Abstract A85: Passenger genes on tumor-associated amplicons function as tumor rejection antigens
Authors: Berk Michael , Modi Arun , Patel Mona , Wang Li-Xin , Plautz Gregory 
Published: May 2015

Abstract
Tumors frequently contain DNA copy number amplifications (amplicons) that are driven by overexpression of an oncogene but often lead to high expression of neighboring passenger genes. We hypothesized that proteins encoded on amplicons which are present at high frequency in particular types of cancer could serve as shared tumor rejection antigens. The ERBB2 (HER2) amplicon on 17q12-21 has a core region of approximately 280kB in size and is frequently present in multiple copies in 10-15% of breast and gastric cancers. Analysis of data from TCGA breast cancer project and multiple other mRNA expression studies show that ERBB2 overexpression is highly correlated with expression of some, but not all, adjacent genes. From the set of amplicon genes we selected MIEN, GRB7, PGAP3, PNMT, ORMDL3, and GSDMB as candidates for stimulating an immune response because these proteins are minimally expressed in normal tissues. Immunohistochemistry of tissue microarrays showed expression of these proteins in HER2+ breast cancer cells but not in HER2 non-amplified breast tumors. We used the epitope identification programs, NetCTLpan and NetMHCII (Center for Biological Sequence Analysis at the Technical University of Denmark) to identify several candidate peptides with high binding affinity &lt;50 nM to common HLA alleles. In vitro sensitization of normal donor PBMC with several of these peptides led to IFN γ production. Moreover, DCs pulsed with tumor lysate from HER2 amplified tumor cells were able to sensitize T cells against epitopes derived from amplicon proteins indicating that the natural abundance of these antigens in tumors is sufficient for cross-presentation to T cells. The TM15 murine breast cancer line has amplification of a syntenic region surrounding Her2/neu and 50-fold over-expression of pnmt by qPCR. Mice vaccinated with recombinant murine PNMT rejected TM15 tumor challenge without apparent toxicity. These findings indicate that tumor genomic data can be used to identify candidate tumor antigens and epitope prediction can be used to engineer a T cell immune response to break tolerance to these normal proteins. It appears that a therapeutic window can be established to target amplicon-derived antigens due to substantially higher expression in tumor cells relative to normal tissues. Moreover, peptide sequence differences between human and murine proteins at sites of immunogenic epitopes provide an opportunity to generate high-affinity TCR in HLA transgenic mice. Although shared tumor antigens such as HER2 have served as targets for cancer immunotherapy there have been some difficulties with off-target toxicity against normal tissues due to broad expression. Selection of over-expressed passenger genes that exceed a threshold for T cell responsiveness only in tumor but not normal tissues might lead to greater efficacy. This strategy potentially could be applied to other tumor-associated amplicons such as MYCN to search for peptide epitopes from passenger genes which are predicted to have high affinity for common HLA alleles.
Citation Format: Michael Berk, Arun Modi, Mona Patel, Li-Xin Wang, Gregory Plautz. Passenger genes on tumor-associated amplicons function as tumor rejection antigens. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A85.