Genotypic analysis of esophageal squamous cell carcinoma by molecular cytogenetics and real-time quantitative polymerase chain reaction

We performed an integrated cytogenetic study using a combination of comparative genomic hybridization (CGH), spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) to analyze chromosomal aberrations associated with 8 human esophageal squamous cell carcinoma (EC-SCC) cell lines, and used real-time quantitative PCR (Q-PCR) to study the copy number changes of two candidate genes of chromosome 3q, PIK3CA and TP63, in 20 primary tumors of EC-SCC. The pooled CGH results revealed frequent gain abnormalities on chromosome arms 1p, 1q, 3q, 5p, 6p, 7p, 7q, 8q, 9q, 11q, 12p, 14q, 15q, 16p, 16q, 17q, 18p, 19q, 20q, 22q, and Xq, while frequent losses were found on 3p, 4, 5q, 6q, 7q, 9p, and 18q. SKY detected 195 translocations, 13 deletions and 2 duplications. Among the 374 breakpoints, most clustered at the centromeric regions, such as 8q10, 13q10, 7q10, 9q10, 14q10, 15q10, 16q10, 21q10, and 22q10, but also at other regions, including 3q (3q21, 3q22, 3q25), 7p (7p22, 7p14, 7p12), 7q (7q21, 7q31, 7q32), 8q (8q21.1, 8q23), 11q (11q21, 11q24), 13q (13q14) and 18q (18q21). There was a good correlation between the number of aberrations identified by CGH and SKY (r=0.667; p=0.035). Combined CGH and SKY analyses indicated that chromosomes 3, 7, 9, 11, 14, 16, 18, 19, 20, and 22 harbored higher frequency of chromosomal aberrations than expected. FISH using BAC clones containing oncogene PIK3CA and TP63 found that both genes were amplified in 6 and 5 cell lines, respectively. Q-PCR analysis of primary tumors revealed amplification of PIK3CA and TP63 in 100% and 80% of the cases. Average copy number of PIK3CA per haploid genome was greater than that of TP63 (6.27 vs 2.73), and the difference showed statistical significance (p<0.001). Combination of CGH, SKY and FISH could reveal detailed chromosomal changes associated with esophageal cancer cells, and Q-PCR could assess the change of the candidate genes in clinical samples in a high throughput way.     

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.     

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.     

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.     

Frequent gains of 20q and losses of 18q are associated with lymph node metastasis in intestinal-type gastric cancer

BACKGROUND: The genetic aberrations associated with development and progression of gastric carcinomas (GCs) are poorly understood. The aim of this study was to identify chromosomal aberrations associated with the development and/or progression of intestinal-type GC. MATERIALS AND METHODS: Comparative genomic hybridization (CGH) analysis was applied to 36 intestinal-type GCs. We compared chromosomal aberrations detected by CGH analysis with clinicopathological parameters. RESULTS: Frequent gains of DNA copy number were found on 8q, 13q, 20q, 3q, 6q and losses were found on 17p, 18q in intestinal-type GCs. No significant differences were observed in the chromosomal aberrations between tumor stage, tumor location, peritoneal dissemination, liver metastasis or other distant metastasis. However, the frequencies of 20q12-13 gain and 18q21-22 loss were significantly higher in tumors with lymph node metastasis than in those without metastasis. CONCLUSION: Gains of 20q and losses of 18q may contribute to lymph node metastasis and the malignant phenotype in intestinal-type GCs.     

Distinct chromosomal aberrations of ampulla of Vater and pancreatic head cancers detected by laser capture microdissection and comparative genomic hybridization

Despite the continuous progress in molecular methodology, the genetic events involved in the carcinogenesis of pancreatic head ductal carcinomas (PHDCs) and ampulla of Vater cancer (AVC) remain largely unknown. Their proximity within the confined region lends them to very similar clinical presentations and operative approaches. However, it is unclear why AVC has a significantly better outcome than PHDCs. The drastic difference may arise from the distinct tumor origins. Therefore, the study of genetic alterations within these two neighboring tumors may elucidate their mechanisms. However, previous genetic analysis of PHDCs may have been influenced by contamination of excessive stromal and inflammatory cells in the background. To date, most comparative genomic hybridization (CGH) studies of pancreatic cancer are based in cell lines or specimens that were not microdissected. Even AVC has a paucity of data. To determine the differences of the genetic alterations of PHDCs and AVC, we used laser capture microdissection combined with degenerated oligonucleotide polymerase chain reaction and CGH to identify the chromosomal aberrations. Frequent gains were found on 5p, 13q, 3q, 8q, 1p, 2p, 2q, 3q, 7p, 8p, 9p, 13q, and 21q, while frequent losses were located on 7p, 18q, 1p, 11q, 16q, 19q, 1q, 2q, 3p, 19p, and 17p in PHDCs. In AVC, chromosomal gains occurred frequently in 7p, 8q, 1q, 2q, 3p, 4p, 7p, 3q, 5p, 20p, 20q, 4q, 5q, 7q, 11p, 12p, 13q, 14q, 18p, 18q, 21q, 9p, 10p, and 15q, and losses frequently in 17q, 1p, 18q, 19p, 5q, 10p, and 10q. This is the first report on the CGH profiles of AVC. These data provide evidence that chromosomal gains/amplifications of AVC differ from those of PHDCs. Among them, chromosomal gains on 1q25, 7p15, 8q23, and 3p21, and losses on 17q24, were statistically higher in AVC than PHDCs (P<0.05). The gains and losses suggest possible putative tumor suppressors and oncogenes that may be involved in the different carcinogenic pathways of AVC.     

Chromosome alterations in breast carcinomas: frequent involvement of DNA losses including chromosomes 4q and 21q.

Comparative genomic hybridization was applied to map DNA gains and losses in 39 invasive ductal breast carcinomas. Frequent abnormalities included gains on chromosomal regions 1q, 8q, 11q12-13, 16p, 19, 20q and X as well as frequent losses on 1p, 5q, 6q, 9p, 11q, 13q and 16q. Furthermore, frequent losses on 4q (20 cases) and 21q (14 cases) were found for the first time in this tumour type. High copy number amplifications were observed at 8q12-24, 11q11-13 and 20q13-ter. Highly differentiated tumours were associated with gains on 1q and 11q12-13 along with losses on 1p21-22, 4q, 13q, 11q21-ter. Undifferentiated breast carcinomas were characterized by additional DNA imbalances, i.e. deletions of 5q13-23, all of chromosome 9, the centromeric part of chromosome 13 including band 13q14 and the overrepresentation of chromosome X. We speculate that these changes are associated with tumour progression of invasive ductal breast cancer.     

Assessment of chromosomal losses and gains in hepatocellular carcinoma

We examined the chromosomal changes of 22 hepatocellular carcinomas (HCCs) by comparative genomic hybridization (CGH) analysis and compared the results with that of allelotype by polymerase chain reaction based loss of heterozygosity (PCR-LOH) analysis. By CGH analysis, frequent chromosomal losses were noted in the chromosomal region of 4q (59%), 8p (77%), and 16q (50%), whereas gains were noted in 1q (86%) and 8q (77%). All of these chromosomal arms were revealed to have frequent allelic imbalances by PCR-LOH analysis, however, 9% of chromosomal aberrations were detected only by CGH analysis and 2% were detected only by PCR-LOH analysis. Our results suggest that CGH analysis gives more precise results for the screening of chromosomal aberrations in HCCs than that of PCR-LOH analysis with randomly selected microsatellite markers.     

Chromosomal changes during development and progression of prostate adenocarcinomas

Chromosomal copy number changes were investigated in 16 prostate carcinomas, 12 prostatic intraepithelial neoplasias (PIN; 4 low-grade and 8 high-grade) adjacent to the invasive tumour areas, and 5 regional lymph node metastases. For this purpose, comparative genomic hybridization (CGH) was performed and a copy number karyotype for each histomorphological entity was created. CGH on microdissected cells from non-neoplastic glands was carried out on 3 different cases to demonstrate the reliability of the overall procedure. None of the non-neoplastic tissue samples revealed chromosome copy number changes. In PIN areas, chromosomal imbalances were detected on chromosomes 7, 8q, Xq (gains), and on 4q, 5q, 8p, 13q and 18q (losses). In the primary tumours, recurrent (at least 25% of cases) gains on chromosomes 12q and 15q, and losses on 2q, 4q, 5q, Xq, 13q and 18q became apparent. Losses on 8p and 6q as well as gains on 8q and of chromosome 7 were also detected at lower frequencies than previously reported. The pooled CGH data from the primary carcinomas revealed a novel region of chromosomal loss on 4q which is also frequently affected in other tumour entities like oesophageal adenocarcinomas and is supposed to harbour a new tumour suppressor gene. Gains on chromosome 9q and of chromosome 16 and loss on chromosome 13q were observed as common aberrations in metastases and primary tumours. These CGH results indicate an accumulation of chromosomal imbalances during the PIN-carcinoma-metastasis sequence and an early origin of tumour-specific aberrations in PIN areas.     

Oncogenetic tree modeling of human hepatocarcinogenesis

Classical comparative genomic hybridization (CGH) has been used to identify recurrent genomic alterations in human HCC. As hepatocarcinogenesis is considered as a stepwise process, we applied oncogenetic tree modeling on all available classical CGH data to determine occurrence of genetic alterations over time. Nine losses (1p, 4q, 6q, 8p, 9p, 13q, 16p, 16q and 17p) and ten gains (1q, 5p, 6p, 7p, 7q, 8q, 17q, 20p, 20q and Xq) of genomic information were used to build the oncogenetic tree model. Whereas gains of 1q and 8q together with losses of 8p formed a cluster that represents early etiology-independent alterations, the associations of gains at 6q and 17q as well as losses of 6p and 9p were observed during tumor progression. HBV-induced HCCs had significantly more chromosomal aberrations compared to HBV-negative tumors. Losses of 1p, 4q and 13q were associated with HBV-induced HCCs, whereas virus-negative HCCs showed an association of gains at 5p, 7, 20q and Xq. Using five aberrations that were significantly associated with tumor dedifferentiation a robust progression model of stepwise human hepatocarcinogensis (gain 1q → gain 8q → loss 4q → loss 16q → loss 13q) was developed. In silico analysis revealed that protumorigenic candidate genes have been identified for each recurrently altered hotspot. Thus, oncogenic candidate genes that are coded on chromosome arms 1q and 8q are promising targets for the prevention of malignant transformation and the development of biomarkers for the early diagnosis of human HCC that may significantly improve the treatment options and thus prognosis of HCC patients.     

Chromosomal alterations during lymphatic and liver metastasis formation of colorectal cancer

Comparative genomic hybridization (CGH) was used to screen colorectal carcinomas for chromosomal aberrations that are associated with metastatic phenotype. In total, 63 tumor specimens from 40 patients were investigated, comprising 30 primary tumors, 22 systemic metastases (12 liver, 6 brain, and 4 abdominal wall metastases) and 11 lymph node tumors. Using statistical analysis and histograms to evaluate the chromosomal imbalances, overrepresentations were detected most frequently at 20q11.2-20q13.2, 7q11.1-7q12, 13q11.2-13q14, 16p12, 19p13, 9q34, and 19q13.1-19q13.2. Deletions were prominent at 18q12-18q23, 4q27-4q28, 4p14, 5q21, 1p21-1p22, 21q21, 6q16-6q21, 3p12, 8p22-8p23, 9p21, 11q22, and 14q13-14q21. Hematogenous metastases showed more alterations than lymph node tumors, particularly more deletions at 1p, 3, 4, 5q, 10q, 14, and 21q21 and gains at 1q, 7p, 12qter, 13, 16, and 22q. Comparing liver metastases with their corresponding primary tumors, particularly deletions at 2q, 5q, 8p, 9p, 10q, and 21q21 and gains at 1q, 11, 12qter, 17q12-q21, 19, and 22q were more often observed. The analysis suggested that the different pathways of tumor dissemination are reflected by a nonrandom accumulation of chromosomal alterations with specific changes being responsible for the different characteristics of the metastatic phenotype.     

Chromosomal abnormalities associated with neck nodal metastasis in nasopharyngeal carcinoma.

Neck lymphatic metastasis represents the single most important clinical prognostic factor in nasopharyngeal carcinoma (NPC), but underlying genetic mechanisms remain ill defined. In this study 23 samples of primary tumor (PT) and 9 of neck lymph node metastasis (NLNM) obtained from NPC patients were analyzed by comparative genomic hybridization (CGH) coupled with tissue microdissection and degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR). A similar pattern of chromosomal abnormalities was seen in PT and NLNM, the common aberrations were gains on 5p, 12p, 12q and 18p and deletions on 1p, 3p, 9q, 14q, 17p and 16q. However, NLNMs, but not PTs, also exhibited frequent losses on 9p, 16p, 17q, 20q, 21p, 21q and 22q and gains on 8q and 8p. The most frequent unique aberration in NLNMs was loss on 16p, observed in 100% (9/9) NLNMs tested, as well as loss of 20q, observed in 77.8% of tumors tested. For the first time, we report that a gain on 8p and a loss at 20q is common to NLNMs. The analysis furthermore suggests that specific alterations, e.g. losses of 9p, 16p, 7q, 20q, 21p, 21q, 22q and gains on 8q and 8p are associated with NLNM of NPC, and that these alterations may be involved in the onset and/or progression of a metastatic phenotype.     

Genome-wide array-based CGH for mantle cell lymphoma: identification of homozygous deletions of the proapoptotic gene BIM

Mantle cell lymphoma (MCL) is characterized by 11q13 chromosomal translocation and CCND1 overexpression, but additional genomic changes are also important for lymphomagenesis. To identify the genomic aberrations of MCL at higher resolutions, we analysed 29 patient samples and seven cell lines using array-based comparative genomic hybridization (array CGH) consisting of 2348 artificial chromosome clones, which cover the whole genome at a 1.3 mega base resolution. The incidence of identified genomic aberrations was generally higher than that determined with chromosomal CGH. The most frequent imbalances detected by array CGH were gains of chromosomes 3q26 (48%), 7p21 (34%), 6p25 (24%), 8q24 (24%), 10p12 (21%) and 17q23 (17%), and losses of chromosomes 2p11 (83%), 11q22 (59%), 13q21 (55%), 1p21-p22 (52%), 13q34 (52%), 9q22 (45%), 17p13 (45%), 9p21 (41%), 9p24 (41%), 6q23-q24 (38%), 1p36 (31%), 8p23 (34%), 10p14 (31%), 19p13 (28%), 5q21 (21%), 22q12 (21%), 1q42 (17%) and 2q13 (17%). Our analyses also detected several novel recurrent regions of loss located at 1p36, 1q42.2-q43, 2p11.2, 2q13, 17p13.3 and 19p13.2-p13.3, as well as recurrent regions of homozygous loss such as 2p11 (Ig(kappa)), 2q13 and 9p21.3-p24.1 (INK4a/ARF). Of the latter, we investigated the 2q13 loss, which led to identification of homozygous deletions of the proapoptotic gene BIM. The high-resolution array CGH technology allowed for the precise identification of genomic aberrations and identification of BIM as a novel candidate tumor suppressor gene in MCL.     

Genetic changes in colorectal carcinoma tumors with liver metastases analyzed by comparative genomic hybridization and DNA ploidy

BACKGROUND: Liver metastases are found in 10% of primary colorectal malignancies, and they affects the prognosis of patients with colorectal carcinoma. The authors investigated DNA copy number aberrations by using comparative genomic hybridization (CGH) and DNA ploidy alterations by using flow cytometry (FCM) in patients with primary colorectal carcinoma (primary tumors). To determine whether there are characteristic DNA copy number alterations that contribute to liver metastasis, cytogenetic aberrations were examined by CGH and FCM. METHODS: The authors analyzed 35 primary tumors, including 16 primary tumors with liver metastasis, by using CGH and FCM. RESULTS: Increases in DNA copy numbers were detected in 6q (5 of 16 tumors), 7q (6 of 16 tumors), 8q (7 of 16 tumors), 9p (5 of 16 tumors), 13q (8 of 16 tumors), 20p (9 of 16 tumors), and 20q (15 of 16 tumors) in primary tumors with liver metastases. Decreases in DNA copy numbers were found in 17p (5 of 16 tumors), 18p (6 of 19 tumors), 18q (8 of 16 tumors), and 22q (5 of 16 tumors). In contrast, primary tumors without liver metastasis showed gains in chromosome arms 8q (2 of 19 tumors), 13q (2 of 19 tumors), 20p (6 of 19 tumors), and 20q (5 of 19 tumors); however, they showed no gains in 6q or 7q and showed losses in chromosome arms 17p (2 of 19 tumors), 18p (4 of 19 tumors), 18q (6 of 19 tumors), and 22q (5 of 19 tumors). There was a significant difference in the frequency of DNA copy number gains and losses in 6q (P < 0.05), 7q (P < 0.01), 8q (P < 0.05), 13q (P < 0.05), and 20q (P < 0.01), respectively, between primary tumors with and without liver metastases. The differences in the DNA index were not significant between the two groups of primary tumors. CONCLUSIONS: In liver metastases of primary tumors from patients with colorectal carcinoma, a correlation between DNA copy number aberrations and gains of chromosome arms 6q, 7q, 8q, 13q, and 20q is suggested.     

Clinical relevance of genomic aberrations in homogeneously treated high-risk stage II/III breast cancer patients

Little is known about the prognostic impact of chromosome aberrations in breast cancer. The aim of our study was to determine whether genomic aberrations of prognostic relevance can be identified in the context of a clinical study using molecular cytogenetics. Paraffin-embedded tumor samples of 44 patients with high-risk stage II/III breast cancer were analyzed by comparative genomic hybridization. All patients received identical therapy including dose-escalated chemotherapy followed by peripheral blood stem cell transplantation. The most frequent chromosomal aberrations were gains on chromosome arms 17q (24 cases), 1q (21 cases), 8q (17 cases), 20q (13 cases), 6p (9 cases) as well as losses on chromosome arms 13q (25 cases), 11q (20 cases), 5q (11 cases), 6q (11 cases), 9p (10 cases), 18q (10 cases), 8p (9 cases) and 16q (9 cases). In univariate analysis, the correlation with the clinical outcome revealed a higher risk for patients with tumors exhibiting 13q losses and a reduced risk for tumors exhibiting 16q losses (p = 0.020), 6q losses (p = 0.041) and estrogen-receptor positivity (0.051). In multivariate analysis using the Cox model, only the loss of 16q exhibited borderline significance (p = 0.065). These data show that comparative genomic hybridization can be performed in the context of a clinical trial. In our subgroup of high-risk breast cancer patients, chromosomal aberrations were valuable prognostic parameters.     

Patterns of chromosomal imbalances in muscle invasive bladder cancer

Cytogenetic investigations of bladder cancer suggested that development and progression is characterized by specific chromosomal aberrations. In order to identify genetic changes linked to muscle invasive tumors and metastatic growth we analyzed 67 bladder carcinomas (30 pT1 and 37 pT2-4) by means of comparative genomic hybridization (CGH). The most frequent changes were gains of chromosome 1q (54%), 8q (54%), 17q (49%), 2p (30%), 12 (30%), 5p (25%), 3q (24%) and 6p (24%) as well as losses of 11p (43%), 8p (42%), 9p (36%), 11q (34%), 2q, 4q, 5q (30% each), 9q (27%) and 10q (27%). Previously not described amplifications were found at 5p11-p13, 7q21-q31, 9p24 and 17q24-q25. Gains of 3q, 7p, and 18p were markedly more frequent in pT2-4 in comparison to pT1 carcinomas but the difference did not reach statistical significance. Non-metastatic tumors showed more aberrations on average than metastatic carcinomas, although no particular change was found to be predominating in either group. Our data confirm previous findings of strong genetic similarities between minimally and deeply invasive bladder carcinomas but argue for differences between metastatic and non-metastatic disease.     

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.     

Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement of 3q amplification

We applied a combination of molecular cytogenetic methods, including comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization, to characterize the genetic aberrations in a panel of 11 cell lines derived from head and neck squamous cell carcinoma and 1 cell line derived from premalignant oral epithelium. CGH identified recurrent chromosomal losses at 1p, 3p, 4, 8p, 10p, and 18q; gains at 3q, 5p, 8q, 9q, and 14q; and high-level amplification at 3q13, 3q25-q26, 5q22-q23, 7q21, 8q24, 11q13-q14, 12p13, 14q24, and 20q13.1. Several recurrent translocations including t(1;13)(q10;q10), t(13;13)(q10;q10), t(14;14)(q10;q10), i(8)(q10), and i(9)(q10) and breakpoint clusters at 1p11, 1q21, 3p11, 5q11, 5q13, 6q23, 8p11, 8q11, 9p13, 9q13, 10q11, 11q13, 13q10, 14q10, and 15q10 were identified by SKY. There was a good correlation between the number of aberrations identified by CGH and SKY (r = 0.69), and the analyses were both confirmatory and complementary in their assessment of genetic aberrations. Amplification at 3q26-q27 was identified in 42% of cases. Although SKY defined the derivation of 3q gain, the precise breakpoint remained unassigned. Positional cloning efforts directed at the amplified region at 3q26-q27 identified three highly overlapping nonchimeric yeast artificial chromosome clones containing the apex of amplification. The use of these yeast artificial chromosome clones as a probe for fluorescence in situ hybridization analysis allowed a detailed characterization and quantification of the 3q amplification and refinement of unassigned SKY breakpoints.     

Genomic profiling of malignant melanoma using tiling-resolution arrayCGH

Malignant melanoma is an aggressive, heterogeneous disease where new biomarkers for diagnosis and clinical outcome are needed. We searched for chromosomal aberrations that characterize its pathogenesis using 47 different melanoma cell lines and tiling-resolution bacterial artificial chromosome-arrays for comparative genomic hybridization. Major melanoma genes, including BRAF, NRAS, CDKN2A, TP53, CTNNB1, CDK4 and PTEN, were examined for mutations. Distinct copy number alterations were detected, including loss or gain of whole chromosomes but also minute amplifications and homozygous deletions. Most common overlapping regions with losses were mapped to 9p24.3-q13, 10 and 11q14.1-qter, whereas copy number gains were most frequent on chromosomes 1q, 7, 17q and 20q. Amplifications were delineated to oncogenes such as MITF (3p14), CCND1 (11q13), MDM2 (12q15), CCNE1 (19q12) and NOTCH2 (1p12). Frequent findings of homozygous deletions on 9p21 and 10q23 confirmed the importance of CDKN2A and PTEN. Pair-wise comparisons revealed distinct sets of alterations, for example, mutually exclusive mutations in BRAF and NRAS, mutual mutations in BRAF and PTEN, concomitant chromosome 7 gain and 10 loss and concomitant chromosome 15q22.2-q26.3 gain and 20 gain. Moreover, alterations of the various melanoma genes were associated with distinct chromosomal imbalances suggestive of specific genomic programs in melanoma development.     

Non-random chromosomal rearrangements in pancreatic cancer cell lines identified by spectral karyotyping

The molecular events involved in pancreatic cancer are becoming increasingly well characterized, with mutations in the dominant oncogene KRAS and the tumour suppressor genes TP53, CDKN2A and MADH4 being typically observed. However, other genetic abnormalities remain to be identified and molecular cytogenetics may be useful to detect chromosomal loci involved in recurrent rearrangements. We have used spectral karyotyping to characterize cytogenetic aberrations in a panel of 20 human pancreatic carcinoma cell lines and confirmed their identities by dual and triple color fluorescence in situ hybridization. The most common partial or whole-arm gains involved 5p, 7q, 12p, 1q, 7p, 5q, 9p, 9q and 11p. The most common partial or whole-arm losses affected 9p, 11q, 18q, 3p, 2q and 1p, as well as the short arms of the acrocentric chromosomes. Spectral karyotyping allowed us to identify a number of recurrent structural aberrations, all of them unbalanced: most frequently i(5)(p10), del(11)(q23), i(12)(p10), i(1)(q10), del(7)(q22) and del(10)(p11). Spectral karyotyping mapped the complex aberrations occurring in pancreatic cancer cell lines and identified non-random patterns of chromosomal rearrangement. This comprehensive characterization should be useful to direct future investigation. The observation that loss at 11q and gains at 5p with i(5)(p10) and 12p with i(12)(p10) are more frequent changes than previously reported would justify more intensive investigation of these chromosomal regions.