Comparative Genomic Hybridization Experiments Research Articles

Heterologous microarray experiments used to identify the early gene response to heat stress in a coral reef fish

Coral reef fishes are expected to experience rising sea surface temperatures due to climate change. How well tropical reef fishes will respond to these increased temperatures and which genes are important in the response to elevated temperatures is not known. Microarray technology provides a powerful tool for gene discovery studies, but the development of microarrays for individual species can be expensive and time-consuming. In this study, we tested the suitability of a Danio rerio oligonucleotide microarray for application in a species with few genomic resources, the coral reef fish Pomacentrus moluccensis. Results from a comparative genomic hybridization experiment and direct sequence comparisons indicate that for most genes there is considerable sequence similarity between the two species, suggesting that the D. rerio array is useful for genomic studies of P. moluccensis. We employed this heterologous microarray approach to characterize the early transcriptional response to heat stress in P. moluccensis. A total of 111 gene loci, many of which are involved in protein processing, transcription, and cell growth, showed significant changes in transcript abundance following exposure to elevated temperatures. Changes in transcript abundance were validated for a selection of candidate genes using quantitative real-time polymerase chain reaction. This study demonstrates that heterologous microarrays can be successfully employed to study species for which specific microarrays have not yet been developed, and so have the potential to greatly enhance the utility of microarray technology to the field of environmental and functional genomics.

A Segmentation/Clustering Model for the Analysis of Array CGH Data

Microarray-CGH (comparative genomic hybridization) experiments are used to detect and map chromosomal imbalances. A CGH profile can be viewed as a succession of segments that represent homogeneous regions in the genome whose representative sequences share the same relative copy number on average. Segmentation methods constitute a natural framework for the analysis, but they do not provide a biological status for the detected segments. We propose a new model for this segmentation/clustering problem, combining a segmentation model with a mixture model. We present a new hybrid algorithm called dynamic programming-expectation maximization (DP-EM) to estimate the parameters of the model by maximum likelihood. This algorithm combines DP and the EM algorithm. We also propose a model selection heuristic to select the number of clusters and the number of segments. An example of our procedure is presented, based on publicly available data sets. We compare our method to segmentation methods and to hidden Markov models, and we show that the new segmentation/clustering model is a promising alternative that can be applied in the more general context of signal processing.

Whole-genome analysis of the methyl tert-butyl ether-degrading beta-proteobacterium Methylibium petroleiphilum PM1.

Methylibium petroleiphilum PM1 is a methylotroph distinguished by its ability to completely metabolize the fuel oxygenate methyl tert-butyl ether (MTBE). Strain PM1 also degrades aromatic (benzene, toluene, and xylene) and straight-chain (C(5) to C(12)) hydrocarbons present in petroleum products. Whole-genome analysis of PM1 revealed an approximately 4-Mb circular chromosome and an approximately 600-kb megaplasmid, containing 3,831 and 646 genes, respectively. Aromatic hydrocarbon and alkane degradation, metal resistance, and methylotrophy are encoded on the chromosome. The megaplasmid contains an unusual t-RNA island, numerous insertion sequences, and large repeated elements, including a 40-kb region also present on the chromosome and a 29-kb tandem repeat encoding phosphonate transport and cobalamin biosynthesis. The megaplasmid also codes for alkane degradation and was shown to play an essential role in MTBE degradation through plasmid-curing experiments. Discrepancies between the insertion sequence element distribution patterns, the distributions of best BLASTP hits among major phylogenetic groups, and the G+C contents of the chromosome (69.2%) and plasmid (66%), together with comparative genome hybridization experiments, suggest that the plasmid was recently acquired and apparently carries the genetic information responsible for PM1's ability to degrade MTBE. Comparative genomic hybridization analysis with two PM1-like MTBE-degrading environmental isolates (approximately 99% identical 16S rRNA gene sequences) showed that the plasmid was highly conserved (ca. 99% identical), whereas the chromosomes were too diverse to conduct resequencing analysis. PM1's genome sequence provides a foundation for investigating MTBE biodegradation and exploring the genetic regulation of multiple biodegradation pathways in M. petroleiphilum and other MTBE-degrading beta-proteobacteria.

A stepwise framework for the normalization of array CGH data

BackgroundIn two-channel competitive genomic hybridization microarray experiments, the ratio of the two fluorescent signal intensities at each spot on the microarray is commonly used to infer the relative amounts of the test and reference sample DNA levels. This ratio may be influenced by systematic measurement effects from non-biological sources that can introduce biases in the estimated ratios. These biases should be removed before drawing conclusions about the relative levels of DNA. The performance of existing gene expression microarray normalization strategies has not been evaluated for removing systematic biases encountered in array-based comparative genomic hybridization (CGH), which aims to detect single copy gains and losses typically in samples with heterogeneous cell populations resulting in only slight shifts in signal ratios. The purpose of this work is to establish a framework for correcting the systematic sources of variation in high density CGH array images, while maintaining the true biological variations.ResultsAfter an investigation of the systematic variations in the data from two array CGH platforms, SMRT (Sub Mega base Resolution Tiling) BAC arrays and cDNA arrays of Pollack et al., we have developed a stepwise normalization framework integrating novel and existing normalization methods in order to reduce intensity, spatial, plate and background biases. We used stringent measures to quantify the performance of this stepwise normalization using data derived from 5 sets of experiments representing self-self hybridizations, replicated experiments, detection of single copy changes, array CGH experiments which mimic cell population heterogeneity, and array CGH experiments simulating different levels of gene amplifications and deletions. Our results demonstrate that the three-step normalization procedure provides significant improvement in the sensitivity of detection of single copy changes compared to conventional single step normalization approaches in both SMRT BAC array and cDNA array platforms.ConclusionThe proposed stepwise normalization framework preserves the minute copy number changes while removing the observed systematic biases.

Involvement of cyclin D3 in liver metastasis of colorectal cancer, revealed by genome-wide copy-number analysis

The question of whether any genetic differences exist between primary and colorectal cancers (CRCs) and their metastatic foci is controversial. To look for genetic aberrations involved in metastasis of CRCs to the liver, we performed subtractive comparative genomic hybridization (CGH) experiments using paired samples from 20 CRC patients with primary tumors and synchronous or metachronous liver metastases. Relatively frequent gains in DNA copy number were detected at 6p, suggesting the presence of one or more metastasis-related genes in the region. Analysis of 11 CRC cell lines using array-based CGH (CGH-array) revealed one 6p candidate gene, CCND3. Quantitative reverse transcriptase-polymerase chain reaction experiments showed that CCND3 was significantly upregulated in liver-metastatic lesions compared with primary lesions (P<0.0152). In addition, immunohistochemical analysis of 120 primary CRC tumors demonstrated that cyclin D3 expression in the region of rolled edge was significantly associated with total recurrence, especially hematogenous recurrence (P=0.0307). The results implied involvement of cyclin D3 in liver metastasis of CRC, and the data may contribute to the development of a novel therapy or diagnostic agent for this currently intractable disease. Our experiments also confirmed the power of subtractive CGH and CGH-array analysis for identifying cancer-related genes.

Data Rotation Improves Genomotyping Efficiency

Unsequenced bacterial strains can be characterized by comparing their genomic DNA to a sequenced reference genome of the same species. This comparative genomic approach, also called genomotyping, is leading to an increased understanding of bacterial evolution and pathogenesis. It is efficiently accomplished by comparative genomic hybridization on custom-designed cDNA microarrays. The microarray experiment results in fluorescence intensities for reference and sample genome for each gene. The log-ratio of these intensities is usually compared to a cut-off, classifying each gene of the sample genome as a candidate for an absent or present gene with respect to the reference genome. Reducing the usually high rate of false positives in the list of candidates for absent genes is decisive for both time and costs of the experiment. We propose a novel method to improve efficiency of genomotyping experiments in this sense, by rotating the normalized intensity data before setting up the list of candidate genes. We analyze simulated genomotyping data and also re-analyze an experimental data set for comparison and illustration. We approximately halve the proportion of false positives in the list of candidate absent genes for the example comparative genomic hybridization experiment as well as for the simulation experiments.

Genetic changes and clonality relationship between primary colorectal cancers and their pulmonary metastases?An analysis by comparative genomic hybridization

About 10% of colorectal carcinoma patients develop pulmonary metastases during their lifetime. We address whether and how the chromosomal abnormalities differ between the primary cancers and their metastatic counterparts, what the clonality relationship (CR) is between them, and whether certain genomic aberrations contribute to this disease progression. Comparative genomic hybridization (CGH) experiments were performed on 18 paired samples of primary and pulmonary metastases obtained from patients who had undergone two consecutive surgeries and from whom clinical data had been collected. The CGH profiles also were used as indexes for determining the CR between the cancers. The overall CGH abnormality profiles were similar for the primary colorectal carcinomas and their pulmonary metastases. Frequent gains were found on chromosome arms 20q, 8q, 13q, and 7q, whereas common losses were found on 18q, 8p, and 18p. The pulmonary metastases, however, contained more CGH abnormalities than did the primary carcinomas (total aberration events per tumor: 12.6 +/- 5.0 vs. 8.3 +/- 5.7, respectively, P = 0.024; gains: 7.6 +/- 3.1 vs. 5.1 +/- 3.5, respectively, P = 0.036; losses: 5.0 +/- 2.8 vs. 3.3 +/- 2.9, respectively, P = 0.076). Comparing CGH profiles between individual primary and metastasis pairs, we found that 10 of the 18 (56%) paired samples examined exhibited a high degree of CR, indicating that they were likely to have originated from the same clone and/or that not many additional chromosomal changes had occurred in the metastases, except for 4q loss, whose incidence was much higher in the metastases than in the primaries (60% vs. 10%; P = 0.030). Also, the primary tumors of the high-CR group carried more genomic aberrations, especially 8p loss, than did the primary tumors in the low-CR group. We found more chromosomal changes associated with the pulmonary metastases of colorectal cancer compared with the corresponding primary tumors. We concluded that primary cancers containing more genomic lesions, especially 8p losses, are more likely to metastasize to the lungs. Loss of 4q is potentially a supplementary factor contributing to the dissemination of this disease.

Genomic DNA Copy Number Alterations Present in AML Bone Marrow Samples with Normal Cytogenetics.

Cytogenetics and comparative genomic hybridization (CGH) have been used to identify large genomic amplifications and deletions in all subtypes of acute myeloid leukemia (AML). Up to 15–20% of AML patients have a normal karyotype at diagnosis. While cytogenetic abnormalities confer important prognostic information for patients with AML, there remain differences in the therapeutic response and outcome among patients with the same cytogenetic profile, implying that other, more subtle, genetic abnormalities may exist. We hypothesized that a subset of AML patients with normal cytogenetics may contain genomic DNA copy number changes that are too small to be detected using standard cytogenetic techniques. To address this possibility, we used high-resolution bacterial artificial chromosome (BAC) array CGH technology to examine 31 AML patients with normal cytogenetics. The BAC arrays contain 2,464 BAC clones spotted in triplicate on glass slides, and provide a 1 Mb resolution of the entire human genome. Technical generation of the arrays, hybridization parameters, and analysis were similar to that reported for murine BAC array CGH (Nat Genet. 2001 Dec;29(4):459–64). The 31 AML samples included 4 M0, 8 M1, 10 M2, and 9 M4 patients. Array CGH experiments were performed using 500 nanograms of Cyanine 5 labeled genomic DNA from unmanipulated AML bone marrow, mixed with an equal amount of control DNA (a pool of DNA from 4 cancer-free individuals) labeled with Cyanine 3. Using the human 1 Mb BAC arrays, we identified amplifications and deletions from multiple samples that were confirmed with G-banding cytogenetics [del(7)(q31), del(7)(p11.2), +8, del(11)(q13q23), +21, add(21)(q22), −X, −Y, +Y]. In addition, BAC arrays robustly detected copy number alterations that were identified in as few as 4/21 metaphases. We identified 5/31 (16%) patients with normal cytogenetics that contained altered genomic DNA copy numbers using BAC array CGH. Copy number changes were confirmed for several of these genomic loci using a dye-swap experiment, where the AML DNA was labeled with Cyanine 3, and the control DNA with Cyanine 5. Two of the 5 patients with abnormalities detected using array CGH would be reclassified from “intermediate” to “unfavorable” cytogenetics [del(7)(q31.31q34), add(11)(q23.3qter), and 17(p12pter)]. These results suggest that a subset of AML patients with normal cytogenetics contain genomic copy number alterations that may effect treatment and outcome.Patient #FAB subtypeGenomic locationGain or lossSize (Megabase)Dye-Swap confirmed1M07(q31.31q34)loss2.0Not done111(q23.3qter)gain16.5Not done2M111(p14)loss7.4Yes3M111(q13.2q14.1)gain15.8Yes319(p)gain64.0Yes4M217(p12pter)gain8.6Not Done5M219(p13.1pter)loss14.8Yes512(q13)loss5.0Yes

Comprehensive conventional and molecular cytogenetic characterization of B-CPAP, a human papillary thyroid carcinoma-derived cell line

Cell lines derived from different thyroid tumor histotypes are useful for the in vitro study of both the phenotypic and genetic features of these cancers. Although karyotypic changes are known to be associated with thyroid lesions, the chromosome patterns of only a few cell lines have been published. Herein, we report an extensive conventional and molecular cytogenetic investigation of the human papillary thyroid carcinoma derived cell line B-CPAP. Morphological studies and expression of tumor markers in this cell line have been reported previously, but no detailed characterization on the origin of the chromosome markers is available. B-CPAP cells have a rather stable hypertriploid karyotype, with chromosome polysomies and structural chromosome abnormalities featuring whole chromosome arm imbalances. Chromosome banding revealed a main clone with nine chromosome markers, and fluorescence in situ hybridization (FISH) with whole chromosome paint (wcp), partial chromosome paint (pcp), and centromeric probes clarifyied their origin. The use of centromeric probes provided accurate refinement of the rearrangements classified as whole-arm translocations by banding and FISH with wcp probes. Both chromosomal and array-based comparative genomic hybridization experiments confirmed the cytogenetic characterization of this cell line. Moreover, the use of fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) technique, which simultaneously shows nuclear ploidy and cytoplasmic immunofluorescence, detailed the oncocytic feature of the cells. Intriguingly, despite their origin, they lack most of the features expressed in papillary thyroid tumor cells and have a chromosomal pattern reminiscent of that of a subgroup of oncocytic malignant thyroid tumors.

Application of bacterial artificial chromosome array-based comparative genomic hybridization and spectral karyotyping to the analysis of glioblastoma multiforme

Identification of genetic losses and gains is valuable in analysis of brain tumors. Locus-by-locus analyses have revealed correlations between prognosis and response to chemotherapy and loss or gain of specific genes and loci. These approaches are labor intensive and do not provide a global view of the genetic changes within the tumor cells. Bacterial artificial chromosome (BAC) arrays, which cover the genome with an average resolution of less than 1 MbP, allow defining the sum total of these genetic changes in a single comparative genomic hybridization (CGH) experiment. These changes are directly overlaid on the human genome sequence, thus providing the extent of the amplification or deletion, reflected by a megabase position, and gene content of the abnormal region. Although this array-based CGH approach (CGHa) seems to detect the extent of the genetic changes in tumors reliably, it has not been robustly tested. We compared genetic changes in four newly derived, early-passage glioma cell lines, using spectral karyotyping (SKY) and CGHa. Chromosome changes seen in cell lines under SKY analysis were also detected with CGHa. In addition, CGHa detected cryptic genetic gains and losses and resolved the nature of subtle marker chromosomes that could not be resolved with SKY, thus providing distinct advantages over previous technologies. There was remarkable general concordance between the CGHa results comparing the cell lines to the original tumor, except that the magnitude of the changes seen in the tumor sample was generally suppressed compared with the cell lines, a consequence of normal cells contaminating the tumor sample. CGHa revealed changes in cell lines that were not present in the original tumors and vice versa, even when analyzed at the earliest passage possible, which highlights the adaptation of the cells to in vitro culture. CGHa proved to be highly accurate and efficient for identifying genetic changes in tumor cells. This approach can accurately identify subtle, novel genetic abnormalities in tumors directly linked to the human genome sequence. CGHa far surpasses the resolution and information provided by conventional metaphase CGH, without relying on in vitro culture of tumors for metaphase spreads.

S26.4: Reducing false positives rates by rotating microarray‐based genomotyping data

Microarray-based comparative genomic hybridization is leading to an increased understanding of bacterial evolution and patho-genesis by efficiently comparing whole genomes. Here, a sample genome is compared to a reference genome via comparative hybridization using two different fluorescent dyes. The logratio of the fluorescence intensities for sample and reference genome for each gene on the array is then usually compared to a cutoff classifying the belonging gene as absent or present with respect to the sample genome. The resulting list of candidate absent genes then undergoes confirmational analyses, i.e. PCR or sequencing, which are decisive with respect to both time and costs of the experiment. Thus, there is vital interest to reduce the rate of false positives in the list of candidate absent genes from the comparative genomic hybridization step. Our approach to accomplish this task uses the expected relationship between the logratio and the mean log intensities for absent genes to linear transform the inten-sity data before comparing to a cutoff. For validated data from a series of comparative genomic hybridizations for two Bartonella species we assessed significance and efficacy of the new approach. We show that we are able to halve the rate of false positives in the list of candidate absent genes for a typical comparative genomic hybridization experiment, thus significantly reducing time and costs of necessary confirmational analyses.

Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization.

The use of metaphase comparative genomic hybridization (CGH) to screen all human chromosomes for aneuploidy in preimplantation embryos is hindered by the time required to perform the analysis. We report in this paper a novel approach to manufacture a DNA microarray for CGH for the detection of aneuploidy in single cells. We spotted human chromosome-specific libraries on glass slides that were depleted of repetitive sequences and tested our array CGH method in 14 experiments using either single male and/or single female lymphocytes. For the autosomes, the mean normalized ratios were all close to the expected ratio of 1.0 with overall 300/308 (97%) of the normalized ratios falling within the range 0.75 to 1.25. It was possible to deduce the correct copy number of the X chromosome in 13/14 (92.9%) separate array CGH experiments but the Y chromosome in only 4/14 (29%). We tested our microarray CGH method on a single fibroblast from each of three cell lines containing a specific chromosome aneuploidy (trisomy 13, 15 or 18) and in each case our microarray analysis was able to obtain a diagnosis based on the fact that the aneuploid chromosome gave the highest ratio (1.32, 1.27 and 1.27 respectively) with the ratios of all other chromosomes falling within the range 0.75-1.25. Requiring just 30 h, our method may be more suitable for PGD aneuploidy screening than metaphase CGH.

Significant Hybridization Differences in Comparative Genomic Hybridization due to Nucleotides Used for DNA Labelling and to DNA Chosen for Cohybridization

Objective: Comparative genomic hybridization (CGH) has been established as an informative technique in genetic analysis. However, differences in the ratio of hybridization intensities were reported for particular chromosomes, which may affect CGH results. The aim of this study was to define these differences in more detail. For this purpose, CGH results of 70 samples of bone marrow cells (BMC) with normal karyotype in conventional cytogenetics (CC) were evaluated using seven different reference DNAs and two different DNA labeling systems. Methods and Results: CGH using fluorochrome-conjugated nucleotides for DNA labeling indicated signal deviations in 21/70 BMC samples. Deviations affected chromosomes 1 (n = 21), 2 (n = 11), 4 (n = 11), 5 (n = 9), 6 (n = 7), 7 (n = 2), 8 (n = 2), 12 (n = 5), 13 (n = 15), 14 (n = 1), 16 (n = 17), 17 (n = 11), 19 (n = 21), 20 (n = 12), and/or 22 (n = 17). None of the imbalances were confirmed by fluorescence in situ hybridization (FISH). Using digoxigenin and biotin-conjugated nucleotides in exemplary cases (n = 5) led to the disappearance of the signal deviations. Repeated CGH experiments using seven different reference DNAs showed remarkable variations in the signal deviations. Conclusion: Hybridization differences depend not only on the hapten or fluorochrome-labeled nucleotides used for DNA labeling, but also on the reference DNA chosen. Therefore, close control of CGH experiments is mandatory, and additional techniques such as FISH should be performed to confirm the results obtained by CGH.

Fully automatic quantification of microarray image data.

DNA microarrays are now widely used to measure expression levels and DNA copy number in biological samples. Ratios of relative abundance of nucleic acids are derived from images of regular arrays of spots containing target genetic material to which fluorescently labeled samples are hybridized. Whereas there are a number of methods in use for the quantification of images, many of the software systems in wide use either encourage or require extensive human interaction at the level of individual spots on arrays. We present a fully automatic system for microarray image quantification. The system automatically locates both subarray grids and individual spots, requiring no user identification of any image coordinates. Ratios are computed based on explicit segmentation of each spot. On a typical image of 6000 spots, the entire process takes less than 20 sec. We present a quantitative assessment of performance on multiple replicates of genome-wide array-based comparative genomic hybridization experiments. By explicitly identifying the pixels in each spot, the system yields more accurate estimates of ratios than systems assuming spot circularity. The software, called, runs on Windows platforms and is available free of charge for academic use.

Comparative genomic hybridization analysis of chromosomal alterations in patients with long-standing ulcerative colitis

Patients with ulcerative colitis (UC) are prone to develop colorectal cancer which is related to the duration and extent of the disease. One of the earliest events in tumor progression is the development of aneuploidy. Aneuploidy is correlated with the grade of dysplasia which serves as a common but not always reproducible marker for the prediction of UC associated formation of cancer. We analyzed 48 biopsy samples from 5 patients with long-standing ulcerative colitis by comparative genomic hybridization (CGH). The majority of these samples represented premalignant stages which are not well characterized at the molecular level as yet. We compared biopsy samples from different colon locations in regard to chromosomal alterations, dysplasia status and DNA index. Besides chromosomal changes occurring only in certain patients in restricted areas of the colon we also detected amplifications and deletions which were common in all persons throughout the colon. The stage of dysplasia seems to have no influence on the number and appearance of chromosomal changes. Amplifications in 2, 3, 6, 9, 11, 12 and 15 were found in almost all cases. In dysplastic samples chromosomal regions 3, 6 and 11 revealed gains of DNA. Deletions were detected within 8q, 15, 18q, 20p and 22q. The affected chromosomal regions may contain yet unknown oncogenes or tumor suppressor genes participating in UC associated carcinogenesis. The conspicuous regions found in the CGH experiments allow the selective and detailed characterization at a molecular level.

Genetic abnormalities detected by comparative genomic hybridization in a human endometriosis-derived cell line.

Comparative genomic hybridization (CGH) was used in parallel with fluorescence in-situ hybridization (FISH) and conventional karyotyping to perform a genome-wide survey of DNA gains and losses in the endometriosis-derived permanent cell line, FbEM-1. The cytogenetic analysis showed a complex karyotype with numerical changes and multiple chromosome aberrations, including the der(1) complement marker exhibiting a large homogenous staining region (HSR). The chromosomal rearrangement interpreted as der(5) t(5;6)(q34;p11) was found in the majority of the metaphases indicating a clonal abnormality. Repeated CGH experiments demonstrated over-representation of chromosomes 1, 2, 3, 5, 6p, 7, 16, 17q, 20, 21q and 22q, while chromosomes 6q, 9, 11p, 12, 13q, 18 and X were under-represented. Using DNA from the original endometriotic tissues, including a peritoneal implant and ovarian endometrioma, CGH analysis revealed loss of DNA copy number on 1p, 22q and chromosome X, while gain was found on chromosomal arms 6p and 17q. FISH analysis confirmed that the gain at 17q includes amplification of the proto-oncogene HER-2/neu in 16% of the FbEM-1 nuclei and in 12% of cells from the primary ovarian endometrioma tissue. These findings demonstrate that FbEM-1 cells share certain molecular cytogenetic features with the original tissue and suggest that chromosomal instability is important in the development of endometriosis.

Mean and variance of ratio estimators used in fluorescence ratio imaging This research was presented at ASCI'95, First Annual Conference of the Advanced School for Computing and Imaging, Heijen, The Netherlands, 16–18 May 1995.

Background The ratio of two measured fluorescence signals (called x and y) is used in different applications in fluorescence microscopy. Multiple instances of both signals can be combined in different ways to construct different ratio estimators. Methods The mean and variance of three estimators for the ratio between two random variables, x and y, are discussed. Given n samples of x and y, we can intuitively construct two different estimators: the mean of the ratio of each x and y and the ratio between the mean of x and the mean of y. The former is biased and the latter is only asymptotically unbiased. Using the statistical characteristics of this estimator, a third, unbiased estimator can be constructed. Results We tested the three estimators on simulated data, real-world fluorescence test images, and comparative genome hybridization (CGH) data. The results on the simulated and real-world test images confirm the presented theory. The CGH experiments show that our new estimator performs better than the existing estimators. Conclusions We have derived an unbiased ratio estimator that outperforms intuitive ratio estimators. Cytometry 39:300–305, 2000 © 2000 Wiley-Liss, Inc.

Comparative Genomic Hybridization Reveals Extensive Variation Among Different MCF-7 Cell Stocks

Comparative genomic hybridization (CGH) allows the detection of DNA sequence copy number changes on a genome-wide scale in a single hybridization reaction. The ability of CGH to be applied to formalin-fixed, paraffin-embedded tumor samples has lead to its widespread application in the cytogenetic analysis of archival material. When setting up CGH in the laboratory, rigorous control experiments must be carried out to ensure that the losses and gains are scored correctly. Groups interested in breast cancer frequently use the MCF-7 cell line as a positive control in these experiments, comparing the results to previously described genetic alterations. Here we present the results of CGH carried out with three stocks of MCF-7 cells. The cells differ widely in their proliferative response to 17- β estradiol and show extensive variation in copy number changes affecting specific chromosomal regions. We suggest that care must be taken, therefore, when choosing a cell line as a positive control for CGH experiments.

Recurrent Chromosomal Imbalances Detected in Biopsy Material from Oral Premalignant and Malignant Lesions by Combined Tissue Microdissection, Universal DNA Amplification, and Comparative Genomic Hybridization

Biopsies routinely performed for the histopathological diagnosis of oral epithelial lesions before treatment were screened for chromosomal imbalances by comparative genomic hybridization. Comparative genomic hybridization was performed on 12 oral premalignant lesions (OPLs; dysplasias and carcinomas in situ) and 14 oral squamous cell carcinomas (OSCCs). Eight biopsies displayed areas of different histopathological appearance, so that OPLs and OSCCs from the same patient were analyzed. To avoid contamination with nonneoplastic cells, defined cell populations were isolated by micromanipulation with a glass needle. Before comparative genomic hybridization analysis, universal DNA amplification was performed using the DOP-polymerase chain reaction protocol. In the 14 OSCCs examined, the average number of chromosomal imbalances was significantly higher than in the 12 OPLs (mean +/- SEM: 11.9 +/- 1.9 versus 3.2 +/- 1.2; P = 0.003). The DNA copy number changes identified in more than one OPL were gains on 8q (3 of 12) and 16p (2 of 12), as well as losses on 3p (5 of 12); 5q (4 of 12); 13q (3 of 12); and 4q, 8p, and 9p (2 of 12 each). In more than 30% of OSCCs, gains of chromosomal material were identified on 20q (8 of 14); 8q, 11q, 22q (7 of 14 each); 3q, 15q, and 17p (6 of 14 each); and 14q, 17q, and 20p (5 of 14 each), and losses were identified on 3p and 4q (9 of 14 each), 5q (7 of 14), 13q (6 of 14), and 2q and 9p (5 of 14 each). These results were validated by positive and negative control comparative genomic hybridization experiments and microsatellite analysis for the detection of allelic loss. The vast majority of genomic alterations found in OPLs were again identified in OSCCs from the same biopsy, supporting the hypothesis that multiple lesions in the same patient are clonally related. In summary, we show that comprehensive information on the genomic alterations in oral epithelial lesions can be obtained from small biopsies. Such data may identify prognostic indicators that could eventually assist in designing therapeutic strategies.

Comparison of fluorescein isothiocyanate- and Texas red-conjugated nucleotides for direct labeling in comparative genomic hybridization

In this study, we investigated whether fluorescein isothiocyanate (FITC)-labeling of test DNA and Texas-red (TR) labeling of reference DNA in comparative genomic hybridization (CGH) experiments cause the results to differ from those obtained using the opposite combination (reverse labeling). Analysis was performed on a total of 20 DNA specimens consisting of 13 frozen bone marrow aspirates from patients with acute myeloid leukemia, and fresh peripheral blood samples from seven healthy donors. For CGH, one aliquot from each test DNA sample was labeled using nick-translation with FITC-dUTP and another with TR-dUTP. Afterwards, the FITC-dUTP and TR-dUTP-labeled test DNAs were hybridized to TR-dUTP- and FITC-dUTP-labeled normal reference DNAs, respectively. The results using the two combinations were compared with each other and with the results of G-banding karyotype analysis. Karyotype data was used to detect artifacts known to occur in some chromosome regions in CGH analysis. The control DNAs labeled with FITC or TR showed no DNA copy number changes. Regardless of the fluorochrome employed for labeling, no DNA copy number changes were detected using CGH in patients with normal karyotypes, nor in patients whose karyotype aberrations were present in less than 40% of cells. In the remaining patients, CGH revealed DNA copy number changes that coincided with the results of the G-banding analysis. Hybridization artifacts known to occur in CGH experiments affecting chromosome regions 1p33-pter, 16p, 17p, 19, and 22 were observed in 15-23% of the tumor samples labeled with FITC, but not in samples labeled with TR. In addition, other previously unreported overrepresentations affecting 7q21, 9q34, 16q, 17q, and chromosome 20 were observed at very low frequencies in up to 10% of the samples when FITC was used to label test DNA. However, when TR was used, overrepresentations were observed at 4q13-q21, 11q21-q23, 13q21-qter, and Xq21-q22, whereas 19p was underrepresented. The results demonstrate that TR-labeling confirms abnormalities detected using FITC-labeling and reduces hybridization artifacts in the known problematic regions of the human genome.