Immune Checkpoint Blockade Response Research Articles

Loss of BLK expression as a potential predictor of poor prognosis and immune checkpoint blockade response in NSCLC and contribute to tumor progression

BackgroundImmune checkpoint blockade (ICB) has been proved to have significant anti-tumor effect in the clinical treatment of non-small cell lung cancer (NSCLC). Therefore, biomarkers predicting ICB response can provide better treatment for patients with NSCLC. MethodsDifferential expression genes (DEGs) were identified by ImmuCellAI database. Copy number alteration (CNA) was analyzed by cBioPortal. The predicted efficiency of 4 genes on cancer immunotherapy was assessed by ROC analysis. The survival value of BLK was analyzed by Kaplan-Meier plotter and Prognoscan analysis. Clinical significance of BLK IHC-TMA score in NSCLC was also explored. The CCK-8 assay, wound healing assay, western blot assay in vitro and subcutaneous xenograft experiments in vivo were used for investigating the functions of BLK. The RNA-sequencing were performed to screen BLK regulated genes and conducted for GO/KEGG enrichment analysis. The transcriptional regulatory factor of BLK promoter region was predicted by ChIP-seq analysis. Results39 common DEGs between ICB Response (R) group and No Response (NR) group with NSCLC were identified, in which the CNA frequency of BLK deletion (> 6%) was found. The predicted efficiency of BLK on immunotherapy was performed best in NSCLC (AUC>0.7). Low expression of BLK was related to NSCLC with significantly poor prognosis. BLK overexpression can inhibit growth of NSCLC via activating apoptosis pathway, inhibiting the G2M checkpoint and Glycolysis pathway. The enrichment analysis indicated that BLK regulated genes related to oncogenic potential in NSCLC. Besides, BLK expression was inhibited via H3K27me3 modification in A549 and H1299 cells. BLK mRNA level was negatively correlated with methylation and positively correlated with the tumor purity in NSCLC. ConclusionOur study provides strong evidence that low expression of BLK may serve as a biomarker for poor prognosis in NSCLC, while response to ICB therapy and contributes to NSCLC tumor progression.

Neoadjuvant immune checkpoint blockade triggers persistent and systemic Treg activation which blunts therapeutic efficacy against metastatic spread of breast tumors

ABSTRACT The clinical successes of immune checkpoint blockade (ICB) in advanced cancer patients have recently spurred the clinical implementation of ICB in the neoadjuvant and perioperative setting. However, how neoadjuvant ICB therapy affects the systemic immune landscape and metastatic spread remains to be established. Tumors promote both local and systemic expansion of regulatory T cells (Tregs), which are key orchestrators of tumor-induced immunosuppression, contributing to immune evasion, tumor progression and metastasis. Tregs express inhibitory immune checkpoint molecules and thus may be unintended targets for ICB therapy counteracting its efficacy. Using ICB-refractory models of spontaneous primary and metastatic breast cancer that recapitulate the poor ICB response of breast cancer patients, we observed that combined anti-PD-1 and anti-CTLA-4 therapy inadvertently promotes proliferation and activation of Tregs in the tumor, tumor-draining lymph node and circulation. Also in breast cancer patients, Treg levels were elevated upon ICB. Depletion of Tregs during neoadjuvant ICB in tumor-bearing mice not only reshaped the intratumoral immune landscape into a state favorable for ICB response but also induced profound and persistent alterations in systemic immunity, characterized by elevated CD8+ T cells and NK cells and durable T cell activation that was maintained after treatment cessation. While depletion of Tregs in combination with neoadjuvant ICB did not inhibit primary tumor growth, it prolonged metastasis-related survival driven predominantly by CD8+ T cells. This study demonstrates that neoadjuvant ICB therapy of breast cancer can be empowered by simultaneous targeting of Tregs, extending metastasis-related survival, independent of a primary tumor response.

Small-molecule PIK-93 modulates the tumor microenvironment to improve immune checkpoint blockade response

Immune checkpoint inhibitors (ICIs) targeting PD-L1 immunotherapy are state-of-the-art treatments for advanced non-small cell lung cancer (NSCLC). However, the treatment response of certain patients with NSCLC is unsatisfactory because of an unfavorable tumor microenvironment (TME) and poor permeability of antibody-based ICIs. In this study, we aimed to discover small-molecule drugs that can modulate the TME to enhance ICI treatment efficacy in NSCLC in vitro and in vivo. We identified a PD-L1 protein-modulating small molecule, PIK-93, using a cell-based global protein stability (GPS) screening system. PIK-93 mediated PD-L1 ubiquitination by enhancing the PD-L1-Cullin-4A interaction. PIK-93 reduced PD-L1 levels on M1 macrophages and enhanced M1 antitumor cytotoxicity. Combined PIK-93 and anti-PD-L1 antibody treatment enhanced T cell activation, inhibited tumor growth, and increased tumor-infiltrating lymphocyte (TIL) recruitment in syngeneic and human peripheral blood mononuclear cell (PBMC) line-derived xenograft mouse models. PIK-93 facilitates a treatment-favorable TME when combined with anti-PD-L1 antibodies, thereby enhancing PD-1/PD-L1 blockade cancer immunotherapy.

Abstract 6548: Bioinformatic characterization of dendritic and T cell subpopulations in the tumor microenvironment across different cancer types by single-cell RNA-Seq analysis

Abstract Although the development of single-cell RNAseq (scRNAseq) has improved the knowledge of the tumor microenvironment (TME), there is not a clear understanding of specific cell subtypes, markers that define these cell populations, nor the interaction between them. Previously, we showed that inflammasome activation is correlated with antitumor responses triggered by immune checkpoint blockade (ICB). This highlights a central role for TMEM176B, mostly expressed in myeloid cells, that impairs the recruitment of cytotoxic immune cells in the TME by inhibiting NLRP3 inflammasome activation. Still, the roles of the different subsets of myeloid and T cells in the TME and to what extent they are connected remain controversial. Here, we used scRNAseq data from human tumors to identify and characterize cDC2, Th17 and exhausted CD8+ T cell subpopulations in cancer. Five scRNAseq data sets of melanoma, basal cell carcinoma, breast, lung, colorectal, and ovary cancer patients were integrated, accounting for 382,019 immune cells in total. We applied and compared different bioinformatic tools for scRNASeq data normalization and data sets integration to find conserved immune cell populations across samples, data sets, and cancer types. Then, we evaluated different parameters using well-established methods for clustering and cell type classification to identify subtypes of DCs and T cells in the integrated data. We combined automatic methods with manual analysis of cluster markers to improve robustness and confidence in the cell populations identified. Three clusters of cDC2 were defined and characterized using different scRNAseq analysis tools for pathway enrichment analysis and transcription factors (TF) activation prediction. We found a subset of inflammatory cDC2 with high gene expression of IL1B and NLRP3, in contrast with another cluster characterized by high expression of TMEM176B and CD14. A third cluster showed an intermediate transcriptomic profile. The TMEM176B+ CD14+ cluster was characterized by the activation of JAK-STAT and hypoxia pathways, and the TF FOXL2, ETS2, and ESR2. The NLRP3+ IL1B+ cluster showed more activation of MAPK, Trail, and PI3K pathways, and the TF CEBPE. Moreover, the activation of TNFα and NFkB signaling pathways was found in both clusters. In addition, a conserved population of Th17 cells in the integrated data was observed. Th17 cells were heterogeneous, making the subpopulations and transcriptome signature difficult to define. Further analysis is being performed to identify Th17 subsets and their potential interactions with the cDC2 subclusters. A robust identification and characterization of these immune cell subsets will build towards our understanding of the interactions between these subpopulations triggered by inflammasome activation within the TME and the mechanisms behind ICB response. Citation Format: Yamil D. Mahmoud, Natalia Rego, Marcela Vilarino, Florencia Veigas, Maria R. Girotti, Juan M. Perez Saez, Gabriel A. Rabinovich, Marcelo Hill. Bioinformatic characterization of dendritic and T cell subpopulations in the tumor microenvironment across different cancer types by single-cell RNA-Seq analysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6548.

Abstract 5830: Presence of androgens improves efficacy of PD1 blockade

Abstract Immune therapies have revolutionized the treatment of cancer, however, until now only a minority of patients derive long-term benefit. Notably, despite widely reported significant differences between the immune system of males and females, there continues to exist a knowledge gap regarding sex disparities in anti-cancer immune responses. Recent meta-analyses indicate that immune checkpoint blockade (ICB) treatment has higher efficacy in male patients compared to females, regardless of the cancer type. To investigate the mechanisms underlying these sex-specific differences we utilized the syngeneic colorectal cancer model MC38 because it mirrors the clinical situation yielding responding and non-responding mice. Therefore, we inoculated tumor cells subcutaneously (s.c.) into male and female mice, and treated them with either IgG2a control Ab or anti-PD1 Ab. When analyzing the tumor response to anti-PD1 treatment we observed significant sex-biased growth kinetics upon ICB favoring males. To asses a potential role of androgens in impacting observed sex differences to ICB response in vivo, we performed surgical androgen deprivation therapy (orchiectomy) in male mice, as well as sham-surgery in male and female mice as control. After a recovery period, we proceeded with s.c. cell injection and ICB treatment as previously described. Sham male mice showed a very high response rate to anti-PD1 (92%) compared to sham females (58%), but more importantly, testosterone suppression by surgical androgen deprivation therapy led to a lower response rate of castrated mice (71%). To further confirm whether the presence of testosterone was beneficial for ICB response, we implanted s.c. sham or testosterone pumps into male and female mice and proceeded as before. Males with sham pumps showed a very high response rate to anti-PD1 (90%) compared to females with sham pumps (50%), while testosterone supplementation in females led to enhanced response rate (85%). Overall, this demonstrated that regardless of biological sex, presence of testosterone is sufficient to positively impact ICB therapy outcome. Furthermore, immunophenotyping analysis by flow cytometry showed that females supplemented with testosterone and treated with anti-PD1 had significantly increased intratumoral stem-like CD8+TCF1+PD1+ T cells compared to sham females. Additionally, these findings also correlated with enhanced intratumoral terminal differentiated effector CD8+TCF1-PD1+ T cells in both sham males and females supplemented with testosterone, compared to sham females. In summary, androgens can modulate anti-cancer immune responses by contributing to a more sustained anti-tumor CD8+ T cell response and consequently better responses upon treatment with anti-PD1. These findings are in concordance with male cancer patients responding better to ICB and warrant therapy escalation in female patients. Citation Format: María Elena Vargas-Delgado, Lara Meier, Jonas Waizenegger, Julia Oberbauer, Nikolaus Berenbrok, Janik Engelmann, Victoria Gensch, Franziska Heilmann, Jochim Reinert, Kristoffer Riecken, Boris Fehse, Hannelore Lotter, Dorothee Schwinge, Christoph Schramm, Hans-Willi Mittruecker, Isabel Ben-Batalla, Sonja Loges. Presence of androgens improves efficacy of PD1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5830.

Abstract 5904: Intra-tumor microbes identified by RNAseq associated with response to immune checkpoint blockade in metastatic melanoma

Abstract The tumor microbiome has recently been shown to play a key role in the context of oncogenesis, cancer immune phenotype, cancer progression and treatment outcomes in a variety of cancers. We investigated the possible associations between tumor microbiome and successful treatment outcomes with immune checkpoint blockade (ICB) in patients with metastatic melanoma. We evaluated RNAseq from tumor samples, collected prior to the start of treatment with ICB, from 71 patients with metastatic melanoma. Samples were provided by eight members of the Oncology Research Information Exchange Network (ORIEN). Non-response was determined as change in treatment after less than 12 months. Patients maintaining the same treatment regimen for greater than 12 months were classified as responders. We applied our custom tool, {exotic} (Exogenous sequences in Tumor and Immune Cells), to carefully identify non-human sequences within the RNAseq data. After filtering reads aligning to the human reference genome, reads were further filtered of common laboratory contaminants, taxa inversely correlated with input RNA quantity, and taxa frequently found in the negative controls of microbiome experiments. A differential abundance analysis was performed on the response groups at every taxonomic level utilizing DESeq2. We calculated expression signatures using {tmesig}, and related them to ICB response using {IOSig}. We observed significantly enriched taxa (p-value < 0.05) with a high (>1.00) fold-difference in abundance between responders and non-responders found within the tumor RNAseq data, including Fusobacterium nucleatum and several viruses in responders, and Delftia lacustris and Fungi in non-responders. These microbes were associated with immune cell expression signatures, including Th17 cells and CD8+ T-cells. We calculated the gene expression scores of 30 signatures with literature precedence for the ability to predict ICB treatment outcomes in melanoma. The receiver operator characteristic (ROC) curve of the random forest classification model for prediction of response to ICB using the combined expression signature scores resulted in an AUC of 0.8750. Combining expression signature scores with microbe relative abundances at the genus level improved the ability to predict ICB response (AUC = 0.8958). Combining tumor expression signatures with curated tumor microbiome relative abundances improves the performance of predictive models for treatment outcomes with ICB in melanoma. Citation Format: Caroline E. Wheeler, Samuel Coleman, Rebecca Hoyd, Afaf Osman, Louis Denko, Aik Choon Tan, Daniel Spakowicz, Ahmad Tarhini. Intra-tumor microbes identified by RNAseq associated with response to immune checkpoint blockade in metastatic melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5904.

Abstract 2176: A cell mass-based platform for characterizing immune cell function and response to checkpoint blockade in primary blood and malignant fluid samples

Abstract Existing predictive biomarkers for immune checkpoint blockade (ICB) therapies that rely on characterization of tumor genomics or immunohistological assessment of the tumor microenvironment have demonstrated mixed success, with no single biomarker proving to be universally predictive of ICB treatment outcome. To complement these existing approaches, we present a workflow that relies on high-resolution single-cell mass measurements curated with CNN-based image classification to characterize T cell activation and functional response to treatment with ICB therapies. As a highly integrative measure of cellular phenotype, cell mass can be used as a label-free biomarker for characterizing immune cell function. For instance, upon activation with anti-CD3/CD28, T cells isolated from human PBMC showed a rapid and significant increase in cell mass. However, in T cells that had been repeatedly stimulated in vitro to generate a PD-1+ exhausted-like phenotype, this mass response to activation was blunted. We hypothesized that this variability in activation-induced mass response was the result of an altered balance of stimulatory and inhibitory “signal two” cues seen by the T cells. Consistent with this hypothesis, purified T cells stimulated with anti-CD3 alone (no co-stimulation) demonstrated a significantly smaller increase in cell mass when compared with those stimulated with both anti-CD3 and anti-CD28. Furthermore, the T cell response measured when unpurified PBMC were activated with anti-CD3 demonstrated an intermediate mass increase, suggesting contributions of both stimulatory and inhibitory signaling from other cells in the milieu. To explore how ICB treatment disrupts this co-stimulatory/inhibitory signaling balance, we measured T cell mass responses for samples treated with anti-CD3 alone or in combination with pembrolizumab, atezolizumab, nivolumab, or dostarlimab. These samples included PBMC isolated from blood and tumor/immune cell milieus isolated from malignant fluids (pleural effusions or abdominal ascites) collected from patients with advanced cancer. We found that T cells isolated from samples treated with both anti-CD3 and an ICB drug often showed a significantly greater mass increase than the same samples treated with anti-CD3 alone. This mass response showed significant heterogeneity across patients, with many samples showing no significant treatment effect. Despite all four of these therapies targeting ostensibly the same PD-1/PD-L1 signaling axis, we also found significant response heterogeneity to these different treatments within individual samples.These results suggest that mass response measurements capture biological heterogeneity associated with ICB response and may serve as a valuable complementary readout to existing biomarkers. Citation Format: Robert Kimmerling, Mark Stevens, Selim Olcum, Anthony Minnah, Madeleine Vacha, Rachel LaBella, Katie Katsis, Audrey Guo, Juanita Fujii, Zayna Shaheen, Srividya Sundaresan, Anobel Tamrazi, Cliff Reid. A cell mass-based platform for characterizing immune cell function and response to checkpoint blockade in primary blood and malignant fluid samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2176.

Abstract 141: Interrogating subclonal mechanisms of immune evasion and immunotherapy resistance in melanoma

Abstract Immune checkpoint blockade (ICB) therapies are the current first-line melanoma treatments within the clinic; however, their specific mechanisms for combatting tumor progression remain unclear and determinants for efficacious and durable responses within patients remain unknown. Intratumor heterogeneity (ITH) provides cancers with significant adaptability, leading to metastatic progression and resistance to treatment, but its complexity often complicates its study. To understand how ITH contributes to ICB response, we derived 24 clonal sublines from single cells of an ICB-sensitive and genetically and phenotypically heterogenous mouse model of melanoma. Genomic and transcriptomic analyses uncovered the diversity of these sublines and their plasticity while in vivo studies demonstrated a wide range of responses to ICB therapy including resistant, partially sensitive, and fully responsive. Distinct responses to anti-CTLA-4 monotherapy compared to combination (anti-CTLA-4 plus anti-PD-L1) therapy were also observed, and further exploration of untreated tumors revealed correlations between highly inflamed and differentiated phenotypes with response to anti-CTLA-4. To better understand changes in plasticity and immune profile throughout the course of treatment, tumors were harvested from the parental cell line prior to treatment and at several timepoints after treatment with anti-CTLA-4 monotherapy. These samples are undergoing high throughput spatial transcriptomics analysis to map the melanoma phenotypes characterized in the clonal sublines with changes in the immune compartment. Furthermore, blood samples were collected to investigate inflammatory profiles of cytokines and chemokines to describe the evolution of the tumor microenvironment throughout the course of immunotherapy. Further studies will allow us to identify predictive and prognostic biomarkers of resistance and relapse upon ICB therapy, respectively. Overall, the 24 clonal sublines serve as a valuable tool to model tumor dynamics associated with ICB efficacy and understand the complex role melanoma plasticity plays in immune evasion mechanisms. Citation Format: Isabella Church, Charli Gruen, Antonella Sassano, Julio Valencia, Emily Wu, Howard Yang, Gabe Needle, Cari Smith, Sung Chin, Jessica Ebersole, Christina Marcelus, Anyen Fon, Huaitian Liu, Chi-Ping Day, Howard Young, Maxwell Lee, Glenn Merlino, Eva Pérez-Guijarro. Interrogating subclonal mechanisms of immune evasion and immunotherapy resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 141.

Abstract 4157: Tumor context dictates reliance on TCF1 for response to immunotherapy

Abstract Stem-like CD8 T cells are regulated by the transcription factor TCF1 and are key players in the response to immune checkpoint blockade (ICB). However, recent findings indicate that the dependance on TCF1+ stem-like T cells for ICB efficacy may not be equal across patients or in different tumor contexts. Here we leveraged TCF1 conditional knock-out (TCF1 cKO) mice to investigate how TCF1 instructs the early fate and functions of CD8 T cells upon ICB therapy in tumors that differ for immunogenicity and levels of tumor antigen expression. Strikingly, we discovered that TCF1 expression in CD8 T cells is required for ICB efficacy in poorly immunogenic B16OVA melanomas but is dispensable in highly immunogenic MC38OVA colorectal tumors. Single-cell RNA sequencing and immunophenotyping in the tumor draining lymph node (TDLN) revealed defective priming and expansion of tumor-specific TCF1 cKO T cells in B16OVA- but not MC38OVA-bearing mice treated with ICB. Conversely, ICB therapy efficiently expanded tumor-specific TCF1 WT T cells in the TDLN of both tumor models. In vitro, we found defective proliferation, reduced PD1 and CD28 up-regulation and reduced phosphorylation of key molecules downstream the T cell receptor pathway in TCF1 cKO T cells stimulated with low but not high TCR signals. These data indicate that TCF1 poises T cells for optimal responsiveness in suboptimal priming conditions such as those found in low antigen expressing tumors. Furthermore, in the absence of TCF1 we found the accumulation in the TDLN of a subset of tumor-specific naïve T cells poised to give rise to short-lived effectors, which are less suited to sustain anti-tumor responses in poorly immunogenic tumors where expansion of T cells retaining memory potential is required for durable responses. Single-cell RNA sequencing and immunophenotyping within the tumor microenvironment showed that in MC38OVA tumors both WT and TCF1 cKO mice expanded a CD8 subset sharing a signature with transitory effectors, which were shown to mediate ICB efficacy in chronic viral infection models. Expansion of cytotoxic CD8 T cells likely accounted for the strong anti-tumor response observed in both WT and TCF1 cKO mice. Conversely, B16OVA tumors failed to expand transitory effectors and accumulated Tox+ dysfunctional CD8 T cells. Importantly, in the absence of TCF1 dysfunctional T cells became destabilized, failed to persist and shared features with CD8 T cells found in patients that fail ICB. Altogether, the reduced priming of stem-like T cells in the TDLN combined with a destabilized dysfunctional T cell state in the tumor contributed to the failure of TCF1 cKO mice to sustain effective ICB responses in poorly immunogenic tumors. Our study highlights a role for TCF1 during the priming and early stages of the anti-tumor CD8 T cell response with important implications for guiding optimal therapeutic interventions in cancers with low frequency of TCF1+ CD8 T cells and low neoantigen expression. Citation Format: Giulia Escobar, Katherine Tooley, Joan Pages Oliveras, Linglin Huang, Hanning Cheng, Chang Xue, Davide Mangani, Natanael Hazel, Carola Rutigliani, Luca Biasco, Ana C. Anderson. Tumor context dictates reliance on TCF1 for response to immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4157.

Abstract 1393: Persistent mutation burden drives sustained anti-tumor immune responses in human cancers

Abstract INTRODUCTION: Tumor mutation burden (TMB) is a commonly used biomarker for cancer immunotherapy however TMB only partially captures tumor foreignness. We hypothesized that mutations in single-copy regions of the genome or mutations present in multiple copies (hereafter referred to as persistent mutations) are retained during cancer evolution and immunoediting, may render the tumor continuously visible to the immune system and promote sustained tumor control during immune checkpoint blockade (ICB). METHODS: We performed pan-cancer analyses of whole exome sequencing data across 31 tumor types in TCGA to quantify the landscape of persistent mutations (n=9,242). We then evaluated the association between persistent tumor mutation burden (pTMB) and ICB response compared to TMB in eight ICB-treated cohorts of patients with NSCLC, melanoma, mesothelioma, and head and neck cancer (n=524). To investigate the clonal evolution of persistent mutations we serially analyzed whole exome sequence data from NSCLCs prior to and at emergence of acquired resistance to ICB. Finally, we evaluated the composition of the tumor microenvironment (TME) in baseline and on-ICB melanomas by RNA sequencing differential enrichment analyses and deconvolution. RESULTS: Integration of sequence alterations in only-copy and multi-copy states for 9,242 tumors across 31 tumor types revealed a cancer lineage-dependent distribution of persistent mutations that was largely independent of the overall TMB. In evaluating differential classification based on pTMB- vs TMB-high, we found re-classification rates as high as 53% in individual tumor types, with a median reclassification rate of 33% across all tumor types (range 15% - 53%). We then evaluated the clonal composition of persistent mutations and found a wide range of correlations between pTMB and fraction of clonal mutations (Spearman ρ range: -0.11 - 0.59). In ICB-treated cohorts, pTMB better distinguished responding tumors compared to TMB, and a number of mutation and copy-number related features including tumor aneuploidy (melanoma: Mann-Whitney p=2.3e-06, NSLC: p<2.0e-03, mesothelioma p=0.03, HNSCC p=0.05). Using in silico simulations, we found a similar advantage for pTMB when estimated from gene-panel targeted next generation sequencing. To support the biological plausibility of pTMB in the context of tumor evolution, we evaluated the rate loss of persistent mutations in longitudinal analyses of pre- and post-ICB NSCLC and found that a rate of loss significantly lower for persistent compared to loss-prone mutations (odds ratio 61.43, p<2.2e-16). Consistent with our hypothesis, pTMB-high tumors had a more inflamed TME (p<1e-11). CONCLUSIONS: Persistent mutations represent a biologically distinct subset within the overall TMB that is unlikely to be lost under selective pressure of ICB and may function as an intrinsic driver of sustained immunologic tumor control. Citation Format: Noushin Niknafs, Archana Balan, Christopher Cherry, Karlijn Hummelink, Kim Monkhorst, Xiaoshan M. Shao, Zineb Belcaid, Kristen A. Marrone, Joseph Murray, Kellie N. Smith, Benjamin Levy, Josephine Feliciano, Christine L. Hann, Vincent Lam, Drew M. Pardoll, Rachel Karchin, Tanguy Y. Seiwert, Julie R. Brahmer, Patrick M. Forde, Victor E. Velculescu, Valsamo K. Anagnostou. Persistent mutation burden drives sustained anti-tumor immune responses in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1393.

Abstract 5472: Utilizing response in immune checkpoint inhibitor treated cohorts improves clinical applicability of neoantigen immunogenicity predictions

Abstract Introduction: Neoantigen-based biomarkers have improved predictions of response to immune checkpoint blockade (ICB) therapy, highlighting the importance of accurate prediction of immunogenic neoantigen candidates. A challenge in developing robust immunogenicity prediction models is limited availability of sequencing-associated immunogenicity data for evaluating methods due to the complexity of generating such datasets. We propose a novel approach to optimize prediction models of immunogenic neoantigens using a meta-analysis framework based on multiple ICB cohorts. Methods: To build on the 110 mono-allelic immunopeptidomics-derived SHERPA® MHC binding prediction framework, we engineered T-cell recognition features on two datasets: peptide-centric data aggregated by Schmidt et al. and patient-specific exome and transcriptome sequencing data from the TESLA consortium. We developed two-tiered models based on the feature landscapes of both datasets to predict peptide-MHC (pMHC) immunogenicity, incorporating features with significant performance gains. We systematically re-processed publicly available DNA and RNA sequencing data from over 500 ICB treated patients spanning 12 different cohorts across five different cancer types with a harmonized bioinformatics pipeline. We then evaluated the performance of each model consisting of a unique combination of immunogenic features across the ICB training (N=7) and validation (N=5) cohorts using a meta-analysis framework. Results: We evaluated iterations of SHERPA-Immunogenicity (SI) models using the Schmidt et al. and TESLA datasets, resulting in a range of performance metrics (area under the precision recall curves of 0.74-0.84 and positive predictive values of 0.32-0.54). After aggregating pMHC predictions into patient-specific scores based on the most immunogenic peptide present (SHERPA-Immunogenicity Maximum - SIM) or the quantity of immunogenic peptides identified (SHERPA-Immunogenicity Burden - SIB), we observed that responders had higher SIM and SIB scores compared to non-responders across the melanoma training cohorts. We found SIM scores outperformed SIB scores, suggesting the degree of epitope immunogenicity may be a critical factor in predicting response. The model with the most significant meta p-value for ICB response in melanoma cohorts (OR=2.43, p=0.006) also predicted overall survival in 3/5 melanoma cohorts (p&lt0.05). Conclusions: We developed a novel framework to predict neoantigen immunogenicity utilizing meta-analysis of ICB cohorts to overcome dataset limitations and gain prediction performance confidence. We look forward to supporting personalized cancer vaccine development with our pMHC immunogenicity predictions and applying our predictive biomarker on additional ICB cohorts. Citation Format: Hima Anbunathan, Neeraja Ravi, Rachel Marty Pyke, Steven Dea, Richard O. Chen, Sean Michael Boyle. Utilizing response in immune checkpoint inhibitor treated cohorts improves clinical applicability of neoantigen immunogenicity predictions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5472.

Abstract 6667: Pan-cancer assessment of tumour and peripheral T-cell receptor repertoire dynamics in patients treated with immune checkpoint inhibitors

Abstract Introduction: Clinical benefit from Immune Checkpoint Blockade (ICB) is a function of local T-cell specificity for tumor-associated antigens. However, overcoming local T-cell dysfunction necessitates systemic immunity engagement. Therefore, studying the dynamics of both local and peripheral T-cell repertoires in response to ICB is required to identify features of T-cell repertoires associated with pathological response. Methods: We conducted TCRβ-sequencing on tumor-residing T-cells (n=59), Peripheral Blood Mononuclear Cells (PBMCs) (n=306) and cell-free DNA (cfDNA) (n=73) from pre- and multiple on-ICB timepoints collected from patients enrolled in the pan-cancer INvestigator-initiated Phase II Study of Pembrolizumab Immunological Response Evaluation (INSPIRE; NCT02644369) trial. To assess specificity-agnostic shifts in TCR repertoires, we first compared TCR diversity and clonal expansion in longitudinal tumor and PBMC samples. Then, to temporally track the specificity-associated features of local and systemic TCR repertoires, we leveraged a Graph Neural Network (GNN) model that took in unique TCRβ chains as nodes. The connectivity between the nodes was defined by multi-relational edges that represented VJ-gene usage and GLIPHII-identified (Grouping Lymphocyte Interactions by Paratope Hotspots) specificities derived from a compendium of TCR sequences with empirically confirmed specificities. Results: While absolute diversity and clonal expansion values in baseline tumor (n=33) were not associated with response to ICB, changes in these values were informative between pre- and on-ICB tumors. All patients (n=4) with low baseline tumor TCR diversity and lack of clonotypic re-structuring in tumor TCR repertoire on-ICB had either progressive or short-term stable disease. Furthermore, pairwise comparison of pre- and on-ICB tumors for each patient (n=17) revealed that all the patients, irrespective of their pathological response, experienced emergence of new TCR clonotypes (i.e., clonal replacement) in response to ICB, suggesting only a minority of these TCRs might consist of tumor-associated clonotypes. Patients with clinical benefit also had higher degree of GLIPHII-identified clustering at baseline tumor, highlighting the role of both specificity-agnostic and specificity-centric TCR analysis in determining the response to ICB. Analysis of TCR sequences in blood plasma found cfDNA contains a small number of TCR sequences (median 32, range 12-89) enriched for TCRs found in matched tumor tissues, suggesting that cfDNA TCR repertoire may provide an indirect measurement of tumor-residing T-cells. Conclusions: TCR diversity and functional clonal annotation are emerging biomarkers of ICB response and cfDNA TCR repertoire can potentially be exploited for clinical diagnostics and monitoring. Citation Format: Shirin Soleimani, Ben X. Wang, Stephanie Pedersen, Jenna Eagles, Jacob Brick, Marcus O. Butler, Scott V. Bratman, Lillian L. Siu, Pamela S. Ohashi, Trevor J. Pugh. Pan-cancer assessment of tumour and peripheral T-cell receptor repertoire dynamics in patients treated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6667.

Abstract 5188: Interrogating the role of the immune microenvironment in the response of brain metastases to immunotherapy using new preclinical melanoma models

Abstract Brain metastases (BrM) remain an intractable, deadly complication for advanced melanoma patients and efficient therapeutic strategies are desperately needed. The tumor microenvironment (TME) plays an important role in response to therapy. However, studies addressing the contribution of the TME to therapy efficacy for BrM are lacking, mostly due to limited access to human samples and scarcity of appropriate preclinical models. Here, we describe two novel isogenic immunocompetent BrM models generated by intracardiac injection of UV-induced mouse melanoma cell lines, representative of mutant-RAS human melanoma subtypes. We used these models to test immune checkpoint blockade (ICB) therapy and to interrogate the role of the TME in therapeutic efficacy. To evaluate response, we developed and applied a new machine-learning method to quantify metastatic burden. We investigated the TME by high-parametric flow cytometry and single-cell RNA sequencing (scRNA-seq). We showed that the models have distinct metastatic behaviors, with BR1 being mostly brain tropic and BR3 displaying widespread metastases. Notably, BR1 BrM were sensitive to ICB with a better response to anti-PD-L1/anti-CTLA-4 combination therapy as compared to monotherapies. In contrast, BR3 BrM were resistant to both mono- and combination therapies. Interestingly, we found that ICB efficacy on extracranial BR3 metastases is organ-dependent. Characterization of the BrM immune microenvironment before and after treatment revealed dramatic differences between the models. Untreated BR1 BrM showed significant recruitment of T cells, dendritic cells, and natural killer cells, while neutrophils were enriched in untreated ICB-resistant BR3 BrM. Moreover, we uncovered phenotypically distinct microglia populations exclusively present in ICB-sensitive BR1 BrM that positively correlated with T cell infiltration. Consistent with this finding, scRNA-seq showed upregulation of genes encoding for T cell-attracting chemokines and antigen presentation uniquely in the BR1-associated microglia. Post-treatment analysis of the brain TME highlighted beneficial changes induced by ICB in the responsive BR1 model, including increased recruitment of CD8 T cells with an activated phenotype, while a mild recruitment of exhausted T cells was observed in the resistant BR3 model. Altogether, our data emphasize the importance of interrogating the BrM TME to understand therapeutic response. Our unique BrM models, mirroring the diversity of ICB response observed in patients, provide a robust platform for the much-needed mechanistic studies to optimize BrM therapy. Deciphering the contribution of the newly identified BR1 BrM-associated microglia to ICB efficacy will be crucial to the identification of novel therapeutic targets. Citation Format: Amélie Lopès, Jessica Rappaport, Eva Pérez Guijarro, Quanyi Chen, Emily Wu, Isabella Church, April Huang, Jessica Bridge, Sung Chin, Cari Smith, Charli Gruen, Khiem C. Lam, Romina E. Araya, Antonella Sassano, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid. Interrogating the role of the immune microenvironment in the response of brain metastases to immunotherapy using new preclinical melanoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5188.

Abstract 5390: Robust prediction of pan-cancer immune checkpoint blockade response using machine learning

Abstract Immune checkpoint blockade (ICB) has revolutionized our approach to cancer treatment. However, the response rate is still low. With the accumulation of data, efforts to use these data to build machine learning predictors of ICB response are rising. However, as most datasets are still quite small, pertaining machine learning models may often ‘overfit’ the data, i.e., have a much weaker performance on independent test data than on the data they were learned upon. Here we analyzed ~2000 patient samples from multiple cohorts across 16 solid tumor types. Based on 10 genomic & clinical features, we built different machine learning models, comparing their performance in terms of the Area Under ROC Curve (AUC) and importantly, in terms of the AUC difference between training vs validation sets, using a standard cross validation procedure. As a result, the Linear LASSO Regression (LLR) model outperformed all other models by having the highest AUC on the validation set (0.74) and notably, the smallest AUC difference between training and validation sets (0.02). In contrast, these two AUC values for the FDA approved tumor mutational burden (TMB) biomarker are 0.63 and 0.03. The highest predictive features were, receiving chemotherapy before ICB treatment or not, TMB, cancer type, blood albumin level, blood neutrophil-to-lymphocyte ratio, and patient age. The LLR score also consistently predicted overall survival and progression-free survival across all individual cancer types. More importantly, ICB response probability increased near-monotonically from 0% to > 43% with the LLR score, which is an important feature for patient exclusion. In contrast, ICB response probability is ~25% when TMB = 0 and not always higher with higher TMB. In summary, the LLR model identifies key features predicting pan-cancer ICB response and survival. The use of combined genomic and clinical features holds potential to further facilitate clinical ICB patient stratification beyond TMB. Citation Format: Tiangen Chang, Saugato R. Dhruba, Yingying Cao, Luc G. Morris, Eytan Ruppin. Robust prediction of pan-cancer immune checkpoint blockade response using machine learning. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5390.

PD-L1 Tumor Expression as a Predictive Biomarker of Immune Checkpoint Inhibitors' Response and Survival in Advanced Melanoma Patients in Brazil.

Immune checkpoint blockade (ICB) agents are prominent immunotherapies for the treatment of advanced melanoma. However, they fail to promote any durable clinical benefit in a large cohort of patients. This study assessed clinical and molecular predictors of ICB response and survival in advanced melanoma. A retrospective analysis was performed on 210 patients treated with PD-1 or CTLA-4 inhibitors at Barretos Cancer Hospital, Brazil. PD-L1 expression was assessed by immunohistochemistry using formalin-fixed paraffin-embedded tumor tissues collected prior to ICB therapy. Patients were divided into responders (complete and partial response and stable disease for more than 6 months) and non-responders (stable disease for less than 6 months and progressive disease). Among them, about 82% underwent anti-PD-1 immunotherapy, and 60.5% progressed after the ICB treatment. Patients that received ICB as first-line therapy showed higher response rates than previously treated patients. Higher response rates were further associated with superficial spreading melanomas and positive PD-L1 expression (>1%). Likewise, PD-L1 positive expression and BRAF V600 mutations were associated with a higher overall survival after ICB therapy. Since ICBs are expensive therapies, evaluation of PD-L1 tumor expression in melanoma patients should be routinely assessed to select patients that are most likely to respond.

Tertiary Lymphoid Structure Raises Survival and Immunotherapy in HPV− HNSCC

Immune checkpoint blockade (ICB) targeting PD-1/PD-L1 has been used for the treatment of head and neck squamous cell carcinoma (HNSCC). However, the overall response rate to ICB therapy for HNSCC remains less than 20%. It has recently been reported that the appearance of tertiary lymphoid structures (TLSs) in tumor tissue is correlated with better prognosis and response to ICB treatment. Here, we demonstrated an immune classification for the tumor microenvironment (TME) of HNSCC by analyzing The Cancer Genome Atlas (TCGA)–HNSCC data set and found that immunotype D with TLS enrichment had a better prognosis and response to ICB treatment. Furthermore, we observed that TLSs were present in a part of tumor samples of human papillomavirus (HPV) infection negative HNSCC (HPV− HNSCC) and were associated with the densities of dendritic cell (DC)–LAMP+ DCs, CD4+ T cells, CD8+ T cells, and progenitor T cells in TME. We established an HPV− HNSCC mouse model with TLS-enriched TME by overexpressing LIGHT in a mouse HNSCC cell line. We found that the induction of TLS formation enhanced the response to PD-1 blockade treatment in the HPV− HNSCC mouse model, accompanied by increases in DCs and progenitor exhausted CD8+ T cells in the TME. Elimination of CD20+ B cells attenuated the therapeutic effect of PD-1 pathway blockade in TLS+ HPV− HNSCC mouse models. These results indicate that TLSs contribute to the favorable prognosis and antitumor immunity of HPV− HNSCC. Inducing TLS formation in HPV− HNSCC tumors is a potential therapeutic method for improving the ICB response rate in patients with HPV− HNSCC.

Single-cell and bulk RNA sequencing identifies T cell marker genes score to predict the prognosis of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies, with limited biomarkers identified to predict its prognosis and treatment response of immune checkpoint blockade (ICB). This study aimed to explore the predictive ability of T cell marker genes score (TMGS) to predict their overall survival (OS) and treatment response to ICB by integrating single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data. Multi-omics data of PDAC were applied in this study. The uniform manifold approximation and projection (UMAP) was utilized for dimensionality reduction and cluster identification. The non-negative matrix factorization (NMF) algorithm was applied to molecular subtypes clustering. The Least Absolute Shrinkage and Selection Operator (LASSO)-Cox regression was adopted for TMGS construction. The prognosis, biological characteristics, mutation profile, and immune function status between different groups were compared. Two molecular subtypes were identified via NMF: proliferative PDAC (C1) and immune PDAC (C2). Distinct prognoses and biological characteristics were observed between them. TMGS was developed based on 10 T cell marker genes (TMGs) through LASSO-Cox regression. TMGS is an independent prognostic factor of OS in PDAC. Enrichment analysis indicated that cell cycle and cell proliferation-related pathways are significantly enriched in the high-TMGS group. Besides, high-TMGS is related to more frequent KRAS, TP53, and CDKN2A germline mutations than the low-TMGS group. Furthermore, high-TMGS is significantly associated with attenuated antitumor immunity and reduced immune cell infiltration compared to the low-TMGS group. However, high TMGS is correlated to higher tumor mutation burden (TMB), a low expression level of inhibitory immune checkpoint molecules, and a low immune dysfunction score, thus having a higher ICB response rate. On the contrary, low TMGS is related to a favorable response rate to chemotherapeutic agents and targeted therapy. By combining scRNA-seq and bulk RNA-seq data, we identified a novel biomarker, TMGS, which has remarkable performance in predicting the prognosis and guiding the treatment pattern for patients with PDAC.

RTP4 silencing provokes tumor-intrinsic resistance to immune checkpoint blockade in colorectal cancer.

Recent advances in immunecheckpoint blockade (ICB) have improved patient prognosis in mismatch repair-deficient and microsatellite instability-high colorectal cancer (dMMR/MSI-H CRC); however, PD-1 blockade has faced a challenge in early progressive disease. We aimed to understand the early event in ICB resistance using an in vivo model. We subcutaneously transplanted the MC38 colon cancer cells into C57BL/6 mice, intraperitoneally injected anti-PD-1 antibody and then isolated ICB-resistant subclones from the recurrent tumors. Comparative gene expression analysis discovered seven genes significantly downregulated in the ICB-resistant cells. Tumorigenicity assay of the MC38 cells knocked out each of the seven candidate genes into C57BL/6 mice treated with anti-PD-1 antibody and bioinformatics analysis of the relationship between the expression of the seven candidate genes and the outcome of cancer patients receiving immunotherapy identified Rtp4, an interferon-stimulated gene and a chaperon protein of G protein-coupled receptors, as a gene involved in ICB resistance. Immunohistochemical analysis of transplanted tumor tissues demonstrated that anti-PD-1 antibody failed to recruit T lymphocytes in the Rtp4-KO MC38 cells. Mouse and human RTP4 expression could be silenced via histone H3 lysine 9 (H3K9)trimethylation, and public transcriptome data indicated the high expression level of RTP4 in most but not all of dMMR/MSI-H CRC. We clarified that RTP4 could be silenced by histone H3K9 methylation as the early event of ICB resistance. RTP4 expression could be a promising biomarker for predicting ICB response, and thecombination of epigenetic drugs and immunecheckpoint inhibitors might exhibit synergistic effects on dMMR/MSI-H CRC.

Multi-Omics Analysis Reveals Intratumor Microbes as Immunomodulators in Colorectal Cancer.

Recent studies indicated that intratumor microbes are an essential part of the tumor microenvironment. Here, we performed an integrated analysis of genetic, epigenetic, and intratumor microbial factors to unravel the potential remodeling mechanisms of immune-cell infiltration (ICI) and tumorigenesis of colorectal cancer (CRC). We identified the components and structure of the intratumor microbiome as primary contributors to the difference in survival between ICI subtypes. Multiple tumor-infiltrating immune cells (TIICs) and immune-related genes were associated with intratumor microbial abundance. Additionally, we found that Clostridium was enriched in CRC patients who were nonsensitive to immune checkpoint blockade (ICB) therapy. We further provided clues that the intratumor microbes might influence the response to ICB therapy by mediating TIICs, especially MAIT (mucosa-associated invariant T) cells. Finally, three ICB-related TIICs and 22 of their associated microbes showed the potential to predict the response to ICB therapy (area under the receiver operating characteristic curve [AUC] = 89%). Our findings highlight the crucial role of intratumor microbes in affecting immune-cell infiltration patterns, prognosis, and therapy response of CRC and provide insights for improving current immunotherapeutic treatment strategies and prognosis for CRC patients. IMPORTANCE Using the multi-omics data from The Cancer Genome Atlas (TCGA) colorectal cancer (CRC) cohort, we estimated the tumor microenvironment (TME) infiltration patterns of patients and unraveled the interplay of gene expression, epigenetic modification, and the intratumor microbiome. This study suggests the impact of intratumor microbes on maintaining the tumor immune microenvironment in the pathogenesis of CRC and modulating the response to immune checkpoint blockade (ICB) therapy. We identified a set of combined features, including 3 ICB-related tumor-infiltrating immune cells (TIICs) and 22 of their associated microbes, that are predictive of ICB responses.

A Dual-Responsive STAT3 Inhibitor Nanoprodrug Combined with Oncolytic Virus Elicits Synergistic Antitumor Immune Responses by Igniting Pyroptosis.

Immune checkpoint blockade (ICB) therapy shows excellent efficacy against malignancies; however, insufficient tumor immunogenicity and the immunosuppressive tumor microenvironment (TME) are considered as the two major stumbling blocks to a broad ICB response. Here, a combinational therapeutic strategy is reported, wherein TME-reactive oxygen species/pH dual-responsive signal transducers and activators of transcription 3 inhibitor nanoprodrugs MPNPs are combined with oncolytic herpes simplex virus 1 virotherapy to synergistically ignite pyroptosis for enhancing immunotherapy. MPNPs exhibit a certain level of tumor accumulation, reduce tumor cell stemness, and enhance antitumor immune responses. Furthermore, the simultaneous application of oncolytic viruses (OVs) confers MPNPs with higher tumor penetration capacity and remarkable gasdermin-E-mediated pyroptosis, thereby reshaping the TME and transforming "cold" tumors into "hot" ones. This "fire of immunity" strategy successfully activates robust T-cell-dependent antitumor responses, potentiating ICB effects against local recurrence and pulmonary metastasis in preclinical "cold" murine triple-negative breast cancer and syngeneic oral cancer models. Collectively, this work may pave a new way and offer an unprecedented opportunity for the combination of OVs with nanomedicine for cancer immunotherapy.