Prioritization of immunogenic neoantigens is key to enhancing cancer immunotherapy through the development of personalized vaccines, adoptive T cell therapy, and the prediction of response to immune ...checkpoint inhibition. Neoantigens are tumor-specific proteins that allow the immune system to recognize and destroy a tumor. Cancer immunotherapies, such as personalized cancer vaccines, adoptive T cell therapy, and immune checkpoint inhibition, rely on an understanding of the patient-specific neoantigen profile in order to guide personalized therapeutic strategies. Genomic approaches to predicting and prioritizing immunogenic neoantigens are rapidly expanding, raising new opportunities to advance these tools and enhance their clinical relevance. Predicting neoantigens requires acquisition of high-quality samples and sequencing data, followed by variant calling and variant annotation. Subsequently, prioritizing which of these neoantigens may elicit a tumor-specific immune response requires application and integration of tools to predict the expression, processing, binding, and recognition potentials of the neoantigen. Finally, improvement of the computational tools is held in constant tension with the availability of datasets with validated immunogenic neoantigens. The goal of this review article is to summarize the current knowledge and limitations in neoantigen prediction, prioritization, and validation and propose future directions that will improve personalized cancer treatment.
Accurate prioritization of immunogenic neoantigens is key to developing personalized cancer vaccines and distinguishing those patients likely to respond to immune checkpoint inhibition. However, ...there is no consensus regarding which characteristics best predict neoantigen immunogenicity, and no model to date has both high sensitivity and specificity and a significant association with survival in response to immunotherapy. We address these challenges in the prioritization of immunogenic neoantigens by (1) identifying which neoantigen characteristics best predict immunogenicity; (2) integrating these characteristics into an immunogenicity score, the NeoScore; and (3) demonstrating a significant association of the NeoScore with survival in response to immune checkpoint inhibition. One thousand random and evenly split combinations of immunogenic and nonimmunogenic neoantigens from a validated dataset were analyzed using a regularized regression model for characteristic selection. The selected characteristics, the dissociation constant and binding stability of the neoantigen:MHC class I complex and expression of the mutated gene in the tumor, were integrated into the NeoScore. A web application is provided for calculation of the NeoScore. The NeoScore results in improved, or equivalent, performance in four test datasets as measured by sensitivity, specificity, and area under the receiver operator characteristics curve compared with previous models. Among cutaneous melanoma patients treated with immune checkpoint inhibition, a high maximum NeoScore was associated with improved survival. Overall, the NeoScore has the potential to improve neoantigen prioritization for the development of personalized vaccines and contribute to the determination of which patients are likely to respond to immunotherapy.
There is a need to identify molecular biomarkers of melanoma progression to assist the development of chemoprevention strategies to lower melanoma incidence. Using datasets containing gene expression ...for dysplastic nevi and melanoma or melanoma arising in a nevus, we performed differential gene expression analysis and regularized regression models to identify genes and pathways that were associated with progression from nevi to melanoma. A small number of genes distinguished nevi from melanoma. Differential expression of seven genes was identified between nevi and melanoma in three independent datasets. C1QB, CXCL9, CXCL10, DFNA5 (GSDME), FCGR1B, and PRAME were increased in melanoma, and SCGB1D2 was decreased in melanoma, compared to dysplastic nevi or nevi that progressed to melanoma. Further supporting an association with melanomagenesis, these genes demonstrated a linear change in expression from benign nevi to dysplastic nevi to radial growth phase melanoma to vertical growth phase melanoma. The genes associated with melanoma progression showed significant enrichment of multiple pathways related to the immune system. This study demonstrates (1) a novel application of bioinformatic approaches to aid clinical trials of melanoma chemoprevention and (2) the feasibility of determining a gene signature biomarker of melanomagenesis.
A low percentage of actinic keratoses progress to develop into cutaneous squamous cell carcinoma. The immune mechanisms that successfully control or eliminate the majority of actinic keratoses and ...the mechanisms of immune escape by invasive squamous cell carcinoma are not well-understood. Here, we took a systematic approach to evaluate the neoantigens present in actinic keratosis and cutaneous squamous cell carcinoma specimens. We compared the number of mutations, the number of neoantigens predicted to bind MHC class I, and the number of neoantigens that are predicted to bind MHC class I and be recognized by a T cell receptor in actinic keratoses and cutaneous squamous cell carcinomas. We also considered the relative binding strengths to both MHC class I and the T cell receptor in a fitness cost model that allows for a comparison of the immune recognition potential of the neoantigens in actinic keratosis and cutaneous squamous cell carcinoma samples. The fitness cost was subsequently adjusted by the expression rates of the neoantigens to examine the role of neoantigen expression in tumor immune evasion. Our analyses indicate that, while the number of mutations and neoantigens are not significantly different between actinic keratoses and cutaneous squamous cell carcinomas, the predicted immune recognition of the neoantigen with the highest expression-adjusted fitness cost is lower for cutaneous squamous cell carcinomas compared with actinic keratoses. These findings suggest a role for the down-regulation of expression of highly immunogenic neoantigens in the immune escape of cutaneous squamous cell carcinomas. Furthermore, these findings highlight the importance of incorporating additional factors, such as the quality and expression of the neoantigens, rather than focusing solely on tumor mutational burden, in assessing immune recognition potential.
Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II restricted presentation of multiple melanoma antigens. There is variable GILT protein expression in malignant ...melanocytes in melanoma specimens. High GILT mRNA expression in melanoma specimens is associated with improved overall survival, before the advent of immune checkpoint inhibitors (ICI). However, the association of GILT in metastatic melanoma with survival in patients treated with ICI and the cell type expressing GILT associated with survival have not been determined. Using RNA sequencing datasets, high GILT mRNA expression in metastatic melanoma specimens was associated with improved progression-free and overall survival in patients treated with ICI. A clinical dataset of metastatic melanoma specimens was generated and annotated with clinical information. Positive GILT immunohistochemical staining in antigen presenting cells and melanoma cells was observed in 100% and 65% of metastatic melanoma specimens, respectively. In the subset of patients treated with ICI in the clinical dataset, high GILT protein expression within melanoma cells was associated with improved overall survival. The association of GILT mRNA and protein expression with survival was independent of cancer stage. These studies support that high GILT mRNA expression in bulk tumor samples and high GILT protein expression in melanoma cells is associated with improved survival in ICI-treated patients. These findings support further investigation of GILT as a biomarker to predict the response to ICI.
BackgroundNon-melanoma skin cancer, which includes cutaneous squamous cell carcinoma (cSCC), is the most common cancer. In the US, there are more than a million cases of cSCC diagnosed annually. ...Although most early stage cSCC are successfully treated with excision, cSCC results in significant morbidity, and approximately 4% of cSCC patients develop metastases and 2% die. T cell-mediated immunotherapy with immune checkpoint inhibitors improves survival in cSCC patients, but about one half of patients do not respond. To improve immunotherapeutic approaches, there is a need to better understand T cell-mediated control of cSCC.MethodsTo investigate the neoantigen-specific T cell responses that control cSCC, we generated a panel of transplantable cSCC clonal cell lines from solar simulated light-induced tumors in BALB/c mice.1 Whole exome and RNA sequencing was performed on the cSCC cell lines. Mice lacking T cells and T cell depleting antibodies were used to study the role of T cells. MHC class I neoantigens anticipated to elicit T cell responses were prioritized based on predicted MHC class I binding affinity, presentation on MHC class I, and mRNA variant allele-specific expression.ELISPOT assays were used to quantify IFN-γ secreting T cells specific for each predicted neoantigen. To test the ability of vaccination to constrain cSCC, mice were vaccinated with irradiated tumor cells or an immunogenic neoantigen.ResultsThe cSCC cell lines constitutively express MHC class I, and thus, can be targeted for destruction by CD8 T cells. CSCC tumors grow faster in athymic mice, lacking mature T cells, compared to wild-type mice, supporting that T cells constrain cSCC growth. In vivo depletion of CD8 and CD4 T cells demonstrates a major role for CD8 T cells and a contributing role for CD4 T cells in controlling cSCC growth. Vaccination with irradiated cSCC cells completely protects from tumor challenge, and this response is dependent on CD8 T cells. Vaccination with irradiated tumor cells from another cSCC cell line does not protect from tumor challenge, demonstrating that vaccination-induced protection is cell line specific. IFN-γ-secreting CD8 T cells that recognize a mutated Kars peptide are present within cSCC tumors. Prophylactic vaccination with the mutated Kars peptide decreases cSCC growth, demonstrating that an immune response to mutated Kars controls tumor growth.ConclusionsNeoantigen-specific CD8 T cells constrain cSCC growth in a clinically relevant mouse model. Neoantigen-specific approaches may improve immunotherapy outcomes for cSCC patients.AcknowledgementsThis work was supported by the American Skin Association (A.C.A), NIH (T32CA009213, A.C.A; F30CA257622, A.C.A; F30CA281056, E.S.B; P30CA023074, D.J.R; P01CA229112, K.T.H), and Training Oncology Physician Scientists Award from the University of Arizona Cancer Center (E.S.B).ReferenceAdams AC, Macy AM, Saboda K, et al. Solar Simulated Light Induces Cutaneous Squamous Cell Carcinoma in Inbred Mice: A Clinically Relevant Model to Investigate T-Cell Responses. J Invest Dermatol. 2021;141(12):2990–2993.e2996.Ethics ApprovalThis study was approved by the University of Arizona’s Institutional Animal Care and Use Committee (Protocol #13–469).
BackgroundDetermining the shared and unique neoantigen profiles in cutaneous squamous cell carcinoma (cSCC) is critical to selecting immunotherapeutic approaches for improved prevention and treatment ...of cSCC. Nonmelanoma skin cancer, including cSCC, is the most common cancer in the United States. Immune checkpoint inhibitors have been approved for treatment of cSCC, but are only effective in 32–46% of patients. Determining the extent to which neoantigens are shared between tumors will guide future development and selection of immunotherapeutic approaches for cSCC.MethodsWe characterized the mutational profiles of human cSCC and a physiologically-relevant murine model to determine the likelihood of shared neoantigens in cSCC. Variant calls from 88 publicly available human cSCC tumors1 and three solar-simulated UV light-induced murine cSCC cell lines generated in our laboratory2 were analyzed for shared mutations. The murine cSCC cell lines were characterized to determine if their mutational profile and driver mutations recapitulated that of human cSCC. Finally, two clonal murine cSCC cell lines were analyzed for cross-protection with a prophylactic irradiated tumor cell vaccine.ResultsAcross the 88 human cSCC tumors, the majority of mutations are unique to individual tumors with only 27 (0.075%) shared in three or four tumors and no mutations recurring in greater than four tumors. The combination of the 27 shared mutations covers 53.4% of the tumors. The murine cSCC cell lines have a mutational signature that recapitulates that of human disease with an equivalent proportion of C>T and G>A mutations. Each murine cell line contains putative driver mutations in TP53 that overlap with human cSCC tumors. The shared driver mutations and mutational signature support the physiological relevance of the mouse model. Despite the shared genetic background and UV exposure of the murine cell lines, there were no missense mutations shared across all three cell lines and only 17 missense mutations shared across any two cell lines. Prophylactic vaccination with the same cell line provides complete protection from tumor challenge, but vaccination with an independent cell line, sharing three mutations and the highest number of shared predicted binding neoantigens, does not constrain tumor growth.ConclusionsThis work demonstrates a low number of shared neoantigens in human cSCC as well as in a physiologically-relevant mouse model. The lack of shared mutations and ineffective cross-protection suggests that immunotherapeutic approaches for cSCC need to be agnostic to the neoantigen or personalized.AcknowledgementsThis work was supported by the Training Oncology Physician Scientists Award from the University of Arizona Cancer Center (E.S.B), NIH (F30CA281056, E.S.B; T32CA009213, A.C.A: F30CA257622, A.C.A; P30CA023074, D.J.R; P01CA229112, K.T.H), and American Skin Association (A.C.A).ReferencesChang D and Shain AH. The Landscape of Driver Mutations in Cutaneous Squamous Cell Carcinoma. NPJ Genomic Medicine. 2021;6(1):61. https://doi.org/10.1038/s41525-021-00226-4Adams AC, Macy AM, Saboda K, et al. Solar Simulated Light Induces Cutaneous Squamous Cell Carcinoma in Inbred Mice: A Clinically Relevant Model to Investigate T-Cell Responses. J Invest Dermatol. 2021;141(12):2990–2993.e2996. doi:10.1016/j.jid.2021.06.005Ethics ApprovalThis study was approved by the University of Arizona’s Institutional Animal Care and Use Committee (Protocol #13–469).
A prototype multicomponent resistance index was constructed for use in the selection of parent trees for a Sitka spruce, Picea sitchensis Bong (Carr.), breeding program and screening of progeny for ...resistance against the white pine weevil, Pissodes strobi (Peck). Data describing density of outer resin ducts, bark thickness, amount of volatile foliar terpenes, amount of cortical diterpene resin acids, feeding and oviposition deterrent, and repellency were collected from a clonal trial on Vancouver Island. These data were combined into a resistance index based on their hypothesized importance in weevil host selection. Clones differed in their complements of resistance traits, indicating that resistance is probably polygenic. There was a significant negative relationship between the resistance index and number of weevil attacks for the clones examined, suggesting that the resistance index could predict the resistance status of the trees. This relationship was improved if feeding deterrency and repellency were removed from the index. Our results suggest that a multicomponent resistance index is a promising tool for screening for resistance to P. strobi
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