Publications by authors named "Luca Malcovati"

164 Publications

Investigating the Molecular Mechanism of H3B-8800: A Splicing Modulator Inducing Preferential Lethality in Spliceosome-Mutant Cancers.

Int J Mol Sci 2021 Oct 18;22(20). Epub 2021 Oct 18.

National Research Council of Italy, Institute of Materials Foundry (CNR-IOM) c/o SISSA, Via Bonomea 265, 34136 Trieste, Italy.

The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre-mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B-8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B-8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome-mutant cancer cells remains elusive. Here, microsecond-long all-atom simulations addressed the binding/dissociation mechanism of H3B-8800 to wild type and K700E SF3B1-containing SF3b (SB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B-8800 binding. This supports the hypothesis that the selectivity of H3B-8800 towards mutant cancer cells is unrelated to its preferential targeting of SB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B-8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug-discovery efforts to specifically target mutant SF3b.
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http://dx.doi.org/10.3390/ijms222011222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8540225PMC
October 2021

A geno-clinical decision model for the diagnosis of myelodysplastic syndromes.

Blood Adv 2021 11;5(21):4361-4369

Leukemia Program, Department of Hematology and Medical Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH.

The differential diagnosis of myeloid malignancies is challenging and subject to interobserver variability. We used clinical and next-generation sequencing (NGS) data to develop a machine learning model for the diagnosis of myeloid malignancies independent of bone marrow biopsy data based on a 3-institution, international cohort of patients. The model achieves high performance, with model interpretations indicating that it relies on factors similar to those used by clinicians. In addition, we describe associations between NGS findings and clinically important phenotypes and introduce the use of machine learning algorithms to elucidate clinicogenomic relationships.
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http://dx.doi.org/10.1182/bloodadvances.2021004755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8579270PMC
November 2021

is mutated in clonal hematopoiesis and myelodysplastic syndromes and impacts RNA splicing.

Blood Cancer Discov 2021 Sep 14;2(5):500-517. Epub 2021 Jul 14.

The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Clonal hematopoiesis results from somatic mutations in cancer driver genes in hematopoietic stem cells. We sought to identify novel drivers of clonal expansion using an unbiased analysis of sequencing data from 84,683 persons and identified common mutations in the 5-methylcytosine reader, , as well as in , , and . We also identified these mutations at low frequency in myelodysplastic syndrome patients. edited mouse hematopoietic stem and progenitor cells exhibited a competitive advantage and increased genome-wide intron retention. mutations potentially link DNA methylation and RNA splicing, the two most commonly mutated pathways in clonal hematopoiesis and MDS.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8462124PMC
September 2021

Refined diagnostic criteria for bone marrow mastocytosis: a proposal of the European competence network on mastocytosis.

Leukemia 2021 Sep 20. Epub 2021 Sep 20.

KU Leuven Department of Microbiology, Immunology and Transplantation, Allergy and Clinical Immunology Research Group and MASTeL, University Hospitals Leuven, Leuven, Belgium.

In the current classification of the World Health Organization (WHO), bone marrow mastocytosis (BMM) is a provisional variant of indolent systemic mastocytosis (ISM) defined by bone marrow involvement and absence of skin lesions. However, no additional diagnostic criteria for BMM have been proposed. Within the registry dataset of the European Competence Network on Mastocytosis, we compared characteristics and outcomes of 390 patients with BMM and 1175 patients with typical ISM. BMM patients were significantly older, predominantly male, had lower tryptase and lower burden of neoplastic mast cells, and displayed a higher frequency of allergic reactions, mainly triggered by Hymenoptera, than patients with typical ISM. The estimated 10-year progression-free survival of BMM and typical ISM was 95.9% and 92.6%, respectively. In BMM patients defined by WHO-based criteria, the presence of one B-Finding and tryptase level ≥125 ng/mL were identified as risk factors for progression in multivariate analyses. BMM patients without any of these risk factors were found to have better progression-free survival (p < 0.05) and better overall survival (p < 0.05) than other ISM patients. These data support the proposal to define BMM as a separate SM variant characterized by SM criteria, absence of skin lesions, absence of B-Findings, and tryptase levels <125 ng/mL.
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http://dx.doi.org/10.1038/s41375-021-01406-yDOI Listing
September 2021

The journey of a thousand miles begins with 1 step.

Authors:
Luca Malcovati

Blood 2021 09;138(10):824-826

University of Pavia and IRCCS S Matteo Hospital Foundation.

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http://dx.doi.org/10.1182/blood.2021012304DOI Listing
September 2021

EHA Endorsement of the European Guidelines for Myelodysplastic Syndromes, MDS-RIGHT.

Hemasphere 2021 Sep 30;5(9):e631. Epub 2021 Aug 30.

Department of Molecular Medicine, University of Pavia & Department of Hematology Oncology, IRCCS S. Matteo Hospital Foundation, Pavia, Italy.

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http://dx.doi.org/10.1097/HS9.0000000000000631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405128PMC
September 2021

Peripheral blood cytopenias in the aging general population and risk of incident hematological disease and mortality.

Blood Adv 2021 09;5(17):3266-3278

Department of Hematology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Peripheral blood cytopenias may precede the development of hematological malignancies and frequently pose clinical challenges in the older population. The natural course of (mild) cytopenias during aging and their association with hematological disorders in community-dwelling individuals are not well studied. Within the population-based Lifelines cohort (n = 167729), we studied changes in peripheral blood counts, occurrence of cytopenias, and associated hematological outcomes in the context of aging. Development of hematological malignancies and (cause-specific) mortality were evaluated by linkage to nationwide registries. Anemia and thrombocytopenia emerged with older age, in line with a general age-related decline in these blood counts. For neutropenia, no increase in prevalence with older age was observed. Using standard reference limits to define cytopenias, anemia (hazard ratio [HR], 1.84; 95% confidence interval [CI], 1.59-2.12), thrombocytopenia (HR, 1.58; 95% CI, 1.32-1.89), and, especially the concomitant presence of anemia and thrombocytopenia (HR, 4.75; 95% CI, 2.98-7.55) were associated with inferior overall survival. Only a minor proportion of deaths was explained by diagnosed hematological malignancies, with the majority attributable to other causes. Neutropenia, either isolated (HR, 0.88; 95% CI, 0.73-1.06) or combined with another cytopenia, did not affect overall survival. For individuals aged ≥60 years, 5-year cumulative incidence of hematological malignancies was 0.60% (95% CI, 0.50-0.70), with higher incidences among those with anemia (P < .001) or thrombocytopenia (P < .001) but not neutropenia (P = .201). Highest cumulative incidences of diagnoses and mortality from hematological malignancies were observed in individuals with >1 cytopenia. We conclude that anemia and thrombocytopenia, but not neutropenia, are associated with inferior overall survival of community-dwelling individuals. Hematological malignancies develop in a small fraction of these cases.
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http://dx.doi.org/10.1182/bloodadvances.2021004355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525229PMC
September 2021

A predictive algorithm using clinical and laboratory parameters may assist in ruling out and in diagnosing MDS.

Blood Adv 2021 08;5(16):3066-3075

Department of Medicine, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.

We present a noninvasive Web-based app to help exclude or diagnose myelodysplastic syndrome (MDS), a bone marrow (BM) disorder with cytopenias and leukemic risk, diagnosed by BM examination. A sample of 502 MDS patients from the European MDS (EUMDS) registry (n > 2600) was combined with 502 controls (all BM proven). Gradient-boosted models (GBMs) were used to predict/exclude MDS using demographic, clinical, and laboratory variables. Area under the receiver operating characteristic curve (AUC), sensitivity, and specificity were used to evaluate the models, and performance was validated using 100 times fivefold cross-validation. Model stability was assessed by repeating its fit using different randomly chosen groups of 502 EUMDS cases. AUC was 0.96 (95% confidence interval, 0.95-0.97). MDS is predicted/excluded accurately in 86% of patients with unexplained anemia. A GBM score (range, 0-1) of less than 0.68 (GBM < 0.68) resulted in a negative predictive value of 0.94, that is, MDS was excluded. GBM ≥ 0.82 provided a positive predictive value of 0.88, that is, MDS. The diagnosis of the remaining patients (0.68 ≤ GBM < 0.82) is indeterminate. The discriminating variables: age, sex, hemoglobin, white blood cells, platelets, mean corpuscular volume, neutrophils, monocytes, glucose, and creatinine. A Web-based app was developed; physicians could use it to exclude or predict MDS noninvasively in most patients without a BM examination. Future work will add peripheral blood cytogenetics/genetics, EUMDS-based prospective validation, and prognostication.
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http://dx.doi.org/10.1182/bloodadvances.2020004055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405190PMC
August 2021

Clonal hematopoiesis and myeloid malignancies: clonal dynamics and clinical implications.

Curr Opin Hematol 2021 09;28(5):347-355

Department of Hematology Oncology, IRCCS San Matteo Hospital Foundation.

Purpose Of Review: Clinical and experimental studies have uncovered relevant clinical implications of clonal hematopoiesis. However, the true magnitude of this process, clonal dynamics over time and mechanisms of progression into overt malignancy remain to be largely elucidated. In this article, the consequences of clonal hematopoiesis, its significance in the context of cytopenia, and its implications in the clinical management of patients with myeloid malignancies are reviewed and discussed.

Recent Findings: Clonal hematopoiesis has been associated with higher risk of hematologic cancers, as well as of death from cardiovascular causes. Clonal hematopoiesis has been proven clinically relevant in the context of disorders characterized by peripheral blood cytopenia, including aplastic anemia, cytopenia of undetermined significance, as well as unexplained anemia of the elderly.

Summary: The available evidence has been proving the utility of somatic mutational analysis in patients with unexplained cytopenia, as well as in those receiving a diagnosis of myeloid neoplasm, enabling more accurate diagnosis, risk assessment, effective therapeutic strategies and residual disease monitoring. The access to a minimally invasive assessment is paving the way for screening programs of clonal hematopoiesis in individuals with absent or mild hematologic phenotype, as well as for therapeutic targeting of preleukemia cells.
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http://dx.doi.org/10.1097/MOH.0000000000000675DOI Listing
September 2021

Relationship between clone metrics and clinical outcome in clonal cytopenia.

Blood 2021 09;138(11):965-976

Department of Hematology Oncology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Fondazione Policlinico San Matteo, Pavia, Italy.

Clonal cytopenia of undetermined significance (CCUS) is associated with an increased risk of developing a myeloid neoplasm with myelodysplasia (MN). To identify the features of the mutant clone(s) that is associated with clinical phenotype and progression, we studied the following cohorts of individuals: 311 patients with idiopathic cytopenia of undetermined significance (ICUS), 532 community-dwelling individuals without hematologic phenotype (n = 355) or with unexplained anemia (n = 177), and 592 patients with overt MN. Ninety-two of 311 (30%) patients with ICUS carried a somatic genetic lesion that signaled CCUS. Clonal hematopoiesis (CH) was detected in 19.7% and 27.7% of nonanemic and anemic community-dwelling individuals, respectively. Different mutation patterns and variant allele frequencies (VAFs) (clone metrics parameters) were observed in the conditions studied. Recurrent mutation patterns exhibited different VAFs associated with marrow dysplasia (0.17-0.48), indicating variable clinical expressivity of mutant clones. Unsupervised clustering analysis based on mutation profiles identified 2 major clusters, characterized by isolated DNMT3A mutations (CH-like cluster) or combinatorial mutation patterns (MN-like cluster), and showing different overall survival (HR, 1.8). In patients with CCUS, the 2 clusters had different risk of progression to MN (HR, 2.7). Within the MN-like cluster, distinct subsets with different risk of progression to MN were identified based on clone metrics. These findings unveil marked variability in the clinical expressivity of myeloid driver genes and underline the limitations of morphologic dysplasia for clinical staging of mutant hematopoietic clones. Clone metrics appears to be critical for informing clinical decision-making in patients with clonal cytopenia.
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http://dx.doi.org/10.1182/blood.2021011323DOI Listing
September 2021

Incidence and prognosis of clonal hematopoiesis in patients with chronic idiopathic neutropenia.

Blood 2021 10;138(14):1249-1257

Hemopoiesis Research Laboratory, School of Medicine, University of Crete-Department of Hematology, University Hospital of Heraklion, Heraklion, Greece.

The incidence and prognosis of clonal hematopoiesis in patients with isolated neutropenia among patients with idiopathic cytopenia of undetermined significance (ICUS), known as ICUS-N or chronic idiopathic neutropenia (CIN) patients, is poorly defined. The current study sought to investigate the frequency and clinical significance of mutations of genes implicated in myeloid malignancies using next-generation sequencing in patients with CIN (n = 185) with a long follow-up. We found that 21 (11.35%) of 185 patients carried a total of 25 somatic mutations in 6 genes with a median variant allele frequency of 12.75%. The most frequently mutated genes were DNMT3A and TET2 involving >80% of patients, followed by IDH1/2, SRSF2, and ZRSR2. The frequency of transformation to a myeloid malignancy was low in the total group of patients (5 of 185 patients [2.70%]). However, from the transformed patients, 4 belonged to the clonal group (4 of 21 [19.05%]) and 1 to the nonclonal group (1 of 164 [0.61%]), indicating that the presence of mutation(s) confers a relative risk for transformation of 31.24 (P = .0017). The variant allele frequency of the mutant clones in the transformed patients was >10% in all cases, and the genes most frequently associated with malignant transformation were SRSF2 and IDH1. No significant differences were identified between the clonal and nonclonal groups in the severity of neutropenia. Patients with clonal disease were older compared with nonclonal patients. These data contribute to the better understanding of the heterogeneous entities underlying ICUS and highlight the importance of mutation analysis for the diagnosis and prognosis of patients with unexplained neutropenias.
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http://dx.doi.org/10.1182/blood.2021010815DOI Listing
October 2021

The Immunomodulatory Effect and Clinical Efficacy of Daratumumab in a Patient With Cold Agglutinin Disease.

Front Immunol 2021 1;12:649441. Epub 2021 Mar 1.

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy.

Daratumumab is a monoclonal antibody directed against the transmembrane glycoprotein CD38 expressed on plasma cells and lymphoplasmocytes, with a proven efficacy in multiple myeloma. Here we show its clinical efficacy in a patient with cold agglutinin disease (CAD) relapsed after multiple lines of therapy. CAD is caused by cold reactive autoantibodies that induce complement mediated hemolysis and peripheral circulatory symptoms. The disease is also characterized by the presence of monoclonal IgM gammopathy and of a lymphoid bone marrow infiltration that benefits from B-cell targeting therapies (i.e., rituximab) but also from plasma cell directed therapies, such as proteasome inhibitors. In the patient described, we also show that daratumumab therapy influenced the dynamics of several immunoregulatory cytokine levels (IL-6, IL-10, IL-17, IFN-γ, TNF-α, TGF-β) indicating an immunomodulatory effect of the drug beyond plasma cell depletion. In addition, we provide a literature review on the use of daratumumab in autoimmune conditions, including multi-treated and refractory patients with autoimmune hemolytic anemia (both CAD and warm forms), Evans syndrome (association of autoimmune hemolytic anemia and immune thrombocytopenia) and non-hematologic autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.
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http://dx.doi.org/10.3389/fimmu.2021.649441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956980PMC
September 2021

Gene expression profile correlates with molecular and clinical features in patients with myelofibrosis.

Blood Adv 2021 03;5(5):1452-1462

Center for Genome Research.

Myelofibrosis (MF) belongs to the family of classic Philadelphia-negative myeloproliferative neoplasms (MPNs). It can be primary myelofibrosis (PMF) or secondary myelofibrosis (SMF) evolving from polycythemia vera (PV) or essential thrombocythemia (ET). Despite the differences, PMF and SMF patients are currently managed in the same way, and prediction of survival is based on the same clinical and genetic features. In the last few years, interest has grown concerning the ability of gene expression profiles (GEPs) to provide valuable prognostic information. Here, we studied the GEPs of granulocytes from 114 patients with MF, using a microarray platform to identify correlations with patient characteristics and outcomes. Cox regression analysis led to the identification of 201 survival-related transcripts characterizing patients who are at high risk for death. High-risk patients identified by this gene signature displayed an inferior overall survival and leukemia-free survival, together with clinical and molecular detrimental features included in contemporary prognostic models, such as the presence of high molecular risk mutations. The high-risk group was enriched in post-PV and post-ET MF and JAK2V617F homozygous patients, whereas pre-PMF was more frequent in the low-risk group. These results demonstrate that GEPs in MF patients correlate with their molecular and clinical features, particularly their survival, and represent the proof of concept that GEPs might provide complementary prognostic information to be applied in clinical decision making.
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http://dx.doi.org/10.1182/bloodadvances.2020003614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948267PMC
March 2021

Cytogenetic and molecular aberrations and worse outcome for male patients in systemic mastocytosis.

Theranostics 2021 1;11(1):292-303. Epub 2021 Jan 1.

Department of Dermatology and Venereology, Allergy Centrr, Kepler University Hospital, Linz, Austria.

In systemic mastocytosis (SM), the clinical features and survival vary greatly. Patient-related factors determining the outcome in SM are largely unknown. We examined the impact of sex on the clinical features, progression-free survival (PFS), and overall survival (OS) in 3403 patients with mastocytosis collected in the registry of the European Competence Network on Mastocytosis (ECNM). The impact of cytogenetic and molecular genetic aberrations on sex differences was analyzed in a subset of patients. Of all patients enrolled, 55.3% were females. However, a male predominance was found in a subset of advanced SM (AdvSM) patients, namely SM with an associated hematologic neoplasm (SM-AHN, 70%; 0.001). Correspondingly, organomegaly (male: 23% female: 13%, 0.007) was more, whereas skin involvement (male: 71% female: 86%, 0.001) was less frequent in males. In all patients together, OS ( 0.0001) was significantly inferior in males, and also within the WHO sub-categories indolent SM, aggressive SM (ASM) and SM-AHN. PFS was significantly ( 0.0002) worse in males when all patients were grouped together; due to low numbers of events, this significance persisted only in the subcategory smoldering SM. Finally, prognostically relevant cytogenetic abnormalities (10% 5%, 0.006) or molecular aberrations (// profile; 63% 40%, 0.003) were more frequently present in males. Male sex has a major impact on clinical features, disease progression, and survival in mastocytosis. Male patients have an inferior survival, which seems related to the fact that they more frequently develop a multi-mutated AdvSM associated with a high-risk molecular background.
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http://dx.doi.org/10.7150/thno.51872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681091PMC
August 2021

Multicenter Next-Generation Sequencing Studies between Theory and Practice: Harmonization of Data Analysis Using Real-World Myelodysplastic Syndrome Data.

J Mol Diagn 2021 03 23;23(3):347-357. Epub 2020 Dec 23.

Institute of Medical Informatics, University of Münster, Münster, Germany.

In the age of personalized medicine, genetic testing by means of targeted sequencing has taken a key role. However, when comparing different sets of targeted sequencing data, these are often characterized by a considerable lack of harmonization. Laboratories follow their own best practices, analyzing their own target regions. The question on how to best integrate data from different sites remains unanswered. Studying the example of myelodysplastic syndrome (MDS), we analyzed 11 targeted sequencing sets, collected from six different centers (n = 831). An intersecting target region of 43,076 bp (30 genes) was identified; whereas, the original target regions covered up to 499,097 bp (117 genes). Considering a region of interest in the context of MDS, a target region of 55,969 bp (31 genes) was identified. For each gene, coverage and sequencing data quality was evaluated, calculating a sequencing score. Analyses revealed huge differences between different data sets as well as between different genes. Analysis of the relation between sequencing score and mutation frequency in MDS revealed that most genes with high frequency in MDS could be sequenced without expecting low coverage or quality. Still, no gene appeared consistently unproblematic for all data sets. To allow for comparable results in a multicenter setting analyzing MDS, we propose to use a predefined target region of interest and to perform centralized data analysis using harmonized criteria.
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http://dx.doi.org/10.1016/j.jmoldx.2020.12.001DOI Listing
March 2021

Co-mutation pattern, clonal hierarchy, and clone size concur to determine disease phenotype of SRSF2-mutated neoplasms.

Leukemia 2021 08 21;35(8):2371-2381. Epub 2020 Dec 21.

Department of Molecular Medicine, University of Pavia, Pavia, Italy.

Somatic mutations in splicing factor genes frequently occur in myeloid neoplasms. While SF3B1 mutations are associated with myelodysplastic syndromes (MDS) with ring sideroblasts, SRSF2 mutations are found in different disease categories, including MDS, myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). To identify molecular determinants of this phenotypic heterogeneity, we explored molecular and clinical features of a prospective cohort of 279 SRSF2-mutated cases selected from a population of 2663 patients with myeloid neoplasms. Median number of somatic mutations per subject was 3. Multivariate regression analysis showed associations between co-mutated genes and clinical phenotype, including JAK2 or MPL with myelofibrosis (OR = 26.9); TET2 with monocytosis (OR = 5.2); RAS-pathway genes with leukocytosis (OR = 5.1); and STAG2, RUNX1, or IDH1/2 with blast phenotype (MDS or AML) (OR = 3.4, 1.9, and 2.1, respectively). Within patients with SRSF2-JAK2 co-mutation, JAK2 dominance was invariably associated with clinical feature of MPN, whereas SRSF2 mutation was dominant in MDS/MPN. Within patients with SRSF2-TET2 co-mutation, clinical expressivity of monocytosis was positively associated with co-mutated clone size. This study provides evidence that co-mutation pattern, clone size, and hierarchy concur to determine clinical phenotype, tracing relevant genotype-phenotype associations across disease entities and giving insight on unaccountable clinical heterogeneity within current WHO classification categories.
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http://dx.doi.org/10.1038/s41375-020-01106-zDOI Listing
August 2021

Atomic-Level Mechanism of Pre-mRNA Splicing in Health and Disease.

Acc Chem Res 2021 01 14;54(1):144-154. Epub 2020 Dec 14.

CNR-IOM-Democritos National Simulation Center c/o SISSA, Trieste 34136, Italy.

Intron removal from premature-mRNA (pre-mRNA splicing) is an essential part of gene expression and regulation that is required for the production of mature, protein-coding mRNA. The spliceosome (SPL), a majestic machine composed of five small nuclear RNAs and hundreds of proteins, behaves as an eminent transcriptome tailor, efficiently performing splicing as a protein-directed metallo-ribozyme. To select and excise long and diverse intronic sequences with single-nucleotide precision, the SPL undergoes a continuous compositional and conformational remodeling, forming eight distinct complexes throughout each splicing cycle. Splicing fidelity is of paramount importance to preserve the integrity of the proteome. Mutations in splicing factors can severely compromise the accuracy of this machinery, leading to aberrant splicing and altered gene expression. Decades of biochemical and genetic studies have provided insights into the SPL's composition and function, but its complexity and plasticity have prevented an in-depth mechanistic understanding. Single-particle cryogenic electron microscopy techniques have ushered in a new era for comprehending eukaryotic gene regulation, providing several near-atomic resolution structures of the SPL from yeast and humans. Nevertheless, these structures represent isolated snapshots of the splicing process and are insufficient to exhaustively assess the function of each SPL component and to unravel particular facets of the splicing mechanism in a dynamic environment.In this Account, building upon our contributions in this field, we discuss the role of biomolecular simulations in uncovering the mechanistic intricacies of eukaryotic splicing in health and disease. Specifically, we showcase previous applications to illustrate the role of atomic-level simulations in elucidating the function of specific proteins involved in the architectural reorganization of the SPL along the splicing cycle. Moreover, molecular dynamics applications have uniquely contributed to decrypting the channels of communication required for critical functional transitions of the SPL assemblies. They have also shed light on the role of carcinogenic mutations in the faithful selection of key intronic regions and the molecular mechanism of splicing modulators. Additionally, we emphasize the role of quantum-classical molecular dynamics in unraveling the chemical details of pre-mRNA cleavage in the SPL and in its evolutionary ancestors, group II intron ribozymes. We discuss methodological pitfalls of multiscale calculations currently used to dissect the splicing mechanism, presenting future challenges in this field. The results highlight how atomic-level simulations can enrich the interpretation of experimental results. We envision that the synergy between computational and experimental approaches will aid in developing innovative therapeutic strategies and revolutionary gene modulation tools to fight the over 200 human diseases associated with splicing misregulation, including cancer and neurodegeneration.
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http://dx.doi.org/10.1021/acs.accounts.0c00578DOI Listing
January 2021

Guideline-based indicators for adult patients with myelodysplastic syndromes.

Blood Adv 2020 08;4(16):4029-4044

Department of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.

Myelodysplastic syndromes (MDSs) represent a heterogeneous group of hematological stem cell disorders with an increasing burden on health care systems. Evidence-based MDS guidelines and recommendations (G/Rs) are published but do not necessarily translate into better quality of care if adherence is not maintained in daily clinical practice. Guideline-based indicators (GBIs) are measurable elements for the standardized assessment of quality of care and, thus far, have not been developed for adult MDS patients. To this end, we screened relevant G/Rs published between 1999 and 2018 and aggregated all available information as candidate GBIs into a formalized handbook as the basis for the subsequent consensus rating procedure. An international multidisciplinary expert panel group (EPG) of acknowledged MDS experts (n = 17), health professionals (n = 7), and patient advocates (n = 5) was appointed. The EPG feedback rates for the first and second round were 82% (23 of 28) and 96% (26 of 27), respectively. A final set of 29 GBIs for the 3 domains of diagnosis (n = 14), therapy (n = 8), and provider/infrastructural characteristics (n = 7) achieved the predefined agreement score for selection (>70%). We identified shortcomings in standardization of patient-reported outcomes, toxicity, and geriatric assessments that need to be optimized in the future. Our GBIs represent the first comprehensive consensus on measurable elements addressing best practice performance, outcomes, and structural resources. They can be used as a standardized instrument with the goal of assessing, comparing, and fostering good quality of care within clinical development cycles in the daily care of adult MDS patients.
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http://dx.doi.org/10.1182/bloodadvances.2020002314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448599PMC
August 2020

Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes.

Nat Med 2020 10 3;26(10):1549-1556. Epub 2020 Aug 3.

Department of Hematology, Democritus University of Thrace Medical School, Alexandroupolis, Greece.

Tumor protein p53 (TP53) is the most frequently mutated gene in cancer. In patients with myelodysplastic syndromes (MDS), TP53 mutations are associated with high-risk disease, rapid transformation to acute myeloid leukemia (AML), resistance to conventional therapies and dismal outcomes. Consistent with the tumor-suppressive role of TP53, patients harbor both mono- and biallelic mutations. However, the biological and clinical implications of TP53 allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS for TP53 mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third of TP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R). Surprisingly, monoallelic patients did not differ from TP53 wild-type patients in outcomes and response to therapy. This study shows that consideration of TP53 allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response.
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http://dx.doi.org/10.1038/s41591-020-1008-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8381722PMC
October 2020

Combined loss of function of two different loci of miR-15/16 drives the pathogenesis of acute myeloid leukemia.

Proc Natl Acad Sci U S A 2020 06 18;117(22):12332-12340. Epub 2020 May 18.

Department of Cancer Biology and Genetics, The Ohio State University, Columbus, OH 43210;

Double knockout of the two miR-15/16 loci in mouse resulted in the development of acute myeloid leukemia (AML). This result suggested that, at least, a fraction of human AMLs could be due to a similar mechanism. We analyzed the role of the two miR-15/16 clusters in 93 myelodysplastic syndrome (MDS) patients divided in three subgroups: patients with MDS, patients with MDS before transforming into AML (MDS-T), and patients with AML evolving from MDS (MDS-AML). Then, we tested 139 AML cases and 14 different AML cell lines by assessing microRNA (miRNA) expression, target protein expression, genetic loss, and silencing. MDS-T and MDS-AML patients show a reduction of the expression of miR-15a/-15b/-16 compared to MDS patients. Each miRNA can significantly predict MDS and MDS-T groups. Then, 79% of primary AMLs show a reduced expression of miR-15a and/or miR-15b. The expression of miR-15a/-15b/-16 significantly stratified AML patients in two prognostic classes. Furthermore, 40% of AML cell lines showed a combined loss of the expression of miR-15a/-15b and overexpression of their direct/indirect targets. As potential mechanisms involved in the silencing of the two miR-15/16 loci, we identified a genetic loss of miR-15a and miR-15b and silencing of these two loci by methylation. We identified a potential driver oncogenic role in the loss of expression of both miR-15/16 clusters in the progression of MDS into AML and in AML pathogenesis. The stratification of AML patients, based on miR-15/16 expression, can lead to targeted and combination therapies for the treatment of this incurable disease.
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http://dx.doi.org/10.1073/pnas.2003597117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275703PMC
June 2020

SF3B1-mutant MDS as a distinct disease subtype: a proposal from the International Working Group for the Prognosis of MDS.

Blood 2020 07;136(2):157-170

Amsterdam University Medical Center, Amsterdam, The Netherlands.

The 2016 revision of the World Health Organization classification of tumors of hematopoietic and lymphoid tissues is characterized by a closer integration of morphology and molecular genetics. Notwithstanding, the myelodysplastic syndrome (MDS) with isolated del(5q) remains so far the only MDS subtype defined by a genetic abnormality. Approximately half of MDS patients carry somatic mutations in spliceosome genes, with SF3B1 being the most commonly mutated one. SF3B1 mutation identifies a condition characterized by ring sideroblasts (RS), ineffective erythropoiesis, and indolent clinical course. A large body of evidence supports recognition of SF3B1-mutant MDS as a distinct nosologic entity. To further validate this notion, we interrogated the data set of the International Working Group for the Prognosis of MDS (IWG-PM). Based on the findings of our analyses, we propose the following diagnostic criteria for SF3B1-mutant MDS: (1) cytopenia as defined by standard hematologic values, (2) somatic SF3B1 mutation, (3) morphologic dysplasia (with or without RS), and (4) bone marrow blasts <5% and peripheral blood blasts <1%. Selected concomitant genetic lesions represent exclusion criteria for the proposed entity. In patients with clonal cytopenia of undetermined significance, SF3B1 mutation is almost invariably associated with subsequent development of overt MDS with RS, suggesting that this genetic lesion might provide presumptive evidence of MDS in the setting of persistent unexplained cytopenia. Diagnosis of SF3B1-mutant MDS has considerable clinical implications in terms of risk stratification and therapeutic decision making. In fact, this condition has a relatively good prognosis and may respond to luspatercept with abolishment of the transfusion requirement.
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http://dx.doi.org/10.1182/blood.2020004850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362582PMC
July 2020

Loss of lenalidomide-induced megakaryocytic differentiation leads to therapy resistance in del(5q) myelodysplastic syndrome.

Nat Cell Biol 2020 05 6;22(5):526-533. Epub 2020 Apr 6.

Michael Smith Genome Sciences Centre, BC Cancer Research Centre, Vancouver, British Columbia, Canada.

Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common structural genomic variant in myelodysplastic syndromes (MDS). Lenalidomide (LEN) is the treatment of choice for patients with del(5q) MDS, but half of the responding patients become resistant within 2 years. TP53 mutations are detected in ~20% of LEN-resistant patients. Here we show that patients who become resistant to LEN harbour recurrent variants of TP53 or RUNX1. LEN upregulated RUNX1 protein and function in a CRBN- and TP53-dependent manner in del(5q) cells, and mutation or downregulation of RUNX1 rendered cells resistant to LEN. LEN induced megakaryocytic differentiation of del(5q) cells followed by cell death that was dependent on calpain activation and CSNK1A1 degradation. We also identified GATA2 as a LEN-responsive gene that is required for LEN-induced megakaryocyte differentiation. Megakaryocytic gene-promoter analyses suggested that LEN-induced degradation of IKZF1 enables a RUNX1-GATA2 complex to drive megakaryocytic differentiation. Overexpression of GATA2 restored LEN sensitivity in the context of RUNX1 or TP53 mutations by enhancing LEN-induced megakaryocytic differentiation. Screening for mutations that block LEN-induced megakaryocytic differentiation should identify patients who are resistant to LEN.
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http://dx.doi.org/10.1038/s41556-020-0497-9DOI Listing
May 2020

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

Cancer Discov 2020 06 5;10(6):836-853. Epub 2020 Apr 5.

Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including . However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in -cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency..
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http://dx.doi.org/10.1158/2159-8290.CD-19-0982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269820PMC
June 2020

Mutational spectrum and dynamics of clonal hematopoiesis in anemia of older individuals.

Blood 2020 04;135(14):1161-1170

Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Anemia is a major and currently poorly understood clinical manifestation of hematopoietic aging. Upon aging, hematopoietic clones harboring acquired leukemia-associated mutations expand and become detectable, now referred to as clonal hematopoiesis (CH). To investigate the relationship between CH and anemia of the elderly, we explored the landscape and dynamics of CH in older individuals with anemia. From the prospective, population-based Lifelines cohort (n = 167 729), we selected all individuals at least 60 years old who have anemia according to World Health Organization criteria (n = 676) and 1:1 matched control participants. Peripheral blood of 1298 individuals was analyzed for acquired mutations at a variant allele frequency (VAF) of 1% or higher in 27 driver genes. To track clonal evolution over time, we included all available follow-up samples (n = 943). CH was more frequently detected in individuals with anemia (46.6%) compared with control individuals (39.1%; P = .007). Although no differences were observed regarding commonly detected DTA mutations (DNMT3A, TET2, ASXL1) in individuals with anemia compared with control individuals, other mutations were enriched in the anemia cohort, including TP53 and SF3B1. Unlike individuals with nutrient deficiency (P = .84), individuals with anemia of chronic inflammation and unexplained anemia revealed a higher prevalence of CH (P = .035 and P = .017, respectively) compared with their matched control individuals. Follow-up analyses revealed that clones may expand and decline, generally showing only a subtle increase in VAF (mean, 0.56%) over the course of 44 months, irrespective of the presence of anemia. Specific mutations were associated with different growth rates and propensities to acquire an additional hit. In contrast to smaller clones (<5% VAF), which did not affect overall survival, larger clones were associated with increased risk for death.
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http://dx.doi.org/10.1182/blood.2019004362DOI Listing
April 2020

Clinical features and survival of patients with indolent systemic mastocytosis defined by the updated WHO classification.

Allergy 2020 08 16;75(8):1927-1938. Epub 2020 Mar 16.

Departement of Hematology, Centre National de Référence des Mastocytoses, Imagine Institute, INSERM U1123, Université Paris Descartes, Sorbonne, Paris Cité, Hôpital Necker, Assistance Publique des Hôpitaux de Paris (APHP), Paris, France.

Background: In indolent systemic mastocytosis (ISM), several risk factors of disease progression have been identified. Previous studies, performed with limited patient numbers, have also shown that the clinical course in ISM is stable and comparable to that of cutaneous mastocytosis (CM). The aim of this project was to compare the prognosis of patients with ISM with that of patients with CM.

Methods: We employed a dataset of 1993 patients from the registry of the European Competence Network on Mastocytosis (ECNM) to compare outcomes of ISM and CM.

Results: We found that overall survival (OS) is worse in ISM compared to CM. Moreover, in patients with typical ISM, bone marrow mastocytosis (BMM), and smoldering SM (SSM), 4.1% of disease progressions have been observed (4.9% of progressions in typical ISM group, 1.7% in BMM, and 9.4% in SSM). Progressions to advanced SM were observed in 2.9% of these patients. In contrast, six patients with CM (1.7%) converted to ISM and no definitive progression to advanced SM was found. No significant differences in OS and event-free survival (EFS) were found when comparing ISM, BMM, and SSM. Higher risk of both progression and death was significantly associated with male gender, worse performance status, and organomegaly.

Conclusion: Our data confirm the clinical impact of the WHO classification that separates ISM from CM and from other SM variants.
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http://dx.doi.org/10.1111/all.14248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115854PMC
August 2020
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