Glioma growth is often accompanied by a hypoxic microenvironment favorable for the induction and maintenance of the glioma stem cell (GSC) phenotype. Due to the paucity of cell models of Isocitrate Dehydrogenase 1 mutant (IDH1mut) GSCs, biology under hypoxic conditions has not been sufficiently studied as compared to IDH1 wildtype (IDH1wt) GSCs. We therefore grew well-characterized IDH1mut (n = 4) and IDH1wt (n = 4) GSC lines under normoxic (20%) and hypoxic (1.5%) culture conditions and harvested mRNA after 72 h. Transcriptome analyses were performed and hypoxia regulated genes were further analyzed using the expression and clinical data of the lower grade glioma cohort of The Cancer Genome Atlas (LGG TCGA) in a confirmatory approach and to test for possible survival associations. Results show that global expression changes were more pronounced in IDH1wt than in IDH1mut GSCs. However, when focusing on known hypoxia-regulated gene sets, enrichment analyses showed a comparable regulation in both IDH1mut and IDH1wt GSCs. Of 272 significantly up-regulated genes under hypoxic conditions in IDH1mut GSCs a hypoxia-related survival score (HRS-score) of five genes (LYVE1, FAM162A, WNT6, OTP, PLOD1) was identified by the Least Absolute Shrinkage and Selection Operator (LASSO) algorithm which was able to predict survival independent of age, 1p19q co-deletion status and WHO grade (II vs. III) in the LGG TCGA cohort and in the Rembrandt dataset. Altogether, we were able to identify and validate a novel hypoxia-related survival score in IDH1mut GSCs consisting of five hypoxia-regulated genes which was significantly associated with patient survival independent of known prognostic confounders.

No abstract

No abstract

The prognosis of colorectal carcinoma (CRC) patients is dependent on the establishment of distant metastasis, which have been shown to arise from a small population of long term tumor initiating cells (LT-TIC). Another major prognostic parameter is the T cell infiltrate in the primary tumor of CRC patients, suggesting that tumor specific T cell responses occur and that the T cell mediated immune surveillance might play an important role in controlling tumor relapse, metastasis and response to treatment. Based on these observations we hypothesize that T cell responses against antigens expressed on TIC might trigger more efficient immune surveillance. So far little is known about the target antigens of the spontaneous T cell responses in CRC patients. In order to identify those antigens in a systematic and unbiased manner, we established a two-dimensional protein separation technique (PF2D). This technique allowed to fractionate lysates from primary and metastatic tumor tissue and to select the fractions which were recognized by the patient's T cell repertoire, as assessed by IFN-γ ELISpot assays. The reactive fractions were further characterized with mass spectrometry (MS) and candidate proteins were verified by use of synthetic peptides in ELISpot. As a result, we have identified 21 novel CRC-associated target antigens of spontaneous T cell responses. We have shown in a cohort of 20 patients that the newly identified target antigens not only triggered responses more frequently than “canonical” tumor antigens that are commonly used for immunotherapy (up to 50% of the tested patients), but the frequency of the T cells specific for the novel tumor associated antigens (TAA) was significantly higher. Furthermore, as shown by gene expression analysis some of the newly identified TAAs were selectively overexpressed on LT-TIC of CRC, and by means of PF2D of LT-TIC enriched spheroid cultures and subsequent MS of the immunogenic fractions, we have demonstrated that they represent target antigens of patients' endogenous T cell responses in all of the tested LT-TIC cultures. In addition, we have identified 30 amino acids long sequences of the these TAA, which elicit CD8+ or CD4+ T cell responses and we are currently working on identification of T cell epitopes and generation of T cell clones specific for these antigens. These would be further used to test the hypothesis that LT-TIC specific T cell clones have the capability to reduce the metastatic potential and tumorigenicity of TIC enriched spheroids in vitro and in vivo. In conclusion we have shown that novel set of antigens expressed in a therapeutically relevant subset of CRC cells, can be highly immunogenic and might serve as better targets for immune monitoring and could be utilized for more efficient T cell based immunotherapy. Citation Format: Slava Stamova, Christoph Schlude, Saskia Rösch, Christel Herold-Mende, Taronish D. Dubash, Claudia R. Ball, Hanno Glimm, Martin A. Schneider, Philipp Beckhove. Identification of novel tumor associated antigens in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2896. doi:10.1158/1538-7445.AM2014-2896