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Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is an extremely rare, aggressive cancer affecting children and young women. We identified germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 75% (9/12) of SCCOHT cases in addition to SMARCA4 protein loss in 82% (14/17) of SCCOHT tumors but in only 0.4% (2/485) of other primary ovarian tumors. These data implicate SMARCA4 in SCCOHT oncogenesis.

Microarray technology makes it possible to simultaneously study the expression of thousands of genes during a single experiment. We have developed an information system, ArrayDB, to manage and analyse large-scale expression data. The underlying relational database was designed to allow flexibility in the nature and structure of data input and also in the generation of standard or customized reports through a web-browser interface. ArrayDB provides varied options for data retrieval and analysis tools that should facilitate the interpretation of complex hybridization results. A sampling of ArrayDB storage, retrieval and analysis capabilities is available ( http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/ ), along with information on a set of approximately 15,000 genes used to fabricate several widely used microarrays. Information stored in ArrayDB is used to provide integrated gene expression reports by linking array target sequences with NCBI's Entrez retrieval system, UniGene and KEGG pathway views. The integration of external information resources is essential in interpreting intrinsic patterns and relationships in large-scale gene expression data.

We have performed microdissection of 16 putative homogeneously staining regions (hsrs) from nine different breast cancer cell lines in order to determine their chromosomal origin and composition. As expected, the most commonly amplified chromosomal band-region was 17q12 (containing ERBB2). However, regions not containing known oncogenes were also identified, including 13q31 (5/9 cases) and 20q12–13.2 (4/9 cases). The chromosomal composition of the integrated amplified DNA within each hsr was determined and in 13/16 cases (81 %), hsrs were shown to be composed of two or more chromosomal regions. These studies shed light on the mechanism of formation of hsrs, and identify chromosomal regions likely to harbour genes amplified in breast cancer.

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

The strategy presented here to identify unequivocally cryptic chromosomal rearrangements has relevance to both prenatal and postnatal cytogenetic analysis as well as the analysis of tumour-associated chromosome rearrangements. Microdissection and in vitro amplification of specific chromosomal regions are performed, followed by labelling for fluorescent in situ hybridization (FISH) to normal metaphase chromosomes (Micro-FISH). Micro-FISH probes have been used successfully to determine the derivation of chromosome segments unidentifiable by standard chromosome banding analysis. Micro-FISH probes (created in less than 24 hours) now make it possible to identify explicitly the chromosome constitution of virtually all cytologically visible chromosome rearrangements.

In the film What About Bob, comic wizard Bill Murray plays Bob Wiley, a mental patient with a multi-phobic personality whose fear of almost everything leaves him in a constant state of panic. Richard Dreyfus plays Dr Leo Marvin, an eminent psychiatrist whose therapy goals for Bob are summed up in his crowning professional accomplishment, a book entitled Baby Steps, in which he advocates setting small, reasonable goals one day a time—one tiny step at a time. The multiphobic character played by Murray will resonate all too well with the geneticist who faces the rapid evolution of bioinformatics driven by the massive quantities of data produced by genomescale technologies. Impressive though the evolution of bioinformatic analysis may be, the problems associated with mapping and sequencing are dwarfed in comparison with the mathematical challenges generated by recent progress in parallel gene expression analysis using microarrays 1 . This new type of expression data brings us face to face with the

We carried out a fine-mapping study in the HNF1B gene at 17q12 in two study populations and identified a second locus associated with prostate cancer risk, approximately 26 kb centromeric to the first known locus (rs4430796); these loci are separated by a recombination hot spot. We confirmed the association with a SNP in the second locus (rs11649743) in five additional populations, with P = 1.7 x 10(-9) for an allelic test of the seven studies combined. The association at each SNP remained significant after adjustment for the other SNP.

We conducted a genome-wide association pooling study for cutaneous melanoma and performed validation in samples totaling 2,019 cases and 2,105 controls. Using pooling, we identified a new melanoma risk locus on chromosome 20 (rs910873 and rs1885120), with replication in two further samples (combined P < 1 x 10(-15)). The per allele odds ratio was 1.75 (1.53, 2.01), with evidence for stronger association in early-onset cases.

The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. We combined nonsense-mediated RNA decay microarrays and array-based comparative genomic hybridization for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled us to identify previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, our findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

RNASEL (encoding ribonuclease L) has recently been proposed as a candidate for the hereditary prostate cancer (HPC1) gene. We determined that the RNASEL variant Arg462Gln has three times less enzymatic activity than the wildtype and is significantly associated with prostate cancer risk (P = 0.007). At least one copy of the mutated allele that causes this substitution is carried by nearly 60% of the men in our study. Men that are heterozygous with respect to the mutated allele have 50% greater risk of prostate cancer than non-carriers, and homozygotes have more than double the risk.

Deletions on human chromosome 8p22-23 in prostate cancer cells and linkage studies in families affected with hereditary prostate cancer (HPC) have implicated this region in the development of prostate cancer. The macrophage scavenger receptor 1 gene (MSR1, also known as SR-A) is located at 8p22 and functions in several processes proposed to be relevant to prostate carcinogenesis. Here we report the results of genetic analyses that indicate that mutations in MSR1 may be associated with risk of prostate cancer. Among families affected with HPC, we identified six rare missense mutations and one nonsense mutation in MSR1. A family-based linkage and association test indicated that these mutations co-segregate with prostate cancer (P = 0.0007). In addition, among men of European descent, MSR1 mutations were detected in 4.4% of individuals affected with non-HPC as compared with 0.8% of unaffected men (P = 0.009). Among African American men, these values were 12.5% and 1.8%, respectively (P = 0.01). These results show that MSR1 may be important in susceptibility to prostate cancer in men of both African American and European descent.

Although prostate cancer is the most common non-cutaneous malignancy diagnosed in men in the United States1,2, little is known about inherited factors that influence its genetic predisposition3,4,5. Here we report that germline mutations in the gene encoding 2′-5′-oligoadenylate(2-5A)–dependent RNase L (RNASEL)6,7,8 segregate in prostate cancer families that show linkage to the HPC1 (hereditary prostate cancer 1) region at 1q24–25 (ref. 9). We identified RNASEL by a positional cloning/candidate gene method, and show that a nonsense mutation and a mutation in an initiation codon of RNASEL segregate independently in two HPC1-linked families. Inactive RNASEL alleles are present at a low frequency in the general population. RNASEL regulates cell proliferation and apoptosis through the interferon-regulated 2-5A pathway and has been suggested to be a candidate tumor suppressor gene10,11,12. We found that microdissected tumors with a germline mutation showed loss of heterozygosity and loss of RNase L protein, and that RNASEL activity was reduced in lymphoblasts from heterozyogous individuals compared with family members who were homozygous with respect to the wildtype allele. Thus, germline mutations in RNASEL may be of diagnostic value, and the 2-5A pathway might provide opportunities for developing therapies for those with prostate cancer.

The study of complex genetic traits in humans is limited by the expense and difficulty of ascertaining populations of sufficient sample size to detect subtle genetic contributions to disease. Here we introduce an application of a somatic cell hybrid construction strategy called conversion1,2,3,4 that maximizes the genotypic information from each sampled individual. The approach permits direct observation of individual haplotypes, thereby eliminating the need for collecting and genotyping DNA from family members for haplotype-based analyses. We describe experimental data that validate the use of conversion as a whole-genome haplotyping tool and evaluate the theoretical efficiency of using conversion-derived haplotypes instead of conventional genotypes in the context of haplotype-frequency estimation. We show that, particularly when phenotyping is expensive, conversion-based haplotyping can be more efficient and cost-effective than standard genotyping.

Altered growth and function of synoviocytes, the intimal cells which line joint cavities and tendon sheaths, occur in a number of skeletal diseases. Hyperplasia of synoviocytes is found in both rheumatoid arthritis and osteoarthritis, despite differences in the underlying aetiologies of the two disorders. We have studied the autosomal recessive disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP; MIM 208250) to identify biological pathways that lead to synoviocyte hyperplasia, the principal pathological feature of this syndrome. Using a positional-candidate approach, we identified mutations in a gene (CACP) encoding a secreted proteoglycan as the cause of CACP. The CACP protein, which has previously been identified as both 'megakaryocyte stimulating factor precursor' and 'superficial zone protein', contains domains that have homology to somatomedin B, heparin-binding proteins, mucins and haemopexins. In addition to expression in joint synovium and cartilage, CACP is expressed in non-skeletal tissues including liver and pericardium. The similarity of CACP sequence to that of other protein families and the expression of CACP in non-skeletal tissues suggest it may have diverse biological activities.

cDNA microarrays are capable of profiling gene expression patterns of tens of thousands of genes in a single experiment. DNA targets, in the form of 3' expressed sequence tags (ESTs), are arrayed onto glass slides (or membranes) and probed with fluorescent- or radioactively-labelled cDNAs. Here, we review technical aspects of cDNA microarrays, including the general principles, fabrication of the arrays, target labelling, image analysis and data extraction, management and mining.

Over 200,000 new prostate cancer cases are diagnosed in the United States each year, accounting for more than 35% of all cancer cases affecting men, and resulting in 40,000 deaths annually. Attempts to characterize genes predisposing to prostate cancer have been hampered by a high phenocopy rate, the late age of onset of the disease and, in the absence of distinguishing clinical features, the inability to stratify patients into subgroups relative to suspected genetic locus heterogeneity. We previously performed a genome-wide search for hereditary prostate cancer (HPC) genes, finding evidence of a prostate cancer susceptibility locus on chromosome 1 (termed HPC1; ref. 2). Here we present evidence for the location of a second prostate cancer susceptibility gene, which by heterogeneity estimates accounts for approximately 16% of HPC cases. This HPC locus resides on the X chromosome (Xq27-28), a finding consistent with results of previous population-based studies suggesting an X-linked mode of HPC inheritance. Linkage to Xq27-28 was observed in a combined study population of 360 prostate cancer families collected at four independent sites in North America, Finland and Sweden. A maximum two-point lod score of 4.60 was observed at DXS1113, theta=0.26, in the combined data set. Parametric multipoint and non-parametric analyses provided results consistent with the two-point analysis. Significant evidence for genetic locus heterogeneity was observed, with similar estimates of the proportion of linked families in each separate family collection. Genetic mapping of the locus represents an important initial step in the identification of an X-linked gene implicated in the aetiology of HPC.

To evaluate the timing of mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) during melanocytic neoplasia, we carried out mutation analysis on microdissected melanoma and nevi samples. We observed mutations resulting in the V599E amino-acid substitution in 41 of 60 (68%) melanoma metastases, 4 of 5 (80%) primary melanomas and, unexpectedly, in 63 of 77 (82%) nevi. These data suggest that mutational activation of the RAS/RAF/MAPK pathway in nevi is a critical step in the initiation of melanocytic neoplasia but alone is insufficient for melanoma tumorigenesis.

Terminal deletions are found frequently in both malignancies and clinically recognizable deletion syndromes in man. Little is known, particularly in cancer, of the specific mechanisms which lead to the generation of deleted chromosomes or the process by which these broken chromosomes are stabilized. We demonstrate that several examples of apparent terminal deletions are, in fact, subtelomeric translocations which were not detectable using conventional cytogenetics. The unexpectedly high frequency of this phenomenon and the diversity of partner chromosomes involved in the subtelomeric translocations is consistent with a model in which telomere capture can stabilize chromosome breakage in man.

Identification of suppressor genes associated with the chromosome 6-mediated suppressed melanoma cell UACC903(+6) by cDNA microarray

An integrated approach to the extraction, storage, processing and analysis of microarray gene expression data

Molecular cloning and expression analysis of human and mouse homologs of a new transcriptional regulator as a candidate tumor suppresor gene

Microarray analysis discriminates BRCA2 mutation-positive from BRCA1 and sporadic breast cancer patient tumour biopsies

Drug resistance in cancer is a major obstacle to successful chemotherapy. Cancer cells exposed to antitumour drugs may be directly induced to express a subset of genes that could confer resistance, thus allowing a population of cells to survive and form the relapsed resistant tumour. Alternatively, some cancer cells may be expressing an array of genes that could confer intrinsic resistance, and exposure to cytotoxic drugs select for the survival of these cells that form the relapsed tumour. To assess whether there are differences in the mechanism of drug resistance by induction or selection, we used complementary DNA (cDNA) microarray to monitor mRNA expression in breast cancer cells that were either transiently treated with or selected for resistance to doxorubicin. Expression profiles of the human breast carcinoma MCF-7 cells transiently treated with doxorubicin were compared with those obtained from a MCF-7 cell line selected for resistance to doxorubicin. Transient treatment with doxorubicin altered the expression of a diverse group of genes in a time-dependent manner. We found a subset of transiently induced genes constitutively overexpressed in cells selected for resistance to doxorubicin, suggesting that cancer cells activate a distinct set of genes in acquiring drug resistance by either induction or selection. Our studies demonstrate the feasibility of obtaining molecular profiles of drug resistance in cancer cells by cDNA microarray, which might yield insights into the mechanisms of resistance during chemotherapy, and suggest alternative methods of treatment.

cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene

We have recently observed that tissue sections from uveal melanoma and metastatic cutaneous melanoma generally lack evidence of significant necrosis and contain patterned networks of interconnected loops of extracellular matrix. Similar networks are formed in tissue cultures of highly invasive primary and metastatic melanoma. These networks, which are not formed by melanocytes or poorly invasive melanomas, have the properties of vascular channels and suggest that certain tumours may have an intrinsic ability to undergo vasculogenesis without a contribution from host endothelial cells. Previous studies have identified abnormal co-expression in aggressive melanoma cells of intermediate filaments (an interconverted phenotype), suggesting reversion to a pluripotent cell. We sought to extend the definition of this phenotype at the gene expression level by cDNA microarray analysis. Comparison of aggressive melanomas that form vascular channels with less aggressive cells that do not exhibit this phenotype reveal differences in their gene expression profile. The specific genes expressed in the highly invasive melanoma cultures include those encoding extracellular matrix proteins, signalling and regulatory molecules which are highly consistent with the vasculogenic phenotype exhibited by these cells. The results of this study support the concept that some tumours generate vascular channels independently of conventional angiogenesis and have important implications for the development of therapeutic interventions directed at this novel property of aggressive melanomas.

Jeffrey M. Trent, Ph.D., is Scientific Director of the National Human Genome Research Institute (NHGRI), and also serves as Chief of NHGRI's Cancer Genetics Branch. He is also an Adjunct Professor of Oncology at the Johns Hopkins University. He specializes in studying the molecular changes related to the predisposition and progression of cancer. After earning his undergraduate degree from Indiana University, Dr. Trent received M.S. and Ph.D. degrees in genetics from the University of Arizona. Prior to his arrival at the NIH, Dr. Trent was the Emanuel N. Maisel Professor of Oncology and Professor of Radiation Oncology and Human Genetics at the University of Michigan, Ann Arbor. He also served as Director of the Division of Cancer Biology, and Director of Basic Sciences in the University of Michigan's Comprehensive Cancer Center. In 1993, Dr. Trent came to the National Human Genome Research Institute to establish and direct its Division of Intramural Research. Under his guidance, the division has become a major research center in human genetics.

The ‘stress chip’: a specialized functional genomics application for the study of genotoxic stress response