Checkpoint Blockade Treatment Research Articles

Antimony Component Schottky Nanoheterojunctions as Ultrasound‐Heightened Pyroptosis Initiators for Sonocatalytic Immunotherapy

Sonocatalytic therapy (SCT) holds promise due to its exceptional penetration depth; however, the rapid recombination of electron‐hole (e−‐h+) pairs and the complex tumor microenvironment (TME) impede its broader application. Herein, we discovered that antimony (Sb)‐based nanomaterials induced pyroptosis in cancer cells. Therefore, a Schottky heterojunction containing a Sb component (Sb2Se3@Pt) was effectively designed and constructed via in‐situ growth of platinum (Pt) nanoparticles (NPs) on a Sb2Se3 semiconductor with narrow bandgaps, which were utilized as US‐heightened pyroptosis initiators to induce highly effective pyroptosis in cancer cells to boost SCT‐immunotherapy. The biological effects of the Sb2Se3@Pt nanoheterojunction itself combined with the sonocatalytic amplification of oxidative stress significantly induced Caspase‐1/GSDMD‐dependent pyroptosis in cancer cells. Therefore, SCT treatment with Sb2Se3@Pt not only effectively restrained tumor proliferation but also induced potent immune memory responses and suppressed tumor recurrence. Furthermore, the integration of this innovative strategy with immune checkpoint blockade (ICB) treatment elicited a systemic immune response, effectively augmenting therapeutic effects and impeding the growth of abscopal tumors. Overall, this study provides further opportunities to explore pyroptosis‐mediated SCT‐immunotherapy.

Identification of a novel prognostic model for gastric cancer utilizing glutamine-related genes

BackgroundGlutamine metabolism presents a promising avenue for cancer prevention and treatment, but the underlying mechanisms in gastric cancer (GC) progression remain elusive. MethodsThe TCGA-STAD and GEO GSE62254 datasets, containing gene expression, clinical information, and survival outcomes of GC, were meticulously examined. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were employed to excavate a key module (MEturquoise), which was used to intersect with glutamine metabolism-related genes (GMRGs) and differentially expressed genes (DEGs) to identify differentially expressed GMRGs (DE-GMRGs). LASSO and Cox Univariate analyses were implemented to determine risk model genes. Correlation of the risk model with clinical parameters, pathways, and tumor immune microenvironments, was analyzed, and its prognostic independence was validated by Cox analyses. Finally, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to validate the expression levels of MYB, LRFN4, LMNB2, and SLC1A5 in GC and para-carcinoma tissue. ResultsThe excavation of 4521 DEGs led to the discovery of the key MEturquoise module, which exhibited robust correlations with GC traits. The intersection analysis identified 42 DE-GMRGs, among which six genes showed consistency. Further LASSO analysis established MYB, LRFN4, LMNB2, and SLC1A5 as pivotal risk model genes. The risk model demonstrated associations with oncogenic and metabolism-related pathways, inversely correlating with responses to immune checkpoint blockade therapies. This risk model, together with “age”, was validated to be an independent prognostic factor for GC. RT-qPCR result indicated that MYB, LRFN4, LMNB2, and SLC1A5 expressions were remarkably up-regulated in GC tissues comparison with para-carcinoma tissue. ConclusionThe present study has generated a novel risk module containing four DE-GMRGs for predicting the prognosis and the response to immune checkpoint blockade treatments for GC. This risk model provides new insights into the involvement of glutamine metabolism in GC, warranting further investigation.

Predicting Resistance to Immunotherapy in Melanoma, Glioblastoma, Renal, Stomach and Bladder Cancers by Machine Learning on Immune Profiles

Strategies for tackling cancer involve surgery, radiotherapy, chemotherapy, and immune checkpoint inhibitors (ICB). However, the effectiveness of ICB remains constrained, prompting the need for a proactive strategy to foresee treatment responses and resistances. This study undertook an analysis across diverse cancer patient cohorts (including melanoma, clear cell renal carcinoma, glioblastoma, bladder, and stomach cancers) subjected to various immune checkpoint blockade treatments. Surprisingly, our findings unveiled that over 38% of patients demonstrated resistance and persistent disease progression despite undergoing ICB intervention. To unravel the intricacies of resistance, we scrutinized the immune profiles of cancer patients experiencing ongoing disease progression and resistance post-ICB therapy. These profiles delineated multifaceted defects, including compromised macrophage, monocyte, and T cell responses, impaired antigen presentation, aberrant regulatory T cell (Tregs) responses, and an elevated expression of immunosuppressive and G protein-coupled receptor molecules (TGFB1, IL2RA, IL1B, EDNRB, ADORA2A, SELP, and CD276). Building upon these insights into resistance profiles, we harnessed machine learning algorithms to construct models predicting the response and resistance to ICB and developed the accompanying software. While previous work on glioblastoma with only one type of algorithm had an accuracy of 0.82, we managed to develop 20 models that provided estimates of future events of resistance or response in five cancer types, with accuracies ranging between 0.79 and 1, based on their distinct immune characteristics. In conclusion, our approach advocates for the personalized application of immunotherapy in cancer patients based on patient-specific attributes and computational models.

Construction of a prognostic model for colon cancer by combining endoplasmic reticulum stress responsive genes

Endoplasmic reticulum stress may affect the occurrence and development of cancer. However, its effect on the prognosis of colon cancer (CC) patients is not clear yet. Herein, based on TCGA database, we screened 15 endoplasmic reticulum stress responsive genes (ERSRGs) associated with the prognosis of CC patients by Cox regression. By LASSO and multivariate Cox regression analyses, a prognostic risk assessment model involving 12 genes (DNAJB2, EIF4A1, YPEL4, COQ10A, IRX3, ASPHD1, NTRK2, TRIM39, XBP1, GRIN2B, LRRC59, and RORC) was built. The survival curves indicated that patients in the low-risk group had good prognosis. ROC curves demonstrated a good performance of this 12-gene prognostic model, and the Riskscore could be considered as an independent prognostic factor. Patients in low-risk group benefit more from immune checkpoint inhibitor and immune checkpoint blockade (ICB) treatment. Besides, the enrichment analysis suggested a remarkable difference in Ca2+ signaling in both groups. Finally, based on the cMAP database, we identified several potential drugs that could target high-risk groups, such as Dasatinib, GNF-2, Saracatinib, and WZ-1-84. To sum up, our research constructed an ERSRGs-characteristic prognostic model. The model is a promising biomarker for prediction of clinical outcomes and immune therapy response of CC patients. SignificanceBased on the transcriptomic data of colon cancer in the TCGA database, this study screens 12 endoplasmic reticulum stress-related genes (ERSRGs), including DNAJB2, EIF4A1, YPEL4, COQ10A, IRX3, ASPHD1, NTRK2, TRIM39, XBP1, asphD1, NTRK2. GRIN2B, LRRC59, and RORC, and a prognostic model was constructed. This model can be used as a predictor of prognosis and immunotherapy response in colon cancer patients. At the same time, model-based prediction of drugs can also be a potential option for colon cancer treatment in the future.

Antitumor efficacy and potential mechanism of FAP-targeted radioligand therapy combined with immune checkpoint blockade

Radiotherapy combined with immune checkpoint blockade holds great promise for synergistic antitumor efficacy. Targeted radionuclide therapy delivers radiation directly to tumor sites. LNC1004 is a fibroblast activation protein (FAP)-targeting radiopharmaceutical, conjugated with the albumin binder Evans Blue, which has demonstrated enhanced tumor uptake and retention in previous preclinical and clinical studies. Herein, we demonstrate that 68Ga/177Lu-labeled LNC1004 exhibits increased uptake and prolonged retention in MC38/NIH3T3-FAP and CT26/NIH3T3-FAP tumor xenografts. Radionuclide therapy with 177Lu-LNC1004 induced a transient upregulation of PD-L1 expression in tumor cells. The combination of 177Lu-LNC1004 and anti-PD-L1 immunotherapy led to complete eradication of all tumors in MC38/NIH3T3-FAP tumor-bearing mice, with mice showing 100% tumor rejection upon rechallenge. Immunohistochemistry, single-cell RNA sequencing (scRNA-seq), and TCR sequencing revealed that combination therapy reprogrammed the tumor microenvironment in mice to foster antitumor immunity by suppressing malignant progression and increasing cell-to-cell communication, CD8+ T-cell activation and expansion, M1 macrophage counts, antitumor activity of neutrophils, and T-cell receptor diversity. A preliminary clinical study demonstrated that 177Lu-LNC1004 was well-tolerated and effective in patients with refractory cancers. Further, scRNA-seq of peripheral blood mononuclear cells underscored the importance of addressing immune evasion through immune checkpoint blockade treatment. This was emphasized by the observed increase in antigen processing and presentation juxtaposed with T cell inactivation. In conclusion, our data supported the efficacy of immunotherapy combined with 177Lu-LNC1004 for cancer patients with FAP-positive tumors.

Characterization of the prognostic, diagnostic, and immunological roles of DSCC1 and its genomic alteration and instability in human cancers.

DNA replication and sister chromatid cohesion 1 (DSCC1) exerts various functions including sister chromatid cohesion. DSCC1 overexpression plays an important role in cancer development, such as in colorectal, breast, and hepatocellular cancers. The specific role of DSCC1 in tumor progression remains largely unknown, necessitating a pan-cancer investigation to understand the potential function of DSCC1 in various cancers. In this study, we obtained data on physiological conditions, transcriptional expression, survival prognosis, genomic alteration, genomic instability, enriched pathways, immune infiltration, and immunotherapy from The Cancer Genome Atlas, The Genotype-Tissue Expression, cBioPortal, and other publicly available databases to systematically characterize the oncogenic and immunological roles of DSCC1 in 33 different cancers. We found that DSCC1 expression was upregulated at both mRNA and protein levels in various cancers. Additionally, DSCC1 expression was associated with higher tumor stage and grade in specific cancers. DSCC1 was a potential pan-cancer prognostic biomarker for its close association with patient prognosis and a diagnostic biomarker for its high predictive value in distinguishing tumor tissues from normal tissues. DSCC1 was universally amplified across different cancers and tightly associated with genomic instability. Moreover, DSCC1 had a close relationship with tumor immune cell infiltration; thus, it could be used as a potential biomarker for predicting the response and survival of patients with cancer who receive immune checkpoint blockade treatment. To sum up, our study revealed that DSCC1 is a promising target for tumor therapy.

Organ preservation in locally advanced colorectal cancer with microsatellite instability-high after immunotherapy

Neoadjuvant immunotherapy has achieved exciting efficacy with high clinical complete response (cCR) and pathologic complete response (pCR) rates and durable long-term effects. PD-1 checkpoint blockade-based immunotherapy has been highly successful in microsatellite instability high (MSI-H)/mismatch repair deficiency (dMMR) colorectal cancer and has been recommended as the first-line treatment for metastatic colorectal cancer by domestic and international guidelines. Several studies have shown that immunotherapy can be a potentially curable treatment for MSI-H rectal cancer and has even shown promise in organ preservation in colon cancer. In this study, we first clarified the feasibility of the watch-and-wait strategy after PD-1 checkpoint blockade treatment by indirect and direct evidence. Then from the assessment tools (including digital rectal examination, endoscopy, radiology, and lymph node assessment), the viable assessment methods of cCR for immunotherapy and related difficulties are proposed. Finally, the medication choices of immunotherapy, the treatment regimen, and the follow-up strategy are further discussed. We hope that neoadjuvant immunotherapy could be appropriately applied in MSI-H/dMMR colorectal cancer so that more patients can achieve organ preservation.

Tumor immune dysfunction and exclusion subtypes in bladder cancer and pan-cancer: a novel molecular subtyping strategy and immunotherapeutic prediction model

BackgroundMolecular subtyping is expected to enable precise treatment. However, reliable subtyping strategies for clinical application remains defective and controversial. Given the significance of tumor immune dysfunction and exclusion (TIDE), we aimed to develop a novel TIDE-based subtyping strategy to guide personalized immunotherapy in the bladder cancer (BC).MethodsTranscriptome data of BC was used to evaluate the heterogeneity and the status of TIDE patterns. Subsequently, consensus clustering was applied to classify BC patients based on TIDE marker-genes. Patients’ clinicopathological, molecular features and signaling pathways of the different TIDE subtypes were well characterized. We also utilize the deconvolution algorithms to analyze the tumor microenvironment, and further explore the sensitivity and mechanisms of each subtype to immunotherapy. Furthermore, BC patient clinical information, real-world BC samples and urine samples were collected for the validation of our findings, which were used for RNA-seq analysis, H&E staining, immunohistochemistry and immunofluorescence staining, and enzyme-linked immunosorbent assay. Finally, we also explored the conservation of our novel TIDE subtypes in pan-cancers.ResultsWe identified 69 TIDE biomarker genes and classified BC samples into three subtypes using consensus clustering. Subtype I showed the lowest TIDE status and malignancy with the best prognosis and highest sensitivity to immune checkpoint blockade (ICB) treatment, which was enriched of metabolic related signaling pathways. Subtype III represented the highest TIDE status and malignancy with the poorest prognosis and resistance to ICB treatment, resulting from its inhibitory immune microenvironment and T cell terminal exhaustion. Subtype II was in a transitional state with intermediate TIDE level, malignancy, and prognosis. We further confirmed the existence and characteristics of our novel TIDE subtypes using real-world BC samples and collected patient clinical data. This subtyping method was proved to be more efficient than previous known methods in identifying non-responders to immunotherapy. We also propose that combining our TIDE subtypes with known biomarkers can potentially improve the sensitivity and specificity of these biomarkers. Moreover, besides guiding ICB treatment, this classification approach can assist in selecting the frontline or recommended drugs. Finally, we confirmed that the TIDE subtypes are conserved across the pan-tumors.ConclusionsOur novel TIDE-based subtyping method can serve as a powerful clinical tool for BC and pan-cancer patients, and potentially guiding personalized therapy decisions for selecting potential beneficiaries and excluding resistant patients of ICB therapy.

Inhibition of autophagy-related protein 7 enhances anti-tumor immune response and improves efficacy of immune checkpoint blockade in microsatellite instability colorectal cancer

BackgroundThe efficacy of anti-PD-1 therapy is primarily hindered by the limited T-cell immune response rate and immune evasion capacity of tumor cells. Autophagy-related protein 7 (ATG7) plays an important role in autophagy and it has been linked to cancer. However, the role of ATG7 in the effect of immune checkpoint blockade (ICB) treatment on high microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) CRC is still poorly understood.MethodsIn this study, patients from the cancer genome altas (TCGA) COAD/READ cohorts were used to investigate the biological mechanism driving ATG7 development. Several assays were conducted including the colony formation, cell viability, qRT-PCR, western blot, immunofluorescence, flow cytometry, ELISA, immunohistochemistry staining and in vivo tumorigenicity tests.ResultsWe found that ATG7 plays a crucial role in MSI-H CRC. Its knockdown decreased tumor growth and caused an infiltration of CD8+ T effector cells in vivo. ATG7 inhibition restored surface major histocompatibility complex I (MHC-I) levels, causing improved antigen presentation and anti-tumor T cell response by activating reactive oxygen species (ROS)/NF-κB pathway. Meanwhile, ATG7 inhibition also suppressed cholesterol accumulation and augmentation of anti-tumor immune responses. Combining ATG7 inhibition and statins improved the therapeutic benefit of anti-PD-1 in MSI-H CRC. Importantly, CRC patients with high expression of both ATG7 and recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) experienced worse prognosis compared to those with low ATG7 and HMGCR expression.ConclusionsInhibition of ATG7 leads to upregulation of MHC-I expression, augments immune response and suppresses cholesterol accumulation. These findings demonstrate that ATG7 inhibition has therapeutic potential and application of statins can increase the sensitivity to immune checkpoint inhibitors.

Abstract 3914: IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target

Abstract Background: Loss of MHC-I antigen presentation is often associated with T-cell excluded tumors, and represents a common mechanism of both primary and acquired resistance to immune checkpoint blockade (ICB) treatment. MHC-I normally acts as a marker of “self” and cells that lack MHC-I are recognized and killed by innate immunity. In this study, we investigated the mechanism of innate immune evasion in tumors with MHC-I antigen presentation defects. Methods: Integrating functional genomics, big data, and artificial intelligence, we discovered that IGSF8 expressed on cancer cells is an evolutionarily conserved innate immune checkpoint. IGSF8 is normally expressed in neuronal tissues and is not essential in vitro or in vivo. However, knockout of IGSF8 in B16-F10 melanoma cell line decreased tumor growth in vivo. For clinical relevance of IGSF8 in tumor immunity, we analyzed genomics, transcriptomics, and clinical data from The Cancer Genome Atlas (TCGA). We developed an antibody (GV20-0251) against IGSF8 and tested its function to induce immune cell killing of cancer cells in vitro and inhibit tumor growth in vivo. Results: IGSF8 has receptors on both natural killer (NK) cells and dendritic cells (DC) to strongly suppress NK cytotoxicity and antigen presentation. In the TCGA tumor profiles, IGSF8 has highest mRNA expression in melanoma and is significantly overexpressed in many cancer types. IGSF8 also shows frequent copy number amplifications. In addition, IGSF8 expression is associated with low antigen presentation, low immune infiltration, and worse overall survival in patients with low MHC class I expression. Immunohistochemistry staining of various cancers showed that IGSF8 expression was primarily observed in malignant cells with low MHC-I. We developed a cross-species reactive antibody against IGSF8 (GV20-0251) which blocks IGSF8 interaction with NK and DC. Flow cytometry of the tumor infiltrating CD45+ cells revealed that IGSF8 inhibition significantly increased NK and dendritic cell infiltration. GV20-0251 enhances NK killing of cancer cells in vitro and increases antigen presentation, NK-mediated cytotoxicity, and T cell signaling in vivo. In multiple syngeneic tumor models (B16-F10, CT26, LLC, 4T1 and EMT6), anti-IGSF8 shows single-agent efficacy and is synergistic with anti-PD1 in controlling tumor growth. Conclusions: IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target. A phase 1 study is ongoing to explore the IGSF8 inhibitor GV20-0251 in patients with advanced or metastatic solid tumors (NCT05669430). Citation Format: X. Shirley Liu, Yulong Li, Xiangyang Wu, Hailing Liu, Huizhu Liu, Caibin Sheng, Qingyang Ding, Yixuan Tang, Chao Liu, Bin Xie, Xi Xiao, Quan Yu, Rongbin Zheng, Xihao Hu, Tengfei Xiao. IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target [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 3914.

Anti-PD-L1 nanobody guided in situ self-assembly and release of IOX1 and FDU for enhanced cancer chemo-immunotherapy

The clinical outcomes of immunotherapy are limited by the immunosuppressive tumor microenvironment (TME) and immune-related adverse effects. To address these issues, here we develop a tumor-specific chemo-immunotherapy prodrug FDU-IOX1-FF-Nb for highly-effective and low-toxic cancer therapy. This prodrug incorporates fluorouracil (FDU) to induce immunogenic cell death and reshape the TME, while the checkpoint inhibitor 5-carboxy-8-hydroxyquinoline (IOX1) provides immune checkpoint blockade (ICB) treatment and inhibits the expression of P-glycoproteins to overcome drug resistance. Furthermore, a small-sized anti-PD-L1 nanobody selectively delivers the prodrug to PD-L1 overexpressing tumor cells and blocks the PD-1/PD-L1 pathway for improved ICB treatment effect. The prodrug self-assembles into large nanoparticles within tumor cells via the π-π stacking interaction between FF after the reduction of glutathione, accumulating and preserving the drugs in the tumor sites. Finally, the esterase that are highly expressed in tumor cells cleave the ester bonds on nanoparticles, releasing free IOX1 and FDU for the chemotherapy and immunotherapy. Through the process of PD-L1 targeting, glutathione-triggered self-assembly, and esterase-mediated release of IOX1 and FDU, efficient cancer chemo-immunotherapy and precise tumor suppression with minimal non-target toxicity are achieved.

Abstract 6200: Single-cell analyses unravel novel immune and tumor cell characteristics linked to the response to neoadjuvant immune checkpoint blockade in patients with gastric cancer

Abstract Background: Gastric cancer remains the leading cause of cancer-related deaths worldwide, ranking fifth for incidence and fourth for mortality. Combining neoadjuvant therapy with immune checkpoint inhibitors has demonstrated improved outcomes for patients with gastric cancer. However, responses exhibit heterogeneity, and the understanding of the mediators influencing therapy response is limited. Methods: In this study, we collected 80 blood and tumor samples from 22 gastric cancer patients treated with XELOX/SOX alone or its combination with Nivolumab (anti-PD-1). Paired single-cell RNA-, TCR-, and BCR-sequencing were performed on pre- and post-treatment samples to investigate the dynamics of immune and tumor cells during the development of response/resistance to therapy. Results: Our findings revealed that elevated levels of baseline CD4 and CD8+ tissue-resident memory T (TRM) cells, in activated and stressed states, are associated with a superior response to combined therapy, and treatment further amplifies this cell population in responsive patients. Tracking T cell receptor clones across pre-/post-treated blood and tumor samples disclosed that the expanded clonotypes of TRM cells are exclusively detected in tumors, and considerable preexisting clones contribute to the expansion of these T cell populations following treatment. The simultaneous presence of TRM, B, and dendritic cells in pre-treated tumors predicted a favorable response, and they concertedly expanded in tumors responsive to anti-PD-1 therapy. Conclusions: Our findings highlight the significance of TRM cells in effective responses to anti-PD-1 therapies and indicate that their cellular communication with B and dendritic cells play a pivotal role in shaping the antitumor immunity elicited by combination therapy. Together, this study illuminates vital immune cell subsets during immune checkpoint blockade treatment, provides biological insights into the mechanism of successful combination therapy, and unravels potential targets for improving treatment efficacy in gastric cancer. Citation Format: Enyu Dai, Jiang-Jiang Qin, Can Hu, Zhiyuan Xu, Guangchun Han, Yanshuo Chu, Ruiping Wang, Minghao Dang, Yunhe Liu, Guangsheng Pei, Jiahui Jiang, Fuduan Peng, Yuanyuan Zhang, Andre Luis Faustino, Jaffer A. Ajani, Linghua Wang, Xiangdong Cheng. Single-cell analyses unravel novel immune and tumor cell characteristics linked to the response to neoadjuvant immune checkpoint blockade in patients with gastric cancer [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 6200.

Abstract 6397: Immune profiling for improving responsiveness to ipilimumab plus nivolumab checkpoint blockade treatment in patients with metastatic melanoma

Abstract Background:aCTLA-4 and aPD-1 combination therapy has significantly improved clinical outcomes in patients with metastatic melanoma, with 50%-60% of patients responding to treatment, but predictors of response and treatment induced immunological effects are poorly characterized. Identifying circulating immune populations as biomarkers could reveal crucial insights into the mechanisms driving treatment success. Methods:Using unbiased single-cell profiling (with cytometry by Time-Of-Flight, CyTOF), we characterized the systemic immune compartments of over 250 peripheral blood mononuclear samples from 89 patients with advanced melanoma before and during combination therapy. We evaluated the association between circulating immune populations and clinical outcome using nonparametric Mann-Whitney tests, and investigated the prognostic values of these features using survival analysis. Additionally, we assessed treatment-induced immunological effects, providing insights into the dynamic changes within the immune system during therapy. Results:We characterized circulating T cells, NK cells, B cells, and monocytes from peripheral blood. Following combination therapy, there was a shift from naïve and central memory T cells to effector populations in both CD4+ and CD8+ T cells compartments. There was also a broad activation observed in both adaptive and innate populations, characterized by an elevation of multiple co-stimulatory and co-inhibitory molecules. Lower CD8+ NK cell populations prior to combination therapy were predictive of durable clinical benefit (DCB). Pre-treatment measurements of Tregs were not predictive of outcome, but higher Tregs one month after starting therapy, as well as increases of Tregs from pre to post treatment, predicted better progression free survival (PFS) and overall survival (OS). Starting from early on treatment, higher “exhausted-like” CD8+ T cells, characterized by positive expression of at least two co-inhibitory markers, is associated with resistance. Conclusions:aCTLA-4 and aPD-1 combination therapy led to a longitudinal shift from naïve and central memory to effector phenotypes in T cells, along with a broad activation in both adaptive and innate populations. Within innate immune compartment, favorable clinical outcome was associated with lower CD8+ NK cells, capable of regulating proliferation and activation of CD4+ T cells. Interestingly, one month after the start of the therapy, the increase in Tregs and a decrease in 'exhausted-like' CD8+ T populations are associated with a better response. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in future. Citation Format: Joanna Baginska, Jiajia Chen, Giuseppe Tarantino, Anita Giobbie-Hurder, Jason L. Weirather, Mariano Severgnini, Michael Manos, Kelly Burke, Janice D. Russell, Martha Brainard, Elizabeth Gabriel, Ryan Brennick, Matthew Nazzaro, Emma Hathaway, Marta Holovatska, Claire Manuszak, Srinika Ranashinge, David Liu, F. Stephen Hodi. Immune profiling for improving responsiveness to ipilimumab plus nivolumab checkpoint blockade treatment in patients with metastatic melanoma [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 6397.

Abstract 2422: Liquid biopsy approaches for monitoring metastatic pancreatic cancer in immunotherapy treated patients

Abstract Introduction: Pancreatic cancer has a poor prognosis especially when identified at advanced stages. Globally in 2020, >450,000 people died from the disease. For those with locally advanced or metastatic cancer, the standard of care treatment is chemotherapy. Preliminary studies show that some patients with advanced-stage disease respond to immune checkpoint blockade treatment. Determining response to therapy using imaging techniques can be challenging. There is a clinical unmet need for noninvasive approaches that can provide a real-time assessment of response to therapy in these patients. We evaluated two methods for assessing response to therapy using circulating cell-free DNA (cfDNA) in patients with metastatic pancreatic cancer treated with immune checkpoint inhibition and radiation as part of the CheckPAC study (NCT02866383). Methods: For all patients with available samples (n=40), we evaluated one pre-treatment blood draw and one on-treatment blood draw (26-65 days after treatment start). We extracted cfDNA from each sample and performed low-coverage 1-2x whole genome sequencing (WGS). We evaluated tumor and matched buffy coat for the 34 patients who had > 10% tumor purity using WGS with at least 60x and 30x coverage, respectively. We compiled the mutated bases in the tumor genome and examined these positions in the cfDNA. We counted the number of mutant observations from cfDNA WGS and divided them by the total number of observations at these bases to calculate whole-genome mutant allele frequency (MAF). We categorized responders based on >30% drop in MAF from baseline. In a tissue independent approach, we detected changes in genome-wide cfDNA fragmentation patterns using the DNA evaluation of fragments for early interception (DELFI) approach. We evaluated survival using DELFI scores of on-treatment samples. Samples were designated DELFI high (above median) or DELFI low (below median.) Log-Rank tests were used to compare survival curves. Results: For the mutation based approach, molecular responders (n=7) had a median progression free survival (PFS) of 485 days compared to 57 days for non-responders (n=9) (p=0.02). Molecular responders had a median overall survival (OS) of 694 days compared to 200 days for non-responders (p=0.062). Patients with DELFI low scores after therapy (n=14) had a longer median PFS than those with DELFI high scores (n=14) (307 days versus 70 days, respectively) (p=0.013). DELFI low patients also had a longer median OS than DELFI high patients (627 vs 158 days, respectively) (p<0.0001). Conclusions: These analyses suggest that WGS mutation-based and fragmentation cfDNA approaches can identify individuals with metastatic pancreatic cancer who respond to immune checkpoint inhibition. Incorporation of these molecular methods for evaluation of tumor burden may provide information for patient-physician decision making to improve patient quality of life. Citation Format: Carolyn Anna Hruban, Inna Chen, Daniel C. Bruhm, Shashikant Koul, Jennie Yao, Susann Theile, Kavya Boyapati, Nicholas A. Vulpescu, Akshaya Annapragada, Leonardo Ferreira, Stephen Cristiano, Zachariah H. Foda, Vilmos Adleff, Julia Johansen, Jillian Phallen, Robert B. Scharpf, Victor E. Velculescu. Liquid biopsy approaches for monitoring metastatic pancreatic cancer in immunotherapy treated patients [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 2422.

Establishment and Validation of Novel Prognostic Subtypes in Hepatocellular Carcinoma Based on Bile Acid Metabolism Gene Signatures Using Bulk and Single-Cell RNA-Seq Data.

Hepatocellular carcinoma (HCC) is a highly detrimental cancer type and has limited therapeutic options, posing significant threats to human health. The development of HCC has been associated with a disorder in bile acid (BA) metabolism. In this study, we employed an integrative approach, combining various datasets and omics analyses, to comprehensively characterize the tumor microenvironment in HCC based on genes related to BA metabolism. Our analysis resulted in the classification of HCC samples into four subtypes (C1, C2a, C2b, and C3). Notably, subtype C2a, characterized by the highest bile acid metabolism score (BAMS), exhibited the highest survival probability. This subtype also demonstrated increased immune cell infiltration, lower cell cycle scores, reduced AFP levels, and a lower risk of metastasis compared to subtypes C1 and C3. Subtype C1 displayed poorer survival probability and elevated cell cycle scores. Importantly, the identified subtypes based on BAMS showed potential relevance to the gene expression of drug targets in currently approved drugs and those under clinical research. Genes encoding VEGFR (FLT4 and KDR) and MET were elevated in C2, while genes such as TGFBR1, TGFB1, ADORA3, SRC, BRAF, RET, FLT3, KIT, PDGFRA, and PDGFRB were elevated in C1. Additionally, FGFR2 and FGFR3, along with immune target genes including PDCD1 and CTLA4, were higher in C3. This suggests that subtypes C1, C2, and C3 might represent distinct potential candidates for TGFB1 inhibitors, VEGFR inhibitors, and immune checkpoint blockade treatments, respectively. Significantly, both bulk and single-cell transcriptome analyses unveiled a negative correlation between BA metabolism and cell cycle-related pathways. In vitro experiments further confirmed that the treatment of HCC cell lines with BA receptor agonist ursodeoxycholic acid led to the downregulation of the expression of cell cycle-related genes. Our findings suggest a plausible involvement of BA metabolism in liver carcinogenesis, potentially mediated through the regulation of tumor cell cycles and the immune microenvironment. This preliminary understanding lays the groundwork for future investigations to validate and elucidate the specific mechanisms underlying this potential association. Furthermore, this study provides a novel foundation for future precise molecular typing and the design of systemic clinical trials for HCC therapy.

A large-scale study of peptide features defining immunogenicity of cancer neo-epitopes.

Accurate prediction of immunogenicity for neo-epitopes arising from a cancer associated mutation is a crucial step in many bioinformatics pipelines that predict outcome of checkpoint blockade treatments or that aim to design personalised cancer immunotherapies and vaccines. In this study, we performed a comprehensive analysis of peptide features relevant for prediction of immunogenicity using the Cancer Epitope Database and Analysis Resource (CEDAR), a curated database of cancer epitopes with experimentally validated immunogenicity annotations from peer-reviewed publications. The developed model, ICERFIRE (ICore-based Ensemble Random Forest for neo-epitope Immunogenicity pREdiction), extracts the predicted ICORE from the full neo-epitope as input, i.e. the nested peptide with the highest predicted major histocompatibility complex (MHC) binding potential combined with its predicted likelihood of antigen presentation (%Rank). Key additional features integrated into the model include assessment of the BLOSUM mutation score of the neo-epitope, and antigen expression levels of the wild-type counterpart which is often reflecting a neo-epitope's abundance. We demonstrate improved and robust performance of ICERFIRE over existing immunogenicity and epitope prediction models, both in cross-validation and on external validation datasets.

Abstract B035: The contribution of inflammasomes and cGAS-STING in RT-induced cell fate

Abstract Radiotherapy (RT) is a curative treatment modality for many solid cancers. Our group has previously demonstrated that activation of the cGAS-STING pathway by RT induces inflammatory cytokine production and subsequent stimulation of anti-tumour immunity in concert with immune checkpoint blockade treatment. We have identified three other proteins that contribute to inflammatory signalling after RT: AIM2 (Absent in Melanoma 2), NLRP3 (NOD-Like Receptor Pyrin Domain-Containing 3), and IFI16 (Interferon Gamma Inducible Protein 16). Each of these proteins contribute to form distinct protein complexes called inflammasomes that, like cGAS-STING, sense cytoplasmic DNA. Little is known about how inflammasome signalling may change after RT, or how inflammasomes may influence RT-ICB induced anti-tumour immunity. We hypothesize that RT induces inflammasome signalling to influence specific gene expression and cytokine profiles, in tandem with alterations in cellular fate with implications for systemic anti-tumour immunity triggered by radiotherapy. We will present ongoing work in which we show that pharmacological or genetic disruption of inflammasomes alters cytokine production post-RT. We also demonstrate that manipulation of cell fate through senolytic therapy or manipulation of caspase activity alters the balance of cytokine production post-RT. Together our data strongly implicates the role of several inflammasome complexes in the response of epithelial cells to RT. Our work will also highlight the possible crosstalk between the cGAS-STING pathway and inflammasome complex pathway, both triggered by RT. Understanding and subsequent manipulation of cell fate, in part through inflammasome and cGAS-STING mediated activity, may be a therapeutic strategy to influence treatment responses, such as anti-tumour immunity, in cancer. Citation Format: Cindy T Ha, Shirony Nicholson-Puthenveedu, Shane M Harding. The contribution of inflammasomes and cGAS-STING in RT-induced cell fate [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B035.

Serum ferritin level is an effective prognostic factor for lung cancer immunotherapy

ABSTRACT Immunotherapy of lung cancer has achieved promising clinical results. However, it is urgent to develop predictive biomarkers for effective immunotherapy. While ferroptosis plays a critical role in immunotherapy efficacy, ferritin is an important regulatory factor. We, therefore, hypothesize that basal serum ferritin levels before immunotherapy and their corresponding changes during immunotherapy can be useful predictors of immunotherapy response in patients with lung cancer. We measured serum ferritin levels in 107 patients with lung cancer before and during immune checkpoint blockade treatments and studied the correlation between ferritin levels, response rate, and survival. Moreover, the correlation between basal ferritin and PD-L1 expression, tumor stages and pathological types was also analyzed. Patients with lower basal serum ferritin levels before immunotherapy had longer progression-free survival (PFS) (median 7 vs 4 months, P = .023) and higher disease control rate (DCR) (X2 = 4.837, P = .028), those with downregulated serum ferritin levels during immunotherapy correlated with longer PFS (median 9.5 vs 4 months, P < .001) and higher DCR (X2 = 6.475, P = .011). However, the “integrated factor”, which was calculated as the combination of lower basal serum ferritin levels before immunotherapy and downregulated serum ferritin levels during immunotherapy, correlated with prolonged PFS (P < .001). Multivariate analyses revealed that the basal serum ferritin levels before immunotherapy and the corresponding changes during immunotherapy were both strong independent prognostic factors (hazard ratio (HR) = 1.60, P = .041; HR = 2.65, P = .001). These findings suggest that serum ferritin levels can be used as a prognostic biomarker for lung cancer in predicting immunotherapy efficacy.

Inhibition of SF3B1 improves the immune microenvironment through pyroptosis and synergizes with αPDL1 in ovarian cancer

Ovarian cancer is resistant to immune checkpoint blockade (ICB) treatment. Combination of targeted therapy and immunotherapy is a promising strategy for ovarian cancer treatment benefit from an improved immune microenvironment. In this study, Clinical Proteomic Tumor Analysis Consortium (CPTAC) and The Cancer Genome Atlas (TCGA) cohorts were used to screen prognosis and cytotoxic lymphocyte infiltration-associated genes in upregulated genes of ovarian cancer, tissue microarrays were built for further verification. In vitro experiments and mouse (C57/BL6) ovarian tumor (ID8) models were built to evaluate the synergistic effect of the combination of SF3B1 inhibitor and PD-L1 antibody in the treatment of ovarian cancer. The results show that SF3B1 is shown to be overexpressed and related to low cytotoxic immune cell infiltration in ovarian cancer. Inhibition of SF3B1 induces pyroptosis in ovarian cancer cells and releases mitochondrial DNA (mtDNA), which is englobed by macrophages and subsequently activates them (polarization to M1). Moreover, pladienolide B increases cytotoxic immune cell infiltration in the ID8 mouse model as a SF3B1 inhibitor and increases the expression of PD-L1 which can enhance the antitumor effect of αPDL1 in ovarian cancer. The data suggests that inhibition of SF3B1 improves the immune microenvironment of ovarian cancer and synergizes ICB immunotherapy, which provides preclinical evidence for the combination of SF3B1 inhibitor and ICB to ovarian cancer treatment.

Neural network models for sequence-based TCR and HLA association prediction.

T cells rely on their T cell receptors (TCRs) to discern foreign antigens presented by human leukocyte antigen (HLA) proteins. The TCRs of an individual contain a record of this individual's past immune activities, such as immune response to infections or vaccines. Mining the TCR data may recover useful information or biomarkers for immune related diseases or conditions. Some TCRs are observed only in the individuals with certain HLA alleles, and thus characterizing TCRs requires a thorough understanding of TCR-HLA associations. The extensive diversity of HLA alleles and the rareness of some HLA alleles present a formidable challenge for this task. Existing methods either treat HLA as a categorical variable or represent an HLA by its alphanumeric name, and have limited ability to generalize to the HLAs that are not seen in the training process. To address this challenge, we propose a neural network-based method named Deep learning Prediction of TCR-HLA association (DePTH) to predict TCR-HLA associations based on their amino acid sequences. We demonstrate that DePTH is capable of making reasonable predictions for TCR-HLA associations, even when neither the HLA nor the TCR have been included in the training dataset. Furthermore, we establish that DePTH can be used to quantify the functional similarities among HLA alleles, and that these HLA similarities are associated with the survival outcomes of cancer patients who received immune checkpoint blockade treatments.