Mis-splicing of mitotic regulators sensitizes SF3B1-mutated human HSCs to CHK1 inhibition.

Splicing factor SF3B1 mutations are frequent somatic lesions in myeloid neoplasms that transform hematopoietic stem cells (HSCs) by inducing mis-splicing of target genes. However, the molecular and functional consequences of SF3B1 mutations in human HSCs remain unclear. Here, we identify the mis-splicing program in human HSCs as a targetable vulnerability by precise gene editing of SF3B1 K700E mutations in primary CD34+ cells. Mutant SF3B1 induced pervasive mis-splicing and reduced expression of genes regulating mitosis and genome maintenance leading to altered differentiation, delayed G2/M progression, and profound sensitivity to CHK1 inhibition (CHK1i). Mis-splicing or reduced expression of mitotic regulators BUBR1 and CDC27 delayed G2/M transit and promoted CHK1i sensitivity. Clinical CHK1i prexasertib selectively targeted SF3B1-mutant HSCs and abrogated engraftment in vivo. These findings identify mis-splicing of mitotic regulators in SF3B1-mutant HSCs as a targetable vulnerability engaged by pharmacological CHK1 inhibition.

Early transfusion patterns improve the Molecular International Prognostic Scoring System (IPSS-M) prediction in myelodysplastic syndromes.

BACKGROUND: The Molecular International Prognostic Scoring System (IPSS-M) is the new gold standard for diagnostic outcome prediction in patients with myelodysplastic syndromes (MDS). This study was designed to assess the additive prognostic impact of dynamic transfusion parameters during early follow-up. METHODS: We retrieved complete transfusion data from 677 adult Swedish MDS patients included in the IPSS-M cohort. Time-dependent erythrocyte transfusion dependency (E-TD) was added to IPSS-M features and analyzed regarding overall survival and leukemic transformation (acute myeloid leukemia). A multistate Markov model was applied to assess the prognostic value of early changes in transfusion patterns. RESULTS: Specific clinical and genetic features were predicted for diagnostic and time-dependent transfusion patterns. Importantly, transfusion state both at diagnosis and within the first year strongly predicts outcomes in both lower (LR) and higher-risk (HR) MDSs. In multivariable analysis, 8-month landmark E-TD predicted shorter survival independently of IPSS-M (p < 0.001). A predictive model based on IPSS-M and 8-month landmark E-TD performed significantly better than a model including only IPSS-M. Similar trends were observed in an independent validation cohort (n = 218). Early transfusion patterns impacted both future transfusion requirements and outcomes in a multistate Markov model. CONCLUSION: The transfusion requirement is a robust and available clinical parameter incorporating the effects of first-line management. In MDS, it provides dynamic risk information independently of diagnostic IPSS-M and, in particular, clinical guidance to LR MDS patients eligible for potentially curative therapeutic intervention.

In-vitro dehydration kinetics coefficient of Kalifilcon A and other contact lens materials.

In contact lens (CL) wear, dehydration needs to be tailored to avoid dryness and related symptoms. In this view, this work aims to assess and compare the in-vitro dehydration kinetics of five CL materials, including the newly developed Kalifilcon A CL. At 36 °C and 60% relative humidity, the in-vitro dehydration kinetics of the different CLs were compared using a gravimetric method. CLs were analyzed either after a rinse of a few seconds in preservative-free saline solution or after a 24-h incubation in the same solution. A model based on the Fick diffusion equation was employed to deduce a water kinetics coefficient, providing insights into water diffusion within the polymeric matrix. The study reveals that all materials exhibit a non-Fickian dehydration behavior, with significant differences in dehydration kinetics coefficients and dehydration rate slopes. Etafilcon A and Omafilcon A, both hydrogel CLs, exhibit a similar behavior, different compared to the pattern shown by Senofilcon A and Delefilcon A, silicone-hydrogel CLs. Notably, Kalifilcon A, despite being a silicone-hydrogel, displays a hydration behavior reminiscent of hydrogel CLs.

Prevalence and clinical expression of germ line predisposition to myeloid neoplasms in adults with marrow hypocellularity.

Systematic studies of germ line genetic predisposition to myeloid neoplasms in adult patients are still limited. In this work, we performed germ line and somatic targeted sequencing in a cohort of adult patients with hypoplastic bone marrow (BM) to study germ line predisposition variants and their clinical correlates. The study population included 402 consecutive adult patients investigated for unexplained cytopenia and reduced age-adjusted BM cellularity. Germ line mutation analysis was performed using a panel of 60 genes, and variants were interpreted per the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines; somatic mutation analysis was performed using a panel of 54 genes. Of the 402 patients, 27 (6.7%) carried germ line variants that caused a predisposition syndrome/disorder. The most frequent disorders were DDX41-associated predisposition, Fanconi anemia, GATA2-deficiency syndrome, severe congenital neutropenia, RASopathy, and Diamond-Blackfan anemia. Eighteen of 27 patients (67%) with causative germ line genotype were diagnosed with myeloid neoplasm, and the remaining with cytopenia of undetermined significance. Patients with a predisposition syndrome/disorder were younger than the remaining patients and had a higher risk of severe or multiple cytopenias and advanced myeloid malignancy. In patients with myeloid neoplasm, causative germ line mutations were associated with increased risk of progression into acute myeloid leukemia. Family or personal history of cancer did not show significant association with a predisposition syndrome/disorder. The findings of this study unveil the spectrum, clinical expressivity, and prevalence of germ line predisposition mutations in an unselected cohort of adult patients with cytopenia and hypoplastic BM.

Haematological malignancies in relatives of patients affected with myeloproliferative neoplasms.

In a cohort of 3131 patients with myeloproliferative neoplasms (MPNs), we identified 200 patients (6.4%) who reported a second case of haematological malignancies (HM) in first- or second-degree relatives. The occurrence of a second HM in the family was not influenced by MPN subtype, sex or driver mutation, while it was associated with age at MPN diagnosis: 8.5% of patients diagnosed with MPN younger than 45 years had a second relative affected with HM compared to 5.5% of those diagnosed at the age of 45 years or older (p = 0.003), thus suggesting a genetic predisposition to HM with early onset.

Rare variants in Toll-like receptor 7 results in functional impairment and downregulation of cytokine-mediated signaling in COVID-19 patients.

Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.

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

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.

Enrichment of Double RUNX1 Mutations in Acute Leukemias of Ambiguous Lineage.

Acute leukemia of ambiguous lineage (ALAL) is a rare type of leukemia and represents an unmet clinical need. In fact, due to heterogeneity, substantial rarity and absence of clinical trials, there are no therapeutic guidelines available. We investigated the genetic basis of 10 cases of ALAL diagnosed at our centre from 2008 and 2020, through a targeted myeloid and lymphoid sequencing approach. We show that this rare group of acute leukemias is enriched in myeloid-gene mutations. In particular we found that RUNX1 mutations, which have been found double mutated in 40% of patients and tend to involve both alleles, are associated with an undifferentiated phenotype and with lineage ambiguity. Furthermore, because this feature is typical of acute myeloid leukemia with minimal differentiation, we believe that our data strengthen the idea that acute leukemia with ambiguous lineage, especially those with an undifferentiated phenotype, might be genetically more closer to acute myeloid leukemia rather than acute lymphoblastic leukemia. These data enrich the knowledge on the genetic basis of ALAL and could have clinical implications as an acute myeloid leukemia (AML) - oriented chemotherapeutic approach might be more appropriate.

ZBTB33 is mutated in clonal hematopoiesis and myelodysplastic syndromes and impacts RNA splicing.

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, ZBTB33, as well as in YLPM1, SRCAP, and ZNF318. We also identified these mutations at low frequency in myelodysplastic syndrome patients. Zbtb33 edited mouse hematopoietic stem and progenitor cells exhibited a competitive advantage in vivo and increased genome-wide intron retention. ZBTB33 mutations potentially link DNA methylation and RNA splicing, the two most commonly mutated pathways in clonal hematopoiesis and MDS.

Dysphagic disorder in a cohort of COVID-19 patients: Evaluation and evolution.

BACKGROUND: COVID-19 is a multisystem disease complicated by respiratory failure requiring sustanined mechanical ventilation (MV). Prolongued oro-tracheal intubation is associated to an increased risk of dysphagia and bronchial aspiration. Purpose of this study was to investigate swallowing disorders in critically ill COVID-19 patients. MATERIAL AND METHODS: This was a retrospective study analysing a consecutive cohort of COVID-19 patients admitted to the Intensive Care Unit (ICU) of our hospital. Data concerning dysphagia were collected according to the Gugging Swallowing Screen (GUSS) and related to demographic characteristics, clinical data, ICU Length-Of-Stay (LOS) and MV parameters. RESULTS: From March 2 to April 30, 2020, 31 consecutive critically ill COVID-19 patients admitted to ICU were evaluated by speech and language therapists (SLT). Twenty-five of them were on MV (61% through endotracheal tube and 19% through tracheostomy); median MV length was 11 days. Seventeen (54.8%) patients presented dysphagia; a correlation was found between first GUSS severity stratification and MV days (p < 0.001), ICU LOS (p < 0.001), age (p = 0.03) and tracheostomy (p = 0.042). No other correlations were found. At 16 days, 90% of patients had fully recovered; a significant improvement was registered especially during the first week (p < 0.001). CONCLUSION: Compared to non-COVID-19 patiens, a higher rate of dysphagia was reported in COVID-19 patients, with a more rapid and complete recovery. A systematic early SLT evaluation of COVID-19 patients on MV may thus be useful to prevent dysphagia-related complications.

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

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.

Applying Hyperspectral Reflectance Imaging to Investigate the Palettes and the Techniques of Painters.

Reflectance Spectroscopy (RS) and Fiber Optics Reflectance Spectroscopy (FORS) are well-established techniques for the investigation of works of art with particular attention to paintings. Most modern museums put at the disposal of their research groups portable equipment that, together with the intrinsic non-invasiveness of RS and FORS, makes possible the in situ collection of reflectance spectra from the surface of artefacts. The comparison, performed by experts in pigments and painting materials, of the experimental data with databases of reference spectra drives the characterization of the palettes and of the techniques used by the artists. However, this approach requires specific skills and it is time consuming especially if the number of the spectra to be investigated becomes large as is the case of Hyperspectral Reflectance Imaging (HRI) datasets. The HRI experimental setups are multi-dimensional cameras that associate the spectral information, given by the reflectance spectra, with the spatial localization of the spectra over the painted surface. The resulting datasets are 3D-cubes (called hypercubes or data-cubes) where the first two dimensions locate the spectrum over the painting and the third is the spectrum itself (i.e., the reflectance of that point of the painted surface versus the wavelength in the operative range of the detector). The capability of the detector to simultaneously collect a great number of spectra (typically much more than 10,000 for each hypercube) makes the HRI datasets large reservoirs of information and justifies the need for the development of robust and, possibly, automated protocols to analyze the data. After the description of the procedure designed for the data acquisition, we present an analysis method that systematically exploits the potential of the hypercubes. Based on Spectral Angle Mapper (SAM) and on the manipulation of the collected spectra, the algorithm handles and analyzes thousands of spectra while at the same time it supports the user to unveil the features of the samples under investigation. The power of the approach is illustrated by applying it to Quarto Stato, the iconic masterpiece by Giuseppe Pellizza da Volpedo, held in the Museo del Novecento in Milan (Italy).

Relationship between clone metrics and clinical outcome in clonal cytopenia.

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.

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

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.

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

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-ß) 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.

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

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.

Co-mutation pattern, clonal hierarchy, and clone size concur to determine disease phenotype of SRSF2P95-mutated neoplasms.

Somatic mutations in splicing factor genes frequently occur in myeloid neoplasms. While SF3B1 mutations are associated with myelodysplastic syndromes (MDS) with ring sideroblasts, SRSF2P95 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 SRSF2P95-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.

Behind the Scene of "The Holy Family with St. Anne and the Young St. John" by Bernardino Luini: A Computer-Assisted Method to Unveil the Underdrawings.

Uncovering the underdrawings (UDs), the preliminary sketch made by the painter on the grounded preparatory support, is a keystone for understanding the painting's history including the original project of the artist, the pentimenti (an underlying image in a painting providing evidence of revision by the artist) or the possible presence of co-workers' contributions. The application of infrared reflectography (IRR) has made the dream of discovering the UDs come true: since its introduction, there has been a growing interest in the technology, which therefore has evolved leading to advanced instruments. Most of the literature either report on the technological advances in IRR devices or present case studies, but a straightforward method to improve the visibility of the UDs has not been presented yet. Most of the data handling methods are devoted to a specific painting or they are not user-friendly enough to be applied by non-specialized users, hampering, thus, their widespread application in areas other than the scientific one, e.g., in the art history field. We developed a computer-assisted method, based on principal component analysis (PCA) and image processing, to enhance the visibility of UDs and to support the art-historians and curators' work. Based on ImageJ/Fiji, one of the most widespread image analysis software, the algorithm is very easy to use and, in principle, can be applied to any multi- or hyper-spectral image data set. In the present paper, after describing the method, we accurately present the extraction of the UD for the panel "The Holy Family with St. Anne and the Young St. John" and for other four paintings by Luini and his workshop paying particular attention to the painting known as "The Child with the Lamb".