Paroxysmal Nocturnal Hemoglobinuria Clone Research Articles

The metabolic fuel of paroxysmal nocturnal haemoglobinuria.

Metabolic reprogramming has been investigated in haematological malignancies. To date, a few studies have analysed the metabolic profile of paroxysmal nocturnal haemoglobinuria (PNH). The study by Chen and colleagues sheds light on the involvement of metabolic changes in the proliferation of PNH clones. Commentary on: Chen etal. The histone demethylase JMJD1C regulates CPS1 expression and promotes the proliferation of PNH clones through cell metabolic reprogramming. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19477.

The histone demethylase JMJD1C regulates CPS1 expression and promotes the proliferation of paroxysmal nocturnal haemoglobinuria clones through cell metabolic reprogramming.

Paroxysmal nocturnal haemoglobinuria (PNH) is a disorder resulting from erythrocyte membrane deficiencies caused by PIG-A gene mutations. While current treatments alleviate symptoms, they fail to address the underlying cause of the disease-the pathogenic PNH clones. In this study, we found that the expression of carbamoyl phosphate synthetase 1 (CPS1) was downregulated in PNH clones, and the level of CPS1 was negatively correlated with the proportion of PNH clones. Using PIG-A knockout K562 (K562 KO) cells, we demonstrated that CPS1 knockdown increased cell proliferation and altered cell metabolism, suggesting that CPS1 participates in PNH clonal proliferation through metabolic reprogramming. Furthermore, we observed an increase in the expression levels of the histone demethylase JMJD1C in PNH clones, and JMJD1C expression was negatively correlated with CPS1 expression. Knocking down JMJD1C in K562 KO cells upregulated CPS1 and H3K36me3 expression, decreased cell proliferation and increased cell apoptosis. Chromatin immunoprecipitation analysis further demonstrated that H3K36me3 regulated CPS1 expression. Finally, we demonstrated that histone demethylase inhibitor JIB-04 can suppressed K562 KO cell proliferation and reduced the proportion of PNH clones in PNH mice. In conclusion, aberrant regulation of the JMJD1C-H3K36me3-CPS1 axis contributes to PNH clonal proliferation. Targeting JMJD1C with a specific inhibitor unveils a potential strategy for treating PNH patients.

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.

Abnormal expression of CUX1 influences autophagy activation in paroxysmal nocturnal hemoglobinuria.

The mechanism underlying autophagy in paroxysmal nocturnal hemoglobinuria (PNH) remains largely unknown. We previously sequenced the entire genome exon of the CD59- cells from 13 patients with PNH and found genes such as CUX1 encoding Cut-like homeobox 1. Peripheral blood samples from 9 patients with PNH and 7 healthy control subjects were obtained to measure CUX1 expression. The correlation between CUX1 messenger RNA expression and PNH clinical indicators was analyzed. To simulate CUX1 expression in patients with PNH, we generated a panel of PNH cell lines by knocking out PIGA in K562 cell lines and transfected lentivirus with CUX1. CCK-8 and EDU assay assessed cell proliferation. Western blotting was used to detect Beclin-1, LC3A, LC3B, ULK1, PI3K, AKT, p-AKT, mTOR, and p-mTOR protein levels. Autophagosomes were observed with transmission electron microscopy. Chloroquine was used to observe CUX1 expression in PNH after autophagy inhibition. Leukocytes from patients with PNH had lower levels of CUX1 messenger RNA expression and protein content than healthy control subjects. The lactose dehydrogenase level and the percentage of PNH clones were negatively correlated with CUX1 relative expression. We reduced CUX1 expression in a PIGA knockout K562 cell line, leading to increased cell proliferation. Levels of autophagy markers Beclin-1, LC3B, LC3A, and ULK1 increased, and autophagosomes increased. Furthermore, PI3K/AKT/mTOR protein phosphorylation levels were lower. CUX1 expression did not change and cell proliferation decreased in CUX1 knocked down PNH cells after inhibition of autophagy by chloroquine. In brief, CUX1 loss-of-function mutation resulted in stronger autophagy in PNH.

Case report on &amp;quot;Spontaneous Remission in Paroxysmal Nocturnal Hemoglobinuria: A Rare Phenomenon

ABSTRACT Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disorder characterized by a deficiency of GPI-anchored proteins on blood cells. It is associated with hemolysis, thromboembolic events, and bone marrow failure. Management includes complement inhibitors, anticoagulation, and stem cell transplantation. Spontaneous remission of PNH is rarely reported. Case Presentation: A 28-year-old male initially diagnosed with aplastic anemia developed PVT and was subsequently diagnosed with PNH. Due to the unavailability of complement inhibitors, anticoagulation therapy and supportive care were employed. Splenectomy with splenorenal shunt was performed for chronic thrombosis and associated complications. The patient achieved spontaneous remission with normalized blood counts and diminished PNH clone. Conclusion: Managing PNH in resource-limited settings presents challenges due to the unavailability of complement inhibitors. Spontaneous remission of PNH is rarely reported and needs further research. Multidisciplinary approach, accessibility to diagnostic tests and advanced treatments will enhance PNH management in resource limited settings.

FLAER as a standalone reagent for paroxysmal nocturnal hemoglobinuria: Do we need to reconsider the guidelines for testing?

Flow cytometry-based paroxysmal nocturnal hemoglobinuria (PNH) testing involves utilization of monoclonal antibodies against GPI-linked proteins and FLAER. The ability of FLAER to bind to a wide variety of GPI-linked structures and to be utilized across different leukocyte subsets is remarkable. We hypothesize that FLAER as a standalone reagent may be equally effective for detecting PNH clones. The present study intends to compare the results of a FLAER alone-based strategy to the recommended FLAER+GPI-linked protein-based approach for applicability in clinical settings. EDTA-anticoagulated blood samples from patients for PNH workup were tested for PNH by multiparametric flow cytometry. A conventional panel comprising gating markers (CD45 for WBC, CD15 for granulocytes, and CD64 for monocytes) and a combination of FLAER and GPI-linked markers, such as CD24 and CD14, henceforth referred to as the "routine panel," was employed. Second, a "FLAER-only panel" comprising the gating markers and FLAER alone (excluding the GPI-linked markers CD24 and CD14) was set up. The samples were processed using the lyse-wash-stain-wash technique, and events were acquired on BC Navios Ex flow cytometer (Beckman Coulter, Inc., USA) and analyzed on Kaluza Software 2.1. The presence of a PNH clone was reported at a value of ≥0.01%. A total of 209 patients were tested. Both panels found a PNH clone in 20.1% of patients (n = 42/209) with a 100% concordance rate. The PNH clone range for granulocytes was 0.01%-89.68%, and for monocyte was 0.04%-96.09% in the routine panel. The range in the FLAER-only panel for granulocytes was 0.01%-89.61%, and for monocytes, it was 0.01%-96.05%. Pearson correlation statistics revealed a significant correlation between the size of the PNH clone of granulocytes and monocytes among the two panels tested (granulocytes r = 0.9999, p < 0.0001, 95% CI = 0.9999 to 1.000; monocytes r = 0.9974, p < 0.0001, 95% CI = 0.9966-0.9980). Based on our results, FLAER as a standalone marker is specific and sensitive for identifying PNH clones in granulocytes and monocytes, even for high-sensitivity PNH assay. The proposed "FLAER-only panel" panel is efficient and cost-effective for highly sensitive PNH testing in two different cell lineages, especially in resource-limited clinical settings.

Distinct Immune and Molecular Profiles of Hepatitis-Associated Aplastic Anemia

Background: Hepatitis-associated aplastic anemia (HAAA) is characterized by the development of hematopoietic failure within months following an acute episode of non-infectious hepatitis. Although disease presentation with pancytopenia and a hypocellular marrow in HAAA is similar to non-hepatitis immune AA (non-HAAA), the immune and molecular profile in HAAA has not been completely elucidated. Here, we aimed to assess HAAA patients for cytokines and presence of clonal events linked to AA, the presence of paroxysmal nocturnal hemoglobinuria (PNH) clones and somatic mutations in peripheral blood mononuclear cells (PBMC) subpopulations, and compared to a non-HAAA cohort. Methods: A cohort of HAAA patients (n=30), age and sex-matched non-HAAA patients (n=24) and healthy controls (n=23) seen at the National Institutes of Health since 2003 were retrospectively screened for a panel of inflammatory cytokines and growth factors by Luminex, and somatic mutations in sorted CD3-, CD3+CD4+, and CD3+CD8+ PBMC fractions by error-corrected DNA sequencing. A customized panel with 42 myeloid-related and 49 autoimmunity-related genes at minimum limited of detection of 0.1% were used. All samples were before any immunosuppressive treatment (IST). Cytokine levels and frequencies of PNH clones and clonal hematopoiesis (CH) in different fractions were compared among groups. Results: HAAA patients were in average young (median age=23); 37% were at age &amp;lt; 18 (Table 1). Although disease severity and blood counts were similar to the non-HAAA cohort, immune and molecular markers of immune AA were distinct among cohorts. The frequency of PNH clones was significantly lower in HAAA compared to the non-HAAA cohort (15% v 58%, p=0.0008). The median size of PNH clones in HAAA and non-HAAA was 1.4% and 7.4%, respectively (p =0.03). The cytokine profiles performed in the sera of 22 HAAA before IST were marked by significantly higher levels of IL-1ra (p=0.0008), CXCL-10 (p=0.0038), and VEGF (p=0.0023) in comparison to 31 non-HAAA assessed patients; IL-6 (p=0.0002) and CD40L (p=0.0001) levels were significantly lower than in non-HAAA. As reported in immune AA, HAAA patients had higher levels of TPO (p=0.0001), G-CSF (p=0.0001), and EPO (p=0.0001) than controls; lower levels of CCL-5 (p=0.0001) and CCL-11 (p=0.0025) were seen in the HAAA. FLT3-L levels were markedly elevated in both AA cohorts when compared to HC. The clonal landscape was also strikingly different between HAAA and non-HAAA in all fractions evaluated (Figure 1). In HAAA, 3 out of 21 assessed patients (14%) had CH in the CD3- fraction at maximum variant allele frequency (VAF) of 1.8%; one patient had two RUNX1 mutations while a JAK2 and DNMT3A mutation were found in the other two patients. No mutations were found in CD4+ and CD8+ compartments in subjects with HAAA. In contrast, 13 of 24 non-HAAA patients (54%) had CH in the CD3- populations, which was dominated by PIGA, BCOR, and DNMT3A clones, a pattern consistent with the known clonal landscape of immune AA. The frequency of non-HAAA with CH in the CD4+ and CD8+ compartments was 7/24 (29%) and 8/24 (30%) patients, respectively; CH in these fractions was similar to those found in CD3- dominated by mutations in PIGA, BCOR, and DNMT3A at VAFs ranging from 0.2% to 15% (Figure 1). CH in lymphoid-related genes was only found in a single patient with a STAT3 p.S614Rmutation at VAF of 3% exclusive to the CD8+ fraction. Conclusion: HAAA has a distinct immunologic and molecular profile than non-HAAA patients. PNH clones were less frequent in HAAA and patterns of cytokines were significantly different than the non-HAAA cohort. CH in PIGA and BCOR, highly associated with AA, were not present in our small HAAA cohort. Our results indicate a distinct underlying pathophysiology that initiates the immune destruction resulting in marrow failure in HAAA patients. Confirming these findings in a larger cohort and correlating with clinical outcomes is ongoing.

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.

Ahemolytic PNH: Clinical Features of a Distinct Phenotype of Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem/progenitor cell disorder caused by PIGA mutations. All hematopoietic lineages derived from the affected clone exhibit the PNH phenotype [deficiency of glycosyl phosphatidylinositol linked membrane proteins (GPI-APs)]. Diagnosis is made by flow cytometric analysis of red blood cells (RBC), granulocytes, and monocytes. The percentage of granulocytes and monocytes with absent expression of GPI-APs is a measure of the size of the PNH clone. Typically, the percentage of GPI-AP deficient RBCs is somewhat less than that of granulocytes and monocytes due to selective destruction by complement-mediated intravascular hemolysis, manifested by high LDH, bilirubin, reticulocyte count and low haptoglobin. Herein, we report five patients with large PNH clones based on percentage of GPI-AP deficient neutrophils and monocytes, but with absent or near absent PNH RBCs and no biochemical evidence of hemolysis. We call this unique disease subtype, ahemolytic PNH. Methods: The medical records of PNH patients (174 patients with PNH granulocytes &amp;gt;20%) at the University of Texas Southwestern, Cleveland Clinic Foundation, and National Institutes of Health from 2000 to 2022 were examined. Information pertaining to their clinical, laboratory, and molecular attributes (NGS sequencing) was collected. Five patients with granulocyte clones &amp;gt;50% and RBC clones &amp;lt;5% were identified (Table). Results: Patient 1 is a 63-year-old male with a remote history of non-severe aplastic anemia (AA) diagnosed at 11 years old. The first PNH flow cytometry, obtained at age 61 years, showed 0.02% type II PNH RBC, 0.5% type III PNH RBC, 96% PNH monocyte and 97% PNH granulocytes (Table). There was no biochemical evidence of hemolysis at the time of diagnosis or in the preceding 5 years. Patient 2 is a 50-year-old male who presented with pancytopenia and was diagnosed with acute myeloid leukemia with myelodysplastic-related mutations. Flow cytometry showed 85% PNH monocytes and 59% PNH with no detectable PNH RBCs and no biochemical evidence of hemolysis at diagnosis or in 3 years of follow-up (Table). Patient 3 is a 73-year-old female who presented with pancytopenia and marrow aplasia with unremarkable morphology, and a PNH granulocyte clone of 93.6% with 1.3% type III PNH RBCs and 1.3% and 0.5% type II PNH RBCs, with no evidence of hemolysis (Table). Patient 4 is a 21-year-old female who presented with Budd-Chiari syndrome. Flow cytometry showed 73% PNH granulocytes with 3.6% type III PNH RBCs and 1.8% type II PNH RBCs. There was no biochemical evidence of hemolysis, but because of thrombosis, she was treated complement inhibitor therapy (Table). Patient 5 is a 61-year-old female with history of severe aplastic anemia treated with immunosuppression (IST) and eltrombopag. PNH flow cytometry showed PNH granulocytes of 92.4% and 2.1% PNH type II RBCs 2.1% post IST treatment with no biochemical evidence of hemolysis (Table). Conclusion: Herein, we present five patients with a distinct PNH phenotype that we call ahemolytic PNH. Except for the absence or near absence of PNH erythrocytes, this entity shares clinical and pathophysiological features with classic PNH, including thrombophilia (Budd-Chiari Syndrome, Table). The basis of this phenotype is speculative but may be determined by the genotype of the affected HSPC in which the somatic mutation of PIGA first occurred. According to this hypothesis, somatic mutations other than PIGA skew differentiation toward granulocyte/monocyte lineages. The International PNH Interest Group recognizes the following three categories of PNH: classic PNH, PNH in the setting of another define bone marrow abnormality, and subclinical PNH. Like ahemolytic PNH, subclinical PNH has no biochemical evidence of hemolysis. In this case, however, the absence of hemolysis is due to the small size of the PNH clone (median clone size &amp;lt;1%) as determined by flow cytometry analysis of peripheral blood granulocytes and monocytes. We propose that ahemolytic PNH, defined as patients having less than &amp;lt;5% PNH red cells and &amp;gt;50% PNH granulocytes/monocytes, be recognized as a distinct category of PNH.

Neutrophil Functionalities in Patients with Paroxysmal Nocturnal Haemoglobinuria Are Not Affected By Complement C5- Inhibition

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired haematological stem cell disorder characterized by haemolysis, thrombophilia, and cytopenia. PNH is caused by a somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene at the level of pluripotent, hematopoietic stem cells leading to a lack of GPI anchored cell surface proteins, such as complement-inactivating proteins, (e.g., CD55, CD59) on stem cells, polymorphonuclear neutrophil granulocytes (PMN), erythrocytes, and platelets. Subsequently, uncontrolled complement overactivity leads to the systemic complications. Phenotypic mosaicism is a distinct characteristic of PNH. For diagnostic purposes, the absence of the GPI-anchor on granulocytes and monocytes in the flow cytometry is considered the gold standard. However, the exact consequences of GPI deficiency in PMNs and monocytes have not been studied sufficiently yet. Since the implementation of complement inhibitors, the primary cause of morbidity and mortality in PNH has been significantly reduced. Despite this, the tendency towards thrombosis, as well as the consequences of the deficiency of GPI-linked proteins on PMNs and monocytes, remain largely unclear. Therefore, the main objective of this project is to gain a deeper understanding of the impact of PNH-PMNs and monocytes on thrombo-inflammation in PNH. Specifically, the impact of complement inhibitors on these factors has been compared. Blood from PNH patients (n=14) with a median clone size of 74 % were compared with an age- and sex-matched control group (n =20). Various cellular markers indicating a state of activation (CD11b, TREM1, CD64) or showing platelet adherence (CD42), were measured using flow cytometry. Unmutated PMNs and monocytes from PNH patients (PMN) served as intrinsic control, GPI positive PMNs (WT-PMN (PNH)) and monocytes from a control group served as extrinsic control. We compared unstimulated PNH and WT cells for their basal phenotype, and then functional changes upon stimulation (phorbol 12-myristate 13-acetate (PMA)). Our data present a contrary perspective to our initial assumptions, indicating that PNH-PMNs have a direct impact on unmutated PMNs, resulting in the generation of a thrombogenic state and increased levels of activation. The unmutated GPI-positive PMNs and monocytes exhibit a significantly stronger direct aggregation with platelets compared to GPI-deficient clones. It is hypothesized that the PNH clones influence the activity and aggregation propensity of the remaining unmutated granulocytes through additional cellular mechanisms. In a subgroup analysis, the impact of complement factor 5 inhibitors (C5i) on the aforementioned factors was investigated detecting no significant differences in terms of PMN activation or platelet aggregation. Thus, it can be hypothesized that thrombosis and hyper-inflammation are not solely driven by the PNH clones directly and modulated C5 inhibition. Additional cellular mechanisms and signaling pathways appear to play an important role, and these aspects have not been adequately addressed by the current therapy.

Branching Trajectories and Diversification of Clonal Escape in Aplastic Anemia Revealed By Single-Cell Genomics

Acquired aplastic anemia (AA) is a bone marrow failure resulting from an autoimmune T-cell attack against hematopoietic stem and progenitor cells (HSPCs). Several escape mechanisms within the HSPC compartment have been reported: i) adaptive alterations causing loss of high-risk HLA allele of AA development [deletion or uniparental disomy (UPD) of chromosome 6p including HLA cluster region, and loss-of-function (LOF) mutations in HLA gene], ii) clonal expansion of paroxysmal nocturnal hemoglobinuria (PNH) clone resulting from PIGA mutation, and iii) presence of clonal hematopoiesis of indeterminate potential (CHIP) mutations (i.e., BCOR/ BCORL1, DNMT3A, ASXL1). Multiple types of alterations often co-occur, however due to the inherent limitations of conventional methods, it is not known if i) these alterations are in the same or independent clones, ii) what is the mutational hierarchy, and iii) what is the cell of origin. We addressed these questions using single-cell and bulk genomic sequencing. We developed custom single-cell panels covering HLA class I and II genes, along with 14 genes commonly mutated in AA and myelodysplastic syndromes. To identify copy number variations and UPD, we included 197 single nucleotide variants (SNVs) frequent across all ethnic backgrounds on chromosomes 6, 7, 8, 20, and 21. The TotalSeq™-D panel (BioLegend), was included to target 42 surface antigens and the samples were prepared on the Tapestri platform (Missionbio). High-resolution HLA genotyping was done using AlloSeq Tx 17 hybrid capture NGS assay (CareDx), and for confirmatory bulk sequencing we applied a custom duplex UMI NGS panel (TWIST Bioscience). We examined ten patients with severe AA (pediatric: n=6, age range 2-15 years; adult: n=4, 26-67), seven patients with refractory cytopenia of childhood (RCC, age range 2-16 years), who presented a hypocellular marrow and absence of a genetic predisposition syndrome, and one healthy control. All 17 patients had at least one of the HLA-A or HLA-B risk alleles linked to AA development. A total of 136,505 cells (median 4,467/run) were successfully genotyped across the samples. Single-cell trajectory analysis revealed that all identified clones arose independent of each other (Figure 1). UPD6p clones were found in five patients with three having multiple UPD6p clones carrying different breakpoints, pointing to clonal diversification. Additionally, three cases had LOF mutations in HLA-A or HLA-B (two having coexisting UPD6p). In a patient with two UPD6p and two HLA-A mutant clones, we verified that HLA-A risk allele (A*02:01) was lost in each instance. Multi-omic analysis revealed that these clones had both granulocytic and non-granulocytic (incl. lymphocytic) surface phenotypes, suggesting their early origin within hematopoietic ontogeny. Seven patients had nine independent clones with PIGA mutations (0.3-11.3% of the cells), consistent with its tendency to generate clonal mosaicism. Clones with mutations in BCOR/BCORL1, ASXL1, DNMT3A, and TET2 were identified in six, seven, four, and two patients, respectively. We next analyzed how many patients carry multiple clones and found that 65% (11/17) have concomitant clones, with the majority (9/11) belonging to different types of clonal escape (Figure 1). Finally, we found that 4/6 patients with adaptive HLA loss (representing true clonal escape from T-cell autoimmunity) also carried CHIP clones, indicating that adaptive hematopoiesis does not protect from the acquisition of CHIP mutations. In conclusion, we show that patients with AA across age spectrum and some patients with RCC share similar clonal backgrounds, with multiple functionally redundant alterations arising independently from coexisting CHIP clones. For the first time at a single-cell level, we were able to resolve the multifaceted clonal mosaic generated by the immunological forces at play in AA, and precisely unravel their immunoediting consequences.

Superior Outcome of Bone Marrow Transplantation Using Fludarabine and Cyclophosphamide with ATG over Fludarabine and Cyclophosphamide without ATG Conditioning Regimen in Severe Aplastic Anemia

ABSTRACT Background: Matched sibling donor (MSD) allogenic hematopoietic stem cell transplantation (allo-HSCT) is the standard of care as the first line therapy for young fit patients with severe aplastic anemia (SAA). There are several conditioning regimens that have been evaluated and used for these patients. Fludarabine (Flu)/cyclophosphamide (Cy)/antithymocyte globulin (ATG) or Cy/ATG led to the best survival. The 5-year probabilities of survival after Flu/Cy/ATG, Cy/ATG, Cy ± Flu, and Busulfan /Cy were 91% (95% CI, 85-96), 91% (95% CI, 89-94), 80% (95% CI, 73-87), and 84% (95% CI, 75-91) (P = .001), (Bejanyan et al. 2019). Objective: To report the outcome of a homogeneous cohort of SAA patients who received a uniform transplantation conditioning regimen protocol (Flu/Cy/ATG) and compare it with the historical protocol (Flu/Cy) from the same transplant center for the same patients population. Methods: This study is a 10-year retrospective analysis of SAA patients who underwent MSD allo-HSCT at King Faisal Specialist Hospital &amp; Research Centre, Riyadh City, Saudi Arabia during the period from 2013 to 2022. The conditioning regimen was consistent of Fludarabine 150 mg/m 2/Cyclophosphamide 200 mg/kg/ Anti-thymocyte globulin (rabbit) 2.5 mg/kg (Flu/Cy/ATG). Graft-versus-host disease (GVHD) prophylaxis was methotrexate (MTX)/tacrolimus (FK) or methotrexate/cyclosporin (CsA). The outcome was compared with the historical published data of 82 patients with SAA received Flu/Cy conditioning regimen for MSD allo-HSCT, (El Fakih et al. 2021). Results: The study included 28 patients with SAA, 46.2% (n=12) patients were females, paroxysmal nocturnal hemoglobinuria (PNH) clone detected in 57.1% (n=16). Total body irradiation (TBI) was added to the conditioning regimen in 25% (n=7) for patients with heavily blood products transfusion pre-HSCT. A total of twenty-six patients (93%) has fully MSD, while 2 (7%) has matched unrelated donors (MUD). Bone marrow (BM) was the stem cell source in 60.7% while 28.6% received peripheral blood (PB) stem cell and 10.7 % received combined (BM/PB). There were twelve patients with donor-recipient sex mismatch. Major ABO mismatch counted for 17.9% (n=5). The median age at transplantation was 25 ±10.2 years. During the observational period, there was no graft failure (GF) and none of the patients died. At 60 days of HSCT; 61 % (n=17) patients achieved full donor whole blood (WB) chimerism, while 11 (39 %) patients had mixed chimerism. At both 180 days and 365 days of HSCT; 69.2% (n=18) patients had full donor chimerism and 30.8% (n=8) of the patients had mixed donor chimerism. Acute GVHD occurs in five cases (17.9%) and none had developed grade III or IV, notably; 80% of GVHD cases (4/5) received PB or BM/PB stem cell source. Chronic GVHD was observed in six cases (21.7%), 66% (4/6) of them had received PB or PB/BM stem cell source. The use of PB was due to either donor related reasons like recipient-donor weight discrepancy or patient's medical condition to achieve early engraftment. Hemorrhagic cystitis occurred in one case (3.6%), none had sinusoidal obstruction syndrome (SOS). CMV viremia without disease occurs in eleven patients (39 %). The median follow-up time for survivors was 34 months, the 2 years overall survival (OS) was 100%, the 2 years event free survival (EFS) defined with GF, or death was 100% compared with the previous result published (El Fakih et al., 2021) which had showen lower OS and EFS of 87.8% and 82.9% respectively with median follow-up of 100 months, Figure1&amp;2. Table-1 and table-2 summarize the comparison of patient's characteristics and outcome of both regimens (Flu/Cy vs Flu/Cy/ATG). Conclusions: The results of the present study showed better outcomes for SAA patients using Flu/Cy/ATG compared with the previous study (El Fakih et al., 2021). Early MSD allo-HSCT for SAA patients using Flu/Cy/ATG conditioning regimen is associated with a favorable outcome of excellent graft function, low rate GVHD and no GF. BM should be the stem cell source in severe aplastic anemia HSCT procedure. Key Words: Severe Aplastic Anemia, Matched Sibling Donor, Conditioning, Bone Marrow Graft, Flu/Cy/ATG.

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.

The Role of Red Blood Cell Lifespan Testing in the Evaluation of Patients with Paroxysmal Nocturnal Hemoglobinuria

Objective To explore the role of red blood cell(RBC) lifespan testing in the evaluation of patients with paroxysmal nocturnal hemoglobinuria (PNH). Methods The endogenous breath carbon monoxide (CO) test was used to detect the RBC lifespan of PNH patient with the treatment of crovalimab（ Roche） or iptacopan（ Novartis.），for comparison of different hemolysis indices in the assessment of patients' condition. Results Nine patients have been enrolled in the study at our centre from June to December 2021, 4 with the treatment of crovalimab and 5 with iptacopan. The median lifespan in the untreated patients was 17 (range 12-38) days, median hemoglobin(Hb) 83(69-94) g/l, median lactic dehydrogenase（LDH）590.8(446.1-935.5)U/L, median PNH clones 96.0(33.2-99.9)%. By 6 months post-treatment, 8 patients achieved hematologic improvement without breakthrough hemolysis. In all patients, LDH levels decreased rapidly and stabilized at lower levels after starting treatment. But the degree of Hb recovery varied，median 60(20-85)g/L. Analyzing the correlation between different hemolytic indices (LDH, RBC lifespan, total bilirubin, indirect bilirubin, etc.) and Hb changes, RBC lifespan showed good correlation（P=0.002，R=0.867）. In the crovalimab group, three patients presented with a positive Coombs test and the negative patient didn't obtain hematologic improvement. No patients with a positive Coombs test were seen in the iptacopan group. Conclusion Different (upstream/downstream) complement inhibitors have different effects on the condition of PNH patients, and different indicators can reflect different results; LDH responds to intravascular hemolysis, and red blood cell lifespan also quantifies extravascular hemolysis, and is applied to monitor hemolysis. The application of RBC lifespan testing can provide an accurate scientific basis for the assessment of PNH conditions. Keywords: red blood cell lifespa, paroxysmal nocturnal hemoglobinuria ,red blood cell lifespan,lactic dehydrogenase

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

Sars-Cov-2 Antibody and T-Cell Responses in Patients with Paroxysmal Nocturnal Hemoglobinuria and Aplastic Anemia after Four COVID-19 Vaccinations

Introduction: There is limited data on SARS-CoV-2 vaccination responses in the rare hematological disorders paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA). These patients were expected to have more severe COVID-19 infection and reduced vaccination responses due to their underlying disease and immunosuppressive treatment. We previously reported limited anti-spike IgA/G/M responses after first COVID-19 vaccination which improved following second vaccination (Pike et al, Lancet Haematology 2022). Here we report spike-specific IgG, in vitro viral neutralization and T-cell responses in 244 patients with PNH and/or AA and in 49 healthy volunteers, following the first four vaccinations. Methods: In 2021, the United Kingdom National PNH service centre based in Leeds initiated a prospective observational non-interventional study evaluating immune responses to COVID-19 vaccinations in patients with PNH and/or AA. All patients were consented to the Leeds PNH Research Tissue Bank. At baseline, the patient cohort comprised 94 patients with classic PNH, 75 with AA-PNH overlap and 75 with AA with asymptomatic PNH clones &amp;lt;50%. Serological humoral responses were evaluated in blood samples using a quantitative spike-specific IgG SARS-CoV-2 ELISA (EuroImmun). Samples were further analyzed using a high-throughput live-virus neutralization assay performed at the Francis Crick Institute, London, against wild-type SARS-CoV-2 and against omicron B.1.1.529 and delta B.1.617.2 variants. Adaptive T-cell immune responses were assessed by incubating cryopreserved peripheral blood mononuclear cells with SARS-CoV-2 spike and nucleocapsid peptides in an ELISpot interferon-gamma release assay (Oxford Immunotec). Results: Prior to vaccination (baseline) in-vitro anti-spike IgG response was not significantly different between patients and healthy volunteers (p&amp;gt;0.99). After first vaccination, 2/40 (5%) healthy volunteers and 66/155 (42.6%) PNH and AA patients failed to mount a detectible spike-specific IgG. The antibody titre was also significantly reduced (median spike-specific IgG titre in patients 37.9 BAU/ml [IQR 11.56-120.7] versus 289.4 BAU/ml in healthy volunteers [IQR 177.7-1326], p&amp;lt;0.0001). The magnitude of IgG response improved after second vaccination but remained lower than healthy volunteers (median IgG titre in patients 408.6 BAU/ml [IQR 184.1-942.6] versus healthy volunteers 2639 BAU/ml [IQR 894.0-3706], p&amp;lt;0.001). However, responses improved sequentially following repeated vaccinations and after the third dose were equivalent to healthy volunteers (p&amp;gt;0.99). Viral neutralizing antibody activity elicited by vaccination showed similar results, with significantly reduced titres post first vaccination against wild-type SARS-CoV-2 in patients compared with healthy volunteers (p=0.0001). After second vaccination, neutralizing antibody titres in patients versus healthy volunteers remained significantly reduced against wild-type (p=0.0088) and the delta variant (p=0.0001) but not the omicron variant (p=0.078). Reassuringly, responses improved after 3 rd dose of vaccine following which there was no significant difference detected for all three strains (p&amp;gt;0.99). Of the evaluable samples, anti-spike T-cell responses were reduced at all timepoints in patients compared with healthy volunteers but improved with each vaccination. After first vaccination, a reactive result was seen in 32.1% (n=27/84) versus 40% in healthy volunteers (n=16/40) and remained low after second vaccination (35.0% (n=43/123) in patients versus 64.1% (n=24/39) in healthy volunteers). However, after third vaccination, responses had improved to 58.7% (n=44/75) in patients versus 75% (n=24/32) in healthy volunteers. After 4 th vaccination, spike-specific responses were detectable in 67.6% (n=25/37) of patients. There was limited reactivity to nucleocapsid antigens in both groups at all timepoints highlighting that the observed responses were due to vaccination rather than COVID-19 infection. To date there have been no deaths due to COVID-19 infection in patients in this study or in our wider service population since the introduction of the vaccination programme. Conclusion: This study demonstrates the importance of repeated SARS-Cov-2 vaccinations in patients with PNH and/or AA in order to improve humoral and cellular immune responses.

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.

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.

Paroxysmal Nocturnal Haemoglobinuria (PNH) Arising from Non-Canonical Mutations

Introduction Acquired somatic mutations in the X-linked PIGA gene are the most commonly described pathological mutations found in the serious haematopoietic disorder paroxysmal nocturnal haemoglobinuria (PNH), though incidental cases of mutations in PIGT, PIGB and PIGV have been reported. Mutations in the family of 20 PIG (phosphatidylinositol glycan) genes can disrupt biosynthesis of glycosylphosphatidylinositol (GPI) anchors, critical for tethering many important proteins, including the vital complement regulators CD55 and CD59, to the cell membrane. Their absence in PNH leads to complement dysregulation and complications including potentially life-threatening haemolysis and thrombosis, which may be mitigated by lifelong anti-complement therapy. From the cohort of 273 PNH patients currently on complement inhibitor therapy at the Leeds National PNH Centre we have identified 37 patients whose diagnostic flow cytometry report or clinical course differs significantly from expectation, particularly with evidence of inflammatory presentations, which may be indicative of an alternative, non- PIGA, aetiology. Methods Patients consented to the Leeds PNH Research Tissue Bank with unusual clinical and laboratory presentations were identified, genomic DNA was extracted from peripheral blood and initially screened by targeted high sensitivity NGS of the PIGA gene. From the first ten patient samples sequenced, three lacked any evidence of PIGA mutations and so whole exome sequencing (WES) was carried out. Results Patient 1:An 80 year old man, diagnosed with MDS with rearrangement of chromosomes 3 and 7 resulting in a dicentric chromosome and loss of chromosome 20q, was diagnosed with PNH following an increase in transfusion requirement. In addition to classical symptoms of PNH he was diagnosed with seronegative rheumatoid arthritis. Flow cytometry showed an unusual PNH phenotype with a large population of partially GPI-anchor deficient cells only (Table 1). Symptoms improved on eculizumab but he remained transfusion dependent and the disease later transformed to acute myeloid leukaemia. WES: exonic frameshift insertion in PIGT (figure1), pathogenic variant rs760395465. Patient 2:A 34 year old woman presented with a 13 year history of intermittent episodes of generalized urticaria associated with nausea, vomiting, general malaise and myalgia, increasing in intensity and latterly being associated with haemoglobinuria, lethargy and jaundice leading to a PNH diagnosis. Episodes were triggered by pregnancy or infections, but she remained asymptomatic between attacks. Flow cytometry revealed a PNH clone with a majority of cells being partially GPI deficient. She responds well to C5 inhibition with no further symptoms, including urticaria. WES: exonic frameshift insertion in PIGT (figure 1), pathogenic variant rs760395465. Patient 3: A 14 year old girl presented with intermittent episodes of urticaria and severe lethargy and was diagnosed with symptomatic significant transfusion-dependent PNH, with completely GPI-deficient cells. Diagnosis followed an episode of massive haemolysis resulting in transient acute renal failure requiring haemodialysis. She commenced anticoagulation initially until eculizumab was approved 2 years later; despite occasional episodes of breakthrough haemolysis she displays no further urticaria. She has also had symptoms of diffuse arthralgia. WES: stopgain mutation in PIGV (figure1), pathogenic variant rs145160045. Conclusions These cases add to the emerging picture of non- PIGA associated PNH cases and suggest that the presence of joint problems and episodic urticaria may be sufficient indication to prompt screening for alternative causative mutations. Similar to the previously reported PIGB mutated case, the patients with PIGT mutations only had partially deficient cells. It is important to fully characterise these emerging non-canonical variants in order to improve our understanding of the pathophysiology of this disorder and to provide more individualised care.

Contribution of Genetic Screening to the Diagnosis of Patients with Bone Marrow Failure and Short Telomeres

Telomeres are structures responsible for the maintenance of chromosomal stability. Telomere length is maintained by the telomerase complex, shelterin proteins, and others involved in telomere biology. Germline variants in telomere-biology genes may cause excessive telomere shortening, cell senescence, and genomic instability, which in humans may clinically translate into bone marrow failure, liver and lung fibrosis, skin lesions, and a proclivity for cancer. The prevalence of telomere diseases is unknown, partly due to variable penetrance. Patients without phenotypical signs or a family history may be misdiagnosed as having acquired aplastic anemia (AAA). Other inherited bone marrow failure syndromes (IBMF) and AAA may also present with short telomeres. The aim of this study was to determine the genetic variants in patients with bone marrow failure (BMF) and short telomeres. The study was conducted at a tertiary hospital in Sao Paulo, Brazil. One-hundred forty-four patients with BMF followed between 2016 and 2022 were enrolled. Ethical approval was obtained from the local Ethics Committee. Telomere length was determined by Southern blot or flow-FISH in peripheral blood leukocytes. Telomeres were interpreted as short when below the 10th percentile according to age. All samples were submitted to genomic analysis by Next Generation Sequencing (NGS), using a panel for 154 genes associated with hematologic malignancies and BMF syndromes. A presumptive diagnosis was made based on clinical and laboratorial data. Liver or pulmonary fibrosis, early greying, dystrophic nails, leukoplakia, hypopigmented skin, solid malignant tumors, retinopathy, family history of hematological diseases and consanguinity were considered as red flag symptoms for telomeropathies. Patients with no red flags who had severe cytopenias were considered as having possible AAA. Patients with non-severe cytopenias starting at an early age were considered as possibly having other IBMF syndromes. Thirty patients (21%) out of 144 analyzed had short telomere length and were included. They had their telomeres measured at diagnosis (53%) or at follow-up (47%).Combining clinical, laboratory, and NGS data, 10 patients (34%) were diagnosed with Telomeropathies [TERT (N=4), RTEL1 (N=3), DKC1 (N=1), TERC (N=1), ACD (N=1)]. Six patients (20%) were diagnosed with other IBMF syndromes [4 Fanconi anemia (FANCA, FANCG, BRCA2), 1 GATA2 deficiency (GATA2), 1 Blackfan-Diamond anemia (RPS19)]. Seven patients (23%) were considered as having AAA, not having any germline variants, and 7 (23%) as undetermined cases, with some suggestive clinical feature but no identified germline variant (figure 1). Comparing with NSG data, the clinical and laboratorial presumptive diagnosis had a 95% accuracy, 100% sensitivity and 92% specificity. In the group with confirmed telomeropathy (N=10), 70% were male, 80% were white, and the median age at diagnosis was 25 years. All of them had at least one red flag symptom: hepatic fibrosis (60%), early graying (60%), family history of hematologic diseases (40%) and one patient had the classic dermatologic triad of dyskeratosis congenital. Only one had severe cytopenia and none had significant paroxysmal nocturnal hemoglobinuria clone. Most of them had hypoplastic bone marrow without significant dysplasia (N=8). In conclusion, clinical features combined with laboratory analysis had high accuracy when compared with NGS. Despite having access to NGS, a final diagnosis was not achieved for 23% of patients, who would benefit from further genetic analysis.