Spectral karyotyping analysis of head and neck squamous cell carcinoma

Objectives/Hypothesis: The genetic content of head and neck squamous cell c arcinomas is ill defined. Spectral karyotyping (SKY) is a new technique tha t allows the simultaneous detection of all chromosomal translocations by la beling each individual chromosome with different fluorescent agents. In the current study we used SKY to analyze cell lines and a primary tumor derive d from head and neck squamous cell carcinomas (HNSCC) to delineate recurren t translocations and breakpoints. Study Design: Spectral karyotyping analys is 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 gla ss slides. Painting probes for each of the autosomes and the sex chromosome s were generated from flow-sorted human chromosomes using sequence-independ ent DNA amplification. The probes were labeled using a polymerase chain rea ction-based reaction and hybridized to metaphase preparations for 2 days at 37 degreesC. Biotinylated probes were detected using avidin Cy5 and digoxi genin-labeled probes with an anti-mouse digoxigenin antibody followed by go at 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 ch romosomes were determined by comparison of corresponding DAPI banding. Resu lts. Spectral karyotyping analysis revealed a complex pattern of chromosoma l 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 identifi ed in these cases. Overall, 96 breakpoints were assigned to metaphase chrom osomes and another 74 breakpoints could not be assigned. Breakpoints most c ommonly involved chromosomes in genetic rearrangements were 1, 3, 5, 8, 13, 16, and 17. Conclusions. Spectral karyotyping analysis reveals the true co mplexity of chromosomal aberrations in cell lines derived from head and nec k squamous cell carcinomas. The use of SKY, in combination with other techn iques, may allow for a more complete assessment of the genetic abnormalitie s of head and neck cancers and serve as a starting point for gene identific ation.