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A generalized clustering problem, with application to DNA microarrays.

We think of cluster analysis as class discovery. That is, we assume that there is an unknown mapping called clustering structure that assigns a class label to each observation, and the goal of cluster analysis is to estimate this clustering structure, that is, to estimate the number of clusters and cluster assignments. In traditional cluster analysis, it is assumed that such unknown mapping is unique. However, since the observations may cluster in more than one way depending on the variables used, it is natural to permit the existence of more than one clustering structure. This generalized clustering problem of estimating multiple clustering structures is the focus of this paper. We propose an algorithm for finding multiple clustering structures of observations which involves clustering both variables and observations. The number of clustering structures is determined by the number of variable clusters. The dissimilarity measure for clustering variables is based on nearest-neighbor graphs. The observations are clustered using weighted distances with weights determined by the clusters of the variables. The motivating application is to gene expression data.

Algorithms↗

Global analysis of shear stress-responsive genes in vascular endothelial cells.

DNA microarray gene expression analysis was conducted in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) exposed to laminar or turbulent shear stress. Approximately 3% of the total 5600 gene in HUVECs and HCAECs increased their expression more than two-fold or decreased it to less than half the static control in response to an arterial level of laminar shear stress (15 dynes/cm(2) for 24 hours). The proportions of shear-stress-responsive genes decreased to around 2% under the venous level of laminar shear stress (1.5 dynes/cm(2)) in both cell lines. Turbulent shear stress of 1.5 dynes/cm(2) altered the expression of 1.1% of all genes in the HCAECs. Laminar shear stress, but not turbulent shear stress, decreased the expression of a number of genes involved in DNA synthesis and the cell cycle in both HUVECs and HCAECs. Clustering analysis showed a variety of temporal profiles of gene expression in HUVECs exposed to laminar shear stress of 15 dynes/cm(2) for 3, 6, 12, 24, and 48 hours. Turbulent shear stress affected expression of many genes that play a role in vascular remodeling, including genes encoding plasminogen activators and their inhibitor, endothelin-1, transforming growth factor-beta, collagen type IV, and ephrin A1.

Arteriosclerosis↗

Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma.

The pathological distinction between malignant pleural mesothelioma (MPM)and adenocarcinoma (ADCA) of the lung can be cumbersome using established methods. We propose that a simple technique, based on the expression levels of a small number of genes, can be useful in the early and accurate diagnosis of MPM and lung cancer. This method is designed to accurately distinguish between genetically disparate tissues using gene expression ratios and rationally chosen thresholds. Here we have tested the fidelity of ratio-based diagnosis in differentiating between MPM and lung cancer in 181 tissue samples (31 MPM and 150 ADCA). A training set of 32 samples (16 MPM and 16 ADCA) was used to identify pairs of genes with highly significant, inversely correlated expression levels to form a total of 15 diagnostic ratios using expression profiling data. Any single ratio of the 15 examined was at least 90% accurate in predicting diagnosis for the remaining 149 samples (e.g., test set). We then examined (in the test set) the accuracy of multiple ratios combined to form a simple diagnostic tool. Using two and three expression ratios, we found that the differential diagnoses of MPM and lung ADCA were 95% and 99% accurate, respectively. We propose that using gene expression ratios is an accurate and inexpensive technique with direct clinical applicability for distinguishing between MPM and lung cancer. Furthermore, we provide evidence suggesting that this technique can be equally accurate in other clinical scenarios.

Adenocarcinoma↗

Genome-wide analysis of gene expression in synovial sarcomas using a cDNA microarray.

Among a histologically heterogeneous group of soft tissue sarcomas, synovial sarcoma (SS) is regarded as a "miscellaneous" entity of uncertain origin. Although recent molecular analysis has disclosed involvement of a specific chromosomal translocation in the pathogenesis of SS, its genetic features remain largely unclear. In the work reported here we examined genome-wide gene expression profiles of 13 SS cases and 34 other spindle-cell sarcoma cases by cDNA microarray consisting of 23,040 genes. A hierarchical clustering analysis grouped SS and malignant peripheral nerve sheath tumor into the same category, and these two types of tumor shared expression patterns of numerous genes relating to neural differentiation. Several genes were up-regulated in almost all SS cases, and the presumed functions of known genes among them were related to migration or differentiation of neural crest cells, suggesting the possibility of neuroectodermal origin of SS. Moreover, we identified a set of genes that divided SS cases into two putative subclasses, a feature that may shed light on novel biological aspects of SS in addition to those having to do with epithelial differentiation. These data have provided clues for understanding the origin and tumorigenesis of SS.

Cluster Analysis↗

Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse.

Current models of prostate cancer classification are poor at distinguishing between tumors that have similar histopathological features but vary in clinical course and outcome. Here, we applied classical survival analysis to genome-wide gene expression profiles of prostate cancers and preoperative prostate-specific antigen (PSA) levels from each patient, to identify prognostic markers of disease relapse that provide additional predictive value relative to PSA concentration. Three of approximately 200 probesets showing strongest correlation with relapse were identified as the gene for the putative calcium channel protein, trp-p8, with loss of trp-p8 mRNA expression associated with a significantly shorter time to PSA relapse-free survival. We observed subsequently that trp-p8 is lost in the transition to androgen independence in a prostate cancer xenograft model and in prostate cancer tissue from patients treated preoperatively with antiandrogen therapy, suggesting that trp-p8 is androgen regulated, and its loss may be associated with more advanced disease. The identification of trp-p8 and other proteins implicated in the phosphatidylinositol signal transduction pathway that are associated with prostate cancer outcome, both here and in other published work, suggests an integral role for this pathway in prostate carcinogenesis. Thus, our findings demonstrate that multivariable survival analysis can be applied to gene expression profiles of prostate cancers with censored follow-up data and used to identify molecular markers of prostate cancer relapse with strong predictive power and relevance to the etiology of this disease.

Animals↗

A comparison of cDNA, oligonucleotide, and Affymetrix GeneChip gene expression microarray platforms.

We have conducted a study to compare the variability in measured gene expression levels associated with three types of microarray platforms. Total RNA samples were obtained from liver tissue of four male mice, two each from inbred strains A/J and C57BL/6J. The same four samples were assayed on Affymetrix Mouse Genome Expression Set 430 GeneChips (MOE430A and MOE430B), spotted cDNA microarrays, and spotted oligonucleotide microarrays using eight arrays of each type. Variances associated with measurement error were observed to be comparable across all microarray platforms. The MOE430A GeneChips and cDNA arrays had higher precision across technical replicates than the MOE430B GeneChips and oligonucleotide arrays. The Affymetrix platform showed the greatest range in the magnitude of expression levels followed by the oligonucleotide arrays. We observed good concordance in both estimated expression level and statistical significance of common genes between the Affymetrix MOE430A GeneChip and the oligonucleotide arrays. Despite their apparently high precision, cDNA arrays showed poor concordance with other platforms.

Analysis of Variance↗

[The diagnosis of acute myeloid leukaemia enhanced by using DNA microarrays].

Recently, two studies have shown that the use ofgene-expression profiling using DNA microarrays or DNA chips may improve the classification of acute myeloid leukaemia (AML). In both studies, cluster analyses based on the molecular signatures defined known subgroups as well as novel subgroups of AML. Chromosomal lesions, mutations, and abnormal gene expression with prognostic value determined the clustering. In fact, gene-expression profiling recognized leukaemias with certain chromosomal aberrations that had been missed by routine cytogenetics. Thus, gene-expression profiling allows a comprehensive classification of AML that includes previously-identified genetically-defined as well as novel prognostically-relevant subgroups. One comprehensive DNA chip may in the future replace a variety of cytogenetic, immunological and molecular techniques that are currently used in combination.

Acute Disease↗

Fast and sensitive probe selection for DNA chips using jumps in matching statistics.

The design of large scale DNA microarrays is a challenging problem. So far, probe selection algorithms must trade the ability to cope with large scale problems for a loss of accuracy in the estimation of probe quality. We present an approach based on jumps in matching statistics that combines the best of both worlds. This article consists of two parts. The first part is theoretical. We introduce the notion of jumps in matching statistics between two strings and derive their properties. We estimate the frequency of jumps for random strings in a non-uniform Bernoulli model and present a new heuristic argument to find the center of the length distribution of the longest substring that two random strings have in common. The results are generalized to near-perfect matches with a small number of mismatches. In the second part, we use the concept of jumps to improve the accuracy of the longest common factor approach for probe selection by moving from a string-based to an energy-based specificity measure, while only slightly more than doubling the selection time.

Algorithms↗

RNA isolation from yeast using silica matrices.

RNA isolation from yeast is complicated by the need to initially break the cell wall. While this can be accomplished by glass bead disruption or enzyme treatment, these approaches result in DNA contamination and/or the need for incubation periods. We have developed a protocol for the isolation of RNA samples from yeast that minimizes degradation by RNases and incorporates two purification steps: acid phenol extraction and binding to a silica matrix. The procedure requires no precipitation steps, facilitating automation, and can be completed in less than 90 min. The RNA quality is ideal for microarray analysis.

Biotechnology↗

SpotWhatR: a user-friendly microarray data analysis system.

SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely available R statistical language (http://www.r-project.org) for Windows and Linux operational systems. The aim of SpotWhatR is to help the researcher to analyze microarray data by providing basic tools for data visualization, normalization, determination of differentially expressed genes, summarization by Gene Ontology terms, and clustering analysis. SpotWhatR allows researchers who are not familiar with computational programming to choose the most suitable analysis for their microarray dataset. Along with well-known procedures used in microarray data analysis, we have introduced a stand-alone implementation of the HTself method, especially designed to find differentially expressed genes in low-replication contexts. This approach is more compatible with our local reality than the usual statistical methods. We provide several examples derived from the Blastocladiella emersonii and Xylella fastidiosa Microarray Projects. SpotWhatR is freely available at http://blasto.iq.usp.br/~tkoide/SpotWhatR, in English and Portuguese versions. In addition, the user can choose between "single experiment" and "batch processing" versions.

Blastocladiella↗