Silent cerebral infarction during immune TTP remission: prevalence, predictors, and impact on cognition.

Immune thrombotic thrombocytopenic purpura (iTTP) survivors have increased risk of cardiovascular disease, including strokes, and report persistent cognitive difficulties during remission. We conducted this prospective study involving iTTP survivors during clinical remission to determine the prevalence of silent cerebral infarction (SCI), defined as magnetic resonance imaging (MRI) evidence of brain infarction without corresponding overt neurodeficits. We also tested the hypothesis that SCI is associated with cognitive impairment, assessed using the National Institutes of Health ToolBox Cognition Battery. For cognitive assessments, we used fully corrected T scores adjusted for age, sex, race, and education. Based on the diagnostic and statistical manual 5 criteria, we defined mild and major cognitive impairment as T scores with a 1 or 2 standard deviation (SD) and >2 SD below the mean on at least 1 test, respectively. Forty-two patients were enrolled, with 36 completing MRIs. SCI was present in 50% of the patients (18), of which 8 (44.4%) had prior overt stroke including during acute iTTP. Patients with SCI had higher rates of cognitive impairment (66.7% vs 27.7%; P = .026), including major cognitive impairment (50% vs 5.6%; P = .010). In separate logistic regression models, SCI was associated with any (mild or major) cognitive impairment (odds ratio [OR] 10.5 [95% confidence interval (95% CI), 1.45-76.63]; P = .020) and major cognitive impairment (OR 7.98 [95% CI, 1.11-57.27]; P = .039) after adjusting for history of stroke and Beck depression inventory scores. MRI evidence of brain infarction is common in iTTP survivors; the strong association of SCI with impaired cognition suggests that these silent infarcts are neither silent nor innocuous.

Anti-PF4 antibodies associated with disease severity in COVID-19.

Severe COVID-19 is characterized by a prothrombotic state associated with thrombocytopenia, with microvascular thrombosis being almost invariably present in the lung and other organs at postmortem examination. We evaluated the presence of antibodies to platelet factor 4 (PF4)-polyanion complexes using a clinically validated immunoassay in 100 hospitalized patients with COVID-19 with moderate or severe disease (World Health Organization score, 4 to 10), 25 patients with acute COVID-19 visiting the emergency department, and 65 convalescent individuals. Anti-PF4 antibodies were detected in 95 of 100 hospitalized patients with COVID-19 (95.0%) irrespective of prior heparin treatment, with a mean optical density value of 0.871 ± 0.405 SD (range, 0.177 to 2.706). In contrast, patients hospitalized for severe acute respiratory disease unrelated to COVID-19 had markedly lower levels of the antibodies. In a high proportion of patients with COVID-19, levels of all three immunoglobulin (Ig) isotypes tested (IgG, IgM, and IgA) were simultaneously elevated. Antibody levels were higher in male than in female patients and higher in African Americans and Hispanics than in White patients. Anti-PF4 antibody levels were correlated with the maximum disease severity score and with significant reductions in circulating platelet counts during hospitalization. In individuals convalescent from COVID-19, the antibody levels returned to near-normal values. Sera from patients with COVID-19 induced higher levels of platelet activation than did sera from healthy blood donors, but the results were not correlated with the levels of anti-PF4 antibodies. These results demonstrate that the vast majority of patients with severe COVID-19 develop anti-PF4 antibodies, which may play a role in the clinical complications of COVID-19.

Characterization of myeloproliferative neoplasms in the paediatric and young adult population.

The aim of this study was to compare the genomic features and clinical outcomes between paediatric and young adult patients (PAYA, <40 years) and older adults (OA, ≥40 years) with myeloproliferative neoplasms (MPN) to gain insight into pathogenesis, disease prognosis and management. Of 630 MPN patients, 171 (27%) were PAYA with an average age at diagnosis of 31 years. Females were more prevalent in PAYA than OA (71% vs 58%; p = 0.002), and PAYA more frequently presented with essential thrombocytosis (ET) at diagnosis (67% vs 39%; p < 0.001). The presence of a JAK2 somatic mutation was higher in OA (80.4% vs 64.3%; p < 0.001), while a CALR mutation or lack of any traditional driver mutation was more common in PAYA (20.5% vs 10.5%; p = 0.001, 8.8% vs 3.7%; p = 0.01 respectively). Venous thrombosis was more common in PAYA compared to OA (19.8% vs 10.7%; p = 0.002). PAYA had a higher prevalence of familial MPN and familial cancer predisposition, and two PAYA patients harboured pathogenic germline JAK2 lesions. PAYA demonstrated longer survival from diagnosis than OA (median not reached vs 13 years), while disease transformation was less frequent (19.3% vs 37.9%).

Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein-treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins.

Complement activity and complement regulatory gene mutations are associated with thrombosis in APS and CAPS.

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies, including anti-ß2-glycoprotein-I (anti-ß2GPI), that are considered central to APS pathogenesis. Based on animal studies showing a role of complement in APS-related clinical events, we used the modified Ham (mHam) assay (complement-dependent cell killing) and cell-surface deposition of C5b-9 to test the hypothesis that complement activation is associated with thrombotic events in APS. A positive mHam (and corresponding C5b-9 deposition) were present in 85.7% of catastrophic APS (CAPS), 35.6% of APS (and 68.5% of samples collected within 1 year of thrombosis), and only 6.8% of systemic lupus erythematosus (SLE) sera. A positive mHam assay was associated with triple positivity (for lupus anticoagulant, anticardiolipin, and anti-ß2GPI antibodies) and recurrent thrombosis. Patient-derived anti-ß2GPI antibodies also induced C5b-9 deposition, which was blocked completely by an anti-C5 monoclonal antibody, but not by a factor D inhibitor, indicating that complement activation by anti-ß2GPI antibodies occurs primarily through the classical complement pathway. Finally, patients with CAPS have high rates of rare germline variants in complement regulatory genes (60%), compared with patients with APS (21.8%) or SLE (28.6%) or normal controls (23.3%), and have mutations at a rate similar to that of patients with atypical hemolytic uremic syndrome (51.5%). Taken together, our data suggest that anti-ß2GPI antibodies activate complement and contribute to thrombosis in APS, whereas patients with CAPS have underlying mutations in complement regulatory genes that serve as a "second hit," leading to uncontrolled complement activation and a more severe thrombotic phenotype.

Complement dysregulation is associated with severe COVID-19 illness.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may manifest as thrombosis, stroke, renal failure, myocardial infarction, and thrombocytopenia, reminiscent of other complement- mediated diseases. Multiple clinical and preclinical studies have implicated complement in the pathogenesis of COVID-19 illness. We previously found that the SARS-CoV-2 spike protein activates the alternative pathway of complement (APC) in vitro through interfering with the function of complement factor H, a key negative regulator of APC. Here, we demonstrated that serum from 58 COVID-19 patients (32 patients with minimal oxygen requirement, 7 on high flow oxygen, 17 requiring mechanical ventilation and 2 deaths) can induce complementmediated cell death in a functional assay (the modified Ham test) and increase membrane attack complex (C5b-9) deposition on the cell surface. A positive modified Ham assay (>20% cell-killing) was present in 41.2% COVID-19 patients requiring intubation (n=7/17) and only 6.3% in COVID-19 patients requiring minimal oxygen support (n=2/32). C5 and factor D inhibition effectively mitigated the complement amplification induced by COVID-19 patient serum. Increased serum factor Bb level was associated with disease severity in COVID-19 patients, suggesting that APC dysregulation plays an important role. Moreover, SARS-CoV-2 spike proteins directly block complement factor H from binding to heparin, which may lead to complement dysregulation on the cell surface. Taken together, our data suggest that complement dysregulation contributes to the pathogenesis of COVID-19 and may be a marker of disease severity.

Reduced ADAMTS13 activity during TTP remission is associated with stroke in TTP survivors.

With timely and effective treatment, most patients with thrombotic thrombocytopenic purpura (TTP) survive the acute TTP episode. In addition to the risk of relapse, TTP survivors have higher all-cause mortality than the general population and increased rates of chronic morbidities, including hypertension, depression, and mild cognitive impairment. We conducted this retrospective-prospective cohort study to determine the incidence and prevalence of stroke after recovery from acute TTP and to test the hypothesis that lower ADAMTS13 activity after recovery from TTP is associated with an increased risk of stroke during remission. Of 170 consecutive patients treated for TTP at The Johns Hopkins Hospital from 1995 through 2018, 14 (8.2%) died during the index episode and 19 were observed for less than 1 month after recovery. Of the remaining 137 patients, 18 (13.1%) developed stroke unrelated to an acute TTP episode over a median observation period of 3.08 years, which is fivefold higher than the expected prevalence of 2.6% from an age- and sex-matched reference population (P = .002). ADAMTS13 activity during remission was measured in 52 patients and was >70% in 44.2%, 40% to 70% in 23.1%, 10% to 39% in 25%, and <10% in 7.7%. Stroke after recovery from acute TTP occurred in 0% (0 of 22) of patients with normal remission ADAMTS13 activity (>70%) and in 27.6% (8 of 29) of patients with low ADAMTS13 activity (≤70%; P = .007). In conclusion, stroke is common after recovery from TTP and is associated with reduced ADAMTS13 activity during remission.

Major adverse cardiovascular events in survivors of immune-mediated thrombotic thrombocytopenic purpura.

Cardiovascular disease is a leading cause of death in survivors of immune-mediated thrombotic thrombocytopenic purpura (iTTP), but the epidemiology of major adverse cardiovascular events (MACE) in iTTP survivors is unknown. We evaluated the prevalence and risk factors for MACE, defined as the composite of non-fatal or fatal myocardial infarction (MI), stroke, and cardiac revascularization, during clinical remission in two large iTTP cohorts (Johns Hopkins University and Ohio State University). Of 181 patients followed for ≥ 3 months after recovery from acute iTTP, 28.6% had a MACE event over a median follow up of 7.6 years. Stroke was the most common type of MACE (18.2%), followed by non-fatal MI (6.6%), cardiac revascularization (4.9%) and fatal MI (0.6%). Compared to the general United States population, iTTP survivors were younger at first stroke in remission (males [56.5 years vs. 68.6 years, p = 0.031], females [49.7 years vs. 72.9 years, p < 0.001]) or MI in remission (males [56.5 years vs. 65.6 years, p < 0.001] and females [53.1 years vs. 72.0 years, p < 0.001]). Age (HR 1.03 [95% CI 1.002-1.054]), race (Black/Other vs. White) (HR 2.32 [95% CI 1.12-4.82]), and diabetes mellitus (HR 2.37 [95% CI 1.09-0.03]) were associated with MACE in a Cox regression model also adjusted for sex, hypertension, obesity, hyperlipidemia, chronic kidney disease, atrial fibrillation, autoimmune disease, and relapsing iTTP. Remission ADAMTS13 activity was not significantly associated with MACE. In conclusion, iTTP survivors experience high rates of MACE and may benefit from aggressively screening for and managing cardiovascular risk factors.

Ex vivo assays to detect complement activation in complementopathies.

In complement-driven thrombotic microangiopathies, failure to regulate complement activation leads to end-organ damage. The modified Ham (mHam) test measures complement-mediated killing of a nucleated cell in vitro but lacks a confirmatory assay and reliable positive controls. We demonstrate that C5b-9 accumulation on the surface of TF1 PIGAnull cells correlates with cell killing in the mHam. We also show that Sialidase treatment of cells or addition of Shiga toxin 1 to human serum serve as a more reliable positive control for the mHam than cobra venom factor or lipopolysaccharide. Simultaneously performing the mHam and measuring C5b-9 accumulation either in GVB++ or GVB0 MgEGTA buffer with the addition of complement pathway specific inhibitors (anti-C5 antibody or a factor D inhibitor, ACH-145951) can be used to localize defects in complement regulation. As more targeted complement inhibitors become available, these assays may aid in the selection of personalized treatments for patients with complement-mediated diseases.

Genomic analysis of germ line and somatic variants in familial myelodysplasia/acute myeloid leukemia.

Familial clustering of myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) can be caused by inherited factors. We screened 59 individuals from 17 families with 2 or more biological relatives with MDS/AML for variants in 12 genes with established roles in predisposition to MDS/AML, and identified a pathogenic germ line variant in 5 families (29%). Extending the screen with a panel of 264 genes that are recurrently mutated in de novo AML, we identified rare, nonsynonymous germ line variants in 4 genes, each segregating with MDS/AML in 2 families. Somatic mutations are required for progression to MDS/AML in these familial cases. Using a combination of targeted and exome sequencing of tumor and matched normal samples from 26 familial MDS/AML cases and asymptomatic carriers, we identified recurrent frameshift mutations in the cohesin-associated factor PDS5B, co-occurrence of somatic ASXL1 mutations with germ line GATA2 mutations, and recurrent mutations in other known MDS/AML drivers. Mutations in genes that are recurrently mutated in de novo AML were underrepresented in the familial MDS/AML cases, although the total number of somatic mutations per exome was the same. Lastly, clonal skewing of hematopoiesis was detected in 67% of young, asymptomatic RUNX1 carriers, providing a potential biomarker that could be used for surveillance in these high-risk families.

Efficient and allele-specific genome editing of disease loci in human iPSCs.

Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

Chronic myeloid leukemia (CML) evolves from Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) with unexpected frequency.

Myeloproliferative neoplasms (MPN) are chronic clonal disorders characterized by overproduction of myeloid-lineage blood cells and potential risk of evolution to acute myeloid leukemia (AML). Chronic myeloid leukemia (CML) is distinct from other MPNs in that its pathophysiology stems from the BCR-ABL fusion protein of the Philadelphia chromosome (Ph +). Though there are known cases of Ph- and Ph + MPNs coexisting in a single patient, overall prevalence has never been quantified in a prospective cohort. Here, we review our center's MPN registry, which shows 0.6% of Ph- MPN patients later developed CML. This development occurred no less than 10 and up to 36 years after Ph- MPN diagnosis. This rate of chronic transformation exceeds what is expected, as the incidence of CML in the United States is 2 per 100,000 people-years. The probability of this CML case rate in an average-risk population is less than 0.001%, suggesting there are shared risk factors between Ph- and Ph + MPNs. We speculate that these risk factors may include exposures, genetic predispositions, or be inherent to disease biology. Abrupt-onset leukocytosis heralded post-MPN CML in all cases here and suggests this salient clinical feature should trigger hematologists to consider this diagnosis and perform appropriate testing.

Recurrent germline variant in ATM associated with familial myeloproliferative neoplasms.

Genetic predisposition (familial risk) in the myeloproliferative neoplasms (MPNs) is more common than the risk observed in most other cancers, including breast, prostate, and colon. Up to 10% of MPNs are considered to be familial. Recent genome-wide association studies have identified genomic loci associated with an MPN diagnosis. However, the identification of variants with functional contributions to the development of MPN remains limited. In this study, we have included 630 MPN patients and whole genome sequencing was performed in 64 individuals with familial MPN to uncover recurrent germline predisposition variants. Both targeted and unbiased filtering of single nucleotide variants (SNVs) was performed, with a comparison to 218 individuals with MPN unselected for familial status. This approach identified an ATM L2307F SNV occurring in nearly 8% of individuals with familial MPN. Structural protein modeling of this variant suggested stabilization of inactive ATM dimer, and alteration of the endogenous ATM locus in a human myeloid cell line resulted in decreased phosphorylation of the downstream tumor suppressor CHEK2. These results implicate ATM, and the DNA-damage response pathway, in predisposition to MPN.

The roles of sex and genetics in the MPN.

The Philadelphia chromosome negative myeloproliferative neoplasms(MPNs), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are acquired hematopoietic stem cell disorders driven by activating mutations of intracellular signal transduction pathways that control the production of circulating blood cells. The MPN are characterized clinically by marked variation in degrees of vascular risk, familial clustering, and evolution to myelofibrosis and acute leukemia. MPN disease presentations and outcomes are highly variable, and are markedly influenced by both sex and germline genetic variation. This chapter will focus on the evidence of sex and germline genetic background as modifiers of MPN development and outcomes. Large population genome wide association studies in both clonal hematopoiesis and MPN have revealed novel mechanisms, including inflammatory pathways and genomic instability, which further our understanding of how sex and genetic background mediate MPN risk. Recent advances in our understanding of clonal hematopoiesis and MPN development in various contexts informs the mechanisms by which sex, inflammation, exposures and genetics influence MPN incidence and outcomes, and provide opportunities to develop new strategies for prognostics and therapeutics in the MPN.

Antiphospholipid syndrome: Complement activation, complement gene mutations, and therapeutic implications.

Antiphospholipid syndrome (APS) is an acquired thromboinflammatory disorder characterized by the presence of antiphospholipid antibodies as well as an increased frequency of venous or arterial thrombosis and/or obstetrical morbidity. The spectrum of disease varies from asymptomatic to a severe form characterized by widespread thrombosis and multiorgan failure, termed catastrophic APS (CAPS). CAPS affects only about ∼1% of APS patients, often presents as a thrombotic microangiopathy and has a fulminant course with >40% mortality, despite the best available therapy. Animal models have implicated complement in the pathophysiology of thrombosis in APS, with more recent data from human studies confirming the interaction between the coagulation and complement pathways. Activation of the complement cascade via antiphospholipid antibodies can cause cellular injury and promote coagulation via multiple mechanisms. Finally, analogous to classic complement-mediated diseases such as atypical hemolytic uremic syndrome, a subset of patients with APS may be at increased risk for development of CAPS because of the presence of germline variants in genes crucial for complement regulation. Together, these data make complement inhibition an attractive and potentially lifesaving therapy to mitigate morbidity and mortality in severe thrombotic APS and CAPS.

Outcomes of a clinician-directed protocol for discontinuation of complement inhibition therapy in atypical hemolytic uremic syndrome.

Terminal complement inhibition is the standard of care for atypical hemolytic uremic syndrome (aHUS). The optimal duration of complement inhibition is unknown, although indefinite therapy is common. Here, we present the outcomes of a physician-directed eculizumab discontinuation and monitoring protocol in a prospective cohort of 31 patients that started eculizumab for acute aHUS (and without a history of renal transplant). Twenty-five (80.6%) discontinued eculizumab therapy after a median duration on therapy of 2.37 (interquartile range: 1.06, 9.70) months. Eighteen patients discontinued per protocol and 7 because of nonadherence. Of these, 5 (20%) relapsed; however, relapse rate was higher in the case of nonadherence (42.8%) vs clinician-directed discontinuation and monitoring (11.1%). Four of 5 patients who relapsed were successfully retreated without a decline in renal function. One patient died because of recurrent aHUS and hypertensive emergency in the setting of nonadherence. Nonadherence to therapy (odds ratio, 8.25; 95% confidence interval, 1.02-66.19; P = .047) was associated with relapse, whereas the presence of complement gene variants (odds ratio, 1.39; 95% confidence interval, 0.39-4.87; P = .598) was not significantly associated with relapse. Relapse occurred in 40% (2 of 5) with a CFH or MCP variant, 33.3% (2 of 6) with other complement variants, and 0% (0 of 6) with no variants (P = .217). There was no decline in mean glomerular filtration rate from the date of stopping eculizumab until end of follow-up. In summary, eculizumab discontinuation with close monitoring is safe in most patients, with low rates of aHUS relapse and effective salvage with eculizumab retreatment in the event of recurrence.