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Michael Wessolly,1,2,* Susann Stephan-Falkenau,3,* Anna Streubel,3 Robert Werner,3 Sabrina Borchert,1,2 Sergej Griff,3 Elena Mairinger,1 Robert FH Walter,1,4 Torsten Bauer,5 Wilfried EE Eberhardt,4,6 Torsten G Blum,5 Kurt W Schmid,1 Jens Kollmeier,5 Thomas Mairinger,1,* Fabian D Mairinger1,2,* 1Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2German Cancer Consortium (DKTK), Partner Site University Hospital Essen, Essen, Germany; 3Department of Tissue Diagnostics, Helios Klinikum Emil Von Behring, Berlin, Germany; 4Ruhrlandklinik, West German Lung Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 5Lungenklinik Heckeshorn, Helios Klinikum Emil Von Behring, Berlin, Germany; 6Department of Medical Oncology, West German Cancer Center, University, Hospital Essen, Essen, Germany*These authors contributed equally to this workCorrespondence: Michael Wessolly Research Group: Translational Cancer Research, Institute for PathologyUniversity Hospital Essen, Hufelandstraße 55, Essen D-45147, GermanyTel +49 201 723 – 83035Fax +49 201 723 – 3880Email michael.wessolly@uk-essen.deBackground: Immune checkpoint inhibition, especially the blockade of PD-1 and PD-L1, has become one of the most thriving therapeutic approaches in modern oncology. Immune evasion caused by altered tumor epitope processing (so-called processing escapes) may be one way to explain immune checkpoint inhibition therapy failure. In the present study, we aim to demonstrate the effects of processing escapes on immunotherapy outcome in NSCLC patients.Patients and Methods: Whole exome sequencing data of 400 NSCLC patients (AdC and SCC) were extracted from the TCGA database. The ICB cohort was composed of primary tumor probes from 48 NSCLC patients treated with nivolumab. Mutations were identified by targeted amplicon-based sequencing including hotspots and whole exomes of 22 genes. The effect of mutations on proteasomal processing was evaluated by deep learning methods previously trained on 1260 known MHC-I ligands. Cox regression modelling was used to determine the influence on overall survival.Results: In the TCGA cohort, processing escapes were associated with decreased overall survival (p= 0.0140). In the ICB cohort, patients showing processing escapes in combination with high levels of PD-L1 (n=8/48) also showed significantly decreased overall survival, independently of mutational load or PD-L1 status.Conclusion: The concept of altered epitope processing may help to understand immunotherapy failure. Especially when combined with PD-L1 status, this method can be used as a biomarker to identify patients not suitable for immunotherapy.Keywords: massive parallel sequencing, NSCLC, immunotherapy, epitope, processing escape, deep learning

Robert Fred Henry Walter,1,2 Saskia Roxanne Sydow,3 Erika Berg,3 Jens Kollmeier,4 Daniel Christian Christoph,5,6 Sandra Christoph,7 Wilfried Ernst Erich Eberhardt,1 Thomas Mairinger,4 Jeremias Wohlschlaeger,2 Kurt Werner Schmid,2 Fabian Dominik Mairinger2 1Ruhrlandklinik, West German Lung Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 3Institute of Pathology, Charité Universitaetsmedizin, Berlin, Germany; 4Institute of Pathology, Helios Klinikum Emil Von Behring, Berlin, Germany; 5Department of Oncology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 6Department of Internistic Oncology, Kliniken Essen Mitte, Essen, Germany; 7Department of Bone Marrow Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, GermanyCorrespondence: Robert Fred Henry WalterUniversity Hospital Essenm, Institute of Pathology, Hufelandstraße 55, Essen D-45147, GermanyTel +49 0 201/723 84828Fax +49 0 201/723 3880Email robert.walter@ruhrlandklinik.uk-essen.deBackground: Bortezomib is an approved proteasome inhibitor for the treatment of certain lymphoma subtypes. Two clinical trials investigated bortezomib in patients with malignant pleural mesothelioma (MPM) and failed to improve outcome. We present a potential explanation for this event.Methods: 171 patients with MPM were analyzed for their mRNA expression of proteasomal subunits PSMA1, PSMA5, PSMB1, PSMB2, PSMB4 and PSMB5 via qPCR (n=84) or sequencing (n=87 TCGA/cBioPortal data set “Mesothelioma”). Outcome and subunit expression were correlated. Four mesothelial and one fibroblast cell line were treated with bortezomib and cisplatin. Cellular response was measured after 0, 6, 12, 24, 48 and 72 hrs. Enzyme activity of proteasomal subunits was assessed via functional enzyme activity assays.Results: Patients with MPM presented with elevated expression of proteasomal subunits compared to benign controls (p