Abstract Background: Of all possible sources of antigens for cancer vaccines, frameshift (FS) peptides are attractive in that they may represent neo-antigens. FS peptides may be induced by insertions/deletions or by chimeric transcripts that result from mis-splicing, chromosomal translocations or trans-splicing of mRNA. Aim: In order to discover FS neo-antigens, we have taken a bioinformatical approach at identifying tumor specific, neo-peptides that are generated when chimeric transcripts are translated. Specifically we have focused our attention on identifying chimeric transcripts that create a frameshift when the exons of two different genes are joined. As a result of this gene fusion, approximately 50% of the time a frameshift occurs thus creating a neo-peptide. Method: In order to quickly identify these types of transcripts that are specific to cancer and not present in normal tissue, we have developed an algorithm that can be used to screen publically available databases and high-throughput sequencing data. As proof of principle we have applied this algorithm to the NCBI Expressed Sequence Tag database that contains cDNA sequencing information for normal and cancer tissues. Results: Based on this approach, we have identified and validated 35 chimeric transcripts that are present in breast cancer cell lines and in primary breast tumors. In addition, 9 chimeric transcripts are also present in seven different pancreatic cancer cell lines. Of the 35 chimeric transcripts, 11 chimeric transcripts produce frameshift peptides that are at least nine amino acids or longer and are predicted by SYFPEITHI and Rankpep to be immunogenic and capable of being presented in the most frequent human class I MHC alleles. Moreover, our analyses also identified chimeric transcripts that have already been confirmed by 454 sequencing for breast and prostate cancer. Discussion: Defining the chimeric transcriptome in both healthy and cancer patients could be critical for the development of new therapeutic agents. Of more interest to us, is their potential contribution to a prophylactic cancer vaccine. (This work is supported by a DoD Innovator Award and a Keck Foundation grant to SAJ) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4767.