Spectral karyotyping analysis of head and neck squamous cell carcinoma.
OBJECTIVE
The genetic content of head and neck squamous cell carcinomas is ill defined. Spectral karyotyping (SKY) is a new technique that allows the simultaneous detection of all chromosomal translocations by labeling each individual chromosome with different fluorescent agents. In the current study we used SKY to analyze cell lines and a primary tumor derived from head and neck squamous cell carcinomas (HNSCC) to delineate recurrent translocations and breakpoints.


METHODS
Spectral karyotyping analysis of head and neck cancer.


METHODS
Two cell lines (MDA886 and MSK922) and one primary tumor in short-term culture were subjected to metaphase growth arrest with colcemide in their exponential growth phase and fixed onto glass slides. Painting probes for each of the autosomes and the sex chromosomes were generated from flow-sorted human chromosomes using sequence-independent DNA amplification. The probes were labeled using a polymerase chain reaction-based reaction and hybridized to metaphase preparations for 2 days at 37 degrees C. Biotinylated probes were detected using avidin Cy5 and digoxigenin-labeled probes with an anti-mouse digoxigenin antibody followed by goat anti-mouse antibody conjugated to Cy5.5. Chromosomes were counterstained with 4,6-diamino-2-phenyliodole (DAPI), and a minimum of five metaphases were captured and analyzed for each case. Breakpoints on the SKY-painted chromosomes were determined by comparison of corresponding DAPI banding.


RESULTS
Spectral karyotyping analysis revealed a complex pattern of chromosomal abnormalities. A total of 66 translocations were identified in the three cases, with one new recurrent translocation at (der(4)t(4;20)(q35;?)). Nine complex translocations, involving three or more chromosomes, were identified in these cases. Overall, 96 breakpoints were assigned to metaphase chromosomes and another 74 breakpoints could not be assigned. Breakpoints most commonly involved chromosomes in genetic rearrangements were 1, 3, 5, 8, 13, 16, and 17.


CONCLUSIONS
Spectral karyotyping analysis reveals the true complexity of chromosomal aberrations in cell lines derived from head and neck squamous cell carcinomas. The use of SKY, in combination with other techniques, may allow for a more complete assessment of the genetic abnormalities of head and neck cancers and serve as a starting point for gene identification.
Singh
B
B
Laboratory of Epithelial Cancer Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. singhb@mskcc.org
Gogineni
S
S
Goberdhan
A
A
Sacks
P
P
Shaha
A
A
Shah
J
J
Rao
P
P
D002294:Carcinoma, Squamous Cell / Q000235:genetics*; D002869:Chromosome Aberrations / Q000175:diagnosis* / Q000235:genetics*; D025063:Chromosome Disorders; D003582:Cytogenetics / Q000379:methods / Q000592:standards; D015342:DNA Probes; D003703:Demecolcine; D015321:Gene Rearrangement / Q000235:genetics*; D006258:Head and Neck Neoplasms / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods / Q000592:standards; D007621:Karyotyping / Q000379:methods*; D008677:Metaphase / Q000235:genetics; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods* / Q000592:standards; D014178:Translocation, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D003703:Demecolcine