High-resolution analysis of genomic copy number alterations in bladder cancer by microarray-based comparative genomic hybridization

We have screened 22 bladder tumour-derived cell lines and one normal urothelium-derived cell line for genome-wide copy number changes using array comparative genomic hybridization (CGH). Comparison of array CGH with existing multiplex-fluorescence in situ hybridization (M-FISH) results revealed excellent concordance. Regions of gain and loss were defined more accurately by array CGH, and several small regions of deletion were detected that were not identified by M-FISH. Numerous genetic changes were identified, many of which were compatible with previous results from conventional CGH and loss of heterozygosity analyses on bladder tumours. The most frequent changes involved complete or partial loss of 4q (83%) and gain of 20q (78%). Other frequent losses were of 18q (65%), 8p (65%), 2q (61%), 6q (61%), 3p (56%), 13q (56%), 4p (52%), 6p (52%), 10p (52%), 10q (52%) and 5p (43%). We have refined the localization of a region of deletion at 8p21.2-p21.3 to an interval of approximately 1 Mb. Five homozygous deletions of tumour suppressor genes were confirmed, and several potentially novel homozygous deletions were identified. In all, 15 high-level amplifications were detected, with a previously reported amplification at 6p22.3 being the most frequent. Real-time PCR analysis revealed a novel candidate gene with consistent overexpression in all cell lines with the 6p22.3 amplicon.     

Different chromosomal imbalances in metastasized and nonmetastasized tongue carcinomas identified by comparative genomic hybridization

Tumors of different metastatic behavior possibly differ in genomic constitution. We identified molecular cytogenetic differences between a group of metastasized and nonmetastasized primary tongue tumors by comparative genomic hybridization. Most frequent chromosome copy number changes for metastasized and nonmetastasized tumors were +8q (100% and 71%, respectively) and +3q (56% and 43%, respectively). Metastasized tumors showed significantly more chromosome copy number changes than nonmetastasized tumors. High copy number gains were exclusively found in metastasized tumors for 3q23-qter, 5p, 12p and 13q21-q22. Genomic imbalances occurring in metastasized tumors but not in nonmetastasized tumours were +7q21 (44%), +14q (33%), and -15q (33%). The genetic constitution of primary tongue tumors that metastasize differs from tongue tumors that do not metastasize. Our data, although obtained from a relative small group of tumors, spotlights copy number gain of chromosome region 7q21 as a potential marker for metastatic behavior.     

Progression analysis of lung squamous cell carcinomas by comparative genomic hybridization.

OBJECTIVE: The genetic mechanisms underlying the development and progression of lung squamous cell carcinoma (SCC), the major subtype of non-small cell lung cancer, are still unknown. To better understand this disease, we studied the association between genetic alterations and the progression of lung SCC. METHODS: Chromosomal aberrations in 39 samples of lung SCC, including 21 nonmetastatic and 18 metastatic carcinomas, were characterized by comparative genomic hybridization and statistically correlated to clinical staging and metastatic ability. RESULTS: The average gains and losses per patient were significantly higher in the advanced-stage lung SCC and metastatic SCC group compared to the early-stage lung SCC and nonmetastatic SCC group. Gains of 2p, 20p and losses of 2q, 4q, 5q, 9q, 13p, 18q correlated with advanced-stage lung SCC. Losses on 2q, 4q, 6p, 16p, 16q, 18q, 20q, 21q and gains on 2p, 7p, 7q, 20p were more frequent in the metastatic SCC group, which was significantly different from the nonmetastatic SCC group. Gains on 2p, 20p and losses on 2q, 4q, 18q were not only associated with an advanced clinical stage but also with metastases of lung SCC. CONCLUSIONS: The results suggest that several chromosomal aberrations (e.g. gains on 2p, 20p and losses on 2q, 4q, 18q) may contribute to the progression of lung SCC.     

Comparison of chromosomal and array-based comparative genomic hybridization for the detection of genomic imbalances in primary prostate carcinomas

Background  

In order to gain new insights into the molecular mechanisms involved in prostate cancer, we performed array-based comparative genomic hybridization (aCGH) on a series of 46 primary prostate carcinomas using a 1 Mbp whole-genome coverage platform. As chromosomal comparative genomic hybridization (cCGH) data was available for these samples, we compared the sensitivity and overall concordance of the two methodologies, and used the combined information to infer the best of three different aCGH scoring approaches.     

Genetic alterations in lobular breast cancer by comparative genomic hybridization

Infiltrating lobular carcinoma (ILC) and infiltrating ductal carcinoma (IDC) are distinguished by their histopathological appearance. However, little is known about the differences in genetic changes between lobular cancers and ductal cancers. We used comparative genomic hybridization (CGH) and compared aberrations in 19 ILCs and 46 IDCs. The total number of aberrations was lower in ILC than in IDC. While the average number of DNA copy number losses did not reach significance between them, copy number gains were significantly lower in ILCs. Fifteen of 19 ILCs (79%) showed increased copy number of 1q, and 12 cases (63%) revealed loss of 16q. The presence of these aberrations was independent of nodal status, histologic subtypes (pleomorphic or classic ILC), or BrdUrd-labeling index. ILCs had a higher frequency of 16q loss than did ductal cancers, and a lower frequency of 8q and 20q gains. Our data suggest that the altered growth pattern and clinical presentation which characterize infiltrating lobular cancers are correlated with distinct genetic alterations. Int. J. Cancer 74:513–517, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of genetic changes in schistosome-related transitional and squamous bladder cancers using comparative genomic hybridization

The development of bladder tumors has been associated with a number of causative agents, including schistosomiasis. Schistosome-related cancers show different clinical and pathological features compared with non-schistosome-related bladder cancers, occurring in younger patients, and being predominantly of squamous cell type. This study addresses the difference between squamous and transitional tumor types in the presence of schistosome infection as a measure of the relationship between tumor genotype and phenotype. We have used comparative genomic hybridization to analyze primary muscleinvasive schistosome-related bladder tumors in 54 patients. Twenty-six of these tumors were squamous cell carcinomas; the remaining 28 were of transitional cell type. On average, transitional cell tumors showed 1.8 times the number of chromosomal aberrations as squamous cell tumors (14.4 versus 8.2, P: < 0.001). For both groups combined, the most prevalent genetic alterations were losses of 8p and 18q, and gains of 8q. Transitional cell cancers also showed frequent losses involving 5q, 9p, 10q, 11p and 11q, and gains at 1q and 17q. Loss of 11p was significantly more frequent in TCC than in SCC tumors (50 versus 4%, P: = 0.01). Squamous cell cancers showed more frequent losses of 17p and 18p than transitional tumors, which was clearly significant given the overall reduced frequency of changes in squamous cancers (P: = 0.001 and P: = 0.03, respectively). These data show that different histologic subgroups of bladder tumors are characterized by distinct patterns of chromosomal alterations. The genetic changes found in the transitional cell group are similar to those reported in non-schistosome-related transitional cell tumors, but differ from tumors exhibiting squamous differentiation.     

High incidence of unbalanced chromosomal changes in mantle cell lymphoma detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) was carried out in 30 mantle cell lymphoma (MCL) patients at the time of diagnosis. CGH results were supported by conventional cytogenetics (CC), FISH, molecular genetic PCR methods and 2 patients were examined by array CGH. Using all cytogenetic, molecular cytogenetic and PCR methods, chromosomal changes were detected in 28 (93%) patients. Using CGH, unbalanced chromosomal changes were detected in 24 (80%) cases. The most frequent aberrations were losses of 1p (8 cases), 8p (10 cases), 9q (6 cases), 11q (11 cases), 13q (10 cases) and 17p (9 cases), and gains of chromosome 3 and 3q (12 cases) and 8q (7 cases). Total number of 60 gains and 116 losses were detected. The primary chromosomal change t(11;14) was detected using FISH and/or PCR in 20 (66.6%) patients, and in 9 of them, the breakpoint was determined using PCR in the major translocation cluster (MTC). The evaluation of the frequencies of CGH changes in groups of patients with and without t(11;14) revealed the differences only in losses 6q and 9q, which were only found in patient with t(11;14). An important result was obtained using array CGH method. In a patient without the primary t(11;14), the gain of CCND1 gene was found. Our results show high heterogeneity of the additional chromosomal changes in MCL cases, which involved specific chromosomal subregions. We did not confirm the importance of subdividing of MCL cases with and without t(11;14). Also, statistical significance in survival rates between both subgroups was not confirmed.     

Additional chromosomal abnormalities detected by array comparative genomic hybridization in AML

Improved outcome of acute myeloid leukemia (AML) depends on the better differentiation of subtypes to predict treatment response and the identification of new target for treatment. In this study, array comparative genomic hybridization (aCGH) was used to distinguish eight cases of AML cases. Validation was performed by FISH and quantitative genomic PCR. The aCGH revealed new large and small recurrent genomic imbalances, such as gains of 1p36, 10q26, 11p15, 20q13, 22q23, harboring many proto-oncogenes. These results better define genetically the studied cases and could be used to understand the multiple phenomena involved in leukemogenesis.     

采用比较基因组杂交方法分析原发性食管癌染色体异常

背景与目的:有研究表明原发性食管癌常有染色体的改变,包括染色体基因组异常扩增和缺失.比较基因组杂交技术可以显示这些染色体的异常变化.本实验采用比较基因组杂交技术研究和分析原发性食管癌染色体基因组的变化特点及其与预后的关系.方法:采用比较基因组杂交技术检测16例食管癌组织中染色体的异常改变,并分析染色体异常与预后的关系.研究的病例中7例食管癌术后2年内死亡(对照组),9例术后生存3年以上(生存组).结果:食管癌患者中多数染色体基因组发生改变,最常见的染色体基因组高扩增频率发生在1q/p,2q/p,3q,5q/p,8q/p,9q/p,11q/p,17和20q/p染色体区段上,在染色体1q/p,4p,9p,18q和xp中常见染色体基因缺失.染色体7q/p和19扩增频率和染色体4q/p和18q缺失频率,生存组与对照组间存在显著性差异.结论:食管癌患者染色体区段基因易发生异常扩增和缺失,生存组与对照组存在明显差异.     

Detection of invasion-related chromosomal changes in highly and weakly invasive melanoma cell clones by a modified comparative genomic hybridization approach

Invasion is a key step in the systemic spread of tumour cells. The aim of this study was to investigate whether specific chromosomal aberrations in melanoma occur during acquisition of a strongly invasive phenotype. We previously selected highly and weakly invasive cell clones from the human melanoma cell line Mel Im. Both cell clones retained a stable phenotype over more than 40 passages, revealing a five-fold difference in their invasive potential in vitro. Direct comparative genomic hybridization (CGH) (modified CGH) of the two cell clones was used as a powerful tool to screen for different chromosomal aberrations in both clones. Standard CGH and fluorescence in situ hybridization (FISH) was performed to verify the results of this improved technique. In general, the CGH pattern showed a high degree of identity consistent with the fact that the cell lines represent subclones of the same cell line. However, a few defined changes between the two cell clones were observed, including loss of 1q21-qter, 4q, 11p14-pter, 19q and 20p in the highly invasive cell clone. Two of the regions (1q and 11p) have already been suggested to be involved in melanoma progression, whereas changes in the others have not been detected before. In summary, our direct CGH approach proved to be suitable for fast and direct comparison of two cell types and allowed the identification of two known and three novel chromosomal changes involved in the acquisition of a strongly invasive melanoma cell phenotype.     

Detection of chromosomal aberrations by comparative genomic hybridization during transformation of human breast epithelial cells in vitro

Breast cancer is the most frequent malignancy in women. It is well recognized that tumorigenesis is a multistep process resulting from the accumulation of sequential genetic alterations. In breast cancers LOH has been described on one or both arms of multiple chromosomes. Comparative genomic hybridization (CGH) analysis was performed to identify chromosomal imbalances in the breast epithelial cells (HBEC). We have used a human in vitro-in vivo system in which the environmental carcinogen benz(a)pyrene (BP) and the c-Ha-ras oncogene were utilized for inducing in vitro transformation of HBEC. Immortal MCF-10F cells were treated with BP which resulted in the transformed cell line BP-1 that was further enhanced by transfection with the c-Ha-ras to generate the cell line BP-1-Tras. This cell line is tumorigenic when injected in severe combined immunodeficient (SCID) mice, generating the tumor cell line BP-1-Tras T J#4. Our comparative genomic hybridization analysis indicates that the most overrepresented segment after cell transformation and in the BP-1, BP-1-Tras and in the tumor cell line were 1p (80%), 5q21-ter (80%), 8q24.1 (90%) and Xq27-28 (60%). DNA sequence amplification at 10p14-15 was observed in BP-1-Tras T J#4 cells. Allelic losses of chromosome 4, 8p11-21 and 15q11-12, occur after cell transformation and are maintained consistently during tumorigenesis.     

Array comparative genomic hybridization analysis of genomic alterations in breast cancer subtypes

In this study, we performed high-resolution array comparative genomic hybridization with an array of 4153 bacterial artificial chromosome clones to assess copy number changes in 44 archival breast cancers. The tumors were flow sorted to exclude non-tumor DNA and increase our ability to detect gene copy number changes. In these tumors, losses were more frequent than gains, and gains in 1q and loss in 16q were the most frequent alterations. We compared gene copy number changes in the tumors based on histologic subtype and estrogen receptor (ER) status, i.e., ER-negative infiltrating ductal carcinoma, ER-positive infiltrating ductal carcinoma, and ER-positive infiltrating lobular carcinoma. We observed a consistent association between loss in regions of 5q and ER-negative infiltrating ductal carcinoma, as well as more frequent loss in 4p16, 8p23, 8p21, 10q25, and 17p11.2 in ER-negative infiltrating ductal carcinoma compared with ER-positive infiltrating ductal carcinoma (adjusted P values < or = 0.05). We also observed high-level amplifications in ER-negative infiltrating ductal carcinoma in regions of 8q24 and 17q12 encompassing the c-myc and c-erbB-2 genes and apparent homozygous deletions in 3p21, 5q33, 8p23, 8p21, 9q34, 16q24, and 19q13. ER-positive infiltrating ductal carcinoma showed a higher frequency of gain in 16p13 and loss in 16q21 than ER-negative infiltrating ductal carcinoma. Correlation analysis highlighted regions of change commonly seen together in ER-negative infiltrating ductal carcinoma. ER-positive infiltrating lobular carcinoma differed from ER-positive infiltrating ductal carcinoma in the frequency of gain in 1q and loss in 11q and showed high-level amplifications in 1q32, 8p23, 11q13, and 11q14. These results indicate that array comparative genomic hybridization can identify significant differences in the genomic alterations between subtypes of breast cancer.     

Array comparative genomic hybridization analysis of colorectal cancer cell lines and primary carcinomas

Array comparative genomic hybridization, with a genome-wide resolution of approximately 1 Mb, has been used to investigate copy number changes in 48 colorectal cancer (CRC) cell lines and 37 primary CRCs. The samples were divided for analysis according to the type of genomic instability that they exhibit, microsatellite instability (MSI) or chromosomal instability (CIN). Consistent copy number changes were identified, including gain of chromosomes 20, 13, and 8q and smaller regions of amplification such as chromosome 17q11.2-q12. Loss of chromosome 18q was a recurrent finding along with deletion of discrete regions such as chromosome 4q34-q35. The overall pattern of copy number change was strikingly similar between cell lines and primary cancers with a few obvious exceptions such as loss of chromosome 6 and gain of chromosomes 15 and 12p in the former. A greater number of aberrations were detected in CIN+ than MSI+ samples as well as differences in the type and extent of change reported. For example, loss of chromosome 8p was a common event in CIN+ cell lines and cancers but was often found to be gained in MSI+ cancers. In addition, the target of amplification on chromosome 8q appeared to differ, with 8q24.21 amplified frequently in CIN+ samples but 8q24.3 amplification a common finding in MSI+ samples. A number of genes of interest are located within the frequently aberrated regions, which are likely to be of importance in the development and progression of CRC.     

比较基因组杂交技术检测肝细胞癌染色体异常及其临床意义

目的探讨肝细胞癌染色体异常及其临床意义。方法运用比较基因组杂交技术检测25例肝细胞癌染色体DNA异常情况,并与临床指标作相关分析。结果25例肝癌均存在不同程度的染色体DNA拷贝数的扩增或缺失,较为常见的染色体DNA异常是＋1q（72％）、＋1p（64％）、＋2q（、48％）、＋2p（48％）、＋5q（48％）、＋Xq（48％）、＋7q（44％）、-4q（48％）、-16p（48％）、-8p（40％）、-17p（36％）;相关分析显示＋17p、＋18p、-8p、-13q、-11q、-8q染色体异常事件与临床指标部分相关。结论肝细胞癌存在明显的染色体异常,部分染色体异常事件是非随机性的,可能与肝癌的发生、发展有关,并与肿瘤的生物学行为和预后相关。     

比较基因组杂交技术检测肝细胞癌染色体异常及其临床意义

目的探讨肝细胞癌染色体异常及其临床意义。方法运用比较基因组杂交技术检测25例肝细胞癌染色体DNA异常情况,并与临床指标作相关分析。结果 25例肝癌均存在不同程度的染色体DNA拷贝数的扩增或缺失,较为常见的染色体DNA异常是+1q(72%)、+1p(64%)、+2q(48%)、+2p(48%)、+5q(48%)、+Xq(48%)、+7q(44%)、-4q(48%)、-16p(48%)、-8p(40%)、-17p(36%);相关分析显示+17p、+18p、-8p、-13q、-11q、-8q染色体异常事件与临床指标部分相关。结论肝细胞癌存在明显的染色体异常,部分染色体异常事件是非随机性的,可能与肝癌的发生、发展有关,并与肿瘤的生物学行为和预后相关。     

Analysis of genetic alterations in uterine leiomyomas and leiomyosarcomas by comparative genomic hybridization

Uterine leiomyomas are the most prevalent tumor type in women of reproductive age and are the most common reason for hysterectomies. Although uterine leiomyomas are considered to be benign, they are a major public health concern for women. In contrast, leiomyosarcomas are rare but highly malignant uterine tumors. They may arise in uteri with preexisting leiomyomas and histologically sometimes resemble leiomyomas, thus causing controversy about whether leiomyosarcomas arise within leiomyomas. In this study, we used comparative genomic hybridization (CGH) to identify genetic alterations unique to each tumor type and alterations that are common between the two tumors. We analyzed 14 cases of uterine leiomyomas and eight cases of uterine leiomyosarcomas. Only two of the 14 leiomyomas exhibited genetic alterations, and those were restricted to gains on chromosomes 14 and 19 and losses on chromosomes 1 and 4. In addition, 68 leiomyomas were examined for loss of heterozygosity on chromosomes 1 and 4, and only three tumors exhibited any losses. In contrast, all eight leiomyosarcomas showed gains and losses of DNA by CGH, and in many cases multiple changes were observed. The most commonly observed genetic aberration, occurring in five tumors, was gains on both arms of chromosome 1, suggesting that this chromosome contains loci involved in the development of leiomyosarcoma. Our results do not provide evidence for the progression from benign leiomyoma to malignant leiomyosarcoma. Moreover, the large number of random chromosomal alterations in the leiomyosarcomas suggests that increased genetic instability plays a role in the formation of these tumors. Mol. Carcinog. 19:273–279, 1997. © 1997 Wiley-Liss, Inc. This article is a US Government work and, as such, is the public domain in the United States of America.     

Comparative genomic hybridization array analysis and real time PCR reveals genomic alterations in squamous cell carcinomas of the lung

Genomic alterations have been identified in lung cancer tissues and reported in numerous studies. To analyze genomic aberrations in lung cancer patients, we used array comparative genomic hybridization (array CGH) in 14 squamous cell lung carcinoma (SqC) tissues. Copy number gain and loss in chromosomal regions were detected, and the corresponding genes were confirmed by real time PCR. Several frequently altered loci, including gain of 3q (36% of samples), were found. The most frequently identified losses were found at 14q32.33 (21% of samples). The relative degree of chromosomal change was analyzed using log2 ratios. High-level DNA amplifications (>0.8 log2 ratio) were detected at 20 regions in 1p, 2q, 3q, 4q, 6q, 7p, 8q, 9p, 10q, 12q, 14q and 19p. We found that the fold change levels were highest at EVI1 (3q26.2), LPP (3q27-28) and FHF-1 (3q28) gene loci. Our results show that array CGH is a useful tool for identification of gene alteration in lung cancer, and that the above-mentioned genes might represent potential candidate genes for pathogenesis and diagnosis of lung cancer.     

Use of complete coverage array comparative genomic hybridization to define copy number alterations on chromosome 3p in oral squamous cell carcinomas

Loss of 3p has been associated with oral cancer progression and is common in many cancers. However, regions of alteration on 3p are poorly defined. We have constructed a high-resolution chromosomal array using a tiling set of 535 human bacterial artificial chromosomes that provides near complete coverage of 3p. Array comparative genomic hybridization analysis of 20 microdissected oral squamous cell carcinomas showed multiple and recurrent segments of copy number changes. These include a deletion containing the FHIT gene; novel segments of copy decrease at 3p22, 3p24, and 3p26; and an unexpected approximately 0.7 Mbp segmental increase at 3p21. These data strongly support the value of using chromosomal array comparative genomic hybridization for detailed profiling of oral squamous cell carcinomas.     

Prognostic value of genomic alterations in invasive cervical squamous cell carcinoma of clinical stage IB detected by comparative genomic hybridization.

The clinical behavior of invasive cervical carcinoma of clinical stage IB varies considerably in tumors presenting without regional lymph node metastases. The early identification of patients at higher risk for poor outcome may prove useful because these patients would benefit from aggressive adjuvant treatments. In this study, comparative genomic hybridization was applied to evaluate whether genomic aberrations have prognostic significance in cervical carcinoma. Genomic alterations were evaluated in 62 cervical carcinomas of clinical stage IB. DNA sequence losses were most prevalent at chromosomes 4q (53%), 3p (52%), 13q (45%), 4p (44%), Xq (44%), 5q (40%), 18q (37%), and 6q (35%). Several genomic alterations were associated with poor clinical outcome or metastasis. The total number of DNA aberrations/tumor (P < 0.02) and the number of DNA sequence losses/tumor (P < 0.04) were associated with disease-specific survival. 9p deletions were significantly more frequent in carcinomas with lymph node metastasis than in node-negative tumors (P < 0.03). Losses of chromosome 11p (P < 0.0001) and 18q (P < 0.01) were associated with poor prognosis in cervical carcinomas without lymph node metastasis. These data suggest that inactivation of tumor suppressor genes on chromosomes 9p, 11p, and 18q may play a role in the progression of cervical carcinoma.     

Microarray comparative genomic hybridization detection of chromosomal imbalances in uterine cervix carcinoma

Background

Sarcomas are one of the most refractory diseases among malignant tumors. More effective therapies based on an increased understanding of the molecular biology of sarcomas are needed as current forms of therapy remain inadequate. Recently, it has been reported that Wnt-1/β-catenin signaling inhibits apoptosis in several cancers. In this study, we investigated the efficacy of a monoclonal anti-Wnt-1 antibody in sarcoma cells.

Methods

We treated cell lines A-204, SJSA-1, and fresh primary cultures of lung metastasis of sarcoma with a monoclonal anti-Wnt-1 antibody. Wnt-1 siRNA treatment was carried out in A-204. We assessed cell death using Crystal Violet staining. Apoptosis induction was estimated by flow cytometry analysis (Annexin V and PI staining). Cell signaling changes were determined by western blotting analysis.

Results

We detected Wnt-1 expression in all tissue samples and cell lines. Significant apoptosis induction was found in monoclonal anti-Wnt-1 antibody treated cells compared to control monoclonal antibody treated cells (p < 0.02). Similarly, we observed increased apoptosis in Wnt-1 siRNA treated cells. Blockade of Wnt-1 signaling in both experiments was confirmed by analyzing intracellular levels of Dishevelled-3 and of cytosolic β-catenin. Furthermore, the monoclonal anti-Wnt-1 antibody also induced cell death in fresh primary cultures of metastatic sarcoma in which Wnt-1 signaling was active.

Conclusion

Our results indicate that Wnt-1 blockade by either monoclonal antibody or siRNA induces cell death in sarcoma cells. These data suggest that Wnt-1 may be a novel therapeutic target for the treatment of a subset of sarcoma cells in which Wnt-1/β-catenin signaling is active.