Abstract B24: A systematic search for cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets.

Background: A wide range of therapeutic cancer vaccines that target diverse tumor-associated antigens are currently been evaluated in early phase clinical trials. However the effectiveness of cancer vaccines is so far limited, potentially due in part to the limited availability of candidate targets with broad expression in a tumor type. Cancer/testis (CT) antigens are a major target of cancer vaccines, because CT antigens are specifically expressed in immune-privileged testis and placenta, and exhibit aberrant expression in various types of cancer, resulting in high immunogenicity with no self-tolerance and reduced adverse effects. However expression of particular antigens is limited to a subset of tumors of a given type. Thus there is a need to identify antigens with complementary expression patterns for more effective therapeutic intervention. Methods: Gene expression data of BioGPS, and DNA methylation and RNA-seq data of TCGA lung cancer samples were downloaded. All lung cancer cell lines were cultured in RPMI1640 containing 10% FBS. A total of 83 pairs of treatment naive lung adenocarcinoma tissues and adjacent non-tumor lung tissues had been obtained earlier with informed consent. Gene expression data for cell lines and tissues were obtained using the Illumina Human WG-6 v3.0 Expression BeadChips. For mass spectrometric analysis, 38 non-small cell lung cancer (NSCLC) cell lines were cultured according to the standard SILAC protocol. The same batch of cells was used for analysis of whole cell extract, conditioned media and cell surface proteins. Tissue microarrays comprised 463 surgically resected NSCLC tumor specimens collected under an IRB protocol. Monoclonal anti-mouse VCX3A antibody was used for Western blot and immunohistochemistry analysis. Results: With mining the public database BioGPS, we identified VCX/Y gene family as novel CT antigens in lung cancer. Our findings also suggested epigenetic regulation of VCX/Y gene expression, and potential oncogenic roles of VCX/Y genes. Expression of one of the VCX/Y family genes, VCX3A, was further confirmed at the protein level in ~20% of lung adenocarcinoma and ~35% of squamous cell carcinoma tumors. We further examined gene expression patterns of CT antigens to determine whether a panel could be assembled with broad representation in lung adenocarcinoma. Of 255 known CT antigens, 109 CT antigens were profiled at both mRNA and protein levels in 38 NSCLC cell lines. mRNA expression levels of 10 CT antigens were significantly and positively correlated with protein expression levels. We examined mRNA expression profiles of these 10 CT antigens, together with VCX and MAGEA3 which is a current target for vaccine immunotherapy, in 83 lung adenocarcinoma tumors. We selected XAGE1, VCX, IL13RA2, and SYCE1 to form a CT antigen panel and compared mRNA expression levels of these four genes between tumor and adjacent non-tumor tissue. One or more of the CT antigens in the panel were overexpressed in 66 (79.5%) of lung adenocarcinomas. Conclusions: The present study identifies for the first time a novel CT antigen VCX/Y gene family in lung cancer. A CT antigen panel was found to provide a broad coverage of lung adenocarcinoma tumors tested, suggestive of a potential for more effective immunotherapy through targeting the combined antigen panel compared to a single antigen target. Citation Format: Ayumu Taguchi, Allen D. Taylor, Jaime Rodriguez, Muge Celiktas, Hui Liu, Xiaotu Ma, Qing Zhang, Chee-Hong Wong, Alice Chin, Luc Girard, Carmen Behrens, Wan Lam, Stephen Lam, John D. Minna, Ignacio I. Wistuba, Adi F. Gazdar, Samir M. Hanash. A systematic search for cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B24.