Chromosomal alterations in breast cancer revealed by multicolour fluorescence in situ hybridization.

The aim of this study was to characterise cytogenetically, breast cancer cell lines and primary tumours to identify chromosomal regions of interest in breast cancer. Multicolour fluorescence in situ hybridization (MFISH) and comparative genomic hybridization (CGH) were used to karyotype five established breast cancer cell lines and two short-term primary tumour cultures. Chromosome 8 was identified as a frequent target for aberrations in all cell lines and one primary culture by MFISH and CGH. CGH identified frequent gains of 1q (all samples) and 14q (all cell lines) and deletion of 22q (all samples). MFISH revealed a t(9;17) translocation in both primary tumours and the T47D cell line. MFISH analysis of the cell lines revealed a significant number of translocations previously unidentified in other studies using similar techniques, highlighting the necessity of utilising data from both primary cultures and established cell lines when investigating complex cytogenetic aberrations using MFISH and CGH.

Can a genetic signature for metastatic head and neck squamous cell carcinoma be characterised by comparative genomic hybridisation?

Survival from head and neck squamous cell carcinoma (HNSCC) has remained static for the last 20 years. The development of lymph node metastasis (LNM) significantly reduces the 5-year survival rate, thus the ability to identify tumours with the potential to metastasise would allow more aggressive treatment regimes to be directed at these patients regardless of negative clinical and radiological findings at the time of presentation. Comparative genomic hybridisation (CGH) can identify chromosomal aberrations that may lead to metastasis. DNA from 23-paired specimens of primary tumour (PT) and LNM were analysed. Nonrandom copy number changes were identified in all paired samples. Similar numbers of aberrations were identified on PT and LNM samples. The most common aberrations were 3q (90%), 8q (65%), 1q (50%), 5p (43%), 2q (41%) and 11q (41%) and deletions 3p (57%), 1p (54%), 4p (48%), 13q (48%), 11q (41%) and 10q (37%). A number of differences were also detected. No aberration was found to be preferentially associated with the LNM, although gains on 6q (48 vs 22%) and 22q (26 vs 9%) were found at higher frequencies. Clonality studies demonstrated that LNM develop from the dominant population of cells in the PT. These results were compared with two similar publications. No combination of chromosomal aberrations, as detected by CGH, was associated with metastatic progression in HNSCC.

Chromosomal analysis of non-small-cell lung cancer by multicolour fluorescent in situ hybridisation.

The cytogenetic abnormalities in non-small-cell lung cancer remain elusive due primarily to the difficulty in obtaining metaphase spreads from solid tumours. We have used the molecular cytogenetic techniques of multicolour fluorescent in situ hybridisation (M-FISH) and comparative genomic hybridisation (CGH) to analyse four primary non-small-cell lung cancer samples and two established cell lines (COR-L23 and COR-L105) in order to identify common chromosomal aberrations. CGH revealed regions on 5p, 3q, 8q, 11q, 2q, 12p and 12q to be commonly over-represented and regions on 9p, 3p, 6q, 17p, 22q, 8p, 10p, 10q and 19p to be commonly under-represented. M-FISH revealed numerous complex chromosomal rearrangements. Translocations between chromosomes 5 and 14, 5 and 11 and 1 and 6 were observed in three of the six samples, with a further 14 translocations being observed in two samples each. Loss of the Y chromosome and gains of chromosomes 20 and 5p were also frequent. Chromosomes 4, 5, 8, 11, 12 and 19 were most frequently involved in interchromosomal translocations. Further investigation of the recurrent aberrations will be necessary to identify the specific breakpoints involved and any role they may have in the aetiology, diagnosis and prognosis of non-small-cell lung cancer.

Prognostic value of genomic alterations in head and neck squamous cell carcinoma detected by comparative genomic hybridisation.

A total of 45 primary head and neck squamous cell carcinomas were analysed by comparative genomic hybridisation to identify regions of chromosomal deletion and gain. Multiple regions of copy number aberration were identified including gains affecting chromosomes 3q, 8q, 5p, 7q, 12p and 11q and deletion of material from chromosomes 3p, 11q, 4p, 5q, 8p, 10q, 13q and 21. Kaplan-Meier survival analysis revealed significant correlations between gain of 3q25-27 and deletion of 22q with reduced disease-specific survival. In addition, gain of 17q and 20q, deletion of 19p and 22q and amplification of 11q13 were significantly associated with reduced disease-free survival. A Cox proportional hazards regression model identified deletion of 22q as an independent prognostic marker. The data presented here provide further evidence that the creation of a genetically based tumour classification system will soon be possible, complementing current histopathological characterisation.

Chromosomal alterations in small cell lung cancer revealed by multicolour fluorescence in situ hybridization.

Small cell lung cancer (SCLC) is a major cause of cancer related morbidity and mortality. Karyotypic studies have revealed numerous chromosomal aberrations in most SCLC however, classical G-banding analysis is unable to fully characterise complex marker chromosomes. Recent developments in molecular cytogenetics now allow accurate identification of the chromosomal components of complicated rearrangements. We have applied the technique of multicolour fluorescence in situ hybridization (M-FISH) in combination with comparative genomic hybridization (CGH) to the analysis of 5 SCLC cell lines and 1 primary tumour specimen to characterise the chromosomal abnormalities. CGH analysis identified many similarities between specimens, with frequent DNA copy number decreases on chromosomes 3p, 5q, 10, 16q, 17p and frequent gains on 3q, 1p, 1q and 14q. In contrast, M-FISH analysis revealed a large number of structural abnormalities, with each specimen demonstrating an individual pattern of chromosomal translocations. Forty different translocations were identified with the vast majority (39) being unbalanced. Chromosome 5 was the most frequently rearranged chromosome (9 translocations) followed by chromosomes 2, 10 and 16 (6 translocations each). Further investigation of these frequently involved chromosomes is warranted to establish whether consistent break points are involved in these translocations, causing dysregulation of specific genes that are crucial for tumour progression and secondly to identify the affected genes.

Karyotypic variation between independently cultured strains of the cell line MCF-7 identified by multicolour fluorescence in situ hybridization.

The breast cancer cell line MCF-7 is a widely used model in breast cancer research however a number of conflicting reports have been published regarding its biological properties. We hypothesised that there will be significant in vitro mutation and genotypic evolution over time in this cell line. To assess the genetic divergence of MCF-7 at the chromosomal level, we analysed MCF-7 cell lines grown independently at three different laboratories using M-FISH and CGH. In addition, MCF-7 cells from our own laboratory were also analysed at two time points 18 months apart. Several common chromosomal translocations were identified in all variants of the cell lines. In addition, a significant number of unique abnormalities were identified, characterising each of the variants studied. Genotypic differences between cell lines grown independently in different laboratories would significantly alter the phenotypic characteristics of each cell line rendering biological properties inconsistent between laboratories.

Genetic analysis of head and neck squamous cell carcinoma and surrounding mucosa.

OBJECTIVE: Comparative genomic hybridization was performed on head and neck squamous cell carcinoma and surrounding mucosa to determine whether common chromosomal aberrations could be detected that would predispose an individual to developing a second primary tumor. DESIGN: Biopsy specimens were taken from 19 patients with squamous cell carcinoma of the head and neck, 3 samples from each person: 1 specimen from the tumor site and 1 each from 1 and 5 cm from the macroscopic tumor margin. Samples were snap frozen in liquid nitrogen. A portion of each distant sample and tissue taken from immediately adjacent to the site of the tumor specimen were sectioned and stained with hematoxylin-eosin, either to search by light microscopy for tumor cells or signs of dysplasia in the distant samples, or to determine whether the tumor specimen had substantial non-tumor cell content. Tissue adjacent to the tumor biopsy site was used because the biopsy specimens were relatively small. Comparative genomic hybridization was performed on all samples. SUBJECTS: Nineteen patients with newly diagnosed carcinomas of the head and neck. RESULTS: The tumor biopsy specimens showed no substantial nontumor cell content, and the distant specimens were all histologically normal. The tumors showed multiple mutations: mean (SD) number of deletions, 5.4 (4.3); amplifications, 5.2 (4.6). Deletion of chromosome 3p was seen in 13 of 19 cases and was associated with amplification of 3q in 10 cases. No mutations were seen in the distant biopsy specimens. CONCLUSIONS: Frequently occurring chromosomal aberrations were seen in the tumor cells, suggesting a key role for these mutations in tumor development. Screening histologically normal upper aerodigestive tract mucosa with comparative genomic hybridization does not provide information on early genetic events that predispose a patient to developing a second primary tumor.

Multiple cell populations in colorectal carcinomas: analysis by 3-colour fluorescence in situ hybridization.

A three-colour FISH approach using centromere-specific DNA probes was used to analyse the number of chromosomes 7, 17 and 18 found within individual tumour cells and the results were correlated with total DNA ploidy determined by image analysis. FISH analysis showed a high level of heterogeneity in the majority of tumour samples with only 7 out of 44 samples having a single chromosome profile occurring in greater than 40% of the cells. Analysis of the modal chromosome number showed that a diploid 2/2/2 profile for chromosome 7, 17 and 18 respectively occurred most commonly. The DNA ploidy index for biopsies with a 2/2/2 profile varied between 0.93-2.06. No gain of chromosome was observed in the adenoma samples or Dukes A tumours but a loss of chromosome 18 was seen in 50% of these early carcinomas. A modal chromosome profile of 4/2/2 was commonly found in Dukes B and C tumours suggesting that endoreduplication with the relative loss of chromosome 17 and 18 is common in advanced cancers. The DNA ploidy index for the more advanced tumours was also variable but significantly higher than that found in the early tumours and non-tumour controls. In conclusion, this work shows that tumours are highly heterogeneous and that the majority of tumours consist of a large number of cell sub-populations with respect to the expression of chromosomes 7, 17 and 18.