Amplification at 7q22 Targets CDK6 in T-Cell Lymphoma.

Genomic amplification is microscopically visible extrachromosomally as double-minute chromosomes (DMIN) or intrachromosomally as homogeneously stained regions (HSR). Both DMIN and HSR comprise iterated copies of discrete genomic regions (amplicons) effecting copy number increases associated with upregulation of target genes. In cancer cells, these usually comprise oncogenes such as MYC. Cytogenetic analysis of the anaplastic large cell lymphoma (ALCL) cell line SU-DHL-1 revealed an HSR on the long arm region of chromosome 7 (7q). Detailled analysis of this region by fluorescence in situ hybridization (FISH) using tilepath BAC clones identified an amplicon corresponding to 86–95 Mb (NCBI Build 36.1) at 7q22. Expression analysis using reverse transcription (RT)-PCR of candidate oncogenes mapped within the amplicon, comprising DBF4, SRI, AKAP9, GATAD1, CDK6 and PPP1R9A, highlighted cyclin-dependent kinase 6 (CDK6) as a plausible amplification target. With reference to other ALCL cell lines, including SR-786, lacking this amplicon, SU-DHL-1 displays upregulation of CDK6 at both the RNA and protein levels as indicated by semiquantitative RT-PCR, real-time quantitative (RQ)-PCR and immuno-cytochemistry. Another ALCL cell line, L-82, containing a larger amplicon (7q21-31), also showed enhanced CDK6 expression as analyzed by RQ-PCR. For a functional test both SU-DHL-1 and SR-786 cells were treated with rapamycin, an inhibitor of the AKT-pathway which is involved in degradation of CDK-inhibitor p27. In contrast to SR-786, SU-DHL-1 cells are resistant to the effects of rapamycin on both proliferation and G1 cell cycle arrest, implying that CDK6 overexpression may confer proliferative advantage. Copy number analysis of several tumor-relevant genes using a commercial PCR-based approach (MLPA) determined a nearly 5-fold amplification of the CDK6 gene in SU-DHL-1. Interphase FISH confirmed copy number increase in SU-DHL-1 and identified CDK6 amplification in 1/77 ALCL and 4/38 peripheral T-cell lymphoma patient samples, indicating that this genetic event is relatively rare among T-cell lymphomas. Taken together, we have identified an amplicon at 7q22 in T-cell lymphoma cells targeting CDK6 which is an important cell cycle regulator and probably connected to the increased proliferative capacity of these malignant cells. Although CDK6 amplification seems to be infrequent overall, it strengthens the importance of this gene in regulating proliferation among T-cell lymphomas and may, therefore, represent a potential molecular target for therapeutic intervention in these disease entities.