PNH Clone Size By Flow Cytometry and Its Correlation with PIG Gene Mutation

Abstract Background: Routine diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) is based on flow cytometric measurement of PNH clone in RBC and granulocyte with sensitivity of 1%, and recent introduction of high-sensitive flow cytometry (FCM) makes it possible to detect 0.01% clone. However, the clinical significance of small PNH clone has not been elucidated, so even the necessity to treat or not is controversial. We investigated whether quantitative results of PNH clone size measured by FCM correlate with mutant burden of PIG gene. Methods: A total of 44 specimens from 20 patients whose PNH clone size was >1% in either RBC or granulocyte by routine FCM were enrolled (classical PNH n=10, AA/PNH n=8, MDS/PNH n=2). To detect small quantity of cells with PIG gene mutation, we performed ultra-deep sequencing (average depth 3000X) for PIGA, PIGM, PIGT, and PIGX genes on these 44 consecutive specimens. Results: Sixteen patients (80.0%) were found to harbor PIG gene mutations: 15 patients had PIGA mutation and 1 patient had PIGM mutation. Granulocyte PNH clone size and variant allele frequency (VAF) of PIG gene mutation showed higher correlation (Spearman's r=0.73, p=0.0002) than that of RBC (Spearman's r=0.61, p=0.0073). All patients harboring PIG gene mutation showed more than 10% PNH clone by FCM. In contrast, 4 patients who did not have PIG gene mutation showed less than 10% PNH clone by FCM. Conclusion: Considering the mechanism of PNH development, the presence of PIG gene mutation is a definitive evidence supporting PNH diagnosis. PNH granulocyte clone size more than 10% by FCM seems to be clinically significant in relevance to PIG gene mutation. We suggest that neither high sensitivity FCM nor the detection of PIG gene mutation is a requisite for routine clinical diagnosis of PNH. Disclosures No relevant conflicts of interest to declare.

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Observational Study Of PNH Clone Size By High Sensitivity Flow Cytometry In High-Risk Patients InA Large Russian Population

Blood ◽

10.1182/blood.v122.21.4867.4867 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4867-4867

Author(s):

Elena Babenko ◽

Alexandra Sipol ◽

Vyacheslav Borisov ◽

Elena Naumova ◽

Elena Boyakova ◽

...

Keyword(s):

Flow Cytometry ◽

High Risk ◽

Research Funding ◽

Surface Membrane ◽

High Risk Patient ◽

High Sensitivity ◽

Clone Size ◽

Hematopoietic Stem ◽

Pnh Clone

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and life-threatening hematopoietic stem cell disease caused by a partial or absolute deficiency of proteins linked to the cell surface membrane via a glycosylphosphatidyl-inositol anchor, which leads to complement-induced intravascular hemolysis mediated via the membrane attack complex. Multiparameter high-sensitivity flow cytometric measurement of PNH clones is the method of choice for the diagnosis of PNH, as recommended by the International Clinical Cytometry Society (ICCS). After publication of the ICCS guidelines, screening of patients considered at high risk of PNH was commenced in Russia. Data are presented on PNH clone size distribution across patients with relevant ICD-10 diagnostic codes (based on patients′ initial assumed diagnoses). Methods Patients were tested for the presence and size of PNH clones using high-sensitivity flow cytometry across nine laboratories. PNH clone evaluations were performed as described in the ICCS guidelines: CD59/CD235a monoclonal antibodies for RBC; CD45/CD15/CD24/FLAER for granulocytes and; CD45/CD64/CD14/FLAER or CD45/CD33/CD14/FLAER for monocytes. The sensitivity for PNH clone detection was 0.01%. Changes in PNH clone size were evaluated among patients who had follow-up studies after initial measurements. Results 1889 patients were assessed between October 2011 and June 2013 (Table 1). Suspected PNH and bone-marrow disorders (AA, MDS, cytopenia) were the most common reasons for PNH testing. The greatest proportions of patients with PNH clones were among those with of an initial assumed diagnosis of AA or PNH. Notably, around 40% of patients with an initial assumed diagnosis of PNH actually had no detectable PNH clones. Most patients with small clone sizes (< 1%) were in the AA, MDS and hemolytic anemia groups. Overall, mean clone sizes were slightly higher in monocytes (31.5%) than in granulocytes (30.1%) across the diagnostic categories. While there was generally a good correlation between clone size measurements in granulocytes and monocytes (linear regression r2 = 0.9851), 10% of PNH-positive patients had detectable clones only in one of these leucocyte populations (i.e. either in monocytes or in granulocytes, but not both). PNH clones in RBCs were generally lower than in granulocytes. Repeat clone size measurements were performed in 316 patients over a mean follow-up period of 7.8 months. In patients with initial clone sizes <50% the PNH clones tended to decrease over time, whereas in patients with initial clone sizes >50%, clones tended to increase. PNH clones were not changed at all in 98 patients at follow-up, among whom 48% were patients with AA. Conclusion These screening data confirm the utility of high-sensitivity flow cytometry testing in high-risk patient groups to ensure early and accurate diagnosis and to aid in the effective clinical management of patients. Disclosures: Babenko: Alexion: Research Funding. Sipol:Alexion: Research Funding. Borisov:Alexion: Employment. Naumova:Alexion: Research Funding. Boyakova:Alexion: Research Funding. Glazanova:Alexion: Research Funding. Chubukina:Alexion: Research Funding. Pronkina:Alexion: Research Funding. Popov:Alexion: Research Funding. Mustafin:Alexion: Research Funding. Fidarova:Alexion: Honoraria. Lisukov:Alexion: Honoraria. Kulagin:Alexion: Honoraria.

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Ultradeep Sequencing Analysis of Paroxysmal Nocturnal Hemoglobinuria Clones Detected by Flow Cytometry: PIG Mutation in Small PNH Clones

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqaa211 ◽

2020 ◽

Author(s):

Dajeong Jeong ◽

Hee Sue Park ◽

Sung-Min Kim ◽

Kyongok Im ◽

Jiwon Yun ◽

...

Keyword(s):

Flow Cytometry ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Gene Mutations ◽

Sequencing Analysis ◽

Strong Positive Correlation ◽

Variant Allele Frequency ◽

Clone Size ◽

Positive Correlation ◽

Quantitative Correlation ◽

Pnh Clone

Abstract Objectives We aimed to determine whether small paroxysmal nocturnal hemoglobinuria (PNH) clones detected by flow cytometry (FCM) harbor PIG gene mutations with quantitative correlation. Methods We analyzed 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM. We performed ultradeep sequencing for the PIGA, PIGM, PIGT, and PIGX genes in these specimens. Results A strong positive correlation between PNH clone size by FCM and variant allele frequency (VAF) of PIG gene mutation was identified (RBCs: r = 0.77, P < .001; granulocytes: r = 0.68, P < .001). Granulocyte clone size of 2.5% or greater and RBCs 0.4% or greater by FCM always harbored PIG gene mutations. Meanwhile, in patients with clone sizes of less than 2.5% in granulocytes or less than 0.4% in RBCs, PIG gene mutations were present in only 15.9% and 12.2% of cases, respectively. In addition, there was not a statistically significant positive correlation between FCM clone size and VAF or the presence or absence of a PIG mutation. Conclusions Our results showed that in small PNH clones PIG gene mutations were present in only a small portion without significant correlation to VAF or the presence or absence of a PIG mutation.

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PS1119 PNH CLONE SIZE BY FLOW CYTOMETRY AND ITS CORRELATION TO PIG GENE MUTATION

HemaSphere ◽

10.1097/01.hs9.0000562760.46451.20 ◽

2019 ◽

Vol 3 (S1) ◽

pp. 506-507

Author(s):

D. Jeong ◽

H.S. Park ◽

S. Choi ◽

H. Seo ◽

S.-M. Kim ◽

...

Keyword(s):

Flow Cytometry ◽

Gene Mutation ◽

Clone Size ◽

Pnh Clone

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Distribution of PNH Clone Sizes within High Risk Diagnostic Categories Among 481 PNH Positive Patients Identified by High Sensitivity Flow Cytometry

Blood ◽

10.1182/blood.v120.21.1271.1271 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1271-1271 ◽

Cited By ~ 4

Author(s):

Mayur K Movalia ◽

Andrea Illingworth

Keyword(s):

Flow Cytometry ◽

High Risk ◽

Aplastic Anemia ◽

High Risk Patient ◽

High Sensitivity ◽

Risk Patient ◽

Clone Size ◽

Diagnostic Categories ◽

Patient Groups ◽

Pnh Clone

Abstract Abstract 1271 Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder associated with patients (pts) with aplastic anemia (AA), myelodysplastic syndrome (MDS), unexplained cytopenias, unexplained thrombosis, and hemolysis. High sensitivity flow cytometry (HSFC) is recommended by The International Clinical Cytometry Society (ICCS) as the method of choice for diagnosing PNH. The incidence of PNH clones in these patient groups has been previously reported, but the distribution of clinically significant PNH clones not been previously explored. The purpose of this study is to analyze the distribution of PNH clone sizes using HSFC with sensitivity up to 0.01% among 7,699 pts who were screened for PNH clones utilizing CD235a/CD59 for RBCs, FLAER/CD24/CD15/CD45 for neutrophils and FLAER/CD14/CD64/CD45 for monocytes. We evaluated the distribution of PNH clones sizes against the provided ICD-9 diagnostic (DX) codes and evaluated the significance of hemolysis on PNH clone size. Based on a sensitivity of at least 0.01%, 6.2% of all pts (481/7,699) were found to be PNH positive. Of those pts, 3.8% (293/7,699) were found to have a PNH clone size of >1%, while 2.4% (188/7,699) were found to have a PNH clone size of <1%. Of the 481 PNH positive patients, the distribution of PNH clone sizes among high risk diagnostic categories is shown in Table 1. Aplastic anemia (AA) and hemolysis were more commonly associated with PNH clone sizes >20%. In 32 patients reported to have both aplastic anemia and hemolysis, 20 pts (63%) had PNH clone sizes >20%, while 30 pts (94%) had PNH clone sizes >1%. Pts with MDS, cytopenias and thrombosis more frequently showed PNH clones sizes of <1%. However, in 18 pts reported to have either MDS or cytopenias and hemolysis, 4 pts (22%) showed PNH clone sizes >20%, while 10 pts (56%) showed PNH clone sizes >1%. In this single-laboratory experience, we evaluated the distribution of PNH clone sizes among high risk patient groups based on ICD-9 diagnostic code. Pts with large PNH clone sizes are more likely to have clinical symptoms, particularly those associated with hemolysis, and thus most likely to benefit from therapy. In this study, pts with hemolysis showed a higher incidence of PNH clone sizes >20%, underscoring the need to test for hemolysis in these high risk patient groups. In addition, this study confirmed the need to continue actively testing high risk patient populations, including aplastic anemia, myelodysplastic syndrome, unexplained cytopenias, unexplained thrombosis and hemolysis for PNH based on the ICCS recommendations to ensure accurate diagnosis and appropriate therapy. Table 1. Distribution of PNH Clone Sizes within high risk diagnostic categories among 481 PNH+ Patients at Dahl-Chase Diagnostic Services PNH Clone Sizes Total <1% 1–20% >20% Aplastic Anemia 32 39 40 25.8% (111) MDS 20 9 7 8.4% (36) Cytopenias (including pancytopenia, leukopenia and non-hemolytic anemia) 78 27 10 26.7% (115) Hemolysis (including hemolytic anemia and hemoglobinuria) 24 29 129 42.3% (182) Thrombosis 12 4 5 4.9% (21) Miscellaneous 15 2 5 5.1% (22) Not provided 17 6 28 11.9% (51) Note: Table reflects patients who had more than one associated ICD-9 code. Disclosures: Illingworth: Alexion: Consultancy, Honoraria.

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

Blood ◽

10.1182/blood.v114.22.3015.3015 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3015-3015 ◽

Cited By ~ 4

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.

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Paroxysmal Nocturnal Haemoglobinuria Clone In Aplastic Anaemia Patients At The Beginning Of Disease and In Remission

Blood ◽

10.1182/blood.v122.21.4868.4868 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4868-4868

Author(s):

Zalina Fidarova ◽

Elena Mikhailova ◽

Svetlana Lugovskaia ◽

Elena Naumova ◽

Vera Troitskaia ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Aplastic Anaemia ◽

Partial Remission ◽

De Novo ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Paroxysmal Nocturnal Haemoglobinuria ◽

Clone Size ◽

Pnh Clone

Abstract Introduction Aplastic Anaemia (AA) and Paroxysmal Nocturnal Haemoglobinuria (PNH) are severe hematological diseases accompanied by bone marrow failure syndromes. The high-sensitivity flow cytometry standartised methods helped us to detect the PNH clone incedence at AA patients at the different stages of disease and of treatement and to reveal its’ influence on the immunosuppressive therapy (IST) effectiveness. Objective to detect the PNH clone at AA patients at different stages of disease and to reveal its’ influence on the IST effectiveness. Methods 63 patients with severe AA (SAA) who received combined IST with antithymocytic globulin (hATG) and cyclosporin A (CsA) have been included into the study. Mediane age – 26 years (16-65). All 63 patients were divided into 2 groups. The 1st one included de novo AA patients (n=28); the 2nd group – AA patients in complete remission (CR) after IST (n=35). The median remission duration was 3 years (2-6 y). The results of the de novo AA treatment (1stgroup) were evaluated at 3, 6 and 12 months from the start of IST. We used the flow cytometry (Becton Dickinson (BD) FACS Canto II and Beckman Coulter (BC) FC 500) to evaluate the PNH clone. Peripheral blood samples were analyzed with antibodies CD45(BD), CD15(BD), CD64(BD), CD235a(BC), GPI-tying antibodies CD59 (Invitrogen), CD14(BC), CD24(BC) and FLAER (Cedarlane). Minor PNH clone was detected when the count of GPI- deficient cells did not exceed 1%. Results The PNH clone was found in 18 patients among 28 (64%) from the 1st group. The minor clone was found in 4 patients, in 3 patients the clone size exceeded 50%. Median (Me) clone size on the Red Blood Cells (RBC: type II + type III) was 0,25% (0,03-25,3%), Granulocytes (GR) – 1,7% (0,02- 93,92%), Monocytes (Mon)- 23,2% (0,05-95,66%). 7/18 SAA patients (38,9%) with the PNH clone, showed a haematological response at 3 months from the treatment start, including 3 patients with the minor PNH clone. 2/18 patients (11.1%) underwent allogenic bone marrow transplantation (alloBMT). 9/18 did not get the remission by the 3d month. 4/18 patients (22,2%), without response at 6 months, received the second course of IST; 5/18 patients (27.7%) are being followed up and can‘t be analyzed at 6 months. It worth to note, that PNH clone disappeared after allo-BMT (n=2) and in 1 patient with CR after IST. None of 10 patients without PNH clone attained response at 3 months (p=0,02). 4 out of 10 (40%) achieved only partial remission at 6 months. In these cases minor PNH clone appeared after hematological response and persisting from 6 till 18 months. 6 other patients are still on the treatment (ATG). In the 2nd group the PNH clone was detected in 26 of 35 cases (74,3%), only 11 of them had a minor clone. The Me of PNH clone size on RBC - 1,4% (0,02 to 3,76%), Gr – 25,2% (0,01-93,73%), Mon - 23,52% (0,01-54,32%) Conclusion The PNH clone has been detected in more than 60 % of de novo SAA patients. The disease was characterized by pancytopenia and aplasia of the bone marrow without clinical signs of intravascular hemolysis. In our study we observed the quick IST response at 3 months in SAA patients with the PNH clone (38,9%), in patients without PNH clone at time of diagnosis achievement of partial remission at 6 months was followed by PNH clone appearance and persistence. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v124.21.5162.5162 ◽

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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Prevalence of paroxysmal nocturnal hemoglobinuria (PNH) cells in patients with myelodysplastic syndromes (MDS), aplastic anemia (AA), or other bone marrow failure (BMF) syndromes: Interim results from the EXPLORE trial

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.7082 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 7082-7082 ◽

Cited By ~ 2

Author(s):

N. Galili ◽

F. Ravandi ◽

G. Palermo ◽

J. Bubis ◽

A. Illingworth ◽

...

Keyword(s):

Flow Cytometry ◽

Interim Analysis ◽

Bone Marrow Failure ◽

Treatment Options ◽

White Blood Cells ◽

High Sensitivity ◽

Clone Size ◽

Hematopoietic Stem ◽

Point Prevalence Study ◽

Pnh Clone

7082 Background: In patients with PNH, lack of the glycophosphatidylinositol (GPI)-anchored terminal complement inhibitor CD59 on hematopoietic stem cells results in chronic intravascular hemolysis, kidney and pulmonary disorders, thrombosis, and shortened life span. Presence of even minor populations of PNH cells in AA or MDS patients is medically important as it may indicate a higher likelihood of response to immunosuppressive therapy. We conducted the first large multicenter, point-prevalence study (EXamination of PNH, by Level Of CD59 on REd and white blood cells [EXPLORE]) of PNH cells in patients with AA, MDS, or other BMF syndromes. Here we report an interim analysis. Methods: A central laboratory conducted high-sensitivity flow cytometry utilizing a combination of GPI-linked antibodies (CD59, CD24, and CD14) and fluorescent aerolysin (FLAER) to identify GPI anchor-deficient PNH cells in RBC and WBC resulting in 0.01% sensitivity. The primary endpoint was percentage of patients who had a PNH WBC clone ≥1%. Results: Among 5,212 patients screened, 4,500 (86.3%) were MDS patients, 413 (7.9%) were AA patients, and 356 (6.8%) had other BMF syndromes. Approximately 1/4 (24.5%) of patients with AA, 1.2% with MDS, and 4.6% with other BMF were newly found to have a significant PNH clone ≥ 1%. Many of the newly identified clones were of clinical significance as the median PNH clone size was 11.1% in AA patients, 16.3% in MDS patients, and 32.6% in patients with other BMF. Presence of PNH cells (≥ 0.01%) was common in all examined BMF types: 70% of AA patients, 55% of MDS patients and 55% of patients with other BMF. PNH cells were identified in all MDS subtypes represented in the trial. Conclusions: Interim analysis from this first large multicenter study demonstrates that PNH cells are present in a majority of patients with AA, MDS, and other BMF. A spectrum of PNH clone sizes was noted in patients with each form of BMF. Screening patients with BMF with high-sensitivity flow cytometry for PNH cells may guide treatment options for the underlying BMF and/or PNH. The EXPLORE trial continues to enroll patients with AA. (EXPLORE Clinical Study Abstract 1/6/2009) [Table: see text]

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Paroxysmal Nocturnal Hemoglobinuria Clones Are Infrequent in Hepatitis-Associated Aplastic Anemia at the Time of Diagnosis

Blood ◽

10.1182/blood-2019-125620 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5016-5016

Author(s):

Wenrui Yang ◽

Xin Zhao ◽

Guangxin Peng ◽

Li Zhang ◽

Liping Jing ◽

...

Keyword(s):

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Clonal Evolution ◽

Extrinsic Factors ◽

Clone Size ◽

Hematopoietic Stem ◽

Over Time ◽

Pnh Clone

Aplastic anemia (AA) is an immune-mediated bone marrow failure, resulting in reduced number of hematopoietic stem and progenitor cells and pancytopenia. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clone in AA usually suggests an immunopathogenesis in patients. However, when and how PNH clone emerge in AA is still unclear. Hepatitis associated aplastic anemia (HAAA) is a special variant of AA with a clear disease course and relatively explicit immune pathogenesis, thus serves as a good model to explore the emergence and expansion of PNH clone. To evaluate the frequency and clonal evolution of PNH clones in AA, we retrospectively analyzed the clinical data of 90 HAAA patients that were consecutively diagnosed between August 2006 and March 2018 in Blood Diseases Hospital, and we included 403 idiopathic AA (IAA) patients as control. PNH clones were detected in 8 HAAA patients (8.9%,8/90) at the time of diagnosis, compared to 18.1% (73/403) in IAA. Eight HAAA patients had PNH clone in granulocytes with a median clone size of 3.90% (1.09-12.33%), and 3 patients had PNH clone in erythrocytes (median 4.29%, range 2.99-10.8%). Only one HAAA patients (1/8, 12.5%) had a PNH clone larger than 10%, while 24 out of 73 IAA patients (32.9%) had larger PNH clones. Taken together, we observed a less frequent PNH clone with smaller clone size in HAAA patients, compared to that in IAAs. We next attempted to find out factors that associated with PNH clones. We first split patients with HAAA into two groups based on the length of disease history (≥3 mo and < 3mo). There were more patients carried PNH clone in HAAA with longer history (21.4%, 3/14) than patients with shorter history (6.6%, 5/76), in line with higher incidence of PNH clone in IAA patients who had longer disease history. Then we compared the PNH clone incidence between HAAA patients with higher absolute neutrophil counts (ANC, ≥0.2*109/L) and lower ANC (< 0.2*109/L). Interestingly, very few VSAA patients developed PNH clone (5%, 3/60), while 16.7% (5/30) of non-VSAA patients had PNH clone at diagnosis. We monitored the evolution of PNH clones after immunosuppressive therapy, and found increased incidence of PNH clone over time. The overall frequency of PNH clone in HAAA was 20.8% (15/72), which was comparable to that in IAA (27.8%, 112/403). Two thirds of those new PNH clones occurred in non-responders in HAAA. In conclusion, PNH clones are infrequent in HAAA compared to IAA at the time of diagnosis, but the overall frequency over time are comparable between the two groups of patients. In SAA/VSAA patients who are under the activated abnormal immunity, longer clinical course and relatively adequate residual hematopoietic cells serve as two important extrinsic factors for HSCs with PIGA-mutation to escape from immune attack and to expand. Disclosures No relevant conflicts of interest to declare.

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Clonal Evolution and Devolution Following Chemotherapy in Adult Acute Myelogenous Leukemia.

Blood ◽

10.1182/blood.v120.21.2487.2487 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2487-2487

Author(s):

Brian Parkin ◽

Peter Ouillette ◽

Yifeng Li ◽

Cheng Li ◽

Kerby Shedden ◽

...

Keyword(s):

Complete Remission ◽

Gene Mutation ◽

Induction Chemotherapy ◽

Acute Myelogenous Leukemia ◽

Conflicts Of Interest ◽

Gene Mutations ◽

Myelogenous Leukemia ◽

Stable Gene ◽

Paired Data ◽

Acute Myelogenous

Abstract Abstract 2487 Introduction: Despite significant advances in the understanding of the biology of adult acute myelogenous leukemia (AML), overall survival remains poor due chiefly to the high rate of relapse after achieving complete remission as well as primary failure of induction chemotherapy. Efforts to further unravel the mechanisms leading to relapse and primary refractory disease are critical in order to guide the development of effective and durable treatment strategies for AML. To that end, this study seeks to elucidate the clonal relationship of AML in various disease phases. Methods: We employed SNP 6.0 array-based genomic profiling of acquired copy number aberrations (aCNA) and copy neutral LOH (cnLOH) together with sequence analysis of recurrently mutated genes to characterize paired AML genomes. We analyzed 28 AML sample pairs from patients that achieved complete remission with chemotherapy and subsequently relapsed (median remission duration 272 days [range 25 – 1249 days]) and 11 sample pairs from patients with persistent disease following induction chemotherapy. AML cell samples were isolated with a Ficoll gradient, negatively selected using Miltenyi microbead columns, and then further purified with flow cytometric cell sorting. Processed DNA isolated from highly purified AML blasts and paired buccal DNA was hybridized to Affymetrix SNP 6.0 arrays. aCNA were visually identified using the dChip program in paired data displays and corroborated by algorithmic lesion scoring, and cnLOH was detected using internally developed software. In addition, 11 genes known to be recurrently mutated in AML (CEBPA, DNMT3A, IDH1, IDH2, RUNX1, BCORL1, NPM1, NRAS, KRAS, FLT3 and TP53) were resequenced in all 39 presentation samples to identify somatically acquired mutations. Genes found mutated in individual AML cases were subsequently tested for the persistence of the mutation in paired samples. Results: For the 28 paired specimens in the relapsed cohort, comparison of aCNA and cnLOH occurrences, gene mutation patterns and karyotypes revealed 6 cases that carried no aCNA/cnLOH at either presentation or relapse, but at presentation carried at least 1 gene mutation, all of which but one were stable in relapse (1 case lost a RUNX1 mutation but carried a t(8;21) in both disease stages); 11 cases that were characterized by the presence of aCNA/cnLOH at presentation, of which 55% (6 of 11) gained additional aCNA/cnLOH at relapse; 6 cases without aCNA/cnLOH at presentation that gained aCNA/cnLOH at relapse, of which 2 concurrently lost a FLT3-ITD or CEPBA mutation; and 5 cases that carried no informative genomic events. For the 11 paired specimens in the persistent AML cohort, the same comparison revealed 2 cases without aCNA/cnLOH before or after chemotherapy and stable gene mutations; 5 cases with aCNA/cnLOH at presentation that carried the same genomic lesions and gene mutations before and after chemotherapy; 3 cases with aCNA/cnLOH present at enrollment that lost some but not all of these aCNA/cnLOH and gained none after initial induction therapy; and 1 additional case that lost a FLT3-ITD. Comparative analysis of these patterns demonstrates that relapsed AML invariably represents reemergence or evolution of an antecedent clone. Furthermore, all individual aCNA or cnLOH detected at presentation persisted at relapse indicating that this lesion type is proximally involved in AML evolution. Analysis of informative paired persistent AML disease samples uncovered at least two coexisting dominant clones of which at least one was chemotherapy sensitive and one resistant. Conclusion: This detailed genomic analysis supports the conclusion that incomplete eradication of AML founder clones rather than stochastic emergence of fully unrelated novel clones underlies AML relapse and persistence with direct implications for clinical AML research. Disclosures: No relevant conflicts of interest to declare.

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