Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification

This study presents an optimized proteogenomic workflow for the discovery of neoantigens (in particular RNA-derived neoantigens) in a pan-cancer cohort and sheds light on the entity-agnostic prevalence of HLA-I peptide presentation of RNA processing events for tumor targeting.
Published in Cancer

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Targeted immunotherapy has become an important avenue for the treatment of cancer. Recent successes of cellular therapies with genetically engineered T cells carrying chimeric antigen receptors (CARs) or transgenic t cell receptors (TCRs)1 are very promising. Moreover, cancer vaccines in combination with immune checkpoint inhibitors showed positive results in early clinical studies2,3. However, all these therapies are dependent on tumor-associated or – preferably – tumor-specific antigens, that we are often lacking.

This is why the discovery of such antigens is paramount for the success of these therapies in the future. Previously, we have developed a pipeline for the discovery of tumor-specific so-called neoantigens in melanoma patients4 that utilizes a proteogenomic approach: whole-exome sequencing (WES) and mass spectrometry (MS)-based analysis of the human leukocyte antigen (HLA) class I immunopeptidome are combined to identify neoantigens that are not only present at the DNA level but are also translated and presented on the tumor cells. With this work we could show for the first time in melanoma patients that MS-based neoantigen identification is feasible in patient-derived solid tumors.

In the current study5, we used our proteogenomic pipeline as a foundation and innovated upon it by adding additional algorithms for their bioinformatical identification. To investigate the importance of neoantigens as a result of RNA aberrations in the tumor, we included tumor transcriptomics to our optimized workflow and tested it in a diverse cohort of patients with different solid tumors (Figure 1). This allowed us to test if our approach of neoantigen discovery works independent of the tumor entity. Our pipeline was able to identify 90 neoantigens in 75% of patients independent of the tumor entity. Interestingly, the vast majority of neoantigens that we discovered were causes of RNA aberrations and showed an unaltered sequence at the respective DNA locus. When focusing more on these RNA-derived neoantigens, we noticed that many of these RNA aberrations showed typical adenosine (A) to inosine (I) (detected as guanosine (G) during sequencing) nucleotide exchanges that are characteristic for A-to-I RNA-editing. Moreover, a portion of mainly RNA-derived neoantigens elicited an immune response of CD8+ T cells in vitro. This might open up a treasure trove of potential tumor targets for immunotherapy in the future that are caused by RNA altering mechanisms. Recent publications also discovered that alternative splicing can result in the formation of neoantigens6 and support this notion.

To validate our neoantigen candidates, we implemented extensive analyses for MS-based peptide sequence verification and assessment of their prevalence in normal tissues. We defined criteria that could guide the selection of candidates for clinical testing and we could validate 32 neoantigens according to these criteria with our pipeline. These criteria could be helpful for clinical application by narrowing down the selection of candidates to a pool of highly promising neoantigens to reduce excessive testing. Most interestingly, the fact that the majority of potential neoantigens were discovered from RNA sources in our study highlights that antigens derived from RNA aberrations could represent new targets for future immunotherapies in cancer.

Figure 1: Schematic of the proteogenomic pipeline for the discovery of neoantigens in a pan-cancer cohort from Tretter et al.5. 32 patients with diverse tumor entities and treatment history were included in the study. Variant calling was performed with MuTect2 on whole exome sequencing (WES) data and with Strelka2 on RNA-seq data from tumor samples. WES data of matched blood samples served as a control and extensive post-processing was performed to exclude common population single nucleotide polymorphisms (SNPs). Human leukocyte antigen (HLA)-I peptides (pHLA-I) were immunoprecipitated from tumor samples and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). By combining the sequencing data with the pHLA-I immunopeptidomics data for each patient, tumor-specific neoantigens were predominantly discovered from RNA sources. Immunogenicity of the potential neoantigens was assessed in vitro with autologous or allogeneic-matched T cells. Extensive validation was performed by verifying the peptide sequence via MS of the synthetic or predicted peptides as well as an assessment of the expression of neoantigen candidates in normal tissues (data obtained from the Genotype-Tissue Expression (GTEx) project7)



  1. Hiltensperger, M. & Krackhardt, A. M. Current and future concepts for the generation and application of genetically engineered CAR-T and TCR-T cells. Front. Immunol. 14 (2023)
  2. Sahin, U. et al. Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 547, 222–226 (2017).
  3. Balachandran, V. P. et al. Phase I trial of adjuvant autogene cevumeran, an individualized mRNA neoantigen vaccine, for pancreatic ductal adenocarcinoma. J. Clin. Oncol. 40, 2516–2516 (2022). ).
  4. Bassani-Sternberg, M. et al. Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry. Nat Commun 7, 13404 (2016).
  5. Tretter, C., et al. Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification. Nat Commun 14, 4632 (2023).
  6. Merlotti, A. et al. Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in patients with lung cancer. Sci. Immunol. 8, eabm6359 (2023).
  7. Lonsdale, J. et al. The genotype-tissue expression (GTEx) project. Nat. Genet. 45, 580–585 (2013).

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