Analysis of gene expression data using self-organizing maps

There is a growing need for a method to analyze massive gene expression data obtained from DNA microarrays. We introduce a method of combining a self-organizing map (SOM) and a k-means clustering for analyzing and categorizing the gene expression data. The SOM is an unsupervised neural network learning algorithm and forms a mapping the high-dimensional data to two-dimensional space. However, it is difficult to find clustering boundaries from results of the SOM. On the other hand, the k-means clustering can partition the data into the clusters under the assumption of the known number of clusters. In order to understand easily the results of SOM, we apply the k-means clustering to finding the clustering boundaries from results of SOM. We have applied the proposed method to the published data of yeast gene expression and show that the method is effective for categorizing the data.

BackgroundSerial Analysis of Gene Expression (SAGE) is a DNA sequencing-based method for large-scale gene expression profiling that provides an alternative to microarray analysis. Most analyses of SAGE data aimed at identifying co-expressed genes have been accomplished using various versions of clustering approaches that often result in a number of false positives.Principal FindingsHere we explore the use of seriation, a statistical approach for ordering sets of objects based on their similarity, for large-scale expression pattern discovery in SAGE data. For this specific task we implement a seriation heuristic we term ‘progressive construction of contigs’ that constructs local chains of related elements by sequentially rearranging margins of the correlation matrix. We apply the heuristic to the analysis of simulated and experimental SAGE data and compare our results to those obtained with a clustering algorithm developed specifically for SAGE data. We show using simulations that the performance of seriation compares favorably to that of the clustering algorithm on noisy SAGE data.ConclusionsWe explore the use of a seriation approach for visualization-based pattern discovery in SAGE data. Using both simulations and experimental data, we demonstrate that seriation is able to identify groups of co-expressed genes more accurately than a clustering algorithm developed specifically for SAGE data. Our results suggest that seriation is a useful method for the analysis of gene expression data whose applicability should be further pursued.

Methods and approaches in the analysis of gene expression data

Background - In the NeoTRIPaPDL1 phase III trial, triple-negative breast cancer (TNBC) patients were randomized to receive nab-paclitaxel/carboplatin for 8 cycles (CT arm) with or without atezolizumab (CT/A arm). We previously reported that the majority of patients (65%) with PD-L1- at baseline converted to PD-L1+ (the majority with IC2/IC3) after the first cycle of treatment in the atezolizumab arm. Here we studied treatment associated changes in the molecular tumour features and immune microenvironment by PD-L1 expression. Methods – A total of 158 (56.4%) of patients enrolled in the NeoTRIPaPDL1 trial (76 and 82 from CT/A and CT arm, respectively) were included in this analysis by satisfying the following criteria: i) availability of a paired core biopsy at baseline and at day 1 of the second treatment cycle (D1C2); ii) evaluation of stromal TILs (sTILs) and staining for PD-L1 (Ventana SP142); iii) successful RNA-seq gene expression profiling. PD-L1 groups were defined as IC0/IC1 (<5% PD-L1low) vs IC2/IC3 (>=5% PD-L1high). Presence of different immune cell populations was quantified by gene expression profile deconvolution using ConsensusTME R package. Cancer hallmark gene set collection and custom signature activation status were estimated in each sample using singscore R package. Differences in score distribution were evaluated by 2-sided t-test. Results At baseline, both sTILs and immune cell signatures were upregulated in PD-L1high compared to PD-L1low tumours (p<0.001). No significant differences were found between the two treatment arms in the PD-L1high subpopulation. In the PD-L1low cases, both sTILs and immune related signatures were slightly downregulated in the CT/A arm (p<0.05), suggesting a modest unbalance among treatment arms with CT arm being slightly more inflamed than CT/A arm.At D1C2, PD-L1high tumours in the CT arm systematically had high sTILs (median=70%, range=30-90%), while PD-L1low tumours receiving CT/A had low sTILs in a significant proportion of cases (median=30%, range=0-90%; p<0.001). Similarly, at D1C2 several gene expression-estimated immune cell populations and immune-related signatures were upregulated in the CT arm compared to CT/A arm in PD-L1high tumours, with the weakest association observed for M2 macrophages (p=0.058). No tumour-related signatures were differentially expressed among the two treatment arms within groups with PD-L1high, suggesting that different treatment. modulate PD-L1 by engaging a different immune mileau instead of modulating tumor related features.Considering PD-L1low groups at D1C2, in the CT arm 21 tumors had >30% sTILs (n=21/69, 30.4%), while in the CT/A arm only 2 had sTILs >30% (2/27, 7.4%) (p<0.001). Analysis of gene expression data identified IFN-related signatures as the most upregulated in the CT compared to CT/A arm in PD-L1low cases at D1C2 (p<0.05). Conclusions Integrated dynamic analysis of PD-L1 expression and gene expression data highlighted significant treatment-specific changes of the immune landscape according to PD-L1 expression, when this biomarker is assessed during treatment. This indicates that the immune milieu associated with PD-L1 status is strongly dependent on the time of assessment in relationship to treatment received. Such observation may explain why in the NeoTRIP trial, baseline PD-L1 but not on-treatment PD-L1 was predictive of pCR in CT/A arm. In addition, our findings could have implications related to the use of PD-L1 as a predictive biomarker in pre-treated patients, especially when assessed early on during treatment. Citation Format: Maurizio Callari, Chiun-Sheng Huang, Daniel Egle, Begoña Bermejo, Claudio Zamagni, Matteo Dugo, Marc Thill, Antonio Anton, Marco Barreca, Stefania Russo, Eva Maria Ciruelos, Richard Greil, Stefania Zambelli, Balázs Gyorffy, Chanel Smart, Olivia Biasi, Pinuccia Valagussa, Giuseppe Viale, Luca Gianni, Giampaolo Bianchini. Immune milieu associated with PD-L1 status in TNBC is dependent on time of biomarker assessment and treatment received: A secondary analysis of the NeoTRIPaPDL1 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-02.

We present a method for individual and integrative analysis of high dimension, low sample size data that capitalizes on the recurring theme in multivariate analysis of projecting higher dimensional data onto a few meaningful directions that are solutions to a generalized eigenvalue problem. We propose a general framework, called SELP (Sparse Estimation with Linear Programming), with which one can obtain a sparse estimate for a solution vector of a generalized eigenvalue problem. We demonstrate the utility of SELP on canonical correlation analysis for an integrative analysis of methylation and gene expression profiles from a breast cancer study, and we identify some genes known to be associated with breast carcinogenesis, which indicates that the proposed method is capable of generating biologically meaningful insights. Simulation studies suggest that the proposed method performs competitive in comparison with some existing methods in identifying true signals in various underlying covariance structures.

Recent research has focused on incorporating biological function and pathway information into the analysis of gene expression data, partly as a means of compensating for insufficient experimental replications, low signal to noise, lack of reproducibility and/or multiple testing confounds. A Bayesian approach seems to be ideal for incorporating functional information into gene expression data analysis. In this study, we tested the feasibility of using literature derived gene relationships in a Bayesian model to analyze gene expression data. Prior distributions were constructed based on gene associations derived from the biomedical literature using Latent Semantic Indexing (LSI). The LSI model was built using more than 1 million Medline abstracts corresponding to 22,000 human and mouse genes. A key advantage of LSI is that both explicit and implicit gene relationships can be derived from the literature. Gene neighborhoods were determined using latent Gaussian Markov random fields and logistic transformation of the latent variables. We tested the procedure on a microarray dataset for interferon-stimulated genes in mouse embryonic fibroblasts. By integrating functional information from literature, Bayesian approach identified relevant genes that previously did not meet the 0.05 significance level. In comparison to a standard mixture model, spatial mixture model has more power for identifying direct and indirect interferon regulated genes. The spatial model enhanced the ranks of some genes which are known to be affected by interferon treatment, such as Nmi (NMI N-myc and STAT interactor) and ifi35 (interferon-induced protein 35). It also identified some genes that previously were ignored because of the marginal p-values, such as dpysl2, map2k1, msn, Psck5, and Il6st. Interestingly, these genes appear to be indirectly related to interferon treatment. In summary, we show that our procedure increases statistical power and produces more biologically meaningful gene lists. These results suggest that Bayesian methods which incorporate functional information from the literature may improve analysis of gene expression data.

Today, DNA microarray technology is the most widely employed functional genomics approach in the bioinformatics domain. Disclosing the patterns masked in gene expression data provides an opportunity for an enhanced comprehension of functional genomics. But, the large number of genes and the intricate biological networks has greatly increased the challenges of comprehending and interpreting the resulting mass of data, which often contains millions of measurements. So, clustering techniques are used to tackle this challenge. Several clustering techniques have been developed and used in the analysis of microarray data. But, such techniques are not much efficient in analyzing the gene expression data. In this paper, we propose a novel efficient hybrid clustering approach based on quad tree data structure. In the hierarchical clustering process, the creation of the closest pair data structure in each level is an important time factor that provides the processing time of clustering. Here, a quad tree based data structure is used for identifying the closest pair elements. Thus, the proposed approach reduces the processing time successfully and offers a better analysis of gene expression data.

The emergence of large-scale multi-omics data warrants method development for data integration. Genomic studies from cancer patients have identified epigenetic and genetic regulators – such as methylation marks, somatic mutations, and somatic copy number alterations (SCNAs), among others – as predictive features of cancer outcome. However, identification of “driver genes” associated with a given alteration remains a challenge. To this end, we developed a computational tool, iEDGE, to model cis and trans effects of (epi-)DNA alterations and identify potential cis driver genes, where cis and trans genes denote those genes falling within and outside the genomic boundaries of a given (epi-)genetic alteration, respectively. iEDGE first identifies the cis and trans gene expression signatures associated with the presence/absence of a particular epi-DNA alteration across samples. It then applies tests of statistical mediation to determine the cis genes predictive of the trans gene expression. Finally, cis and trans effects are annotated by pathway enrichment analysis to gain insights into the underlying regulatory networks. We used iEDGE to perform integrative analysis of SCNAs and gene expression data from breast cancer and 18 additional cancer types included in The Cancer Genome Atlas (TCGA). Notably, cis gene drivers identified by iEDGE were found to be significantly enriched for known driver genes from multiple compendia of validated oncogenes and tumor suppressors, suggesting that the remainder are of equal importance. Furthermore, predicted drivers were enriched for functionally relevant cancer genes with amplification-driven dependencies, which are of potential prognostic and therapeutic value. All the analyses results are accessible at https://montilab.bu.edu/iEDGE. In summary, integrative analysis of SCNAs and gene expression using iEDGE successfully identified known cancer driver genes and putative cancer therapeutic targets across 19 cancer types in the TCGA. The proposed method can easily be applied to the integration of gene expression profiles with other epi-DNA assays in a variety of disease contexts.

In order to understand even the simplest cellular processes, we need to integrate proteomic, gene expression and other biomolecular data. To date, most computational approaches aimed at integrating proteomics and gene expression data use direct gene/protein correlation measures. However, due to post-transcriptional and translational regulations, the correspondence between the expression of a gene and its protein is complicated. We apply a multivariate statistical method, co-inertia analysis (CIA), to visualise gene and proteomic expression data stemming from the same biological samples. Principal components analysis or correspondence analysis can be used for data exploration on single datasets. CIA is then used to explore the relationships between two or more datasets. We further explore the data by projecting gene ontology (GO) information onto these plots to describe the cellular processes in action. We apply these techniques to gene expression and protein abundance data from studies of the human malarial parasite life cycle and the NCI-60 cancer cell lines. In each case, we visualise gene expression, protein abundance and GO classes in the same low dimensional projections and identify GO classes that are likely to be of biological importance.

The accumulated gene expression and DNA methylation data provide a great opportunity to exploit the mechanisms of biological systems. Current algorithms for the integration of gene expression and methylation data are characterized for undesirable performance because they fail to address the latent relations in the heterogeneous data. To solve this problem, we propose a novel multi-view clustering with self-representation learning and low-rank tensor constraint (MCSL-LTC), where the gene expression and DNA methylation data are treated as complementary views, and MCSL-LTC obtains a consensus partitioning reflecting the structure and features of various views. Specifically, self-representation learning is employed to explore the low-dimensional subspace structures embedded in different views, where the tensor norm is adopted to smooth different views, therefore improving the quality of features. Experimental results demonstrate that the proposed approach outperforms state-of-the-art baselines in terms of accuracy on both the social and cancer data, provides an effective and efficient method for the integration of heterogeneous genomic data.

Analysis of temporal gene expression data poses a significant challenge due to the combination of high dimensionality and low sample size. The purpose of this paper is to present a methodology for classification, modelling, and analysis of short time-series gene expression data using spiking neural networks (SNN) and to uncover temporal expression patterns for knowledge discovery. The classification is based on the NeuCube SNN model. Time-series gene expression data of mouse primary cortical neurons is examined as a case study. The results of the analysis are promising, indicating that SNN methodologies can be effectively used to model and analyse temporal gene expression data with surpassing performance over traditional machine learning algorithms. Additionally, a gene interaction network is constructed from the temporal gene activity modelled using the NeuCube architecture offering a new way of knowledge discovery. Future work will be directed towards using gene interactions networks to help guide pharmacological research for dementia.

Genome-wide analysis of cellular transcriptomes using RNA-seq or expression arrays is a major mainstay of current biological and biomedical research. EXPANDER (EXPression ANalyzer and DisplayER) is a comprehensive software package for analysis of expression data, with built-in support for 18 different organisms. It is designed as a “one-stop shop” platform for transcriptomic analysis, allowing for execution of all analysis steps starting with gene expression data matrix. Analyses offered include low-level preprocessing and normalization, differential expression analysis, clustering, bi-clustering, supervised grouping, high-level functional and pathway enrichment tests, and networks and motif analyses. A variety of options is offered for each step, using established algorithms, including many developed and published by our laboratory.EXPANDER has been continuously developed since 2003, having to date over 18,000 downloads and 540 citations. One of the innovations in the recent version is support for combined analysis of gene expression and ChIP-seq data to enhance the inference of transcriptional networks and their functional interpretation. EXPANDER implements cutting-edge algorithms and makes them accessible to users through user-friendly interface and intuitive visualizations. It is freely available to users at http://acgt.cs.tau.ac.il/expander/.

Changes in gene expression programs play a central role in cancer. Chromosomal aberrations such as deletions, duplications and translocations of DNA segments can lead to highly significant positive correlations of gene expression levels of neighboring genes. This should be utilized to improve the analysis of tumor expression profiles. Here, we develop a novel model class of autoregressive higher-order Hidden Markov Models (HMMs) that carefully exploit local data-dependent chromosomal dependencies to improve the identification of differentially expressed genes in tumor. Autoregressive higher-order HMMs overcome generally existing limitations of standard first-order HMMs in the modeling of dependencies between genes in close chromosomal proximity by the simultaneous usage of higher-order state-transitions and autoregressive emissions as novel model features. We apply autoregressive higher-order HMMs to the analysis of breast cancer and glioma gene expression data and perform in-depth model evaluation studies. We find that autoregressive higher-order HMMs clearly improve the identification of overexpressed genes with underlying gene copy number duplications in breast cancer in comparison to mixture models, standard first- and higher-order HMMs, and other related methods. The performance benefit is attributed to the simultaneous usage of higher-order state-transitions in combination with autoregressive emissions. This benefit could not be reached by using each of these two features independently. We also find that autoregressive higher-order HMMs are better able to identify differentially expressed genes in tumors independent of the underlying gene copy number status in comparison to the majority of related methods. This is further supported by the identification of well-known and of previously unreported hotspots of differential expression in glioblastomas demonstrating the efficacy of autoregressive higher-order HMMs for the analysis of individual tumor expression profiles. Moreover, we reveal interesting novel details of systematic alterations of gene expression levels in known cancer signaling pathways distinguishing oligodendrogliomas, astrocytomas and glioblastomas. An implementation is available under www.jstacs.de/index.php/ARHMM.

The application of mathematics in the field of bioinformatics has been widely developed. For example Support Vector Machines (SVM) and Random Forest (RF) are state of the art for classification of cancer in many applications. One of them is Chronic Kidney Disease (CKD). CKD is one of the kidney diseases that sufferers are increasing and have symptoms that are difficult to detect at first. Later, microarrays in gene expression are important tools for this approach. Microarrays gene expression provides an overview of all transcription activities in biological samples. The purpose of this research is a hybrid model combining Random Forest (RF) and Support Vector Machine (SVM) can be used to classify gene expression data. RF can highly accurate, generelize better and are interpretable and SVM (called RF-SVM) to effectively predict gene expression data with very high dimensions. In addition, from the simulation results on data from the Gene Expression Omnibus (GEO) database, it is shown that the proposed RF-SVM is a more accurate algorithm on CKD data than RFE-SVM.

Introduction: Molecularly targeted agents, such as the BRAF inhibitor vemurafenib, may produce short-term responses in some patients; however, most patients are intrinsically resistant, or develop resistance through restructuring of signal transduction pathways. SnapPath™ is a live-cell-processing platform that utilizes ex vivo signal transduction modulation of live tumor samples to produce Functional Signaling Profiles (FSPs). Application of this technology to Champions TumorGraft models may provide novel insights to guide oncology drug development as these models preserve the biological properties of the original human tumor. Methods: Fresh melanoma tumor specimens were collected from patients and implanted into immunodeficient mice. Fine needle aspiration biopsies were performed on each melanoma TumorGraft model and processed on the SnapPath™ platform (BioMarker Strategies) to modulate tumor cell signal transduction networks through brief ex vivo exposure to the vemurafenib tool compound PLX-4720. Cell lysates were then analyzed using a multiplexed immunoassay to assess the inhibition of the downstream MAPK markers pMEK1 and pERK-1/2. FSPs were then created for each TumorGraft model based on baseline and modulated levels of each phosphoprotein. In parallel, the in vivo sensitivity to vemurafenib and BRAF mutation status was evaluated in each Champions TumorGraft model. FSPs were then compared with in vivo efficacy, gene expression and genotype data. Results: Functional profiling stratified the TumorGraft models into two distinct groups upon ex vivo exposure to a BRAF inhibitor: 1) MAPK markers suppressed and 2) MAPK markers not suppressed. As anticipated, TumorGraft models that showed resistance to ex vivo BRAF inhibition demonstrated vemurafenib resistance in vivo and were BRAF wild type. There were other models that displayed MAPK suppression with ex vivo BRAF inhibition and vemurafenib sensitivity in vivo or MAPK suppression with ex vivo BRAF inhibition but demonstrated vemurafenib resistance in vivo. One of these TumorGrafts contained a BRAF V600E mutation, suggesting the activation of an alternate pathway that conferred resistance. The other TumorGraft contained a novel BRAF insertion. The functional profiling suggests that this insertion may activate BRAF and is susceptible to vemurafenib inhibition, but the tumor may contain an alternate pathway that confers resistance. Analysis of gene expression data demonstrated hierarchical clustering of BRAF mutated TumorGraft models. Conclusions: These results demonstrate the capability of the SnapPath™ platform to generate FSPs from FNAs of Champions melanoma TumorGraft models. Overall, the combination of Champions TumorGraft models with functional profiling represents a powerful tool for pharmacodynamic assessment of targeted therapeutics in clinically relevant models and has the potential to guide oncology therapy. Citation Format: Elizabeth M. Bruckheimer, Adam Schayowitz, Kala Barnes, Greg Bertenshaw, Tin Khor, James Cotton, Jay Friedman, Dhanrajan Tiruchinapalli, Douglas P. Clark. Functional profiling of Champions TumorGraft™ models from metastatic melanoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3498. doi:10.1158/1538-7445.AM2013-3498