La Función Oblicua de Paul Virilio Y Claude Parent

pipeline

The analysis pipeline developed in this study adds valuable information to the anal- ysis of cancer patient data and the exploration of additional treatment options. The pipeline is already being used to assess the immunogenicity of tumor samples from sev- eral large cohorts of patients with rare cancers, which have not been analyzed in this manner previously. These studies will reveal the potential immunogenicity of those tu- mor entities and provide new insights on whether immunotherapy would be feasible for affected patients. Additionally, the pipeline is being applied to samples from patients with refractory or rare cancers to explore new therapeutic strategies. The obtained de- tailed data about tumor-host interactions may provide information on clinical outcome and help identify patients who are most likely to benefit from immunotherapy. The developed pipelines nicely complement the available genomics pipelines. The pipelines are fast and robust and have been nicely integrated into the clinical setting. From the point where the whole-exome-sequencing data is available, prioritized epitope lists can be generated within 48 hours.

4.4.1

Case studies

We applied our analysis workflow and performed immunomic profiling of an extremely rare metastatic neuroendocrine tumor of the rectum. This analysis, for the first time, provides comprehensive details about the immunogenicity and microenvironment of a neuroendocrine tumor. A manuscript describing this case report is currently in revision for publication in Oncoimmunology.

In this thesis, two case studies of patients with metastatic colorectal cancer have been included to demonstrate and discuss the application of the analysis pipeline (section 3.7). The HLA typing pipeline was used with the whole- exome-sequencing data of

the patients to infer the corresponding HLA genotypes. Subsequently, the epitope pre- diction and selection pipeline was used to select suitable candidates to include in a potential therapeutic vaccine. We then synthesized selected peptides containing HLA class I or class II epitopes, or both, and analyzed them for existing T cell reactivity via ELISpot assays with blood from the corresponding patient. For patient B8G6 short peptides (9mers) only containing the predicted neoepitope, as well as long peptides (29mers), which have the neoepitope centered and additional flanking amino acids on each side, were analyzed in the ELISpot assay. All mutated short peptides showed significantly higher spot counts than the IgG control and it can be considered that these peptides elicit a T cell response. Also, all mutated peptides had higher spot counts compared to the corresponding wildtype peptide. These results indicate that there is a preexisting T cell response against all of the analyzed neoepitopes, and that they elicit stronger T cell responses than their wildtype counterparts. These peptides would be ideal candidates for therapeutic vaccination aiming to boost and reactivate T cells specific for them. Interestingly, when the spot counts of the short mutated peptides are compared to spot counts of the corresponding long peptides, it is striking that the long peptides have lower counts. Surprisingly, the long peptides seem less reactive when compared to the short peptides, although they contain the exact same predicted neoepitope. An explanation for this might be that the long peptides are not processed as expected, and that the predicted neoepitope is not presented properly on HLA. For patient VF77 only long peptides, containing mainly both, predicted HLA class I and class II epitopes, were synthesized and tested. Here, the spot counts of all tested peptides, mutated and wildtype, strongly exceeded the counts of the IgG con- trol, indicating strong T cell reactivity. Also, almost all wildtype peptides had stronger spot counts than the corresponding mutated peptides, although the predicted affinities of the mutated peptides were all stronger. However, a vaccination with mutated pep- tides would lead to an oversaturation with them, so that they may be presented more frequently increasing the chance of specific T cells detecting the presented neoepitopes, boosting the neoepitope-specific T cell response.

For a long time, it was controversially discussed among researchers whether enhance- ment of an existing T cell response or generation of de novo responses is clinically relevant for an effective tumor vaccine (Fritsch et al., 2014). Carreno et al. recently reported that neoepitope vaccination not only can amplify existing CD8+ _{T cell re-}

sponses but also can produce responses that might have been silent prior to vaccination (Carreno et al., 2015; Delamarre et al., 2015). Thus, even if no T cell reactivity was seen for certain neoepitopes in the ELISpot assays, vaccination with them might still generate potent antitumor T cell responses.

If a therapeutic vaccine is going to be administered, including multiple neoepitopes, for both CD4+ _{and CD8}+ _{T cells, is of advantage. In doing so, the likelihood of}

generating an immune response against at least some of the neoantigens increases, and the likelihood of the tumor escaping the immune response by immunoediting decreases. Case studies like these were conducted with several patients. In doing so, a working logistics for the clinical setting was established, and the results provided insights into

the feasibility of the approach. Based on these findings, clinical studies with neoepitope- based vaccines are already in development in Professor J¨ager’s group.