Two SERPINC1 variants affecting N-glycosylation of Asn224 cause severe thrombophilia not detected by functional assays.

Antithrombin deficiency, the most severe congenital thrombophilia, might be underestimated, as some pathogenic variants are not detected by routine functional methods. We have identified 2 new SERPINC1 variants, p.Glu227Lys and p.Asn224His, in 4 unrelated thrombophilic patients with early and recurrent thrombosis that had normal antithrombin activity. In one case, the mutation was identified by whole genome sequencing, while in the 3 remaining cases, the mutation was identified by sequencing SERPINC1 based on a single functional positive finding supporting deficiency. The 2 variants shared a common functional defect, an impaired or null N-glycosylation of Asn224 according to a eukaryotic expression model. Carriers had normal anti-FXa or anti-FIIa activities but impaired anti-FVIIa activity and a detectable loss of inhibitory function when incubating the plasma for 1 hour at 41°C. Moreover, the ß glycoform of the variants, lacking 2 N-glycans, had reduced secretion, increased heparin affinity, no inhibitory activity, and a potential dominant-negative effect. These results explain the increased thrombin generation observed in carriers. Mutation experiments reflected the role that Lysine residues close to the N-glycosylation sequon have in impairing the efficacy of N-glycosylation. Our study shows new elements involved in the regulation of N-glycosylation, a key posttranslational modification that, according to our results, affects folding, secretion, and function, providing new evidence of the pathogenic consequence of an incorrect N-glycosylation of antithrombin. This study supports that antithrombin deficiency is underestimated and encourages the development of new functional and genetic tests to diagnose this severe thrombophilia.

Usefulness and Limitations of Multiple Ligation-Dependent Probe Amplification in Antithrombin Deficiency.

Multiplex ligation-dependent probe amplification (MLPA) identifies genetic structural variants in SERPINC1 in 5% of cases with antithrombin deficiency (ATD), the most severe congenital thrombophilia. Our aim was to unravel the utility and limitations of MLPA in a large cohort of unrelated patients with ATD (N = 341). MLPA identified 22 structural variants (SVs) causing ATD (6.5%). MLPA did not detect SVs affecting introns (four cases), and the diagnosis was inaccurate in two cases according to long-range PCR or nanopore sequencing. MLPA was used to detect possible hidden SVs in 61 cases with type I deficiency with single nucleotide variations (SNVs) or small insertion/deletion (INDEL). One case had a false deletion of exon 7, as the 29-bp deletion affected an MLPA probe. We evaluated 32 variants affecting MLPA probes: 27 SNVs and 5 small INDELs. In three cases, MLPA gave false-positive results, all diagnosed as deletions of the affected exon: a small INDEL complex, and two SNVs affecting MLPA probes. Our study confirms the utility of MLPA to detect SVs in ATD, but also shows some limitations in detecting intronic SVs. MLPA renders imprecise and false-positive results for genetic defects which affect MLPA probes. Our results encourage the validation of MLPA results.

Paroxysmal Nocturnal Hemoglobinuria: Biology and Treatment.

Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal hematopoietic disorder characterized by the lack of glycosylphosphatidylinositol-anchored proteins (GPI-APs) as a consequence of somatic mutations in the phosphatidylinositol glycan anchor biosynthesis class A (PIGA) gene. Clinical manifestations of PNH are intravascular hemolysis, thrombophilia, and bone marrow failure. Treatment of PNH mainly relies on the use of complement-targeted therapy (C5 inhibitors), with the newest agents being explored against other factors involved in the complement cascade to alleviate unresolved intravascular hemolysis and extravascular hemolysis. This review summarizes the biology and current treatment strategies for PNH with the aim of reaching a general audience with an interest in hematologic disorders.

Molecular and clinical characterization of transient antithrombin deficiency: A new concept in congenital thrombophilia.

Antithrombin deficiency, the most severe thrombophilia, might be underestimated, since it is only investigated in cases with consistent functional deficiency or family history. We have analyzed 444 consecutive, unrelated cases, from 1998 to 2021, with functional results supporting antithrombin deficiency in at least one sample. Plasma antithrombin was evaluated by functional and biochemical methods in at least two samples. SERPINC1 gene was analyzed by sequencing and MPLA. Hypoglycosylation was studied by electrophoresis and high-performance liquid chromatography (HPLC). In 260 of 305 cases (85.2%) with constitutive deficiency (activity < 80% in all samples), a SERPINC1 (N = 250), or N-glycosylation defect (N = 10) was observed, while 45 remained undetermined. The other 139 cases had normal antithrombin activity (≥ 80%) in at least one sample, what we called transient deficiency. Sixty-one of these cases (43.9%) had molecular defects: 48 had SERPINC1 variants, with two recurrent mutations (p.Ala416Ser[Cambridge II], N = 15; p.Val30Glu[Dublin], N = 12), and 13 hypoglycosylation. Thrombotic complications occurred in transient deficiency, but were less frequent, latter-onset, and had a higher proportion of arterial events than in constitutive deficiency. Two mechanisms explained transient deficiency: The limitation of functional methods to detect some variants and the influence of external factors on the pathogenic consequences of these mutations. Our study reveals a molecular defect in a significant proportion of cases with transient antithrombin deficiency, and changes the paradigm of thrombophilia, as the pathogenic effect of some mutations might depend on external factors and be present only at certain timepoints. Antithrombin deficiency is underestimated, and molecular screening might be appropriate in cases with fluctuating laboratory findings.

High penetrance of inferior vena cava system atresia in severe thrombophilia caused by homozygous antithrombin Budapest 3 variant: Description of a new syndrome.

Atresia of inferior vena cava (IVC) is a rare congenital malformation associated with high risk of venous thrombosis that still has unknown etiology, although intrauterine IVC thrombosis has been suggested to be involved. The identification of IVC atresia in a case with early idiopathic venous thrombosis and antithrombin deficiency caused by the homozygous SERPINC1 c.391C > T variant (p.Leu131Phe; antithrombin Budapest 3) encouraged us to evaluate the role of this severe thrombophilia in this vascular abnormality. We have done a cross-sectional study in previously identified cohorts of patients homozygous for the Budapest 3 variant (N = 61) selected from 1118 patients with congenital antithrombin deficiency identified in two different populations: Spain (N = 692) and Hungary (N = 426). Image analysis included computed tomography and phlebography. Atresia of the IVC system was observed in 17/24 cases (70.8%, 95% confidence interval [CI]: 48.9%-87.3%) homozygous for antithrombin Budapest 3 with available computed tomography (5/8 and 12/16 in the Spanish and Hungarian cohorts, respectively), 16 had an absence of infrarenal IVC and one had atresia of the left common iliac vein. All cases with vascular defects had compensatory mechanisms, azygos-hemiazygos continuation or double IVC, and seven also had other congenital anomalies. Short tandem repeat analysis supported the specific association of the IVC system atresia with SERPINC1. We show the first evidence of the association of a severe thrombophilia with IVC system atresia, supporting the possibility that a thrombosis in the developing fetal vessels is the reason for this anomaly. Our hypothesis-generating results encourage further studies to investigate severe thrombophilic states in patients with atresia of IVC.

Enhanced therapeutic effect using sequential administration of antigenically distinct oncolytic viruses expressing oncostatin M in a Syrian hamster orthotopic pancreatic cancer model.

BACKGROUND: The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed. METHODS: We have developed a protocol consisting of sequential intratumor administrations of new Adenovirus (Ad) and Newcastle Disease Virus (NDV)-based OV encoding the immunostimulatory cytokine oncostatin M (OSM). Transgene expression, toxicity and antitumor effect were evaluated using an aggressive orthotopic pancreatic cancer model in Syrian hamsters, which are sensitive to OSM and permissive for replication of both OVs. RESULTS: NDV-OSM was more cytolytic, whereas Ad-OSM caused higher OSM expression in vivo. Both viruses achieved only a marginal antitumor effect in monotherapy. In addition, strong secretion of OSM in serum limited the maximal tolerated dose of Ad-OSM. In contrast, moderate doses of Ad-OSM followed one week later by NDV-OSM were safe, showed a significant antitumor effect and stimulated immune responses against cancer cells. Similar efficacy was observed when the order of virus administrations was reversed. CONCLUSION: Sequential administration of oncolytic Ad and NDV encoding OSM is a promising approach against pancreatic cancer.

A tower of babel of acronyms? The shadowlands of MGUS/MBL/CHIP/TCUS.

With the advent of outperforming and massive laboratory tools, such as multiparameter flow cytometry and next-generation sequencing, hematopoietic cell clones with putative abnormalities for a variety of blood malignancies have been appreciated in otherwise healthy individuals. These conditions do not fulfill the criteria of their presumed cancer counterparts, and thus have been recognized as their precursor states. This is the case of monoclonal gammopathy of unknown significance (MGUS), the first blood premalignancy state described, preceding multiple myeloma (MM) or Waldenström macroglobulinemia (WM). However, in the last 2 decades, an increasing list of clonopathies has been recognized, including monoclonal B cell lymphocytosis (MBL), which antecedes chronic lymphocytic leukemia (CLL), clonal hematopoiesis of indeterminate potential (CHIP) for myeloid neoplasms (MN), and T-cell clones of uncertain significance (TCUS) for T-cell large chronic lymphocytic leukemia (LGLL). While for some of these entities diagnostic boundaries are precisely set, for others these are yet to be fully defined. Moreover, despite mostly considered of "uncertain significance," they have not only appeared to predispose to malignancy, but also to be capable of provoking set of immunological and cardiovascular complications that may require specialized management. The clinical implications of the aberrant clones, together with the extensive knowledge generated on the pathogenetic events driving their evolution, raises the question whether earlier interventions may alter the natural history of the disease. Herein, we review this Tower of Babel of acronyms pinpointing diagnostic definitions, differential diagnosis, and the role of genomic profiling of these precursor states, as well as potential interventional strategies.

Thrombosis in Myeloid Malignancies, from CHIP to AML.

The development of myeloid malignancies is a multi-step process starting from pre-malignant stages. Large-scale studies on clonal hematopoiesis of indeterminate potential (CHIP) identified this condition as a risk factor for developing hematologic malignancies, in particular myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). In parallel, CHIP was found to confer an enhanced thrombotic risk, in particular for cardiovascular diseases. In a similar fashion, in recent years, alongside their life-threatening features, increasing attention has been drawn toward thrombotic complications in myeloid malignancies. The purpose of this review is to gather growing body of evidence on incidence, pathogenesis and clinical impact of thrombosis in myeloid malignancies at every step of malignant progression, from CHIP to AML.

Thrombotic risk and features of patients with inferior vena cava agenesis: a multicentre, retrospective, observational study.

BACKGROUND: Inferior vena cava agenesis (IVCA) is a rare anomaly predisposing affected people to lower-limb venous thrombosis with low frequency of pulmonary embolism. Antenatal thrombosis and inherited thrombophilia have been suggested as causes of IVCA. However, there is little evidence on the clinical course and management of this condition. We designed a patient registry to assess the thrombotic risk and features of IVCA. METHODS: In this this multicentre, retrospective, observational study, we included patients with IVCA diagnosed by routine imaging from 20 hospitals in Spain (n=18), Portugal (n=1), and Italy (n=1). Patients were identified from a systematic search in radiology databases using data extraction software (cohort A) and alternative searches in medical records for confirmed IVCA (cohort B; option allowed when systematic approaches were unapplicable). Primary outcomes were clinical and imaging features, thrombotic risk, phenotype of IVCA-associated thrombosis, anticoagulant treatment, and the results of thrombophilia testing. FINDINGS: We included patients with IVCA diagnosed by routine imaging studies done between Jan 1, 2010, and Dec 31, 2022. In the systematic search, 4 341 333 imaging exams were screened from the radiology databases of eight centres. 122 eligible patients were enrolled in cohort A. A further 95 patients were identified by screening medical records at 12 centres, of whom 88 were eligible and included in cohort B, making a combined cohort of 210 patients. 96 (46%) of 210 patients were female and 200 (95%) were European or Hispanic. 60 (29%) of 210 patients had hepatic IVC interruption, whereas 150 (71%) had extrahepatic IVCA. In cohort A, 65 (53%) of 122 patients had venous thrombosis, with an estimated annual risk of 1·15% (95% CI 0·89-1·46). Extrahepatic IVCA was associated with a greater risk of venous thrombosis than hepatic IVCA (56 [67%] of 84 patients vs nine [24%] of 38 patients, odds ratio 5·31, 95% CI 2·27-12·43; p<0·0001). Analysis of 126 patients with venous thrombosis pooled from cohorts A and B showed early-onset (median age 34·6 years, IQR 23·3-54·3) and recurrent events (50 [40%] of 126 patients). Patients with extrahepatic IVCA had greater proportions of lower-limb venous thrombosis (95 [87%] of 109 vs nine [53%] of 17, p=0·0010) and recurrence (48 [44%] of 109 vs two [12%] of 17, p=0·015), but lower rates of pulmonary embolism (10 [10%] of 99 vs four [33%] of 12, p=0·044) than did patients with hepatic IVCA. 77 (63%) of 122 patients with thrombosis underwent indefinite anticoagulation. 32 (29%) of 111 patients (29 [34%] of 86 with thrombosis) had coexisting thrombophilias. The recurrence risk was lower for patients receiving indefinite anticoagulation (adjusted odds ratio 0·24, 95% CI 0·08-0·61; p=0·010), and greater for thrombophilias (3·19, 1·09-9·32; p=0·034). INTERPRETATION: This evaluation of a large patient cohort demonstrates the high thrombotic burden of IVCA. We have identified two distinct forms of IVCA, hepatic and extrahepatic, suggesting different underlying mechanisms. Beyond clinical characterisation, we draw attention to this orphan disease and highlight the need for its study and improved care. FUNDING: Spanish Society of Thrombosis and Haemostasis, Instituto de Salud Carlos III, FEDER, Fundación Séneca.

Immunotherapy in Acute Myeloid Leukemia: A Literature Review of Emerging Strategies.

In the last twenty years, we have witnessed a paradigm shift in the treatment and prognosis of acute myeloid leukemia (AML), thanks to the introduction of new efficient drugs or approaches to refine old therapies, such as Gemtuzumab Ozogamicin, CPX 3-5-1, hypomethylating agents, and Venetoclax, the optimization of conditioning regimens in allogeneic hematopoietic stem cell transplantation and the improvement of supportive care. However, the long-term survival of non-M3 and non-core binding factor-AML is still dismal. For this reason, the expectations for the recently developed immunotherapies, such as antibody-based therapy, checkpoint inhibitors, and chimeric antigen receptor strategies, successfully tested in other hematologic malignancies, were very high. The inherent characteristics of AML blasts hampered the development of these treatments, and the path of immunotherapy in AML has been bumpy. Herein, we provide a detailed review of potential antigenic targets, available data from pre-clinical and clinical trials, and future directions of immunotherapies in AML.

Antithrombin Deficiency Is Associated with Prothrombotic Plasma Fibrin Clot Phenotype.

BACKGROUND: Deficiency of antithrombin increases risk of venous thromboembolism. We hypothesized that antithrombin deficiency affects fibrin clot structure and function. METHODS: We evaluated 148 patients (age: 38 [32-50] years; 70% women) with genetically confirmed antithrombin deficiency and 50 healthy controls. Fibrin clot permeability (Ks) and clot lysis time (CLT) along with thrombin generation capacity were assessed before and after antithrombin activity normalization in vitro. RESULTS: Antithrombin-deficient patients had lower antithrombin activity (-39%) and antigen levels (-23%) compared with controls (both p < 0.01). Prothrombin fragment 1 + 2 levels were 26.5% higher in patients with antithrombin deficiency than in controls along with 94% increased endogenous thrombin potential (ETP) and 108% higher peak thrombin (all p < 0.01). Antithrombin deficiency was associated with 18% reduced Ks and 35% prolonged CLT (both p < 0.001). Patients with type I (n = 65; 43.9%) compared with type II antithrombin deficiency (n = 83; 56.1%) had 22.5% lower antithrombin activity (p < 0.001) and despite similar fibrinogen levels, 8.4% reduced Ks, 18% prolonged CLT, and 30% higher ETP (all p < 0.01). Reduced Ks was associated with lower antithrombin antigen level (ß = - 6.1, 95% confidence interval [CI]: -1.7 to -10.5), while prolonged CLT was associated with lower antithrombin antigen (ß = - 69.6, 95% CI: -9.6 to -129.7), activity (ß = - 2.4, 95% CI: -0.3 to -4.5), higher PAI-1 (ß = 12.1, 95% CI: 7.7-16.5), and thrombin-activatable fibrinolysis inhibitor levels (ß = 3.8, 95% CI: 1.9-5.7). Addition of exogenous antithrombin reduced ETP (-42%) and peak thrombin (-21%), and improved Ks (+8%) and CLT (-12%; all p < 0.01). CONCLUSION: Our study suggests that enhanced thrombin generation and prothrombotic plasma fibrin clot phenotype can contribute to increased risk of thrombosis in patients with antithrombin deficiency.

Impact of genetic structural variants in factor XI deficiency: identification, accurate characterization, and inferred mechanism by long-read sequencing.

BACKGROUND: Congenital factor XI (FXI) deficiency is a probably underestimated coagulopathy that confers antithrombotic protection. Characterization of genetic defects in F11 is mainly focused on the identification of single-nucleotide variants and small insertion/deletions because they represent up to 99% of the alterations accounting for factor deficiency, with only 3 gross gene defects of structural variants (SVs) having been described. OBJECTIVES: To identify and characterize the SVs affecting F11. METHODS: The study was performed in 93 unrelated subjects with FXI deficiency recruited in Spanish hospitals over a period of 25 years (1997-2022). F11 was analyzed by next-generation sequencing, multiplex ligand probe amplification, and long-read sequencing. RESULTS: Our study identified 30 different genetic variants. Interestingly, we found 3 SVs, all heterozygous: a complex duplication affecting exons 8 and 9, a tandem duplication of exon 14, and a large deletion affecting the whole gene. Nucleotide resolution obtained by long-read sequencing revealed Alu repetitive elements involved in all breakpoints. The large deletion was probably generated de novo in the paternal allele during gametogenesis, and despite affecting 30 additional genes, no syndromic features were described. CONCLUSION: SVs may account for a high proportion of F11 genetic defects implicated in the molecular pathology of congenital FXI deficiency. These SVs, likely caused by a nonallelic homologous recombination involving repetitive elements, are heterogeneous in both type and length and may be de novo. These data support the inclusion of methods to detect SVs in this disorder, with long-read-based methods being the most appropriate because they detect all SVs and achieve adequate nucleotide resolution.

Scalable production of tissue-like vascularized liver organoids from human PSCs.

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

Persistent Large Granular Lymphocyte Clonal Expansions: "The Root of Many Evils"-And of Some Goodness.

Large granular lymphocyte leukemia (LGLL) is a chronic disease of either mature phenotype cytotoxic CD3+ T lymphocytes or CD3- NK cells. LGLL diagnosis is hampered by the fact that reactive persistent clonal LGL expansions may fulfill the current criteria for LGLL diagnoses. In addition to the presence of characteristic clinical and hematological signs such as anemia or neutropenia, LGLL/LGL clonal expansions have been associated with an array of conditions/disorders. We review here the presence of these persistent clonal expansions in autoimmune, hematological disorders and solid neoplasms and after hematopoietic stem cell transplantation. These associations are a unique translational research framework to discern whether these persistently expanded LGL clones are causes or consequences of the concomitant clinical settings and, more importantly, when they should be targeted.