Paroxysmal nocturnal hemoglobinuria-related thrombosis in the era of novel therapies: a 2043-patient-year analysis.

ABSTRACT: Thrombophilia is one of the principal features of paroxysmal nocturnal hemoglobinuria (PNH) and constitutes the main cause of disease morbidity/mortality. Anticomplement treatment has revolutionized the natural history of PNH, with control of the hemolytic process and abolition of thrombotic events (TEs). However, no guidelines exist for the management of thromboembolic complications in this setting, with type and duration of anticoagulation depending on individual practices. Besides, a scarcity of data is present on the efficacy of direct oral anticoagulants (DOACs). Herein, we accrued a large real-world cohort of patients with PNH from 4 US centers to explore features, predictors of TE, and anticoagulation strategies. Among 267 patients followed up for a total of 2043 patient-years, 56 (21%) developed TEs. These occurred at disease onset in 43% of cases, involving more frequently the venous system, typically as Budd-Chiari syndrome. Rate of TEs was halved in patients receiving complement inhibitors (21 vs 40 TEs per 1000 patient-years in untreated cases, with a 2-year cumulative incidence of thrombosis of 3.9% vs 18.3%, respectively), and varied according to PNH granulocytes and erythrocytes clone size, type, disease activity parameters, as well as number (≥2 mutations, or less) and variant allelic frequency of PIGA mutations. Anticoagulation with warfarin (39%), DOACs (37%), and low-molecular weight heparin (16%) was administered for a median of 29 months (interquartile range [IQR], 9-61.8). No thrombotic recurrence was observed in 19 patients treated with DOACs at a median observation of 17.1 months (IQR, 8.9-45) whereas 14 cases discontinued anticoagulation without TE recurrence at a median time of 51.4 months (IQR, 29.9-86.8).

The metabolic fuel of paroxysmal nocturnal haemoglobinuria.

Metabolic reprogramming has been investigated in haematological malignancies. To date, a few studies have analysed the metabolic profile of paroxysmal nocturnal haemoglobinuria (PNH). The study by Chen and colleagues sheds light on the involvement of metabolic changes in the proliferation of PNH clones. Commentary on: Chen et al. The histone demethylase JMJD1C regulates CPS1 expression and promotes the proliferation of PNH clones through cell metabolic reprogramming. Br J Haematol 2024;204:2468-2479.

Splenectomy outcomes in immune cytopenias: Treatment outcomes and determinants of response.

BACKGROUND: Splenectomy is commonly used to treat refractory immune-mediated cytopenia, but there are no established factors that are associated with response to the procedure. OBJECTIVES: A cohort study was conducted to evaluate the hematologic and surgical outcomes of splenectomy in adult patients with immune cytopenias and identify preoperative factors associated with response. METHODS: Data from the Cleveland Clinic Foundation for 1824 patients aged over 18 who underwent splenectomy from 2002 to 2020 were analyzed. RESULTS: The study found that the most common indications for splenectomy were immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia, with a median age of 55 years and median time from diagnosis to splenectomy of 11 months. Hematologic response rates were 74% overall, with relapse in 12% of cases. Postsplenectomy discordant diagnoses were present in 13% of patients, associated with higher relapse rates. Surgery-related complications occurred in 12% of cases, whereas only 3% of patients died from disease complications. On univariate analysis, preoperative factors associated with splenectomy treatment failure were ≥3 lines of pharmacologic treatment, whereas isolated thrombocytopenia, primary ITP, and age ≤40 years had a strong association with response. The multivariable regression confirmed that treatment failure with multiple lines of medical therapy was associated with the failure to respond to splenectomy. CONCLUSION: Overall, the study demonstrates that splenectomy is an effective treatment option for immune-mediated cytopenias with a low complication rate.

KMT2A Rearrangements in Leukemias: Molecular Aspects and Therapeutic Perspectives.

KMT2A (alias: mixed-lineage leukemia [MLL]) gene mapping on chromosome 11q23 encodes the lysine-specific histone N-methyltransferase 2A and promotes transcription by inducing an open chromatin conformation. Numerous genomic breakpoints within the KMT2A gene have been reported in young children and adults with hematologic disorders and are present in up to 10% of acute leukemias. These rearrangements describe distinct features and worse prognosis depending on the fusion partner, characterized by chemotherapy resistance and high rates of relapse, with a progression-free survival of 30-40% and overall survival below 25%. Less intensive regimens are used in pediatric patients, while new combination therapies and targeted immunotherapeutic agents are being explored in adults. Beneficial therapeutic effects, and even cure, can be reached with hematopoietic stem cell transplantation, mainly in young children with dismal molecular lesions; however, delayed related toxicities represent a concern. Herein, we summarize the translocation partner genes and partial tandem duplications of the KMT2A gene, their molecular impact, clinical aspects, and novel targeted therapies.

The clinicopathologic significance of NPM1 mutation and ability to detect mutated NPM1 by immunohistochemistry in non-AML myeloid neoplasms.

NPM1 mutated non-AML myeloid neoplasms (MN; <20% blasts) are characterized by an aggressive clinical course in a few studies. In this retrospective study, we evaluate the clinicopathologic and immunohistochemical features of non-AML MN patients with NPM1 mutations. We assessed NPM1 mutation by targeted next generation sequencing (NGS). Cytoplasmic NPM1 expression was assessed by immunohistochemistry (IHC) on formalin-fixed, formic acid-decalcified bone marrow biopsy specimens. We evaluated 34 non-AML MN patients with NPM1 mutations comprising MDS (22), MPN (3) and MDS/MPN (9). They commonly presented with anemia (88%), thrombocytopenia (58%) and leukopenia (50%). Bone marrow dysplasia was common (79%). The karyotype was often normal (64%). NGS for MN-associated mutations performed in a subset of the patients showed a median of 3 mutations. NPM1 mutations were more often missense (c.859C > T p. L287F; 65%) than frameshift insertion/duplication (35%) with median variant allele frequency (VAF; 9.7%, range 5.1%-49.8%). Mutated NPM1 by IHC showed cytoplasmic positivity in 48% and positivity was associated with higher VAF. The median overall survival (OS) in this cohort was 70 months. Nine patients (26%) progressed to AML. OS in patients who progressed to AML was significantly shorter than the one of patients without progression to AML (OS 20 vs. 128 months, respectively, log rank p = 0.05). NPM1 mutated non-AML MN patients commonly had cytopenias, dysplasia, normal karyotype, mutations in multiple genes, and an unfavorable clinical outcome, including progression to AML. Our data demonstrated that IHC for NPM1 can be a useful supplementary tool to predict NPM1 mutation in some non-AML MN; however, genetic testing cannot be replaced by IHC assessment.

Machine learning integrates genomic signatures for subclassification beyond primary and secondary acute myeloid leukemia.

Although genomic alterations drive the pathogenesis of acute myeloid leukemia (AML), traditional classifications are largely based on morphology, and prototypic genetic founder lesions define only a small proportion of AML patients. The historical subdivision of primary/de novo AML and secondary AML has shown to variably correlate with genetic patterns. The combinatorial complexity and heterogeneity of AML genomic architecture may have thus far precluded genomic-based subclassification to identify distinct molecularly defined subtypes more reflective of shared pathogenesis. We integrated cytogenetic and gene sequencing data from a multicenter cohort of 6788 AML patients that were analyzed using standard and machine learning methods to generate a novel AML molecular subclassification with biologic correlates corresponding to underlying pathogenesis. Standard supervised analyses resulted in modest cross-validation accuracy when attempting to use molecular patterns to predict traditional pathomorphologic AML classifications. We performed unsupervised analysis by applying the Bayesian latent class method that identified 4 unique genomic clusters of distinct prognoses. Invariant genomic features driving each cluster were extracted and resulted in 97% cross-validation accuracy when used for genomic subclassification. Subclasses of AML defined by molecular signatures overlapped current pathomorphologic and clinically defined AML subtypes. We internally and externally validated our results and share an open-access molecular classification scheme for AML patients. Although the heterogeneity inherent in the genomic changes across nearly 7000 AML patients was too vast for traditional prediction methods, machine learning methods allowed for the definition of novel genomic AML subclasses, indicating that traditional pathomorphologic definitions may be less reflective of overlapping pathogenesis.

The similarity of class II HLA genotypes defines patterns of autoreactivity in idiopathic bone marrow failure disorders.

Idiopathic aplastic anemia (IAA) is a rare autoimmune bone marrow failure (BMF) disorder initiated by a human leukocyte antigen (HLA)-restricted T-cell response to unknown antigens. As in other autoimmune disorders, the predilection for certain HLA profiles seems to represent an etiologic factor; however, the structure-function patterns involved in the self-presentation in this disease remain unclear. Herein, we analyzed the molecular landscape of HLA complexes of a cohort of 300 IAA patients and almost 3000 healthy and disease controls by deeply dissecting their genotypic configurations, functional divergence, self-antigen binding capabilities, and T-cell receptor (TCR) repertoire specificities. Specifically, analysis of the evolutionary divergence of HLA genotypes (HED) showed that IAA patients carried class II HLA molecules whose antigen-binding sites were characterized by a high level of structural homology, only partially explained by specific risk allele profiles. This pattern implies reduced HLA binding capabilities, confirmed by binding analysis of hematopoietic stem cell (HSC)-derived self-peptides. IAA phenotype was associated with the enrichment in a few amino acids at specific positions within the peptide-binding groove of DRB1 molecules, affecting the interface HLA-antigen-TCR ß and potentially constituting the basis of T-cell dysfunction and autoreactivity. When analyzing associations with clinical outcomes, low HED was associated with risk of malignant progression and worse survival, underlying reduced tumor surveillance in clearing potential neoantigens derived from mechanisms of clonal hematopoiesis. Our data shed light on the immunogenetic risk associated with IAA etiology and clonal evolution and on general pathophysiological mechanisms potentially involved in other autoimmune disorders.

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.

HHEX promotes myeloid transformation in cooperation with mutant ASXL1.

Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knockin (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we used retrovirus-mediated insertional mutagenesis, which exhibited the susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified the hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT-expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and chromatin immunoprecipitation-next-generation sequencing analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicate that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.

A study of Telomerase Reverse Transcriptase rare variants in myeloid neoplasia.

Telomere dysfunctions are associated with several hematopoietic stem cell (HSC) malignancies. Recent findings have indicated that the occurrence of rare variants of unknown significance (VUS) in the Telomerase Reverse Transcriptase (TERT) gene influences the outcomes of patients with myelodysplastic syndromes undergoing allogeneic HSC transplantation. However, the role of TERT variants has been historically controversial as initially considered pathogenic variants (H412Y, A202T) presenting functional consequences, were found very frequent in general population questioning their pathogenicity and risk allele significance. Herein, we show that overall TERT VUS are non-recurrent in myeloid disorders and cannot be considered risk alleles individually nor can their biological impact.

Personalized Risk Schemes and Machine Learning to Empower Genomic Prognostication Models in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are characterized by variable clinical manifestations and outcomes. Several prognostic systems relying on clinical factors and cytogenetic abnormalities have been developed to help stratify MDS patients into different risk categories of distinct prognoses and therapeutic implications. The current abundance of molecular information poses the challenges of precisely defining patients' molecular profiles and their incorporation in clinically established diagnostic and prognostic schemes. Perhaps the prognostic power of the current systems can be boosted by incorporating molecular features. Machine learning (ML) algorithms can be helpful in developing more precise prognostication models that integrate complex genomic interactions at a higher dimensional level. These techniques can potentially generate automated diagnostic and prognostic models and assist in advancing personalized therapies. This review highlights the current prognostication models used in MDS while shedding light on the latest achievements in ML-based research.

Comprehensive Transcriptomic Analysis of VISTA in Acute Myeloid Leukemia: Insights into Its Prognostic Value.

The V-domain Ig suppressor of T-cell activation (VISTA) has been recognized as a critical negative regulator of antitumor immune response and is gaining growing interest as a potential pharmacological target in immunotherapy. This molecule is highly expressed in hematopoietic stem cells and myeloid compartment, and it has been found upmodulated in acute myeloid leukemia (AML). However, VISTA-associated immune features are relatively unexplored in myeloid malignancies. Herein, we aimed to explore whether this immune checkpoint regulator could play a role in the generation of an immune escape environment in AML patients. We characterized VISTA mRNA expression levels in leukemia cell lines and in large publicly available cohorts of specimens from bone marrow of healthy individuals and AML patients at diagnosis by deploying bulk and single-cell RNA sequencing. We also defined the correlations with leukemia-associated burden using results of whole-exome sequencing of AML samples at disease onset. We showed that VISTA expression linearly increased across the myeloid differentiation tree in normal hematopoiesis. Accordingly, its transcript was highly enriched in AML cell lines as well as in AML patients at diagnosis presenting with myelomonocytic and monocytic differentiation. A strong correlation was seen with NPM1 mutations regardless of the presence of FLT3 lesions. Furthermore, VISTA expression levels at baseline correlated with disease recurrence in patients with normal karyotype and NPM1 mutations, a subgroup traditionally considered as favorable according to current diagnostic schemes. Indeed, when compared to patients with long-term remission (>5 years after standard chemotherapy regimens), cases relapsing within 2 years from diagnosis had increased VISTA expression in both leukemia and T cells. Our results suggest a rationale for developing VISTA-targeted therapeutic strategies to treat molecularly defined subgroups of AML patients to prevent disease recurrence and treatment resistance.

Friend or foe? The case of Wilms' Tumor 1 (WT1) mutations in acute myeloid leukemia.

Wilms tumor 1 (WT1) gene is commonly mutated in acute myeloid leukemia (AML), particularly in younger age population. The mechanism through which WT1 mutations drive leukemogenesis have not been fully elucidated; however, recent studies reported an association with the epigenetic pathway. Here, we studied the phenotypic characteristics and somatic mutational profile of 114 WT1-mutant AML patients and focused on potential WT1 gene relations to other cooperative genomic events that may impact disease prognosis. Invariant phenotypic and genomic associations of WT1 mutations in AML were uncovered and rigorously described. Our findings help improving the current understanding and definition of WT1-mutant AML patients' characteristics and clinical outcomes.

Frequency and perturbations of various peripheral blood cell populations before and after eculizumab treatment in paroxysmal nocturnal hemoglobinuria.

While red blood cells (RBCs) and granulocytes have been more studied, platelets and reticulocytes are not commonly used in paroxysmal nocturnal hemoglobinuria (PNH) flow-cytometry and less is known about susceptibility to complement-mediated destruction and effects of anti-complement therapy on these populations. We performed flow-cytometry of RBCs and granulocytes in 90 PNH patients and of platelets and reticulocytes in a subgroup (N = 36), to unveil perturbations of these populations during PNH disease course before and after anti-complement treatment. We found that platelets and reticulocytes were less sensitive to complement-mediated lysis than RBCs but not as resistant as granulocytes, as shown by mean sensitive fraction (difference in a given PNH population vs. PNH granulocyte clone size). In treated patients, reticulocytes, platelets, RBCs (with differences between type II and III) and granulocytes significantly increased post-treatment, confirming the role of PNH hematopoiesis within the context of anti-complement therapy. Moreover, we found that PNH platelet clone size reflects PNH granulocyte clone size. Finally, we established correlations between sensitive fraction of PNH cell-types and thrombosis. In sum, we applied a flow-cytometry panel for investigation of PNH peripheral blood populations' perturbations before and after eculizumab treatment to explore complement-sensitivity and kinetics of these cells during the disease course.

The Interactome between Metabolism and Gene Mutations in Myeloid Malignancies.

The study of metabolic deregulation in myeloid malignancies has led to the investigation of metabolic-targeted therapies considering that cells undergoing leukemic transformation have excessive energy demands for growth and proliferation. However, the most difficult challenge in agents targeting metabolism is to determine a window of therapeutic opportunities between normal and neoplastic cells, considering that all or most of the metabolic pathways important for cancer ontogeny may also regulate physiological cell functions. Targeted therapies have used the properties of leukemic cells to produce altered metabolic products when mutated. This is the case of IDH1/2 mutations generating the abnormal conversion of α-ketoglutarate (KG) to 2-hydroxyglutarate, an oncometabolite inhibiting KG-dependent enzymes, such as the TET family of genes (pivotal in characterizing leukemia cells either by mutations, e.g., TET2, or by altered expression, e.g., TET1/2/3). Additional observations derive from the high sensitivity of leukemic cells to oxidative phosphorylation and its amelioration using BCL-2 inhibitors (Venetoclax) or by disrupting the mitochondrial respiration. More recently, nicotinamide metabolism has been described to mediate resistance to Venetoclax in patients with acute myeloid leukemia. Herein, we will provide an overview of the latest research on the link between metabolic pathways interactome and leukemogenesis with a comprehensive analysis of the metabolic consequences of driver genetic lesions and exemplificative druggable pathways.

Extended experience with a non-cytotoxic DNMT1-targeting regimen of decitabine to treat myeloid malignancies.

The nucleoside analogue decitabine can deplete the epigenetic regulator DNA methyltransferase 1 (DNMT1), an effect that occurs, and is saturated at, low concentrations/doses. A reason to pursue this molecular-targeted effect instead of the DNA damage/cytotoxicity produced with high concentrations/doses, is that non-cytotoxic DNMT1-depletion can cytoreduce even p53-null myeloid malignancies while sparing normal haematopoiesis. We thus identified minimum doses of decitabine (0·1-0·2 mg/kg) that deplete DNMT1 without off-target anti-metabolite effects/cytotoxicity, and then administered these well-tolerated doses frequently 1-2X/week to increase S-phase dependent DNMT1-depletion, and used a Myeloid Malignancy Registry to evaluate long-term outcomes in 69 patients treated this way. Consistent with the scientific rationale, treatment was well-tolerated and durable responses were produced (~40%) in genetically heterogeneous disease and the very elderly.

Large granular lymphocytic leukaemia after solid organ and haematopoietic stem cell transplantation.

T-cell large granular lymphocytic leukaemia (T-LGLL) is a chronic clonal lymphoproliferative disorder of cytotoxic T lymphocytes which commonly occurs in older patients and is often associated with autoimmune diseases. Among 246 patients with T-LGLL seen at our institution over the last 10 years, we encountered 15 cases following solid organ or haematopoietic stem cell transplantation. Here, we studied the clinical characterization of these cases and compared them to de novo T-LGLL. This experience represented a clear picture of the intricate nature of the disease manifestation and the complexities of several immune mechanisms triggering the clonal expansion.

Deciphering the Therapeutic Resistance in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a clonal hematopoietic disorder characterized by abnormal proliferation, lack of cellular differentiation, and infiltration of bone marrow, peripheral blood, or other organs. Induction failure and in general resistance to chemotherapeutic agents represent a hindrance for improving survival outcomes in AML. Here, we review the latest insights in AML biology concerning refractoriness to therapies with a specific focus on cytarabine and daunorubicin which still represent milestones agents for inducing therapeutic response and disease eradication. However, failure to achieve complete remission in AML is still high especially in elderly patients (40-60% in patients >65 years old). Several lines of basic and clinical research have been employed to improve the achievement of complete remission. These lines of research include molecular targeted therapy and more recently immunotherapy. In terms of molecular targeted therapies, specific attention is given to DNMT3A and TP53 mutant AML by reviewing the mechanisms underlying epigenetic therapies' (e.g., hypomethylating agents) resistance and providing critical points and hints for possible future therapies overcoming AML refractoriness.