Gregory J. Riggins

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS–RAF–MEK–ERK–MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.

Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome

Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27.3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal of the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome.

The genetic landscape of the childhood cancer medulloblastoma

Genomic analysis of a childhood cancer reveals markedly fewer mutations than what is typically seen in adult cancers. Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

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.

Gene discovery using the serial analysis of gene expression technique: Implications for cancer research

Cancer is a genetic disease. As such, our understanding of the pathobiology of tumors derives from analyses of the genes whose mutations are responsible for those tumors. The cancer phenotype, however, likely reflects the changes in the expression patterns of hundreds or even thousands of genes that occur as a consequence of the primary mutation of an oncogene or a tumor suppressor gene. Recently developed functional genomic approaches, such as DNA microarrays and serial analysis of gene expression (SAGE), have enabled researchers to determine the expression level of every gene in a given cell population, which represents that cell populations entire transcriptome. The most attractive feature of SAGE is its ability to evaluate the expression pattern of thousands of genes in a quantitative manner without prior sequence information. This feature has been exploited in three extremely powerful applications of the technology: the definition of transcriptomes, the analysis of differences between the gene expression patterns of cancer cells and their normal counterparts, and the identification of downstream targets of oncogenes and tumor suppressor genes. Comprehensive analyses of gene expression not only will further understanding of growth regulatory pathways and the processes of tumorigenesis but also may identify new diagnostic and prognostic markers as well as potential targets for therapeutic intervention.

Comprehensive sampling of gene expression in human cell lines with massively parallel signature sequencing

Whereas information is rapidly accumulating about the structure and position of genes encoded in the human genome, less is known about the complexity and relative abundance of their expression in individual human cells and tissues. Here, we describe the characteristics of the transcriptomes of two cultured cell lines, HB4a (normal breast epithelium) and HCT-116 (colon adenocarcinoma), using massively parallel signature sequencing (MPSS). We generated in excess of 107 short signature sequences per cell line, thus providing a comprehensive snapshot of gene expression, within the technical limitations of the method. The number of genes expressed at one copy per cell or more in either of the lines was estimated to be between 10,000 and 15,000. The vast majority of the transcripts found in these cells can be mapped to known genes and their polyadenylation variants. Among the genes that could be identified from their signature sequences, ≈8,500 were expressed by both cell lines, whereas 6,000 showed cellular specificity. Taking into account sequence tags that map uniquely to the genome but not to known transcripts, overall the data are consistent with an upper limit of 17,000 for the total number of genes expressed at more than one copy per cell in one or both of the two cell lines examined.

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define ≈23,500 genes, of which only ≈1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

HER-2 Gene Amplification Correlates with Higher Levels of Angiogenesis and Lower Levels of Hypoxia in Primary Breast Tumors

Purpose: This study investigated the connection among HER-2 gene amplification, HER-2 protein expression, and markers of tumor angiogenesis and oxygenation in patients with operable, invasive breast tumors. Experimental Design: From 1988 to 1995, 425 patients with metastatic breast cancer were enrolled in a study of high-dose chemotherapy with autologous transplant. Primary tumor blocks were obtained and evaluated using immunohistochemistry (IHC) staining of vessels with von Willebrand factor antibody. Mean microvessel densities (MVD) were determined by counting von Willebrand factor stained cells in three separate “vascular hot spots” using image analysis. Tumor samples were also stained for HER-2 by IHC, HER-2 gene amplification by fluorescence in situ hybridization, carbonic anhydrase 9 by IHC, and vascular endothelial growth factor (VEGF) by IHC. Plasma from 36 patients with primary tumor samples had VEGF (R&D Systems, MN) and d-dimer (American Diagnostica, Greenwich, CT) levels determined. Results: There was a significant positive correlation between HER-2 gene amplification and both maximum and average MVD (Spearman coefficient = 0.51 and 0.50; P = 0.03 and 0.05, respectively). There was an inverse correlation with HER-2 gene amplification and expression of the tumor hypoxia marker CA-9 (χ2 P = 0.02). The level of HER-2 gene amplification correlated with plasma d-dimer levels (Spearman coefficient = 0.43; P = 0.021). Interestingly, tumors with HER-2 by IHC had decreased amounts of VEGF staining (χ2 = 5.81; P = 0.01). There was no correlation between HER-2 by IHC and MVD or d-dimer. Of all of the variables examined, only average (P = 0.0016) and maximum MVD (P = 0.0128) predicted disease-free survival (Cox univariate model). Conclusions: HER-2-amplified breast cancers have increased amounts of angiogenesis, decreased amounts of hypoxia, and increased markers of fibrin degradation. These findings have prognostic, predictive, and therapeutic implications in breast cancer treatment.

Navigating the human transcriptome

The potential coding capacity of the human genome is currently a topic of great interest. The number of genes predicted from the recent human-genome analysis was at the lower end of previous estimates, which had ranged between about 30,000 and 120,000 (1, 2). Whereas estimates of gene number are likely to increase based on additional experimental evidence and improved gene-finding algorithms, it is clear that gene number is only one mechanism for creating the genetic diversity required to encode the full complement of human proteins. The scientific literature richly describes the presence and functional significance of alternatively processed forms of human transcripts that are derived from different transcription initiation sites, alternative exon splicing, and multiple polyadenylation sites (3–5). Determining the various transcript forms and investigating the purpose of these complex mixtures of instructions will be the next great endeavor toward understanding human biology.

Using Serial Analysis of Gene Expression to identify tumor markers and antigens

Tumor markers and antigens are normally highly expressed in malignant tissue, but not in the surrounding normal tissue. Serial Analysis of Gene Expression (SAGE) is a technology that counts mRNA transcripts and can be used to find those genes most highly induced in malignant tissues. SAGE produces a comprehensive profile of gene expression and can be used to search for tumor biomarkers in a limited number of samples. Public sources of SAGE data, in particular through the Cancer Genome Anatomy Project, increase the value of this technology by making a large source of information on many tumors and normal tissues available for comparison. Although the perfect tumor-specific gene does not exist, the differences in gene expression between tumor and normal can be exploited for therapeutic or diagnostic purposes.