Abstract 1559: Proteogenomic analysis of alternative splicing in colorectal adenoma-to-carcinoma progression

Abstract Background Early diagnosis of colorectal cancer (CRC) and identification of its precursor lesions (adenomas) is crucial in reducing CRC mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test that detects human protein hemoglobin. Although FIT is beneficial in its current form with a sensitivity of ~65% for detection of CRC and ~27% for adenomas, its performance is still suboptimal and needs to be further improved. Adenoma-to-carcinoma progression is accompanied by alternative splicing, which results in expression of tumor-specific protein variants. These may yield novel biomarkers suitable for improving detection of progressive adenomas and CRCs. Aim We aim to identify novel biomarkers to improve early detection of CRC. Materials and methods RNA was isolated from 3D organoid cultures derived from 5 adenomas and 4 CRC tissues. RNA and proteins were isolated from 18 healthy human colon tissues, 30 adenomas and 30 CRCs. Samples were analyzed by RNA sequencing (Illumina) and in-depth tandem mass spectrometry proteomics (QExactive). For both organoid- and tissue-datasets differential splicing analysis was performed on RNA level to enrich the sequence database, against which mass spectra were searched, with predicted protein isoforms. Results Comparative splicing analysis between CRC and adenoma organoids revealed ~90 differentially spliced genes, yielding candidate biomarkers from epithelial origin. In the tissues, differential splicing analysis between CRCs and controls and between CRCs and adenomas identified over 1000 of splice variants. These include known alternatively spliced genes involved in cancer such as CD44 and VEGFA and a number of candidates overlapping with the isoforms derived from the organoids. Proteomics analysis revealed that approximately 150 of the splice variants were expressed on protein level. Conclusion and Discussion We have confirmed that adenoma-to-carcinoma progression is accompanied by aberrant splicing. Analysis of the organoid cultures allowed us to identify gene isoforms from (neoplastic) epithelial origin. Tissue analysis yielded tumor-specific splice variants that represent novel protein candidate biomarkers for early detection of CRC. The diagnostic performance of these splice variant proteins will be validated in series of stool and FIT samples. Citation Format: Malgorzata A. Komor, Thang V. Pham, Sander R. Piersma, Anne S. Bolijn, Tim Schelfhorst, Pien M. Delis-van Diemen, Marianne Tijssen, Annemieke C. Hiemstra, Meike de Wit, Beatriz Carvalho, Gerrit A. Meijer, Connie R. Jimenez, Remond J. Fijneman. Proteogenomic analysis of alternative splicing in colorectal adenoma-to-carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1559. doi:10.1158/1538-7445.AM2017-1559