Faculty Bibliography

This study developed and explored a novel composite endpoint to assess the overall impact that treatment can have on patients living with paroxysmal nocturnal hemoglobinuria (PNH). Candidate composite endpoint variables were selected by a group of experts and included: lactate dehydrogenase levels as a measure of intravascular hemolysis; complete terminal complement inhibition; absence of major adverse vascular events, including thrombosis; absence of any adverse events leading to death or discontinuation of study treatment; transfusion avoidance; and improvements in fatigue-related quality of life as determined by the Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue score. From these variables a novel composite endpoint was constructed and explored using data collected in the ravulizumab PNH Study 301 (NCT02946463). Thresholds were defined and reported for each candidate variable. Five of the six candidate variables were included in the final composite endpoint; the FACIT-Fatigue score was excluded. Composite endpoint criterion was defined as patients meeting all five selected individual component thresholds. All patients in the ravulizumab arm achieved complete terminal complement inhibition and a reduction in lactate dehydrogenase levels; 51.2% and 41.3% of patients in the ravulizumab arm and eculizumab arm, respectively, achieved all composite endpoint component thresholds (treatment difference: 9.4%; 95% confidence interval: -3.0, 21.5). The composite endpoint provided a single and simultaneous measurement of overall benefit for patients receiving treatment for PNH. Use of the composite endpoint in future PNH research is recommended to determine clinical benefit, and its use in health technology assessments should be evaluated.

Purpose/UNASSIGNED:Most patients with paroxysmal nocturnal hemoglobinuria (PNH) treated with a complement protein 5 (C5) inhibitor achieve full control of terminal complement activity and intravascular hemolysis. The minority remains anemic and transfusion dependent despite this control. Etiology for ongoing anemia is multifactorial and includes bone marrow failure, breakthrough hemolysis, extravascular hemolysis (EVH) and nutritional deficiencies. Patients and Methods/UNASSIGNED:/L and hemoglobin level ≤9.5 g/dL. Safety data were not collected. Mean treatment duration was 26.5±17.2 months. Results/UNASSIGNED:Treatment with C5 inhibitors significantly improved hemoglobin, lactate dehydrogenase, and number of transfusions versus baseline. Among the patients with hemoglobin <10 g/dL during the last 6 months of treatment (n=38), one patient (eculizumab) had clinically evident EVH, and 10 patients had active concomitant bone marrow failure. Bone marrow failure was a major contributor to hemoglobin <10 g/dL and transfusion dependence; clinically evident EVH was uncommon. Conclusion/UNASSIGNED:A range of hematologic causes need to be considered when evaluating anemia in the presence of treatment with a C5 inhibitor.

The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement. Here, we describe paired gRNA deletions along the PIGA locus on the human X chromosome ranging from 17 kb to 2 Mb. We found no compelling linear correlation between deletion size and the deletion efficiency, and there is no substantial impact of topologically associating domains on deletion frequency. Using this precise deletion technique, we have engineered a series of designer deletion cell lines, including one with deletions of two X-chromosomal counterselectable (negative selection) markers, PIGA and HPRT1, and additional cell lines bearing each individual deletion. PIGA encodes a component of the glycosylphosphatidylinositol (GPI) anchor biosynthetic apparatus. The PIGA gene counterselectable marker has unique features, including existing single cell level assays for both function and loss of function of PIGA and the existence of a potent counterselectable agent, proaerolysin, which we use routinely for selection against cells expressing PIGA. These designer cell lines may serve as a general platform with multiple selection markers and may be particularly useful for large scale genome engineering projects such as Genome Project-Write (GP-write).

OBJECTIVES/OBJECTIVE:To evaluate the effects of eculizumab on transfusions and thrombotic events (TEs) in patients with and without prior history of transfusion in the International Paroxysmal Nocturnal Hemoglobinuria (PNH) Registry. METHODS:Registry patients enrolled on or before January 1, 2018, initiated on eculizumab no more than 12 months prior to enrollment, having known transfusion status for the 12 months before eculizumab initiation, and ≥12 months of Registry follow-up after eculizumab initiation, were included. RESULTS:Eculizumab treatment was associated with a 50% reduction in transfusions in patients with a transfusion history (10.6 units/patient-year before eculizumab vs 5.4 after; P < 0.0001), with greater reduction observed in those with no history of bone marrow disease versus those with bone marrow disease. Mean lactate dehydrogenase levels decreased from a mean of 6.7 to 1.4 times the upper limit of normal (ULN) in patients with transfusion history and from 5.1 to 1.2 times ULN in those with no transfusion history. TE and major adverse vascular event rates also decreased by 70% in patients with and without history of transfusion. CONCLUSION/CONCLUSIONS:The benefit of eculizumab therapy does not appear to be limited to any group defined by transfusion history or bone marrow disease history.

BACKGROUND Patients receiving immunosuppressive therapies might be more susceptible to COVID-19. Conversely, an exaggerated inflammatory response to the SARS-CoV-2 infection might be blunted by certain forms of immunosuppression, which could be protective. Indeed, there are data from animal models demonstrating that complement may be a part of the pathophysiology of coronavirus infections. There is also evidence from an autopsy series demonstrating complement deposition in the lungs of patients with COVID-19. This raises the question of whether patients on anti-complement therapy could be protected from COVID-19. CASE REPORT Case 1 is a 39-year-old woman with an approximately 20-year history of paroxysmal nocturnal hemoglobinuria (PNH), who had recently been switched from treatment with eculizumab to ravulizumab prior to SARS-CoV-2 infection. Case 2 is a 54-year-old woman with a cadaveric renal transplant for lupus nephritis, complicated by thrombotic microangiopathy, who was maintained on eculizumab, which she started several months before she developed the SARS-CoV-2 infection. Case 3 is a 60-year-old woman with a 14-year history of PNH, who had been treated with eculizumab since 2012, and was diagnosed with COVID-19 at the time of her scheduled infusion. All 3 patients had a relatively mild course of COVID-19. CONCLUSIONS We see no evidence of increased susceptibility to SARS-CoV-2 in these patients on anti-complement therapy, which might actually have accounted for the mild course of infection. The effect of anti-complement therapy on COVID-19 disease needs to be determined in clinical trials.

The International Paroxysmal Nocturnal Hemoglobinuria (PNH) Registry (NCT01374360) was initiated to optimize patient management by collecting data regarding disease burden, progression, and clinical outcomes. Herein, we report updated baseline demographics, clinical characteristics, disease burden data, and observed trends regarding clone size in the largest cohort of Registry patients. Patients with available data as of July 2017 were stratified by glycosylphosphatidylinositol (GPI)-deficient granulocyte clone size (< 10%, ≥ 10%-< 50%, and ≥ 50%). All patients were untreated with eculizumab at baseline, defined as date of eculizumab initiation or date of Registry enrollment (if never treated with eculizumab). Outcomes assessed in the current analysis included proportions of patients with high disease activity (HDA), history of major adverse vascular events (MAVEs; including thrombotic events [TEs]), bone marrow failure (BMF), red blood cell (RBC) transfusions, and PNH-related symptoms. A total of 4439 patients were included, of whom 2701 (60.8%) had available GPI-deficient granulocyte clone size data. Among these, median clone size was 31.8% (1002 had < 10%; 526 had ≥ 10%-< 50%; 1173 had ≥ 50%). There were high proportions of patients with HDA (51.6%), history of MAVEs (18.8%), BMF (62.6%), RBC transfusion (61.3%), and impaired renal function (42.8%). All measures except RBC transfusion history significantly correlated with GPI-deficient granulocyte clone size. A large proportion of patients with GPI-deficient granulocyte clone size < 10% had hemolysis (9.7%), MAVEs (10.2%), HDA (9.1%), and/or PNH-related symptoms. Although larger GPI-deficient granulocyte clone sizes were associated with higher disease burden, a substantial proportion of patients with smaller clone sizes had history of MAVEs/TEs.

Background: The clinical features of paroxysmal nocturnal hemoglobinuria (PNH) are largely complement mediated and include anemia, fatigue, thrombosis, and dyspnea due to intravascular hemolysis. Currently, ravulizumab and eculizumab are the only approved drugs for adults with PNH. Clinical studies have traditionally used single, separate endpoints that captured several outcomes to describe benefits of PNH treatment. However, a composite endpoint (CoE) combining relevant variables for PNH may better capture overall treatment benefits.
Aim(s): The aim of this study was to explore and develop a CoE for PNH that could be used for further clinical development, health economic evaluation, and research. The CoE was then used to evaluate the clinical benefits of the 2 approved drugs for PNH.
Method(s): Components of a CoE were chosen for their relevance to PNH by advice from 4 hematologists and 2 patients. Clinical variables selected for evaluation included transfusion avoidance, lactate dehydrogenate (LDH) levels, complement inhibition, major adverse vascular events (MAVE), absence of any adverse events (AEs) leading to death or discontinuation from study treatment, and Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue scores. Thresholds for binary assessment of these components were defined by consensus of a multidisciplinary panel of 6 experts after evaluation of candidate thresholds that were applied to data from a phase 3, open-label, interventional study evaluating the effects of ravulizumab versus eculizumab in complement inhibitor-naive patients with PNH (Lee JW et al, Blood 2019 133(6):530-539; NCT02946463) using descriptive statistical analysis.
Result(s): Endpoint thresholds for variables in a PNH CoE were selected from candidate thresholds applied to data from 246 adults with PNH treated with ravulizumab (n = 125) or eculizumab (n = 121) for 26 weeks (wks). The threshold for transfusion avoidance, defined as patients remaining transfusion free for 26 wks, was met by 73.6% of patients in the ravulizumab arm and 66.1% of patients in the eculizumab arm. Hemolysis was evaluated by LDH levels and the threshold for LDH (all LDH values from day 29-183 below 1.5 times the upper limit of normal) was reached by 68% of patients in the ravulizumab arm and 57% of patients in the eculizumab arm. The threshold for complement inhibition (free C5 concentration values < 0.5 ug/mL after first dose through 26 wks) was met by all patients receiving ravulizumab (100%) and 87.5% of patients receiving eculizumab. The threshold for MAVE, defined as the absence of MAVE throughout 26 wks, was achieved by almost all patients (ravulizumab arm = 98.4%; eculizumab arm = 99.2%). The final variable and threshold, absence of any AEs leading to death or discontinuation from study treatment, was achieved in all patients receiving ravulizumab (100%) and by 99.2% of patients receiving eculizumab. Due to uncertainties concerning the threshold criteria, the FACIT-Fatigue score was not selected for inclusion in the CoE but should be evaluated separately in clinical trials of PNH. The combined CoE (each patient meeting all 5 endpoint thresholds) was achieved by 51.2% of ravulizumab-treated patients and 41.3% of eculizumab-treated patients (Figure). Summary/Conclusion: This CoE for PNH is a novel tool that provides a single measurement encompassing the key clinical benefits of ravulizumab, eculizumab, and potentially other therapies for PNH and may be used in future research in PNH. (Figure Presented)

The marked pro-thrombotic tendency in PNH is likely to be at least partly due to the population of platelets derived from the abnormal stem cell clone. However, identification of GPI (-) platelets by flow cytometry can be technically difficult. Here we describe a technique that involves the addition of aspirin immediately after the separation of platelet rich plasma and the use of gel filtration to isolate platelets away from plasma proteins and other blood cells. In a study of 92 analyses of samples from 50 patients, we have demonstrated that the percentage of PNH platelets correlates well with the percentage of PNH granulocytes. We also provide data on several cases where there was an extreme discrepancy between the proportion of PNH granulocytes and red cells; in these cases, the demonstration of abnormal platelets suggests that the patient is likely to be at risk of thrombosis. We believe this test will be potentially useful in the evaluation of samples from such patients and may serve as a tool to investigate the causes of hypercoagulability in PNH.

Background/UNASSIGNED:as a candidate gene in which mutations might be contributing to clonal expansion. Methods/UNASSIGNED:genes in 19 patients with large PNH clones. Results/UNASSIGNED:in multiple hematopoietic lineages, which was detectable upon repeat testing. This patient has had severe thromboses and has relatively higher peripheral blood counts compared with the other patients-but does not have other features of a myeloproliferative neoplasm. Conclusions/UNASSIGNED:may contribute to clonal expansion in exceptional cases of PNH.