Interferon-gamma Signaling Research Articles

Identification of candidate genes for endometrial cancer in multi-omics: a Mendelian randomization analysis

Endometrial cancer is the most common malignant tumor of the uterus, but the underlying genetic mechanisms of EC remain unclear. To identify candidate genes and investigate genetic mechanisms for endometrial cancer, we utilized the summary-data-based Mendelian randomization (SMR) method to investigate causal associations between genetic variants, gene expression, DNA methylation, and endometrial cancer. Three main analyses were conducted utilizing cis-expression and methylation quantitative trait loci (eQTLs and mQTLs) as instrumental variables to examine causal relationships with endometrial cancer, and assessing the causal relationship between DNA methylation and gene expression. Data sources included genetic association data from O'Mara et al. eQTL data from the GTEx database, and mQTL data from McRae et al. Analysis involved the HEIDI test to distinguish pleiotropy, SMR analysis with multiple testing correction, and colocalization analysis to assess associations driven by linkage disequilibrium. Functional enrichment analysis was performed by the Metascape tool. Our study showed that three genes, SNX11, LINC00243, and EVI2A, were identified as causally related to endometrial cancer. SNX11 exhibited a positive causal relationship, while LINC00243 and EVI2A showed negative ones. Furthermore, 24 CpG sites were identified as causally related to endometrial cancer, with cg14424631 (CYP19A1) being the most significant. The study revealed common genes implicated in endometrial cancer, gene expression, and methylation sites, with LINC00243 playing a key role. Colocalization analysis confirmed significant causal relationships between LINC00243, SNX11, and endometrial cancer. Enrichment analysis uncovered pathways like interferon gamma signaling enriched in both endometrial cancer GWAS and e/mQTL. These findings shed light on the molecular mechanisms underlying endometrial cancer development. The study identified candidate genes and DNA methylation loci causally associated with endometrial cancer, which are expected to serve as potential targets for treatment.

8449 A Chronic Treatment of Interleukin-12 Restores Diet-Induced Hepatic Insulin Resistance and Liver Fibrosis in Mice with Interferon-gamma Signaling Deficient Macrophages

8449 A Chronic Treatment of Interleukin-12 Restores Diet-Induced Hepatic Insulin Resistance and Liver Fibrosis in Mice with Interferon-gamma Signaling Deficient Macrophages

Mast cells promote pathology and susceptibility in tuberculosis.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world's population. While most infected individuals are asymptomatic, latent TB infection (LTBI) can progress to cause pulmonary TB (PTB). We recently reported an increased accumulation of mast cells (MCs) in lungs of macaques with PTB, compared with LTBI in macaques. MCs respond in vitro to Mtb exposure via degranulation and by inducing proinflammatory cytokines. In the current study, we show the dominant production of chymase by MCs in granulomas of humans and macaques with PTB. Using scRNA seq analysis, we show that MCs found in LTBI and healthy lungs in macaques are enriched in genes involved in tumor necrosis factor alpha, cholesterol and transforming growth factor beta signaling. In contrast, MCs clusters found in PTB express transcriptional signatures associated with interferon gamma, oxidative phosphorylation, and MYC signaling. Additionally, MC deficiency in the mouse model showed improved control of Mtb infection that coincided with reduced accumulation of lung myeloid cells and diminished inflammation at chronic stages. Thus, these collective results provide novel evidence for the pathological contribution of MCs during Mtb infection and may represent a novel target for host directive therapy for TB.

Integrative analysis of the immunological significances of guanylate binding protein family genes in microsatellite stability colorectal cancer

BackgroundMicrosatellite stability (MSS) colorectal cancer (CRC) has poor sensitivity to immunotherapy and its underlying mechanisms are still unclear. Guanylate binding proteins (GBPs) are a family of GTPase involving innate immune responses by providing defense against invading microbes and pathogens. However, the immunological significances of GBPs in MSS CRC remain unknown. MethodsWe utilized bioinformatic tools to comprehensively analysis the expression pattern, clinical relevance, prognostic value, biological function, and immunoregulation effect of distinct GBP members in MSS CRC. ResultsThe expression of all seven GBPs in MSS samples are remarkably decreased compared to microsatellite instability-high (MSI-H) samples. Among them, GBP1/2/4/5 are obviously correlated with distant metastasis status. High expression of GBP1/4/5/6 was remarkably related to favorable overall survival (OS) and progression-free survival (PFS) in CRC patients with MSS tumor. Subsequent enrichment analysis revealed that Interferon-gamma (IFN-γ) and NOD-like receptor signaling are the most relevant functions. Besides, the expression patterns of GBPs are remarkably associated with several tumor infiltrated immune cells (e.g. regulatory T cells, CD4+ T cells, and macrophages) and diverse immunoregulatory molecules (e.g. immune checkpoint biomarkers (ICBs) and major histocompatibility complex (MHC) molecules). Moreover, high GBP1/2/4/5 expression predicted better immunotherapy responsiveness in immunotherapy cohorts. ConclusionThese findings might provide novel insights for the identification of therapeutic targets and potential prognostic biomarkers of GBP family in CRC with MSS samples.

Interferon-gamma signaling drives epithelial TNF-alpha receptor-2 expression during colonic tissue repair.

Interferon-gamma (IFNγ) is traditionally recognized for its pro-inflammatory role during intestinal inflammation. Here, we demonstrate that IFNγ also functions as a pro-repair molecule by increasing TNFα receptor 2 (TNFR2 protein/TNFRSF1B gene) expression on intestinal epithelial cells (IEC) following injury invitro and invivo. In silico analyses identified binding sites for the IFNγ signaling transcription factor STAT1 in the promoter region of TNFRSF1B. Scratch-wounded IEC exposed to IFNγ exhibited a STAT1-dependent increase in TNFR2 expression. Insitu hybridization revealed elevated Tnfrsf1b mRNA levels in biopsy-induced colonic mucosal wounds, while intraperitoneal administration of IFNγ neutralizing antibodies following mucosal injury resulted in impaired IEC Tnfrsf1b mRNA and inhibited colonic mucosal repair. These findings challenge conventional notions that "pro-inflammatory" mediators solely exacerbate damage by highlighting latent pro-repair functions. Moreover, these results emphasize the critical importance of timing and amount in the synthesis and release of IFNγ and TNFα during the inflammatory process, as they are pivotal in restoring tissue homeostasis.

Identification of host immune-related biomarkers in active tuberculosis: A comprehensive analysis of differentially expressed genes

Tuberculosis (TB) is a serious public health issue in India. Numerous molecular mechanisms and immunological responses play significant roles in the pathogenesis of tuberculosis. This study aimed to identify host immune-related biomarkers that are significantly differentially expressed in active TB and that play a vital role in disease progression. The methodology employed in this study included data collection, pre-processing, analysis, and interpretation of the results. Six microarray datasets were used to identify differentially expressed genes (DEGs), and only the common DEGs were used for further downstream analysis, such as hub gene identification, gene ontology, pathway enrichment analysis, and drug-gene interaction analysis. The study identified 1728 DEGs, including 906 upregulated and 822 downregulated genes. Five hub genes were identified that were: STAT1, GBP5, GBP1, FCGR1A, and BATF2. Gene ontology and pathway enrichment revealed that most of the genes were involved in interferon-gamma signaling. In addition, through drug-gene interactions, known drugs have been identified for STAT1, FCGR1A and GBP1. The findings of this study may contribute to early detection and treatment of active TB.

EPEN-08. MULTI-OMICS PROFILES REVEAL UNIQUE DEFINING MOLECULAR FEATURES OF POSTERIOR FOSSA EPENDYMOMA SUBTYPE A FROM INFANTS

Abstract BACKGROUND Ependymoma is a heterogeneous brain cancer with multiple subgroups based on locations and molecular profiles. Tumours of the methylation class, posterior fossa group A (PF-EPN-A) represent the most common and aggressive neoplasms and have a poor outcome. PF-EPN-A tumours in infants (under three years of age) are understudied; this project aimed to characterise these infant tumours. METHODS We collected DNA methylation (n=570) and gene expression (n=56) profiles of PF-EPN-A tumours, and single-cell RNA sequencing data of human fetal cerebellum from published studies. RESULTS Analysis of copy number profiles derived from DNA methylation arrays showed that infants with PF-EPN-A have relatively quiescent genomes when compared to children aged four to eighteen years (mean fraction of changes in infants (n=300) = 0.018 and children (n=270) = 0.055; p-value < 2.2 × 10-16). No recurrent focal amplifications or losses were detected, however, copy number loss involving chromosome arms, 12q (12%), 7q (9.7%), 5p (8.3%), and 16p (6%) were relatively common in infants, while 1q gain, 6q loss, and 22q loss were more frequent in children. When we stratified PF-EPN-A based on copy number alterations, infants with altered genomes were associated with poor outcomes compared to infants with stable genomes (fraction of changes = 0), which in turn showed poor overall survival compared to children with stable genomes. Gene set enrichment analysis using data from microarrays showed that infant tumours were specifically enriched with genes involved in the extracellular matrix organization, interferon gamma signaling, neuronal system, autophagy, and signaling by Interleukins, VEGF and PDGF. Integration with single-cell data from the human fetal cerebellum revealed that infants in PF-EPN-A had significantly higher proportions of glial progenitor cells and decreased granule cell progenitor subpopulations. CONCLUSIONS Together, our study identifies distinct genetic and transcriptional programs among infants in PF-EPN-A tumours, representing opportunities to refine future treatment.

Proteomic analysis of pleomorphic dermal sarcoma reveals a fibroblastic cell of origin and distinct immune evasion mechanisms

Pleomorphic dermal sarcomas are infrequent neoplastic skin tumors, manifesting in regions of the skin exposed to ultraviolet radiation. Diagnosing the entity can be challenging and therapeutic options are limited. We analyzed 20 samples of normal healthy skin tissue (SNT), 27 malignant melanomas (MM), 20 cutaneous squamous cell carcinomas (cSCC), and 24 pleomorphic dermal sarcomas (PDS) using mass spectrometry. We explored a potential cell of origin in PDS and validated our findings using publicly available single-cell sequencing data. By correlating tumor purity (TP), inferred by both RNA- and DNA-sequencing, to protein abundance, we found that fibroblasts shared most of the proteins correlating to TP. This observation could also be made using publicly available SNT single cell sequencing data. Moreover, we studied relevant pathways of receptor/ligand (R/L) interactions. Analysis of R/L interactions revealed distinct pathways in cSCC, MM and PDS, with a prominent role of PDGFRB-PDGFD R/L interactions and upregulation of PI3K/AKT signaling pathway. By studying differentially expressed proteins between cSCC and PDS, markers such as MAP1B could differentiate between these two entities. To this end, we studied proteins associated with immunosuppression in PDS, uncovering that immunologically cold PDS cases shared a “negative regulation of interferon-gamma signaling” according to overrepresentation analysis.

Immunotherapy-relevance of a candidate prognostic score for Acute Myeloid Leukemia

BackgroundAcute Myeloid Leukemia (AML) exhibits a wide array of phenotypic manifestations, progression patterns, and heterogeneous responses to immunotherapies, suggesting involvement of complex immunobiological mechanisms. This investigation aimed to develop an integrated prognostic model for AML by incorporating cancer driver genes, along with clinical and phenotypic characteristics of the disease, and to assess its implications for immunotherapy responsiveness. MethodsCritical oncogenic driver genes linked to survival were identified by screening primary effector and corresponding gene pairs using data from The Cancer Genome Atlas (TCGA), through univariate Cox proportional hazard regression analysis. This was independently verified using dataset GSE37642. Primary effector genes were further refined using LASSO regression. Transcriptomic profiling was quantified using multivariate Cox regression, and the derived prognostic score was subsequently validated. Finally, a multivariate Cox regression model was developed, incorporating the transcriptomic score along with clinical parameters such as age, gender, and French-American-British (FAB) classification subtype. The ‘Accurate Prediction Model of AML Overall Survival Score’ (APMAO) was developed and subsequently validated. Investigations were conducted into functional pathway enrichment, alterations in the gene mutational landscape, and the extent of immune cell infiltration associated with varying APMAO scores. To further investigate the potential of APMAO scores as a predictive biomarker for responsiveness to cancer immunotherapy, we conducted a series of analyses. These included examining the expression profiles of genes related to immune checkpoints, the interferon-gamma signaling pathway, and m6A regulation. Additionally, we explored the relationship between these gene expression patterns and the Tumor Immune Dysfunction and Exclusion (TIDE) dysfunction scores. ResultsThrough the screening of 95 cancer genes associated with survival and 313 interacting gene pairs, seven genes (ACSL6, MAP3K1, CHIC2, HIP1, PTPN6, TFEB, and DAXX) were identified, leading to the derivation of a transcriptional score. Age and the transcriptional score were significant predictors in Cox regression analysis and were integral to the development of the final APMAO model, which exhibited an AUC greater than 0.75 and was successfully validated. Notable differences were observed in the distribution of the transcriptional score, age, cytogenetic risk categories, and French-American-British (FAB) classification between high and low APMAO groups. Samples with high APMAO scores demonstrated significantly higher mutation rates and pathway enrichments in NFKB, TNF, JAK-STAT, and NOTCH signaling. Additionally, variations in immune cell infiltration and immune checkpoint expression, activation of the interferon-γ pathway, and expression of m6A regulators were noted, including a negative correlation between CD160, m6A expression, and APMAO scores. ConclusionThe combined APMAO score integrating transcriptional and clinical parameters demonstrated robust prognostic performance in predicting AML survival outcomes. It was linked to unique phenotypic characteristics, distinctive immune and mutational profiles, and patterns of expression for markers related to immunotherapy sensitivity. These observations suggest the potential for facilitating precision immunotherapy and advocate for its exploration in upcoming clinical trials.

Molecular patterns of resistance to immune checkpoint blockade in melanoma

Immune checkpoint blockade (ICB) has improved outcome for patients with metastatic melanoma but not all benefit from treatment. Several immune- and tumor intrinsic features are associated with clinical response at baseline. However, we need to further understand the molecular changes occurring during development of ICB resistance. Here, we collect biopsies from a cohort of 44 patients with melanoma after progression on anti-CTLA4 or anti-PD1 monotherapy. Genetic alterations of antigen presentation and interferon gamma signaling pathways are observed in approximately 25% of ICB resistant cases. Anti-CTLA4 resistant lesions have a sustained immune response, including immune-regulatory features, as suggested by multiplex spatial and T cell receptor (TCR) clonality analyses. One anti-PD1 resistant lesion harbors a distinct immune cell niche, however, anti-PD1 resistant tumors are generally immune poor with non-expanded TCR clones. Such immune poor microenvironments are associated with melanoma cells having a de-differentiated phenotype lacking expression of MHC-I molecules. In addition, anti-PD1 resistant tumors have reduced fractions of PD1+ CD8+ T cells as compared to ICB naïve metastases. Collectively, these data show the complexity of ICB resistance and highlight differences between anti-CTLA4 and anti-PD1 resistance that may underlie differential clinical outcomes of therapy sequence and combination.

Interferon-gamma signaling promotes cartilage regeneration after injury

Osteoarthritis is a common chronic disease and major cause of disability and chronic pain in ageing populations. In this pathology, the entire joint is involved, and the regeneration of articular cartilage still remains one of the main challenges. Here, we investigated the molecular mechanisms underlying cartilage regeneration in young mice using a full-thickness cartilage injury (FTCI) model. FTCI-induced cartilage defects were created in the femoral trochlea of young and adult C57BL/6 mice. To identify key molecules and pathways involved in the early response to cartilage injury, we performed RNA sequencing (RNA-seq) analysis of cartilage RNA at 3 days after injury. Young mice showed superior cartilage regeneration compared to adult mice after cartilage injury. RNA-seq analysis revealed significant upregulation of genes associated with the immune response, particularly in the IFN-γ signaling pathway and qRT-PCR analysis showed macrophage polarization in the early phase of cartilage regeneration (3 days) in young mice after injury, which might promote the removal of damaged or necrotic cells and initiate cartilage regeneration in response to injury. IFN-γR1- and IFN-γ-deficient mice exhibited impaired cartilage regeneration following cartilage injury. DMM-induced and spontaneous OA phenotypes were exacerbated in IFN-γR1−/− mice than in wild-type mice. Our data support the hypothesis that IFN-γ signaling is necessary for cartilage regeneration, as well as for the amelioration of post-traumatic and age-induced OA.

Single-cell profiling of response to neoadjuvant chemo-immunotherapy in surgically resectable esophageal squamous cell carcinoma

BackgroundThe efficacy of neoadjuvant chemo-immunotherapy (NAT) in esophageal squamous cell carcinoma (ESCC) is challenged by the intricate interplay within the tumor microenvironment (TME). Unveiling the immune landscape of ESCC in the context of NAT could shed light on heterogeneity and optimize therapeutic strategies for patients.MethodsWe analyzed single cells from 22 baseline and 24 post-NAT treatment samples of stage II/III ESCC patients to explore the association between the immune landscape and pathological response to neoadjuvant anti-PD-1 combination therapy, including pathological complete response (pCR), major pathological response (MPR), and incomplete pathological response (IPR).ResultsSingle-cell profiling identified 14 major cell subsets of cancer, immune, and stromal cells. Trajectory analysis unveiled an interesting link between cancer cell differentiation and pathological response to NAT. ESCC tumors enriched with less differentiated cancer cells exhibited a potentially favorable pathological response to NAT, while tumors enriched with clusters of more differentiated cancer cells may resist treatment. Deconvolution of transcriptomes in pre-treatment tumors identified gene signatures in response to NAT contributed by specific immune cell populations. Upregulated genes associated with better pathological responses in CD8 + effector T cells primarily involved interferon-gamma (IFNγ) signaling, neutrophil degranulation, and negative regulation of the T cell apoptotic process, whereas downregulated genes were dominated by those in the immune response-activating cell surface receptor signaling pathway. Natural killer cells in pre-treatment tumors from pCR patients showed a similar upregulation of gene expression in response to IFNγ but a downregulation of genes in the neutrophil-mediated immunity pathways. A decreased cellular contexture of regulatory T cells in ESCC TME indicated a potentially favorable pathological response to NAT. Cell–cell communication analysis revealed extensive interactions between CCL5 and its receptor CCR5 in various immune cells of baseline pCR tumors. Immune checkpoint interaction pairs, including CTLA4-CD86, TIGIT-PVR, LGALS9-HAVCR2, and TNFSF4-TNFRSF4, might serve as additional therapeutic targets for ICI therapy in ESCC.ConclusionsThis pioneering study unveiled an intriguing association between cancer cell differentiation and pathological response in esophageal cancer patients, revealing distinct subgroups of tumors for which neoadjuvant chemo-immunotherapy might be effective. We also delineated the immune landscape of ESCC tumors in the context of clinical response to NAT, which provides clinical insights for better understanding how patients respond to the treatment and further identifying novel therapeutic targets for ESCC patients in the future.

Abstract 6212: A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden that leads to the generation of few neo-epitopes derived from mutated genes that can be recognized by cytotoxic T cells. The immunosuppressive microenvironment of PDAC comprises suppressive cell populations that include myeloid cells, cancer associated fibroblast subpopulations, and regulatory T cells that reduce T cell infiltration and cytotoxic function. We have developed a murine personalized cancer vaccine to enhance cytotoxic T cell function against PDAC, PancVAX (Kinkead et al, JCI Insight 2018). Survival benefit with PancVAX was dependent on the addition of immune checkpoint inhibitor (ICI) therapy to increase the number of cytotoxic CD8 T cells infiltrating tumors and maintain their cytotoxic function (Huff et al, JCI Insight 2023). Yet, the impact of cell-cell communication on effector T cell function arising from the immunosuppressive microenvironment of PDAC remains unknown. We previously developed a ligand-receptor inference method called Domino that infers inter- and intra-cellular signaling networks in single-cell RNA-seq data (Cherry et al, Nat Biomed Eng 2021). However, this technique is limited to data from single conditions. Understanding how this combination immunotherapy alters cellular signaling networks requires new computational tools that identify changes in regulatory networks between treatment conditions. We developed capabilities for assessment of differential signaling with Domino founded on a basis of differential signal receipt. Active receptors are identified by receptor expression in recipient cells that is correlated with downstream transcription factors in the intracellular signaling cascade. Identification of causative ligands for receptor activation is assessed by a probability of interaction for ligand-receptor interactions between cell types. This probability metric considers mean expression of ligand and receptor genes as well as the number of sender cells for comparison across treatment groups. Comparing communication networks in PancVAX alone to PancVAX with ICI, PancVAX with ICI enhances cell-cell adhesion and interferon gamma signaling in cytotoxic CD8 T cells towards exhausted CD8 T cells. Macrophages in PancVAX with ICI enhance expression of immune checkpoint ligands towards CD8 T cells, despite the presence of blocking ICI antibodies. Intracellular signaling downstream of PD-1 in exhausted T cells shows diminished numbers of transcription factors linked to PD-1 signaling. This software informs prioritization of inhibitory signals for modulation in future immunotherapy regimens and can be applied generally to other tumor subtypes and treatment contexts. Citation Format: Jacob T. Mitchell, Atul Deshpande, Amanda L. Huff, Chris Cherry, Sushma Nagaraj, Kavita Krishnan, Dmitrijs Lvovs, Jennifer H. Elisseeff, Elizabeth M. Jaffee, Neeha Zaidi, Elana J. Fertig. A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6212.

Abstract 1581: Colony-stimulating factor-1 receptor as a potential therapeutic target in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis, rising incidence and suffers from limited existing treatment options. Currently, salient features of PDAC are being investigated that can potentially predict immunotherapy outcomes, to identify small patient populations that can benefit from such strategies. Preliminary studies show that colony-stimulating factor-1 receptor (CSF1R), a surface receptor tyrosine kinase that drives survival, function, proliferation and differentiation of several myeloid lineage cell types can be a potentially interesting target for combination immunotherapy. Methods: PDAC patients from the TCGA PanCancer Atlas were stratified into CSF1Rhi and CSF1Rlo cohorts and clinically relevant characteristics were compared between the two. The mRNA expression of CSF1R and immunological markers of interest were evaluated and Gene Set Enrichment Analysis (GSEA) was run on the two groups to analyze differential expression of gene sets. Results: mRNA expression z-scores were used to classify 129 patient samples in the TCGA PanCancer Atlas dataset into CSF1Rhi (z-score>1, n=20) and CSF1Rlo (z-score<0, n=109) cohorts. Both have similar disease staging, MSI MANTIS scores, tumor mutation burden, median diagnosis age, sex and race distribution. However, the CSF1Rhi cohort has a distinct, non-conventional PDAC driver gene mutation profile (genomic alteration frequencies in CSF1Rhi vs CSF1Rlo cohorts respectively: a) KRAS: 30% vs 73% b) CDKN2A: 15% vs 57% and c) SMAD4: 5% vs 42%) (p-value<0.01 for each). CSF1R correlates with markers of CD8+ T-cell function (R2 values: CD8A: 0.683, GZMB: 0.508, PRF1: 0.673), CD8+ T-cell infiltration (CXCL9: 0.596, CXCL10: 0.483, CCL5: 0.666) and expression of immune checkpoint genes (PDCD1: 0.602, CD274: 0.544, CTLA4: 0.613). CD8A, GZMB, PRF1, CXCL9, CXCL10, CCL5, PDCD1, CD274, CTLA4 are significantly upregulated in the CSF1Rhi group (log2Fold changes respectively: 2.07, 1.57, 1.48, 2.51, 1.71, 1.75, 1.63, 1.34, 1.80). GSEA against the hallmark gene set database shows that KRAS signaling, Inflammatory response, IL6 JAK STAT3 and Interferon-gamma signaling are significantly upregulated in the CSF1R high patient group (FDR q-value <0.01 for each). Conclusion: Preliminary in silico analysis suggests that PDAC patients with higher levels of CSF1R may be more immune infiltrated. Future studies will evaluate the potential of CSF1R as a biomarker to identify an immune infiltrated subset of PDAC patients ex vivo and the mechanism of immune infiltration in CSF1Rhi PDACs in vivo. Citation Format: Somak Chaudhuri, Dustin A. Deming, Cheri A. Pasch, Katherine A. Johnson, Chelsie K. Sievers, Philip B. Emmerich. Colony-stimulating factor-1 receptor as a potential therapeutic target in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1581.

Abstract 5100: Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade

Abstract Background: Why some patients fail or have short lived response to immune checkpoint blockade (ICB) immunotherapy remains largely unknown. While baseline molecular assessments have provided clues to prognostic factors, insights into resistance drivers remains elusive. This is partially due to the difficulty in getting access to post progression samples from patients that were either primary resistant or developed acquired resistance after an initial response to ICB. Thus, the tumor-intrinsic and -extrinsic features that are selected for during progression and potentially drive primary and acquired resistance to immunotherapy remain underexplored. Methods: To compare clinical features and immunogenomic drivers of acquired and primary resistance to ICB across major cancers, we analysed and annotated de-identified patient records in the Tempus real-world database. We built an immuno-oncology cohort consisting of >2500 multimodal (DNA, RNA and clinical outcome data) pre-treatment baseline with >1500 post-treatment tumour biopsy samples from mainly NSCLC, TNBC, HNC and Bladder cancer patients. We used bulk RNA-seq data to estimate activation of the hallmark oncogenic pathways and immune cell composition and used panel DNA-seq data (>500 genes) to quantify mutation selection at the gene and pathway levels using dndscv. Results: Compared to acquired, primary resistant patients tended to have a higher observation of liver lesions at progression. Post-ICB, acquired resistant NSCLC and HNC patients showed a significantly inflamed tumour microenvironment (TME) characterised by higher estimation of infiltration of T cells and myeloid cells and higher activation of interferon gamma (IFNg) signalling as compared to primary resistant patients. In addition, in post-ICB acquired resistance in NSCLC we observed selection for mutations in genes involved in known immunomodulatory pathways, including loss-of-function mutations in B2M. Consistently, acquired resistance patients showed stronger selection for mutations in Hedgehog and Notch pathways as compared to primary resistance patients across NSCLC, HNC and TNBC post-ICB. Conclusions: Acquired and primary ICB resistant patients have distinct clinical and molecular features at progression. Their tumours’ TME is fundamentally different with acquired resistance TMEs being infiltrated with immune cells albeit escaped post progression. In addition, ICB selects mutations that potentially activate pathways such as Notch and Hedgehog. This multi-modal Real-World Data with post therapy biopsies has given insights for patient selection strategies and provides rational into combination treatment options for acquired resistant patients. Citation Format: Mohamed Reda Keddar, Sebastian Carrasco Pro, Kathleen Burke, Ana Camelo Stewart, Mark Cobbold, Ross Stewart, Sajan Khosla, Ben Sidders, Scott Hammond, Douglas C. Palmer, Jonathan Dry, Martin L. Miller. Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5100.

Abstract 6870: Immunological aging in triple negative breast cancer leads to a chronic interferon phenotype and immune cell exhaustion that impedes response to therapy

Abstract Age is the biggest risk factor for developing most forms of cancer, and understanding how age impacts cancer progression is critical to improving responses to therapy. As we age immune function declines suggesting that therapeutic responses, particularly to immunotherapies, will also change with age. Early clinical trial data suggests that there are age-associated responses to immunotherapies. Clinical trials, however, are not designed to reflect patient chronological and immunological ages - over 50% of breast cancer patients are over the age of 60, yet less than 25% of patients in clinical trials are in this age range. Here, we utilize a preclinical mouse model of immunological aging to understand how aging impacts immune responses to triple-negative breast cancer (TNBC). Immunologically young (8 weeks old) and aged (~12 months old) FVB mice were orthotopically injected with syngeneic Met1 TNBC cells. We previously established that this preclinical model mimics human immunological aging and enables us to better understand how age impacts disease progression and therapeutic responses. Once tumors became palpable, mice were treated with paclitaxel or vehicle control and/or anti-PDL1 to mimic first-line therapeutic strategies. Tumors grew significantly faster in young control mice compared to aged, but young mice had a more robust response to therapy. Notably, only young mice had a significant benefit of combo therapy (paclitaxel + anti-PDL1) over monotherapy alone. Tumors in aged mice had significantly more infiltration of CD8+ cytotoxic T cells and FOXP3+ inhibitory T-regulatory cells compared to young control mice, yet only tumors in young mice exhibited an increase in T cell infiltration with chemo or immunotherapy. Depletion of either CD8+ or CD4+ T cells in immune-competent FVB mice both led to a complete loss of age-associated differences in tumor growth. RNA sequencing analysis indicated that the infiltrating T cells in aged mice may be exhausted due to a chronic interferon phenotype, as indicated by enrichments of interferon alpha, interferon gamma, and IL2 signaling pathways, as well as expression of RIPK1, TNFa, and the immune exhaustion markers PD1, CTLA4, TIM3, and LAG3. Analysis of published single cell RNAseq data indicated the same interferon response pathways enriched with age in human TNBC, particularly in the T cell compartment. Our findings show that the aging immune microenvironment significantly impacts tumor progression and that a chronic interferon phenotype with age may lead to T cell exhaustion and impaired therapeutic responses. This work suggests that targeting interferon signaling may lead to improved therapeutic responses. Together these findings demonstrate how immunological aging can impact disease progression and response to therapy and highlight the importance of developing age-stratified treatment strategies. Citation Format: Milos Spasic, Adrienne Parsons, Gregory Goreczny, Qiuchen Guo, Abigail Recko, Elizabeth Mittendorf, Peter van Galen, Sandra McAllister. Immunological aging in triple negative breast cancer leads to a chronic interferon phenotype and immune cell exhaustion that impedes response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6870.

Abstract 2415: Resistance to immune checkpoint inhibitor (ICI) therapy in metastatic melanoma is revealed by deep circulating tumor DNA tracking in concert with tumor immune profiling

Abstract Monitoring clinically actionable and emerging mutations in circulating tumor DNA (ctDNA) may be crucial for guiding treatment, but adoption has been slow due to the narrow scope and reproducibility of current approaches. Here, we investigate melanoma patients receiving ICI over several years, beginning with extensive tumor mutational and immune profiling followed by deep, whole-exome ctDNA monitoring.Here, 66 plasma samples from 23 advanced stage melanoma patients that were collected throughout ICI treatment (up to 40 months) were profiled using the NeXT Liquid Biopsy® platform (NeXT LB). NeXT LB monitors exome-wide ctDNA by leveraging a patient’s tumor, PBMCs and plasma samples, detecting both tumor-derived and emerging mutations. In addition to ctDNA tracking, we also used the ImmunoID NeXT Platform® to immunologically profile tumor tissue; including gene expression quantification, HLA profiling (typing, mutation, and loss of heterozygosity), T-cell receptor and tumor microenvironment profiling, and neoantigen prediction. ctDNA dynamics and tumor immunity were correlated with clinical outcome. Patients who progressed had lower tumor mutational burden (TMB, Mann-Whitney (MW); p=0.03), and lower neoantigen burden (MW; p=0.04). Responding tumors were enriched for interferon gamma and inflammatory signaling (Wald; adjusted p<0.01) and myeloid dendritic cells (MW; p=0.01), as measured by baseline RNA expression profiling. With an average coverage of 2260X SD +/-157, plasma samples yielded from 4 to 4081 variants. The number of on-treatment tumor variants detected in plasma predicted increased OS hazard (HR=2.95, Wald p=0.03; Kaplan-Meier (KM) p=0.0003) and progression hazard (HR=1.74, Wald p=0.04; KM p=0.0003). Overall, 10% (4277) of all variants were novel mutations observed only in the plasma. Novel variants were less likely to emerge in genes conferring sensitivity to ICI therapy (mixed-model; p<0.001). Dynamic shifts in allelic fraction between baseline and the first available on-treatment timepoint predicted increased OS hazard (HR=3.02, Wald p=0.05) and progression hazard (HR=2.90, Wald p=0.02). In ctDNA clonal phylogenies, 100% of BRAF and NRAS mutations belonged to the dominant clone. By combining immune and ctDNA features, a multivariable model predicted resistance to ICI treatment (log-likelihood p=0.002; c-index=0.89). Using a single liquid biopsy platform, we profiled tumor immunity and tracked existing and emerging ctDNA variants over the course of ICI treatment. We demonstrate that, even though counts of on-treatment ctDNA variants were sufficient for significant survival models, deep profiling of both tumor immunity and ctDNA dynamics provided deeper understanding of ICI resistance. Subsequent studies will determine if ctDNA-guided intervention can improve clinical outcomes. Citation Format: Charles W. Abbott, Laura Keller, Isabel Heidrich, Julian Kött, Glenn Geidel, Daniel J. Smit, Ronald Simon, Stefan Schneider, Jason Pugh, Sean M. Boyle, Richard O. Chen, Klaus Pantel, Christoffer Gebhardt. Resistance to immune checkpoint inhibitor (ICI) therapy in metastatic melanoma is revealed by deep circulating tumor DNA tracking in concert with tumor immune profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2415.

Abstract 7363: Clinical, genomic, transcriptomic and immunologic profile of BRAF mutant colorectal tumors

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. BRAF, a protein kinase of the Mitogen-Activated Protein Kinase (MAPK) pathway, is mutated in 12% of colorectal cancers and is a poor prognostic factor for patients. BRAF mutations at the V600 codon are most studied but non-V600 BRAF mutations also exist and behave differently. BRAF V600 CRC treated with BRAF+MEK inhibitors show 12% response rates while we observe 65% response rates in BRAF V600 Melanoma. Additionally, no approved targeted therapies exist for non-V600 BRAF mutant cancers and retrospective studies show poorer PFS with BRAF+MEK inhibitors vs BRAF V600 cancers. We interrogated publicly available data of BRAF mutant CRC to uncover differences in the tumor microenvironment and signalling pathways of BRAF V600 and non-V600 tumors in hopes of uncovering better treatments. Methods: We analyzed the clinical and genomic profile of BRAF mutant CRC tumors from the GENIE v12 data. Gene Set Enrichment Analysis was performed on RNAseq from the TCGA data (discovery cohort) and the combined data from CPTAC and Sidra-LUMC data (validation cohort). We investigated the composition of tumor infiltrating immune cells using CIBERSORTx (deconvolution algorithm for bulk RNAseq). Results: Non-V600 mutations (n=219/1061) were most represented in patients of Asian or Black race compared to White patients (p<0.0001) and importantly, were found in younger patients (p<0.0001). We identified n=67 and n=74 BRAF mutant RNAseq samples in the discovery and validation cohorts, respectively. In the discovery cohort: BRAF V600 CRC were enriched for Interferon Gamma Signaling, Complement pathway, and IL6 JAK STAT3 Signaling (p<0.05) while the non-V600 BRAF mutant CRC showed an enrichment for Wnt-beta Catenin, Notch, and Hedgehog Signaling pathways (p<0.05). In the validation cohorts these same pathways were enriched in V600 and non-V600 BRAF mutant tumors. Interestingly, enrichment of genes co-mutated (GENIE data) in non-V600 BRAF mutant CRC also revealed the Wnt pathway. Non-V600 CRC tumors have a higher proportion of M0 macrophages (p=0.0035) and CD4 memory resting T cells (p<0.0001) while patients with V600 CRC have higher proportions of CD8 T cells (p=0.006) and activated mast cells (p=0.0341). Conclusion: No optimal therapeutic strategies have been defined for these potentially actionable non-V600 BRAF mutant CRCs. By exploring the multi-omic landscape of these tumors we have identified unique characteristics of non-V600 BRAF mutant CRC that could be therapeutically exploited. Citation Format: Emmanuelle Rousselle, Suzanne Kazandjian, April Rose. Clinical, genomic, transcriptomic and immunologic profile of BRAF mutant colorectal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7363.

Abstract 1: Biomarkers of response and acquired resistance to TIL-ACT in solid tumors

Abstract Background: Cancer immunotherapy has revolutionized cancer treatment in the last decade. However, most patients either do not respond to immunotherapy or eventually relapse so there is a need to identify biomarkers of response and understand mechanisms of resistance. Most biomarker studies have analyzed patients treated with immune checkpoint inhibitors (CPI) therapy while the few studies of adoptive cell transfer of tumor infiltrating lymphocytes (TIL-ACT) have analyzed relatively small patient cohorts. Methods: To systematically identify biomarkers of response and resistance to TIL-ACT, we collected published and unpublished whole-exome and transcriptomic data for tumors from 400 patients treated with TIL-ACT from five tumor types across four institutions and processed these datasets using a standardized bioinformatic pipeline. Results: To identify biomarkers of response in pre-treatment tumors, we analyzed previously reported biomarkers and performed biomarker discovery in our cohorts. For the subset of 200 patients with T cell reactivities identified from the infusion products and fresh tumor, we refined our biomarkers to include neo-antigen reactivities in addition to identifying both mutational processes and transcriptional signatures associated with neo-antigens reactivity. To identify mechanisms of acquired resistance to TIL-ACT, we analyzed 15 patients with paired pre- and post-treatment tumors and found multiple mechanisms of immune evasion specific to infused reactive T cells including loss of heterozygosity of the HLA allele presenting the neo-antigen, genomic loss of the neo-antigen and inactivation of interferon-gamma signaling pathway. Conclusions: In summary, we will present the biomarkers landscape of TIL-ACT therapy including differences between histology and across treatment modalities to improve our understanding of the mechanisms of treatment response. Citation Format: Welles Robinson, Andrea Castro, Maise Al-Bakir, Jared J. Gartner, Christopher Aled Chamberlain, Anders H. Kverneland, Troels H. Borch, Inge Marie Svane, Marco Donia, Paul Robbins, Kevin Litchfield. Biomarkers of response and acquired resistance to TIL-ACT in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1.

Role of CD4+ T-cells for regulating splenic myelopoiesis and monocyte differentiation after experimental myocardial infarction

Myocardial infarction (MI) induces the generation of proinflammatory Ly6Chigh monocytes in the spleen and the recruitment of these cells to the myocardium. CD4+ Foxp3+ CD25+ T-cells (Tregs) promote the healing process after myocardial infarction by engendering a pro-healing differentiation state in myocardial monocyte-derived macrophages. We aimed to study the effects of CD4+ T-cells on splenic myelopoiesis and monocyte differentiation. We instigated MI in mice and found that MI-induced splenic myelopoiesis is abrogated in CD4+ T-cell deficient animals. Conventional CD4+ T-cells promoted myelopoiesis in vitro by cell–cell-contact and paracrine mechanisms, including interferon-gamma (IFN-γ) signalling. Depletion of regulatory T-cells enhanced myelopoiesis in vivo, as evidenced by increases in progenitor cell numbers and proliferative activity in the spleen 5 days after MI. The frequency of CD4+ T-cells-producing factors that promote myelopoiesis increased within the spleen of Treg-depleted mice. Moreover, depletion of Tregs caused a proinflammatory bias in splenic Ly6Chigh monocytes, which showed predominantly upregulated expression of IFN-γ responsive genes after MI. Our results indicate that conventional CD4+ T-cells promote and Tregs attenuate splenic myelopoiesis and proinflammatory differentiation of monocytes.