PIK3CA is implicated as an oncogene in ovarian cancer.

Ovarian cancer is the leading cause of death from gynecological malignancy and the fourth leading cause of cancer death among American women, yet little is known about its molecular aetiology. Studies using comparative genomic hybridization (CGH) have revealed several regions of recurrent, abnormal, DNA sequence copy number that may encode genes involved in the genesis or progression of the disease. One region at 3q26 found to be increased in copy number in approximately 40% of ovarian and others cancers contains PIK3CA, which encodes the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3-kinase). The association between PIK3CA copy number and PI3-kinase activity makes PIK3CA a candidate oncogene because a broad range of cancer-related functions have been associated with PI3-kinase mediated signalling. These include proliferation, glucose transport and catabolism, cell adhesion, apoptosis, RAS signalling and oncogenic transformation. In addition, downstream effectors of PI3-kinase, AKT1 and AKT2, have been found to be amplified or activated in human tumours, including ovarian cancer. We show here that PIK3CA is frequently increased in copy number in ovarian cancers, that the increased copy number is associated with increased PIK3CA transcription, p110alpha protein expression and PI3-kinase activity and that treatment with the PI3-kinase inhibitor LY294002 decreases proliferation and increases apoptosis. Our observations suggest PIK3CA is an oncogene that has an important role in ovarian cancer.

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.

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.

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.

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.

Bacterial characterization by flow cytometry.

Bacteria were analyzed in a dual-beam flow cytometer after double staining with the fluorescent dyes chromomycin A3 and Hoechst 33258, which bind preferentially to DNA that is rich in guanine-cytosine and adenine-thymine, respectively. The measurements were indicative of the cellular DNA content and base composition, cell concentration, and proliferative state of the population. The ratio of the chromomycin A3 signal to the Hoechst 33258 signal increased with the guanine-cytosine content of the cellular DNA for the six cultured species measured, following expectation. Bacteria in urine from patients with urinary tract infections were characterized without interference from host cell DNA, debris, or other particulates.

Sex preselection in mammals? Separation of sperm bearing Y and "O" chromosomes in the vole Microtus oregoni.

The two sex determining sperm populations of the vole Microtus oregoni were separated according to DNA content by use of flow sorting instrumentation. Although the sperm were not viable, they should be useful for addressing the question of haploid expression of genes linked to sex chromosomes and for efficiently searching for biochemical markers that differentiate the two populations.

Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.

Comparative genomic hybridization produces a map of DNA sequence copy number as a function of chromosomal location throughout the entire genome. Differentially labeled test DNA and normal reference DNA are hybridized simultaneously to normal chromosome spreads. The hybridization is detected with two different fluorochromes. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along the target chromosomes. Analysis of tumor cell lines and primary bladder tumors identified 16 different regions of amplification, many in loci not previously known to be amplified.

Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization.

Chronic myelogeneous leukemia (CML) is genetically characterized by fusion of the bcr and abl genes on chromosomes 22 and 9, respectively. In most cases, the fusion involves a reciprocal translocation t(9;22)(q34;q11), which produces the cytogenetically distinctive Philadelphia chromosome (Ph1). Fusion can be detected by Southern (DNA) analysis or by in vitro amplification of the messenger RNA from the fusion gene with polymerase chain reaction (PCR). These techniques are sensitive but cannot be applied to single cells. Two-color fluorescence in situ hybridization (FISH) was used with probes from portions of the bcr and abl genes to detect the bcr-abl fusion in individual blood and bone marrow cells from six patients. The fusion event was detected in all samples analyzed, of which three were cytogenetically Ph1-negative. One of the Ph1-negative samples was also PCR-negative. This approach is fast and sensitive, and provides potential for determining the frequency of the abnormality in different cell lineages.

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.

Dual inactivation of RB and p53 pathways in RAS-induced melanomas.

The frequent loss of both INK4a and ARF in melanoma raises the question of which INK4a-ARF gene product functions to suppress melanoma genesis in vivo. Moreover, the high incidence of INK4a-ARF inactivation in transformed melanocytes, along with the lack of p53 mutation, implies a cell type-specific role for INK4a-ARF that may not be complemented by other lesions of the RB and p53 pathways. A mouse model of cutaneous melanoma has been generated previously through the combined effects of INK4a(Delta2/3) deficiency (null for INK4a and ARF) and melanocyte-specific expression of activated RAS (tyrosinase-driven H-RAS(V12G), Tyr-RAS). In this study, we made use of this Tyr-RAS allele to determine whether activated RAS can cooperate with p53 loss in melanoma genesis, whether such melanomas are biologically comparable to those arising in INK4a(Delta2/3-/-) mice, and whether tumor-associated mutations emerge in the p16(INK4a)-RB pathway in such melanomas. Here, we report that p53 inactivation can cooperate with activated RAS to promote the development of cutaneous melanomas that are clinically indistinguishable from those arisen on the INK4a(Delta2/3) null background. Genomewide analysis of RAS-induced p53 mutant melanomas by comparative genomic hybridization and candidate gene surveys revealed alterations of key components governing RB-regulated G(1)/S transition, including c-Myc, cyclin D1, cdc25a, and p21(CIP1). Consistent with the profile of c-Myc dysregulation, the reintroduction of p16(INK4a) profoundly reduced the growth of Tyr-RAS INK4a(Delta2/3-/-) tumor cells but had no effect on tumor cells derived from Tyr-RAS p53(-/-) melanomas. Together, these data validate a role for p53 inactivation in melanomagenesis and suggest that both the RB and p53 pathways function to suppress melanocyte transformation in vivo in the mouse.

E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas.

The E2A gene products, E12 and E47, are critical for proper early B-cell development and commitment to the B-cell lineage. Here we reveal a new role for E2A in T-lymphocyte development. Loss of E2A activity results in a partial block at the earliest stage of T-lineage development. This early T-cell phenotype precedes the development of a T-cell lymphoma which occurs between 3 and 9 months of age. The thymomas are monoclonal and highly malignant and display a cell surface phenotype similar to that of immature thymocytes. In addition, the thymomas generally express high levels of c-myc. As assayed by comparative genomic hybridization, each of the tumor populations analyzed showed a nonrandom gain of chromosome 15, which contains the c-myc gene. Taken together, the data suggest that the E2A gene products play a role early in thymocyte development that is similar to their function in B-lineage determination. Furthermore, the lack of E2A results in development of T-cell malignancies, and we propose that E2A inactivation is a common feature of a wide variety of human T-cell proliferative disorders, including those involving the E2A heterodimeric partners tal-1 and lyl-1.

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.

Measurement of sister chromatid exchanges at very low bromodeoxyuridine substitution levels using a monoclonal antibody in Chinese hamster ovary cells.

A monoclonal antibody to bromodeoxyuridine (BrdUrd) incorporated into DNA allowed visualization of sister chromatid exchanges (SCE) when as little as 0.6% of the thymine in a single DNA strand has been substituted. Measurement of the SCE frequency as a function of BrdUrd substitution in a normal Chinese hamster ovary cell line showed a plateau of six SCEs per cell for substitution levels up to at least 20%. A clear elevation in frequencies was noted at 60% substitution. However, in the mutant line EM9, previously shown to have a highly elevated frequency of SCE, the level of exchanges declined continuously as the percentage of BrdUrd substitution decreased. At 0.6% substitution, the frequency of SCE was still 4-fold higher than that of the parental cells. The antibody procedure described here should be useful in evaluating the extent to which SCEs induced by mutagenic agents result from interactions between the DNA damage caused by the agent and the BrdUrd routinely used for measuring SCE.

Chromosomal gains and losses in primary cutaneous melanomas detected by comparative genomic hybridization.

The analysis of genetic changes in primary cutaneous melanoma has been limited by the need for fixation for diagnostic purposes. However, comparative genomic hybridization is able to analyze such specimens. We have applied comparative genomic hybridization to 32 primary melanomas to discover and map genomic regions with aberrant DNA copy numbers. The analysis was performed on native, nonamplified DNA extracted from manually dissected tumor sections. Loss of chromosome 9 was detected in 81% of the tumors, most commonly affecting the p arm. Additional common losses occurred on chromosomes 10 (63%), 6q (28%), and 8p (22%). Gains in copy number involved chromosomes 7 (50%), 8 (34%), 6p (28%), 1q (25%), 20 (13%), 17 (13%), and 2 (13%). Amplifications indicating areas harboring potential oncogenes were seen at 4q12, 5p14.3-pter, 7q33-qter, 8q12-13, 11q13.3-14.2, and 17q25. Correlations among the regions with copy number changes indicate that losses of chromosomes 9 and 10 occur early on in melanoma progression, whereas gains of chromosome 7 occur later. This sequence of events was further substantiated by an intratumoral comparison of copy number changes in areas with radial and vertical growth phase patterns. The overall pattern of regions affected by copy number changes is consistent with cytogenetic data from metastatic melanoma; however, the frequencies of involvement differ, providing further insight into the course of genetic events.

Mechanisms of inactivation of E-cadherin in breast cancer cell lines.

Loss of E-cadherin (CDH1) function is thought to contribute to progression in breast cancer and other solid tumors by increasing proliferation, invasion, and/or metastasis. In some cases, the restoration of CDH1 function may be an important therapeutic option. This possibility will depend on the mechanism by which CDH1 is inactivated. Here we present analyses of CDH1 expression, genetic mutation, and promoter methylation in CDH1 in 10 commonly used breast cancer cell lines. Five cell lines (BT-474, MCF-7, MDA-MB-361, MDA-MB-468, and T-47D) expressed CDH1 and were genetically normal. Five others (SK-BR-3, 600 MPE, MDA-MB-134 IV, CAMA1, and MDA-MB-435) did not express CDH1. Fluorescence in situ hybridization analyses of each of these cell lines showed evidence for the physical deletion of one allele of CDH1, and three cell lines were found to carry homozygous deletions. SK-BR-3 was deleted from exon 12 through the promoter; exon 6 was deleted in MDA-MB-134 IV cells, and 600 MPE cells carried a 21-bp deletion in the splicing acceptor site for exon 9. CAMA1 seemed to have been inactivated through promoter methylation. No explanation was found for the inactivation of CDH1 in MDA-MB-435.

Gene amplifications characterize acral melanoma and permit the detection of occult tumor cells in the surrounding skin.

Acral melanoma (AM) is commonly distinguished from superficial spreading melanoma (SSM), the most common type of melanoma, by its clinical presentation as well as its ethnic distribution. However, justification for such a distinction is controversial because of histological overlap and lack of prognostic significance. We analyzed chromosomal aberrations of 15 AMs and 15 SSMs that were comparable for tumor thickness and patient age, using comparative genomic hybridization. All AMs had at least one (mean, 2.0) gene amplification, significantly more than the SSMs, in which only 2 of 15 (13%) had one amplification each (P < 0.0001). At least 15 different genomic regions were amplified in AM. These involved small portions of chromosomal arms, sometimes including known oncogenes implicated in melanoma. The most frequently amplified regions in AMs occurred at 11q13 (47%), 22q11-13 (40%), and 5p15 (20%). Comparison of the amplification levels of invasive and noninvasive portions of the tumors using fluorescence in situ hybridization suggested that amplifications occurred before the formation of the invasive portion. The finding of amplifications of 11q13 in three of five additional cases of AM in situ further supports the notion that amplifications arise early in the progression of AM. Very significantly, we found isolated melanocytes with amplifications in the epidermis up to 3 mm beyond the histologically recognizable extent of the melanomas in 5 of 15 invasive AMs. In conclusion, our data show that AM is a distinct type of melanoma characterized by focused gene amplifications occurring early in tumorigenesis, and that malignant cells are present beyond the histologically detectable boundary, thereby revealing one mechanism of local recurrence.

Difluoromethylornithine chemoprevention of epidermal carcinogenesis in K14-HPV16 transgenic mice.

To be informative for chemoprevention, animal models must both closely emulate human disease and possess surrogate endpoint biomarkers that facilitate rapid drug screening. This study elucidated site-specific histopathological and biochemical surrogate endpoint biomarkers of spontaneous epidermal carcinogenesis in K14-HPV16 transgenic mice and demonstrated that the incidence and severity of these markers were decreased by the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO). The cumulative incidence of visible epidermal cancers in 127 untreated transgenic mice was 42% by 52 weeks of age, most frequently affecting the chest as flat lesions in association with chronic ulcers, or in the ear as protuberant masses. Microscopic malignancies were detected in 39% of 32-week-old transgenic mice and were found to emerge from precursor lesions that were of two distinct types: dysplastic sessile ear papillomas and hyperproliferative follicular/interfollicular chest dysplasias. ODC activity and tissue polyamine contents were differentially elevated in ear and chest skin during carcinogenesis, such that there was a marked elevation of both parameters of polyamine metabolism as early as 4 weeks of age in the ear, whereas in the chest, polyamine metabolism was increased significantly only in the late stages of neoplastic progression and in epidermal cancers. Administration of 1.0% DFMO in the drinking water from 4 to 32 weeks of age prevented both visible and microscopic malignancies and significantly decreased the incidence of chest and ear precursor lesions. ODC activity and tissue putrescine content were markedly diminished by DFMO chemoprevention in ear skin, whereas there was a more modest decline of these parameters in chest skin. DFMO treatment of transgenic mice from 28 to 32 weeks of age was associated with an absence of ear cancer and a marked regression of dysplastic papillomas. In contrast, the results in chest skin were complex in that the severity of chest precursors diminished, but their incidence was unchanged, and microscopic cancers were still detectable within these lesions. Collectively, this study highlights the utility of multistage epidermal carcinogenesis in K14-HPV16 transgenic mice both for the study of the biology of, and as a screening tool for, novel drugs and chemopreventive regimens.