Tumor Immune Activity Research Articles

Tumor immune microenvironment dynamics and outcomes of prognosis in non-muscle-invasive bladder cancer.

Agents that target PD-1 and PD-L1 have been developed in the treatment of bladder cancer (BC). However, the diversity of immune cell infiltration in non-muscle-invasive BC (NMIBC) and the dynamics of the microenvironment as it progresses to muscle-invasive/metastatic disease remains unknown. To assess tumor immune activity, hierarchical clustering was applied to 159 BC samples based on cellular positivity for the defined immune cellular markers (CD3/CD4/CD8/FOXP3/CD20/PD-1/PD-L1/LAG3/TIGIT), divided into two clusters. There was a "hot cluster" (25%) consisting of patients with a high expression of these markers and a "cold cluster" (75%) comprising those without. The expression of CD39, CD44, CD68, CD163, IDO1, and Ki67 was significantly higher in tumors in the hot cluster. Immunologically, high-grade T1 tumors were significantly hotter, whereas tumors that had progressed to muscle invasion turned cold. However, a certain number of high-grade NMIBC patients were in the cold cluster, and these patients had a significantly higher risk of disease progression. Using an externally available TCGA dataset, RB1 and TP53 alterations were more frequently observed in TCGA hot cluster; rather FGFR3, KDM6A, and KMT2A alterations were common in TCGA cold/intermediate cluster. Analyses of recurrent tumors after BCG therapy revealed that tumor immune activity was widely maintained before and after treatment, and high FGFR3 expression was detected after recurrence in tumors initially classified into the cold cluster. Collectively, we revealed the dynamics of the tumor microenvironment in BC as a whole and identified candidate molecules as therapeutic targets for recurrent NMIBC, e.g., after BCG therapy.

Tuning Immune-Cold Tumor by Suppressing USP10/B7-H4 Proteolytic Axis Reinvigorates Therapeutic Efficacy of ADCs.

Tuning immune-cold tumor hot has largely attracted attention to improve cancer treatment, including immunotherapy and antibody-drug conjugates (ADCs). Utilizing multiomic analyses and experimental validation, this work identifies a pivotal role for the USP10/B7-H4 proteolytic axis in mediating the interplay between tumor immune responses and ADC efficacy, particularly for sacituzumab govitecan (SG) in treating triple negative breast cancers (TNBCs). Mechanistically, the inhibition of autocrine motility factor receptor (AMFR)-mediated ubiquitylation of B7-H4 by the deubiquitinase USP10 leads to the stabilization of B7-H4, which suppresses tumor immune activity and reduces SG treatment effectiveness. Pharmacological inhibition of USP10 promotes the degradation of B7-H4, enhancing tumor immunogenicity and consequently improving the tumor-killing efficacy of SG. In preclinical TNBC models, suppression of USP10/B7-H4 proteolytic axis is effective in increasing SG killing efficacy and reducing tumor growth, especially for the tumors with the USP10high/B7-H7high signature. Collectively, these findings uncover a novel strategy for targeting the immunosuppressive molecule B7-H4 for cancer therapy.

Prognosis and metabolism with a Golgi apparatus-related genes-based formula in breast cancer.

The Golgi apparatus (GA), an organelle that processes, sorts, and transports proteins synthesized by the endoplasmic reticulum, is also involved in many cellular processes associated with cancer, such as angiogenesis, the innate immune response, and tumor invasion and migration. We aimed to construct a breast cancer (BC) prognosis prediction model based on GA-related genetic information to evaluate the prognosis of patients with BC more accurately than existing models and to stratify patients for clinical therapy. In this study, The Cancer Genome Atlas-breast invasive carcinoma was used as the training cohort, and the Molecular Taxonomy of Breast Cancer International Consortium cohort was used as the validation cohort. Using bioinformatics methods, we constructed a GA-related gene risk score (GRS). The GRS was used to divide BC patients into a high-GRS group and a low-GRS group, and functional analysis, survival analysis, mutation analysis, immune landscape analysis, and metabolic analysis were performed to compare the 2 groups. Finally, a nomogram was constructed for clinical application. The genes in the GRS model were mainly related to the glucose metabolism pathway, and the main mutations in the 2 groups of patients were mutations in TP53 and CHD1. The mutation rate in the high-GRS group was greater than that in the low-GRS group. The high GRS group had higher tumor immune activity glycolysis; the pentose phosphate pathway tended to be the dominant metabolic pathways in this group, while fatty acid oxidation and glutamine catabolism tended to be dominant in the low-GRS group. GA-related genes were used to construct a prediction model for BC patients and had high accuracy in predicting prognosis. The mutations associated with the GRS are mainly TP53 and CDH1. Interestingly, the GRS is correlated with glucose metabolism in terms of gene expression and functional enrichment. In summary, the role of GRS-related genes in glucose metabolism is worthy of further study.

Preliminary exploration of Trop2 ADC in combination with immune checkpoint inhibitors in the different molecular subtypes of invasive breast carcinoma.

e12558 Background: Human trophoblastic cell surface antigen 2 (Trop2) is a transmembrane protein overexpressed in breast cancer tissues. However, it is present at minimal levels in adult somatic tissues, making it an ideal target for treatment with antibody-drug conjugates (ADCs). Sacituzumab govitecan, an anti-Trop2 ADC, has been approved for the treatment of triple-negative breast cancer and urothelial cancer. Recent studies have demonstrated that human cytotoxic T lymphocytes can recognize Trop2. Accordingly, we aimed to assess the role of Trop2 in the tumor immune activity of different subtypes of breast cancer, and laying the foundation for further studies on the efficacy of the combination of Trop2 ADC and immune checkpoint inhibitors in breast cancer. Methods: We retrospectively retrieved tissues from 152 invasive breast carcinoma of no special type and 111 benign breast tumor tissues and undergoing surgical resection at our institution from 2018 to 2023. Immunohistochemistry was used to detect Trop2 and PD-L1 expression and lymphatic invasion in patients, and their correlations with clinicopathological factors were analyzed. Trop2 expression was evaluated using the H-score. The median as the cut-off value, a score of 0–240 indicated low expression, and 240–300 indicated high expression. Cases with CPS ≥ 1% were considered positive for PD-L1. Results: The expression was Trop2 in breast cancer tissues than in benign tumor tissues (P<0.0001). Immune infiltration analysis suggested that Trop2 expression was correlated with immune cell infiltration and the abundance (P<0.001). Kaplan–Meier plotter analysis demonstrated that high Trop2 expression was related to poor prognosis in patients with triple-negative breast cancer (overall survival: HR=1.53, P=0.029; relapse-free survival: HR=1.26, P=0.045). In contrast, Trop2 expression was not significantly associated with prognosis in other breast cancer subtypes. However, there was no statistically significant correlation found between PD-L1 expression and Trop-2 expression in breast cancer. Conclusions: The findings suggest that Trop2 correlates with poor prognosis and immune cell infiltration in breast cancer. This indicates the role of Trop2 as a potential prognostic biomarker, laying the foundation for further research focusing on the immune regulatory role of Trop2. [Table: see text]

Abstract PO1-15-10: Lower pre-treatment B-cell gene expression signatures correspond with improved overall survival with palbociclib + endocrine therapy in HR+/HER2- metastatic breast cancer: a biomarker analysis from the GEICAM/2013-02 PEARL trial

Abstract Introduction: CDK4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) is standard-of-care for HR+/HER2- metastatic breast cancer (MBC), but little is known about which gene expression and microenvironment features are associated with benefit from CDK4/6i or differentiate response compared to chemotherapy. We examined the relationship of gene expression signatures (GES) to progression-free (PFS) and overall survival (OS) in the GEICAM/2013-02 PEARL phase III randomized study of palbociclib + ET vs. capecitabine in aromatase inhibitor-resistant HR+/HER2- MBC (NCT02028507). Methods: RNA sequencing (RNAseq) was performed on baseline tumor samples from 328 patients. Intrinsic subtype was derived by the PAM50 predictor after HER2/estrogen receptor subgroup-specific normalization. Correlations to intrinsic subtype signatures, PAM50 proliferation signature, CCNE1 expression, and 10 GES were selected for analysis. After excluding 15 Normal-like samples, Cox models were fitted for PFS and OS endpoints on each selected variable for the combined study population as well as for each treatment arm, adjusted for disease site, prior ET sensitivity, prior chemotherapy for MBC, number of involved sites, treatment arm, and intrinsic subtype. P-values were adjusted based on the Bonferroni method, with an adjusted p-value < 0.05 defined as significant. Results: PAM50 intrinsic subtype (54.95% Luminal A, 38.98% Luminal B and 6.07% non-luminal (4.79% Her2-enriched and 1.28% Basal-like)) was associated with worse PFS with palbociclib + ET for non-luminal vs. Luminal A tumors (HR 3.19, 95% CI 1.65-6.16) and for Luminal B vs. Luminal A tumors (HR 1.90, 95% CI 1.28-2.82). PAM50 luminal status (Luminal A vs. Luminal B vs. non-luminal) was significantly associated with PFS with palbociclib + ET (p=0.0003) but not capecitabine (p=0.89). In the palbociclib + ET arm, lower expression of two B-cell-associated signatures (BCAS) correlated with improved OS - the Immune1 TCGA BRCA GES (PMID: 32573490) (HR 1.52, 95% CI 1.20-1.92, p=0.009) and the B-cell/T-cell cooperativity GES (PMID: 31730857) (HR 1.42, 95% CI 1.14-1.79, p=0.04). Interaction analysis for OS showed that when stratified by low and high expression using the median, the Immune1 TCGA BRCA GES had a significant interaction with treatment arm (p=0.014). On comparison of outcomes between treatment arms, low Immune1 TCGA BRCA GES expression was significantly associated with improved OS with palbociclib + ET compared to capecitabine (HR 0.44, 95% CI 0.26-0.74, p=0.03), whereas high expression was not associated with a significant OS difference (HR 1.73, 95% CI 1.07-2.79, p=0.40). Further exploratory analysis of 164 immune GES found that, after adjustment for confounders, the top eight GES associated with OS with palbociclib + ET were BCAS (unadjusted p-values < 0.005), including our pre-selected Immune1 TCGA BRCA and B-cell/T-cell cooperativity GES, and all eight negatively correlated with OS. In the capecitabine arm, this exploratory analysis failed to demonstrate a similar association between the immune GES and OS. Conclusion: PAM50 intrinsic subtype was significantly associated with PFS with second-line palbociclib + ET. Lower expression of BCAS was associated with improved OS outcomes with palbociclib + ET independent of intrinsic subtype, and for the Immune1 TCGA BRCA GES, lower expression corresponded with improved OS with palbociclib + ET compared to capecitabine. This informs recent findings that in HR+/HER2- breast cancer, tumor immune activity may be a negative predictor of CDK4/6i benefit. Citation Format: Yash Agrawal, Aranzazu Fernandez-Martinez, Miguel Gil-Gil, Christoph Zielinski, Manuel Ruíz- Borrego, Eva Ciruelos, Montserrat Muñoz, Mireia Margelí, Begoña Bermejo, Antonio Antón, Zsuzsanna Kahan, Tibor Csöszi, José L Alonso-Romero, Jose Angel García-Sáenz, Pedro Sánchez-Rovira, Elena Álvarez, J. Ignacio Chacón, Santiago González-Santiago, César A Rodríguez, Sonia Servitja, Adam D. Pfefferle, Jesús Herranz, Yuan Liu, Lisa A. Carey, Isabel Romero - Camarero, Rosalía Caballero, Charles M. Perou, Miguel Martín. Lower pre-treatment B-cell gene expression signatures correspond with improved overall survival with palbociclib + endocrine therapy in HR+/HER2- metastatic breast cancer: a biomarker analysis from the GEICAM/2013-02 PEARL trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-10.

PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

BackgroundPhosducin-like 3 (PDCL3) is a member of the photoreceptor family, characterized by a thioredoxin-like structural domain and evolutionary conservation. It plays roles in angiogenesis and apoptosis. Despite its significance, research on the biological role of PDCL3 in liver hepatocellular carcinoma (LIHC) remains limited. This study aims to explore the prognostic value and potential mechanisms of PDCL3 in cancer, particularly in LIHC, through bioinformatics analysis.MethodsRNA-seq data and corresponding clinical information for pan-cancer and LIHC were extracted from the TCGA database to analyze PDCL3 expression and survival prognosis. Differential expression of PDCL3 was analyzed using the HPA database. GO and KEGG enrichment analysis were performed for PDCL3-associated genes. The relationship between PDCL3 expression and various immune cell types was examined using the TIMER website. Clinical samples were collected, and immunohistochemistry and immunofluorescence experiments were conducted to validate the differential expression of PDCL3 in LIHC and normal tissues. In vitro assays, including CCK-8, wound healing, Transwell, and colony formation experiments, were employed to determine the biological functions of PDCL3 in LIHC cells.ResultsAnalysis from TIMER, GEPIA, UALCAN, and HPA databases revealed differential expression of PDCL3 in various tumors. Prognostic analysis from GEPIA and TCGA databases indicated that high PDCL3 expression was associated with poorer clinical staging and prognosis in LIHC. Enrichment analysis of PDCL3-associated genes revealed its involvement in various immune responses. TCGA and TIMER databases showed that high PDCL3 expression in LIHC decreased tumor immune activity by reducing macrophage infiltration. PDCL3 exhibited positive correlations with multiple immune checkpoint genes. Immunohistochemistry (IHC) and immunofluorescence (IF) experiments confirmed elevated PDCL3 expression in LIHC tissues compared to adjacent normal tissues. In vitro experiments demonstrated that PDCL3 promoted LIHC cell proliferation, migration, invasion, and colony-forming ability.ConclusionPDCL3 is highly expressed in various cancer types. Our study suggests that elevated PDCL3 expression in hepatocellular carcinoma is associated with poorer prognosis and may serve as a potential diagnostic biomarker for LIHC. PDCL3 may regulate the biological functions of LIHC by modulating immune infiltration. However, the precise regulatory mechanisms of PDCL3 in cancer warrant further investigation.

Targeting adipocyte-immune cell crosstalk to control breast cancer progression.

Adipocytes are crucial components of breast cancer and are involved in regulating the progression, therapeutic efficacy, and prognosis of breast cancer patients. Characterized by storing energy and producing a variety of secretory factors, adipocytes are responsible for inducing obesity and regulating the tumor immune activity. Adipocytes communicate with tumor infiltrating immune cells through the secreted adipokines, cytokines, and exosomes in the breast cancer TIME, which shapes the tumor supporting environment to facilitate the immune escape of tumor cells. In-depth studies of the crosstalk between adipocytes and TIME can not only provide a more comprehensive regulatory landscape of TIME, but also be conducive to screening novel targets for future precision targeted therapy. The aim of this review is to discuss recent studies for understanding the role of crosstalk between adipocytes and immune cells in shaping the breast cancer immune microenvironment.

A novel DNA damage repair gene-related prognostic model for evaluating the prognosis and tumor microenvironment infiltration of esophageal squamous cell carcinoma

BackgroundThis study aimed to investigate the potential prognostic value of DNA damage repair genes (DDRGs) in esophageal squamous cell carcinoma (ESCC) and their relationship with immune-related characteristics.MethodsWe analyzed DDRGs of the Gene Expression Omnibus database (GSE53625). Subsequently, the GSE53625 cohort was used to construct a prognostic model based on least absolute shrinkage and selection operator regression, and Cox regression analysis was used to construct a nomogram. The immunological analysis algorithms explored the differences between the potential mechanism, tumor immune activity, and immunosuppressive genes in the high- and low-risk groups. Of the prognosis model-related DDRGs, we selected PPP2R2A for further investigation. Functional experiments were conducted to evaluate the effect on ESCC cells in vitro.ResultsA 5-DDRG (ERCC5, POLK, PPP2R2A, TNP1 and ZNF350) prediction signature was established for ESCC, stratifying patients into two risk groups. Multivariate Cox regression analysis showed that the 5-DDRG signature was an independent predictor of overall survival. Immune cells such as CD4 T cells and monocytes displayed lower infiltration levels in the high-risk group. Additionally, the immune, ESTIMATE, and stromal scores in the high-risk group were all considerably higher than those in the low-risk group. Functionally, knockdown of PPP2R2A significantly suppressed cell proliferation, migration and invasion in two ESCC cell lines (ECA109 and TE1).ConclusionThe clustered subtypes and prognostic model of DDRGs could effectively predict the prognosis and immune activity of ESCC patients.

Clinical verification of the relationship between serum lipid metabolism and immune activity in breast cancer patients treated with neoadjuvant chemotherapy

BackgroundLipid metabolism has been recently reported to affect the prognosis and tumor immune activity in cancer patients. However, the effect of lipid metabolism on chemosensitivity in patients with breast cancer treated with neoadjuvant chemotherapy (NAC) remains unclear.MethodsWe examined 327 patients with breast cancer who were treated with NAC followed by curative surgery. The correlations between the serum levels of total cholesterol (TC) and triglyceride (TG) and the clinicopathological features, including the efficacy of NAC, neutrophil-to-lymphocyte ratio (NLR), and absolute lymphocyte count (ALC), were evaluated retrospectively.ResultsSerum TG levels were increased after NAC in all the subtypes, and the rate of change was the highest, especially in triple-negative breast cancer (TNBC) (21.0% → 48.1%). In addition, only TNBC patients with an objective response (OR) had significantly higher TG levels after NAC than those without (P = 0.049). Patients with a high ALC before NAC had significantly higher TG levels after NAC than patients with all breast cancer (P = 0.001), HER2-enriched breast cancer (P = 0.021), and TNBC (P = 0.008). Patients with a low NLR before NAC had significantly higher TG levels after NAC only among patients with TNBC (P = 0.025). In patients with human epidermal growth factor receptor 2-enriched breast cancer, the group with normal TC levels before NAC had significantly better OS than those with high TC levels (P = 0.013, log-rank test), and in patients with TNBC, the group with high TC levels after NAC had significantly better OS than those with normal TC levels (P = 0.014, log-rank test).ConclusionsGood systemic immune activity and chemosensitivity may be associated with lipid metabolism regulated by NAC in TNBC patients.

Clinicopathological Significance, Related Molecular Changes and Tumor Immune Response Analysis of the Abnormal SWI/SNF Complex Subunit PBRM1 in Gastric Adenocarcinoma.

Background: PBRM1 gene abnormalities were recently found to play a role in tumor development and tumor immune activity. This article will explore the clinicopathological and molecular changes and tumor immune activity of the abnormal SWI/SNF complex subunit PBRM1 in gastric adenocarcinoma (GAC) and its significance. Methods: The cBioPortal, LinkedOmics and TISIDB datasets were used to analyze the abnormality of the PBRM1 gene in GAC and its relationship with prognosis, related molecular changes and tumor-infiltrating lymphocytes (TILs). In addition, 198 GAC cases were collected to further study the relationship between the loss/attenuation of PBRM1 expression and clinicopathology, prognosis, microsatellite stability, PD-L1 expression and TIL in GAC. DNA whole-exome sequencing was performed on 7 cases of gastric cancer with loss of PBRM1 expression. Results: The cBioPortal data showed that PBRM1 deletion/mutation accounted for 7.32% of GAC and was significantly associated with several molecular changes, such as molecular subtypes of GAC. The LinkedOmics dataset showed that PBRM1 mutation and its promoter DNA methylation showed lower PBRM1 mRNA expression, and PBRM1 mutation cases showed significantly higher mRNA expression of PD-L1 (CD274). TISIDB data showed that PBRM1 abnormalities were significantly positively associated with multiple TILs. In our group of 198 cases, the loss/attenuation of PBRM1 expression was significantly positively correlated with intra-tumoral tumor infiltrating lymphocytes (iTILs) and deficient MMR and PD-L1 expression. Kaplan–Meier survival analysis showed that the overall survival of GAC patients with loss/attenuation of PBRM1 expression was significantly better (p = 0.023). iTIL was an independent prognostic factor of GAC. Loss of PBRM1 expression often co-occurs with mutations in other SWI/SNF complex subunit genes, and there are some repetitive KEGG signaling changes. Conclusion: Abnormality of the PBRM1 gene may be related to the occurrence of some GACs and can affect tumor immune activity, thereby affecting clinicopathology and prognosis. It may be a potentially effective predictive marker for immunotherapy and a novel therapeutic approach associated with synthetic lethality.

Glucose-6-phosphate dehydrogenase correlates with tumor immune activity and programmed death ligand-1 expression in Merkel cell carcinoma

BackgroundMerkel cell carcinoma (MCC) is a rare and highly malignant skin cancer. Some cases have a good prognosis and spontaneous regression can occur. Reported prognostic markers, such as Merkel cell...

Excess glucocorticoid activity may suppress tumor immune activity in adrenocortical carcinoma

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract LB-130: Suppression of tumor immune activity in adrenocortical carcinoma with excess glucocorticoid

Abstract Introduction: Elevated glucocorticoid (GC) activity, while difficult to accurately quantify, has been implicated in the pathophysiology of multiple cancer types. Approximately half of adrenocortical carcinoma (ACC) patients exhibit excess GC (GC+), which provides a unique test case to assess correlates of GC activity. The broad immunosuppressive effects of GC may limit tumor immune response and immune checkpoint inhibitor (ICI) efficacy. ACC multi-omics were analyzed to identify the molecular consequences of GC activity and assess the rationale for combining relacorilant, a glucocorticoid receptor (GR) antagonist, with an ICI in GC+ ACC. Methods: GC status, mRNA expression, DNA mutation, and DNA methylation data from distinct adrenal resections (n=71) were accessed via The Cancer Genome Atlas (TCGA). To deconvolute immune cell type abundance, xCell was applied to the mRNA data. Random forest was used to derive gene signatures. Results: The expression of 858 genes differed significantly between GC- and GC+ ACC cases. KEGG pathway analysis showed higher gene expression of 7 pathways involved in steroid synthesis and secretion in GC+ cases. Nineteen pathways showed lower expression, most of which were related to natural killer cells, T-cells, and immune activity. Hypomethylation was primarily observed in the steroid synthesis pathways. Tumor-infiltrating CD4+ memory (P=.003), CD8+ memory (P<.001), and NKT-cells (P=.014) were depleted in GC+ cases, while tumor-associated neutrophils were enriched (P<.001). Higher tumor mutation burden (TMB) was observed in the GC+ cases (P=.029). A gene signature predictive of GC status in ACC was derived and applied to other cancer types to identify tumors with GC+-like transcriptional profiles. Conclusions: Reduced abundance of immune cells and immune-related transcripts in GC+ ACC provides insight into the mechanisms by which GC may limit response to ICI therapy. GR antagonism may increase immune related transcripts, thus promoting tumor immune response in GC+ ACC and other malignancies with elevated GC activity. This hypothesis will be tested in a Phase 1 trial of relacorilant + ICI. Citation Format: Andrew E. Greenstein, Mouhammed Amir Habra, Marzena Mura, Paweł Biernat, Marek J. Piatek, Andreas Grauer, Stacie Peacock Shepherd. Suppression of tumor immune activity in adrenocortical carcinoma with excess glucocorticoid [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-130.

Abstract P4-05-24: A stroma-adjusted ZEB1 expression signature is associated with decreased immune cell infiltration and poor overall survival in breast cancer

Abstract The epithelial-to-mesenchymal transition (EMT) is a critical physiologic process which can be activated by cancer cells to enhance metastatic and chemoresistant properties in experimental models. Recent studies have identified positive associations between the expression of EMT-associated gene signatures with both the immune response and survival in multiple cancer types. Although these studies relied upon whole-tumor RNA sequencing data, which can be confounded by infiltrating immune and stromal cells, they failed to investigate this potential confounding effect. In this study, we comprehensively dissected the associations between the expression of multiple EMT transcription factors with stromal and immune cell tumor infiltration in breast cancer. We demonstrated that previously-reported associations between the expression of EMT marker genes and immune activity were confounded by stromal cell infiltration. Adjusting for this confounding effect eliminated these reported associations. Interestingly, we discovered that stroma-adjusted ZEB1 expression has a unique, inverse association with immune cell activity in multiple cancer types, most significantly in breast cancer. Further analysis revealed that ZEB1 is significantly correlated with reduced tumor infiltration of multiple immune cell types. By deriving a high-confidence stroma-adjusted ZEB1-activated gene signature, we confirmed that ZEB1 transcriptional activity has similar associations with both tumor immune activity and stromal content as ZEB1, thus identifying potential mechanisms underlying decreased immune cell infiltration. Finally, multivariate survival analysis reveals that the ZEB1 gene signature has significant associations with reduced overall survival in breast cancer patients. In conclusion, this study identifies a novel association between stroma-adjusted ZEB1 expression and tumor immune activity and addresses the critical issue of confounding between EMT-associated genes and tumor stromal content. Citation Format: C James Block, Guojun Wu. A stroma-adjusted ZEB1 expression signature is associated with decreased immune cell infiltration and poor overall survival in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-24.

A stroma-corrected ZEB1 transcriptional signature is inversely associated with antitumor immune activity in breast cancer

The epithelial-to-mesenchymal transition (EMT) is an essential developmental process which can be hijacked by cancer cells, leading to enhanced metastasis and chemoresistance in experimental models. Recent studies have linked gene expression of EMT-associated gene signatures to increased inflammatory immune response in multiple cancer types. However, these studies did not account for the potential confounding effects of gene expression by tumor-infiltrating mesenchymal stromal cells. In this study, we comprehensively dissect the associations between multiple EMT transcription factors and EMT markers with stromal and immune tumor infiltration. We find that EMT-related genes are highly correlated with intratumoral stromal cell abundance and identify a specific relationship between stroma-corrected ZEB1 expression and decreased immune activity in multiple cancer types. We derive a stroma-corrected ZEB1-activated transcriptional signature and demonstrate that this signature includes several known inhibitors of inflammation, including BMPR2. Finally, multivariate survival analysis reveals that ZEB1 and its expression signature are significantly associated with reduced overall survival in breast cancer patients. In conclusion, this study identifies a novel association between stroma-adjusted ZEB1 expression and tumor immune activity and addresses the critical issue of confounding between EMT-associated genes and tumor stromal content.

Abstract 3416: APOBEC3C-H gene expression and APOBEC3-mediated RNA editing in breast cancer tumors are associated with immune response and improved outcome

Abstract APOBEC enzymes are strong mutagens in breast cancer. High APOBEC3B (A3B) expression in tumors is associated with poor prognosis, but clinical significance of the other APOBEC3s (A3A, C-H) is unclear. We investigated the associations of A3A and C-H with survival and tumor immune activity in 1091 primary breast cancer tumors of The Cancer Genome Atlas project. Tumor cytolytic activity, T cell receptor (TCR) diversity, and immune cell fractions were estimated from RNA sequencing data. Patients were divided into 3 equal groups by gene expression to compare high and low expressors. Cox, Spearman and t tests were used for survival, correlation and comparison analyses, respectively. Hallmark gene-sets were used for enrichment analysis. RNA editing was determined from whole exome and RNA sequencing data for 5,208 sites of 3,630 gene transcripts that are known to undergo APOBEC3-mediated RNA editing. Examination of 55 breast cancer cell-lines of Cancer Cell Line Encyclopedia showed that A3B and A3C represented 91% of A3 gene expression. Examination of Illumina Human Body Map showed that all APOBEC3s are expressed in leukocytes at a 4-450x higher level compared to breast. In patients, expression of A3B but not other APOBEC3s was higher in tumors compared to normal tissue (4.5x). A3C-H expression levels correlated positively with both leukocyte and lymphocyte fractions in tumor (r = 0.29-0.70 and 0.20-0.50, resp.). Expression of genes related to immune function like interferon response and complement activation was enriched in high A3C-H expressors, which also had significantly more CD4 and CD8 T cells, and TCR diversity (2.3-4.0x, 2.1-5.4x and 1.3-2.1x, resp.). Concordantly, for each of A3C-H, expression correlated with tumor immune cytolytic activity (r = 0.31-0.79), which was increased 3.1-7.9x in high expressors. A3B or A3A gene expression levels had no effect on overall survival (OS), but higher expression for each of A3C-H was significantly associated with improved OS (HR = 0.45-0.66). To obtain insights on any causative mechanism for the prognostic value of A3C-H expression, we examined APOBEC3-mediated C>U RNA editing in the tumors. RNA editing at a mean level of 6.3% (SD = 4.3%) was observed among tumors for 767 genes. Editing was not associated with tumor subtype or pathologic stage but tumors with high editing had 1.4-11x higher gene expression of A3A and C-H (P <0.05 for all). TCR diversity and immune cytolytic activity in tumors correlated with RNA editing (P <0.001 for both), and increased editing was associated with improved disease-free interval (P <0.05). Even though APOBEC3C-H are DNA mutators, their high expression in tumor is associated with a strong immune response and improved survival in breast cancer. This is also observed for APOBEC3-mediated RNA editing, suggesting that RNA editing of specific genes by APOBEC3s may be promoting immune activity against cancer. Note: This abstract was not presented at the meeting. Citation Format: Mariko Asaoka, Santosh K. Patnaik, Eriko Katsuta, Takashi Ishikawa, Kazuaki Takabe. APOBEC3C-H gene expression and APOBEC3-mediated RNA editing in breast cancer tumors are associated with immune response and improved outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3416.

Evidence for selective silencing of MHC-binding neoepitopes to avoid immune surveillance.

2591 Background: Overall response rates to immune checkpoint inhibition (ICI) are < 50% even in TMB-high patients (e.g. Checkmate-227), suggesting other mechanisms of immune escape exist beyond expressing checkpoints. At least 18% of somatic-specific exonic DNA variants are not expressed into mRNA (Rabizadeh, 2018), yet the selection criteria for which variants to silence remains unclear. We sought to determine if immunogenicity of variants factors into their suppression. Methods: Somatic-specific single nucleotide variants (SNVs) were identified from paired tumor/normal whole-exome sequencing (WES), and annotated as expressed if observed in > = 2 RNAseq reads. MHC1 binding affinity for 9-mer neoepitope peptides resulting from said SNVs were predicted using NetMHC within presented HLA-types. Cases with > 200 non-synonymous exonic mutations were designated as TMB-high in accordance with Rizvi et al, 2015. Tumor immune activity was inferred by RNAseq expression of 6 checkpoint/TME markers, as well as by estimating immune infiltration using RNAseq deconvolution of immune genesets (Bindea et al 2013). Significant associations between TMB, neoantigen-load, expressed neoepitope binding affinities, and immune activity were analyzed. Results: Within a clinical database of 1,363 cases with T/N/R sequencing, a total of 147,015 potential neoepitopes were identified. A small but significant enrichment was observed for silencing neoepitopes that are predicted to bind MHC1 (OR = 1.22, p = 2.4e-78 one-sided Fishers test). The silencing rate was similar between the 17% of patients with high TMB vs others, but was increased in 35% of all patients with high inferred immune infiltration (N = 490, OR = 1.30, p = 1.8e-31). A further silencing enrichment was observed in 19% of all patients displaying high immune activity but low PDL1 expression (N = 263, OR = 1.44, p = 4.0e-45). Conclusions: We observe significant preferential silencing of MHC binding neoepitopes. Specifically, when tumor infiltrating immune cells are activated, silencing neoepitopes may be an alternative to checkpoint expression for avoiding an immune cascade. Patients with TILs and silenced neoepitopes may benefit from epigenetic priming therapy prior to ICI therapy.