Kenneth H. Buetow

Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http://mgc.nci.nih.gov).

PID: the Pathway Interaction Database

The Pathway Interaction Database (PID, http://pid.nci.nih.gov) is a freely available collection of curated and peer-reviewed pathways composed of human molecular signaling and regulatory events and key cellular processes. Created in a collaboration between the US National Cancer Institute and Nature Publishing Group, the database serves as a research tool for the cancer research community and others interested in cellular pathways, such as neuroscientists, developmental biologists and immunologists. PID offers a range of search features to facilitate pathway exploration. Users can browse the predefined set of pathways or create interaction network maps centered on a single molecule or cellular process of interest. In addition, the batch query tool allows users to upload long list(s) of molecules, such as those derived from microarray experiments, and either overlay these molecules onto predefined pathways or visualize the complete molecular connectivity map. Users can also download molecule lists, citation lists and complete database content in extensible markup language (XML) and Biological Pathways Exchange (BioPAX) Level 2 format. The database is updated with new pathway content every month and supplemented by specially commissioned articles on the practical uses of other relevant online tools.

Global transcription in pluripotent embryonic stem cells.

The molecular mechanisms underlying pluripotency and lineage specification from embryonic stem cells (ESCs) are largely unclear. Differentiation pathways may be determined by the targeted activation of lineage-specific genes or by selective silencing of genome regions. Here we show that the ESC genome is transcriptionally globally hyperactive and undergoes large-scale silencing as cells differentiate. Normally silent repeat regions are active in ESCs, and tissue-specific genes are sporadically expressed at low levels. Whole-genome tiling arrays demonstrate widespread transcription in coding and noncoding regions in ESCs, whereas the transcriptional landscape becomes more discrete as differentiation proceeds. The transcriptional hyperactivity in ESCs is accompanied by disproportionate expression of chromatin-remodeling genes and the general transcription machinery. We propose that global transcription is a hallmark of pluripotent ESCs, contributing to their plasticity, and that lineage specification is driven by reduction of the transcribed portion of the genome.

The BioPAX community standard for pathway data sharing

Biological Pathway Exchange (BioPAX) is a standard language to represent biological pathways at the molecular and cellular level and to facilitate the exchange of pathway data. The rapid growth of the volume of pathway data has spurred the development of databases and computational tools to aid interpretation; however, use of these data is hampered by the current fragmentation of pathway information across many databases with incompatible formats. BioPAX, which was created through a community process, solves this problem by making pathway data substantially easier to collect, index, interpret and share. BioPAX can represent metabolic and signaling pathways, molecular and genetic interactions and gene regulation networks. Using BioPAX, millions of interactions, organized into thousands of pathways, from many organisms are available from a growing number of databases. This large amount of pathway data in a computable form will support visualization, analysis and biological discovery.

CREBBP mutations in relapsed acute lymphoblastic leukaemia

Relapsed acute lymphoblastic leukaemia (ALL) is a leading cause of death due to disease in young people, but the biological determinants of treatment failure remain poorly understood. Recent genome-wide profiling of structural DNA alterations in ALL have identified multiple submicroscopic somatic mutations targeting key cellular pathways, and have demonstrated substantial evolution in genetic alterations from diagnosis to relapse. However, DNA sequence mutations in ALL have not been analysed in detail. To identify novel mutations in relapsed ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 71 diagnosis–relapse cases and 270 acute leukaemia cases that did not relapse found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CREBBP, also known as CBP). The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the histone acetyltransferase domain. Functionally, the mutations impaired histone acetylation and transcriptional regulation of CREBBP targets, including glucocorticoid responsive genes. Several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations may confer resistance to therapy. These results extend the landscape of genetic alterations in leukaemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL.

An anatomy of normal and malignant gene expression

A genes expression pattern provides clues to its role in normal physiology and disease. To provide quantitative expression levels on a genome-wide scale, the Cancer Genome Anatomy Project (CGAP) uses serial analysis of gene expression (SAGE). Over 5 million transcript tags from more than 100 human cell types have been assembled. To enhance the utility of this data, the CGAP SAGE project created SAGE Genie, a web site for the analysis and presentation of SAGE data (http://cgap.nci.nih.gov/SAGE). SAGE Genie provides an automatic link between gene names and SAGE transcript levels, accounting for alternative transcription and many potential errors. These informatics advances provide a rapid and intuitive view of transcript expression in the human body or brain, displayed on the SAGE Anatomic Viewer. We report here an easily accessible view of nearly any genes expression in a wide variety of malignant and normal tissues.

Rembrandt: Helping Personalized Medicine Become a Reality Through Integrative Translational Research

Finding better therapies for the treatment of brain tumors is hampered by the lack of consistently obtained molecular data in a large sample set and the ability to integrate biomedical data from disparate sources enabling translation of therapies from bench to bedside. Hence, a critical factor in the advancement of biomedical research and clinical translation is the ease with which data can be integrated, redistributed, and analyzed both within and across functional domains. Novel biomedical informatics infrastructure and tools are essential for developing individualized patient treatment based on the specific genomic signatures in each patients tumor. Here, we present Repository of Molecular Brain Neoplasia Data (Rembrandt), a cancer clinical genomics database and a Web-based data mining and analysis platform aimed at facilitating discovery by connecting the dots between clinical information and genomic characterization data. To date, Rembrandt contains data generated through the Glioma Molecular Diagnostic Initiative from 874 glioma specimens comprising ∼566 gene expression arrays, 834 copy number arrays, and 13,472 clinical phenotype data points. Data can be queried and visualized for a selected gene across all data platforms or for multiple genes in a selected platform. Additionally, gene sets can be limited to clinically important annotations including secreted, kinase, membrane, and known gene-anomaly pairs to facilitate the discovery of novel biomarkers and therapeutic targets. We believe that Rembrandt represents a prototype of how high-throughput genomic and clinical data can be integrated in a way that will allow expeditious and efficient translation of laboratory discoveries to the clinic. (Mol Cancer Res 2009;7(2):157–67)

Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group.

We sequenced 120 candidate genes in 187 high-risk childhood B-precursor acute lymphoblastic leukemias, the largest pediatric cancer genome sequencing effort reported to date. Integrated analysis of 179 validated somatic sequence mutations with genome-wide copy number alterations and gene expression profiles revealed a high frequency of recurrent somatic alterations in key signaling pathways, including B-cell development/differentiation (68% of cases), the TP53/RB tumor suppressor pathway (54%), Ras signaling (50%), and Janus kinases (11%). Recurrent mutations were also found in ETV6 (6 cases), TBL1XR1 (3), CREBBP (3), MUC4 (2), ASMTL (2), and ADARB2 (2). The frequency of mutations within the 4 major pathways varied markedly across genetic subtypes. Among 23 leukemias expressing a BCR-ABL1-like gene expression profile, 96% had somatic alterations in B-cell development/differentiation, 57% in JAK, and 52% in both pathways, whereas only 9% had Ras pathway mutations. In contrast, 21 cases defined by a distinct gene expression profile coupled with focal ERG deletion rarely had B-cell development/differentiation or JAK kinase alterations but had a high frequency (62%) of Ras signaling pathway mutations. These data extend the range of genes that are recurrently mutated in high-risk childhood B-precursor acute lymphoblastic leukemia and highlight important new therapeutic targets for selected patient subsets.

Identification of inbred mouse strains harboring genetic modifiers of mammary tumor age of onset and metastatic progression

Metastasis is one of the most important and complex processes in human neoplastic disease. A large number of both positive and negative events must occur to permit a tumor cell to colonize a distant site successfully. To identify mouse strains that harbor dominant genetic modifiers of this process, a strain survey was initiated utilizing a transgenic mouse mammary tumor model that exhibits a high incidence of pulmonary metastases. The transgenic animal was bred to 27 different inbred strains of mice and scored for the metastatic organ tropism and metastatic density. Thirteen strains were identified that had a statistically significant reduction in the numbers of pulmonary metastases. In addition, 10 strains were identified that altered the kinetics of induction of the primary mammary tumor. These strains will likely provide useful model systems for the analysis of genetic interactions in the initiation and progression of mammary adenocarcinomas. Int. J. Cancer 77:640–644, 1998.

Chromatin Remodeling Factors and BRM/BRG1 Expression as Prognostic Indicators in Non-Small Cell Lung Cancer

We immunohistochemically examined 12 core proteins involved in the chromatin remodeling machinery using a tissue microarray composed of 150 lung adenocarcinoma (AD) and 150 squamous cell carcinoma (SCC) cases. Most of the proteins showed nuclear staining, whereas some also showed cytoplasmic or membranous staining. When the expression patterns of all tested antigens were considered, proteins with nuclear staining clustered into two major groups. Nuclear signals of BRM, Ini-1, retinoblastoma, mSin3A, HDAC1, and HAT1 clustered together, whereas nuclear signals of BRG1, BAF155, HDAC2, BAF170, and RbAP48 formed a second cluster. Additionally, two thirds of the cases on the lung tissue array had follow-up information, and survival analysis was performed for each of the tested proteins. Positive nuclear BRM (N-BRM) staining correlated with a favorable prognosis in SCC and AD patients with a 5 year-survival of 53.5% compared with 32.3% for those whose tumors were negative for N-BRM (P = 0.015). Furthermore, patients whose tumors stained positive for both N-BRM and nuclear BRG1 had a 5 year-survival of 72% compared with 33.6% (P = 0.013) for those whose tumors were positive for either or negative for both markers. In contrast, membranous BRM (M-BRM) staining correlated with a poorer prognosis in AD patients with a 5 year-survival of 16.7% compared with those without M-BRM staining (38.1%; P = 0.016). These results support the notion that BRM and BRG1 participate in two distinct chromosome remodeling complexes that are functionally complementary and that the nuclear presence of BRM, its coexpression with nuclear BRG1, and the altered cellular localization of BRM (M-BRM) are useful markers for non-small cell lung cancer prognosis.