Publications

Increased processor and storage capacities have supported the computational sciences, but have simultaneously unleashed a data avalanche on the scientific community. As a result, scientific research is limited by data analysis and visualization capabilities. These new bottlenecks have been the driving motivation behind the Princeton scalable display wall project. To create a scalable and easy-to-use large-format display system for collaborative visualization, the authors have developed various techniques, software tools, and applications.

In recent years, multiple types of high-throughput functional genomic data that facilitate rapid functional annotation of sequenced genomes have become available. Gene expression microarrays are the most commonly available source of such data. However, genomic data often sacrifice specificity for scale, yielding very large quantities of relatively lower-quality data than traditional experimental methods. Thus sophisticated analysis methods are necessary to make accurate functional interpretation of these large-scale data sets. This review presents an overview of recently developed methods that integrate the analysis of microarray data with sequence, interaction, localisation and literature data, and further outlines current challenges in the field. The focus of this review is on the use of such methods for gene function prediction, understanding of protein regulation and modelling of biological networks.

Background
Chromosomal copy number changes (aneuploidies) play a key role in cancer progression and molecular evolution. These copy number changes can be studied using microarray-based comparative genomic hybridization (array CGH) or gene expression microarrays. However, accurate identification of amplified or deleted regions requires a combination of visual and computational analysis of these microarray data.

Results
We have developed ChARMView, a visualization and analysis system for guided discovery of chromosomal abnormalities from microarray data. Our system facilitates manual or automated discovery of aneuploidies through dynamic visualization and integrated statistical analysis. ChARMView can be used with array CGH and gene expression microarray data, and multiple experiments can be viewed and analyzed simultaneously.

Conclusion
ChARMView is an effective and accurate visualization and analysis system for recognizing even small aneuploidies or subtle expression biases, identifying recurring aberrations in sets of experiments, and pinpointing functionally relevant copy number changes. ChARMView is freely available under the GNU GPL at http://function.princeton.edu/ChARMView.

MOTIVATION:
Chromosomal copy number changes (aneuploidies) are common in cell populations that undergo multiple cell divisions including yeast strains, cell lines and tumor cells. Identification of aneuploidies is critical in evolutionary studies, where changes in copy number serve an adaptive purpose, as well as in cancer studies, where amplifications and deletions of chromosomal regions have been identified as a major pathogenetic mechanism. Aneuploidies can be studied on whole-genome level using array CGH (a microarray-based method that measures the DNA content), but their presence also affects gene expression. In gene expression microarray analysis, identification of copy number changes is especially important in preventing aberrant biological conclusions based on spurious gene expression correlation or masked phenotypes that arise due to aneuploidies. Previously suggested approaches for aneuploidy detection from microarray data mostly focus on array CGH, address only whole-chromosome or whole-arm copy number changes, and rely on thresholds or other heuristics, making them unsuitable for fully automated general application to gene expression datasets. There is a need for a general and robust method for identification of aneuploidies of any size from both array CGH and gene expression microarray data.
RESULTS:
We present ChARM (Chromosomal Aberration Region Miner), a robust and accurate expectation-maximization based method for identification of segmental aneuploidies (partial chromosome changes) from gene expression and array CGH microarray data. Systematic evaluation of the algorithm on synthetic and biological data shows that the method is robust to noise, aneuploidal segment size and P-value cutoff. Using our approach, we identify known chromosomal changes and predict novel potential segmental aneuploidies in commonly used yeast deletion strains and in breast cancer. ChARM can be routinely used to identify aneuploidies in array CGH datasets and to screen gene expression data for aneuploidies or array biases. Our methodology is sensitive enough to detect statistically significant and biologically relevant aneuploidies even when expression or DNA content changes are subtle as in mixed populations of cells.
AVAILABILITY:
Code available by request from the authors and on Web supplement at http://function.cs.princeton.edu/ChARM/

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.

We used DNA microarrays representing >12,000 human genes to characterize gene expression patterns in skin biopsies from individuals with a diagnosis of systemic sclerosis with diffuse scleroderma. We found consistent differences in the patterns of gene expression between skin biopsies from individuals with scleroderma and those from normal, unaffected individuals. The biopsies from affected individuals showed nearly indistinguishable patterns of gene expression in clinically affected and clinically unaffected tissue, even though these were clearly distinguishable from the patterns found in similar tissue from unaffected individuals. Genes characteristically expressed in endothelial cells, B lymphocytes, and fibroblasts showed differential expression between scleroderma and normal biopsies. Analysis of lymphocyte populations in scleroderma skin biopsies by immunohistochemistry suggest the B lymphocyte signature observed on our arrays is from CD20+ B cells. These results provide evidence that scleroderma has systemic manifestations that affect multiple cell types and suggests genes that could be used as potential markers for the disease.

Gastric cancer is the world's second most common cause of cancer death. We analyzed gene expression patterns in 90 primary gastric cancers, 14 metastatic gastric cancers, and 22 nonneoplastic gastric tissues, using cDNA microarrays representing ∼30,300 genes. Gastric cancers were distinguished from nonneoplastic gastric tissues by characteristic differences in their gene expression patterns. We found a diversity of gene expression patterns in gastric cancer, reflecting variation in intrinsic properties of tumor and normal cells and variation in the cellular composition of these complex tissues. We identified several genes whose expression levels were significantly correlated with patient survival. The variations in gene expression patterns among cancers in different patients suggest differences in pathogenetic pathways and potential therapeutic strategies.

The vascular system is locally specialized to accommodate widely varying blood flow and pressure and the distinct needs of individual tissues. The endothelial cells (ECs) that line the lumens of blood and lymphatic vessels play an integral role in the regional specialization of vascular structure and physiology. However, our understanding of EC diversity is limited. To explore EC specialization on a global scale, we used DNA microarrays to determine the expression profile of 53 cultured ECs. We found that ECs from different blood vessels and microvascular ECs from different tissues have distinct and characteristic gene expression profiles. Pervasive differences in gene expression patterns distinguish the ECs of large vessels from microvascular ECs. We identified groups of genes characteristic of arterial and venous endothelium. Hey2, the human homologue of the zebrafish gene gridlock, was selectively expressed in arterial ECs and induced the expression of several arterial-specific genes. Several genes critical in the establishment of left/right asymmetry were expressed preferentially in venous ECs, suggesting coordination between vascular differentiation and body plan development. Tissue-specific expression patterns in different tissue microvascular ECs suggest they are distinct differentiated cell types that play roles in the local physiology of their respective organs and tissues.

Genomic sequencing is no longer a novelty, but gene function annotation remains a key challenge in modern biology. A variety of functional genomics experimental techniques are available, from classic methods such as affinity precipitation to advanced high-throughput techniques such as gene expression microarrays. In the future, more disparate methods will be developed, further increasing the need for integrated computational analysis of data generated by these studies. We address this problem with MAGIC (Multisource Association of Genes by Integration of Clusters), a general framework that uses formal Bayesian reasoning to integrate heterogeneous types of high-throughput biological data (such as large-scale two-hybrid screens and multiple microarray analyses) for accurate gene function prediction. The system formally incorporates expert knowledge about relative accuracies of data sources to combine them within a normative framework. MAGIC provides a belief level with its output that allows the user to vary the stringency of predictions. We applied MAGIC to Saccharomyces cerevisiae genetic and physical interactions, microarray, and transcription factor binding sites data and assessed the biological relevance of gene groupings using Gene Ontology annotations produced by the Saccaromyces Genome Database. We found that by creating functional groupings based on heterogeneous data types, MAGIC improved accuracy of the groupings compared with microarray analysis alone. We describe several of the biological gene groupings identified.

Analysis of the patterns of gene expression in follicular lymphomas from 24 patients suggested that two groups of tumors might be distinguished. All patients, whose biopsies were obtained before any treatment, were treated with rituximab, a monoclonal antibody directed against the B cell antigen, CD20. Gene expression patterns in the tumors that subsequently failed to respond to rituximab appeared more similar to those of normal lymphoid tissues than to gene expression patterns of tumors from rituximab responders. These findings suggest the possibility that the response of follicular lymphoma to rituximab treatment may be predicted from the gene expression pattern of tumors.

Follicular non-Hodgkin's lymphoma (NHL) is an indolent B cell malignancy with an annual incidence exceeding 10,000 cases in the United States. Although follicular lymphoma (FL) is frequently responsive to treatment, therapy is very rarely, if ever, curative. Rituximab, a chimeric IgG1 monoclonal antibody directed at the B cell antigen CD20, has become a mainstay of treatment for low-grade NHL; >400,000 patients worldwide have been treated with rituximab. Phase II trials of rituximab in patients with refractory or relapsed low grade or follicular NHL demonstrated a 50% response rate (1).

Despite this extensive clinical experience, the mechanism of action of rituximab remains unclear, as does the nature of resistance (2). Among the proposed mechanisms are antibody-dependent cell-mediated cytotoxicity (3), complement-mediated cytotoxicity (4), and direct cytotoxicity through modulating CD20 function (5–7). The association with resistance to rituximab treatment of a low-affinity variant of the Fc receptor (8) is suggestive of an immune mechanism, and remains the only plausible hint about the nature of resistance.

In this study, we examined whether gene expression profiling using cDNA microarrays could reveal biological diversity among follicular lymphomas and, more specifically, whether gene expression patterns in tumors might predict sensitivity to rituximab treatment.