Paroxysmal Nocturnal Hemoglobinuria Clone Size Research Articles

Comparison of human leukocyte antigen in patients with paroxysmal nocturnal hemoglobinuria of different clone sizes.

Glycosylphosphatidylinositol-anchored protein-deficient hematopoietic stem and progenitor cell development caused by PIGA mutations cannot fully explain the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Herein, patients newly diagnosed with PNH at our hospital between April 2019 and April 2021 were recruited. The human leukocyte antigen (HLA) class I and II loci were analyzed, and patients were stratified by PNH clone sizes: small (< 50%) and large (≥ 50%). In 40 patients (29 males; 72.5%), the median PNH clone size was 72%. Thirteen (32.5%) and twenty-seven (67.5%) patients harbored small and large PNH clones, respectively. DRB1*15:01 and DQB1*06:02 had higher frequencies in patients with PNH than in healthy controls (adjusted P-value = 4.10 × 10-4 and 4.10 × 10-4, respectively). Whole HLA class I and II allele contributions differed (P = 0.046 and 0.065, not significant difference) when comparing patients with small and large PNH clones. B*13:01 and C*04:01 allelic frequencies were significantly higher in patients with small clones (P = 0.032 and P = 0.032, respectively). Patients with small clones had higher class II HLA evolutionary divergence (HED) (P = 0.041) and global class I and II HED (P = 0.019). In the entire cohort, 17 HLA aberrations were found in 11 (27.5%) patients. No significant differences in HLA aberrations were found between patients with small or large clones. In conclusion, patients with small clones tended to have a higher frequency of immune attack-associated alleles. A higher HED in patients with small clones may reflect a propensity for T cell-mediated autoimmunity. HLA aberrations were similar between patients with small and large clones.

Insights from Trajectories of PIGA-Mutated Clones in Paroxysmal Nocturnal Hemoglobinuria

Background: Patients with paroxysmal nocturnal hemoglobinuria (PNH) exhibit clonal expansion of hematopoietic cells deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs). Mutations in PIGA gene, involved in the biosynthesis of GPI-APs, result in the loss of GPI-APs from hematopoietic cells. However, PIGA mutations alone are insufficient to drive this clonal expansion. The pathogenesis of clonal expansion in PNH remains elusive, while several hypotheses or factors have been proposed, including immune attacks with concomitant aplastic anemia (AA) or additional mutations offering intrinsic growth advantage. Recently high-sensitivity flow cytometry and next-generation sequencing have allowed us to evaluate the clonal dynamics and architecture more precisely. The method has the potential to clarify the trajectories of PIGA mutated clones before the significant clonal expansion or the diagnosis, thereby to gain insight into the pathogenesis of the clonal expansion in PNH. Methods: To estimate the trajectories of PIGA-mutated clones, we constructed phylogenetic trees using whole genome sequencing (WGS) data of single cell-derived colonies of bone marrow samples from two PNH patients (53 colonies for Case 1 and 75 for Case 2). We used the targeted-sequencing and exome sequencing to detect driver mutations after isolating GPI-deficient and GPI-positive cells using flow cytometry. Results: Case 1 was diagnosed with AA at the age of 28. He developed hemoglobinuria at the age of 40 and was diagnosed with PNH at the age of 45. Bone marrow and peripheral blood samples were collected when he was 47 years old. The PNH clone sizes in granulocytes and red blood cells were 88.7% and 46.1%, respectively. Targeted-sequencing revealed six PIGA mutations in GPI-deficient cells and three BCOR mutations in GPI-positive cells. Case 2 was diagnosed with AA at the age of 33 and with PNH at the age of 34. Bone marrow and peripheral blood samples were collected when she was 59 years old. The PNH clone sizes in granulocytes and red blood cells were 98.6% and 96.3%, respectively. We previously identified chromosomal abnormalities in chr12, with the breakpoint located in an HMGA2 gene (Inoue et al. Blood. 2006). Targeted-sequencing detected no other driver mutations than single PIGA mutation and the HMGA2 mutation.In Case 1, Phylogenetic tree analysis constructed with WGS of single cell-derived colonies revealed multiple PIGA-mutated and BCOR-mutated clones that expanded independently of each other. All BCOR-mutated clones were identified in the GPI-positive population. In Case 2, all PIGA-mutated clones harbored the HMGA2 mutation, while no GPI-positive populations acquired the HMGA2 mutation.In both cases, the numbers of passenger single nucleotide variants per year were higher in PIGA-mutated clones than in PIGA-unmutated clones (16 vs. 11 mutations/year for Case 1 and 14 vs. 12 for Case 2). Assuming that variant acquisition rates per cell division are similar in both clones, the result suggests that PIGA-mutated clones had growth advantage against PIGA-unmutated clones.The acquisition of PIGA mutations was estimated to have occurred more than ten years prior to the PNH diagnosis. In Case 1, following the AA diagnosis, both PIGA-mutated clones and BCOR-mutated clones started expansion simultaneously, which suggests an association with immune attacks. Interestingly, even wild type clones expanded after AA diagnosis, suggesting bottle-neck effects of post-AA hematopoietic recovery. In Case 2, PIGA-mutated clones began expanding more than ten years before the AA diagnosis. Either a subclinical immune attack that might have existed before the diagnosis of clinical AA or the concomitant HMGA2 mutation conferred survival advantage for PIGA-mutated clones in Case 2. Conclusions: This estimation of the trajectories of the PIGA-mutated clone indicates that the pathogenesis of clonal expansion exhibits heterogeneity between two PNH patients.

Identification of HLA Class I Alleles in Patients with Paroxysmal Nocturnal Hemoglobinuria with Different Clone Sizes

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a hematological disease characterized by complement-mediated hemolysis, thrombosis, and bone marrow failure. Human leukocyte antigen (HLA) evolutionary divergence and mutations were widely analyzed in aplastic anemia (AA). However, there were few studies in PNH thus far. Methods Patients with newly diagnosed PNH at Peking Union Medical College Hospital (PUMCH) were recruited from April 2019 to April 2021 according to International PNH Interest Group standards. They were analyzed the genotypes of classical HLA class I loci (A, B, C) using whole-exome sequencing (WES). Patients were further stratified into four groups according to the clone size (Group A: &amp;lt;10%; Group B: 10% ≤ to ≤50%; Group C: 50% &amp;lt; to ≤80%; Group D: &amp;gt;80%) to identify the association between HLA class I alleles and PNH clone size. Result Samples from 40 patients were enrolled, including 20 classic PNH and 20 bone marrow failure (BMF)/PNH. There were 29 males and 11 females. The median age was 32 years (range, 16-70). The median PNH clone size measured by flaer-granulocyte ratio was 72% (range 1-99%). Among the 59 identified alleles in PNH, HLA-B genotypes, which were reported to be associated with severe AA, accounted for the highest percentage (46%). A*02:01, the most common one and found in all the four groups, was reported as one of the risk alleles overrepresents in AA. The alleles found in group A were significantly different from those in group B, C, and D ( P = 0.004, 0.006, and &amp;lt; 0.001, respectively). But there was no significant difference between B, C, and D groups ( P &amp;gt; 0.05). 47% of the alleles identified in group A were reported in AA, whereas only 26%, 23%, and 26% of the detected alleles in Group B, C, and D were reported in AA, respectively. The other alleles in B, C, D groups were mainly for the susceptibility of autoimmune diseases or drug-related adverse reactions. Among them, HLA-B*46:01, the most common and found in B, C and D groups, may indicate weak antigen presentation and immune ability. B*44, B*57:01, C*07:01 were unique in Group B, B*38:02 were identified in Group C and HLA-B*27 expressed most in Group D, which were mainly on cell survival, drug hypersensitivity, and complement activation. Conclusion Significantly different alleles were observed between PNH with smaller clone sizes (&amp;lt; 10%) and PNH with larger clone sizes (≥10%). Patients with smaller clone sizes had similar HLA class Ⅰ alleles to AA; whereas those with larger clone sizes had numerous unique alleles which may contribute to the pathogenesis of PNH. Keywords: paroxysmal nocturnal hemoglobinuria, human leukocyte antigens, clone size, class I allele, pathogenesis

Biomarker Analyses in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Treated with Crovalimab and Eculizumab: Results from the Phase III Randomized COMMODORE 2 Trial

Introduction PNH is a hematopoietic stem cell disorder characterized by hemolytic anemia, hemoglobinuria, and increased risk of thromboembolic events, and is associated with bone marrow failure. Crovalimab (crova) is a novel complement C5 inhibitor engineered with recycling technology, with a long half-life and high solubility that enables low-volume subcutaneous (SC) administration every 4 weeks (q4w). The global Phase III COMMODORE 2 study (NCT04434092) in C5 inhibitor-naive patients (pts) with PNH demonstrated non-inferiority of crova vs eculizumab (ecu) for hemolysis control, transfusion avoidance, breakthrough hemolysis, and hemoglobin stabilization, and a safety profile that was consistent between crova and ecu (Röth EHA 2023; #S181). Here we report results of the exploratory biomarker analyses in COMMODORE 2. Study Design and Methods In COMMODORE 2, C5 inhibitor-naive pts were randomized 2:1 to receive a weight-based tiered crova regimen or ecu. Regimens included loading doses, followed by maintenance dosing (crova: SC injection q4w; ecu: intravenous infusion every 2 weeks). Co-primary efficacy endpoints were proportion of pts with hemolysis control (central lactate dehydrogenase [LDH] ≤1.5×upper limit of normal [ULN]) from Week (W)5 through W25 and proportion of pts with transfusion avoidance from baseline through W25. Exploratory biomarker analyses included terminal complement activity per 50% hemolytic complement (CH50) liposome immunoassay, free hemoglobin, additional exploration of LDH levels, and flow cytometry of the PNH clone size/C3d levels. Additional descriptive analyses of hemoglobin data compared pts with and without transfusions. All biomarker data were assessed from baseline to W25. Results Data forCH50 and LDH were available for all 204 randomized pts (crova: 135; ecu: 69) and data for free hemoglobin, PNH clone size and C3d were available for 126 pts (crova: 85; ecu: 41). At baseline, mean (standard deviation [SD]) LDH levels were 7.6×ULN (3.4) in crova pts and 7.8×ULN (3.5) in ecu pts. 77% of crova pts and 74% of ecu pts had ≥1 packed red blood cell transfusion ≤12 months before screening. 39% of crova pts and 38% of ecu pts had a history of aplastic anemia; 4% and 9% had myelodysplastic syndrome, respectively. Inhibition of complement activity (per CH50) to below the lower limit of quantification was reached at W2 (first post-dose pharmacodynamic sample) in 93% and 46% of pts in the crova and ecu arms, respectively, and in 90% and 73% at W25 (post-dose) (Figure). With regards to intravascular hemolysis markers, the proportion of pts with LDH ≤1.5×ULN was 81% at W5 and 78% at W25 in crova pts, and 84% at W5 and 77% at W25 in ecu pts. Free hemoglobin, another intravascular hemolysis marker, showed a similar initial decrease from baseline in both arms; however, crova pts consistently showed numerically lower levels than ecu pts from W13 to W25. Mean (SD) increase in hemoglobin level from baseline to W25 was 1.2 g/dL (2.0) in the crova arm (baseline: 8.7 g/dL [1.4]) and 0.8 g/dL (1.9) in the ecu arm (baseline: 8.9 g/dL [1.4]). In both arms, a greater increase in mean hemoglobin level from baseline was observed in pts who achieved transfusion avoidance vs those requiring ≥1 transfusion up to W25 (crova: 1.7 g/dL [SD, 1.8; n=88] vs 0.2 g/dL [SD, 1.9; n=40]; ecu: 1.3 g/dL [SD, 1.6; n=47] vs −0.4 g/dL [SD, 2.1; n=20]). Both arms showed a similar increase from baseline to W25 in the mean (SD) percentage of C3d-positive PNH erythrocytes (crova: 0.7 [1.5] at baseline to 20.8 [19.1] at W25; ecu: 0.6 [0.9] to 22.3 [15.0]). Conclusions Treatment with crova or ecu reduced terminal complement activity effectively, with a higher proportion of crova than ecu pts having undetectable terminal complement activity. However, this did not translate into significant differences in other biomarkers (LDH and free hemoglobin) or in the clinical efficacy between crova and ecu arms. Hemoglobin levels increased in both crova and ecu arms. This improvement was more pronounced in pts not receiving a transfusion from baseline to W25; additional analysis is needed to elucidate the impact of bone marrow failure status. Similar increases in C3d level in each arm were expected, as this is a C5 inhibitor class phenomenon. Overall, biomarker data from COMMODORE 2 support the non-inferior efficacy of crova vs ecu in patients with PNH. Further analyses correlating additional biomarkers with clinical outcomes is warranted.

Ravulizumab Provides Durable Control of Intravascular Hemolysis and Improves Survival in Patients with Paroxysmal Nocturnal Hemoglobinuria: Long-Term Follow-up of Study 301 and Comparisons with Patients of the International PNH Registry

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare chronic blood disorder characterized by uncontrolled terminal complement activation, leading to intravascular hemolysis (IVH), major adverse vascular events (MAVEs; including thrombotic events), organ damage and increased morbidity and mortality. Where available, ravulizumab is considered the standard of care for patients with PNH, providing immediate, complete and sustained inhibition of terminal complement activity. In study 301 (NCT02946463), complement component 5 (C5) inhibitor-naïve patients with PNH reported elevated levels of lactate dehydrogenase (LDH) and low hemoglobin (Hb) concentrations at baseline, alongside a history of MAVEs and need for transfusions. In such patients, ravulizumab provides durable control of IVH, reported for up to 2 years. Objectives: To report ravulizumab treatment outcomes for up to 6 years in C5 inhibitor-naïve patients with PNH from study 301 and compare survival with untreated patients of the International PNH Registry (NCT01374360). Methods: C5 inhibitor-naïve patients with PNH and high-disease activity (HDA; LDH level ≥ 1.5 × the upper limit of normal [ULN] and at least one sign or symptom of PNH) (N = 246) were randomized to receive ravulizumab or eculizumab. After the primary evaluation period (26 weeks), patients received ravulizumab for the 5-year open-label extension (OLE) period. LDH level change from baseline, the proportion of patients experiencing MAVEs, and transfusion avoidance were evaluated. Cox-proportional hazards regression analyses compared survival of patients treated with ravulizumab with untreated patients with HDA and clone size ≥ 5% at PNH Registry enrollment. Covariates comprised age at PNH diagnosis, gender, Hb concentration, estimated glomerular filtration rate, PNH clone size, transfusion history and medical history of MAVEs and bone marrow disease at baseline (i.e., date of ravulizumab treatment initiation [study 301] or PNH Registry enrollment). Transfusion history ( p = 0.05), age at PNH diagnosis and gender were included in the final adjusted model. Results: Ravulizumab treatment data were available for 244/246 patients (median [range] follow-up: 46.8 [0.4-69.3] months). At baseline, all 246 patients had LDH levels &amp;gt; 1.5 × ULN; at 26 weeks, 87.1% of patients achieved LDH levels ≤ 1.5 × ULN, which was maintained in 79.3% of patients at last follow-up (day 2045 [n/N = 42/53]; highest proportion reported at day 225 [n/N = 92/97; 94.9%]). At baseline, 17.1% of patients had a history of MAVEs (MAVE rate: 3.4 per 100 patient-years [PYs]). The proportion of ravulizumab-treated patients who experienced MAVEs was low throughout the study (n = 11 [4.5%], 13 events, MAVE rate: 1.4 per 100 PYs), with 9 patients (3.7%) reporting MAVEs during the OLE (11 events, MAVE rate: 1.3 per 100 PYs). Prior to randomization (≤ 6 months of study entry), 24.8% of patients did not need transfusions; at 26 weeks, this increased to 73.6% and was maintained in 53.9% of patients for the entire OLE with the highest proportion not requiring transfusions reported during the final 6 months of the OLE (75.7%). In addition, units transfused reduced from 1019 prior to randomization to 110 during the final 6 months of the OLE. Overall, 33 patients (13.4%) discontinued ravulizumab treatment during the entire study period (30 [12.2%] during the OLE). Reasons for discontinuation were death (n = 8), patient choice (n = 7), physician decision (n = 5), pregnancy (n = 4), adverse event (n = 3), lost to follow-up (n = 1) and other (n = 5). When compared with 413 untreated patients, ravulizumab was associated with an adjusted survival probability (95% confidence interval [CI]) of 84.1% (77.4, 91.3) at 4 years (hazard ratio [95%CI]: 0.14 (0.06, 0.32), p &amp;lt; 0.001; Figure 1) and mortality was 3.5-fold lower than that observed in untreated patients in the PNH Registry. Conclusions: This study reports the longest duration of ravulizumab treatment exposure in C5-inhibitor-naïve patients with PNH (925.7 PYs) to date. For up to 6 years, ravulizumab provided effective long-term control of IVH, evidenced by the maintenance of LDH ≤ 1.5 × ULN and low incidence of MAVEs and death. No new safety signals were identified and ravulizumab improved survival compared with untreated patients in the PNH Registry, further supporting the use of ravulizumab as the first-line treatment of choice for patients with PNH, where available.

Characterization of Hematopoietic Stem and Progenitor Cells of PNH Patients Using Single-Cell RNA Sequencing Analysis

The etiology of paroxysmal nocturnal hemoglobinuria (PNH) is somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs), and affected PNH-type cells lack cell surface expression of the glycosylphosphatidylinositol-anchored proteins (GPI-APs). In PNH, GPI-AP deficient cells clonally expand and occupy a large proportion of mature cells. Several hypotheses have been proposed to explain clonal expansion of PNH-type HSPCs, including reduced sensitivity to apoptosis and escape from immune-mediated cytotoxicity. However, definitive mechanisms remain unclear. To further understand PNH pathophysiology at high multi-dimensional resolution, we performed single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) cells from PNH patients. Fresh BM samples were obtained from eight PNH patients (male, n = 2, female, n = 6; median age, 51 [range, 21-67] years), who had large PNH-type granulocyte populations and stable clone sizes (median, 45 [range, 16-99] %), and five age- and sex-matched healthy donors (HDs). PNH-type and wild-type (WT) lineage-CD34+HSPCs were enriched from bone marrow mononuclear cells (BMMNCs) by flow cytometric sorting. Sorted cells were processed through the Chromium Controller (10x genomics, Pleasanton, CA, USA). scRNA-Seq libraries were constructed using the Chromium Single Cell 3' Reagent Kits (10x genomics) according to the manufacturer's instructions. The constructed libraries were sequenced with the NovaSeq 6000 system (Illumina, San Diego, CA, USA). We obtained 81,470 high-quality cells from PNH patients and 56,545 cells from HDs for further analysis. Cells were clustered with Seurat and visualized in UMAP. Cell clusters were assigned to 6 HSPC subsets based on expression of canonical marker genes (Figure 1A). To investigate compositional changes in BMMNCs between PNH patients and HDs, we performed differential abundance analysis with Milo and identified a prominent decrease in hematopoietic stem cells (HSCs) and lymphoid progenitors, as compared to HDs (Figure 1B). We then evaluated differential abundance between PNH-type and WT HSPCs in PNH patients, identifying no significant differences in each HSPC subsets (Figure 1B). These data were consistent with flow cytometric profiling of BMMNCs, similar proportions of PNH-type cells in each HSPC subsets in individual patients despite with profound decrease in PNH-type lymphocytes (Figure 1C). We next sought to investigate skewed gene sets in PNH-type HSPCs using gene set enrichment analysis. Comparison between PNH-type and WT HSPCs identified upregulation of inflammatory signaling pathways and downregulation of the cell-cycling pathways in PNH-type HSPCs, as compared to WT HSPCs (Figure 1D). We made comparisons within individual subsets. In HSCs, genes involved in IFN-γ response and oxidative phosphorylation were upregulated in PNH-type HSCs, while cell-cycling genes (ie. E2F targets and G2M checkpoint) were not dysregulated, as compared to WT HSCs. In summary,no significant skewing in lineage differentiation of PNH-type HSPCs was observed, suggesting PNH-type lymphocytes were negatively selected during the maturation process in lymphoid tissues. Upregulation of the inflammatory and immune response pathways in PNH-type HSPCs indicated a proinflammatory phenotype of PNH-type HSPCs. Upregulation of cell cycle-related pathways was not observed in PNH-type HSCs compared to WT HSCs, consistent with stable PNH clone sizes in our cohort of PNH patients.

A Case of PNH with Abdominal Pain, AKI and Hemolytic Anemia

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder affecting 1 to 10 people per million. With an associated 10-year mortality, PNH is a life-threatening disorder where the complement system of the body's innate immune system prematurely lyses red blood cells. It can be characterized by a number of clinical features, the most common being hemolytic anemia, pancytopenia, and thrombosis. 45 year old African American male presented with a chief complaint of shortness of breath x 2-3 days. Reports associated dry, nonproductive cough, congestion, myalgias onset one week prior. He was taking two tablets of Advil daily since symptom onset. He has a past medical history of right sided BKA after motor vehicle accident and currently uses a prosthetic leg. No other significant past medical history noted. On ED admission, the patient was febrile to 103.2F, tachycardic to 110 beats per minute, tachypneic to 26 breaths per minute, and normotensive to 128/64. On physical exam, there was conjunctival pallor, no scleral icterus, and right BKA. The remainder of the physical exam was unremarkable. Labs on admission were notable for anemia (Hgb 3.9, baseline of 9.2, 2 years prior), AKI (Cr 7.3, baseline of 0.9), elevated LDH 2336, and low Haptoglobin <10. Platelet count WNL throughout admission however there was a drop from 242 to 180 between Day 1 and 2 of admission. Peripheral smear did not show schistocytes however ADAMTS13 sent per heme/onc consult. Anemia improved to Hgb 7.6 on Day 3 of admission status post 4 units packed red blood cell transfusion. Workup for hemolytic anemia with AKI was pursued: Direct Coombs test negative, PNH screen sent. SPEP and UPEP unremarkable. On Day 6 of admission, flow cytometry showed evidence of PNH clone upon analysis of RBCs, granulocytes, and monocytes. AKI was attributed to ATN s/t heme-pigment deposition vs NSAID use and Cr trended down to 2.9 prior to discharge. On day 7 of admission, patient developed severe abdominal pain, not associated with fevers, chills, nausea, vomiting, or diarrhea. Pain moderately improved with Norco, ultrasound imaging was negative, and unable to CTA given renal function. Pt was started empirically on therapeutic anticoagulation with Lovenox, as the literature reports a high incidence of venous and/or arterial thrombosis in PNH w/granulocyte count >50% (89.8% in this patient). Patient was discharged the next day with close outpatient hematology follow-up with plan to start eculizumab. Given patient's presentation of fever, symptomatic anemia, and AKI the differential diagnosis included: hemoglobinopathies, red cell membrane disorders, PNH, DIC, TTP, HUS, AIHA, and infections such as malaria or HIV. It was difficult to identify PNH as the cause in this patient on ED presentation as there was no reported urine discoloration. Though classified as an acquired disorder through mutation of a gene on the X chromosome, it occurs in equal distribution among men and women. PNH specifically occurs through the mutation of a gene involved in the synthesis of glycosylphosphatidylinositol (GPI), an anchor protein for complement regulatory proteins that prevent complement-induced hemolysis of red blood cells. The resulting GPI deficiency leads to the unregulated formation of complement attack complexes that damage the cell membrane causing them to lyse. This intravascular hemolysis is seen as decreased levels of Hgb and hemoglobinuria that leads to elevated levels of LDH as seen with this patient. The degree of hemolysis and PNH clone size can increase risk of thrombosis, the leading cause of death in patients with PNH. Strong indicators for the start of PNH therapy are severe anemia, thrombosis, renal dysfunction, and dyspnea. The typical management of PNH in patients with thrombosis is shown in this case as the patient is put on therapeutic anticoagulation and plans for eculizumab, a monoclonal antibody that is a complement inhibitor used to prevent hemolysis. This PNH therapy will likely be administered lifelong and decrease the need for RBC transfusions as well as decrease thromboembolic rate. If the patient is unresponsive to eculizumab they become a strong candidate for allogeneic stem transplantation. This patient meets the popular standard for starting primary prophylaxis for management of thrombosis with a PNH clone size >50%.

Clinico-Molecular Risk Factors and Management of Thrombosis in a Large Real-Life Cohort of Patients with Paroxysmal Nocturnal Hemoglobinuria in the New Era of Anti-Complement Inhibitors and Doacs

The increased propensity for thrombotic events (TE) is well established in patients with paroxysmal nocturnal hemoglobinuria (PNH) and represents the main cause of disease morbidity/mortality. While the precise pathogenesis of TE remains ill-defined and likely multifactorial, cases with fully blown hemolytic PNH clones show the highest risk for such complications, often occurring in atypical localizations including the hepatic circulation (e.g., Budd-Chiari syndrome, BCS). The introduction of anti-complement therapy greatly abated hemolysis and thrombosis, demonstrating the crucial involvement of the underlying hemolytic process in triggering TE. Despite the current availability of PNH-directed treatment, a scarcity of data exist on anticoagulation and specifically on the efficacy of direct oral anticoagulants (DOACs) in this setting. Here, we took advantage of a large, real-life cohort of patients with classical hemolytic PNH to unravel determinants of thrombosis and decipher anticoagulation strategies in cases developing TE. Among 106 PNH cases (M:F ratio 0.86, median age 39 years, IQR 26-58) followed-up for a median of 68 months (95% CI 50-92), 42% were primary and 58% had antecedent aplastic anemia. Median granulocyte clone size at diagnosis was 71% (46-90) with the majority of cases classified as type III dominant red blood cells (RBCs) clone (81%) by flow cytometric analysis.1In total, 77% of cases harbored PIGA mutations by multi-amplicon deep NGS at a median ploidy-adjusted VAF of 20% (9-33), of whom 58% presenting multiple hits (clonal mosaicism).1 A total of 33 patients (31%) experienced TE, of which 84% occurred as the initial disease manifestation prompting to seek medical attention, 13% during a temporary discontinuation of anti-complement treatment (e.g., preparation to allogeneic bone marrow transplant), and only 3% (N=1) during treatment. Overall, the majority of patients had TE originating in the venous system (91%), arterial cerebrovascular district (6%) or both (3%). TE occurred typically in unusual sites with BCS as the most frequent (34%) followed by deep vein thrombosis (DVT, 20%), DVT complicated by pulmonary embolism (14%), splanchnic (14%), cerebral sinuses and superficial veins (9% each). No differences in disease ontogenesis (primary vs secondary to AA), and baseline variables such as age, PNH clone size, gender, PIGA mutations (presence, type and clonal burden), blood counts, reticulocytes, LDH, creatinine and transaminases were observed. Interestingly, patients experiencing TE were more commonly presenting with type II RBC phenotype (36% vs 13%; p=.027), had median lower albumin (4 vs 4.5 g/dL; p<.001) and elevated D-dimers (1520 vs 325 ng/mL; p=.003) as compared to non-TE cases. Patients with superficial TE were promptly started on anti-complement treatment, while the others improved with additional anticoagulation. One patient with portal vein thrombosis had severe thrombocytopenia and eventually died due to progressive BCS. Anticoagulation strategy consisted of warfarin (50%), DOACs (29%) and low molecular weight heparin (LMWH, 21%) with a median treatment duration of 44 months (17-73). LMWH was preferred in patients with thrombocytopenia (median platelets 45 vs 102x109/L in cases receiving other therapeutics; p=.03). In 1 patient, warfarin was switched to DOACs for prolonged treatment, 2 BCS cases underwent TIPS, and in 1 case with DVT an inferior vena cava filter was positioned because of the concomitant diagnosis of myeloproliferative neoplasm with MPLW515L. Recurrence of TE under anticoagulants was noted in only 2 cases receiving warfarin, whereas 7 (N=5 warfarin/ 2 DOACs) experienced bleeding. Two patients on DOACs had acute rectal hemorrhage with additional bleeding risk factors (clopidogel, ulcerative colitis). No thrombotic recurrence was observed in all 9 patients treated with DOACs at a median observation time of 24 months (12-95). While previously TE prompted lifelong anticoagulation, the control of the underlying disease with anti-complement drugs enabled its successful discontinuation in 42% of patients without any TE recurrence at a median time of 50 months (15-90). This is the largest hemolytic PNH series ever reported detailing clinico-molecular risk factors, characteristics of TE and their management in the era of anti-complement therapy and DOACs, whose use in such a setting warrants attention and further studies.

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

Introduction Acquired aplastic anemia (aAA) occurs in both children and adults, though pathophysiology and clinical outcomes differ significantly by age. While clinical testing for paroxysmal nocturnal hemoglobinuria (PNH) clones is routine, targeted next generation sequencing (NGS) for myelodysplastic syndrome (MDS) associated variants has only recently been implemented. Furthermore, single nucleotide polymorphism array (SNP-A) assessing for copy neutral loss of heterozygosity in the human leukocyte antigen (HLA) region on chromosome 6p (6p CN-LOH) and HLA NGS assessing for allele specific loss of function (LoF) mutations, which together are seen in up to 25% of aAA cases, are only now being considered for clinical use. Recent studies have reported on the frequency and overlap of these 3 types of clonal evolution (PNH, MDS-associated, HLA-targeting) in adult aAA cohorts, but none focus specifically on pediatric patients, whose MDS-associated clonal evolution patterns are distinct. Delineating patterns of clonal hematopoiesis in pediatric aAA to determine whether evolution driven by immune escape (PNH and HLA loss) affects likelihood of MDS development is critical for personalizing therapeutic options. Thus, we aimed to describe the frequency, clinical significance, and overlap of clonal evolution including PNH, HLA loss, and MDS-associated gene mutations in a pediatric cohort of aAA patients. Methods This study identified 85 pediatric aAA patients treated at Children's Hospital of Philadelphia (CHOP) and enrolled on the IRB-approved CHOP/Penn BMF Registry and Repository in whom clinical testing for all patterns of clonal hematopoiesis was available or could be performed with biorepository samples. PNH flow cytometry was performed clinically in all cases; subjects with PNH > 0.05% in the granulocyte fraction were considered as having a PNH clone. Peripheral blood and bone marrow samples were used for SNP-A, HLA-NGS, and an institutional somatic mutation NGS panel for hematological malignancies encompassing 118 genes. Descriptive and contingency statistical analyses along with Fisher's Exact Test were performed using SAS (Cary, NC). Results Demographic composition of the 85 pediatric subjects with aAA included a median age of 10.7 years (1.7-20.8), male/female ratio of 59%/41%, and a representative race/ethnicity distribution (Table 1). In total, 66% displayed at least one clonal event. As expected, PNH clones were most common, present in 45% (38/85) of the cohort. An additional 16 (19%) exhibited acquired HLA loss by 6p CN-LOH (n=6), allele specific LoF mutations (n=6), or both (n=4). Fifteen (18%) had clonal hematopoiesis associated with MDS, of whom 3 had cytogenetic lesions that progressed to clinical MDS (3.5%), and 12 developed no morphologic features of MDS but demonstrated a total of 14 mutations in MDS-associated genes (BCOR/BCORL1 n=6, KMT2C/KMT2D n=3, RUNX1 n=1, ASXL1 n=1, SRSF2 n=1, PRPF8 n=1, CALR n=1). Critically, pediatric subjects with aAA who developed HLA loss did not develop MDS-associated mutations, with no patient demonstrating both subtypes of clonal events (Figure 1). In contrast, PNH and MDS-associated clonal events overlapped in 11% of the overall cohort. In total, 60% of patients with MDS-associated clonal hematopoiesis also possessed PNH clones, and 23% of patients with detectable PNH clones demonstrated at least one MDS-associated clonal event. Only 3.53% (n=3) of the cohort demonstrated both PNH and HLA loss, and PNH clone size was < 1% in all 3 patients. Furthermore, patients with HLA loss were significantly less likely to have PNH clones than those without HLA somatic alterations (19% vs 51%, p = 0.026). Correlations between patterns of clonal hematopoiesis and clinical outcomes will be presented. Concalusions Pediatric aAA patients treated with IST are at risk for developing MDS and hemolytic PNH. Presence and evolution of clonal hematopoiesis influenced decisions to pursue IST versus alternative donor transplant. Importantly, our study demonstrates that occurrence of clonal hematopoiesis by HLA loss does not increase and may even decrease the risk of developing MDS-associated or PNH clones. This finding strongly suggests that treatment decisions for aAA patients whose only clonal event is HLA loss need not be considered distinctly from patients with no clonal changes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ECULIZUMAB DOSE ADJUSTMENTS DURING PREGNANCIES IN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: A SINGLE CENTER EXPERIENCE.

Paroxysmal nocturnal hemoglobinuria (PNH) has always been considered a relative contraindication for pregnancy. The use of eculizumab has reduced morbidity and mortality of fetus and mother. Dose adjustments are often required due to emerging breakthrough hemolysis, no standard recommendations are currently available in this setting. Here we report our single hematology center experience on breakthrough hemolysis and eculizumab dose adjustments during PNH pregnancies. Clinical data of four pregnancies from three PNH patients are reported. Two of them were already on standard dosage eculizumab when pregnancies were diagnosed. Their baseline PNH clone sizes were 89.5% and 97.5% on granulocytes and 38% and 90% on erythrocytes, respectively. In both cases low molecular weight heparin-prophylaxis was started when pregnancy was diagnosed. The third 26 year old patient with a baseline PNH clone of 8.8% started treatment at the beginning of her 2 nd trimester. The 26 year old patient was stable on 900 mg Eculizumab until 12 weeks post-partum. The other two developed breakthrough hemolysis during their third trimesters. Eculizumab dosing was increased initially from 900mg bi-weekly to 900 mg weekly and subsequently to 1200 mg biweekly. The 28 year old patient with the largest clone underwent two pregnancies and required even a 1200mg weekly dosage in her first pregnancy until four weeks post-partum. All four babies were delivered without complications. Eculizumab dose adjustments up to 1200 mg biweekly as well as interval shortening from bi-weekly to weekly administration seems effective and safe in breakthrough hemolysis control during pregnancy in PNH. Dose escalations did not impact on birth or development of the babies.

CLONAL EVOLUTION TO ACUTE MYELOID LEUKEMIA FOUR YEARS AFTER ALEMTUZUMAB AND CYCLOSPORINE IMMUNOSUPPRESSION IN A PATIENT WITH VERY SEVERE APLASTIC ANEMIA

Describe a rapid clonal evolution to AML with monossomy 7 in an aplastic anemia (AA) patient. A 17-year-old patient was diagnosed with severe aplastic anemia after a history of fatigue, mucocutaneous bleeding and pancytopenia. Bone marrow (BM) aspirate was hypocellular and had no dysplastic changes. Trephine biopsy confirmed 5% cellularity and absence of increased immature progenitors; reticulin fibrosis was not observed. Conventional cytogenetics showed a 46, XY karyotype; a small paroxysmal nocturnal hemoglobinuria (PNH) clone was present in red cells, neutrophils and monocytes. Chromosome breakage analysis for Fanconi anemia was unremarkable, and telomere length was within the normal range. The patient had no HLA-matched donor, and once hATG was discontinued in Brazil and eltrombopag was unavailable, he was included in a group of patients treated with upfront immunosuppressive therapy (IST) with subcutaneous alemtuzumab and cyclosporine; The patient achieved a partial response after 3 months of treatment. Cyclosporine was gradually tapered and was completely stopped after a year of treatment. This patient was lost to follow-up visits for six months and returned after four years of IST, complaining about worsening of fatigue and gingival bleeding. Complete blood count was notable for a loss of the achieved PR, and the presence of a small population of dysplastic monocytes. Revaluation of BM showed a hypercellular smear, and the presence of 60% of immature myeloid cells composed by myeloblasts and monocytes precursors. BM immunophenotyping was compatible with acute myelomonocytic leukemia. Conventional cytogenetics detected a monosomy of chromosome 7 and a trisomy of chromosome 21. Molecular studies for FLT3 and NPM1 mutations, and RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes were negative. The PNH clone size remained stable. The patient was started on standard AML chemotherapy with cytarabine and daunorubicin. An hematologic complete remission with a negative minimal residual disease was achieved. Due to an acute appendicitis during AML induction, the patient awaits consolidation therapy with an haploidentical hematopoietic stem-cell transplant (HSCT) from his brother. Frontline therapy for severe aplastic anemia patients younger than 40 years who have an HLA-matched sibling donor is HSCT. Triple IST with eltrombopag, hATG and cyclosporine is now considered the standard frontline therapy for patients older than 40 years and those who lack a suitable HLA-matched donor. Once hATG was discontinued in many countries, previous studies have reported outcomes of replacing hATG with rabbit ATG, cyclophosphamide, or alemtuzumab. Clonal evolution to myeloid neoplasms is a recognized complication of IST and patients with AA may develop secondary MDS/AML, with the main associated factors being longer disease duration, presence of mutations with an adverse prognosis, a short telomeric length and the development of cytogenetic abnormalities (CAs). The most recurrent CAs include total or partial loss of chromosome 7, trisomy 8 and 13q deletion, and, less frequently, trisomy 6, trisomy 15 and trisomy 21. T-cell-mediated autoimmune diseases, such as AA, have a higher risk of progression to AML/MDS dur to development of somatic mutations and CAs. Monosomy 7 independently represents a poor prognostic factor due to the high rate of progression to acute leukemia, such as occurred with the patient reported.

P834: REAL-WORLD EVIDENCE OF SAFETY AND EFFECTIVENESS OF ECULIZUMAB AND SWITCH TO RAVULIZUMAB IN A SWISS PATIENT POPULATION WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Background: Eculizumab (ECU) a complement component 5 (C5) inhibitor, is the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH). Ravulizumab (RAV) is engineered from ECU with the main advantage of having an extended 8-week (vs 2-week with ECU) dosing interval, which decreases treatment burden markedly. Hence, RAV is expected to become the new standard of care to treat PNH. In Switzerland, RAV was approved for PNH treatment in 2020. Aims: The objective of this study was to evaluate data from the PNH Swiss Soliris and Ultomiris Reimbursement Registry (SSURR) to assess key parameters for safety and effectiveness of ECU and to assess the adoption of RAV for PNH treatment by physicians under real-world conditions. Methods: The SSURR was designed in 2012 as a prospective, longitudinal, multi-center registry study in Switzerland. Patients were recruited and followed in 9 centers. Health-related data were collected at the first consultation (baseline) and during follow-up visits. In this report, results are based on data collected from 23 February 2012 to 30 September 2021. Data were summarized using descriptive analysis. Ethics approval was obtained. All participants provided written informed consent. Results: In total, 56 patients with a median age of 51 (range 20-84) years were enrolled in the Registry, 52% were female. For 42 patients, the 12-month follow-up (FU) was available: 39 patients on ECU and 3 on RAV (switched from ECU). Median FU was 27 (range 12-108) months. Hemoglobin (Hb) mean was 63 ± 47 g/L at baseline (n=41), and Hb mean was 105 ± 25 g/L (n=26) at the last visit. The lactate dehydrogenase (LDH) decreased from a mean 1457 ± 706 U/L (baseline, n=42) to 403 ± 212 U/L after 12 months FU (n=40; 65% of patients < 1.5 × upper limit of normal [ULN]), and was for 88% of the patients < 1.5 × ULN at their last visit (n=24). Thrombosis events (TE) were reported in 33% of patients prior to treatment start (n=42) and 3 (7%) had thrombosis during FU of which 2 patients had no prior history of TE. Within 12 months prior to treatment start, 21 patients (50%) received one or more red blood cell (RBC) transfusions. At 12 months of FU, 91% (n=42) were RBC transfusion-free. RBC transfusions remained reduced (82%; n=27 at last visit). After 12 months FU, 19% (n=8) of the patients had withdrawn from the registry due to allogenic bone marrow transplantation, low PNH clone size, transformation to hypoplastic MDS, and 5 non-disease related reasons. Eight patients from the 56 enrolled received RAV as initial treatment. They were all C5-inhibitor treatment-naïve with a mean LDH level of 1481 ± 1432 U/L at inclusion, which decreased to 230 ± 62 U/L at the 6-month visit (n=4; all below 1.5 × ULN). Of the 56 enrolled patients with PNH, 43% were switched from ECU to RAV. These patients (58% female) had a median age of 55 (range 26-85) years and 75% of them had LDH < 1.5 × ULN at their last LDH measurement on ECU before RAV administration. There were 7 patients with a 6-month FU after switching at which 6 of them had a LDH below 1.5 × ULN. Summary/Conclusion: The current analysis provides real world evidence for the safety and effectiveness of ECU in the Swiss PNH patient population, demonstrated by pronounced reduction in LDH, low need for RBC transfusion and stable hemoglobin as well as reduced occurrence of thrombosis. The fast adoption of RAV demonstrates RAV may likely become the new standard of care for patients with PNH. In time, more data will become available for safety and effectiveness of RAV under real-world conditions.

CD157 Can Replace CD24 and CD14 in a Single-Tube Flow-Cytometric Assay to Detect Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones on Both Neutrophils and Monocytes: A Prospective Study From North India.

IntroductionAs per current guidelines, detection of paroxysmal nocturnal hematuria (PNH) clones on leucocytes requires the demonstration of the loss of at least two glycosyl-phosphatidyl-inositol (GPI)-linked molecules on both neutrophils and monocytes by flow cytometry. CD24 and CD14 are GPI-linked molecules expressed on neutrophils and monocytes respectively, whereas another GPI-linked molecule, CD157, is expressed on both neutrophils and monocytes. This prospective study evaluated the ability of CD157 to replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes.Materials and methodsPNH clones were newly detected in 52 patients by an existing “standard” single-tube six-color flow-cytometric method, which was routinely performed in our laboratory at the time of undertaking this study. Six antibodies (CD45/CD15/CD64/CD24/CD14/FLAER) were used in this "standard" technique. Subjects were divided into two groups: (i) PNH disease (n=10), and (ii) aplastic anemia/myelodysplastic syndrome (AA/MDS) (n=42). Diagnosis of PNH disease and AA/MDS were made as per standard literature and guidelines. Results were compared with a single-tube five-color “test” assay using the antibodies CD45/CD15/CD64/CD157/FLAER by flow cytometry. Samples from 20 healthy control subjects were used to calculate cut-off values for the “test” assay.ResultsBy the "test" method, cut-off values for detecting PNH clones obtained from receiver operating-characteristic curve analysis were >0.4% for neutrophils (sensitivity=96.15%, specificity=95%), and >0.9% for monocytes (sensitivity=98.08%, specificity=95%). There was significant correlation between PNH clone sizes measured by both the “standard” and “test” assays in neutrophils (PNH disease: r=0.976, p<0.001; AA/MDS: r=0.980, p<0.001) as well as monocytes (PNH disease: r=0.806, p=0.005; AA/MDS: r=0.915, p<0.001). Bland-Altman analysis showed agreement between both assays in all the 52 patients and in individuals with AA/MDS. The cost of the test to the patients was about 15% less in the “test” method than the ”standard” technique, with improved technical efficiency.ConclusionCD157 can replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes, with reduced cost to the patients and improved technical efficiency.

The spectrum of paroxysmal nocturnal hemoglobinuria clinical presentation in a Brazilian single referral center

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder caused by the expansion of a hematopoietic clone harboring a somatic genetic variant in the PIG-A gene translating into a wide spectrum of clinical and laboratory changes, from intravascular hemolysis, thrombosis, and bone marrow failure to subclinical presentation. In this study, we retrospectively analyzed 87 consecutive cases (39 women; median follow-up, 18 months; range, 0–151 months) in whom a PNH clone was detected by flow cytometry between 2006 and 2019 seen at a single Brazilian referral center. The median age at diagnosis was 29 years (range, 8 to 83 years); 29 patients (33%) were initially classified as PNH/bone marrow failure, 13 (15%) as classic PNH, and 45 (52%) as subclinical PNH. The median overall survival (OS) of the entire cohort was not reached during follow-up, without significant differences between groups. At diagnosis, the median PNH clone size was 2.8% (range, 0 to 65%) in erythrocytes and 5.4% (range, 0 to 80%) in neutrophils. Fourteen patients experienced clone expansion during follow-up; in other 14 patients the clone disappeared, and in 18 patients it remained stable throughout the follow-up. A subclinical PNH clone was detected in three telomeropathy patients at diagnosis, but it was persistent and confirmed by DNA sequencing in only one case. In conclusion, PNH presentation was variable, and most patients had subclinical disease or associated with marrow failure and did not require specific anticomplement therapy. Clone size was stable or even disappeared in most cases.

Clinical characteristics of 512 eculizumab-naive paroxysmal nocturnal hemoglobinuria patients in China: a single-center observational study

ABSTRACT Objectives With large patient population and complement inhibitors naïve background, the characteristics patients with paroxysmal nocturnal hemoglobinuria (PNH) in China have not been well studied, especially for different subtypes. Methods We retrospectively reviewed patients with complete data who visited Peking Union Medical College Hospital (PUMCH) from 2009 to 2019 and had been followed up for more than 2 years. Results Five hundred and twelve patients were enrolled including 56.3% males and 43.7% females. The median age at disease onset was 33 (9∼80) years. Most were aged 21∼40 years (50.6%). 52.1%, 46.3% and 1.6% of the patients had classic PNH, bone marrow failure (BMF)/PNH and subclinical PNH, respectively. Symptoms of classic PNH were associated with hemolysis, whereas bleeding was more common in BMF/PNH patients. Classic PNH had higher survival rate, larger PNH clone size, higher lactate dehydrogenase (LDH) level and lower ferritin level than BMF/PNH. Although the rate of thrombosis was similar in the classic PNH and BMF/PNH (P = 0.66), those with BMF/PNH had higher chance of renal impairment (P < 0.05). Immunosuppressive agents was more common use in BMF/PNH (P < 0.05), but glucocorticoids, iron supplements and anticoagulants were more common used in classic PNH (P < 0.05) patients. Less evolution to myeloid malignancies was observed in classic PNH than in BMF/PNH (P = 0.02). The major causes of deaths were thrombosis (29.6%), hemorrhage (18.5%) and infections (18.5%). Conclusion Patients with classic PNH and BMF/PNH have different clinical profiles, and we described a more hemolytic features of PNH in China which might be improved with complement inhibitors.

CT-121: Phase 3 Study of the Efficacy and Safety of Iptacopan (LNP023), an Oral Factor B Inhibitor, in Adult Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Naïve to Complement Inhibitor Therapy

Context Standard of care in PNH is based on complement C5 inhibition with anti-C5 monoclonal antibodies (MAbs), which are generally effective at treating intravascular hemolysis (IVH), but unmet needs remain. Hematological responses to anti-C5 antibody treatment can be heterogeneous, and a substantial number of patients experience residual anemia associated with breakthrough hemolysis (BTH). Iptacopan, a first-in-class, oral, small-molecule, selective, and reversible complement factor B inhibitor, inhibits both IVH and extravascular hemolysis. In an ongoing Ph2 study (NCT03439839), iptacopan has demonstrated improvements in hematological response and biomarkers of disease activity in PNH adult patients who had active hemolysis, despite eculizumab treatment. Objective APPOINT-PNH study (NCT04820530) will assess iptacopan efficacy and safety in PNH adult patients naive to complement inhibitors, including anti-C5 MAbs. Design The study comprises ≤8-wk screening; 24-wk, single-arm, open-label core treatment; and 24-wk, open-label treatment extension. On completion of week 24, patients who benefit from iptacopan treatment can enter extension. Eligible adult patients (N≈40) have confirmed PNH diagnosis (RBCs and WBCs [granulocyte/monocyte] clone size ≥10%); anemia (mean Hb 1.5× ULN). Key exclusion criteria: prior treatment with complement inhibitor; known or suspected hereditary complement deficiency; history of HSCT; laboratory evidence of bone marrow failure; active, systemic infection ≤14 days preceding study treatment; and history of recurrent, invasive infections caused by encapsulated microorganisms. Primary objective will assess hematologic response, defined as achieving sustained increase in Hb ≥2g/dL (in absence of RBC transfusions). Primary endpoint will evaluate proportion of patients achieving an increase from baseline in Hb ≥2g/dL between D126–D168 (in absence of RBC transfusions between D14–D168). Secondary endpoints include proportion of patients reaching Hb ≥12g/dL; absence of RBC transfusions; changes from baseline in Hb; changes in FACIT-Fatigue scores; changes from baseline in reticulocytes; % change from baseline in LDH; occurrence of BTH; and occurrence of major adverse vascular events (including thrombosis). Additional analyses include safety assessments; evolution of PNH clone size and C3d deposition on RBCs; and other PROs. Conclusions Ph3 studies to assess iptacopan efficacy in a wider population of PNH patients, including those naive to anti-C5 MAbs treatment, are needed. Study sponsored by Novartis Pharmaceuticals Corporation.

Baseline Assessment of Patients with Newly-Diagnosed Acquired Bone Marrow Failure Enrolled in a Prospective Observational Study, “Search for Unidentified Links between PNH Clone Size and the Related Clinical Manifestations By High Precision Flow Cytometry (SUPREMACY)”

Baseline Assessment of Patients with Newly-Diagnosed Acquired Bone Marrow Failure Enrolled in a Prospective Observational Study, “Search for Unidentified Links between PNH Clone Size and the Related Clinical Manifestations By High Precision Flow Cytometry (SUPREMACY)”

First Interim Analysis of the French National Observatory of Paroxysmal Nocturnal Hemoglobinuria Clones

Introduction: Following the published International Guidelines (Borowitz, 2010), the French-Belgian biological workshop was initiated in 2010 by the French Paroxysmal Nocturnal Hemoglobinuria (PNH) group (named HPNAFC). This effort to harmonize the diagnostic practices has been pursued through the development of an inter-laboratory comparison program, involving more than 50 French-speaking centers since 2013 (Debliquis, 2015). Thus, the French National Observatory of PNH clones opened for 5 years on the 9th International Rare Disease Day (February 29th 2016), in order to collect all PNH clone cases diagnosed by French cytometrists.Methods: The primary objective of the French National Observatory of PNH clones is to monitor all PNH clones ≥0.01%, along with two secondary objectives according to the PNH clone size: the evolution of minor clones <1% and the proportion and significance of type II and type III cells for clones ≥1%. All patients with a PNH clone ≥0.01% can be included, irrespective of age, provided that the center has validated a PNH flow cytometry quality control. For each patient, the inclusion time point always corresponds with the initial diagnosis date of the PNH clone, even if it was made before the opening of the Observatory, allowing the collection of all cases in the active French PNH file. This Observatory is based on a principle of non-opposition; inclusions are made by the referent cytometrist of each participating center by filling in the e-CRF form available on the HPNAFC website.Results: As of June 30th 2017, 39 cytometry laboratories have registered across France. Of these, 24 centers have submitted 163 inclusion points with a PNH clone above 0.01%, each corresponding to a patient. The cases were reviewed by the 2 principal investigators, who carefully re-examined all of the flow cytometry data. This method enabled the validation and inclusion of 126 cases, with 37 ongoing ones due to missing clinical or biological data. The first extraction led to the preliminary analysis of these 126 cases and showed that patient median age at diagnosis was 45 years [11-87], with 6 pediatric cases, and a M:F sex ratio of 0.83. Diagnosis was made between 1988 and 2017 and clinical information at diagnosis was available for 92% of patients. More than half of them had aplastic anemia (58.7%). Other diagnosis included: hemolytic anemia (with or without aplastic anemia) (15.9%), unexplained cytopenia(s)(7.9%), myelodysplastic syndrome (6.3%), hemoglobinuria (4.8%) and 2 cases (1.6%) of recurrent thrombosis without hemolysis. Biological data showed a median hemoglobin value of 9.3 g/dL [4.7-16.5], 1.4 x109/L [0.1-11] neutrophils and 60 x109/L [2-356] platelets. The median clone size at diagnosis was 1.9% on granulocytes with a very wide range from 0.01% to 99.4%, most of them being less than 10% and mainly composed of type III cells (median type III clone size: 1.6%). A similar distribution of PNH clones was observed on monocytes, with a good correlation between clone sizes on both lineages. RBC analysis, which was performed for less than half of the patients showed a majority of minor clones and no correlation was observed between PNH clone size on granulocytes and RBC, due to an underestimation of the clone size on RBC. Regarding the clinical context, the median PNH clone size on granulocytes in case of aplastic anemia was 1%, yet some patients had very large clones, higher than 99%. Conversely, PNH clones from patients with hemolytic anemia were very large with a median size of 88%, but some patients had smaller clones of less than 20%. The 2 patients with thrombosis displayed a much smaller clone size, below 0.05%.Conclusion: More than one year after its initiation, 126 confirmed patients were enrolled in the French National Observatory of PNH clones. Median PNH clone size was 1.9% on granulocytes and more than half of these patients had aplastic anemia. A good correlation was seen between clone sizes on granulocytes and monocytes, but not on granulocytes and RBCs. This collaborative work has established a strong network of motivated cytometrists as highlighted by the number of registered centers and the increasing inclusions since its recent opening. A comprehensive follow-up of a foreseeable larger number of PNH clones (so far 92 out of the 163 already have at least 1 follow up point) will lead to a better understanding of the physiopathology of minor PNH clones. DisclosuresDrenou:Alexion: Consultancy, Honoraria. Pruvot Debliquis:Alexion: Consultancy, Honoraria. Letestu:Alexion: Consultancy, Honoraria. Chatelain:Alexion: Consultancy, Honoraria. Socié:Alexion Pharmaceuticals, Inc.: Consultancy. Peffault De Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding. Wagner Ballon:Alexion: Consultancy, Honoraria.

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

We aimed to determine whether small paroxysmal nocturnal hemoglobinuria (PNH) clones detected by flow cytometry (FCM) harbor PIG gene mutations with quantitative correlation. 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. 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. 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.

Pediatric Patients with Severe Aplastic Anemia Treated with Combined Immunosuppressive Therapy, Eltrombopag and Eculizumab and Their Clinical Course to Stem Cell Transplant

Acquired aplastic anemia (AA) in children is a rare disorder characterized by pancytopenia and hypocellular bone marrow. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoetic stem cell (HCT) disorder characterized by complemented-mediated hemolysis, thrombosis and bone marrow failure secondary to deficiency of glycosylphosphatidylinositol-anchored proteins (GPI-AP) on hematopoetic stem cells. PNH and acquired AA are closely related; up to 50% of patients with AA have detectable PNH-clones at the time of diagnosis and small percentage of them can have clonal expansion throughout their disease course requiring close monitoring. Current standard therapy for severe aplastic anemia (SAA) patients without matched related donor (MRD) is immunosuppressive therapy (IST) regimen with anti-thymoglobulin (ATG), cyclosporine (CSA), and recent addition of eltrombopag. Eculizumab is a recombinant humanized monoclonal antibody that blocks complement protein C5 and prevents cell lysis. While it has been shown to be effective in children with PNH, concomitant treatment with IST for patients with SAA is unknown. To our knowledge, there has been no pediatric data on combined IST, eltrombopag and eculizumab treatment for children with AA with clinically significant PNH clones. Here we retrospectively reviewed three pediatric patients with SAA with PNH clones treated with IST, eltrombopag and eculizumab and their unique clinical courses. Two out of three patients had high PNH clone size and were started on eculizumab prior to IST with improvement in transfusion intervals. Of the two, one had decrease in PNH clone size after IST but the other patient's clone size continued to increase despite two courses of IST. Finally, the third patient had a minor PNH clone and was not started on eculizumab prior to IST. He remained asymptomatic for over a year until he developed symptomatic PNH with large clone size and aplastic bone marrow. His clone size continued to increase with no improvement in transfusion frequency despite being started on eculizumab. However, steroids were started based on anecdotal literature and his transfusion frequency decreased subsequently. All three patients are in the process of going through matched unrelated donor stem cell transplants. Several studies have reported the presence of a minor PNH clone at the time of AA diagnosis was associated a favorable response to IST. In this case series, the patient with the smallest PNH clone size at the time of diagnosis of SAA had the best response to IST compared to the other two patients who had larger PNH clone populations. These findings suggest that even though minor PNH population may lead to better response to IST compared to absence of the clone, the correlation between clone size and prognosis is unclear. Further study with larger sample size is needed to investigate this relationship. However, regardless of the population size, all three patients were able to prolong transfusion intervals using eculizumab without significant side effects. As the adoption of eltrombopag with standard IST is evolving with ongoing study, the efficacy of incorporating eculizumab to decrease transfusion frequency in patients with PNH-clone in addition to eltrombopag/IST regimen should be further investigated. Disclosures No relevant conflicts of interest to declare.