Abstract 1632: Identification and analysis of EGLN3 as tumor-associated peptide in ccRCC
Authors: Anna Reustle, Moreno Di Marco, Florian Büttner, Stefan Winter, Daniel Kowalewski, Linus Backert, Steffen Rausch, Joerg Hennenlotter, Marcus Scharpf, Falko Fend, Arnulf Stenzl, Jens Bedke, Matthias Schwab, Elke Schaeffeler
Journal: Cancer Research (2017)

Abstract Introduction: Kidney cancer is the 12th most common cancer world-wide with 338.000 newly diagnosed cases in 2012, according to the World Cancer Research Fund International. With around 70-80%, clear cell renal cell carcinomas (ccRCC) make up the vast majority of kidney cancers. Advanced and metastatic ccRCCs are marked by poor survival rates despite the availability of targeted therapies. Currently several immunotherapeutic approaches, including checkpoint-blocking antibodies and dendritic cell vaccines are undergoing clinical investigations. In this study we identified tumor-associated peptides which could be used in cancer vaccines to improve treatment of patients with advanced ccRCC. Methods: To identify suitable peptides for cancer vaccines we set up a tissue collection of paired tumors and normal kidney tissues from 60 ccRCC patients. From these tissues, HLA molecules were isolated by affinity chromatography and bound peptides analyzed by mass spectrometry. HLA ligand profiles of tissues excised from various organ sites of non-ccRCC patients were used to complement the data. Based on these analyses, Egl nine homolog 3 (EGLN3) was selected as a candidate for peptide vaccination and further investigated in our own data cohort comprising the 60 ccRCC patients. Therefore, mRNA and protein expression, as well as DNA methylation data were collected and correlated with HLA ligandomics and clinical data. In addition, we integrated RNA-Seq, DNA methylation, somatic mutation and clinical data from a ccRCC patient cohort from The Cancer Genome Atlas in our analyses. Finally, EGLN3 function was analyzed in 2D and 3D kidney carcinoma cell culture systems. Results: 16 different peptides from EGLN3 were detected in 32 of the 60 analyzed tumor samples, with the most frequent peptide found in tumors of 10 different patients. Only two of the peptides were also detected in normal kidney or other non-ccRCC tissues, rendering EGLN3 peptides promising candidates to be used in ccRCC-directed vaccines. Most of our patients were HLA A*02:01 positive, however using NetMHCpan (http://www.cbs.dtu.dk/services/NetMHCpan/) only one of the peptides identified by HLA ligandomics was also predicted to be a strong binder for that particular HLA molecule. Expression of EGLN3 mRNA was not different in patients with and without HLA-presented peptides. Generally, EGLN3 mRNA expression was strongly induced in ccRCC tumors compared to normal kidney (p&amp;lt;10-6), whereas protein levels remained mainly unchanged in most patients. Functional investigations in different RCC cell culture models indicated involvement of EGLN3 in regulation of apoptosis and cell cycle progression. Conclusion: Advanced ccRCC is marked by poor survival rates due to ineffective therapeutic options. Peptides from EGLN3, as well as other ccRCC-exclusive peptides could represent activators of the immune system to specifically fight ccRCC tumors. The function and targetability of EGLN3 remains to be investigated. Citation Format: Anna Reustle, Moreno Di Marco, Florian Büttner, Stefan Winter, Daniel Kowalewski, Linus Backert, Steffen Rausch, Joerg Hennenlotter, Marcus Scharpf, Falko Fend, Arnulf Stenzl, Jens Bedke, Matthias Schwab, Elke Schaeffeler. Identification and analysis of EGLN3 as tumor-associated peptide in ccRCC [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 1632. doi:10.1158/1538-7445.AM2017-1632