and class II (N=3, Fig.1C) loci were found in 4 patients (67% detected on GPI(+) and 33% on GPI(-) fraction) at a low VAF (mean 3.36%). All these events were insertions or deletion of one or more bases. Class I mutations were located in intron 5, exon 3 and 3' untranslated regions (UTR). Class II were found instead in exon 2 (N=2) and intron 4. A functional and topographical annotation based on IPD-IMGT/HLA database suggested that exonic mutations were disruptive, impairing the bio-functionality of antigen presentation site. The detected intronic mutations instead impair HLA moiety assembling within cellular membrane, possibly altering splicing of the transmembrane domain. Moreover, a computational prediction of the regulatory domains involved in the 3'UTR aberration, showed a possible involvement of the miRNA has-miR-4524a-3p binding site, potentially affecting HLA post-transcriptional regulation. Of note, in 1 patient (UPN 9, Fig.1D) we did not find any PIGA, PIGT or HLA mutation. Finally, myeloid gene mutations analysis revealed the presence of a subclonal ASXL1 mutation in 1/10 patients in the GPI(-) fraction. Of note, this patient (UPN 1) had older age and showed 12 different somatic PIGA hits. This finding is probably explicable with the scenario of PIGA as the initial ancestral event accompanied by secondary mutations previously shown by our group as occurring in 10% of PNH cases in the course of disease evolution.[7-8] In summary, somatic HLA class I/II mutations can be found in patients with PNH. HLA mutations can occur in GPI(+) cells in subclonal fashion but also in GPI(-) cells. The latter clonal mosaicism indicates that various mechanisms of immune escape may play a role. Subclonal HLA mutations may impact the immune pressure on PNH clone dynamics, reflecting an alternative immune escape pathway in patients without PNH clone. (Fig.1E) In addition, detection of occasional "myeloid" hits suggests that various modes of PNH clone maintenance and expansion may be operative. We will present at ASH analysis of a full cohort of these patients including properly powered clinical correlations. Figure 1 Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.

scholarly journals Dysphagia with Blood Disorder? an Unusual Presentation for Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Taha Sheikh ◽  
Rakan Albalawy ◽  
Hina Shuja ◽  
Navkirat M Kahlon ◽  
Danae M Hamouda
Keyword(s):  
Smooth Muscle ◽  
Abdominal Pain ◽  
Erectile Dysfunction ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Red Cells ◽  
Type I ◽  
Type Ii ◽  
Intravascular Hemolysis ◽  
Type Iii ◽  
Blood Disorder

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hemolytic disorder of the multipotent myeloid stem cells, due to underexpression of surface complement inhibitory proteins CD55 and CD59, affecting 0.13/100,000 patients per year. Besides anemia and thrombosis, PNH can also cause smooth muscle dystonias owing to various mechanisms in the body. Studies have described smooth muscle dystonias presenting as recurrent abdominal pain, lethargy and erectile dysfunction, but intermittent dysphagia is rare with PNH. We describe a case of dysphagia with negative work up as initial presentation of PNH. 16-year-old female with no significant past medical history presented to the clinic with 1 week of ongoing fatigue and difficulty swallowing. She never had any similar complaints in the past nor in her family. The dysphagia was progressively worsening, initially for solid, then with liquids, accompanied by nausea, vomiting and globus sensation in the middle chest. No prior psychiatric history, binging-purging behavior, chest pain, shortness of breath, heat/cold intolerance, weight changes, bladder or bowel dysfunction. She also denied any recent travels, allergies, transfusions, sick contacts, or over-the-counter medication use. Initial labwork showed Hb 11.2 g/dL, MCV 80 fL, WBC 9 x103 K/uL, and platelet 219,000 K/uL. Metabolic panel, troponin, BNP, ANA, rheumatoid factor, lyme serology, vitamin B12, folate and iron studies were within normal limits. Mentzer index &lt;13, serum protein electrophoresis ruled out Thalassemia. LDH was elevated and Haptoglobin decreased. Direct and indirect Coomb's antibody test were negative. Peripheral smear showed normocytic normochromic anemia with normal morphology of RBCs. Flow cytometry showed lack of CD55 and CD59. Esophagogastroduodenoscopy, done for dysphagia, to rule out luminal obstruction was unremarkable. CT chest with contrast ruled out external compression on esophagus. Videofluoroscopic swallow study revealed segmental, dynamic obstruction to both solids and liquids in the lower third of the esophagus. Dysphagia was attributed to PNH and patient was started on eculizumab. She responded to the medication in 3 weeks, during which outpatient symptomatic therapy was given for her anemia along with soft semisolid diet. In PNH, the RBCs may have varying degree of CD59 and glycosyl phosphatidylinositol (GPI) anchor protein deficiency, with unaffected cells described as type I, partially affected cells called type II, and complete lack of the proteins deemed type III red cells. PNH red cell type III and type II red cells are 15 to 25 times and 3 to 5 times more sensitive to complement-mediated lysis than the type I normal red cells. This variability in the severity of the deficiency, as well as in the proportion of the cell population affected, defines the extent of the clinical manifestations of the disease. Approximately 40% of patients have a combination of types I, II and III PNH cells With hemolysis, after exceeding haptoglobin's binding capacity, the free hemoglobin binds any available nitrous oxide (NO) in the blood. Hemolysis also reduces endogenous NO generation by conversion of l-arginine, which is a component in NO generation, to ornithine, thereby further reducing the systemic availability of NO. NO is endogenous smooth muscle relaxor via activation of cGMP. Decreases in NO levels are hypothesized to cause smooth muscle contraction, manifesting as abdominal pain, erectile dysfunction and rarely, dysphagia in the lower two-third of the esophagus which is predominantly smooth muscle. These symptoms occur more commonly in patients with large PNH clone sizes and, therefore, higher hemolytic rates. In this case study, our focus was to recognize PNH among differentials for new onset dysphagia due to complement-mediated intravascular hemolysis causing gastrointestinal dysmotility. In doing so, invasive investigations may be avoided. Advance studies may be needed to completely understand the causes and correlate between PNH, intravascular hemolysis and dysphagia. Disclosures No relevant conflicts of interest to declare.

Identification and Clinical Significance of Type II Granulocytes Among Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Identified Using Multiparameter High-Sensitivity Flow Cytometry.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3015-3015 ◽  
Author(s):  
Mayur K Movalia ◽  
Andrea Illingworth
Keyword(s):  
Flow Cytometry ◽  
High Sensitivity ◽  
Type I ◽  
Type Ii ◽  
Flow Cytometry Assay ◽  
Clone Size ◽  
Type Iii ◽  
Gpi Proteins ◽  
Terminal Complement ◽  
Pnh Clone

Abstract Abstract 3015 Poster Board II-991 PNH is a hematopoietic stem cell disorder in which unregulated activation of terminal complement leads to impaired quality of life and significant ischemic morbidities with shortened lifespan. Life-threatening thromboembolism (TE) is the most feared complication of PNH, accounting for 45% of patient deaths. Thrombosis has been observed in PNH patients regardless of the level of hemolysis. Additionally, platelet activation with subsequent consumption and thrombocytopenia are observed more often in PNH patients at risk for thrombosis. Current laboratory PNH diagnostic methods rely on flow cytometry to characterize PNH clones. PNH granulocytes (Gran) are typically detected using antibodies to a variety of GPI-linked markers including CD55, CD59, CD16, CD24, and CD66b. Recently, FLAER, a fluorescent proaerolysin variant that binds directly to the GPI anchor, has been used to identify and quantify GPI-deficient WBCs at a very high level of sensitivity. Although these markers are well established to detect granulocytes with normal expression of GPI proteins (Type I cells) and complete loss of GPI proteins (Type III cells), less is known about their ability to detect granulocytes with partial loss of GPI proteins (Type II cells). The ability to detect both PNH Type II RBCs and WBCs would provide clinically important information since quantitation of only PNH RBC clones can be confounded by transfusion or hemolysis. We evaluated 2,921 consecutive patient peripheral blood samples submitted for PNH diagnostic testing with a high-sensitivity flow cytometry assay for granulocytes that includes the fluorescent proaerolysin variant (FLAER) with confirmatory lineage-specific antibodies to GPI-linked antigens to distinguish Type I, II and III Gran clones. In addition, standard CD235/CD59 analysis was performed on the RBCs and evaluation with FLAER, CD14 and lineage-specific antibodies was performed on the monocytes. 216 patient samples (7.4%) had a detectable PNH gran clone (≥ 0.01% PNH Type III granulocytes and an absolute count of at least 50 cells). Clinical information was available for 162 of these patients (Table I). Of these samples, nineteen (8.8 %) patients demonstrated a distinct Type II Gran population, ranging in size from 1.2 – 65% (median clone size = 7%). In 4/19 patients, this Type II Gran population represented >50% of the total (Type II + Type III) PNH cells. In 10/19 patients (53%), a type II monocyte population was identified. Evaluation of the granulocyte markers (Table II) showed that the type II gran population was detectable in all cases by FLAER and in decreasing percentage by CD66b (88%), CD55 (50%), CD24 (47%) and CD16 (0%). Patients with Type II Gran clones had a significantly larger median total Gran PNH clone size than those without Type II Gran clones (87% vs. 11%; p= 0.0003), as well as larger median Type II and Type III RBC clones, likely a reflection of the ability to detect type II gran PNH clones with overall larger PNH clone sizes. Patients with Type II Gran clones showed significantly lower median platelet (plt) counts (54 ×109/L) than patients without Type II Gran clones (116×109/L; p< 0.01). Patients with Type II Gran clones had similar peripheral WBC, peripheral RBC, absolute neutrophil count, and hemoglobin (Hgb) compared to patients without Type II Gran clones, suggesting that differences in platelet counts are likely not due to differences in underlying marrow blood cell production. Type II PNH cells are an important component of the PNH diagnostic evaluation and both RBC and Gran Type II clones should be enumerated. In a large population of patients tested for the presence of PNH clones using a high sensitivity flow cytometry assay, a significant proportion of patients were identified with Type II PNH Gran clones. This study identified FLAER as the best reagent to identify type II Gran PNH clones and showed CD16 was least useful. This study also identified a clinical association between the presence of significant Type II clones and thrombocytopenia, potentially indicative of terminal complement-mediated platelet consumption. These findings are consistent with an increased risk of thrombosis in patients with significant Type II PNH clones. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria

2020 ◽  
Vol 98 (2) ◽  
pp. 179-192 ◽  
Author(s):  
D. Robert Sutherland ◽  
Stephen J. Richards ◽  
Fernando Ortiz ◽  
Rakesh Nayyar ◽  
Miroslav Benko ◽  
...  
Keyword(s):  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Type Ii ◽  
Type Iii

The Favorable Outcome of Allo-HCT from MUD Following Treosulfan-Based Conditioning in Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4420-4420
Author(s):  
Miroslaw Markiewicz ◽  
Ewa Mendek-Czajkowska ◽  
Barbara Zupanska ◽  
Sebastian Giebel ◽  
Slawomira Kyrcz-Krzemien
Keyword(s):  
Risk Factors ◽  
Bone Marrow ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Therapeutic Option ◽  
Hemorrhagic Cystitis ◽  
Chronic Gvhd ◽  
Complete Disappearance ◽  
Haemorrhagic Cystitis ◽  
Conditioning Treatment ◽  
Pnh Clone

Abstract Although allo-HCT has a potential to cure patients with paroxysmal nocturnal hemoglobinuria (PNH), experience with allo-HCT from matched unrelated donors (MUD) is limited and favorable conditioning treatment for PNH has not been established. We report results of 8 allo-HCTs from MUD (matched in HLA-A,B,C,DR,DQ alleles) performed for PNH with treosulfan-based conditioning in years 2004–2008 in Katowice, Poland. Median age of recipients was 27 years (range 20–35) and donors 33(28–43), median time from diagnosis to transplantation was 20(15–36) months. Median size of PNH clone was 90% granulocytes (range 4%–95%), 4 donors were female and their recipients were male, AB0 incompatibility was minor in 5 pairs, major in 1 and bi-directional in 1 pair. Indication for transplantation despite severe refractory hemolysis with transfusion-dependency (observed in 6 pts) was aplastic or hypoplastic bone marrow and pancytopenia (5 pts), MDS (1 pt), severe course of PNH exacerbating with hemolytic crises (1 pt). Additional risk factors included thrombotic history with Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (1 pt) and additional serological risk factors for hemolysis (1 pt). The preparative regimen consisted of treosulfan 3×14 g/m2 plus fludarabine 5×30 mg/m2 (7 pts) and treosulfan 2×10 g/m2 plus cyclophosphamide 4×40 mg/kg (1 pt). GVHD prophylaxis consisted of thymoglobuline 3×2 mg/kg pretransplant, cyclosporine-A 3 mg/kg/d since day -1 and methotrexate on days 1,3,6,(11). Transplanted cells were harvested from bone marrow (5 donors) and peripheral blood (3 donors) with median numbers 3.0 and 10.9 ×10(8)NC/kg, 3.0 and 6.9 ×10(6)CD34+ cells/kg, 27.5 and 427.7 ×10(6)CD3+ cells/kg, respectively. Myeloablation was complete in all pts with median 10 days (5–14) of absolute agranulocytosis &lt;0.1 G/l. Median number of transfused RBC units was 7 (1–12) and of single-donor platelets units 9 (3–15). All pts engrafted, median granulocyte count of 1.0 G/l was achieved on day 17 (12–22), platelets 50 G/l on day 21 (9–30) and Hb 10 g/dl on day 30 (16–50). No serious acute GVHD was observed, grade I was transiently present in 4 pts and grade II in 3 pts. Transient limited chronic GVHD was observed in 2 pts. Hemolysis gradually decreased. 100% donor chimerism was present in the bone marrow on day +100 at latest in all pts. 1 previously hemodialysed pt died on day 102 in a consequence of nephrotoxicity complicating adenoviral/CMV haemorrhagic cystitis. Complications in survivors included FUO (2 pts), CMV reactivation (2), VOD (1), neurotoxicity (1), venal thrombosis (1), hemorrhagic cystitis (1) and mucositis (1). 7 pts (87.5%) are alive and are currently doing well under ambulatory control with no signs of hemolysis and without need for further hematological treatment, median follow-up is 23 months (2–44). Complete disappearance of PNH clone was confirmed by flow cytometry in all surviving pts. We conclude that MUD allo-HCT with treosulfan-based conditioning can be effectively and relatively safely used in PNH patients and thus it should be considered as a valuable therapeutic option in PNH.

Flow Cytometry Screening for Paroxysmal Nocturnal Hemoglobinuria Abnormal Clones in Saudi Arabia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5162-5162
Author(s):  
Nahlah AlGhasham ◽  
Yasmeen Abulkhair ◽  
Salem Khalil
Keyword(s):  
Flow Cytometry ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Conflicts Of Interest ◽  
Clone Detection ◽  
Sample Type ◽  
Type Ii ◽  
Inhibitory Protein ◽  
Clone Size ◽  
The Past ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease with insidious process, chronic course and life-threatening condition. PNH is clinically defined by the deficiency of the endogenous glycosyl phosphatidylinositol (GPI)-anchored complement inhibitory protein. It has always aroused interest in the medical profession rendering screening and proper diagnosis by flow cytometry (FCM) technology a priority We reviewed all samples submitted for PNH/ FCM screening for the past 2 years (2012-2013) at hematology section, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center (General Organization). We collected the positive cases and reviewed them for age, gender, indication for screening, sample type, size of the PNH clone and cell type affected. Immunophenotypic analysis was performed using gating antibodies CD45, CD15, CD33, CD235a GPI-linked antibodies, CD59, CD14, and CD24 as well as fluorescent Aerolysin (FLAER). In a total of 366 peripheral blood samples submitted for PNH/ FCM screening fifteen samples (4%) were positive for PNH clones but only 12 patients were available for analysis. The median age for our patients was 34 years with approximately equal male to female distribution. 12 cases showed type II and III clones within the RBCs with clone size ranging between 0.04% and 56%. Analysis of granulocytes and monocytes revealed type III clone in 8 cases, type II and III clone in 3 cases and non in one case. The percentage of the clone varies between the granulocytes and monocytes and ranges from 1% up to 100%. Of 12 positive PNH cases, 8 (66.7%) patients were diagnosed as having aplastic anemia (AA), 1 (8.3%) patient with Budd-Chiari syndrome, 1 (8.3%) patient has chronic immune thrombocytopenia (ITP), and 2 (16.7%) patients presented with pancytopenia. This study confirms the rarity of the disease since only 4% of the submitted samples for analysis turned to be positive for PNH. The detection limit for a PNH clone by FCM in the RBC or WBC is 0.01%. Identification of small PNH clone is greater FCM sensitivity relative to old test used for the same purpose (Ham test). The use of the FLAER allowed us to detect granulocytic PNH clone, however, granulocytes PNH clone detection alone without RBCs clone detection is not recommended. This review confirms the previous percentage of positive cases (5.9%) reported from this center on a smaller number of cases during the past few years. Disclosures No relevant conflicts of interest to declare.

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