Comparative Analysis of Cell Mixtures Deconvolution and Gene Signatures Generated for Blood, Immune and Cancer Cells.

In the last two decades, many detailed full transcriptomic studies on complex biological samples have been published and included in large gene expression repositories. These studies primarily provide a bulk expression signal for each sample, including multiple cell-types mixed within the global signal. The cellular heterogeneity in these mixtures does not allow the activity of specific genes in specific cell types to be identified. Therefore, inferring relative cellular composition is a very powerful tool to achieve a more accurate molecular profiling of complex biological samples. In recent decades, computational techniques have been developed to solve this problem by applying deconvolution methods, designed to decompose cell mixtures into their cellular components and calculate the relative proportions of these elements. Some of them only calculate the cell proportions (supervised methods), while other deconvolution algorithms can also identify the gene signatures specific for each cell type (unsupervised methods). In these work, five deconvolution methods (CIBERSORT, FARDEEP, DECONICA, LINSEED and ABIS) were implemented and used to analyze blood and immune cells, and also cancer cells, in complex mixture samples (using three bulk expression datasets). Our study provides three analytical tools (corrplots, cell-signature plots and bar-mixture plots) that allow a thorough comparative analysis of the cell mixture data. The work indicates that CIBERSORT is a robust method optimized for the identification of immune cell-types, but not as efficient in the identification of cancer cells. We also found that LINSEED is a very powerful unsupervised method that provides precise and specific gene signatures for each of the main immune cell types tested: neutrophils and monocytes (of the myeloid lineage), B-cells, NK cells and T-cells (of the lymphoid lineage), and also for cancer cells.

Identification of a gene expression signature associated with breast cancer survival and risk that improves clinical genomic platforms.

Motivation: Modern genomic technologies allow us to perform genome-wide analysis to find gene markers associated with the risk and survival in cancer patients. Accurate risk prediction and patient stratification based on robust gene signatures is a key path forward in personalized treatment and precision medicine. Several authors have proposed the identification of gene signatures to assign risk in patients with breast cancer (BRCA), and some of these signatures have been implemented within commercial platforms in the clinic, such as Oncotype and Prosigna. However, these platforms are black boxes in which the influence of selected genes as survival markers is unclear and where the risk scores provided cannot be clearly related to the standard clinicopathological tumor markers obtained by immunohistochemistry (IHC), which guide clinical and therapeutic decisions in breast cancer. Results: Here, we present a framework to discover a robust list of gene expression markers associated with survival that can be biologically interpreted in terms of the three main biomolecular factors (IHC clinical markers: ER, PR and HER2) that define clinical outcome in BRCA. To test and ensure the reproducibility of the results, we compiled and analyzed two independent datasets with a large number of tumor samples (1024 and 879) that include full genome-wide expression profiles and survival data. Using these two cohorts, we obtained a robust subset of gene survival markers that correlate well with the major IHC clinical markers used in breast cancer. The geneset of survival markers that we identify (which includes 34 genes) significantly improves the risk prediction provided by the genesets included in the commercial platforms: Oncotype (16 genes) and Prosigna (50 genes, i.e. PAM50). Furthermore, some of the genes identified have recently been proposed in the literature as new prognostic markers and may deserve more attention in current clinical trials to improve breast cancer risk prediction. Availability and implementation: All data integrated and analyzed in this research will be available on GitHub (https://github.com/jdelasrivas-lab/breastcancersurvsign), including the R scripts and protocols used for the analyses. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Dose and administration time of indocyanine green in near-infrared fluorescence cholangiography during laparoscopic cholecystectomy (DOTIG): study protocol for a randomised clinical trial.

BACKGROUND: One of the most severe complications in laparoscopic cholecystectomy (LC) is intraoperative bile duct injury (BDI). Despite its low incidence, the medical implications for the patient can be serious. Besides, BDI can also generate significant legal issues in healthcare. Different techniques have been described to reduce the incidence of this complication, and near-infrared fluorescence cholangiography with indocyanine green (NIRFC-ICG) is one of the latest additions. In spite of the great interest aroused by this procedure, there are currently great disparities in the usage or administration protocols of ICG. METHODS AND ANALYSIS: This is a randomised, multicentre, per-protocol analysis, open clinical trial with four arms. The estimated duration of the trial is 12 months. The aim of the study is to analyse whether there are differences between the dose and administration ICG intervals to obtain good-quality NIRFC during LC. The primary outcome is the degree of identification of critical biliary structures during LC. In addition, different factors will be analysed that may have an influence on the results of this technique. ETHICS AND DISSEMINATION: The trial will be conducted according to the recommendations for Clinical Trials in the Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects and the recommendations of the Spanish Agency of Medicines and Medical Devices (AEMPs) for clinical trials. This trial was approved by the local institutional Ethics Committee and the AEMPs. The results of the study will be presented to the scientific community through publications, conferences or other means. EUDRACT NUMBER: 2022-000904-36. PROTOCOL VERSION: V.1.4, 2 June 2022 TRIAL REGISTRATION NUMBER: NCT05419947.

Systematic Evaluation of Antigenic Stimulation in Chronic Lymphocytic Leukemia: Humoral Immunity as Biomarkers for Disease Evolution.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Studies of CLL antibody reactivity have shown differential targets to autoantigens and antimicrobial molecular motifs that support the current hypothesis of CLL pathogenesis. METHODS: In this study, we conducted a quantitative serum analysis of 7 immunoglobulins in CLL and monoclonal B-cell lymphocytosis (MBL) patients (bead-suspension protein arrays) and a serological profile (IgG and IgM) study of autoantibodies and antimicrobial antigens (protein microarrays). RESULTS: Significant differences in the IgA levels were observed according to disease progression and evolution as well as significant alterations in IgG1 according to IGHV mutational status. More representative IgG autoantibodies in the cohort were against nonmutagenic proteins and IgM autoantibodies were against vesicle proteins. Antimicrobial IgG and IgM were detected against microbes associated with respiratory tract infections. CONCLUSIONS: Quantitative differences in immunoglobulin serum levels could be potential biomarkers for disease progression. In the top 5 tumoral antigens, we detected autoantibodies (IgM and IgG) against proteins related to cell homeostasis and metabolism in the studied cohort. The top 5 microbial antigens were associated with respiratory and gastrointestinal infections; moreover, the subsets with better prognostics were characterized by a reactivation of Cytomegalovirus. The viral humoral response could be a potential prognosis biomarker for disease progression.

Exploring High-Throughput Immunoassays for Biomarker Validation in Rheumatic Diseases in the Context of the Human Proteome Project.

Rheumatic diseases are high prevalence pathologies with different etiology and evolution and low sensitivity in clinical diagnosis. Therefore, it is necessary to develop an early diagnosis method which allows personalized treatment, depending on the specific pathology. The biology/disease initiative, at Human Proteome Project, is an integrative approach to identify relevant proteins in the human proteome associated with pathologies. A previously reported literature data mining analysis, which identified proteins related to osteoarthritis (OA), rheumatoid arthritis (RA), and psoriatic arthritis (PSA) was used to establish a systematic prioritization of potential biomarkers candidates for further evaluation by functional proteomics studies. The aim was to study the protein profile of serum samples from patients with rheumatic diseases such as OA, RA, and PSA. To achieve this goal, customized antibody microarrays (containing 151 antibodies targeting 121 specific proteins) were used to identify biomarkers related to early and specific diagnosis in a screening of 960 serum samples (nondepleted) (OA, n = 480; RA, n = 192; PSA, n = 288). This functional proteomics screening has allowed the determination of a panel (30 serum proteins) as potential biomarkers for these rheumatic diseases, displaying receiver operating characteristics curves with area under the curve values of 80-90%.

Light, moderate and vigorous physical activities: New insights into a virtuous circle with happiness.

The study of the physical activity engagement (PA) has given rise to a relevant research agenda in a wide range of fields, such as its close relationship with subjective well-being, self-perceived health and social capital. Previous evidence has identified interrelationships among these variables, but without considering different levels of physical activity. We have thus considered three levels of activity: light (walking), moderate and vigorous. Structural Equation Modelling (SEM) is undertaken on data from Spain's National Health Survey in 2011-2012 to analyse these interrelationships. The SEM shows a simultaneous and bidirectional relationship between different levels of PA (moderate and vigorous activities) and happiness, with a more robust association stemming from happiness to PA than vice versa. This relationship is mediated through health. From a policy perspective, this implies a virtuous circle: involvement in different levels of PA increases happiness and self-perceived health, while happiness involves higher PA and subsequent positive increases in health and happiness. Nevertheless, this virtuous circle does not always run successfully when social capital is considered to mediate the relationship between PA and happiness, which might explain why it has proven to be very difficult for health policymakers to fight against inactivity and a sedentary lifestyle within a great part of the population.HighlightsWe investigate bidirectional interrelationships between different levels of physical activity (PA) and happiness.We consider the mediation role played by self-perceived health (SPH) and social capital.Our results highlight a network of association between different levels of PA, SPH, social capital and happiness.SPH positively mediates this relationship for any type of PA level, whereas social capital only mediates positively when vigorous PA is developed.From a health policy perspective, the simultaneity between PA levels and happiness implies a virtuous circle.

Unravelling soluble immune checkpoints in chronic lymphocytic leukemia: Physiological immunomodulators or immune dysfunction.

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape.

Deciphering Biomarkers for Leptomeningeal Metastasis in Malignant Hemopathies (Lymphoma/Leukemia) Patients by Comprehensive Multipronged Proteomics Characterization of Cerebrospinal Fluid.

In the present work, leptomeningeal disease, a very destructive form of systemic cancer, was characterized from several proteomics points of view. This pathology involves the invasion of the leptomeninges by malignant tumor cells. The tumor spreads to the central nervous system through the cerebrospinal fluid (CSF) and has a very grim prognosis; the average life expectancy of patients who suffer it does not exceed 3 months. The early diagnosis of leptomeningeal disease is a challenge because, in most of the cases, it is an asymptomatic pathology. When the symptoms are clear, the disease is already in the very advanced stages and life expectancy is low. Consequently, there is a pressing need to determine useful CSF proteins to help in the diagnosis and/or prognosis of this disease. For this purpose, a systematic and exhaustive proteomics characterization of CSF by multipronged proteomics approaches was performed to determine different protein profiles as potential biomarkers. Proteins such as PTPRC, SERPINC1, sCD44, sCD14, ANPEP, SPP1, FCGR1A, C9, sCD19, and sCD34, among others, and their functional analysis, reveals that most of them are linked to the pathology and are not detected on normal CSF. Finally, a panel of biomarkers was verified by a prediction model for leptomeningeal disease, showing new insights into the research for potential biomarkers that are easy to translate into the clinic for the diagnosis of this devastating disease.

Tracking the Antibody Immunome in Sporadic Colorectal Cancer by Using Antigen Self-Assembled Protein Arrays.

Sporadic Colorectal Cancer (sCRC) is the third leading cause of cancer death in the Western world, and the sCRC patients presenting with synchronic metastasis have the poorest prognosis. Genetic alterations accumulated in sCRC tumor cells translate into mutated proteins and/or abnormal protein expression levels, which contribute to the development of sCRC. Then, the tumor-associated proteins (TAAs) might induce the production of auto-antibodies (aAb) via humoral immune response. Here, Nucleic Acid Programmable Protein Arrays (NAPPArray) are employed to identify aAb in plasma samples from a set of 50 sCRC patients compared to seven healthy donors. Our goal was to establish a systematic workflow based on NAPPArray to define differential aAb profiles between healthy individuals and sCRC patients as well as between non-metastatic (n = 38) and metastatic (n = 12) sCRC, in order to gain insight into the role of the humoral immune system in controlling the development and progression of sCRC. Our results showed aAb profile based on 141 TAA including TAAs associated with biological cellular processes altered in genesis and progress of sCRC (e.g., FSCN1, VTI2 and RPS28) that discriminated healthy donors vs. sCRC patients. In addition, the potential capacity of discrimination (between non-metastatic vs. metastatic sCRC) of 7 TAAs (USP5, ML4, MARCKSL1, CKMT1B, HMOX2, VTI2, TP53) have been analyzed individually in an independent cohort of sCRC patients, where two of them (VTI2 and TP53) were validated (AUC ~75%). In turn, these findings provided novel insights into the immunome of sCRC, in combination with transcriptomics profiles and protein antigenicity characterizations, wich might lead to the identification of novel sCRC biomarkers that might be of clinical utility for early diagnosis of the tumor. These results explore the immunomic analysis as potent source for biomarkers with diagnostic and prognostic value in CRC. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these novel sCRC immunomic biomarkers.

A Gene Signature Derived from the Loss of CDKN1A (p21) Is Associated with CMS4 Colorectal Cancer.

The epithelial-mesenchymal transition (EMT) is associated with tumor aggressiveness and increased invasion, migration, metastasis, angiogenesis, and drug resistance. Although the HCT116 p21-/- cell line is well known for its EMT-associated phenotype, with high Vimentin and low E-cadherin protein levels, the gene signature of this rather intermediate EMT-like cell line has not been determined so far. In this work, we present a robust molecular and bioinformatics analysis, to reveal the associated gene expression profile and its correlation with different types of colorectal cancer tumors. We compared the quantitative signature obtained with the NanoString platform with the expression profiles of colorectal cancer (CRC) Consensus Molecular Subtypes (CMS) as identified, and validated the results in a large independent cohort of human tumor samples. The expression signature derived from the p21-/- cells showed consistent and reliable numbers of upregulated and downregulated genes, as evaluated with two machine learning methods against the four CRC subtypes (i.e., CMS1, 2, 3, and 4). High concordance was found between the upregulated gene signature of HCT116 p21-/- cells and the signature of the CMS4 mesenchymal subtype. At the same time, the upregulated gene signature of the native HCT116 cells was similar to that of CMS1. Using a multivariate Cox regression model to analyze the survival data in the CRC tumor cohort, we selected genes that have a predictive risk power (with a significant gene risk incidence score). A set of genes of the mesenchymal signature was proven to be significantly associated with poor survival, specifically in the CMS4 CRC human cohort. We suggest that the gene signature of HCT116 p21-/- cells could be a suitable metric for mechanistic studies regarding the CMS4 signature and its functional consequences in CRC. Moreover, this model could help to discover the molecular mechanisms of intermediate EMT, which is known to be associated with extraordinarily high stemness and drug resistance.

Patterns of sports involvement in Spain.

AbstractIn a context of a rise in physical inactivity, this paper aims to provide new insights about the relationship between different forms of sport engagements and their correlates, analysing, for the first time, both active and passive sport involvement for a large sample of individuals. Applying the cluster technique, we identify four different segments of sports involvement among the Spanish population: non-sporty, exclusively practitioner, balanced practitioner, and basically spectator. Also, we develop a multinomial logit model to analyse the main sociodemographic, physical, and sports features that could increase the individual sport involvement, from the non-sporty segment, which assembles the highest number of individuals, to the other clusters. Most of the variables under analysis show a different impact on sport involvement: some of them stimulate active participation (e.g. being member of private sport clubs), whereas others mainly encourage sport attendance (e.g. readers and listeners of sport news, children in the household), and other variables boost sport engagement in general (e.g. being male, educational level, health status, e-sports). The results may have significant implications in terms of developing a general perspective about sport engagement, including both active and passive participation. Particularly, our findings suggest that active and passive sport engagement do not seem to be negatively associated and they can coexist at different levels.

Is Passive Sport Engagement Positively Associated with Happiness?

BACKGROUND: Previous empirical evidence has shown the positive relationship between happiness or subjective well-being (SWB) and sport participation. Nevertheless, passive sport participation has traditionally been ignored as a correlate with happiness. METHODS: Based on a sample of 1,632 Spanish people, one ordered probit model and three extended ordered probit models with an ordinal endogenous covariate technique and robust standard errors were applied. RESULTS: We find that different forms of passive sport participation-such as frequency of attending sporting events and a set of other forms of passive sports participation such as watching sports on TV, listening to sports programmes, reading sports news, and talking to others about sports-are positively associated with happiness. CONCLUSIONS: The results indicate that passive sport participation generally appears to have a closer relationship with individual happiness than active sport participation and emphasise the role played by some forms of sport participation as a source of relational goods. This current research extends the field's understanding of sport participation and happiness, including passive participation, and the relevance of social interactions to account for this association. Finally, the relational aspect of different forms of sport participation offers new implications for the analysis of sport engagement and happiness.

Identification of expression patterns in the progression of disease stages by integration of transcriptomic data.

BACKGROUND: In the study of complex diseases using genome-wide expression data from clinical samples, a difficult case is the identification and mapping of the gene signatures associated to the stages that occur in the progression of a disease. The stages usually correspond to different subtypes or classes of the disease, and the difficulty to identify them often comes from patient heterogeneity and sample variability that can hide the biomedical relevant changes that characterize each stage, making standard differential analysis inadequate or inefficient. RESULTS: We propose a methodology to study diseases or disease stages ordered in a sequential manner (e.g. from early stages with good prognosis to more acute or serious stages associated to poor prognosis). The methodology is applied to diseases that have been studied obtaining genome-wide expression profiling of cohorts of patients at different stages. The approach allows searching for consistent expression patterns along the progression of the disease through two major steps: (i) identifying genes with increasing or decreasing trends in the progression of the disease; (ii) clustering the increasing/decreasing gene expression patterns using an unsupervised approach to reveal whether there are consistent patterns and find genes altered at specific disease stages. The first step is carried out using Gamma rank correlation to identify genes whose expression correlates with a categorical variable that represents the stages of the disease. The second step is done using a Self Organizing Map (SOM) to cluster the genes according to their progressive profiles and identify specific patterns. Both steps are done after normalization of the genomic data to allow the integration of multiple independent datasets. In order to validate the results and evaluate their consistency and biological relevance, the methodology is applied to datasets of three different diseases: myelodysplastic syndrome, colorectal cancer and Alzheimer's disease. A software script written in R, named genediseasePatterns, is provided to allow the use and application of the methodology. CONCLUSION: The method presented allows the analysis of the progression of complex and heterogeneous diseases that can be divided in pathological stages. It identifies gene groups whose expression patterns change along the advance of the disease, and it can be applied to different types of genomic data studying cohorts of patients in different states.

Combined analysis of genome-wide expression and copy number profiles to identify key altered genomic regions in cancer.

BACKGROUND: Analysis of DNA copy number alterations and gene expression changes in human samples have been used to find potential target genes in complex diseases. Recent studies have combined these two types of data using different strategies, but focusing on finding gene-based relationships. However, it has been proposed that these data can be used to identify key genomic regions, which may enclose causal genes under the assumption that disease-associated gene expression changes are caused by genomic alterations.