RNA Dysregulation: An Expanding Source of Cancer Immunotherapy Targets
Authors: Yang Pan, Kathryn E. Kadash-Edmondson, Robert Wang, John Phillips, Song Liu, Antoni Ribas, Richard Aplenc, Owen N. Witte, Yi Xing
Journal: Trends in Pharmacological Sciences (2021)

Discovery of optimal tumor antigens (TAs) is key for developing targeted immunotherapies with safe and effective antitumor immunity. Novel sources of TAs (beyond somatic mutations) are needed to expand the scope of current therapies.Dysregulation of RNA processing reshapes the cancer transcriptome and proteome by changing the abundance and diversity of gene products. Antigens arising from RNA dysregulation are potential cancer immunotherapy targets.Recent advances in high-throughput technologies and rapid accumulation of multiomic cancer profiling data in public repositories have provided opportunities to systematically characterize RNA dysregulation in cancer.To comprehensively discover and prioritize TAs derived from RNA dysregulation, we propose an integrated multiomic and big data strategy incorporating state-of-the-art experimental and computational tools as well as large-scale reference data resources for tumor and normal tissues. Cancer transcriptomes frequently exhibit RNA dysregulation. As the resulting aberrant transcripts may be translated into cancer-specific proteins, there is growing interest in exploiting RNA dysregulation as a source of tumor antigens (TAs) and thus novel immunotherapy targets. Recent advances in high-throughput technologies and rapid accumulation of multiomic cancer profiling data in public repositories have provided opportunities to systematically characterize RNA dysregulation in cancer and identify antigen targets for immunotherapy. However, given the complexity of cancer transcriptomes and proteomes, important conceptual and technological challenges exist. Here, we highlight the expanding repertoire of TAs arising from RNA dysregulation and introduce multiomic and big data strategies for identifying optimal immunotherapy targets. We discuss extant barriers for translating these targets into effective therapies as well as the implications for future research. Cancer transcriptomes frequently exhibit RNA dysregulation. As the resulting aberrant transcripts may be translated into cancer-specific proteins, there is growing interest in exploiting RNA dysregulation as a source of tumor antigens (TAs) and thus novel immunotherapy targets. Recent advances in high-throughput technologies and rapid accumulation of multiomic cancer profiling data in public repositories have provided opportunities to systematically characterize RNA dysregulation in cancer and identify antigen targets for immunotherapy. However, given the complexity of cancer transcriptomes and proteomes, important conceptual and technological challenges exist. Here, we highlight the expanding repertoire of TAs arising from RNA dysregulation and introduce multiomic and big data strategies for identifying optimal immunotherapy targets. We discuss extant barriers for translating these targets into effective therapies as well as the implications for future research. Transcriptomic and proteomic outputs of human cells are controlled by multiple RNA-level regulatory processes. Mechanisms, such as pre-mRNA alternative splicing (AS, see Glossary) and RNA editing, can generate multiple protein isoforms from a single gene, greatly expanding the coding capacity and protein repertoire of human cells. Cancer cells exhibit widespread abnormalities in RNA processing, including driver alterations that functionally contribute to cancer development and progression [1.Obeng E.A. et al.Altered RNA processing in cancer pathogenesis and therapy.Cancer Discov. 2019; 9: 1493-1510Crossref PubMed Scopus (57) Google Scholar,2.Cherry S. Lynch K.W. Alternative splicing and cancer: insights, opportunities, and challenges from an expanding view of the transcriptome.Genes Dev. 2020; 34: 1005-1016Crossref PubMed Google Scholar]. Through prevalent RNA dysregulation, cancer cells can express a distinct set of transcripts and proteins, some of which may represent therapeutic targets. Targeted cancer immunotherapy has had tremendous successes in treating aggressive malignancies; however, important challenges and unmet needs remain [3.Waldman A.D. et al.A guide to cancer immunotherapy: from T cell basic science to clinical practice.Nat. Rev. Immunol. 2020; 20: 651-668Crossref PubMed Scopus (794) Google Scholar,4.Bonifant C.L. et al.Toxicity and management in CAR T-cell therapy.Mol. Ther. Oncolytics. 2016; 316011Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar]. One critical aspect in developing immunotherapies with safe and effective antitumor immunity is to discover targetable tumor antigens (TAs). Current TA discovery efforts focus primarily on somatic mutations in protein-coding regions (i.e., neoantigens) [5.Schumacher T.N. Schreiber R.D. Neoantigens in cancer immunotherapy.Science. 2015; 348: 69-74Crossref PubMed Scopus (2759) Google Scholar] and several well-known alternative sources, such as lineage-specific antigens and cancer/testis antigens [3.Waldman A.D. et al.A guide to cancer immunotherapy: from T cell basic science to clinical practice.Nat. Rev. Immunol. 2020; 20: 651-668Crossref PubMed Scopus (794) Google Scholar,6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar]. These strategies search only a small fraction of proteomic variations in cancer cells and are ineffective for many cancer types [7.Smith C.C. et al.Alternative tumour-specific antigens.Nat. Rev. Cancer. 2019; 19: 465-478Crossref PubMed Scopus (115) Google Scholar]. Compelling evidence now suggests that TAs arising from RNA dysregulation may represent an extensive but largely unexplored repertoire of novel immunotherapy targets [1.Obeng E.A. et al.Altered RNA processing in cancer pathogenesis and therapy.Cancer Discov. 2019; 9: 1493-1510Crossref PubMed Scopus (57) Google Scholar,2.Cherry S. Lynch K.W. Alternative splicing and cancer: insights, opportunities, and challenges from an expanding view of the transcriptome.Genes Dev. 2020; 34: 1005-1016Crossref PubMed Google Scholar,7.Smith C.C. et al.Alternative tumour-specific antigens.Nat. Rev. Cancer. 2019; 19: 465-478Crossref PubMed Scopus (115) Google Scholar]. Through this review, we endeavor to raise awareness of this emerging class of TAs, with the goal of motivating conceptual and technological advances that translate these novel targets into new cancer immunotherapies. By augmenting and engineering patients' antitumor immunity, cancer immunotherapies with immune checkpoint blockade (ICB) and adoptive cell therapies (ACTs) (Figure 1A and Box 1) have created a paradigm shift in cancer treatment. Immunotherapies have achieved improved patient survival, durable response, and other benefits for aggressive and previously untreatable cancers [3.Waldman A.D. et al.A guide to cancer immunotherapy: from T cell basic science to clinical practice.Nat. Rev. Immunol. 2020; 20: 651-668Crossref PubMed Scopus (794) Google Scholar]. TAs play critical roles in inducing strong antitumor immunity [6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar]. The clinical efficacy of ICB is thought to originate from its ability to reactivate T cell responses to TAs [6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar,8.Keenan T.E. et al.Genomic correlates of response to immune checkpoint blockade.Nat. Med. 2019; 25: 389-402Crossref PubMed Scopus (219) Google Scholar]. In ACTs, T cells are engineered to recognize TAs through distinct mechanisms, either by targeting extracellular peptides of cell surface proteins for chimeric antigen receptor T cell (CAR-T) therapy or by targeting peptide epitopes presented on the cell surface via MHC molecules for T cell receptor (TCR) therapy (Figure 1A) [9.Rosenberg S.A. Restifo N.P. Adoptive cell transfer as personalized immunotherapy for human cancer.Science. 2015; 348: 62-68Crossref PubMed Scopus (1410) Google Scholar].Box 1Major Types of Cancer ImmunotherapyThere are two major types of cancer immunotherapy [3.Waldman A.D. et al.A guide to cancer immunotherapy: from T cell basic science to clinical practice.Nat. Rev. Immunol. 2020; 20: 651-668Crossref PubMed Scopus (794) Google Scholar] (see Figure 1A in the main text). ICB works by augmenting the suppressed immune response against tumor cells. The ICB strategy uses immune checkpoint inhibitors [e.g., neutralizing antibodies against programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)] to reactivate tumor-specific T cells. Therapeutic antibodies and ACTs work by directing or engineering immune cells to improve antitumor immunity. Therapeutic antibodies (e.g., anti-CD20 rituximab) bind TAs directly to direct the immune response. For ACTs, the patient's own immune cells are harvested and modified ex vivo to enhance their antitumor ability [9.Rosenberg S.A. Restifo N.P. Adoptive cell transfer as personalized immunotherapy for human cancer.Science. 2015; 348: 62-68Crossref PubMed Scopus (1410) Google Scholar]. CAR-T and TCR therapies are examples of engineered ACTs, in which the patient's T cells are engineered ex vivo to target known TAs. Successful cases of ACTs include using tumor-infiltrating lymphocyte (TIL) therapy to treat metastatic melanoma and CD19 CAR-T therapy to treat B cell acute lymphoblastic leukemia (B-ALL). There are two major types of cancer immunotherapy [3.Waldman A.D. et al.A guide to cancer immunotherapy: from T cell basic science to clinical practice.Nat. Rev. Immunol. 2020; 20: 651-668Crossref PubMed Scopus (794) Google Scholar] (see Figure 1A in the main text). ICB works by augmenting the suppressed immune response against tumor cells. The ICB strategy uses immune checkpoint inhibitors [e.g., neutralizing antibodies against programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)] to reactivate tumor-specific T cells. Therapeutic antibodies and ACTs work by directing or engineering immune cells to improve antitumor immunity. Therapeutic antibodies (e.g., anti-CD20 rituximab) bind TAs directly to direct the immune response. For ACTs, the patient's own immune cells are harvested and modified ex vivo to enhance their antitumor ability [9.Rosenberg S.A. Restifo N.P. Adoptive cell transfer as personalized immunotherapy for human cancer.Science. 2015; 348: 62-68Crossref PubMed Scopus (1410) Google Scholar]. CAR-T and TCR therapies are examples of engineered ACTs, in which the patient's T cells are engineered ex vivo to target known TAs. Successful cases of ACTs include using tumor-infiltrating lymphocyte (TIL) therapy to treat metastatic melanoma and CD19 CAR-T therapy to treat B cell acute lymphoblastic leukemia (B-ALL). TAs can be generated by DNA, RNA, or protein alterations in cancer cells. Certain features of TAs are critical for ensuring immunotherapy success [6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar]. Ideally, the expression of TAs should be exclusive to cancer cells or restricted to nonvital cell lineages, a feature related to their expression selectivity and cancer specificity (Box 2). Another important consideration is the TA foreignness, the extent to which the antigen is recognized by the patient's immune system as 'non-self' [6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar,8.Keenan T.E. et al.Genomic correlates of response to immune checkpoint blockade.Nat. Med. 2019; 25: 389-402Crossref PubMed Scopus (219) Google Scholar,10.Richman L.P. et al.Neoantigen dissimilarity to the self-proteome predicts immunogenicity and response to immune checkpoint blockade.Cell Syst. 2019; 9: 375-382Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar].Box 2Expression Selectivity/Cancer Specificity of TAsThe expression selectivity and cancer specificity of TAs can be grouped into three major classes based on previous definitions in the literature [6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar]. The most specific TAs are the tumor-specific antigens (TSAs), or 'neoantigens', which are expressed exclusively by tumor cells. TSAs offer the highest potential for effective targeting with low off-tumor toxicity but may be difficult to find for certain cancer types. Tumor-associated antigens (TAAs) are overexpressed in tumor cells but are also expressed (to some extent) in normal cells. TAAs are the least tumor-selective TA type. Although immunogenicity and toxicity are concerns, TAAs can be useful targets because they are easy to identify and may be generically expressed across patients and tumor types. ERBB2 (HER2/neu) is a prominent TAA in breast cancer. Cancer/testis antigens (CTAs) are present at elevated levels in tumors and reproductive tissues, while showing limited expression in normal adult tissues. CTAs can be therapeutically targeted by taking advantage of the fact that normal reproductive cells do not express MHC class I molecules. New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a well-known CTA expressed in several cancer types. The expression selectivity and cancer specificity of TAs can be grouped into three major classes based on previous definitions in the literature [6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar]. The most specific TAs are the tumor-specific antigens (TSAs), or 'neoantigens', which are expressed exclusively by tumor cells. TSAs offer the highest potential for effective targeting with low off-tumor toxicity but may be difficult to find for certain cancer types. Tumor-associated antigens (TAAs) are overexpressed in tumor cells but are also expressed (to some extent) in normal cells. TAAs are the least tumor-selective TA type. Although immunogenicity and toxicity are concerns, TAAs can be useful targets because they are easy to identify and may be generically expressed across patients and tumor types. ERBB2 (HER2/neu) is a prominent TAA in breast cancer. Cancer/testis antigens (CTAs) are present at elevated levels in tumors and reproductive tissues, while showing limited expression in normal adult tissues. CTAs can be therapeutically targeted by taking advantage of the fact that normal reproductive cells do not express MHC class I molecules. New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a well-known CTA expressed in several cancer types. Advances in cancer genomic sequencing have enabled a clinically successful strategy for TA discovery focusing on somatic mutations (Figure 1B) [5.Schumacher T.N. Schreiber R.D. Neoantigens in cancer immunotherapy.Science. 2015; 348: 69-74Crossref PubMed Scopus (2759) Google Scholar,6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar,11.Richters M.M. et al.Best practices for bioinformatic characterization of neoantigens for clinical utility.Genome Med. 2019; 11: 56Crossref PubMed Scopus (70) Google Scholar]. Specifically, cancer genomic alterations, including single-nucleotide variations (SNVs), insertion–deletion variations, and gene fusions, can be detected by sequencing and comparing the tumor DNA with normal DNA of a given patient [11.Richters M.M. et al.Best practices for bioinformatic characterization of neoantigens for clinical utility.Genome Med. 2019; 11: 56Crossref PubMed Scopus (70) Google Scholar]. Protein sequence analyses can then identify somatic mutations in protein-coding regions that generate antigenic peptides. Although this strategy is well established, it has important limitations. Somatic mutations are usually specific to a small subset of patients, necessitating therapy development on an individualized ('personalized') basis [6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar]. Furthermore, only a small proportion of somatic mutations alter protein sequences and have strong immunogenicity, thus limiting the utility of this strategy for many cancer types with moderate or low mutation loads [5.Schumacher T.N. Schreiber R.D. Neoantigens in cancer immunotherapy.Science. 2015; 348: 69-74Crossref PubMed Scopus (2759) Google Scholar]. Given these inherent limitations, new types of TAs need to be systematically explored. Post-transcriptional RNA processing plays key roles in regulating gene expression and generating the diverse transcript and protein isoforms required for essential biological functions [12.Licatalosi D.D. Darnell R.B. RNA processing and its regulation: global insights into biological networks.Nat. Rev. Genet. 2010; 11: 75-87Crossref PubMed Scopus (499) Google Scholar]. RNA processing is governed by cis regulatory elements and trans regulatory factors, whose functions can be disrupted by somatic mutations and oncogenic signaling pathways in cancer [1.Obeng E.A. et al.Altered RNA processing in cancer pathogenesis and therapy.Cancer Discov. 2019; 9: 1493-1510Crossref PubMed Scopus (57) Google Scholar,2.Cherry S. Lynch K.W. Alternative splicing and cancer: insights, opportunities, and challenges from an expanding view of the transcriptome.Genes Dev. 2020; 34: 1005-1016Crossref PubMed Google Scholar]. Dysregulation of RNA-related processes can reshape the cancer transcriptomic and proteomic landscape. Consequently, this dysregulation can modulate cellular phenotypes by changing the abundance and diversity of transcripts and proteins (Figure 2). Some of these proteins could harbor cancer-specific antigenic peptides. Here, we summarize the main mechanisms of RNA dysregulation and recent progress in exploring RNA dysregulation as a source of cancer immunotherapy targets. Altered RNA expression is a major source of cancer transcriptome variation, sometimes playing essential oncogenic roles [13.PCAWG Transcriptome Core Group et al.Genomic basis for RNA alterations in cancer.Nature. 2020; 578: 129-136Crossref PubMed Scopus (140) Google Scholar]. RNA overexpression in cancer cells can be caused by transcriptional or post-transcriptional processes that enhance gene transcription or RNA stability. If the corresponding protein product is consistently overexpressed across patients with a cancer type of interest, or even across multiple cancer types, and if protein expression is low and sparse in normal tissues, then this protein may serve as a candidate TA [6.Yarchoan M. et al.Targeting neoantigens to augment antitumour immunity.Nat. Rev. Cancer. 2017; 17: 209-222Crossref PubMed Scopus (480) Google Scholar]. Several clinically targeted TAs follow this pattern, with consistent overexpression in tumors and low or lineage-restricted expression in normal cells (e.g., CD19 [14.Grupp S.A. et al.Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.N. Engl. J. Med. 2013; 368: 1509-1518Crossref PubMed Scopus (2391) Google Scholar] and GPC2 [15.Bosse K.R. et al.Identification of GPC2 as an oncoprotein and candidate immunotherapeutic target in high-risk neuroblastoma.Cancer Cell. 2017; 32: 295-309Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]). However, issues of limited immunogenicity and off-tumor toxicity remain [3.Waldman A.D. et al.A guide to cancer immunotherapy: from T cell basic science to clinical practice.Nat. Rev. Immunol. 2020; 20: 651-668Crossref PubMed Scopus (794) Google Scholar,4.Bonifant C.L. et al.Toxicity and management in CAR T-cell therapy.Mol. Ther. Oncolytics. 2016; 316011Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar]. Pre-mRNA AS is a prevalent mechanism for diversifying gene products in human cells (Figure 2) [16.Park E. et al.The expanding landscape of alternative splicing variation in human populations.Am. J. Hum. Genet. 2018; 102: 11-26Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar]. AS is widespread in cancer transcriptomes and contributes to each of the 'hallmarks of cancer', the key phenotypic characteristics of cancer cells compared with normal cells [17.Oltean S. Bates D.O. Hallmarks of alternative splicing in cancer.Oncogene. 2014; 33: 5311-5318Crossref PubMed Scopus (409) Google Scholar,18.Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (40071) Google Scholar]. Cancer-associated AS can functionally modulate cancer development and progression through multiple mechanisms, such as by producing protein isoforms that promote cell proliferation, inhibit cell death, avoid antitumor immunity, or enable invasion and metastasis [17.Oltean S. Bates D.O. Hallmarks of alternative splicing in cancer.Oncogene. 2014; 33: 5311-5318Crossref PubMed Scopus (409) Google Scholar]. Additionally, AS events may predict patient prognosis and therapy response [2.Cherry S. Lynch K.W. Alternative splicing and cancer: insights, opportunities, and challenges from an expanding view of the transcriptome.Genes Dev. 2020; 34: 1005-1016Crossref PubMed Google Scholar]. Alternatively spliced exons or splice junctions can be translated into antigenic peptides that are enriched in cancer cells [19.Frankiw L. et al.Alternative mRNA splicing in cancer immunotherapy.Nat. Rev. Immunol. 2019; 19: 675-687Crossref PubMed Scopus (87) Google Scholar]. Some of the cancer-associated exons or splice junctions may arise from somatic mutations creating novel splice sites that are not present in normal cells, making them highly cancer specific [20.Jayasinghe R.G. et al.Systematic analysis of splice-site-creating mutations in cancer.Cell Rep. 2018; 23: 270-281Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar]. Additionally, AS introduces larger sequence changes to the final protein products, potentially making AS-derived TAs more 'foreign' and immunogenic than single amino acid changes introduced by somatic mutations. RNA sequencing (RNA-seq) studies of many cancer types have revealed transcriptome-wide changes in AS [13.PCAWG Transcriptome Core Group et al.Genomic basis for RNA alterations in cancer.Nature. 2020; 578: 129-136Crossref PubMed Scopus (140) Google Scholar,20.Jayasinghe R.G. et al.Systematic analysis of splice-site-creating mutations in cancer.Cell Rep. 2018; 23: 270-281Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar,21.Kahles A. et al.Comprehensive analysis of alternative splicing across tumors from 8,705 patients.Cancer Cell. 2018; 34: 211-224Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar]. Comparing tumor tissues with normal tissues, Kahles et al. found elevated levels of AS in tumor tissues, including numerous previously unannotated AS events with the majority being exon skipping and alternative 3′ splice site events [21.Kahles A. et al.Comprehensive analysis of alternative splicing across tumors from 8,705 patients.Cancer Cell. 2018; 34: 211-224Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar]. Considering only splice junction peptides as candidate TAs, the authors validated the expression of some peptides using mass spectrometry (MS) proteomics data and predicted candidate antigens based on expected binding to MHC class I molecules. However, immunogenicity of the candidate TAs was not tested experimentally. A recent pan-cancer study showed that aberrant cancer-specific exons may be introduced by splice site-creating somatic mutations within intronic sequences through a process called exonization [13.PCAWG Transcriptome Core Group et al.Genomic basis for RNA alterations in cancer.Nature. 2020; 578: 129-136Crossref PubMed Scopus (140) Google Scholar]. Dysregulation of intron retention, a specific type of AS, is also common in cancer and provides a mechanism for inactivating tumor suppressors [22.Jung H. et al.Intron retention is a widespread mechanism of tumor-suppressor inactivation.Nat. Genet. 2015; 47: 1242-1248Crossref PubMed Scopus (202) Google Scholar]. Smart et al. experimentally validated MHC class I presentation of intron retention-derived peptides, suggesting their potential immunogenicity [23.Smart A.C. et al.Intron retention is a source of neoepitopes in cancer.Nat. Biotechnol. 2018; 36: 1056-1058Crossref PubMed Scopus (117) Google Scholar]. Collectively, these studies provide compelling evidence for AS as a promising source of novel immunotherapy targets. The repertoire of AS-derived TAs may be particularly large for certain cancer types and patients carrying specific cancer driver mutations, considering that genes encoding splicing regulatory factors are frequently mutated in cancer [2.Cherry S. Lynch K.W. Alternative splicing and cancer: insights, opportunities, and challenges from an expanding view of the transcriptome.Genes Dev. 2020; 34: 1005-1016Crossref PubMed Google Scholar]. Not all AS isoforms are capable of generating protein products. The nonsense-mediated mRNA decay (NMD) pathway is an important mechanism of post-transcriptional regulation, which degrades mRNA transcripts that contain premature termination codons (PTCs) [24.Mendell J.T. et al.Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise.Nat. 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Chimeric RNAs resulting from trans-splicing and cis-SAGe have been found in embryonic stem cells [31.Wu C.S. et al.Integrative transcriptome sequencing identifies trans-splicing events with important roles in human embryonic stem cell pluripotency.Genome Res. 2014; 24: 25-36Crossref PubMed Scopus (76) Google Scholar] and other non-cancer tissues [32.Babiceanu M. et al.Recurrent chimeric fusion RNAs in non-cancer tissues and cells.Nucleic Acids Res. 2016; 44: 2859-2872Crossref PubMed Scopus (110) Google Scholar]. Many chimeric RNAs have been identified as biomarkers or potential therapeutic targets for cancer [30.Jia Y. et al.Intergenically spliced chimeric RNAs in cancer.Trends Cancer. 2016; 2: 475-484Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. If a cancer-specific chimeric RNA is translated into a protein, then the cancer-specific peptide derived from the chimeric RNA may be a candidate TA [33.Cieslik M. Chinnaiyan A.M. Cancer transcriptome profiling at the juncture of clinical translation.Nat. Rev. Genet. 2018; 19: 93-109Crossref PubMed Scopus (114) Google Scholar]. Multiple tools exist to detect chimeric RNAs [34.Haas B.J. et al.Accuracy assessment of fusion transcript detection via read-mapping and de novo fusion transcript assembly-based methods.Genome Biol. 2019; 20: 213Crossref PubMed Scopus (173) Google Scholar]. However, the authenticity of chimeric RNAs detected from cancer RNA-seq data remains a concern, as many such RNAs could represent sequencing or detection artifacts [34.Haas B.J. et al.Accuracy assessment of fusion transcript detection via read-mapping and de novo fusion transcript assembly-based methods.Genome Biol. 2019; 20: 213Crossref PubMed Scopus (173) Google Scholar]. The cancer specificity and immunogenicity of chimeric RNA-derived TAs must be evaluated considering that chimeric RNAs are also expressed in normal tissues [32.Babiceanu M. et al.Recurrent chimeric fusion RNAs in non-cancer tissues and cells.Nucleic Acids Res. 2016; 44: 2859-2872Crossref PubMed Scopus (110) Google Scholar]. Circular RNAs (circRNAs) are a unique class of RNA molecules with a covalently closed loop formed by back-splicing events during pre-mRNA splicing (Figure 2) [35.Kristensen L.S. et al.The biogenesis, biology and characterization of circular RNAs.Nat. Rev. Genet. 2019;