A Journey from Blood Cells to Genes and Back.

I was attracted to hematology because by combining clinical findings with the use of a microscope and simple laboratory tests, one could often make a diagnosis. I was attracted to genetics when I learned about inherited blood disorders, at a time when we had only hints that somatic mutations were also important. It seemed clear that if we understood not only what genetic changes caused what diseases but also the mechanisms through which those genetic changes contribute to cause disease, we could improve management. Thus, I investigated many aspects of the glucose-6-phosphate dehydrogenase system, including cloning of the gene, and in the study of paroxysmal nocturnal hemoglobinuria (PNH), I found that it is a clonal disorder; subsequently, we were able to explain how a nonmalignant clone can expand, and I was involved in the first trial of PNH treatment by complement inhibition. I was fortunate to do clinical and research hematology in five countries; in all of them, I learned from mentors, from colleagues, and from patients.

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.

Upregulation of Checkpoint Ligand Programmed Death-Ligand 1 in Patients with Paroxysmal Nocturnal Hemoglobinuria Explained by Proximal Complement Activation.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.

How I treat paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal, complement-mediated hemolytic anemia with protean manifestations. PNH can present as a hemolytic anemia, a form of bone marrow failure, a thrombophilia, or any combination of the above. Terminal complement inhibition is highly effective for treating intravascular hemolysis from PNH and virtually eliminates the risk of thrombosis, but is not effective for treating bone marrow failure. Here, I present a variety of clinical vignettes that highlight the clinical heterogeneity of PNH and the attributes and limitations of the 2 US Food and Drug Administration-approved C5 inhibitors (eculizumab and ravulizumab) to treat PNH. I review the concept of pharmacokinetic and pharmacodynamic breakthrough hemolysis and briefly discuss new complement inhibitors upstream of C5 that are in clinical development. Last, I discuss the rare indications for bone marrow transplantation in patients with PNH.

Fatigue and health-related quality of life in paroxysmal nocturnal haemoglobinuria: A post hoc analysis of the pegcetacoplan PEGASUS trial data.

OBJECTIVES: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, non-malignant haematological disorder associated with disabling fatigue and reduced health-related quality of life. Post hoc analysis of PEGASUS phase 3 trial (NCT03500549) characterised improvements in patient-reported fatigue measured by functional assessment of chronic illness therapy-fatigue (FACIT-fatigue) instrument item-level ratings for pegcetacoplan and eculizumab for the treatment of PNH. METHODS: Item-level responder analysis was conducted on a ≥2-level change from baseline (CFB) clinically important response (CIR) for the FACIT-fatigue 13 individual items rated on a 5-level Likert scale. We evaluated ≥2-level change against the minimal clinically important difference (MCID) of the FACIT-fatigue total score (≥5 points) and clinical parameters, haemoglobin (Hb; ≥1 g/dL) and normalised absolute reticulocyte count (ARC; 30-100 pg/cells). Logistic regressions estimated baseline-to-Week-16 FACIT-fatigue item-level transitional probabilities; Kaplan-Meier analysis estimated time to FACIT-fatigue item CIR. RESULTS: Pegcetacoplan versus eculizumab was associated with significantly greater odds of Week 16 CIR across 8/13 items and on total score MCID (OR [CI] = 11.19 [3.73, 33.57]) and faster times to responses. The item-level CIR threshold also showed clinical relevance on Hb level and ARC normalization. CONCLUSIONS: Compared with eculizumab, pegcetacoplan was associated with clinically meaningful greater improvements on a majority of FACIT-fatigue items.

Complement Inhibitors in the Management of Complement-Mediated Hemolytic Uremic Syndrome and Paroxysmal Nocturnal Hemoglobinuria.

BACKGROUND: Complement-mediated HUS (CM-HUS) and paroxysmal nocturnal hemoglobinuria (PNH) are rare hematologic disorders that cause dysregulation and hyperactivation of the complement system. Historically, treatment of CM-HUS involved plasma exchange (PLEX), often with limited benefit and variable tolerance. Conversely, PNH was treated with supportive care or hemopoietic stem cell transplant. Within the last decade, monoclonal antibody therapies that block terminal complement pathway activation, have emerged as less invasive and more efficacious options for management of both disorders. This manuscript seeks to discuss a relevant clinical case of CM-HUS and the evolving landscape of complement inhibitor therapies for CM-HUS and PNH. AREAS OF UNCERTAINTY: Eculizumab, the first humanized anti-C5 monoclonal antibody, has been the standard of care in treating CM-HUS and PNH for over a decade. Although eculizumab has remained an effective agent, the variability in ease and frequency of administration has remained an obstacle for patients. The development of novel complement inhibitor therapies with longer half-lives, has allowed for changes in frequency and route of administration, thus improving patient QOL. However, there are limited prospective clinical trial data given disease rarity, and limited information on variable infusion frequency and length of treatment. THERAPEUTIC ADVANCES: Recently, there has been a push to formulate complement inhibitors that improve QOL while maintaining efficacy. Ravulizumab, a derivative of eculizumab, was developed to allow for less frequent administration, while remaining efficacious. In addition, the novel oral and subcutaneous therapies, danicopan and crovalimab, respectively, along with pegcetacoplan are currently undergoing active clinical trials, and poised to further reduce treatment burden. CONCLUSION: Complement inhibitor therapies have changed the treatment landscape for CM-HUS and PNH. With a significant emphasis on patient QOL, novel therapies continue to emerge and require an in-depth review of their appropriate use and efficacy in these rare disorders. CLINICAL CASE: A 47-year-old woman with hypertension and hyperlipidemia presented with shortness of breath and was found to have hypertensive emergency in the setting of acute renal failure. Her serum creatinine was 13.9 mg/dL; elevated from 1.43 mg/dL 2 years before. The differential diagnosis for her acute kidney injury (AKI) included infectious, autoimmune, and hematologic processes. Infectious work-up was negative. ADAMTS13 activity level was not low at 72.9%, ruling out thrombotic thrombocytopenic purpura (TTP). Patient underwent a renal biopsy, which revealed acute on chronic thrombotic microangiopathy (TMA). A trial of eculizumab was initiated with concurrent hemodialysis. The diagnosis of CM-HUS was later confirmed by a heterozygous mutation in complement factor I (CFI), resulting in increased membrane attack complex (MAC) cascade activation. The patient was maintained on biweekly eculizumab and was eventually transitioned to ravulizumab infusions as an outpatient. Her renal failure did not recover, and the patient remains on hemodialysis while awaiting kidney transplantation.

Secondary myelodysplastic syndrome and leukemia in acquired aplastic anemia and paroxysmal nocturnal hemoglobinuria.

Acquired aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are pathogenically related nonmalignant bone marrow failure disorders linked to T-cell-mediated autoimmunity; they are associated with an increased risk of secondary myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Approximately 15% to 20% of AA patients and 2% to 6% of PNH patients go on to develop secondary MDS/AML by 10 years of follow-up. Factors determining an individual patient's risk of malignant transformation remain poorly defined. Recent studies identified nearly ubiquitous clonal hematopoiesis (CH) in AA patients. Similarly, CH with additional, non-PIGA, somatic alterations occurs in the majority of patients with PNH. Factors associated with progression to secondary MDS/AML include longer duration of disease, increased telomere attrition, presence of adverse prognostic mutations, and multiple mutations, particularly when occurring early in the disease course and at a high allelic burden. Here, we will review the prevalence and characteristics of somatic alterations in AA and PNH and will explore their prognostic significance and mechanisms of clonal selection. We will then discuss the available data on post-AA and post-PNH progression to secondary MDS/AML and provide practical guidance for approaching patients with PNH and AA who have CH.

Persistent imbalance, anti-apoptotic, and anti-inflammatory signature of circulating C-C chemokines and cytokines in patients with paroxysmal nocturnal hemoglobinuria.

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal non-malignant disease in which hematopoietic cell apoptosis may play an important pathophysiological role. Previous studies of the content of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) indicated the possibility of remote transmission of anti-apoptotic signals between pathological and normal hematopoietic progenitors. METHODS: The study determined the plasma levels of beta chemokines and cytokines in N = 19 patients with PNH and 31 healthy controls. The research material was peripheral blood plasma (EDTA) stored at -80 °C until the test. Beta chemokine and cytokine concentrations were tested in duplicate with Bio-Plex Pro Human Cytokine Assay (Bio-Rad, Hercules, CA, USA) using a Luminex 200 flow cytometer and xPONENT software (Luminex Corporation, Austin, TX, USA). In peripheral blood CD34+ cells we tested the proportions of PI(3,4,5)P3+ and Annexin binding apoptotic phenotype using FC and phosflow. RESULTS: Compared to the control group, the PNH group showed a significant increase in the plasma concentration of some beta chemokines and cytokines, including MIP-1alpha/CCL3, eotaxin/CCL11, MCP1/CCL2, IL4 and G-CSF. In the group of PNH patients, a significant decrease in the concentration of some cytokines was also observed: RANTES/CCL5, MIP-1beta/CCL4, PDGF-BB and IL9. At the same time, the plasma concentrations of the chemokine IP-10/CXCL10 and the cytokines IFN-gamma, TNF, IL6 and IL10 showed no significant deviations from the values for the control group. Anti-apoptotic phenotype and phosphatidylinositol (3,4,5)-trisphosphate content in PNH clone of CD34+ cells were associated with the level of CCL3 and negatively associated with CCL5, CCL4, PDGF-BB and IL9. CONCLUSIONS: This data suggest the existence of apoptotic and PI(3,4,5)P3 imbalance in PNH CD34+ cells driven by anti-apoptotic cytokine biosignature in PNH. Plasma cytokines and intracellular enzymes that regulate the phosphoinositide pathways may become a therapeutic target in PNH.

Analysis of baseline characteristics, disease burden and long-term follow-up of 167 patients with Paroxysmal Nocturnal Hemoglobinuria at a single center in Brazil.

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure. METHODS: Describe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up. RESULTS: 167 patients, mean age at diagnosis 28.4 (7.1-71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%). CONCLUSION: Large PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

Ravulizumab (ALXN1210) vs eculizumab in C5-inhibitor-experienced adult patients with PNH: the 302 study.

Ravulizumab, a new complement component C5 inhibitor administered every 8 weeks, was noninferior to eculizumab administered every 2 weeks in complement-inhibitor-naive patients with paroxysmal nocturnal hemoglobinuria (PNH). This study assessed noninferiority of ravulizumab to eculizumab in clinically stable PNH patients during previous eculizumab therapy. In this phase 3, open-label, multicenter study, 195 PNH patients on labeled-dose (900 mg every 2 weeks) eculizumab for >6 months were randomly assigned 1:1 to switch to ravulizumab (n = 97) or continue eculizumab (n = 98). Primary efficacy end point was percentage change in lactate dehydrogenase (LDH) from baseline to day 183. Key secondary end points included proportion of patients with breakthrough hemolysis, change in Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue score, transfusion avoidance, and stabilized hemoglobin. In 191 patients completing 183 days of treatment, ravulizumab was noninferior to eculizumab (P inf < .0006 for all end points), including percentage change in LDH (difference, 9.21% [95% confidence interval (CI), -0.42 to 18.84], P = .058 for superiority), breakthrough hemolysis (difference, 5.1 [95% CI, -8.89 to 18.99]), change in FACIT-Fatigue score (difference, 1.47 [95% CI, -0.21 to 3.15]), transfusion avoidance (difference, 5.5 [95% CI, -4.27 to 15.68]), and stabilized hemoglobin (difference, 1.4 [95% CI, -10.41 to 13.31]). The most frequently reported adverse event was headache (26.8%, ravulizumab; 17.3%, eculizumab). No meningococcal infections or discontinuations due to adverse events occurred. Patients with PNH may be safely and effectively switched from labeled-dose eculizumab administered every 2 weeks to ravulizumab administered every 8 weeks. This trial was funded by Alexion Pharmaceuticals, Inc., and is registered at www.clinicaltrials.gov as #NCT03056040.

Ravulizumab (ALXN1210) vs eculizumab in adult patients with PNH naive to complement inhibitors: the 301 study.

Ravulizumab (ALXN1210), a new complement C5 inhibitor, provides immediate, complete, and sustained C5 inhibition. This phase 3, open-label study assessed the noninferiority of ravulizumab to eculizumab in complement inhibitor-naive adults with paroxysmal nocturnal hemoglobinuria (PNH). Patients with lactate dehydrogenase (LDH) ≥1.5 times the upper limit of normal and at least 1 PNH symptom were randomized 1:1 to receive ravulizumab or eculizumab for 183 days (N = 246). Coprimary efficacy end points were proportion of patients remaining transfusion-free and LDH normalization. Secondary end points were percent change from baseline in LDH, change from baseline in Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue score, proportion of patients with breakthrough hemolysis, stabilized hemoglobin, and change in serum free C5. Ravulizumab was noninferior to eculizumab for both coprimary and all key secondary end points (Pinf < .0001): transfusion avoidance (73.6% vs 66.1%; difference of 6.8% [95% confidence interval (CI), -4.66, 18.14]), LDH normalization (53.6% vs 49.4%; odds ratio, 1.19 [0.80, 1.77]), percent reduction in LDH (-76.8% vs -76.0%; difference [95% CI], -0.83% [-5.21, 3.56]), change in FACIT-Fatigue score (7.07 vs 6.40; difference [95% CI], 0.67 [-1.21, 2.55]), breakthrough hemolysis (4.0% vs 10.7%; difference [95% CI], -6.7% [-14.21, 0.18]), and stabilized hemoglobin (68.0% vs 64.5%; difference [95% CI], 2.9 [-8.80, 14.64]). The safety and tolerability of ravulizumab and eculizumab were similar; no meningococcal infections occurred. In conclusion, ravulizumab given every 8 weeks achieved noninferiority compared with eculizumab given every 2 weeks for all efficacy end points, with a similar safety profile. This trial was registered at www.clinicaltrials.gov as #NCT02946463.

The Hematopoietic Cell Transplant Comorbidity Index predicts survival after allogeneic transplant for nonmalignant diseases.

Despite improvements, mortality after allogeneic hematopoietic cell transplantation (HCT) for nonmalignant diseases remains a significant problem. We evaluated whether pre-HCT conditions defined by the HCT Comorbidity Index (HCT-CI) predict probability of posttransplant survival. Using the Center for International Blood and Marrow Transplant Research database, we identified 4083 patients with nonmalignant diseases transplanted between 2007 and 2014. Primary outcome was overall survival (OS) using the Kaplan-Meier method. Hazard ratios (HRs) were estimated by multivariable Cox regression models. Increasing HCT-CI scores translated to decreased 2-year OS of 82.7%, 80.3%, 74%, and 55.8% for patients with HCT-CI scores of 0, 1 to 2, 3 to 4, and ≥5, respectively, regardless of conditioning intensity. HCT-CI scores of 1 to 2 did not differ relative to scores of 0 (HR, 1.12 [95% CI, 0.93-1.34]), but HCT-CI of 3 to 4 and ≥5 posed significantly greater risks of mortality (HR, 1.33 [95% CI, 1.09-1.63]; and HR, 2.31 [95% CI, 1.79-2.96], respectively). The effect of HCT-CI differed by disease indication. Patients with acquired aplastic anemia, primary immune deficiencies, and congenital bone marrow failure syndromes with scores ≥3 had increased risk of death after HCT. However, higher HCT-CI scores among hemoglobinopathy patients did not increase mortality risk. In conclusion, this is the largest study to date reporting on patients with nonmalignant diseases demonstrating HCT-CI scores ≥3 that had inferior survival after HCT, except for patients with hemoglobinopathies. Our findings suggest that using the HCT-CI score, in addition to disease-specific factors, could be useful when developing treatment plans for nonmalignant diseases.

The clinical significance of PNH-phenotype cells accounting for < 0.01% of total granulocytes detected by the Clinical and Laboratory Standards Institute methods in patients with bone marrow failure.

Small populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells accounting for up to 0.01% of total granulocytes can be accurately detected by a high-sensitivity flow cytometry (FCM) assay established by the Clinical and Laboratory Standards Institute (CLSI method) and have a prognostic value in bone marrow failure (BMF); however, the significance of GPI(-) granulocytes accounting for 0.001-0.009% of granulocytes remains unclear. To clarify this issue, we examined the peripheral blood of 21 BMF patients in whom minor (around 0.01%) populations of GPI(-) granulocytes had been previously detected by a different high-resolution FCM method (OPTIMA method, which defines ≥ 0.003% GPI(-) granulocytes as an abnormal increase) using both the CLSI and OPTIMA methods simultaneously. These two methods detected an "abnormal increase" in GPI(-) granulocytes in 10 patients (48%) and 17 patients (81%), respectively. CLSI detected 0.002-0.005% (median, 0.004%) GPI(-) granulocytes in 7 patients who were deemed positive for PNH-type cells according to the OPTIMA method, which detected 0.003-0.012% (median 0.006%) GPI(-) granulocytes. The clone sizes of GPI(-) cells detected by each assay were positively correlated (r = 0.994, p < 0.001). Of the seven patients who were judged positive for PNH-type cells by OPTIMA alone, five received immunosuppressive therapy, and all of them achieved a partial or complete response. GPI(-) granulocytes detected in BMF patients by the CLSI method should thus be considered significant, even at percentages of < 0.01%.

Time and residual hematopoiesis are crucial for PNH clones escape in hepatitis-associated aplastic anemia.

The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in aplastic anemia (AA) suggests immunopathogenesis, but when and how PNH clones emerge and proliferate are unclear. Hepatitis-associated aplastic anemia (HAAA) is a special variant of AA, contrarily to idiopathic AA, in HAAA the trigger for immune activation is clearer and represented by the hepatitis and thus serves as a good model for studying PNH clones. Ninety HAAA patients were enrolled, including 61 males and 29 females (median age 21 years). Four hundred three of idiopathic AA have been included as controls. The median time from hepatitis to cytopenia was 50 days (range 0-180 days) and from cytopenia to AA diagnosis was 26 days (range 2-370 days). PNH clones were detected in 8 HAAA patients (8.9%) at diagnosis and in 73 patients with idiopathic AA (IAA) (18.1%). PNH cells accounted for 4.2% (1.09-12.33%) of red cells and/or granulocytes and were more likely to be detected in patients with longer disease history and less severe disease. During follow-up, the cumulative incidence of PNH clones in HAAA increased to 18.9% (17/90). Nine HAAA patients newly developed PNH clones, including six immunosuppressive therapy (IST) nonresponders. The clone size was mostly stable during follow-up, and only 2 of 14 patients showed increased clone size without proof of hemolysis. In conclusion, PNH clones were infrequent in newly diagnosed HAAA, but their frequency increased to one that was similar to the IAA frequency during follow-up. These results suggest that the PNH clone selection/expansion process is dynamic and takes time to establish, confirming that retesting for PNH clones during follow-up is crucial.

Bone Marrow of Contention: A Rare Case of Recurrent Acute Hepatitis.

Hepatitis-associated aplastic anemia is a well-recognized clinical syndrome in which marrow failure follows the development of hepatitis. Although aplastic anemia is intimately related to paroxysmal nocturnal hemoglobinuria, until now, no cases of PNH-associated hepatitis have been described. We report a case of recurrent acute hepatitis preceding the clinical onset of PNH. Treatment of PNH with the complement inhibitor eculizumab (Soliris®) prevented both recurrences of episodes of intravascular hemolysis and liver enzyme alteration. This is the first known published case of PNH-associated hepatitis.

Presentation clinical, haematological and immunophenotypic features of 1081 patients with GPI-deficient (paroxysmal nocturnal haemoglobinuria) cells detected by flow cytometry.

A retrospective analysis of presentation clinical, laboratory and immunophenotypic features of 1 081 patients with paroxysmal nocturnal haemoglobinuria (PNH) clones [glycosylphosphatidylinositol (GPI)-deficient blood cells] identified at our hospital by flow cytometry over the past 25 years was undertaken. Three distinct clusters of patients were identified and significant correlations between presentation disease type and PNH clone sizes were evident. Smaller PNH clones predominate in cytopenic and myelodysplastic subtypes; large PNH clones were associated with haemolytic, thrombotic and haemolytic/thrombotic subtypes. Rare cases with an associated chronic myeloproliferative disorder had either large or small PNH clones. Cytopenia was a frequent finding, highlighting bone marrow failure as the major underlying feature associated with the detection of PNH clones in the peripheral blood. Red cell PNH clones showed significant correlations between the presence of type II (partial GPI deficiency) red cells and thrombotic disease. Haemolytic PNH was associated with type III (complete GPI deficiency) red cell populations of >20%. Those with both haemolytic and thrombotic features had major type II and type III red cell populations. Distinct patterns of presentation age decade were evident for clinical subtypes with a peak incidence of haemolytic PNH in the 30-49 year age group and a biphasic age distribution for the cytopenia group.

Distinctive and common features of moderate aplastic anaemia.

The therapy algorithm for severe aplastic anaemia (sAA) is established but moderate AA (mAA), which likely reflects a more diverse pathogenic mechanism, often represents a treatment/management conundrum. A cohort of AA patients (n = 325) was queried for those with non-severe disease using stringent criteria including bone marrow hypocellularity and chronic persistence of moderately depressed blood counts. As a result, we have identified and analyzed pathological and clinical features in 85 mAA patients. Progression to sAA and direct clonal evolution (paroxysmal nocturnal haemoglobinuria/acute myeloid leukaemia; PNH/AML) occurred in 16%, 11% and 1% of mAA cases respectively. Of the mAA patients who received immunosuppressive therapy, 67% responded irrespective of time of initiation of therapy while conservatively managed patients showed no spontaneous remissions. Genomic analysis of mAA identified evidence of clonal haematopoiesis with both persisting and remitting patterns at low allelic frequencies; with more pronounced mutational burden in sAA. Most of the mAA patients have autoimmune pathogenesis similar to those with sAA, but mAA contains a mix of patients with diverse aetiologies. Although progression rates differed between mAA and sAA (P = 0·003), cumulative incidences of mortalities were only marginally different (P = 0·095). Our results provide guidance for diagnosis/management of mAA, a condition for which no current standard of care is established.

Macrophage TNF-α licenses donor T cells in murine bone marrow failure and can be implicated in human aplastic anemia.

Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α-/- donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR-/- recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α-/- recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α-/- recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR-/- donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α-producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.

Nationwide study of paroxysmal nocturnal hemoglobinuria in South Korea: paradox of eculizumab.

Eculizumab is effective in managing patients with paroxysmal nocturnal hemoglobinuria (PNH). In South Korea, the financial support for eculizumab therapy is extended by the National Health Insurance Services (NHIS) only to patients with high-risk PNH for approximately 10 years. In this study, we performed a nationwide analysis of the real-world efficacy of eculizumab therapy in patients diagnosed with PNH between January 1, 2002, and December 31, 2016, by using the NHIS database. Patients treated with eculizumab (the eculizumab-treated group) exhibited a significantly higher survival rate than patients not treated with eculizumab (the eculizumab-untreated group), with 4-year survival rates after propensity score matching of 98.31% and 79.67%, respectively (p = 0.0489). The mean red blood cell (RBC) transfusion units per 12 months after eculizumab therapy were significantly lower than that before eculizumab therapy (5.75 units vs. 12.28 units, p < 0.0001). The median time for the first transfusion in the eculizumab-treated group was significantly longer than that in the eculizumab-untreated group. The 4-year transfusion-independence rate for the eculizumab-treated group was significantly higher than that for the eculizumab-untreated group (20.81% vs. 10.24%, p = 0.078). There was no significant difference between the two groups in the incidence of new documented complications related to PNH. In conclusion, eculizumab therapy for patients with high-risk PNH may effectively improve the survival rate and reduce the transfusion requirement. Paradoxically, eculizumab-treated patients with severe PNH exhibit a higher survival rate than eculizumab-untreated patients with less severe PNH.