Tumor genomes can be highly rearranged and non colinear with the host genome.Recurrent genome rearrangements involve genes that are increasingly targeted by anti-tumor therapeutics.Current technologies for studying tumor genomes do not determine their structure and relate it to the underlying sequence.Thus, the role of translocations and inversions in solid tumors is poorly understood.Even the structural organization of amplicons remains largely enigmatic.End Sequence Profiling (ESP) is a sequence-based method for directly determining the structure of tumor genomes, and for cloning all types of rearrangements en masse.ESP begins with the construction of a tumor BAC library.BAC clones are then end-sequenced, and mapped onto the "reference" genome sequence.This process reveals structural aberrations such as copy number changes, translocations, and inversions and identifies BAC clones spanning the genome breakpoints that can be sequenced to identify breakpoint(s) and fusion genes.We applied ESP to the breast cancer cell lines BT474, MCF7, and SKBR3, primary tumors of the brain, breast, ovary, and a metastatic prostate tumor.ESP provides direct evidence for packaging of amplified DNA from multiple loci, extensive rearrangements of amplicons, and for independent mechanisms of amplification within a single tumor genome.This enables the formal testing of hypotheses on tumor genome evolution and function.In addition, ESP can be carried out on tumor transcriptomes for large-scale identification of fusion transcripts.We have demonstrated this by analyzing full length enriched normalized eDNA libraries from MCF7, LnCaP, and a primary brain tumor.Multiple tumor-specific transcripts were identified and analyzed.Preliminary data show that blocking the expression of one of the novel MCF7 transcripts inhibits growth of the MCF7 breast cancer cell line.Being sequence-based, ESP is inherently integrative, thus bringing the power of genetic analysis to interpretation of complex expression microarray and proteomic data.Further, in vitro and in vivo functional studies are possible using tumor BACs for transfections and transgenics, respectively.