Molecular cytogenetic analysis of 17 renal cancer cell lines: increased copy number at 5q31-33 in cell lines from nonpapillary carcinomas.

Comparative genomic hybridization (CGH) was used to screen for genomic imbalances in cell lines derived from 13 nonpapillary renal-cell carcinomas (RCCs), two papillary RCCs, one renal squamous-cell carcinoma, and one transitional-cell carcinoma of the renal pelvis. Aberrations were found in all 17 lines. The most frequent changes in nonpapillary RCC cell lines were gains of 5q (85%), 7q (69%), 8q (69%) and 1q (54%) and losses of 3p (92%), 8p (77%), 4q (62%) and 14q (54%). High-level gains (HLGs) were detected at 4q12, 5p, 5q23-33, 7q22-qter, 8q23-24, 10q21-qter, 12p and 12q13-22. By means of fluorescence in situ hybridization (FISH) we narrowed the smallest common region involving 5q gains to the genomic segment between D5S642 and D5S673, and found that the HLG at 4q12 possibly involved amplifications of c-kit and PDGFRA. Two papillary RCC cell lines showed gains of entire chromosomes 7, 12 and 17. The CGH data reported here should help to facilitate the choice of individual renal-tumor cell lines for exploring target genes in regions of interest.     

Molecular cytogenetic characterization shows higher genetic homogeneity in conventional renal cell carcinoma compared to other kidney cancers

In this study seven primary kidney tumors out of 13 were cytogenetically characterized by comparative genomic hybridization (CGH) on the surgical specimens as well as by spectral karyotyping (SKY) analysis after short-term culturing. In two of the seven cases only a normal karyotype was identified. Non-clonal aberrations were observed in four of the seven cases. Overall numerical alterations were more frequent than structural changes. The two structural alterations identified constituted of a deletion of the short arm of chromosome 3 in a conventional renal cell carcinoma (RCC), and a ring chromosome derived from chromosome 8 in a papillary RCC. By CGH gains of copy number were revealed on chromosomes 3, 5, 7, 8q, and 20, while the losses encompassed 3p and 17p. In the papillary RCCs only gains were found. Comparison between SKY and CGH data suggests that the conventional RCCs are genetically more homogeneous than the other types of kidney cancer. In the two papillary RCCs, trisomies of chromosomes 7 and 17 were typical findings. In the transitional cell carcinoma different findings by CGH and SKY would suggest that these tumors constitute a heterogeneous population of tumor cells which could represent different steps of somatic evolution of tumors.     

人鼻咽癌紫杉醇耐药细胞系与其亲代细胞系比较基因组杂交研究

目的：比较基因组杂交（comparative genomic hybridization, CGH）是一种在荧光原位杂交（fluorescence in situ hybridiza?tion,FISH）技术上发展起来的用于检测两个基因组间相对DNA拷贝数的改变（缺失或扩增）, 并将这些变化在染色体上进行定位的分子细胞遗传学方法。为了解鼻咽癌紫杉醇耐药细胞 （CNE1/Taxol, HNE2/Taxol, 5-8F/Taxol）与亲代细胞（CNE1, HNE2, 5-8F）在基因组DNA水平上可能存在的差异, 以及这种差异在肿瘤获得性耐药产生中的意义, 采用CGH技术对鼻咽癌紫杉醇耐药细胞与亲本细胞的基因组DNA进行检测和分析。方法：三株鼻咽癌紫杉醇耐药细胞采用大剂量冲击与剂量逐渐递加相结合的方法诱导而成。采用集落形成实验测定药物的敏感性,利用比较基因组杂交技术 （CGH） 对耐药细胞和其亲本细胞的基因组DNA进行检测和分析, 比较耐药细胞与亲本细胞在染色体扩增与缺失上的共同点和差异。结果：三株鼻咽癌紫杉醇耐药细胞耐药指数分别为8.43、 8.27和5.26。鼻咽癌亲本细胞存在广泛的染色体改变,主要表现在3q21-qter, 5p13-pter, 12和20q11-qter的共同扩增以及10q11-qter, 18和X染色体的共同缺失,从亲本细胞系诱导的三株鼻咽癌紫杉醇耐药细胞系表现为3q21-qter, 5p13-pter, 12,20q11-qter和8q21-qter的共同扩增, 无明显共同缺失区域。与亲本细胞共同扩增区域相比, 其中最有意义的是8q21-qter区域在三株耐药细胞中出现了新的共同扩增。结论： 3q21-qter, 5p13-pter, 12和20q11-qter的共同扩增以及10q11-qter,18和X染色体的共同缺失可能与鼻咽癌的发生有关, 而8q21-qter染色体扩增可能与鼻咽癌获得性紫杉醇耐药相关,对这些区域的进一步研究, 有可能为肿瘤发生及耐药机制研究提供新的线索。     

Frequent gains and losses of specific chromosome segments in human ovarian carcinomas shown by comparative genomic hybridization

Total genomic DNA obtained from 24 ovarian carcinomas was examined for genomic imbalances by comparative genomic hybridization (CGH). A varying number of gains and losses (1 up to 31) of specific chromosomal segments was detected per tumor. Chromosomal segments which were most often present in increased copy numbers were (in decreasing order): 1q21, 8q24, 8q23, 3q26, 12p12-p13, 20q, 7q31, and 7q33-qter. Loss of material was found most frequently at 16q12, 13q13-q14, Xq, 8p21-p22, 5q13-q14, and 5q21. All these chromosomal segments involved in gains and losses may carry gene loci playing a more or less causal role in the process of ovarian malignancies. Based on these findings CGH can be regarded as a valuable tool for rapid screening of genomic imbalances in human tumors.     

Differentiation between papillary and nonpapillary renal cell carcinomas by DNA analysis

Recent studies have shown that the deletion of chromosome 3p or the loss of DNA sequences at 3p is generally associated with the development of renal cell carcinoma (RCC). However, chromosome analysis of some papillary RCCs suggested that this type of tumor differs genotypically from the most common nonpapillary RCCs. Therefore, by using cytogenetic and molecular genetic approaches, we examined human papillary and nonpapillary RCCs for the loss of heterozygosity or homozygosity at the short arm of chromosome 3. The constitutional heterozygosity for the DNF15S2 locus and for one allele of the c-erbA beta and the c-raf-1 proto-oncogenes was lost in nonpapillary RCCs, whereas both alleles were retained in each papillary RCC analyzed. We conclude that the loss of DNA sequences at the chromosome 3p region is a genomic change occurring consistently in nonpapillary RCCs, but never occurring in papillary RCCs.     

Chromosomal aberrations detected by comparative genomic hybridization predict outcome in patients with colorectal carcinoma

To obtain comprehensive information regarding the correlation between genomic changes and clinicopathological parameters such as disease stage, metastases, and survival, we investigated genomic changes by comparative genomic hybridization (CGH) in 73 patients with colorectal cancer (CRC), and assessed the associations of such charges with clinicopathological parameters. Gains of 8q21-22, 13q21-31 and 20q12-qter and loss of 17p12-pter were detected in >50% of stage I tumors. Gain of 8q23-qter and losses of 8p12-pter and 18q12-qter were observed more frequently in stage III/IV tumors than in stage I tumors (all P<0.05). Loss of 8p12-pter and gain of 8q23-qter were linked to nodal metastasis (all P<0.05). Loss of 18q12-qter and gain of 8q23-qter were associated with distant organ metastasis at diagnosis and/or recurrence after surgery (all P<0.05). Moreover, losses of 8p12-pter and 18q12-qter and gains of 8q23 and 8q24-qter were associated significantly with unfavorable prognosis (all P<0.05). Furthermore, combined examination of the above four changes can provide a more accurate assessment for patient's prognosis. Specifically, 11 of 19 patients with these four changes died, but only 1 of 21 cases without these four changes died during the follow-up period (P<0.0001). Multivariate analysis revealed that loss of 18q12-qter is an independent prognostic marker (P=0.031). Our findings indicate that genetic aberrations detected by CGH may predict outcome in patients with CRC.     

Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line,FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization

Background

Pleomorphic malignant fibrous histiocytoma (MFH) is one of the most frequent malignant soft tissue tumors in adults. Despite the considerable amount of research on MFH cell lines, their characterization at a molecular cytogenetic level has not been extensively analyzed.

Methods and results

We established a new permanent human cell line, FU-MFH-2, from a metastatic pleomorphic MFH of a 72-year-old Japanese man, and applied multicolor fluorescence in situ hybridization (M-FISH), Urovysion™ FISH, and comparative genomic hybridization (CGH) for the characterization of chromosomal aberrations. FU-MFH-2 cells were spindle or polygonal in shape with oval nuclei, and were successfully maintained in vitro for over 80 passages. The histological features of heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original tumor. Cytogenetic and M-FISH analyses displayed a hypotriploid karyotype with numerous structural aberrations. Urovysion™ FISH revealed a homozygous deletion of the p16^INK4A locus on chromosome band 9p21. CGH analysis showed a high-level amplification of 9q31-q34, gains of 1p12-p34.3, 2p21, 2q11.2-q21, 3p, 4p, 6q22-qter, 8p11.2, 8q11.2-q21.1, 9q21-qter, 11q13, 12q24, 15q21-qter, 16p13, 17, 20, and X, and losses of 1q43-qter, 4q32-qter, 5q14-q23, 7q32-qter, 8p21-pter, 8q23, 9p21-pter, 10p11.2-p13, and 10q11.2-q22.

Conclusion

The FU-MFH-2 cell line will be a particularly useful model for studying molecular pathogenesis of human pleomorphic MFH.     

Chromosomal imbalances in malignant peripheral nerve sheath tumor detected by metaphase and microarray comparative genomic hybridization

Malignant peripheral nerve sheath tumors (MPNSTs) are highly malignant tumors affecting adolescents and adults. There have been a few reports on chromosomal aberrations of MPNSTs; however, the tumor-specific alteration remains unknown. We characterized the genomic alterations in 8 MPNSTs and 8 schwannomas by metaphase comparative genomic hybridization (CGH). In 5 of 8 MPNSTs, microarray CGH was added for more detailed analyses. Frequent gains were identified on 3q13-26, 5p13-14, and 12q11-23 and frequent losses were at 1p31, 10p, 11q24-qter, 16, and 17. Microarray CGH revealed frequent gains of EGFR, DAB2, MSH2, KCNK12, DDX15, CDK6, and LAMA3, and losses of CDH1, GLTSCR2, EGR1, CTSB, GATA3, and SULT2A1. These genes seem to be responsible for developing MPNSTs. The concordance rate between metaphase CGH and microarray CGH was 66%. Metaphase CGH was useful for identifying chromosomal alterations before applying microarray CGH.     

Molecular cytogenetic analysis of 11 new breast cancer cell lines

We describe a survey of genetic changes by comparative genomic hybridization (CGH) in 11 human breast cancer cell lines recently established in our laboratory. The most common gains took place at 8q (73%), 1 q (64%), 7q (64%), 3q (45%) and 7p (45%), whereas losses were most frequent at Xp (54%), 8p (45%), 18q (45%) and Xq (45%). Many of the cell lines displayed prominent, localized DNA amplifications by CGH. One-third of these loci affected breast cancer oncogenes, whose amplifications were validated with specific probes: 17q12 (two cell lines with ERBB2 amplifications), 11q13 (two with cyclin-D1), 8p11-p12 (two with FGFR1) and 10q25 (one with FGFR2). Gains and amplifications affecting 8q were the most common genetic alterations in these cell lines with the minimal, common region of involvement at 8q22-q23. No high-level MYC (at 8q24) amplifications were found in any of the cell lines. Two-thirds of the amplification sites took place at loci not associated with established oncogenes, such as 1q41-q43, 7q21-q22, 7q31, 8q23, 9p21-p23, 11p12-p14, 15q12-q14, 16q13-q21, 17q23, 20p11-p12 and 20q13. Several of these locations have not been previously reported and may harbour important genes whose amplification is selected for during cancer development. In summary, this set of breast cancer cell lines displaying prominent DNA amplifications should facilitate discovery and functional analysis of genes and signal transduction pathways contributing to breast cancer development.     

Patterns of chromosomal imbalances in invasive breast cancer

Invasive breast carcinomas are characterized by a complex pattern of chromosomal alterations. We applied comparative genomic hybridization (CGH) to analyze 105 primary breast carcinomas using histograms to indicate the incidence of DNA imbalances of tumor subgroups and difference histograms to compare invasive ductal carcinomas (IDC) with lobular carcinomas (ILC), well and poorly differentiated carcinomas (G1/G3) and estrogen receptor-positive and -negative tumors (ER(+)/ER(-)). Only single imbalances showed a higher incidence in ILC compared with IDC, i.e., gains on chromosomes 4 and 5q13-q23 as well as deletions on chromosomes 6q, 11q14-qter, 12p12-pter, 16q, 17p, 18q, 19, and 22q. Of these, particularly gains of 4 and losses at 16q21-q23, and 18q12-q21 were statistically significant. For most loci, IDC showed more alterations providing a genetic correlate to the fact that ductal carcinoma overall is associated with a worse prognosis than ILC. Of these, many imbalances showing statistical significance were also observed in G3 and ER(-) tumors, i.e., deletions at 2q35-q37, 3p12-p14, 4p15-p16, 5q, 7p15, 8p22-p23, 10q, 11p, 14q21-q31, 15q, and gains at 2p, 3q21-qter, 6p, 8q21-qter, 10p, 18p11-q11, and 20q, suggesting that they contribute to a more aggressive tumor phenotype. By contrast, gains on chromosome 5q13-q23 as well as deletions at 6q, 16q and 22q were more prevalent in G1 and ER(+) tumors. The ratio profiles of all cases as well as histograms are accessible at our CGH online tumor database at http://amba.charite.de/cgh. Our results highlight distinct chromosomal subregions for cancer-associated genes. In addition, these imbalances may serve as markers for a genetic classification of invasive breast cancer.     

Discovery of over-expressed genes and genetic alterations in breast cancer cells using a combination of suppression subtractive hybridization,multiplex FISH and comparative genomic hybridization

To identify genes that are involved in breast cancer, suppression subtractive hybridization (SSH) was utilized to construct a breast cancer subtracted library. Differential screening of the library isolated 28 genes which by Northern analysis were highly expressed in the breast cancer cell line MDA-MB-231 compared to the normal breast cell line MCF12A. Sequence analysis revealed that 15 clones coded for previously described genes such as SNAP43, Cyr61, Thymosin beta4, tra1, elongation factor 1alpha, BSF-2/IL6, BiP, and GDP/GTP exchange protein. The remaining 13 clones did not match sequences in GenBank/EMBL database, indicating that they may be novel genes. SNAP43, a subunit of the TBP-TAF complex, was expressed 20-fold higher in MDA-MB-231 compared to MCF12A and several breast cancer cell lines, implying that SNAP43 may be involved in tumorigenesis of a specific subset of breast cancers. Amplification of SNAP43 was not found by Southern analysis. However, genetic alterations of MDA-MB-231 included a deletion of chromosome 14 with a reciprocal translocation t(6;14) and two additional translocations [t(12;14) and t(14;15)] as determined by fluorescent in situ hybridization (FISH) with YAC 823G8 located at chromosome 14q23 which contained SNAP43. Because of the numerous alterations observed by FISH in MDA-MB-231, we further explored the genetic abnormalities in this breast cancer cell line using multiplex FISH (M-FISH) and comparative genomic hybridization (CGH). These cells were replete with numerous complex structural rearrangements and had DNA copy-number imbalances involving multiple chromosomes including gains on chromosomes 2p, 2q31-q32, 3p14-pter, 5q, 6p, 7q36-qter, 11, 14q21-q24, 17p11.2-pter, 17q21-qter, 19, 20, Xp11-q13 and losses on chromosomes 4pter-q32, 8p, 9p21-p24, 10q26-qter, 16p13-pter, 18q12-qter, 22, Xp11.3-p22.1, Xq13-qter. In summary, SSH revealed a number of genes that were either novel or previously not associated with breast cancer. In addition, we found that breast cancer cells abounded with abnormalities as observed by M-FISH and CGH. Together, these results may facilitate defining the genetic alterations associated with breast cancer progression.     

Comparative genomic hybridization of esophageal squamous cell carcinoma: correlations between chromosomal aberrations and disease progression/prognosis.

BACKGROUND: Esophageal carcinoma is a major cause of cancer-related deaths among males in Taiwan. However, to date, the genetic alterations that accompany this lethal disease are not understood. METHODS: Chromosomal aberrations of 46 samples of esophageal squamous cell carcinoma (EC-SCC) were analyzed by comparative genomic hybridization (CGH), and their correlations with pathologic staging and prognosis were analyzed statistically. RESULTS: In total, 321 gains and 252 losses were found in 46 tumor samples; thus, the average gains and losses per patient were 6.98 and 5.47, respectively. Frequent gain abnormalities were found on chromosome arms 1q, 2q, 3q, 5p, 7p, 7q, 8q, 11q, 12p, 12q, 14q, 17q, 20q, and Xq. Frequent deletions were found on chromosome arms 1p, 3p, 4p, 5q, 8p, 9p, 9q, 11q, 13q, 16p, 17p, 18q, 19p, and 19q. It was found that deletions of 4p and 13q12-q14 and gain of 5p were significantly correlated with pathologic staging. Losses of 8p22-pter and 9p also were found more frequently in patients with advanced disease. Gain of 8q24-qter was seen more frequently in patients with Grade 3 tumors. A univariate analysis found that pathologic staging; gains of 5p and 7q; and deletions of 4p, 9p, and 11q were significant prognostic factors. However, pathologic staging became the only significant factor in a multivariate analysis. CONCLUSIONS: CGH not only revealed novel chromosomal aberrations in EC-SCC, but also found possible genotypic changes associated with disease progression. Despite all of the possible associations of chromosomal aberrations with disease progression, the most important prognostic factor for patients with EC-SCC was pathologic staging.     

Detection of chromosomal alterations in bladder transitional cell carcinomas from Northern China by comparative genomic hybridization

To identify chromosome alterations in Chinese bladder cancer, forty-six transitional cell carcinomas of the bladder were analyzed by comparative genomic hybridization. Frequent gains of DNA copy number were observed on 1p (13/46), 1q (13/46), 5p (8/46), 6p (9/46), 7p (7/46), 8q (12/46), 11q (8/46), 17q (11/46), 19q (7/46), 20q (8/46) and Yq (8/46), with minimal overlapping regions at 1p32-pter (10/46), 1q21-q24 (12/46), 5p (8/46), 6p22-p23 (7/46), 7p11.2-p14 (7/46), 8q22-q24 (12/46), 11q13-q14 (8/46), 17q22-qter (11/46), 19q11-13.2 (7/46), 20q11-q13.2 (8/46) and Yq11 (8/46). Losses were predominantly found on 2q (16/46), 5q (8/46), 8p (7/46), 9p (8/46), 9q (13/46), 11p (7/46), 13q (7/46), 17p (12/46), 18q (7/46), Xp (18/46) and Xq (19/46), with smallest overlapping regions at 2q32-qter (16/46), 5q12-q31 (8/46), 8p12-pter (7/46), 9p21-pter (10/46), 9q (13/46), 11p (7/46), 13q13-q22 (7/46), 17p (12/46), 18q21-qter (7/46), Xp (18/46) and Xq (19/46). There were significantly higher frequencies of gains of 1q21-q24 and 17q22-qter in moderately differentiated tumors as compared with those in well-differentiated tumors, indicating a possible association of these two abnormalities with the dedifferentiation of tumor cells. Gains of 1p32-pter, 5p, 6p22-p23, 11q13-q14, 17q22-qter and losses of 2q32-qter, 9q, 17p were more frequent in pT1 as compared with those in pTa carcinomas. Gains at 1q21-q24, 7p11.2-p14, 8q22-q24, 19q, 20q11-q13.2 and losses at 5q12-q31, 8p12-pter, 9p21-pter, 11p, 13q13-q22 and 18q21-qter were unique to pT1 and higher stage tumors, suggesting that genes responsible for the invasion and progression of bladder cancer might be located at these chromosomal regions. In multiple tumors from the same patients, consistent alterations such as gains of 8q, 11q13-q14, 12q13-q15, 13q12, 20q and losses of 2q32-qter, 8p, 9, 11p, 11q21-qter, 13q13-qter, X were detected. These abnormalities were possibly earlier events, which might play a critical role during the genesis of the tumors. Further detailed studies to the recurrent aberration regions may lead to the identification of oncogenes and tumor suppressor genes involved in the development and progression of Chinese bladder cancer.     

Gains of 8q23-qter and 20q and loss of 11q22-qter in esophageal squamous cell carcinoma associated with lymph node metastasis

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is associated with poor prognosis and lymph node metastasis is one of the critical prognostic factors. Although it is important to elucidate the genetic aberrations underlying its lymph node metastasis, to the authors' knowledge little is known regarding alterations in the primary ESCC that are linked with ESCC metastasis to the lymph nodes. METHODS: To elucidate genetic aberrations involved in the lymph node metastasis of ESCC, comparative genomic hybridization analysis was applied to 36 ESCC specimens, from 12 cases with no lymph node metastasis and 24 cases with lymph node metastasis. RESULTS: Copy number gains frequently were detected at 3q (75%), 8q23-qter (50%), 11q13 (44%), 5p14-pter (25%), 20q (25%), 7q (22%), 2p (19%), 12p (17%), and 20p (17%) and losses were detected at 18q (58%), 3p (50%), 9p (44%), 5q14-23 (39%), 4q (33%), 13q (22%), and 11q22-qter (19%). DNA amplifications were detected at four loci: 11q13, 2q12, 7q21, and 20q11.2 It is interesting to note that the gains of 8q23-qter (P < 0.0005) and 20q (P < 0.02) and loss of 11q22-qter (P < 0.05) were observed in tumors metastatic to the lymph nodes. The gains of 3q and 11q13 and losses of 18q, 3p, 9p, 5q14-23, and 4q were detected in both early and advanced stage ESCCs. CONCLUSIONS: These observations suggest that gains of 8q23-qter and 20q and loss of 11q22-qter allow the prediction of lymph node metastasis, and that gains of 3q and 11q13 and losses of 18q, 3p, 9p, 5q14-23, and 4q are associated with the development of ESCC.     

大肠癌染色体组DNA拷贝数异常的临床病理学意义

目的分析大肠癌染色体组DNA拷贝数异常区域与患者临床病理特征的关系。方法应用比较基因组杂交技术（CGH）检测73例大肠癌患者的标本。结果大肠癌染色体8p12-pter丢失和8q23-qter扩增与淋巴结转移显著相关;而8q23-qter扩增和18q12-qter丢失与远处器官转移和（或）术后复发显著相关;8q23-qter扩增、8p12-pter和18q12-qter丢失,与大肠癌患者不良预后密切相关。Cox多因素分析表明,18q12-qter丢失在大肠癌是一个独立的不良预后生物学指标。结论采用CGH法检测染色体组DNA拷贝数异常能预测大肠癌患者的预后,并为临床治疗提供有意义的信息。     

Mapping of novel regions of DNA gain and loss by comparative genomic hybridization in esophageal carcinoma in the Black and Colored populations of South Africa

Esophageal cancer (EC) is the leading cause of cancer death in the Black male population in South Africa. Although several oncogenes and tumor suppressor genes have previously been found altered in this cancer, many novel genes remain to be identified. To identify the chromosomal location of these unknown genes, we have analyzed DNA of 29 South African EC patients by comparative genomic hybridization. Frequent loss occurred at chromosome 1p (52%), 4p (52%), 18q (48%), 19p (52%), 19q (55%), and 22q (41%). The most common gains were detected at 1q (41%), 2q (52%), 3q (72%), 5p (31%), 7p (48%), 7q (45%), 8q (55%), and Xq (69%). High level amplification was detected at 2q24-33, 6p21.1-q14, 7p12-q21, 7q11.2-31, 8q22-24, 8q13-qter, 13q21-34, and at 13q32-34. The present comparative genomic hybridization study opens the way for additional targeted studies on these particular chromosomal regions to identify the specific genes involved in the higher susceptibility to specific subtypes of esophageal carcinoma in different geographical regions. The loss of 8p (28%) and Xp (17%) in tumors of male individuals may provide clues to the basis of the sex-biased frequency of occurrence of EC favoring men.