Novel Tumor-Specific Antigens for Immunotherapy Identified From Multi-omics Profiling in Thymic Carcinomas

Thymic carcinoma (TC) is the most aggressive thymic epithelial neoplasm. TC patients with microsatellite instability, whole-genome doubling, or alternative tumor-specific antigens from gene fusion are most likely to benefit from immunotherapies. However, due to the rarity of this disease, how to prioritize the putative biomarkers and what constitutes an optimal treatment regimen remains largely unknown. Therefore, we integrated genomic and transcriptomic analyses from TC patients and revealed that frameshift indels in KMT2C and CYLD frequently produce neoantigens. Moreover, a median of 3 fusion-derived neoantigens was predicted across affected patients, especially the CATSPERB-TC2N neoantigens that were recurrently predicted in TC patients. Lastly, potentially actionable alterations with early levels of evidence were uncovered and could be used for designing clinical trials. In summary, this study shed light on our understanding of tumorigenesis and presented new avenues for molecular characterization and immunotherapy in TC.

Genomic Analyses of Metaplastic or Sarcomatoid Carcinomas From Different Organs Revealed Frequent Mutations in KMT2D

Background: Metaplastic or sarcomatoid carcinomas (MSCs) are rare epithelial malignancies with heterologous histological differentiation that can occur in different organs. The objective of the current study was to identify novel somatically mutated genes in MSCs from different organs.Methods: Whole-exome sequencing was performed in 16 paired MSCs originating from the breast (n = 10), esophagus (n = 3), lung (n = 2), and kidney (n = 1). In addition, we collected data on KMT2D mutations from eight independent cohorts (n = 195) diagnosed with MSCs derived from the breast (n = 83), liver (n = 8), esophagus (n = 15), lung (n = 10), and uterus or ovary (n = 79). The expression of KMT2D and its clinical significance were evaluated in our cohort.Results: The most frequently mutated genes were TP53 (13/16, 81%) and KMT2D (5/16,31%). We identified seven somatic KMT2D mutations in the exploratory cohort (n = 16 tumors), including three nonsense mutations, two frameshift indels, one missense mutation, and one splice site mutation. Interestingly, two patients showed double hits on KMT2D with nonsense mutations and frameshift indels. In the eight validation cohorts (n = 195), the average mutation rates for TP53 and KMT2D were 78% (152/195) and 13% (25/195), respectively. Two or more hits on KMT2D were also present in three validation cohorts. Furthermore, KMT2D mutations were associated with low expression of KMT2D, large tumor size and unfavorable prognosis.Conclusions: These findings provide clues for understanding the genetic basis of MSCs originating from different organs and implicate KMT2D alteration as a frequent pathogenic mutation, allowing provision of appropriate treatment for this rare malignant disease in the future.

Neo-Fs Index: A Novel Immunohistochemical Biomarker Panel Predicts Survival and Response to Anti-Angiogenetic Agents in Clear Cell Renal Cell Carcinoma

Background: Frameshift indels have emerged as a predictor of immunotherapy response but were not evaluated yet to predict anti-angiogenetic agent (AAA) response or prognosis in clear cell renal cell carcinoma (ccRCC). Methods: Here, to develop biomarkers that predict survival and response to AAA, we evaluated the immunohistochemical expression of proteins whose genes frequently harbor frameshift indels in 638 ccRCC patients and correlated the individual and integrated markers with prognosis and AAA response. The mutational landscape was evaluated using targeted next-generation sequencing in 12 patients concerning protein markers. Immune gene signatures were retrieved from TCGA RNA seq data. Results: Five proteins (APC, NOTCH1, ARID1A, EYS, and filamin A) were independent adverse prognosticators and were incorporated into the Neo-fs index. Better overall, disease-specific and recurrence-free survival were observed with high Neo-fs index in univariate and multivariate survival analyses. Better AAA responses were observed with a high Neo-fs index, which reflected increased MHC class I, CD8+ T cell, cytolytic activity, and plasmacytoid dendritic cell signatures and decreased type II-IFN response signatures, as well as greater single-nucleotide variant (SNV) and indel counts. Conclusions: Neo-fs index, reflecting antitumor immune signature and more SNVs. and indels, is a powerful predictor of survival and AAA response in ccRCC.

Mosaic Mutagenesis In Vivo Reveals Mutant Blood Stem Cells Intrinsically Resistant to Inflammatory Mediators in Clonal Hematopoiesis

Clonal hematopoiesis (CH) is a state of clonal dominance of a mutant hematopoietic stem and progenitor cell (HSPC) promoted by unknown mechanisms. To study the competitive behavior and the mechanism of such expansion in a native environment, we used a combinatorial approach of HSPCs labeling and mutagenesis in vivo in a technique we called Tissue editing With Inducible Stem cell Tagging via Recombination (TWISTR). TWISTR utilizes Zebrabow zebrafish to fluorescently label endogenous HSPCs, and allows following of the clonal activity and sorting of labeled HSPCs within the hematopoietic system. CRISPR/Cas9 mutagenesis was used to simultaneously induce mosaic insertions/deletions (indels) in zebrafish orthologs of 12 human CH genes during development, and zebrafish were followed for 8 months. As such, mutant stem cells were directly competed against endogenous wildtype stem cells. We achieved a high degree of mosaic mutagenesis with heterozygous edits, with a median of 5 targeted genes per zebrafish in total marrow cells at variant allele frequency (VAF) of 5% or greater. Serial sampling and sequencing of peripheral blood cells showed selective expansion of clones with frameshift indels in exon 12 of asxl1, while clones with other CH mutations did not show a significant change over the 4 months period evaluated. No changes were noted in control gene indels or in the CH gene indels in non-hematopoietic tissue in the same time period. We identified significant enrichment of frameshift indels in asxl1 in sorted dominant clones compared to sorted smaller clones in the same zebrafish (p<0.01) at 8 months, while indels in DNMT3A ortholog dnmt8 were present in clones of various competitive nature. Introduction of asxl1 mutations singly in a mosaic fashion also resulted in clonally dominant states. To identify gene expression signatures associated with the clonal expansion, we used single cell RNA sequencing of marrow cells in zebrafish with a dominant clone with unique sets of mutations. We identified increased expression of inflammatory signaling genes in these zebrafish compared to controls, consistent with recent findings in other organisms. Within sorted dominant clones, mutant neutrophils and macrophages exhibited more than 4-fold increased expression of cytokines, such as il1b and tnfb. In mutant HSPCs and myeloid-biased progenitor cells, we observed elevated levels of genes involved in suppressing responses to cytokines and inflammatory lipids, like socs3a, nr4a1, atf3 and ier2a. This supports a model in which the mutant HSPCs of the dominant clone express anti-inflammatory genes that dampen their response to the inflammatory mediators produced by mature cells of the same clone, limiting the inflammation-induced HSPC exhaustion and providing a selective mechanism for the mutant stem cells to expand over time. We successfully used the Zebrabow color labeling system and prospective endogenous mosaic mutagenesis to show that mutant asxl1 is a potent inducer of CH, and that CH gene mutations lead to clonal dominance by modifying the response of stem cells to inflammatory cues. Disclosures Zon: Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Amagma Therapeutics: Current equity holder in private company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy.

PredCID: prediction of driver frameshift indels in human cancer

The discrimination of driver from passenger mutations has been a hot topic in the field of cancer biology. Although recent advances have improved the identification of driver mutations in cancer genomic research, there is no computational method specific for the cancer frameshift indels (insertions or/and deletions) yet. In addition, existing pathogenic frameshift indel predictors may suffer from plenty of missing values because of different choices of transcripts during the variant annotation processes. In this study, we proposed a computational model, called PredCID (Predictor for Cancer driver frameshift InDels), for accurately predicting cancer driver frameshift indels. Gene, DNA, transcript and protein level features are combined together and selected for classification with eXtreme Gradient Boosting classifier. Benchmarking results on the cross-validation dataset and independent dataset showed that PredCID achieves better and robust performance compared with existing noncancer-specific methods in distinguishing cancer driver frameshift indels from passengers and is therefore a valuable method for deeper understanding of frameshift indels in human cancer. PredCID is freely available for academic research at http://bioinfo.ahu.edu.cn:8080/PredCID.

Higher burden of rare frameshift indels in genes related to synaptic transmission separate familial hemiplegic migraine from common types of migraine

BackgroundFamilial hemiplegic migraine (FHM) is a rare form of migraine with aura that often has an autosomal dominant mode of inheritance. Rare mutations in the CACNA1A, ATP1A2 and SCN1A genes can all cause FHM revealing genetic heterogeneity in the disorder. Furthermore, only a small subset of the affected individuals has a causal mutation. We set out to investigate what differentiates patients with FHM with no mutation in any known FHM gene from patients with common types of migraine in both familial and sporadic cases.Methods2558 male and female participants from a migraine cohort from the Danish Headache Center were included. 112 had FHM; 743 had familial migraine; and 1703 had sporadic migraine. Using a linear regression model, we analysed for over-representation of rare functional variants in FHM versus familial migraine and sporadic migraine. Post hoc analyses included pathway analysis and testing for tissue specificity.ResultsWe found that patients with FHM have significantly more rare frameshift indels compared with patients with familial migraine and sporadic migraine. Pathway analysis revealed that the ‘ligand-gated ion channel activity’ and ‘G protein-coupled receptor downstream signalling’ pathways were significantly associated with mutated genes. We moreover found that the mutated genes showed tissue specificity towards nervous tissue and muscle tissue.ConclusionWe show that patients with FHM compared with patients with common types of migraine suffer from a higher load of rare frameshift indels in genes associated with synaptic signalling in the central nervous system and possibly in muscle tissue contributing to vascular dysfunction.

dbCID: a manually curated resource for exploring the driver indels in human cancer

While recent advances in next-generation sequencing technologies have enabled the creation of a multitude of databases in cancer genomic research, there is no comprehensive database focusing on the annotation of driver indels (insertions and deletions) yet. Therefore, we have developed the database of Cancer driver InDels (dbCID), which is a collection of known coding indels that likely to be engaged in cancer development, progression or therapy. dbCID contains experimentally supported and putative driver indels derived from manual curation of literature and is freely available online at http://bioinfo.ahu.edu.cn:8080/dbCID. Using the data deposited in dbCID, we summarized features of driver indels in four levels (gene, DNA, transcript and protein) through comparing with putative neutral indels. We found that most of the genes containing driver indels in dbCID are known cancer genes playing a role in tumorigenesis. Contrary to the expectation, the sequences affected by driver frameshift indels are not larger than those by neutral ones. In addition, the frameshift and inframe driver indels prefer to disrupt high-conservative regions both in DNA sequences and protein domains. Finally, we developed a computational method for discriminating cancer driver from neutral frameshift indels based on the deposited data in dbCID. The proposed method outperformed other widely used non-cancer-specific predictors on an external test set, which demonstrated the usefulness of the data deposited in dbCID. We hope dbCID will be a benchmark for improving and evaluating prediction algorithms, and the characteristics summarized here may assist with investigating the mechanism of indel–cancer association.

Association of tumor mutational burden in cutaneous squamous cell carcinoma with genomic alterations in Notch family receptors.

e13031 Background: In Cutaneous Squamous Cell Carcinoma (cSqCC), Notch1, Notch2, and Notch3 have been identified as tumor suppressors with a high rate of inactivating mutations early in cSqCC pathogenesis. Despite the high frequency of alterations, the biologic and therapeutic implications of Notch genomic alterations (GAs) in cSqCC are incompletely understood. Methods: 232 FFPE samples of cSqCC were evaluated by comprehensive genomic profiling (CGP) of 315 genes and analyzed for all classes of GAs, with diagnoses confirmed by central pathology review. Notch GAs with unknown functional impact were excluded. Tumor mutational burden (TMB) was calculated from 1.11 Mb of sequenced DNA and reported as mutations/Mb. Results: The cohort of 232 samples was 78% male and 22% female, aged from 17 to 88, with confirmed metastatic disease in 32% of cases. 115 (50%) samples had a Notch family member GA with a known or predicted functional impact. 96 cases had Notch1 GAs, 43 cases had Notch2 GAs, and 14 cases had Notch3 GAs with 40 cases having GAs in multiple Notch family members. These mutations were 41% missense, 34% nonsense, 15% splice site, 9% frameshift indels, and 1% non-frameshift indels alterations. Patients with Notch GAs were significantly older (median 70 years old vs median 64, p < 0.01). TMB was increased among samples with Notch GAs (median TMB of 63 mutations/mb versus 20 mutations/mb, p < 1x10-6), with similar differences for primary and metastatic samples. Cases with Notch GAs had more total GAs per case (mean 10.4 vs 7.4, p < 1.5x10-10); however, the top co-mutated genes were the same (TP53, CDKN2A, FAT1, MLL2). Both groups had a high proportion of C- > T/G- > A transitions, consistent with UV damage; however, the proportion was higher in cases with Notch GAs (median 83% vs 80%) - a difference seen in primary and metastatic samples. Conclusions: 50% of cSqCC cases had a Notch family member loss of function GA, which was associated with increased TMB in the primary and metastatic setting. With pre-clinical models and case studies showing responses to PD-1 inhibitors in cSqCC, further investigations are warranted into the associations of Notch GAs with mutational burden and response to immunotherapy.