Specific Gene Expression Profiles Research Articles

Human pancreatic islet-derived stromal cells reveal combined features of mesenchymal stromal cells and pancreatic stellate cells.

Mesenchymal stromal cells (MSCs) are recognized for their potential in regenerative medicine, attributed to their multipotent differentiation capabilities and immunomodulatory properties. Despite this potential, the classification and detailed characterization of MSCs, especially those derived from specific tissues like the pancreas, remains challenging leading to a proliferation of terminology in the literature. This study aims to address these challenges by providing a thorough characterization of human pancreatic islets-derived mesenchymal stromal cells (hPD-MSCs). hPD-MSCs were isolated from donor islets using enzymatic digestion, immortalized through lentiviral transduction of human telomerase reverse transcriptase (hTERT). Cells were characterized by immunostaining, flow cytometry and multilineage differentiation potential into adipogenic and osteogenic lineages. Further a transcriptomic analysis was done to compare the gene expression profiles of hPD-MSCs with other mesenchymal cells. We show that hPD-MSCs express the classical MSC features, including morphological characteristics, surface markers expression (CD90, CD73, CD105, CD44, and CD106) and the ability to differentiate into both adipogenic and osteogenic lineages. Furthermore, transcriptomic analysis revealed distinct gene expression profiles, showing notable similarities between hPD-MSCs and pancreatic stellate cells (PSCs). The study also identified specific genes that distinguish hPD-MSCs from MSCs of other origins, including genes associated with pancreatic function (e.g., ISL1) and neural development (e.g., NPTX1, ZNF804A). A novel gene with an unknown function (ENSG00000286190) was also discovered. This study enhances the understanding of hPD-MSCs, demonstrating their unique characteristics and potential applications in therapeutic strategies. The identification of specific gene expression profiles differentiates hPD-MSCs from other mesenchymal cells and opens new avenues for research into their role in pancreatic function and neural development.

Prioritizing Maize Metabolic Gene Regulators through Multi-Omic Network Integration.

Elucidating gene regulatory networks (GRNs) is a major area of study within plant systems biology. Phenotypic traits are intricately linked to specific gene expression profiles. These expression patterns arise primarily from regulatory connections between sets of transcription factors (TFs) and their target genes. In this study, we integrated publicly available co-expression networks derived from more than 6,000 RNA-seq samples, 283 protein-DNA interaction assays, and 16 million of SNPs used to identify expression quantitative loci (eQTL), to construct TF-target networks. In total, we analyzed ~4.6M interactions to generate four distinct types of TF-target networks: co-expression, protein-DNA interaction (PDI), trans-expression quantitative loci (trans-eQTL), and cis-eQTL combined with PDIs. To improve the functional annotation of TFs based on its target genes, we implemented three different strategies to integrate these four types of networks. We subsequently evaluated the effectiveness of our method through loss-of function mutant and random networks. The multi-network integration allowed us to identify transcriptional regulators of hormone-, metabolic- and development-related processes. Finally, using the topological properties of the fully integrated network, we identified potentially functional redundant TF paralogs. Our findings retrieved functions previously documented for numerous TFs and revealed novel functions that are crucial for informing the design of future experiments. The approach here-described lays the foundation for the integration of multi-omic datasets in maize and other plant systems.

Comparing gene expression profiles of adults with isolated spinal tuberculosis to disseminated spinal tuberculosis identified by 18FDG-PET/CT at time of diagnosis, 6- and 12-months follow-up: classifying clinical stages of spinal tuberculosis and monitoring treatment response (Spinal TB X cohort study)

BackgroundTuberculosis (TB) is one of the top ten causes of death worldwide, with approximately 10 million cases annually. Focus has been on pulmonary TB, while extrapulmonary TB (EPTB) has received little attention. Diagnosis of EPTB remains challenging due to the invasive procedures required for sample collection. Spinal TB (STB) accounts for 10% of EPTB and often leads to lifelong debilitating disease due to devastating spinal deformation and compression of neural structures. Little is known about the extent of disease, although both isolated STB and a disseminated form of STB have been described. In our Spinal TB X cohort study, we aim to describe the clinical phenotype of STB using whole-body 18FDG-PET/CT, identify a specific gene expression profile for different stages of dissemination and compare findings to previously described gene expression signatures for latent and active pulmonary TB.MethodsA single-centre, prospective cohort study will be established to describe the distributional pattern of STB detected by whole-body 18FDG-PET/CT and gene expression profile of patients with suspected STB on magnetic resonance imaging (MRI) at point of diagnosis, six months, and 12 months. Blood biobanking will be performed at these time points. Specimens for microbiology will be obtained from sputum/urine, from easily accessible sites of disease (e.g., lymph nodes, abscess) identified in the first 18FDG-PET/CT, from CT-guided biopsy and/or surgery. Clinical parameters and functional scores will be collected at every physical visit. Data will be entered into RedCap® database; data cleaning, validation and analysis will be performed by the study team. The University of Cape Town Ethics Committee approved the protocol (243/2022).DiscussionThe Spinal TB X cohort study is the first prospective cohort study using whole-body 18FDG-PET/CT scans in patients with microbiologically confirmed spinal tuberculosis. Dual imaging techniques of the spine using 18FDG-PET/CT and magnetic resonance imaging as well as tissue diagnosis (microbiology and histopathology) will allow us to develop a virtual biopsy model. If successful, a distinct gene-expression profile will aid in blood-based diagnosis (point of care testing) as well as treatment monitoring and would lead to earlier diagnosis of this devastating disease.Trial registration: The study has been registered on ClinicalTrials.gov (NCT05610098).

Multi-level tuberculosis of the spine identified by 18F-FDG-PET/CT and concomitant urogenital tuberculosis: a case report from the spinal TB X cohort.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and typically infects the lungs. However, extrapulmonary forms of TB can be found in approximately 20% of cases. It is suggested, that up to 10% of extrapulmonary TB affects the musculoskeletal system, in which spinal elements (spinal tuberculosis, STB) are involved in approximately 50% of the cases. STB is a debilitating disease with nonspecific symptoms and diagnosis is often delayed for months to years. In our Spinal TB X Cohort, we aim to describe the clinical phenotype of STB using whole-body 18F-fluorodeoxyglucose positron emission tomography computed tomography (PET/CT) and to identify a specific gene expression profile for the different stages of dissemination on PET/CT. Here we report on the first patient recruited into our cohort who underwent PET/CT before treatment initiation, at 6-months and at 12-months - time of TB treatment completion. A 27-year-old immunocompetent male presented with severe thoracolumbar back pain for 9 months with severe antalgic gait and night sweats. Magnetic resonance imaging (MRI) of the whole spine revealed multilevel spinal disease (T5/6, T11/12, L3/4) in keeping with STB. After informed consent and recruitment into the Spinal TB X Cohort, the patient underwent PET/CT as per protocol, which revealed isolated multilevel STB (T4-7, T11/12, L3/4) with no concomitant lung or urogenital lesion. However, sputum and urine were Xpert MTB/RIF Ultra positive and Mtb was cultured from the urine sample. CT-guided biopsy of the T11/12 lesion confirmed drug-sensitive Mtb on Xpert MTB/RIF Ultra and the patient was started on TB treatment according to local guidelines for 12 months. The 6-month follow-up PET/CT revealed new and existing spinal lesions with increased FDG-uptake despite significant improvement of clinical features and laboratory markers. After 9 months of treatment, the patient developed an acute urethral stricture, most likely due to urogenital TB, and a suprapubic catheter was inserted. The 12-month PET/CT showed significantly decreased PET/CT values of all lesions, however, significant persistent spinal inflammation was present at the end of TB treatment. Clinically, the patient was considered cured by the TB control program and currently awaits urethroplasty. In our case, PET/CT emerged as a valuable imaging modality for the initial assessment, surpassing MRI by revealing more comprehensive extensive disease. Subsequent PET/CT scans at 6-month uncovered new lesions and increased inflammation in existing ones, while by the end of TB treatment, all lesions exhibited improvement. However, the interpretation of FDG avidity remains ambiguous, whether it correlates with active infection and viable Mtb. or fibro- and osteoblast activity indicative of the healing process. Additionally, the absence of extraspinal TB lesions on PET/CT despite positive microbiology from sputum and urine maybe explained by paucibacillary, subclinical infection of extraspinal organs. The Spinal TB X Cohort endeavours to shed light on whole-body imaging patterns at diagnosis, their evolution midway through TB treatment, and upon treatment completion. Ultimately, this study aims to advance our understanding of the biology of this complex disease.

Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

Transcriptome profiles of Trypanosoma brucei rhodesiense in Malawi reveal focus specific gene expression profiles associated with pathology.

Sleeping sickness caused by Trypanosoma brucei rhodesiense is a fatal disease and endemic in Southern and Eastern Africa. There is an urgent need to develop novel diagnostic and control tools to achieve elimination of rhodesiense sleeping sickness which might be achieved through a better understanding of trypanosome gene expression and genetics using endemic isolates. Here, we describe transcriptome profiles and population structure of endemic T. b. rhodesiense isolates in human blood in Malawi. Blood samples of r-HAT cases from Nkhotakota and Rumphi foci were collected in PaxGene tubes for RNA extraction before initiation of r-HAT treatment. 100 million reads were obtained per sample, reads were initially mapped to the human genome reference GRCh38 using HiSat2 and then the unmapped reads were mapped against Trypanosoma brucei reference transcriptome (TriTrypDB54_TbruceiTREU927) using HiSat2. Differential gene expression analysis was done using the DeSeq2 package in R. SNP calling from reads that were mapped to the T. brucei genome was done using GATK in order to identify T.b. rhodesiense population structure. 24 samples were collected from r-HAT cases of which 8 were from Rumphi and 16 from Nkhotakota foci. The isolates from Nkhotakota were enriched with transcripts for cell cycle arrest and stumpy form markers, whereas isolates in Rumphi focus were enriched with transcripts for folate biosynthesis and antigenic variation pathways. These parasite focus-specific transcriptome profiles are consistent with the more virulent disease observed in Rumphi and a less symptomatic disease in Nkhotakota associated with the non-dividing stumpy form. Interestingly, the Malawi T.b. rhodesiense isolates expressed genes enriched for reduced cell proliferation compared to the Uganda T.b. rhodesiense isolates. PCA analysis using SNPs called from the RNAseq data showed that T. b. rhodesiense parasites from Nkhotakota are genetically distinct from those collected in Rumphi. Our results suggest that the differences in disease presentation in the two foci is mainly driven by genetic differences in the parasites in the two major endemic foci of Rumphi and Nkhotakota rather than differences in the environment or host response.

Insights into the Interaction between the Biocontrol Agent Bacillus amyloliquefaciens QST 713, the Pathogen Monilinia fructicola and Peach Fruit

Brown rot disease caused by Monilinia fructicola is one of the most important peach fruit threats in the world. The use of biological control agents (BCAs), instead of synthetic fungicides, to successfully inhibit postharvest disease development is a challenge in sustainable and efficient crop management. The commercially available BCA Bacillus amyloliquefaciens QST 713 (formerly Bacillus subtilis QST713) is able to inhibit a variety of fungal pathogens and suppress several plant diseases. Our results showed that this BCA inhibited mycelial growth in vitro, and was able to suppress the disease’s severity in peach fruits via delaying and reducing brown rot symptoms. A transcriptomic analysis of fruits during their pre-treatment with this biocontrol agent following M. fructicola challenge revealed a significant upregulation of specific differentially expressed genes (DEGs) at 48 h after inoculation (HAI). These genes are related to the activation of several transcriptional factors, such as members of the WRKY and NAC families, and receptors that are involved in pathogen recognition and signaling transduction (e.g., LRR-RLKs). Furthermore, the inhibition of M. fructicola by this biocontrol agent was confirmed by analyzing the expression profiles of specific fungal genes, which highlighted the direct antimicrobial impact of this bacterial strain against the fungus. Hence, these findings clearly suggest that B. amyloliquefaciens QST 713 is an efficient BCA against brown rot disease, which can directly inhibit M. fructicola and improve peach fruit tolerance.

Abstract 7045: RNA sequencing approaches enable tissue specific B and T cell gene expression and immune repertoire profiling

Abstract The adaptive immune system defends the human body from pathogens through the recognition ability of antigen receptors from B and T cells. Different organs play various immune function roles, and therefore present varied B and T cell gene expression and immune repertoire profiles. Abnormality of these profiles may reflect immunological disorder, presence of neoplastic tissue, or response to infectious agent. High-throughput sequencing technology has facilitated the understanding of gene expression and related cell functions, but in-depth understanding of B and T cell phenotype and clonotype profiling is an emerging application that has yet to take full advantage of sequencing technology for diagnosis and prognosis of disease. Here, we share results of RNA sequencing of immune system tissues; including B cell and T cell specific gene expression and full-length immune repertoire profiling. Total RNA was extracted from various tissue samples including healthy donor peripheral blood mononuclear cells (PBMC), lymph nodes, bone marrow, spleen, thyroid, thymus and colon, as well as diseased PBMC and colon samples. RNA-seq and Immune-seq libraries were made from these samples and sequenced on Illumina NextSeq2000. Gene expression analysis and immune repertoire profiling from RNA-seq data were performed using edgeR and TRUST4. Immune repertoire sequencing data was processed using pRESTO and IgBlast. We correlated RNA-seq with Immune-seq results for B-cell receptor and T-cell receptor clonotypes. Differential expression and clonotype profiling comparisons were also performed between normal and diseased RNA samples. The effect of reference on immune repertoire detection sensitivity and accuracy was further investigated by comparing IMGT reference and AIRR-C Human IG Reference Sets. This study has identified tissue specific B/T cell phenotype and clonality patterns that relate to tissue functions and disease progression. RNA-seq enables highly multiplex immunophenotyping which is traditionally performed by flow cytometry. RNA-seq can detect high-abundant clones that correlate with Immune-seq, with the latter targeted approach providing more in depth clonality signatures. References comparison results show the importance of continued community effort to develop more representative and accurate immunorepertoire germ line references for clonotype annotation in diverse populations. Integrating both RNA sequencing and immune repertoire sequencing approaches allows accurate phenotype and clonotype determination for immune landscape in various tissues. Citation Format: Chen Song, Evan Janzen, Tessa Devoe, Ariel Erijman, Gautam Naishadham, Li Song, Bradley W. Langhorst. RNA sequencing approaches enable tissue specific B and T cell gene expression and immune repertoire profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7045.

Host-Specific and Homologous Pairs of Melampsora larici-populina Effectors Unveil Novel Nicotiana benthamiana Stromule Induction Factors.

The poplar rust fungus Melampsora larici-populina is part of one of the most devastating group of fungi (Pucciniales) and causes important economic losses to the poplar industry. Because M. larici-populina is a heteroecious obligate biotroph, its spread depends on its ability to carry out its reproductive cycle through larch and then poplar parasitism. Genomic approaches have identified more than 1,000 candidate secreted effector proteins (CSEPs) from the predicted secretome of M. larici-populina that are potentially implicated in the infection process. In this study, we selected CSEP pairs (and one triplet) among CSEP gene families that share high sequence homology but display specific gene expression profiles among the two distinct hosts. We determined their subcellular localization by confocal microscopy through expression in the heterologous plant system Nicotiana benthamiana. Five out of nine showed partial or complete chloroplastic localization. We also screened for potential protein interactors from larch and poplar by yeast two-hybrid assays. One pair of CSEPs and the triplet shared common interactors, whereas the members of the two other pairs did not have common targets from either host. Finally, stromule induction quantification revealed that two pairs and the triplet of CSEPs induced stromules when transiently expressed in N.benthamiana. The use of N.benthamiana eds1 and nrg1 knockout lines showed that CSEPs can induce stromules through an eds1-independent mechanism. However, CSEP homologs shared the same impact on stromule induction and contributed to discovering a new stromule induction cascade that can be partially and/or fully independent of eds1. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Molecular profile of bladder cancer progression to clinically aggressive subtypes.

Bladder cancer is a histologically and clinically heterogenous disease. Most bladder cancers are urothelial carcinomas, which frequently develop distinct histological subtypes. Several urothelial carcinoma histological subtypes, such as micropapillary, plasmacytoid, small-cell carcinoma and sarcomatoid, show highly aggressive behaviour and pose unique challenges in diagnosis and treatment. Comprehensive genomic characterizations of the urothelial carcinoma subtypes have revealed that they probably arise from a precursor subset of conventional urothelial carcinomas that belong to different molecular subtypes - micropapillary and plasmacytoid subtypes develop along the luminal pathway, whereas small-cell and sarcomatoid subtypes evolve along the basal pathway. The subtypes exhibit distinct genomic alterations, but in most cases their biological properties seem to be primarily determined by specific gene expression profiles, including epithelial-mesenchymal transition, urothelial-to-neural lineage plasticity, and immune infiltration with distinct upregulation of immune regulatory genes. These breakthrough studies have transformed our view of bladder cancer histological subtype biology, generated new hypotheses for therapy and chemoresistance, and facilitated the discovery of new therapeutic targets.

Abstract B035: The contribution of inflammasomes and cGAS-STING in RT-induced cell fate

Abstract Radiotherapy (RT) is a curative treatment modality for many solid cancers. Our group has previously demonstrated that activation of the cGAS-STING pathway by RT induces inflammatory cytokine production and subsequent stimulation of anti-tumour immunity in concert with immune checkpoint blockade treatment. We have identified three other proteins that contribute to inflammatory signalling after RT: AIM2 (Absent in Melanoma 2), NLRP3 (NOD-Like Receptor Pyrin Domain-Containing 3), and IFI16 (Interferon Gamma Inducible Protein 16). Each of these proteins contribute to form distinct protein complexes called inflammasomes that, like cGAS-STING, sense cytoplasmic DNA. Little is known about how inflammasome signalling may change after RT, or how inflammasomes may influence RT-ICB induced anti-tumour immunity. We hypothesize that RT induces inflammasome signalling to influence specific gene expression and cytokine profiles, in tandem with alterations in cellular fate with implications for systemic anti-tumour immunity triggered by radiotherapy. We will present ongoing work in which we show that pharmacological or genetic disruption of inflammasomes alters cytokine production post-RT. We also demonstrate that manipulation of cell fate through senolytic therapy or manipulation of caspase activity alters the balance of cytokine production post-RT. Together our data strongly implicates the role of several inflammasome complexes in the response of epithelial cells to RT. Our work will also highlight the possible crosstalk between the cGAS-STING pathway and inflammasome complex pathway, both triggered by RT. Understanding and subsequent manipulation of cell fate, in part through inflammasome and cGAS-STING mediated activity, may be a therapeutic strategy to influence treatment responses, such as anti-tumour immunity, in cancer. Citation Format: Cindy T Ha, Shirony Nicholson-Puthenveedu, Shane M Harding. The contribution of inflammasomes and cGAS-STING in RT-induced cell fate [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B035.

What is the Optimum Endometrium Needed for Implantation?

Implantation most critical step in reproduction. A complex process where blastocyst becomes intimately connected with maternal endometrium/decidua and requires; Competent embryo at blastocyst stage Receptive endometrium Synchronized dialogue between maternal and embryonic tissues Endometrial Changes for Implantation include architectural, morphological, cytochemical, molecular and genetic. These Changes Induce Endometrial receptivity, which is endometrial lining in a state of readiness for blastocyst implantation. It involves acquisition of adhesion ligands and loss of inhibitory components that may act as a barrier to an attaching embryo. Receptive endometrium is capable of selecting good quality embryos and reject the incompetent embryos. Endometrial Evaluation includes 1. Non-Invasive Assessment of Endometrial Receptivity by USG 2. Detection of abnormal Uterine cavity by 3D USG/SIS, HSG and Hysteroscopy 3. Diagnose Chronic Endometritis by hysteroscopy and Molecular tests? 4. Endometrial Microbiome by molecular tests 5. Endometrial asynchrony - Proteomics and transcriptomics 2D and 3D Ultrasound can Identify patients with poor implantation prognosis but has low predictive value in determining endometrial receptivity and IVF outcome. Ideal endometrial thickness for optimal outcome is between 7-14 mm. No Association was seen between clinical pregnancy rates (CPR) and endometrial pattern for women undergoing in IVF with fresh ET and FET. Minimum Endometrial Volume associated with pregnancy is 1.59 mL – 3D. But most pregnancies occur in volumes of 2-13 mL. Evaluation of endometrial volume is particularly useful in synechiae, adenomyosis and uterine anomalies. Vascularity demonstrated within Zone 3 or within Zones 3 and 4 prior to transfer is associated with good implantation rate. Presence of >3 peristaltic contractions of the sub endometrial myometrium/2 minutes interval on Day of hCG administration associated with poor implantation. Diagnosis of chronic endometritis is based on hysteroscopy, endometrial biopsy with plasma cells being identified histologically and specific treatment is determined based on microbial culture. Existence of non-Lactobacillus bacteria in the endometrium is correlated with negative impacts on reproductive function and should be considered as an emerging cause of implantation failure and pregnancy loss. Endometrial Receptivity Array (ERA) is validated for analysing the expression of 238 genes that are intimately related to endometrial receptivity. Abnormality in endometrial receptivity is displayed a specific gene expression profile. Intrauterine secretory profile directly linked to successful implantation and evaluation of endometrial fluid for cytokine, interleukin and growth factor secretions can be used to assess the endometrium. Conclusion: Identification of one or more of endometrial parameters that definitely indicate receptivity for implantation remains an elusive goal. None of the endometrial receptivity markers have sufficient discriminatory value to act as a diagnostic test for endometrial receptivity based on their ability to predict clinical pregnancy. Due to the dynamic, cyclic nature of the endometrium it may be difficult, to reliably assess endometrial function on the basis of a single test.

Somatic Mutations and DNA Hypermethylation at Enhancers and Promoters Identify Distinct Subtypes within Lower-Risk Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of heterogeneous disorders caused by the accumulation of somatic mutations in the hematopoietic stem and progenitor compartment. Besides del(5q), SF3B1 or TP53 mutations, referred to as defining genetic abnormalities, mutation patterning hardly structured the classification of MDS. Mutations in epigenetic factors occur early in the development of clonal hematopoiesis leading to precocious alterations of DNA methylation implicated in oncogenesis. Convergent DNA methylation patterns related to both mutations and microenvironment imprinting may induce specific gene expression profiles and contribute to phenotypic variations. To refine a pathophysiological classification of lower-risk MDS, we combined genetic profiling to DNA methylation and transcriptome data. Methods: We enrolled 543 lower-risk treatment-naïve MDS patients at diagnosis and performed DNA-sequencing of 24 genes. DNA methylation and RNA-sequencing data were generated in a representative subset of 175 cases with available material (75 MDS-SLD/MLD, 40 MDS-EB1, 53 MDS-RS-SLD/MLD, 5 MDS del(5q), 2 MDS-U) and 7 age-matched healthy controls. IPSS-R was very low, low or intermediate in 87.4%. During a median follow-up of 2 years, 16% of MDS cases progressed to AML (MDSp). Infinium EPIC 850K array allowed after filtering the examination of methylation of 723,612 CpG sites with enrichment at enhancers and promoters. Differentially methylated regions with differentially methylated CpG sites (DMR-CpG) between cases and controls were defined with a minimum of 2 probes, a Benjamini-Hochberg-adjusted P-value cut-off of 0.05, a false discovery rate of 0.001 and a mean Δβ-value >20% at CpG sites. Consensus motifs for transcription factors were identified using Homer. Results: Unsupervised clustering on genetic profiling of 543 patients identified distinct subsets of lower-risk MDS with different clinical features: one cluster (group A) was enriched in del(5q), three clusters were characterized by SF3B1 mutations, either isolated (group B) or associated with DNMT3A (group E) or TET2 (group F), while the two remaining clusters were enriched in either complex pattern of mutations (group C) or SRSF2 mutations (group D). Genetic clusters showed significantly different clinical outcomes, clusters C, D and E having lower overall survival and higher risk of progression to high risk MDS or AML compared to other clusters. In the subset of 175 cases, a total of 2,953 unique DMR-CpGs allowed a robust repartition of patient samples in 4 groups using the k-means method. The 4 methylation groups were clinically distinct in terms of age, hemoglobin, neutrophils, monocytes, platelets, BM blasts, WHO classification, IPSS-R, and mutation pattern. A significant correlation was found between genetic and methylation groups ( P<0.001). Hypomethylated CpGs defined group 4 which was enriched in MDS-RSwith low blast count and few co-mutations, while groups 1 and 3 demonstrated hypermethylated profiles driven by TET2/ IDH1-2 mutations and TET2/ SRSF2 mutations, respectively. TET2-mutated cases exhibited an increased proportion of hypermethylated DMR-CpGs at enhancers (DME) significantly enriched in C/EBP and ETS family transcription factor motifs. In MDS without evidence of progression, we identified 1,418 DME and their gene targets of which those significantly deregulated in RNA-seq were involved in the control of translation and response to TGF-β. By contrast, in MDS with rapid progression to AML, DMR-CpGs were significantly enriched at CpG islands and promoters ( P<0.0001) and a set of 10-20 genes downstream of these promoters were specifically deregulated. Conclusion: Somatic mutations and DNA methylation at enhancers and promoters identify distinct subsets of lower-risk MDS with different clinical behavior. Hypermethylation at enhancers with C/EBP or ETS family motifs is a hallmark of MDS and may account for changes in transcription factor recruitment and cell fate. Hypermethylation of promoters with downstream effects on gene expression may indicate a propensity to rapid evolution to AML.

Integrative whole-genome and transcriptome analysis of HER2-amplified metastatic breast cancer

BackgroundIn breast cancer, the advent of anti-HER2 therapies has made HER2+ tumors a highly relevant subgroup. However, the exact characteristics which prohibit clinical response to anti-HER2 therapies and drive disease progression are not yet fully known. Integrative whole-genome and transcriptomic sequencing data from both primary and metastatic HER2-positive breast cancer will enhance our understanding of underlying biological processes.MethodsHere, we used WGS and RNA sequencing data of 700 metastatic breast tumors, of which 68 being HER2+, to search for specific genomic features of HER2+ disease and therapy resistance. Furthermore, we integrated results with transcriptomic data to associate tumors exhibiting a HER2+-specific gene expression profile with ERBB2 mutation status, prior therapy and relevant gene expression signatures.ResultsOverall genomic profiles of primary and metastatic HER2+ breast cancers were similar, and no specific acquired genomics traits connected to prior anti-HER2 treatment were observed. However, specific genomic features were predictive of progression-free survival on post-biopsy anti-HER2 treatment. Furthermore, a HER2-driven expression profile grouped HER2-amplified tumors with ERBB2-mutated cases and cases without HER2 alterations. The latter were reported as ER positive in primary disease, but the metastatic biopsy showed low ESR1 expression and upregulation of the MAPK pathway, suggesting transformation to ER independence.ConclusionsIn summary, although the quantity of variants increased throughout HER2-positive breast cancer progression, the genomic composition remained largely consistent, thus yielding no new major processes beside those already operational in primary disease. Our results suggest that integrated genomic and transcriptomic analyses may be key in establishing therapeutic options.

Rethinking cancer initiation: The role of large-scale mutational events.

Cancer initiation is revisited in light of recent discoveries in cancer pathogenesis. Of note is the detection of mutated cancer genes in benign conditions. More significantly, somatic clones, which harbor mutations in cancer genes, arise in normal tissues from early development through adulthood, but seldom do they transform into cancer. Further, clustered mutational events-kataegis, chromothripsis and chromoplexy-are widespread in cancer, generating point mutations and chromosomal rearrangements in a single cellular catastrophe. These observations are contrary to the prevailing somatic mutation theory, which states that a cancer is caused by the gradual accumulation of mutations over time. A different perspective is proposed within the framework of Waddington's epigenetic landscape wherein tumorigenesis is viewed primarily as a disruption of cell development. Cell types are defined by their specific gene-expression profiles, determined by the gene regulatory network, and can be regarded as attractor states of the network dynamics: they represent specific, self-stabilizing patterns of gene activities across the genome. However, large-scale mutational events reshape the landscape topology, creating abnormal "unphysiological" attractors. This is the crux of the process of initiation. Initiation primes the cell for conversion into a tumor phenotype by oncogenes and tumor suppressor genes, which drive cell proliferation and clonal diversification. This view of tumorigenesis calls for a different approach to therapy.

A novel role of human epicardial adipocyte-derived SPARCL1 in suppression of postoperative atrial fibrillation in patients undergoing cardiovascular surgery

Abstract Background Postoperative atrial fibrillation (POAF) occurs in 20-50% after cardiovascular surgery and is closely associated with the short and long-term morbidity/mortality. The epicardial adipose tissue (EAT) is in contact with the atrial muscle, and may have a potential predisposing factor to induce POAF within it. Purpose We tried to identify the specific gene expression profile of the human epicardial adipocyte associated with POAF. Methods We prospectively evaluated 126 patients without a history of AF (male 86 (68.3%); mean age 68.3 ± 11.7) who underwent scheduled open-heart cardiovascular surgery between May 2020 and November 2022. They were assigned to the POAF group and the Non-POAF group. POAF was defined as AF lasting more than 30 seconds documented by monitor or 12-lead electrocardiogram after the operation. Fresh human EAT samples were obtained from these patients during surgery, and preadipocytes were isolated from these samples. Isolated preadipocytes were terminally differentiated to mature adipocytes and RNAs were extracted. Genome-wide expression profiling of 6 vs. 6 matched samples of the POAF and Non-POAF groups was performed using Human Clariom S Assay-HT 16-Array Plate. The secreted protein levels in the adipocytes-cultured medium were also evaluated using enzyme-linked sorbent assay test to confirm the consistency of mRNA expression levels of adipocytes and its secreted protein levels. Results POAF was observed in 48 patients (38%). Patients in the POAF group were significantly older (p = 0.003) compared to the Non-POAF group. POAF occurred mainly within the first 3 postoperative days (median 2.5 days), and the median duration of POAF was 298 ± 581 minutes. The occurrence of POAF was significantly associated with longer postoperative hospitalization (p = 0.001) and intensive care unit stay (p = 0.04). Microarray analysis revealed that there were 132 down- or up-regulated cording genes (fold change >2.0 or `−2.0, p < 0.05) in epicardial adipocytes of the Non-POAF group compared to the POAF group. Of these genes, 11 genes cording secretable molecules were validated by qPCR, showing that only SPARCL1 had significantly higher expression in the Non-POAF group compared to the POAF group (p = 0.008). The mRNA expression levels of SPARCL1 significantly decreased when the preadipocyte were terminally differentiated to the mature adipocyte (p = 0.0009). SPARCL1 mRNA expression in adipocyte was significantly correlated with the concentration of SPARCL1 protein in the adipocyte-cultured medium (p < 0.0001, r = 0.7476). Conclusion The results of this study suggest that the expression of SPARCL1 in human epicardial adipocytes may play an important role in the suppression of POAF. Our findings suggested that SPARCL1 expression in human epicardial adipocytes may play an important role in the suppression of POAF. SPARCL1 secreted by epicardial adipocytes may be a potential therapeutic target for POAF prevention.Flowchart of patient selectionMicroarray analysis and qPCR varidation

Targeted DNA Damage Boost with Loncastuximab Tesirine in Combination with PARP Inhibitors in Diffuse Large B-Cell Lymphoma

MYC-over expressing lymphomas display inherent resistance to standard chemoimmunotherapy regimens and are characterised by genomic instability and constitutive activation of the DNA damage response (DDR) pathway. Loncastuximab tesirine (Lonca) is an antibody-drug conjugate directed against CD19, a surface antigen broadly expressed in B-cell malignancies. Upon CD19 binding, Lonca induces DNA damage by delivering a pyrrolobenzodiazepine dimer-based, DNA crosslinking warhead into target cells. Here we hypothesised that DDR inhibition could enhance DNA damage induced by Lonca in CD19-positive lymphoma cells. Since MYC-overexpressing cells mainly rely on PARP activity to attenuate endogenous MYC-induced replicative stress, we combined Lonca with Talazoparib (an FDA-approved PARP inhibitor), in a panel of 12 DLBCL cell lines (7 with MYC-rearrangement), and in vivo in a MYC/BCL-2-rearranged DLBCL PDX model. Lonca and Talazoparib showed significant antiproliferative activity in a dose and time dependent manner in most cell lines at clinically achievable concentrations. The highest sensitivity to both compounds was observed in a BRCA2 mutant cell line (DOHH2). These data are in line with the known synthetic lethality between BRCA mutations and platinum derivatives and PARP inhibitors. The addition of Talazoparib significantly enhanced the antiproliferative effects of Lonca in DLBCL cell lines irrespective of MYC/BCL2 rearrangements and TP53 genomic alterations. Of note, the highest efficacy of the combination was observed in MYC-rearranged and MYC-overexpressing cell lines. The combination of Lonca and Talazo sinergistically increased DNA damage as evaluated by comet assay, induced enhanced cell death and caused specific changes in cell cycle dynamics. Enhanced apoptosis was confirmed by Annexin/PI staining and caspase 3/7 cleavage, assessed by caspase Glo assay and Western Blot analyses. As previously shown (Fusani et al., ASH 2021), while Lonca and Talazoparib affected cell cycle inducing G2/M phase arrest, the combination specifically increased the percentage of cells arrested in the BrdU negative S phase of cell cycle. Upon combined treatment, we observed increased DDR activation with the evaluation of several DDR biomarkers such as phospho(p)-ATM, pCHK2 Thr68, pCHK1 Ser345, gH2AX. To better understand the molecular mechanism underlying this synergistic interaction, we performed RNA-Seq analysis in two representative cell lines (OCI-LY18 and SUDHL5). Interestingly while Lonca did not cause significant changes in gene expression profiling (GEP) after 24 hours of incubation, the combination resulted in specific GEP changes such as down-regulation of histone H2B and aurora kinase A (AURKA), genes mainly involved in S and G2/M transition of the cell cycle. Similar results were obtained by combining Talazoparib and different PARP inhibitors with the alkylating agent cisplatin, indicating a class effect. The synergistic interaction of Lonca and Talazoparib was further confirmed in vivo in a MYC/BCL-2 double hit PDX model. As previously shown (Fusani et al., ASH 2021), Lonca/Talazoparib combination did not result in enhanced DNA damage induction in PBMC-derived B cells from healthy donors. Importantly, PBMC-derived T cells from healthy donors did not show any sign of DNA damage accumulation upon exposure to Lonca, Talazoparib and the combination. These data provide the rationale for future therapeutic strategies based on selective induction of DNA damage in neoplastic B cells in combination with DDR inhibition in aggressive MYC-positive B cell lymphoma.

Clinical, Pathologic, and Molecular Characterization of Acanthosis Nigricans Maligna: A Comprehensive Analysis of a Dermatologic Entity and its Association with Underlying Neoplastic Conditions

Acanthosis nigricans maligna (ANM) is a distinctive dermatologic entity presenting with cutaneous hyperpigmentation and epidermal thickening, characteristically located in flexural areas and skin folds. Despite its apparent cutaneous nature, ANM is frequently associated with underlying neoplastic conditions, implying profound clinical significance. In this study, we undertook a comprehensive evaluation of ANM from clinical, pathological and molecular perspectives with the aim of elucidating its etiopathogenic mechanisms and its relationship with neoplastic processes. From a clinical standpoint, we retrospectively analyzed a cohort of patients diagnosed with ANM, exploring the clinical and epidemiologic features that could indicate the presence of an underlying malignant condition. In addition, histopathologic studies were performed on samples of affected skin to discern the microscopic alterations associated with ANM and their correlates with neoplastic processes. In a molecular phase, an in-depth analysis of genetic and molecular markers in skin samples and associated tumor tissues was carried out using advanced sequencing techniques. This allowed the identification of specific mutations, chromosomal alterations and gene expression profiles that could contribute to both the occurrence of ANM and the promotion of the microenvironment conducive to tumor development. The results obtained indicate a significant correlation between the presence of ANM and the abnormal activation of cell signaling pathways, as well as the altered expression of regulatory factors of cell growth and differentiation. Our study highlights the importance of considering ANM as a potential cutaneous marker of underlying neoplastic processes and suggests that its early detection could facilitate the early identification of early stage malignant conditions. Taken together, this comprehensive analysis provides an in-depth view of ANM and its clinical, pathological and molecular relevance in the context of neoplastic diseases.

In Planta Study Localizes an Effector Candidate from Austropuccinia psidii Strain MF-1 to the Nucleus and Demonstrates In Vitro Cuticular Wax-Dependent Differential Expression.

Austropuccinia psidii is a biotrophic fungus that causes myrtle rust. First described in Brazil, it has since spread to become a globally important pathogen that infects more than 480 myrtaceous species. One of the most important commercial crops affected by A. psidii is eucalypt, a widely grown forestry tree. The A. psidii-Eucalyptus spp. interaction is poorly understood, but pathogenesis is likely driven by pathogen-secreted effector molecules. Here, we identified and characterized a total of 255 virulence effector candidates using a genome assembly of A. psidii strain MF-1, which was recovered from Eucalyptus grandis in Brazil. We show that the expression of seven effector candidate genes is modulated by cell wax from leaves sourced from resistant and susceptible hosts. Two effector candidates with different subcellular localization predictions, and with specific gene expression profiles, were transiently expressed with GFP-fusions in Nicotiana benthamiana leaves. Interestingly, we observed the accumulation of an effector candidate, Ap28303, which was upregulated under cell wax from rust susceptible E. grandis and described as a peptidase inhibitor I9 domain-containing protein in the nucleus. This was in accordance with in silico analyses. Few studies have characterized nuclear effectors. Our findings open new perspectives on the study of A. psidii-Eucalyptus interactions by providing a potential entry point to understand how the pathogen manipulates its hosts in modulating physiology, structure, or function with effector proteins.