Genome screening by comparative genomic hybridization.

Comparative genomic hybridization (CGH) provides a molecular cytogenetic approach for genome-wide scanning of differences in DNA sequence copy number. The technique is now attracting wide-spread interest, especially among cancer researchers. The rapidly expanding database of CGH publications already covers about 1500 tumors and is beginning to reveal genetic abnormalities that are characteristic of certain tumor types or stages of tumor progression. Six novel gene amplifications, as well as a locus for a cancer-predisposition syndrome, have been discovered based on CGH data. CGH has now been established as a first-line screening technique for cancer researchers and will serve as a basis for ongoing efforts to develop high-resolution next-generation genome scanning, such as the microarray technology.

Gains and losses of DNA sequences in osteosarcomas by comparative genomic hybridization.

Our aim was to identify chromosomal regions that are likely to harbor previously unknown genes with an important role in the genesis of osteosarcoma. Comparative genomic hybridization was used to screen for losses and gains of DNA sequences along all chromosome arms in 11 tumors. Extensive genetic aberrations, with an average of 11 changes/tumor (range, 1-20), were found in 10 of the 11 specimens. High level amplifications of small chromosomal regions were detected in eight tumors. These involved the 12q12-q13 region (known to contain the SAS-MDM2 locus) and several previously unreported amplification sites such as 17p11-p12, 3q26, and Xq12. When all DNA sequence gains were evaluated, the gains at 8q and Xp were most common (45%). The most common losses of DNA sequences were seen at 2q, 6q, 8p, and 10p (36%). In conclusion, despite the very complex pattern of genetic changes in osteosarcomas, certain chromosomal regions appear to be affected more often than others. Most of these regions have not previously been reported to be implicated in osteosarcomas and may thus highlight locations of novel genes with an important role in the development and progression of these tumors.

Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes.

Studies by comparative genomic hybridization have indicated that a major new locus for DNA amplification in breast cancer is 20q13 and suggested that this genetic event is associated with aggressive clinical behavior. We used interphase fluorescence in situ hybridization with anonymous cosmid probes and gene-specific P1 clones to determine the minimal common region of increased copy number and to study involvement of known genes at 20q13. Based on high-level copy number increases (3 to 10-fold) found with one or more probes in 5 of 14 (35%) breast cancer cell lines and in 3 of 36 (8%) primary tumors, the critical region was narrowed to approximately 1.5 megabases at 20q13.2 defined by fractional length pter values 0.81-0.84. Previously known genes were excluded as candidates, implying that this chromosomal region harbors a novel oncogene that contributes to the malignant progression of breast cancer.

Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization.

Genetic changes leading to the development of prostate cancer and factors that underlie the clinical progression of the disease are poorly characterized. Here, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes along all chromosomes in 31 primary and 9 recurrent uncultured prostate carcinomas. The aim of the study was to identify those chromosome regions that contain genes important for the development of prostate cancer and to identify genetic markers of tumor progression. CGH analysis indicated that 74% of primary prostate carcinoma showed DNA sequence copy number changes. Losses were 5 times more common than gains and most often involved 8p (32%), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). Allelic loss studies with 5 polymorphic microsatellite markers for 4 different chromosomes were done from 13 samples and showed a 76% concordance with CGH results. In local recurrences that developed during endocrine therapy, there were significantly more gains (P < 0.001) and losses (P < 0.05) of DNA sequences than in primary tumors, with gains of 8q (found in 89% of recurrences versus 6% of primary tumors), X (56% versus 0%), and 7 (56% versus 10%), as well as loss of 8p (78% versus 32%), being particularly often involved. In conclusion, our CGH results indicate that losses of several chromosomal regions are common genetic changes in primary tumors, suggesting that deletional inactivation of putative tumor suppressor genes in these chromosomal sites is likely to underlie development of prostate cancer. Furthermore, the pattern of genetic changes seen in recurrent tumors with the frequent gains of 7, 8q, and X suggests that the progression of prostate cancer and development of hormone-independent growth may have a distinct genetic basis. These chromosome aberrations may have diagnostic utility as markers of prostate cancer progression.

Genetic heterogeneity and clonal evolution underlying development of asynchronous metastasis in human breast cancer.

To understand the genetic basis and clonal evolution underlying metastatic progression of human breast cancer in vivo, we analyzed the genetic composition of 29 primary breast carcinomas and their paired asynchronous metastases by comparative genomic hybridization and fluorescence in situ hybridization. The mean number of genetic changes by comparative genomic hybridization was 8.7 +/- 5.3 in primary tumors and 9.0 +/- 5.7 in their metastases. Although most of the genetic changes occurred equally often in the two groups, gains of the Xq12-q22 region were enriched in the metastases. According to a statistical analysis of shared genetic changes and breakpoints in paired specimens, 20 of the metastases (69%) showed a high degree of clonal relationship with the corresponding primary tumor, whereas the genetic composition of 9 metastases (31%) differed almost completely from that of the paired primary tumors. In both groups, however, chromosome X inactivation patterns suggested that the metastatic lesions originated from the same clone as the primary tumor. Fluorescence in situ hybridization analysis with probes specific to metastatic clones usually failed to find such cells in the primary tumor sample. In conclusion, detailed characterization of the in vivo progression pathways of metastatic breast cancer indicates that a linear progression model is unlikely to account for the progression of primary tumors to metastases. An early stem line clone apparently evolves independently in the primary tumor and its metastasis, eventually leading to multiple, genetically almost completely different, clones in the various tumor locations in a given patient. The resulting heterogeneity of metastatic breast cancer may underlie its poor responsiveness to therapy and explain why biomarkers of prognosis or therapy responsiveness measured exclusively from primary tumors give a restricted view of the biological properties of metastatic breast cancer.

Distinct somatic genetic changes associated with tumor progression in carriers of BRCA1 and BRCA2 germ-line mutations.

BRCA1 and BRCA2 mutations confer increased risk for development of breast cancer, but a number of additional, currently largely unknown, somatic genetic defects must also accumulate in the breast epithelial cells before malignancy develops. To evaluate the nature of these additional somatic genetic defects, we performed a genome-wide survey by comparative genomic hybridization on breast cancers from 21 BRCA1 mutation carriers, 15 BRCA2 mutation carriers, and 55 unselected controls. The total number of genetic changes was almost two times higher in tumors from both BRCA1 and BRCA2 mutation carriers than in the control group. In BRCA1 tumors, losses of 5q (86%), 4q (81%), 4p (64%), 2q (40%), and 12q (40%) were significantly more common than in the control group (7-13%). BRCA2 tumors were characterized by a higher frequency of 13q (73%) and 6q (60%) losses and gains of 17q22-q24 (87%) and 20q13 (60%) as compared to the prevalence of these changes in the control group (12-18%). In conclusion, accumulation of somatic genetic changes during tumor progression may follow a unique pathway in individuals genetically predisposed to cancer, especially by the BRCA1 gene. Activation or loss of genes in the affected chromosomal regions may be selected for during tumor progression in cells lacking functional BRCA1 or BRCA2. Identification of such genes could provide targets for therapeutic intervention and early diagnosis.

Gain of 17q24-qter detected by comparative genomic hybridization in malignant tumors from patients with von Recklinghausen's neurofibromatosis.

The genetic changes leading to the development of malignant peripheral nerve sheath tumors (MPNSTs) are largely unknown. The few tumors that have been investigated cytogenetically had highly complex karyotypes and no consistent rearrangements, and the attempts to pinpoint consistent DNA-level changes have met with only limited success. We used comparative genomic hybridization to analyze seven MPNSTs and one dermatofibrosarcoma protuberans from eight patients with von Recklinghausen's disease (neurofibromatosis type 1), as well as three sporadic MPNSTs. Gains and losses of DNA sequences were found in all tumors, with an average of four losses (range, 0-14) and two gains (range, 0-5) per tumor. Two striking observations were made: (a) an increase in copy number of the distal part of the long arm of chromosome 17, with the smallest region of overlap 17q24-qter, was seen in five of seven MPNSTs and in the only dermatofibrosarcoma protuberans, all of which were from patients with neurofibromatosis, whereas none of the three sporadic MPNSTs had this alteration; and (b) loss of 13q, with the smallest region of overlap 13q14-q21, was found in 6 of 10 MPNSTs. The consistent involvement of these two chromosomal regions probably reflects two different pathogenetic mechanisms for MPNSTs.

Genetic aberrations in adrenocortical tumors detected using comparative genomic hybridization correlate with tumor size and malignancy.

The differentiation between malignant and benign adrenocortical tumors is often difficult, and better markers are required. Because the genetic background of adrenocortical tumors is poorly characterized, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes in 8 sporadic primary adrenocortical cancers and 14 adenomas. There was a strong relationship between the number of genetic aberrations detected using CGH and both tumor size and malignancy. No alterations were seen in the smaller adenomas (< 5 cm), whereas the two largest adenomas (5 cm each) and seven of the eight cancers (7-20 cm) showed an increased number of genetic alterations. The presence of genetic aberrations detected using CGH was associated with an aneuploid DNA pattern. In the cancers, losses most often involved the chromosomal regions 2, 11q, and 17p (four of eight tumors), whereas gains took place at chromosomes 4 and 5 (four of eight tumors). In conclusion, our data indicate that genetic changes may help to define the malignant potential of adrenocortical tumors. Furthermore, the CGH results implicate several chromosomal regions that may contain genes with an important role in the development of adrenocortical cancers.

Genotypic stability, segregation and selection in heteroplasmic human cell lines containing np 3243 mutant mtDNA.

The mitochondrial genotype of heteroplasmic human cell lines containing the pathological np 3243 mtDNA mutation, plus or minus its suppressor at np 12300, has been followed over long periods in culture. Cell lines containing various different proportions of mutant mtDNA remained generally at a consistent, average heteroplasmy value over at least 30 wk of culture in nonselective media and exhibited minimal mitotic segregation, with a segregation number comparable with mtDNA copy number (>/=1000). Growth in selective medium of cells at 99% np 3243 mutant mtDNA did, however, allow the isolation of clones with lower levels of the mutation, against a background of massive cell death. As a rare event, cell lines exhibited a sudden and dramatic diversification of heteroplasmy levels, accompanied by a shift in the average heteroplasmy level over a short period (<8 wk), indicating selection. One such episode was associated with a gain of chromosome 9. Analysis of respiratory phenotype and mitochondrial genotype of cell clones from such cultures revealed that stable heteroplasmy values were generally reestablished within a few weeks, in a reproducible but clone-specific fashion. This occurred independently of any straightforward phenotypic selection at the individual cell-clone level. Our findings are consistent with several alternate views of mtDNA organization in mammalian cells. One model that is supported by our data is that mtDNA is found in nucleoids containing many copies of the genome, which can themselves be heteroplasmic, and which are faithfully replicated. We interpret diversification and shifts of heteroplasmy level as resulting from a reorganization of such nucleoids, under nuclear genetic control. Abrupt remodeling of nucleoids in vivo would have major implications for understanding the developmental consequences of heteroplasmy, including mitochondrial disease phenotype and progression.

Gene Copy Number Analysis by Fluorescence in Situ Hybridization and Comparative Genomic Hybridization

Fluorescence in situ hybridization (FISH) with gene- and locus-specific probes provides a rapid means to assess copy numbers of specific sequences in individual interphase nuclei. Recent technical improvements have made FISH applicable to the analysis of both fresh and archival tissue specimens in research as well as in diagnostic laboratories. FISH is limited to analysis of one or a few loci at a time, making genome-wide surveys impractical. Comparative genomic hybridization (CGH) was developed as a means to screen entire genomes for DNA sequence copy number changes. CGH is based on the cohybridization of differentially labeled test and reference DNAs to normal metaphase chromosomes. Measurement of the test to reference fluorescence ratios along all chromosomes provides information on chromosomal regions that are over- or underrepresented in the test genome. The use of these two techniques will be illustrated in the analysis of genetic changes in solid tumors. The techniques are complementary to one another and have proven to be highly useful for identification of previously unknown genetic changes and genes that play an important role in tumor progression.

Genomic structure and chromosomal localization of the rat protein kinase Cdelta-gene.

Protein kinase Cdelta (PKCdelta) is a widely expressed calcium-independent PKC isozyme that is induced at mRNA and protein levels upon stimulation of different cellular pathways. We found the rat PKCdelta gene to consist of 19 exons and to span approximately 29 kb. The exon-intron junctions follow the GT/AG rule. The 5' untranslated region is nearly 12 kb in length, and the transcription initiation site is surrounded by CG-rich sequences. The 5' flanking region contains putative binding sites for activator protein 1 (AP-1), nuclear factor kappa B (NFkappaB), stimulatory protein-1 (Sp-1) and nerve growth factor induced-C (NGFI-C) transcription factors. The PKCdelta gene is localized at the rat chromosome 19p14. The cloned gene will help to elucidate the role of PKCdelta in growth, differentiation and death of mammalian cells.

High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agarose-embedded cells.

Fluorescence in situ hybridization (FISH) and pulse field gel electrophoresis (PFGE) are essential techniques in physical mapping and in positional cloning. We present a technique that utilizes agarose-embedded high molecular weight DNA prepared for PFGE as a target for FISH. The agarose blocks are melted, and the DNA is extended on a poly-L-lysine-coated microscope slide. The resulting DNA fibers appear on the slide as long straight strands and are a suitable target for high resolution FISH mapping as demonstrated here with cosmid and plasmid hybridizations.

Aberrations of chromosome 8 in 16 breast cancer cell lines by comparative genomic hybridization, fluorescence in situ hybridization, and spectral karyotyping.

Comparative genomic hybridization (CGH) studies have shown that chromosome 8 is a frequent target for chromosomal aberrations in breast cancer. We characterized these aberrations of chromosome 8 in 16 breast cancer cell lines (BT-474, BT-549, CAMA-1, DU-4475, MCF-7, MDA-MB-134, MDA-MB-157, MDA-MB-361, MDA-MB-415, MDA-MB-436, MPE600, SK-BR-3, T-47D, UACC-812, UACC-893 and ZR-75-1) by CGH, fluorescence in situ hybridization (FISH) with arm- and locus-specific probes, and spectral karyotyping (SKY). Chromosome 8 was structurally abnormal in 13 of 16 cell lines. Loss of 8p was detected in nine cell lines, gain of entire 8q in six cell lines, 8q21-qter in three, 8q23-qter in two, and 8q12-qter and 8p21-q21 in one cell line. Extra copies of the C-MYC oncogene were found in 11 cell lines, but high-level amplification only in SK-BR-3. Derivative chromosomes including material from chromosomes 8 were complex, and the breakpoints were strikingly dissimilar. Chromosome 11 was the most frequent translocation partner with chromosome 8 (in 7 cell lines). Isochromosomes and/or isoderivative 8q were found in four cell lines. The high frequency and complexity of alterations at 8q indicate a significant pathogenetic role in breast cancer. The high-level amplification of c-myc is less common than previously thought.

Genetic aberrations in pediatric acute lymphoblastic leukemia by comparative genomic hybridization.

Classical cytogenetic analysis plays an important role in the diagnosis and classification of childhood acute lymphoblastic leukemia (ALL). However, poor in vitro growth of the malignant cells and suboptimal quality of metaphase spreads may sometimes cause false-negative findings (normal karyotype). We used comparative genomic hybridization (CGH) to study whether this new method is able to detect and characterize genetic aberrations not detected by karyotyping. CGH showed clonal genetic aberrations in 8 of 13 cases, most of which showed gains of several chromosomes, indicating hyperdiploidy. The sensitivity of CGH was sufficient to detect a small interstitial deletion of 6q. One karyotypically complex case was resolved by CGH showing a high-level amplification of DNA sequences originating from the 12p12-13. Interphase fluorescence in situ hybridization (FISH) analyses confirmed the CGH findings in 2 cases, validating the accuracy of CGH. In conclusion, CGH experiments established the known fact that hyperdiploidy is the most common finding in pediatric ALLs and that CGH may detect aberrations that are not seen in the G-banded karyotype. CGH was also able to further characterize genetic aberrations such as gene amplification, which is occasionally involved in pediatric ALL as well as in other leukemias.

Gains and losses of DNA sequences in malignant mesothelioma by comparative genomic hybridization.

The molecular basis of malignant mesothelioma is poorly known. We examined genetic changes in 11 mesothelioma specimens by comparative genomic hybridization (CGH). Five DNA specimens originated from uncultured tumor tissues and six from cell lines established from the same patients. Findings from the classical karyotypic characterization of both primary tumors and cell lines have been reported previously. In the CGH analyses the most common genetic alterations in the 11 mesothelioma specimens were losses of chromosomal regions in 1p, 8p, 14q, and 22q and gains of 5p, 6p, 8q, 15q, 17q, and 20. The cell lines had on average a much higher total number of genetic changes than the uncultured tumor specimens. Clonal relationship between the cell lines and the uncultured tissue specimens could not usually be demonstrated even though they originated from the same patient. The observed differences may partly be due to high frequency of chromosomal rearrangements, which CGH cannot detect, partly due to contamination of tumor specimens with normal tissue, and partly due to genetic evolution in tumor cell lines.

Effects of pyriproxyfen spray, powder, and oral bait treatments on the relative abundance of fleas (Siphonaptera: Ceratophyllidae) in black-tailed prairie dog (Rodentia: Sciuridae) towns.

Separate black-tailed prairie dog, Cynomys ludovicianus (Ord), towns on the Rocky Mountain Arsenal National Wildlife Refuge, Colorado, were treated with technical pyriproxyfen (Nylar) using spray, powder, and oral bait carriers. Direct combing methods (1997 and 1998) and burrow flagging (1998) were used to estimate relative abundance of the plague vector Oropsylla hirsuta (Baker). Pyriproxyfen spray (0.05%) and powder (0.05%) did not significantly reduce (P > 0.05) O. hirsuta abundance. Pyriproxyfen bait, when applied every 4 wk at a concentration of 286 mg/50 g bait, significantly reduced (P < or = 0.05) O. hirsuta infesting prairie dogs, 4 mo after initial treatment. However, flea populations had recovered to pretreatment levels by the following summer (July 1999).

Effects of pyriproxyfen spray, powder, and oral bait treatments on the relative abundance of nontarget arthropods of black-tailed prairie dog (Rodentia: Sciuridae) towns.

Separate black-tailed prairie dog, Cynomys ludovicianus (Ord), towns on the Rocky Mountain Arsenal National Wildlife Refuge, Colorado, were treated with technical pyriproxyfen (Nylar) spray, powder, and oral bait. The treatments were applied to reduce relative abundance of the plague vector Oropsylla hirsuta (Baker). Because pyriproxyfen is a juvenile hormone analog, we were also concerned with the effects of the treatments on nontarget arthropods, which is the focus of this study. Pitfall traps and sweep net sampling were used to measure relative abundance of arthropod populations pre- and posttreatment. Nontarget arthropod sampling produced a large number of statistical comparisons that indicated significant declines (P < 0.05) in relative arthropod abundance. Many of the significant declines were probably because of natural fluctuations in arthropod populations rather than treatment effects. Because arthropod populations appeared to fluctuate randomly, we only made inferences about highly significant (P < 0.001) declines. In doing so, we hoped to abate some of the confusion created by the natural fluctuation in arthropod abundance and increase our chance of correctly attributing a population reduction to a treatment effect. Only Homoptera at the pyriproxyfen powder site exhibited highly significant reductions that appeared to be attributed to the treatments. Pyriproxyfen spray treatments did not significantly reduce relative arthropod abundance.

High-level amplification at 17q23 leads to coordinated overexpression of multiple adjacent genes in breast cancer.

Increased copy numbers of 17q23 chromosomal region have been shown to occur in different tumour types and to be associated with tumour progression and with poor prognosis. Several genes have earlier been proposed as potential oncogenes at this region largely on the grounds of cell lines studies. In this study, we performed a systematic gene expression survey on 26 primary breast tumours with known 17q23 amplification status by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The 17q23 amplicon is restricted to an approximately 5 MB region in breast cancer and contains 29 known genes. Our survey revealed a statistically significant (P<0.01) difference between the high level and no amplification groups in a set of eleven genes whereas no difference between the moderate and the non-amplified tumour groups were observed. Interestingly, these 11 genes were located adjacent to one another within a 1.56 Mb core region in which all except one of the genes were overexpressed. These data suggest that only high-level amplification at the 17q23 amplicon core leads to elevated gene expression in breast cancer. Moreover, our results highlight the fact that 17q23 amplicon carries multiple candidate genes and that this may be a more common event in gene amplification than previously thought.

Evaluation of camera requirements for comparative genomic hybridization.

Comparative genomic hybridization (CGH) is based on quantitative digital image analysis of fluorescence intensities from metaphase chromosomes. High-quality CCD cameras are commonly used for image acquisition, but the minimal requirements of CCD cameras have not been determined. We first evaluated minimal camera requirements by artificially reducing spatial and dynamic resolution of images produced by a scientific-grade CCD camera (Xillix MicroImager). The results showed that reduction of dynamic resolution from 4,096 to 256 gray levels (12-bit image transformed to an 8-bit image) had negligible effect on CGH profiles and no effect on their interpretation. Similarly, CGH profiles obtained from spatially reduced images (from 1,340 x 1,035 to 670 x 517 pixels) were virtually identical to those obtained from the original image. For a practical test, we compared two 8-bit frame integrating video-rated CCD cameras (Cohu 4910 and Photometrics ImagePoint) to the Xillix Micro-Imager in a real CGH setting. Images collected from the same metaphase cells with all three cameras resulted in the identification of the same genetic changes in the samples studied. We conclude that requirements for camera resolution in CGH analysis are not stringent, and therefore that low-priced video-rated cameras capable of frame integration are sufficient for comparative genomic hybridization.

Four-color CGH: a new method for quality control of comparative genomic hybridization.

Comparative genomic hybridization (CGH) has become a widely used method in molecular cytogenetics to screen for copy number aberrations in human malignancies. Although the hybridization protocol is relatively simple, the validation and quality control of CGH have remained difficult. We describe here a new modification of CGH, four-color CGH, which is based on conventional CGH with an added Cy5-labeled second reference DNA, that serves as an internal standard in every hybridization. The internal standard aids in identifying inconsistently hybridized chromosomal regions (such as 1pter, 19, 22). When using a special second reference DNA (from a sex-mismatched trisomy 13 cell line) for four-color CGH, it is possible to standardize the dynamic range of hybridization. The four-color CGH modification is simple to adopt, requiring only the addition of Cy5-labeled reference DNA to the existing hybridization protocol. The principles and the modifications of the CGH image analysis software are described in detail.