Comparing malignant monocytosis across the updated WHO and ICC classifications of 2022.

ABSTRACT: The World Health Organization (WHO) classification of hematolymphoid tumors and the International Consensus Classification (ICC) of 2022 introduced major changes to the definition of chronic myelomonocytic leukemia (CMML). To assess its qualitative and quantitative implications for patient care, we started with 3311 established CMML cases (according to WHO 2017 criteria) and included 2130 oligomonocytosis cases fulfilling the new CMML diagnostic criteria. Applying both 2022 classification systems, 356 and 241 of oligomonocytosis cases were newly classified as myelodysplastic (MD)-CMML (WHO and ICC 2022, respectively), most of which were diagnosed as myelodysplastic syndrome (MDS) according to the WHO 2017 classification. Importantly, 1.5 times more oligomonocytosis cases were classified as CMML according to WHO 2022 than based on ICC, because of different diagnostic criteria. Genetic analyses of the newly classified CMML cases showed a distinct mutational profile with strong enrichment of MDS-typical alterations, resulting in a transcriptional subgroup separated from established MD and myeloproliferative CMML. Despite a different cytogenetic, molecular, immunophenotypic, and transcriptional landscape, no differences in overall survival were found between newly classified and established MD-CMML cases. To the best of our knowledge, this study represents the most comprehensive analysis of routine CMML cases to date, both in terms of clinical characterization and transcriptomic analysis, placing newly classified CMML cases on a disease continuum between MDS and previously established CMML.

Germ line variant GFI1-36N affects DNA repair and sensitizes AML cells to DNA damage and repair therapy.

ABSTRACT: Growth factor independence 1 (GFI1) is a DNA-binding transcription factor and a key regulator of hematopoiesis. GFI1-36N is a germ line variant, causing a change of serine (S) to asparagine (N) at position 36. We previously reported that the GFI1-36N allele has a prevalence of 10% to 15% among patients with acute myeloid leukemia (AML) and 5% to 7% among healthy Caucasians and promotes the development of this disease. Using a multiomics approach, we show here that GFI1-36N expression is associated with increased frequencies of chromosomal aberrations, mutational burden, and mutational signatures in both murine and human AML and impedes homologous recombination (HR)-directed DNA repair in leukemic cells. GFI1-36N exhibits impaired binding to N-Myc downstream-regulated gene 1 (Ndrg1) regulatory elements, causing decreased NDRG1 levels, which leads to a reduction of O6-methylguanine-DNA-methyltransferase (MGMT) expression levels, as illustrated by both transcriptome and proteome analyses. Targeting MGMT via temozolomide, a DNA alkylating drug, and HR via olaparib, a poly-ADP ribose polymerase 1 inhibitor, caused synthetic lethality in human and murine AML samples expressing GFI1-36N, whereas the effects were insignificant in nonmalignant GFI1-36S or GFI1-36N cells. In addition, mice that received transplantation with GFI1-36N leukemic cells treated with a combination of temozolomide and olaparib had significantly longer AML-free survival than mice that received transplantation with GFI1-36S leukemic cells. This suggests that reduced MGMT expression leaves GFI1-36N leukemic cells particularly vulnerable to DNA damage initiating chemotherapeutics. Our data provide critical insights into novel options to treat patients with AML carrying the GFI1-36N variant.

An Efficient Electron Ptychography Method for Retrieving the Object Spectrum from Only a Few Iterations.

We present an efficient approach for electron ptychography based on a mathematical relationship that differs from that underlying the established algorithms of the ptychography iterative engine or the noniterative algorithms like the Wigner-distribution-deconvolution or the single-side-band method. Three variables are handled in this method-the transfer function of the objective lens, the object spectrum, and the diffraction wave whose phase is unknown. In the case of an aberration-corrected electron microscope, one is able to obtain a well-estimated transfer function of the lens. After reducing the number of three variables down to two, we construct an iterative loop between the object spectrum and the diffraction wave, which retrieves the object spectrum within a small number of iterations. We tested this object spectrum retrieval method on both a calculated and an experimental 4D-STEM datasets. By applying this method, we explore the influence of sampling, dose, and the size of illumination aperture on the reconstructed phase images.

Nintedanib in Asian patients with progressive fibrosing interstitial lung diseases: Results from the INBUILD trial.

BACKGROUND AND OBJECTIVE: In the INBUILD trial in patients with progressive fibrosing interstitial lung diseases (ILDs), nintedanib reduced the rate of decline in forced vital capacity (FVC) with an adverse event profile characterized mainly by gastrointestinal events. We analysed the effects of nintedanib in the subset of Asian subjects. METHODS: Subjects with fibrosing ILDs other than idiopathic pulmonary fibrosis who had shown progression of ILD at any time within the prior 24 months despite management deemed appropriate in clinical practice were randomized to receive nintedanib or placebo. We analysed the rate of decline in FVC (ml/year) over 52 weeks in all Asian subjects and in Asian subjects with a usual interstitial pneumonia (UIP)-like fibrotic pattern on high-resolution computed tomography (HRCT). RESULTS: One hundred sixty-four subjects in the INBUILD trial were of Asian race. The rate of decline in FVC (ml/year) over 52 weeks in this subgroup was -116.8 in the nintedanib group and -207.9 in the placebo group (difference: 91.0 [95% CI: 8.1, 173.9]; nominal p = 0.03). In Asian subjects with a UIP-like fibrotic pattern on HRCT, the rate of decline in FVC (ml/year) over 52 weeks was -130.1 in the nintedanib group and -224.2 in the placebo group (difference: 94.1 [5.5, 182.7]; nominal p = 0.04). Adverse events led to treatment discontinuation in 19.0% of the nintedanib group and 13.8% of the placebo group. CONCLUSION: In Asian patients with progressive fibrosing ILDs, nintedanib reduced the rate of decline in FVC with adverse events that were manageable for most patients.

Focal cavity radiotherapy after neurosurgical resection of brain metastases: sparing neurotoxicity without compromising locoregional control.

PURPOSE: Does focal cavity radiotherapy after resection of brain metastasis "spare" whole-brain radiotherapy, which is associated with toxicity for patients, through the complete course of their disease without compromising long-term local control of the brain? METHODS: We retrospectively analyzed outcomes of patients who underwent adjuvant focal cavity radiotherapy between 2014 and 2021 at our center. RESULTS: A total of 83 patients with 86 resected brain metastases were analyzed. 64% had singular, 36% two to four brain metastases. In cases with multiple metastases, omitted lesions were treated with radiosurgery. Median follow-up was 7.3 months (range 0-71.2 months), 1­year overall survival rate was 57.8% (95% CI 44.9-68.8%). Radiotherapy was administered with a median biologically effective dose (α/ß 10) surrounding the planning target volume of 48 Gy (range 23.4-60 Gy). Estimated 1­year local control rate was 82.7% (95% CI 67.7-91.2%), estimated 1­year distant brain control rate was 55.7% (95% CI 40.5-68.4%), estimated 1­year leptomeningeal disease rate was 16.0% (95% CI 7.3-32.9%). Eleven distant brain recurrences could be salvaged with radiosurgery. In the further course of disease, 14 patients (17%) developed disseminated metastatic disease in the brain. Estimated 1­year free of whole-brain radiotherapy rate was 72.3% (95% CI 57.1-82.9%). All applied treatments led to an estimated 1­year neuro-control rate of 79.1% (95% CI 65.0-88.0%), estimated 1­year radionecrosis rate was 23% (95% CI 12.4-40.5%). CONCLUSION: In our single-center study, focal cavity radiotherapy was associated with high local control. In three out of four patients, whole-brain radiotherapy could be avoided in the complete course of disease, using radiosurgery as salvage approach without compromising neuro-control.

Intraoperative radiotherapy with low-energy x-rays after neurosurgical resection of brain metastases-an Augsburg University Medical Center experience.

PURPOSE: External-beam radiotherapy (EBRT) is the predominant method for localized brain radiotherapy (LBRT) after resection of brain metastases (BM). Intraoperative radiotherapy (IORT) with 50-kV x­rays is an alternative way to focally irradiate the resection cavity after BM surgery, with the option of shortening the overall treatment time and limiting normal tissue irradiation. METHODS: We retrospectively analyzed the outcomes of all patients who underwent neurosurgical resection of BM and 50-kV x­ray IORT between 2013 and 2020 at Augsburg University Medical Center. RESULTS: We identified 40 patients with 44 resected BM treated with 50-kV x­ray IORT. Median diameter of the resected metastases was 2.8 cm (range 1.5-5.9 cm). Median applied dose was 20 Gy. All patients received standardized follow-up (FU) including 3­monthly MRI of the brain. Mean FU was 14.4 months, with a median MRI FU for alive patients of 12.2 months. Median overall survival (OS) of all treated patients was 26.4 months (estimated 1­year OS 61.6%). The observed local control (LC) rate of the resection cavity was 88.6% (estimated 1­year LC 84.3%). Distant brain control (DC) was 47.5% (estimated 1­year DC 33.5%). Only 25% of all patients needed WBI in the further course of disease. The observed radionecrosis rate was 2.5%. CONCLUSION: IORT with 50-kV x­rays is a safe and appealing way to apply LBRT after neurosurgical resection of BM, with low toxicity and excellent LC. Close MRI FU is paramount to detect distant brain failure (DBF) early.

Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis.

The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci. Recent work suggested that rather than up- and downregulating selected groups of genes, Myc targets all active promoters and enhancers in the genome (a phenomenon termed 'invasion') and acts as a general amplifier of transcription. However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations, we detected general increases in total RNA or messenger RNA copies per cell (hereby termed 'amplification') when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome, Myc does not directly act as a global transcriptional amplifier. Instead, our results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.

Degradation dynamics of microRNAs revealed by a novel pulse-chase approach.

The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2> 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2= 4-14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions.

VCF.Filter: interactive prioritization of disease-linked genetic variants from sequencing data.

Next generation sequencing is widely used to link genetic variants to diseases, and it has massively accelerated the diagnosis and characterization of rare genetic diseases. After initial bioinformatic data processing, the interactive analysis of genome, exome, and panel sequencing data typically starts from lists of genetic variants in VCF format. Medical geneticists filter and annotate these lists to identify variants that may be relevant for the disease under investigation, or to select variants that are reported in a clinical diagnostics setting. We developed VCF.Filter to facilitate the search for disease-linked variants, providing a standalone Java program with a user-friendly interface for interactive variant filtering and annotation. VCF.Filter allows the user to define a broad range of filtering criteria through a graphical interface. Common workflows such as trio analysis and cohort-based filtering are pre-configured, and more complex analyses can be performed using VCF.Filter's support for custom annotations and filtering criteria. All filtering is documented in the results file, thus providing traceability of the interactive variant prioritization. VCF.Filter is an open source tool that is freely and openly available at http://vcffilter.rarediseases.at.

Targeted mutation screening of 292 candidate genes in 38 children with inborn haematological cytopenias efficiently identifies novel disease-causing mutations.

Establishing a precise diagnosis is essential in inborn haematological cytopenias to enable appropriate treatment decisions and avoid secondary organ damage. However, both diversity and phenotypic overlap of distinct disease entities may make the identification of underlying genetic aetiologies by classical Sanger sequencing challenging. Instead of exome sequencing, we established a systematic next generation sequencing-based panel targeting 292 candidate genes and screened 38 consecutive patients for disease-associated mutations. Efficient identification of the underlying genetic cause in 17 patients (44·7%), including 13 novel mutations, demonstrates that this approach is time- and cost-efficient, enabling optimal management and genetic counselling.

[Leg ulcers (ulcus cruris): The frequent macrovascular causes]. / Ulcus cruris: Die häufigen, makrovaskulären Ursachen.

Leg ulcers (ulcus cruris): The frequent macrovascular causes Abstract. Four pathologies make up the macrovascular etiologies of leg uclers: Venous leg ulcers (50 %), mixed venous-arterial leg ulcers (20 %), arterial leg ulcers (5 %), and Martorell hypertensive ischemic leg ulcer (5 %). The remaining 20 % concern a large array of other etiologies. Every leg ulcer requires vascular (arterial and venous) work-up, that can be completed with microbiology, biopsy, and more in-depth internal diagnostics, as indicated. Venous leg ulcers are treated with compression therapy. Incompetent saphenous veins and tributaries are abolished if the deep venous system is patent. Occluded iliac veins are recanalised and stented, as possible. Refractory venous leg ulcers are grafted with split skin or punch grafts, depending on their surface. Extensive dermatolipofasciosclerosis may be tangentially removed by shave therapy or fasciectomy, that can be combined with negative pressure wound treatment (NPWT). Skin equivalents are an alternative to treat superficial venous leg ulcers that fail to epithelialise. Their indication in the treatment of more complex leg ulcers still needs to be better investigated and understood. The use of dermal matrices leads to more stable scars. Mixed venous-arterial leg ulcers heal slower and recur more frequently. Compression needs to be reduced. Refractory cases require arterial revascularisation, to transform the mixed venous-arterial into a venous leg ulcer. Arterial leg ulcers require arterial revascularization and split skin graft. Martorell hypertensive ischemic leg ulcer is still underrecognised and often confounded with with pyoderma gangrenosum, which leads therapy into a wrong direction. Necrosectomy, antibiotic treatment in the presence of relevant bacterial superinfection, and repeated split skin grafts eventually heal the vast majority of these extremely painful and potentially mortal wounds.

Genome and network visualization facilitates the analyses of the effects of drugs and mutations on protein-protein and drug-protein networks.

BACKGROUND: Biologists generally interrogate genomics data using web-based genome browsers that have limited analytical potential. New generation genome browsers such as the Integrated Genome Browser (IGB) have largely overcome this limitation and permit customized analyses to be implemented using plugins. We illustrate the use of a plugin for IGB that exploits advanced visualization techniques to integrate the analysis of genomics data with network and structural approaches. RESULTS: We show how visualization technologies that combine both genomics and network biology can facilitate the selection of the key amino acid contacts from protein-protein and protein-drug interactions. Starting from the MDM2-P53 interaction, which is a high-value target for cancer therapy, and Nutlin, the parent small molecule of an MDM2 antagonist that is currently in clinical trials, we show that this method can be generalized to analyze how drugs and mutations can interfere with both protein-protein and drug-protein networks. We illustrate this point by two additional use-cases exploring the molecular basis of tamoxifen side effects and of drug resistance in chronic myeloid leukemia patients. CONCLUSIONS: Combined network and structure biology approaches provide key insights into both the genetic and the edgetic roles of variants in diseases. 3D interactomes facilitate the identification of disease-relevant interactions that can then be specifically targeted by drugs. Recent advances in molecular interaction and structure visualization tools have greatly simplified the mapping of mutated residues to molecular interaction interfaces. Such approaches can now also be integrated with genome visualization tools to enable comparative analyses of interaction contacts.

LowMACA: exploiting protein family analysis for the identification of rare driver mutations in cancer.

BACKGROUND: The increasing availability of resequencing data has led to a better understanding of the most important genes in cancer development. Nevertheless, the mutational landscape of many tumor types is heterogeneous and encompasses a long tail of potential driver genes that are systematically excluded by currently available methods due to the low frequency of their mutations. We developed LowMACA (Low frequency Mutations Analysis via Consensus Alignment), a method that combines the mutations of various proteins sharing the same functional domains to identify conserved residues that harbor clustered mutations in multiple sequence alignments. LowMACA is designed to visualize and statistically assess potential driver genes through the identification of their mutational hotspots. RESULTS: We analyzed the Ras superfamily exploiting the known driver mutations of the trio K-N-HRAS, identifying new putative driver mutations and genes belonging to less known members of the Rho, Rab and Rheb subfamilies. Furthermore, we applied the same concept to a list of known and candidate driver genes, and observed that low confidence genes show similar patterns of mutation compared to high confidence genes of the same protein family. CONCLUSIONS: LowMACA is a software for the identification of gain-of-function mutations in putative oncogenic families, increasing the amount of information on functional domains and their possible role in cancer. In this context LowMACA emphasizes the role of genes mutated at low frequency otherwise undetectable by classical single gene analysis. LowMACA is an R package available at http://www.bioconductor.org/packages/release/bioc/html/LowMACA.html. It is also available as a GUI standalone downloadable at: https://cgsb.genomics.iit.it/wiki/projects/LowMACA.

Cross-sectional and longitudinal changes in DNA methylation with age: an epigenome-wide analysis revealing over 60 novel age-associated CpG sites.

Understanding the role of epigenetic modifications, e.g. DNA methylation, in the process of aging requires the characterization of methylation patterns in large cohorts. We analysed >480 000 CpG sites using Infinium HumanMethylation450 BeadChip (Illumina) in whole blood DNA of 965 participants of a population-based cohort study aged between 50 and 75 years. In an exploratory analysis in 400 individuals, 200 CpG sites with the highest Spearman correlation coefficients for the association between methylation and age were identified. Of these 200 CpGs, 162 were significantly associated with age, which was verified in an independent cohort of 498 individuals using mixed linear regression models adjusted for gender, smoking behaviour, age-related diseases and random batch effect and corrected for multiple testing by Bonferroni. In another independent cohort of 67 individuals without history of major age-related diseases and with a follow-up of 8 years, we observed a gain in methylation at 96% (52%, significant) of the positively age-associated CpGs and a loss at all (89%, significant) of the negatively age-associated CpGs in each individual while getting 8 years older. A regression model for age prediction based on 17 CpGs as predicting variables explained 71% of the variance in age with an average accuracy of 2.6 years. In comparison with cord blood samples obtained from the Ulm Birth Cohort Study, we observed a more than 2-fold change in mean methylation levels from birth to older age at 86 CpGs. We were able to identify 65 novel CpG sites with significant association of methylation with age.

The MI bundle: enabling network and structural biology in genome visualization tools.

UNLABELLED: Prioritization of candidate genes emanating from large-scale screens requires integrated analyses at the genomics, molecular, network and structural biology levels. We have extended the Integrated Genome Browser (IGB) to facilitate these tasks. The graphical user interface greatly simplifies building disease networks and zooming in at atomic resolution to identify variations in molecular complexes that may affect molecular interactions in the context of genomic data. All results are summarized in genome tracks and can be visualized and analyzed at the transcript level. AVAILABILITY AND IMPLEMENTATION: The MI Bundle is a plugin for the IGB. The plugin, help, video and tutorial are available at http://cru.genomics.iit.it/igbmibundle/ and https://github.com/CRUiit/igb-mi-bundle/wiki. The source code is released under the Apache License, Version 2. CONTACT: arnaud.ceol@iit.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

GenoMetric Query Language: a novel approach to large-scale genomic data management.

MOTIVATION: Improvement of sequencing technologies and data processing pipelines is rapidly providing sequencing data, with associated high-level features, of many individual genomes in multiple biological and clinical conditions. They allow for data-driven genomic, transcriptomic and epigenomic characterizations, but require state-of-the-art 'big data' computing strategies, with abstraction levels beyond available tool capabilities. RESULTS: We propose a high-level, declarative GenoMetric Query Language (GMQL) and a toolkit for its use. GMQL operates downstream of raw data preprocessing pipelines and supports queries over thousands of heterogeneous datasets and samples; as such it is key to genomic 'big data' analysis. GMQL leverages a simple data model that provides both abstractions of genomic region data and associated experimental, biological and clinical metadata and interoperability between many data formats. Based on Hadoop framework and Apache Pig platform, GMQL ensures high scalability, expressivity, flexibility and simplicity of use, as demonstrated by several biological query examples on ENCODE and TCGA datasets. AVAILABILITY AND IMPLEMENTATION: The GMQL toolkit is freely available for non-commercial use at http://www.bioinformatics.deib.polimi.it/GMQL/.

Chromatic Aberration Correction for Atomic Resolution TEM Imaging from 20 to 80 kV.

Atomic resolution in transmission electron microscopy of thin and light-atom materials requires a rigorous reduction of the beam energy to reduce knockon damage. However, at the same time, the chromatic aberration deteriorates the resolution of the TEM image dramatically. Within the framework of the SALVE project, we introduce a newly developed C_{c}/C_{s} corrector that is capable of correcting both the chromatic and the spherical aberration in the range of accelerating voltages from 20 to 80 kV. The corrector allows correcting axial aberrations up to fifth order as well as the dominating off-axial aberrations. Over the entire voltage range, optimum phase-contrast imaging conditions for weak signals from light atoms can be adjusted for an optical aperture of at least 55 mrad. The information transfer within this aperture is no longer limited by chromatic aberrations. We demonstrate the performance of the microscope using the examples of 30 kV phase-contrast TEM images of graphene and molybdenum disulfide, showing unprecedented contrast and resolution that matches image calculations.

Evidence of gene-environment interactions between common breast cancer susceptibility loci and established environmental risk factors.

Various common genetic susceptibility loci have been identified for breast cancer; however, it is unclear how they combine with lifestyle/environmental risk factors to influence risk. We undertook an international collaborative study to assess gene-environment interaction for risk of breast cancer. Data from 24 studies of the Breast Cancer Association Consortium were pooled. Using up to 34,793 invasive breast cancers and 41,099 controls, we examined whether the relative risks associated with 23 single nucleotide polymorphisms were modified by 10 established environmental risk factors (age at menarche, parity, breastfeeding, body mass index, height, oral contraceptive use, menopausal hormone therapy use, alcohol consumption, cigarette smoking, physical activity) in women of European ancestry. We used logistic regression models stratified by study and adjusted for age and performed likelihood ratio tests to assess gene-environment interactions. All statistical tests were two-sided. We replicated previously reported potential interactions between LSP1-rs3817198 and parity (Pinteraction = 2.4 × 10(-6)) and between CASP8-rs17468277 and alcohol consumption (Pinteraction = 3.1 × 10(-4)). Overall, the per-allele odds ratio (95% confidence interval) for LSP1-rs3817198 was 1.08 (1.01-1.16) in nulliparous women and ranged from 1.03 (0.96-1.10) in parous women with one birth to 1.26 (1.16-1.37) in women with at least four births. For CASP8-rs17468277, the per-allele OR was 0.91 (0.85-0.98) in those with an alcohol intake of <20 g/day and 1.45 (1.14-1.85) in those who drank ≥ 20 g/day. Additionally, interaction was found between 1p11.2-rs11249433 and ever being parous (Pinteraction = 5.3 × 10(-5)), with a per-allele OR of 1.14 (1.11-1.17) in parous women and 0.98 (0.92-1.05) in nulliparous women. These data provide first strong evidence that the risk of breast cancer associated with some common genetic variants may vary with environmental risk factors.