乳腺叶状肿瘤染色体异常的比较基因组杂交分析初探

乳腺叶状肿瘤是一种较少见的乳腺肿瘤,近年来其发病率有逐渐增高的趋势。2003年WHO新分类中将其分为良性、交界性、恶性3种,其不同的病理类型反应了该瘤由良性发展为恶性的过程。我们应用比较基因组杂交（comparative genomic hybridization,CGH）技术分析乳腺叶状肿瘤不同类型染色体基因组DNA拷贝数的变化,初步探索该肿瘤的分子细胞遗传学变化,为进一步从全基因组染色体角度研究该瘤的恶变机制提供较有价值的资料。     

Microarray comparative genomic hybridization (CGH)-based prenatal diagnosis for chromosome abnormalities using cell-free fetal DNA in amniotic fluid

Cell-free fetal DNA (cffDNA) in the supernatant of amniotic fluid, which is usually discarded, can be used as a sample for prenatal diagnosis. For rapid prenatal diagnosis of frequent chromosome abnormalities, for example trisomies 13, 18, and 21, and monosomy X, using cffDNA, we have developed a targeted microarray-based comparative genomic hybridization (CGH) panel on which BAC clones from chromosomes 13, 18, 21, X, and Y were spotted. Microarray-CGH analysis was performed for a total of 13 fetuses with congenital anomalies using cffDNA from their uncultured amniotic fluid. Microarray CGH with cffDNA led to successful molecular karyotyping for 12 of 13 fetuses within 5 days. Karyotypes of the 12 fetuses (one case of trisomy 13, two of trisomy 18, two of trisomy 21, one of monosomy X, and six of normal karyotype) were later confirmed by conventional chromosome analysis using cultured amniocytes. The one fetus whose molecular-karyotype was indicated as normal by microarray CGH actually had a balanced translocation, 45,XY,der(14;21)(q10;q10). The results indicated that microarray CGH with cffDNA is a useful rapid prenatal diagnostic method at late gestation for chromosome abnormalities with copy-number changes, especially when combined with conventional karyotyping of cultured amniocytes.     

Microarray-based comparative genomic hybridization in the study of constitutional chromosomal abnormalities

Chromosomal aberrations are the first cause of mental impairment and dysmorphism. Rearrangements involving large chromosomal segments can be detected by standard chromosome analysis using GTG-banding, but this technique is not suited for the detection of small chromosome abnormalities. Array comparative genomic hybridisation (array-CGH) is a method used to detect segmental DNA copy number alterations. Recently, advances in this technology have enabled high-resolution examination for identifying genetic alterations and copy number variations on a genome-wide scale. This review describes the current genomic array platforms and CGH methodologies and highlights their applications for studying constitutional disease.     

Identification and analysis of sex chromosomes by comparative genomic hybridization (CGH)

Comparative Genome Hybridization (CGH) can be used as a universal method for the identification of molecularly differentiated sex chromosomes. This is profitable in species with homomorphic sex chromosomes or when chromosomes are unfavourable for cytogenetics, e.g. when size differences are insufficient, chromosomes numerous and/or banding methods fail. In this method, genomic DNA from females competes as a probe with that from males for binding to the chromosome targets. Easy extraction and labelling methods afford a method that can be applied even when few specimens are available, e.g. when specimens for investigation have to be collected in the field – CGH also offers the possibility to obtain a rough estimate of the DNA composition of the sex chromosome.     

Analysis of myelodysplastic syndromes with complex karyotypes by high-resolution comparative genomic hybridization and subtelomeric CGH array

Molecular cytogenetic techniques enabled us to clarify numerical and structural alterations previously detected by conventional cytogenetic techniques in 37 patients who had myelodysplastic syndromes with complex karyotypes. Using high-resolution comparative genomic hybridization (HR-CGH), we found the most recurrent alterations to be deletion of 5q (70%), 18q (35%), 7q (32%), 11q (30%), and 20q (24%), gain of 11q (35%) and 8q (24%), and trisomy of chromosome 8 (19%). Furthermore, in 35% of the patients, 20 amplifications were identified. These amplifications were shown by FISH to involve some genes previously described as amplified in hematological malignancies, such as ERBB2, MLL, and RUNX1. In addition, two other genes, BCL6 and BCL2, which are classically related to apoptosis and non-Hodgkin lymphoma, were shown for the first time to be involved in amplification. Genomic alterations involving different subtelomeric regions with losses in 4p16, 5p15.3, 6q27, 18p11.3, and 18q23 and gains in 1p36.3 and 19p13.3 were detected by HR-CGH. Array CGH analysis of the subtelomeric regions in some samples was able to confirm a number of these alterations and found some additional alterations not detected by conventional CGH.     

DNA in situ hybridization (interphase cytogenetics) versus comparative genomic hybridization (CGH) in human cancer: detection of numerical and structural chromosome aberrations

DNA in situ hybridization techniques for cytogenetic analyses of human solid cancers are nowadays widely used for diagnostic and research purposes. The advantage of this methodology is that it can be applied to cells in the interphase state, thereby circumventing the need for high-quality metaphase preparations for karyotypic evaluation. In situ hybridization (ISH) with chromosome specific (peri)centromeric DNA probes, also termed "interphase cytogenetics", can be used to detect numerical changes, whereas comparative genomic hybridization (CGH) discloses chromosomal gains and losses, i.e. amplifications and deletions. We wanted to compare both methods in human solid tumors, and for this goal we evaluated ISH and CGH within a set of 20 selected prostatic adenocarcinomas. Chromosomes 7 and 8 were chosen for this analysis, since these chromosomes are frequently altered in prostate cancer. ISH with chromosome 7 and 8 specific centromeric DNA probes was applied to standard, formalin-fixed and paraffin-embedded, histological sections for numerical chromosome analysis. CGH with DNA's, extracted from the same histologic area of the archival specimens, was used for screening of gains and losses of 7 and 8. ISH with centromeric probes distinguished a total of 26 numerical aberrations of chromosome 7 and/or 8 in the set of 20 neoplasms. In the same set CGH revealed a total of 35 losses and gains. CGH alterations of 7 and 8 were seen in twenty-two of the 26 chromosomes (85%) that showed aberrations in the ISH analysis. Concordance between ISH and CGH was seen in 11 (of 26; 42%) chromosomes. Eight chromosomes were involved in gains (5 x #7, 3 x #8), three in losses (3 x #8). This included both complete (3/11) and partial (8/11) CGH confirmation of the numerical alteration. Partial CGH confirmation was defined as loss or gain of a chromosome arm with involvement of the centromeric region. In the majority of these cases it concerned a whole chromosome arm, mostly the long arm. We conclude that generally a fair correlation was found between ISH and CGH in interphase preparations of a series of prostate cancers. However, when specified in detail, most of the numerical ISH aberrations were only partly represented in the CGH analysis. On the one hand, it suggests that CGH does not adequately discriminate numerical abnormalities. On the other hand, it likely implies that not all numerical changes, as detected by interphase cytogenetics, are truly involving the whole chromosome. A part of these discrepancies might be caused by structural mechanisms, most notably isochromosome formation.     

Construction of a high-density and high-resolution human chromosome X array for comparative genomic hybridization analysis

The human chromosome X is closely associated with congenital disorders and mental retardation (MR), because it contains a significantly higher number of genes than estimated from the proportion in the human genome. We constructed a high-density and high-resolution human chromosome X array (X-tiling array) for comparative genomic hybridization (CGH). The array contains a total of 1,001 bacterial artificial chromosome (BACs) throughout chromosome X except pseudoautosomal regions and two BACs specific for Y. In four hybridizations using DNA samples from healthy males, the ratio of each spotted DNA was scattered between −3SD and 3SD, corresponding to a log₂ ratio of −0.35 and 0.35, respectively. Using DNA samples from patients with known congenital disorders, our X-tiling array was proven to discriminate one-copy losses and gains together with their physical sizes, and also to estimate the percentage of a mosaicism in a patient with mos 45,X[13]/46,X,r(X)[7]. Furthermore, array-CGH in a patient with atypical Schinzel-Giedion syndrome disclosed a 1.1-Mb duplication at Xq22.3 including a part of the IL1RAPL2 gene as a likely causative aberration. The results indicate our in-house X-tiling array to be useful for the identification of cryptic copy-number aberrations containing novel genes responsible for diseases such as congenital disorders and X-linked MR.     

Characterization of large chromosome markers in a malignant fibrous histiocytoma by spectral karyotyping, comparative genomic hybridization (CGH), and array CGH

In this study, we characterized the chromosomal composition of an intra-abdominal soft tissue sarcoma diagnosed as a malignant fibrous histiocytoma (MFH). By applying a combination of spectral karyotyping, G-banding, and comparative genomic hybridization (CGH), this case was shown to carry large chromosome markers with material mainly from chromosomes 6 and 8. Further characterization of this unique tumor revealed high-level amplifications at the 6q21 approximately q23, 8p21 approximately pter, 8q24 approximately qter, and 12q13 approximately q21 regions. Using array CGH, these amplified regions were found to include MASL1 in 8p, as well s MDM2 and CDK4 in 12q, which have been shown to be amplified in MFH. Similarly, gains of 6q and 8q have also been seen in MFH. In conclusion, our study demonstrates the occurrence of large chromosome markers in MFH and suggests that the regions 6q21 approximately q23, 8p21 approximately pter, 8q24 approximately qter, and 12q13 approximately q21 might harbor oncogenes that could play a role in MFH's tumorigenesis. In addition, gain of 12q13 approximately q21, which is typical of well-differentiated liposarcoma, may also occur in MFH, supporting the previously suggested overlap in genetic etiologies between these two tumor types.     

Genetic aberrations in oligodendroglial tumours: an analysis using comparative genomic hybridization (CGH)

Four low-grade oligodendrogliomas, nine anaplastic oligodendrogliomas and two mixed oligoastrocytomas were investigated for chromosomal aberrations by comparative genomic hybridization on formalin-fixed, paraffin-embedded tissue samples. The most frequent losses observed involved 1p, 9p, 10pq, 14q, 16p, 19q, while the most frequent gains were seen on 7pq, 11pq, 17p, 19pq, and Xp. In one oligodendroglioma, a highly specific amplification of 1q32.1 was seen. The frequent losses of 14q have not been reported previously. In the two cases of mixed oligoastrocytomas multiple gains and losses were found that did not show a clear overlap with the alterations found in the pure oligodendrogliomas. Copyright © 1999 John Wiley & Sons, Ltd.     

Applications of comparative genomic hybridisation in constitutional chromosome studies

G band cytogenetic analysis often leads to the discovery of unbalanced karyotypes that require further characterisation by molecular cytogenetic studies. In particular, G band analysis usually does not show the chromosomal origin of small marker chromosomes or of a small amount of extra material detected on otherwise normal chromosomes. Comparative genomic hybridisation (CGH) is one of several molecular approaches that can be applied to ascertain the origin of extra chromosomal material. CGH is also capable of detecting loss of material and thus is also applicable to confirming or further characterising subtle deletions. We have used comparative genomic hybridisation to analyse 19 constitutional chromosome abnormalities detected by G band analysis, including seven deletions, five supernumerary marker chromosomes, two interstitial duplications, and five chromosomes presenting with abnormal terminal banding patterns. CGH was successful in elucidating the origin of extra chromosomal material in 10 out of 11 non-mosaic cases, and permitted further characterisation of all of the deletions that could be detected by GTG banding. CGH appears to be a useful adjunct tool for either confirming deletions or defining their breakpoints and for determining the origin of extra chromosomal material, even in cases where abnormalities are judged to be subtle. We discuss internal quality control measures, such as the mismatching of test and reference DNA in order to assess the quality of the competitive hybridisation effect on the X chromosome.     

Accuracy of an array comparative genomic hybridization (CGH) technique in detecting DNA copy number aberrations: comparison with conventional CGH and loss of heterozygosity analysis in prostate cancer

Although genomic DNA microarray (array comparative genomic hybridization [CGH]) technique is a rapid and powerful diagnostic tool for the comprehensive analysis of detailed chromosomal alterations of DNA copy numbers, its accuracy has not been well demonstrated. To clarify the accuracy of this technique, we applied array CGH spotted with 283 specific genes to 11 clinical prostate cancers, and the results were compared with comparative genomic hybridization (conventional CGH) and loss of heterozygosity (LOH) analysis using microsatellite DNA markers. The overall rate of correspondence between array CGH and conventional CGH with respect to the loss of DNA sequences was 94.5%. When the results of both CGH techniques were compared with those of LOH analysis, the correspondence rate of array CGH was significantly higher than that of conventional CGH (93.4% vs. 72.2%, P<0.05). In conclusion, the accuracy of array CGH was higher than that of conventional CGH in detecting losses of the DNA sequences. Array CGH is shown to be a promising tool for screening to identify unknown genes involved in tumorigenesis in prostate cancer.     

Identification and characterization of a de novo partial trisomy 10p by comparative genomic hybridization (CGH)

We report the characterization of a de novo unbalanced chromosome rearrangement by comparative genomic hybridization (CGH) in a 15-day-old child with hypotonia and dysmorphia. We describe the combined use of CGH and fluorescence in situ hybridization (FISH) to identify the origin of the additional chromosomal material on the short arm of chromosome 6. Investigation with FISH revealed that the excess material was not derived from chromosome 6. Identification of unknown unbalanced aberrations that could not be identified by traditional cytogenetics procedures is possible by CGH analysis. Visual analysis of digital images from CGH-metaphase spreads revealed a predominantly green signal on the telomeric region of chromosome 10p. After quantitative digital ratio imaging of 10 CGH-metaphase spreads, a region of gain was found in the chromosome band 10p14-pter. The CGH finding was confirmed by FISH analysis, using a whole chromosome 10 paint probe. These results show the usefulness of CGH for a rapid characterization of de novo unbalanced translocation, unidentifiable by karyotype alone.     

Identification of frequent chromosomal aberrations in ductal adenocarcinoma of the pancreas by comparative genomic hybridization (CGH)

Despite the continuous progress in molecular methodology, the genetic events involved in the initiation and progression of ductal adenocarcinoma of the pancreas remain largely unknown. In this study, 33 pancreatic ductal adenocarcinomas were screened for genomic alterations by comparative genomic hybridization (CGH). To date, most CGH studies of pancreatic cancer have been based on cell lines. To emphasize genetic imbalances that are involved in the in vivo development and progression of pancreatic carcinoma only fresh-frozen or paraffin-embedded tumour samples were analysed in the present study. Twenty-two tumours (67%) showed genomic alterations involving up to three (12%) or more (55%) chromosomal regions. The number and nature of the genetic imbalances did not, however, correlate with tumour stage or grade. Chromosome 18 was preferentially altered in the tumours analysed. Frequent chromosomal losses were found at 18q, 10q, 8p, and 13q. Commonly gained regions were located on 8q and 3q. Moreover, high copy number amplifications of the chromosomal regions 5p, 8q22-ter, 12p12-cen, 19q12-13.2, and 20q were identified. These data provide evidence for the occurrence of characteristic genomic alterations which are of biological relevance for the genesis of pancreatic cancer. The identified altered chromosomal regions may harbour tumour genes which involved in the multistep process of pancreatic carcinogenesis.     

Reliability of comparative genomic hybridization to detect chromosome abnormalities in first polar bodies and metaphase II oocytes

BACKGROUND: Preimplantation Genetic Diagnosis (PGD) using FISH to analyze up to nine chromosomes to discard chromosomally abnormal embryos has resulted in an increase of pregnancy rates in certain groups of patients. However, the number of chromosomes that can be analyzed is a clear limitation. We evaluate the reliability of using comparative genomic hybridization (CGH) to detect the whole set of chromosomes, as an alternative to PGD using FISH. METHODS AND RESULTS: We have analysed by CGH both, first polar bodies (1PBs) and metaphase II (MII) oocytes from 30 oocytes donated by 24 women. The aneuploidy rate was 48%. Considering two maternal age groups, a higher number of chromosome abnormalities were detected in the older group of oocytes (23% versus 75%, P < 0.02). About 33% of the 1PB-MII oocyte doublets diagnosed as aneuploid by CGH would have been misdiagnosed as normal if FISH with nine chromosome probes had been used. CONCLUSION: We demonstrate the reliability of 1PB analysis by CGH, to detect almost any chromosome abnormality in oocytes as well as unbalanced segregations of maternal translocations in a time frame compatible with regular in vitro fertilization (IVF). The selection of euploid oocytes could help to increase implantation and pregnancy rates of patients undergoing IVF treatment.     

Array-based comparative genomic hybridization of mapped BAC DNA clones to screen for chromosome 14 copy number abnormalities in meningiomas

Chromosome 14 loss in meningiomas are associated with more aggressive tumour behaviour. To date, no studies have been reported in which the entire chromosome 14q of meningioma tumour cells has been studied by high-resolution array comparative genomic hybridization (a-CGH). Here, we used a high-resolution a-CGH to define the exact localization and extent of numerical changes of chromosome 14 in meningioma patients. An array containing 807 bacterial artificial chromosome clones specific for chromosome 14q (average resolution of approximately 130 Kb) was constructed and applied to the study of 25 meningiomas in parallel to the confirmatory interphase fluorescence in situ hybridization (iFISH) analyses. Overall, abnormalities of chromosome 14q were detected in 10/25 cases (40%). Interestingly, in seven of these cases, loss of chromosome 14q32.3 was detected by iFISH and confirmed to correspond to monosomy 14 by a-CGH. In contrast, discrepant results were found between iFISH and a-CGH in the other three altered cases. In one patient, a diploid background was observed by iFISH, while monosomy 14 was identified by a-CGH. In the remaining two cases, which showed gains of the IGH gene by iFISH, a-CGH did not detected copy number changes in one case showing a tetraploid karyotype, while in the other tumour, varying genetic imbalances along the long arm of chromosome 14 were detected. In summary, here, we report for the first time, the high-resolution a-CGH profiles of chromosome 14q in meningiomas, confirming that monosomy 14 is the most frequent alteration associated with this chromosome; other numerical abnormalities being only sporadically detected.     

Invasive micropapillary carcinoma of the breast is associated with chromosome 8 abnormalities detected by comparative genomic hybridization

Invasive micropapillary carcinoma (IMC) of the breast is a rare variant of invasive ductal carcinoma (IDC) characterized by unique histology and an extremely high incidence of lymph node metastases (approximately 95%). Comparative genomic hybridization (CGH) was used to characterize DNA extracted from 16 archival IMC cases to identify clonal genetic changes associated with this unique and highly metastatic cancer subtype. The average number of chromosomal alterations per IMC tumor was 7.4 +/-2.9 (3.4 gains and 3.9 losses), fewer than the number that we have observed in IDCs not otherwise specified (9.5 +/-6.6), IDCs with erbB-2 gene amplification (12.6 +/-5.9), and invasive lobular carcinomas (8.2 +/-5.5). The mean number of changes in IMC was significantly higher than we have observed in the rarely metastasizing tubular subtype of IDC (3.9 +/-2.3, P = 0.001), but less than the more aggressive subset of erbB-2-amplified IDC (P = 0.02). Remarkably, 100% of IMCs demonstrated loss involving the short arm of chromosome 8 (8p). Six cases showed loss of the entire 8p arm, whereas in 10 cases the loss was limited to the distal portion (8p21-pter) with localized gain of proximal 8p (8p11-p12). A reciprocal gain of 8q was detected in 14 cases (88%). Other common alterations included loss of 17p in 50% of tumors and loss of 16q in 50% of IMC cases. Gains of 17q (38%), 1q (31%), and 16p (25%) were also commonly detected. In comparison, IDCs (not otherwise specified), IDCs of the tubular subtype, and invasive lobular carcinomas showed only modest 8p loss (33%, 28%, and 13%, respectively). This region of chromosome 8 may contain 1 or more genes whose loss leads to this particular histology and/or the lymphotrophic phenotype associated with this histopathologic pattern.     

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. Genes Chromosomes Cancer 24:112–118, 1999. © 1999 Wiley‐Liss, Inc.     

Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization.

PURPOSE: This study was designed to evaluate the feasibility of using a targeted array-CGH strategy for prenatal diagnosis of genomic imbalances in a clinical setting of current pregnancies. METHODS: Women undergoing prenatal diagnosis were counseled and offered array-CGH (BCM V4.0) in addition to routine chromosome analysis. Array-CGH was performed with DNA directly from amniotic fluid cells with whole genome amplification, on chorionic villus samples with amplification as necessary, and on cultured cells without amplification. RESULTS: Ninety-eight pregnancies (56 amniotic fluid and 42 CVS specimens) were studied with complete concordance between karyotype and array results, including 5 positive cases with chromosomal abnormalities. There was complete concordance of array results for direct and cultured cell analysis in 57 cases tested by both methods. In 12 cases, the array detected copy number variation requiring testing of parental samples for optimal interpretation. Array-CGH results were available in an average of 6 and 16 days for direct and cultured cells, respectively. Patient acceptance of array-CGH testing was 74%. CONCLUSION: This study demonstrates the feasibility of using array-CGH for prenatal diagnosis, including reliance on direct analysis without culturing cells. Use of array-CGH should increase the detection of abnormalities relative to the risk, and is an option for an enhanced level of screening for chromosomal abnormalities in high risk pregnancies.