Abstract #5606: Analysis of genomic imbalances to detect gene fusions in cancer
Authors: Nallasivam Palanisamy, Kalpana Ramnarayanan, Thomas Choudary Putti, Kyew Htet Hlaing, Ram Shanker Mani, Chandan Kumar Sinha, Sooryanarayana Varambally, Patrick Tan, Arul Chinnaiyan
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

High level aneuploidy and complex structural rearrangements are the hallmark of solid tumors. Progress on the identification of tumor causing recurrent aberrations within the complex genome is hampered by technical limitations of the conventional cytogenetic and molecular methods. In our quest to identify a molecular biomarker for breast cancer, we used a new approach based on high-resolution oligonucleotide array based comparative genomic hybridization (244K, Agilent) to analyze genomic imbalances to isolate novel fusion genes in MCF7. A systematic analysis of unbalanced copy number changes identified many copy number transition regions (CNTR) or breakpoints within known genes and intergenic regions at an unprecedented resolution of <6kb within genes and <15kb in intergenic regions. We identified 83 loci containing breakpoints affecting known genes and 41 loci in intergenic regions. Gene fusions were identified by further characterization of breakpoints by 5\#8217; and 3\#8217; RACE PCR amplification from the exons close to the breakpoint regions. We validated 48 of the 83 genes containing breakpoints and identified 7 novel gene fusions not previously reported in MCF7 and BT474 cell lines (RPS6KB1/TMEM49; RPS6KB1/EAP30; RPS6KB1/Novel EST; ARGEF2/SULF2; MTAP/Novel EST; ATXN7/Novel EST; BCAS3/ATXN7) from the amplified regions at 17q23 and 20q13. We also identified a novel gene fusion (MTAP/novel EST) by analyzing the boundary of a homozygous deletion region boundary at 9p21. Further validation of the gene fusions identified from MCF7 for their recurrence in primary breast tumors and cell lines we identified ARFGEF2/SULF2 gene fusion in 6 primary tumors (6/35) from an Asian cohort and a promiscuous rearrangement of RPS6KB1 with three different 3\#8217; partner genes in three different samples -RPS6KB1/TMEM49 (MCF7), RPS6KB1/EAP30 (BT474) and RPS6KB1/novel EST (Primary breast tumor). Fusion genes were further confirmed by fluorescence in situ hybridization (FISH). Further integrative analysis of gene expression and copy number change of genomic amplification revealed additional rearranged genes within amplification without obvious breakpoint identified by aCGH. These data suggests that the formation of high level amplification by breakage-fusion-bridge cycle may be the possible mechanism leading to the formation of gene fusions within amplifications and further it provides evidence to show that the genomic amplifications are rich source for identification of gene fusions. We extend this approach to recurrent regions of genomic amplifications in breast cancer ( 17q12, 17q23 and 20q13) to detect recurrent gene fusions in breast cancer, if any. Overall we present a novel approach to discover gene fusions by analyzing the genomic imbalances in solid tumors. 

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5606.