Canine urothelial carcinoma: genomically aberrant and comparatively relevant.

Urothelial carcinoma (UC), also referred to as transitional cell carcinoma (TCC), is the most common bladder malignancy in both human and canine populations. In human UC, numerous studies have demonstrated the prevalence of chromosomal imbalances. Although the histopathology of the disease is similar in both species, studies evaluating the genomic profile of canine UC are lacking, limiting the discovery of key comparative molecular markers associated with driving UC pathogenesis. In the present study, we evaluated 31 primary canine UC biopsies by oligonucleotide array comparative genomic hybridization (oaCGH). Results highlighted the presence of three highly recurrent numerical aberrations: gain of dog chromosome (CFA) 13 and 36 and loss of CFA 19. Regional gains of CFA 13 and 36 were present in 97 % and 84 % of cases, respectively, and losses on CFA 19 were present in 77 % of cases. Fluorescence in situ hybridization (FISH), using targeted bacterial artificial chromosome (BAC) clones and custom Agilent SureFISH probes, was performed to detect and quantify these regions in paraffin-embedded biopsy sections and urine-derived urothelial cells. The data indicate that these three aberrations are potentially diagnostic of UC. Comparison of our canine oaCGH data with that of 285 human cases identified a series of shared copy number aberrations. Using an informatics approach to interrogate the frequency of copy number aberrations across both species, we identified those that had the highest joint probability of association with UC. The most significant joint region contained the gene PABPC1, which should be considered further for its role in UC progression. In addition, cross-species filtering of genome-wide copy number data highlighted several genes as high-profile candidates for further analysis, including CDKN2A, S100A8/9, and LRP1B. We propose that these common aberrations are indicative of an evolutionarily conserved mechanism of pathogenesis and harbor genes key to urothelial neoplasia, warranting investigation for diagnostic, prognostic, and therapeutic applications.

Expression patterns of predicted genes from the C. elegans genome sequence visualized by FISH in whole organisms.

More than 10 megabases of contiguous genome sequence have been submitted to the databases by the Caenorhabditis elegans Genome Sequencing Consortium. To characterize the genes predicted from the sequence, we have developed high resolution FISH for visualization of mRNA distributions in whole animals. The high resolution and sensitivity afforded by the use of directly fluorescently labelled probes and confocal imaging permitted mRNA distributions to be recorded at the cellular and subcellular level. Expression patterns were obtained for 8 out of 10 genes in an initial test set of predicted gene sequences, indicating that FISH is an effective means of characterizing predicted genes in C. elegans.

Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene.

We show here that quantitative measurement of DNA copy number across amplified regions using array comparative genomic hybridization (CGH) may facilitate oncogene identification by providing precise information on the locations of both amplicon boundaries and amplification maxima. Using this analytical capability, we resolved two regions of amplification within an approximately 2-Mb region of recurrent aberration at 20q13.2 in breast cancer. The putative oncogene ZNF217 (ref. 5) mapped to one peak, and CYP24 (encoding vitamin D 24 hydroxylase), whose overexpression is likely to lead to abrogation of growth control mediated by vitamin D, mapped to the other.

High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.

Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization (CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.

Genome scanning with array CGH delineates regional alterations in mouse islet carcinomas.

Carcinomas that develop in the pancreatic islets of transgenic mice expressing the SV40 T-antigens (Tag) under transcriptional control of the rat insulin II promoter (RIP) progress through well-characterized stages that are similar to aspects of human tumor progression, including hyperplastic growth, increased angiogenesis and reduced apoptosis. The latter two stages have been associated with recurrent loss of heterozygosity (LOH) and reduced genome copy number on chromosomes 9 (LOH9) and 16 (LOH16), aberrations which we believe contribute to these phenotypes. Earlier analyses localized LOH9 to approximately 3 Mb and LOH16 to approximately 30 Mb (both syntenic with human 3q21-q25) but were limited by low throughput and a lack of informative polymorphic markers. Here we show that comparative genomic hybridization to DNA microarrays (array CGH) overcomes these limitations by allowing efficient, genome-wide analyses of relative genome copy number. The CGH arrays used in these experiments carried BACs distributed at 2-20-MB intervals across the mouse genome and at higher density in regions of interest. Using array CGH, we further narrowed the loci for LOH9 and LOH16 and defined new or previously unappreciated recurrent regions of copy-number decrease on chromosomes 6, 8 and 14 (syntenic with human chromosomes 12p11-p13, 16q24.3 and 13q11-q32, respectively) and regions of copy-number increase on chromosomes 2 and 4 (syntenic to human chromosomes 20q13.2 and 1p32-p36, respectively). Our analyses of human genome sequences syntenic to these regions suggest that CYP24, PFDN4, STMN1, CDKN1B, PPP2R3 and FSTL1 are candidate oncogenes or tumor-suppressor genes. We also show that irradiation and genetic background influence the spectrum of aberrations present in these tumors.

Assembly of microarrays for genome-wide measurement of DNA copy number.

We have assembled arrays of approximately 2,400 BAC clones for measurement of DNA copy number across the human genome. The arrays provide precise measurement (s.d. of log2 ratios=0.05-0.10) in cell lines and clinical material, so that we can reliably detect and quantify high-level amplifications and single-copy alterations in diploid, polyploid and heterogeneous backgrounds.

The Histopathology of Oral Cancer Pain in a Mouse Model and a Human Cohort.

Oral cancer patients often have severe, chronic, and mechanically induced pain at the site of the primary cancer. Oral cancer pain is initiated and maintained in the cancer microenvironment and attributed to release of mediators that sensitize primary sensory nerves. This study was designed to investigate the histopathology associated with painful oral cancers in a preclinical model. The relationship of pain scores with pathologic variables was also investigated in a cohort of 72 oral cancer patients. Wild-type mice were exposed to the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Nociceptive (pain) behavior was measured with the dolognawmeter, an operant device and assay for measuring functional and mechanical allodynia. Lesions developed on the tongues and esophagi of the 4NQO-treated animals and included hyperkeratoses, papillomas, dysplasias, and cancers. Papillomas included lesions with benign and dysplastic pathological features. Two histologic subtypes of squamous cell carcinomas (SCCs) were identified-SCCs with exophytic and invasive components associated with papillary lesions (pSCCs) and invasive SCCs without exophytic histology (iSCCs). Only the pSCC subtype of tongue cancer was associated with nociceptive behavior. Increased tumor size was associated with greater nociceptive behavior in the mouse model and more pain experienced by oral cancer patients. In addition, depth of invasion was associated with patient-reported pain. The pSCC histology identifies 4NQO-induced tongue cancers that are expected to be enriched for expression and release of nociceptive mediators.

HIM-10 is required for kinetochore structure and function on Caenorhabditis elegans holocentric chromosomes.

Macromolecular structures called kinetochores attach and move chromosomes within the spindle during chromosome segregation. Using electron microscopy, we identified a structure on the holocentric mitotic and meiotic chromosomes of Caenorhabditis elegans that resembles the mammalian kinetochore. This structure faces the poles on mitotic chromosomes but encircles meiotic chromosomes. Worm kinetochores require the evolutionarily conserved HIM-10 protein for their structure and function. HIM-10 localizes to the kinetochores and mediates attachment of chromosomes to the spindle. Depletion of HIM-10 disrupts kinetochore structure, causes a failure of bipolar spindle attachment, and results in chromosome nondisjunction. HIM-10 is related to the Nuf2 kinetochore proteins conserved from yeast to humans. Thus, the extended kinetochores characteristic of C. elegans holocentric chromosomes provide a guide to the structure, molecular architecture, and function of conventional kinetochores.

A large field CCD system for quantitative imaging of microarrays.

We describe a charge-coupled device (CCD) imaging system for microarrays capable of acquiring quantitative, high dynamic range images of very large fields. Illumination is supplied by an arc lamp, and filters are used to define excitation and emission bands. The system is linear down to fluorochrome densities <<1 molecule/microm2. The ratios of the illumination intensity distributions for all excitation wavelengths have a maximum deviation approximately +/-4% over the object field, so that images can be analyzed without computational corrections for the illumination pattern unless higher accuracy is desired. Custom designed detection optics produce achromatic images of the spectral region from approximately 450 to approximately 750 nm. Acquisition of a series of images of multiple fluorochromes from multiple arrays occurs under computer control. The version of the system described in detail provides images of 20 mm square areas using a 27 mm square, 2K x 2K pixel, cooled CCD chip with a well depth of approximately 10(5) electrons, and provides ratio measurements accurate to a few percent over a dynamic range in intensity >1000. Resolution referred to the sample is 10 microm, sufficient for obtaining quantitative multicolor images from >30,000 array elements in an 18 mm x 18 mm square.

Fryns syndrome phenotype caused by chromosome microdeletions at 15q26.2 and 8p23.1.

BACKGROUND: Fryns syndrome (FS) is the commonest autosomal recessive syndrome in which congenital diaphragmatic hernia (CDH) is a cardinal feature. It has been estimated that 10% of patients with CDH have FS. The autosomal recessive inheritance in FS contrasts with the sporadic inheritance for the majority of patients with CDH and renders the correct diagnosis critical for accurate genetic counselling. The cause of FS is unknown. METHODS: We have used array comparative genomic hybridisation (array CGH) to screen patients who have CDH and additional phenotypic anomalies consistent with FS for cryptic chromosome aberrations. RESULTS: We present three probands who were previously diagnosed with FS who had submicroscopic chromosome deletions detected by array CGH after normal karyotyping with G-banded chromosome analysis. Two female infants were found to have microdeletions involving chromosome band 15q26.2 and one male had a deletion of chromosome band 8p23.1. CONCLUSIONS: We conclude that phenotypes similar to FS can be caused by submicroscopic chromosome deletions and that high resolution karyotyping, including array CGH if possible, should be performed prior to the diagnosis of FS to provide an accurate recurrence risk in patients with CDH and physical anomalies consistent with FS.

A novel calmodulin-like gene from the nematode Caenorhabditis elegans.

A novel gene from the nematode Caenorhabditis elegans was isolated by hybridization with a human calmodulin complementary DNA probe. This gene, cal-1, is present at one copy per haploid genome. In-situ hybridization of the cloned gene to metaphase chromosomes allowed us to assign it to the nematode linkage group IV. The polypeptide predicted from the sequence of this gene displays structural features of both calmodulin and troponin C.

Genomic organization of the glyceraldehyde-3-phosphate dehydrogenase gene family of Caenorhabditis elegans.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDHase) is encoded by four genes designated gpd-1 through gpd-4 in the nematode Caenorhabditis elegans. gpd-1 has been isolated and sequenced, and is shown here to have a nearly identical copy (gpd-4) with respect to coding and regulatory flanking sequence information as well as to the placement of its two introns. Both genes, which are separated by 250,000 to 300,000 base-pairs were assigned to chromosome II by in situ hybridization and physically linked to a DNA polymorphism located near unc-4 on the genetic map. The genes gpd-2 and gpd-3 are also nearly identical with each other but differ from the gpd-1 and gpd-4 pair with respect to the positions of their two introns and a cluster of amino acid changes within the amino-terminal region of the enzyme. Furthermore, one gene from each pair (gpd-4 and gpd-2) exhibits a single amino acid substitution at positions heretofore known to be conserved in all other systems so far examined including the extreme thermophiles. gpd-2 and gpd-3 are organized as a direct tandem repeat separated by only 244 base-pairs. They have been assigned to an 85,200 base-pair contig that maps to the left end of the X chromosome. The absence of gpd-3 from C. elegans var. Bergerac was used as a marker to map the gpd-2,3 gene pair near unc-20. Northern analyses have shown that gpd-1 and gpd-4 are preferentially expressed in embryos, while the expression of gpd-2 and gpd-3 increases during postembryonic development. These analyses indicate that the gpd-1,4 gene pair encodes the minor isoenzyme, GAPDHase-1, present in all cells of the nematode while the other gene pair (gpd-2,3) encodes the major isoenzyme, GAPDHase-2, preferentially expressed in the bodywall muscle. The G + T-rich and T-rich regions essential for vertebrate beta-globin polyadenylation were also observed for gpd-3.

Genomic organization of major sperm protein genes and pseudogenes in the nematode Caenorhabditis elegans.

The major sperm proteins (MSPs) are a family of closely related, small, basic proteins comprising 15% of the protein in Caenorhabditis elegans sperm. They are encoded by a multigene family of more than 50 genes, including many pseudogenes. MSP gene transcription occurs only in late primary spermatocytes. In order to study the genomic organization of transcribed MSP genes, probes specific for the 3' untranslated regions of sequenced cDNA clones were used to isolate transcribed genes from genomic libraries. These and other clones of MSP genes were located in overlapping cosmid clones by DNA fingerprinting. These cosmids were aligned with the genetic map by overlap with known genes or in-situ hybridization to chromosomes. Of 40 MSP genes identified, 37, including all those known to be transcribed, are organized into six clusters composed of 3 to 13 genes each. Within each cluster, MSP genes are not in tandem but are separated by at least several thousand bases of DNA. Pseudogenes are interspersed among functional genes. Genes with similar 3' untranslated sequences are in the same cluster. The six MSP clusters are confined to only three chromosomal loci; one on the left arm of chromosome II and two near the middle of chromosome IV. Additional sperm-specific genes are located in one cluster of MSP genes on chromosome IV. The multiplicity of MSP genes appears to be a mechanism for enhancing MSP synthesis in spermatocytes, and the loose clustering of genes could be a result of the mechanism of gene duplication or could play a role in regulation.

Mapping chromosome rearrangement breakpoints to the physical map of Caenorhabditis elegans by fluorescent in situ hybridization.

A scheme for rapidly mapping chromosome rearrangements relative to the physical map of Caenorhabditis elegans is described that is based on hybridization patterns of cloned DNA on meiotic nuclei, as visualized by fluorescent in situ hybridization. From the nearly complete physical map, DNA clones, in yeast artificial chromosomes (YACs), spanning the rearrangement breakpoint were selected. The purified YAC DNAs were first amplified by degenerate oligonucleotide-primed polymerase chain reaction, then reamplified to incorporate fluorescein dUTP or rhodamine dUTP. The site of hybridization was visualized directly (without the use of antibodies) on meiotic bivalents. This allows chromosome rearrangements to be mapped readily if the duplicated, deficient or translocated regions do not pair with a normal homologous region, because the site or sites of hybridization of the probe on meiotic prophase nuclei will be spatially distinct. The pattern, or number, of hybridization signals from probes from within, or adjacent to, the rearranged region of the genome can be predicted from the genetic constitution of the strain. Characterization of the physical extent of the genetically mapped rearrangements places genetic landmarks on the physical map, and so provides linkage between the two types of map.

Isolation of dominant XO-feminizing mutations in Caenorhabditis elegans: new regulatory tra alleles and an X chromosome duplication with implications for primary sex determination.

A strain of Caenorhabditis elegans was constructed that permits selection of dominant or sex-linked mutations that transform XO animals (normally male) into fertile females, using a feminizing mutation, tra-2(e2046gf), which by itself does not sexually transform XO males. Twenty-three mutations were isolated after chemical mutagenesis and found to fall into both expected classes (four dominant tra-1 mutations and eight recessive xol-1 mutations) and novel classes. The novel mutations include 10 second-site mutations of tra-2, which are called eg mutations, for enhanced gain-of-function. The tra-2(gf, eg) alleles lead to complete dominant transformation of XO animals from fertile male into fertile female. Also isolated was a duplication of the left end of the X chromosome, eDp26, which has dominant XO lethal and feminizing properties, unlike all previously isolated duplications of the X chromosome. The properties of eDp26 indicate that it carries copies of one or more numerator elements, which act as part of the primary sex-determination signal, the X:A ratio. The eDp26 duplication is attached to the left tip of the X chromosome in inverted orientation and consequently can be used to generate unstable attached-X chromosomes.

Chromosome rearrangements in Caenorhabditis elegans.

A method for selecting unlinked duplications of a part of the X chromosome of C. elegans is described. Five such duplications have been identified. One of them, Dp (X;V)1, is translocated to linkage group V, where it suppresses crossing over along the left half of linkage group V. Dp(X;V)1 homozygotes grow slowly and are sterile. The other four duplications are associated with chromosome fragments, as observed cytologically by fluorescence microscopy, and tend to be lost. Their frequency of loss is higher in strains homozygous for a mutation that promotes nondisjunction of X chromosomes. The recombination frequencies between two of these duplications and the X have been measured: the frequencies are at least 50 times less than for X-X recombination in the same region. The duplications may prove useful as balancers of recessive lethal mutations.

BAC microarray analysis of 15q11-q13 rearrangements and the impact of segmental duplications.

Chromosome 15q11-q13 is one of the most variable regions of the human genome, with numerous clinical rearrangements involving a dosage imbalance. Multiple clusters of segmental duplications are found in the pericentromeric region of 15q and at the breakpoints of proximal 15q rearrangements. Using sequence maps and previous global analyses of segmental duplications in the human genome, a targeted microarray was developed to detect a wide range of dosage imbalances in clinical samples. Clones were also chosen to assess the effect of paralogous sequences in the array format. In 19 patients analysed, the array data correlated with microsatellite and FISH characterisation. The data showed a linear response with respect to dosage, ranging from one to six copies of the region. Paralogous sequences in arrayed clones appear to respond to the total genomic copy number, and results with such clones may seem aberrant unless the sequence context of the arrayed sequence is well understood. The array CGH method offers exquisite resolution and sensitivity for detecting large scale dosage imbalances. These results indicate that the duplication composition of BAC substrates may affect the sensitivity for detecting dosage variation. They have important implications for effective microarray design, as well as for the detection of segmental aneusomy within the human population.

Septic shock with Micrococcus luteus.

Micrococcus luteus is considered a non-pathogenic saprophyte of human skin and eye. Disease in man caused by this organism is not recorded in medical literature. We present a case of septic shock cause by M luteus. The value of this report is to document the pathogenicity of coagulase-negative staphylococci in patients without valvular heart disease, surgically implanted artifificial prosthetic devices, or polyethylene intravenous catheters.

Technetium-99m-sestamibi SPECT localization of mediastinal parathyroid adenoma.

We report a case in which 99mTc-sestamibi SPECT was used to localize a middle mediastinum parathyroid adenoma that was not detected with planar sestamibi imaging on two previous occasions. Despite prior surgical exploration of the neck and mediastinum, the patient had a 20-yr history of hyperparathyroidism.

Stromal-epithelial interactions in the progression of ovarian cancer: influence and source of tumor stromal cells.

Stromal cells are essential for the progression of many cancers including ovarian tumors. Stromal cell-epithelial cell interactions are important for tumor development, growth, angiogenesis, and metastasis. In the current study, the effects of normal ovarian bovine stromal cells on ovarian tumor progression was investigated. The hypothesis tested is that ovarian stromal cells will alter the onset and progression of ovarian tumors. Conditioned medium from normal bovine ovarian surface stromal cells was found to stimulate the growth of normal ovarian surface epithelium and had no effect on the growth of human tumor cell lines SKOV3 and OCC1. Human ovarian cancer cell lines, SKOV3 and OCC1, were injected subcutaneously into nude mice to examine tumor progression. Tumor growth in the nude mice was dramatically reduced when normal ovarian surface stromal cells were co-injected with SKOV3 or OCC1 cells. Similar results were obtained with normal bovine or human ovarian stromal cells. In contrast, irrelevant testicular stromal cells and epithelial cells had no effect on tumor growth in the nude mouse. Histological examination of these tumors revealed a characteristic stromal cell component adjacent to epithelial cell colonies. Sections of these tumors were hybridized with species specific genomic probes using fluorescence in situ hybridization to identify cell populations. Epithelial cells were shown to be of human origin (i.e. SKOV3 or OCC1), but stromal cells were found to be primarily murine in origin (i.e. host tissue). No detectable bovine cells were observed in the tumors after one week post-injection. Results suggest that stromal cells are an essential component of ovarian tumors. Interestingly, normal ovarian stromal cells had the ability to inhibit tumor growth, but were not able to survive long-term incubation at the tumor site. The developing tumor appears to recruit host (i.e. murine) stromal cells to invade the tumor and support its growth. In summary, normal ovarian stromal cells can inhibit ovarian tumor progression and the developing tumors recruit adjacent host stroma to become "tumor stroma". The tumor stroma likely develop an altered phenotype that cooperates with the tumorigenic epithelial cells to help promote the progression of ovarian cancer.