Checkpoint Immunotherapy Research Articles

Abstract B040: Novel immunotherapy strategies targeting PTEN and TP53 defects in advanced prostate cancer

Abstract Prostate cancer is the most diagnosed cancer in men worldwide and the leading cause of cancer death in men worldwide. Genetic inactivation of PTEN and TP53 are common in advanced prostate cancers. Checkpoint immunotherapy has yielded meaningful responses across many cancers but shown modest activity in advanced prostate cancer. Prior studies showed that overexpression of immune checkpoint B7-H3 (CD276) correlates with the increased risks of clinical recurrence, disease spread, and poor outcomes in various cancer types, including prostate cancer. However, the roles of B7-H3 in prostate cancer development and its tumor microenvironment remain unclear, partially due to the lack of tissue-specific deletion mouse models. This gap in knowledge hinders the application of immunotherapy targeting B7-H3 in prostate cancers. To identify PTEN- and p53-associated immune checkpoints, we performed multi-omics analyses of expression patterns of 51 checkpoint molecules in human prostate cancer samples and found that B7-H3 is one of the most significantly overexpressed immune checkpoints in prostate tumors containing PTEN and TP53 genetic inactivation. Mechanistically, we found that the PTEN-AKT pathway co-operates with p53 pathway in modulating B7-H3 expression in cancer cells. In Pten/Trp53 genetically engineered mouse (GEM) models, prostate-specific deletion of Cd276 resulted in markedly delayed tumor progression and reversed immunosuppression in the tumor microenvironment. Furthermore, we developed and evaluated novel combinatorial immunotherapy strategies targeting B7-H3 in advanced prostate cancer with PTEN and TP53 deficiencies. Citation Format: Wei Shi, Yin Wang, Yuehui Zhao, Di Zhao. Novel immunotherapy strategies targeting PTEN and TP53 defects in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B040.

Product characteristics and clinical trial design for T-Plex: Multiplexed, enhanced T cell receptor engineered T cell therapy for solid tumors.

2554 Background: Checkpoint immunotherapies have revolutionized the treatment of solid tumors yet durably benefit a minority of patients because they rely on endogenous anti-tumor T cells. In patients lacking functional anti-tumor T cells, a solution is engineering their T cells with exogenous T cell receptors (TCRs) to efficiently target and kill tumor cells. Initial clinical trials with TCR engineered T cell therapies (TCR-Ts) only produced partial, short-lasting responses because they targeted single tumor antigens. Solid tumors are notoriously heterogenous not only with highly variable antigen expression levels but also with recently identified HLA gene losses occurring in up to 40% of solid tumors, enabling tumor cells to evade T cell attack. Methods: To overcome this heterogeneity, TScan has developed T-Plex, a multiplexed therapy comprising 2-3 different TCR-Ts, chosen from a collection of TCR-Ts called the ImmunoBank, to target different tumor antigens presented on different HLA types with confirmed tumor expression. Results: To deepen clinical responses, TCR-T cells are engineered to express the CD8α/β co-receptors that, in preclinical experiments, enable CD4+ helper T cells to become >100-fold more cytotoxic and secrete cytokines at >100-fold lower target antigen levels. Finally, to enable T cell persistence despite immunosuppressive TGF-β in the tumor microenvironment, engineered TCR-T cells express the dominant negative TGF-β receptor allowing ~10-fold improved proliferation and ~2-fold improved cytokine production in the presence of TGF-β. The inclusion of these additional genes is enabled by a transposon manufacturing system with no cargo limit. Patient selection for the Phase 1 study uses a separate screening protocol to identify patients any time during standard clinical care so they can rapidly enroll into the treatment protocol upon progression. Screening comprises germline HLA testing, then archival tumor testing for antigen expression and HLA loss. Treatment includes standard lymphodepletion followed by 2 doses of T-Plex infused 28 days apart. T-Plex initially starts with 2 TCR-Ts targeting different MAGE-A1 antigens presented either on HLA-A*02:01 or HLA-C*07:02. Dose escalation starts with testing the single TCR-Ts individually in dose levels 1 and 2. The two TCR-Ts will then be combined and escalated in dose levels 3 and 4. Additional TCR-Ts added to the ImmunoBank will go through dose levels 1 and 2 as single therapies before becoming available for multiplexed dose levels 3 and 4. Primary endpoints include safety and feasibility, secondary endpoints are response rates and duration of response and exploratory endpoints measure T cell persistence. Conclusions: Four additional TCR-Ts are on track to be added to the ImmunoBank in 2023 which makes 50-80% of patients with common solid tumors eligible for multiplexed TCR-T therapy.

Incorporation of triapine (T) with cisplatin chemoradiation (CRT) for locally advanced cervical and vaginal cancer: Results from NRG-GY006, a phase III randomized trial.

5502 Background: Definitive cisplatin-based chemoradiation (CRT) plus brachytherapy for locally advanced cervical cancer (FIGO IB3-IVA) results in sustained survival for 60-70% of patients. Recent studies integrating anti-PD-1 checkpoint immunotherapy (CALLA, NCT03830866) or consolidation chemotherapy (OUTBACK, ACTRN12610000732088) have not demonstrated a survival benefit over CRT. Intrinsic overexpression of ribonucleotide reductase may enhance DNA damage repair due to CRT. We report on the efficacy and tolerability of adding the ribonucleotide reductase inhibitor, triapine, to CRT. Methods: NRG GY006 is a randomized, open-label phase III clinical trial. Eligible patients had FIGO 2009 locally advanced cervical (stages IB2, II, IIIB or IVA without radiographic evidence of para-aortic nodal involvement) or stages II-IV vaginal cancer. Patients were randomly assigned to receive cisplatin (40 mg/m2 weekly) with RT 45 Gy + lymph node boost alone (CRT) or CRT in combination with 15 total infusions of triapine (25 mg/m2 IV) Mon/Wed/Fri (CRT + T). Both image guided IMRT or 3D RT were allowed but needed to be specified and pass a rigorous credentialing process. All RT plans had a pre-treatment review with expert planning feedback to the sites. The primary endpoint was overall survival (OS); secondary endpoint was progression-free survival. Exploratory endpoints included rate of complete metabolic response on post treatment PET/CT imaging at 3 months and knowledge-based planning for image guided IMRT. The target sample was 450 with 127 OS events. The design was to provide 80% power to detect a 10% improvement in OS at 3 years over the control (or HR = 0.6) at 2.5% significance level including one interim futility analysis at 50% information time. Results: Between 1/15/16 and 9/22/22 448 eligible patients were randomized to CRT (n=224) or CRT+T (n=224). The database was locked on 10/18/22 with 69 deaths. Median age was 47 years (range 23-85 years). The majority had cervical cancer (93.3%) and squamous cell carcinoma (82%). 52% had FIGO stage II disease. Racial/ethnic distribution included non-Hispanic white (53.8%), Hispanic/Latina (22.5%), and black (15.2%). IMRT was used in (74.3%) and HDR brachytherapy (98.2%) of cases. No differences in Grade 3-5 toxicities were observed: CRT =52% and CRT +T= 49% with Two G5 toxicities (cardiac arrest and acidosis) in the CRT+T arm. 343 patients have completed all protocol directed therapy. With a median follow-up of 28 months (IQR 15-45 months), the median PFS and OS for both arms were not reached yet. HR for death was 1.018 (95% CI 0.634-1.635), the conditional power was 15% to detect a HR = 0.6 at 100% information time. Conclusions: The addition of triapine to CRT did not improve OS. Clinical trial information: NCT02466971 .

Preoperative pembrolizumab for MSI high, EBV positive or PD-L1 positive locally advanced gastric cancer followed by surgery and adjuvant chemoradiation with pembrolizumab: Interim results of a phase 2 multi-center trial.

e16073 Background: Certain subtypes of gastric cancer are less responsive to perioperative chemotherapy. Preliminary data suggest that patients with microsatellite unstable tumors (MSI-H), EBV expressing tumors, and tumors with high PD-L1 expression may benefit from immunotherapy. This trial evaluates the benefit of PD-1 checkpoint immunotherapy in this subset of patients with operable gastric cancer. Methods: This is an interim analysis of this Phase II multicenter clinical trial (NCT03257163). Patients with clinically staged T2-T4, N0-N3, M0 gastric adenocarcinoma with MSI-H, EBV positive, or PDL1 expression with CPS>/= 1% were included. Patients receive two cycles of neoadjuvant pembrolizumab followed by gastrectomy. Postoperatively, patients receive one cycle of adjuvant capecitabine + pembrolizumab, then chemoradiation (45 Gy in 25 fractions) with capecitabine + pembrolizumab, then two cycles capecitabine + pembrolizumab, followed by 1-year total of pembrolizumab. Primary endpoint is DFS. Results: 33 patients have been enrolled (planned = 40), median age 67.5 years (range, 44 – 88 years), median follow up duration 18.1 months (range, 2.3-42.5 months). Patients were 30.3% Hispanic, 27% Asian, 24% Black, 24% White. Of the 33 patients, 12 were MSI-H, 3 EBV (+), and 27 had PDL1 expression with CPS >/= 1%. Tumors were of advanced clinical stage with 84.8% ≥ cT3 and 57.6% cN(+). All patients received two cycles of neoadjuvant pembrolizumab. Six patients did not undergo surgical resection: peritoneal disease (n=2), soft tissue metastases (n=1), locally unresectable (n=2), and frailty (n=1). Of the 27 patients who completed surgery, treatment effect was seen in 46%, down-staging in 50%, and complete pathologic response seen in 3 patients, 2 of whom has MSI-H tumors and one whose tumor was PDL1+. Two patients did not receive adjuvant therapy due to postoperative complications. A total of 20 patients have received optimal therapy as defined as postoperative RT and >/=2 cycles of adjuvant pembrolizumab. Four patients had adjuvant pembrolizumab monotherapy (after chemoradiation) stopped due to rash (n=2), pneumonia (n=1), and colitis (n=1). The estimated DFS at 24 months is 60% for the entire cohort (n=33). For the optimal therapy cohort (n=20), the DFS at 24 months is 80%. Conclusions: In this biomarker-driven cohort of operable gastric adenocarcinoma patients, results are promising for patients who receive two cycles of preoperative pembrolizumab followed by postoperative concurrent pembrolizumab with chemoradiation therapy, and chemotherapy. This study presents a novel and effective paradigm for the management of localized gastric cancer and warrants additional evaluation. Clinical trial information: NCT03257163 .

Model for predicting prognosis and immunotherapy based on CD+8T cells infiltration in neuroblastoma.

Neuroblastoma (NBL) is an extracranial malignant tumor in children deriving from the neural crest in the sympathetic nervous system. Although various immunotherapy interventions have made significant breakthroughs in many adult cancers, the efficacy of these immunotherapies was still limited in NBL. NBL has low immunogenicity which results in a lack of tumor-infiltrating T lymphocytes in the tumor microenvironment (TME). Moreover, tumor cells can wield many immune evasion strategies both in the TME and systemically to impede lymphocyte infiltration and activation. All these factors hamper the anti-tumor effects of CD8+ T cells during immunotherapy and the levels of infiltrating CD8+ T cells correlate with therapy response. In this study, we utilized multidimensional bioinformatic methods to establish a risk model based on CD8+ T cells -related genes (CD8+ TRGs). We obtained 33 CD8+TRGs with well-predictive ability for prognosis in both GSE49711 and E-MTAB-8248 cohorts. Then, 12 CD8+TRGs including HK2, RP2, HPSE, ELL2, GFI1, SLC22A16, FCGR3A, CTSS, SH2D1A, RBP5, ATF5, and ADAM9 were finally identified for risk model construction and validation. This model revealed a stable performance in prognostic prediction of the overall survival (OS) and event-free survival (EFS) in patients with NBL. Additionally, our research indicated that the immune and stromal scores, immune-related pathways, immune cell infiltration, the expression of major histocompatibility complex (MHC) and immune checkpoint molecules, immunotherapy response, and drug susceptibility revealed significant differences between high and low-risk groups. According to our analyses, the constructed CD8+TRGs-based risk model may be promising for the clinical prediction of anti-tumor therapy responses and prognoses in NBL.

Systemic Alterations in Type-2 Conventional Dendritic Cells Lead to Impaired Tumor Immunity in Pancreatic Cancer.

Intratumoral T-cell dysfunction is a hallmark of pancreatic tumors, and efforts to improve dendritic cell (DC)-mediated T-cell activation may be critical in treating these immune therapy unresponsive tumors. Recent evidence indicates that mechanisms that induce dysfunction of type 1 conventional DCs (cDC1) in pancreatic adenocarcinomas (PDAC) are drivers of the lack of responsiveness to checkpoint immunotherapy. However, the impact of PDAC on systemic type 2 cDC2 development and function has not been well studied. Herein, we report the analysis of 3 cohorts, totaling 106 samples, of human blood and bone marrow (BM) from patients with PDAC for changes in cDCs. We found that circulating cDC2s and their progenitors were significantly decreased in the blood of patients with PDAC, and repressed numbers of cDC2s were associated with poor prognosis. Serum cytokine analyses identified IL6 as significantly elevated in patients with PDAC and negatively correlated with cDC numbers. In vitro, IL6 impaired the differentiation of cDC1s and cDC2s from BM progenitors. Single-cell RNA sequencing analysis of human cDC progenitors in the BM and blood of patients with PDAC showed an upregulation of the IL6/STAT3 pathway and a corresponding impairment of antigen processing and presentation. These results suggested that cDC2s were systemically suppressed by inflammatory cytokines, which was linked to impaired antitumor immunity.

Chemokine Analysis in Patients with Metastatic Uveal Melanoma Suggests a Role for CCL21 Signaling in Combined Epigenetic Therapy and Checkpoint Immunotherapy.

Patients with metastatic uveal melanoma have limited therapeutic options and high mortality rate so new treatment options are needed. We previously reported that patients treated with the PD-1 inhibitor pembrolizumab and the histone deacetylase inhibitor entinostat in the PEMDAC trial, experienced clinical benefits if their tumor originated from iris or was wildtype for BAP1 tumor suppressor gene. Here we present the 2-year follow-up of the patients in the PEMDAC trial and identify additional factors that correlate with response or survival. Durable responses were observed in 4 patients, with additional 8 patients exhibiting a stable disease. The median overall survival was 13.7 months. Grade 3 adverse events were reported in 62% of the patients, but they were all manageable. No fatal toxicity was observed. Activity of thymidine kinase 1 in plasma was higher in patients with stable disease or who progressed on treatment, compared with those with partial response. Chemokines and cytokines were analyzed in plasma. Three chemokines were significantly different when comparing patients with and without response. One of the factors, CCL21, was higher in the plasma of responding patients before treatment initiation but decreased in the same patients upon treatment. In tumors, CCL21 was expressed in areas resembling tertiary lymphoid structures (TLS). High plasma levels of CCL21 and presence of TLS-like regions in the tumor correlated with longer survival. This study provides insight into durable responses in the PEMDAC trial, and describes dynamic changes of chemokines and cytokines in the blood of these patients. The most significant finding from the 2-year follow-up study of the PEMDAC trial was that high CCL21 levels in blood was associated with response and survival. CCL21 was also expressed in TLS-like regions and presence of these regions was associated with longer survival. These analyses of soluble and tumor markers can inform on predictive biomarkers needing validation and become hypothesis generating for experimental research.

Immune checkpoint B7-H3 is a therapeutic vulnerability in prostate cancer harboring PTEN and TP53 deficiencies.

Checkpoint immunotherapy has yielded meaningful responses across many cancers but has shown modest efficacy in advanced prostate cancer. B7 homolog 3 protein (B7-H3/CD276) is an immune checkpoint molecule and has emerged as a promising therapeutic target. However, much remains to be understood regarding B7-H3's role in cancer progression, predictive biomarkers for B7-H3-targeted therapy, and combinatorial strategies. Our multi-omics analyses identified B7-H3 as one of the most abundant immune checkpoints in prostate tumors containing PTEN and TP53 genetic inactivation. Here, we sought in vivo genetic evidence for, and mechanistic understanding of, the role of B7-H3 in PTEN/TP53-deficient prostate cancer. We found that loss of PTEN and TP53 induced B7-H3 expression by activating transcriptional factor Sp1. Prostate-specific deletion of Cd276 resulted in delayed tumor progression and reversed the suppression of tumor-infiltrating T cells and NK cells in Pten/Trp53 genetically engineered mouse models. Furthermore, we tested the efficacy of the B7-H3 inhibitor in preclinical models of castration-resistant prostate cancer (CRPC). We demonstrated that enriched regulatory T cells and elevated programmed cell death ligand 1 (PD-L1) in myeloid cells hinder the therapeutic efficacy of B7-H3 inhibition in prostate tumors. Last, we showed that B7-H3 inhibition combined with blockade of PD-L1 or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) achieved durable antitumor effects and had curative potential in a PTEN/TP53-deficient CRPC model. Given that B7-H3-targeted therapies have been evaluated in early clinical trials, our studies provide insights into the potential of biomarker-driven combinatorial immunotherapy targeting B7-H3 in prostate cancer, among other malignancies.

Predictive value and accuracy of [18F]FDG PET/CT modified response criteria for checkpoint immunotherapy in patients with advanced melanoma

PurposeImmune checkpoint inhibitors (ICIs) are widely used in metastatic melanoma and dramatically alter the treatment of these patients. Given the high cost and potential toxicity, a reliable method for evaluating treatment response is needed. In this study, we assessed tumor response in patients with metastatic melanoma treated with ICIs using three modified response criteria: PET Response Evaluation Criteria for Immunotherapy (PERCIMT), PET Response Criteria in Solid Tumors for up to Five Lesions (PERCIST5), and immunotherapy-modified PET Response Criteria in Solid Tumors for up to Five Lesions (imPERCIST5).MethodsNinety-one patients with non-resectable stage IV metastatic melanoma who received ICIs were retrospectively enrolled in this study. Each patient had two [18F]FDG PET/CT scans performed before and after ICI therapy. Responses at the follow-up scan were evaluated according to PERCIMT, PERCIST5, and imPERCIST5 criteria. Patients were classified into four groups: complete metabolic response (CMR), partial metabolic response (PMR), progressive metabolic disease (PMD), and stable metabolic disease (SMD). To assess the “disease control rate,” two groups have been defined based on each criterion: patients with CMR, PMR, and SMD as “disease-controlled group (i.e., responders)” and PMD as the “uncontrolled-disease group (i.e., non-responders)”. The correspondence between metabolic tumor response defined by these criteria and clinical outcome was assessed and compared.ResultsThe response and the disease control rates were 40.7% and 71.4%, 41.8% and 50.5%, and 54.9% and 74.7% based on the PERCIMT, PERCIST5, and imPERCIST5 criteria, respectively. PERCIMT and imPERCIST5 showed significantly different disease control rates from that of PERCIST5 (P < 0.001), whereas it was not significant between PERCIMT and imPERCIST5. Overall survival was significantly longer in the metabolic responder groups than in the non-responder groups based on PERCIMT and PERCIST5 criteria (PERCIMT: 2.48 versus 1.47 years, P = 0.003; PERCIST5: 2.57 versus 1.81 years. P = 0.017). However, according to imPERCIST5 criterion, this difference was not observed (P = 0.12).ConclusionAlthough the appearance of new lesions can be secondary to an inflammatory response to ICIs and indicative of pseudoprogression, given the higher rate of true progression, the appearance of new lesions should be interpreted deliberately. Of the three assessed modified criteria, PERCIMT appear to provide more reliable metabolic response assessment that correlates strongly with overall patient survival.

Dendritic cell expansion and activation enhances the adaptive immune response against TNBC brain metastases

Abstract Despite initial control by chemotherapy, Triple Negative Breast Cancer (TNBC) quickly acquires resistance and metastasizes to distal organs, including the brain. Median survival following diagnosis of TNBC brain metastases is a meager 7.3 months—indicative of a substantial unmet clinical need, with only a small subpopulation responding to checkpoint immunotherapy. As suboptimal antigen presentation can contribute to the weak T lymphocyte response to intracranial tumors, we hypothesized that enhancing dendritic cell (DC) presence in tumors may support intracranial T cell responses to tumors. Using a murine model of intracranial metastasis, we assessed immune cell frequency and function and in vivocytokine staining for flow cytometry to test whether Flt3L can be used to promote DC presence within tumors, and whether the addition of polyIC further enhances subsequent T cell responses. Flt3L treatment effectively increases the number of DC within the brains and meninges of mice, however these newly expanded populations exhibit lower proportions of the costimulatory molecule CD86 than the untreated control group, indicative of an immature phenotype. 24 hours after polyIC treatment, a higher percentage of DCs in brain tumors express CD86 but do not produce IL-12. Despite the lack of intratumoral IL-12, the combination of Flt3L and polyIC treatments led to a higher presence of T cells within tumors and their production of IFN-γ in the TME than either treatment alone. In conclusion, simply increasing the presence of DC within intracranial tumors is insufficient to promote T cell immunity; rather, deliberate activation of de novo DC is required for this full effect. Supported by grants from NIH (2T32CA009109-46 and 1R21CA226607-01A1) and the Schiff Foundation

Mapping the gene space at single-cell resolution with gene signal pattern analysis

Abstract In single-cell RNA sequencing analysis, several computational methods have been developed to map the cellular state space, but little has been done to map the gene space. A mapping that preserves gene-gene relationships within the dataset is particularly useful for characterizing cellular heterogeneity within cell types, where boundaries between cell subpopulations are often unclear or even arbitrary. Here, we present gene signal pattern analysis, a new paradigm for analyzing single cells. We build a cell-cell graph and design a dictionary of diffusion wavelets, capturing a multiscale view of the cell space. We then transform genes by the dictionary and learn a reduced gene representation. Given the gap in prior research for this problem, we design nine alternative strategies and three benchmarks for evaluating preservation of gene-gene relationships, all of which are outperformed by diffusion wavelet-transformed signals. We also define, calculate, and evaluate localization, a key property of a gene signal on the cellular graph. We demonstrate the utility of gene signal pattern analysis on T cells from a mouse model of peripheral tolerance in skin. The gene space mapping reveals a continuum of gene signals characterized by T cell subtypes and transcriptional programs related to effector function and proliferation. Furthermore, we built a multiscale manifold of 48 melanoma patient samples, demonstrating the ability of our method to characterize differences between responders and non-responders to checkpoint immunotherapy. Together, we show gene signal pattern analysis, through methodology from graph signal processing, spectral graph theory, and machine learning, represents an avenue for future research in scRNA-seq analysis. Gruber Foundation

Identification of TREM2-positive tumor-associated macrophages in esophageal squamous cell carcinoma: implication for poor prognosis and immunotherapy modulation.

It is now understood that the effectiveness of checkpoint immunotherapy can be impaired by immunosuppressive tumor-associated macrophages (TAMs). Nonetheless, the impact of different TAM subpopulations on the antitumor immune response remains unclear, mainly due to their heterogeneity. Herein, we identified a novel TAM subpopulation in esophageal squamous cell carcinoma (ESCC) that might contribute to poor clinical outcomes and immunotherapy modulation. We analyzed two single-cell RNA sequencing (scRNA-seq) datasets (GSE145370 and GSE160269) of esophageal squamous cell carcinoma to identify a novel TREM2-positive TAM subpopulation characterized by upregulation of TREM2, C1QC, C1QB, C1QA, SPP1, and APOE. Quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that these genes were significantly overexpressed in ESCC. Multiplex immunofluorescence validated the infiltration of TREM2+ TAMs in ESCC tissues, which correlated with poorer overall survival (OS). The scRNA-seq analysis in dataset GSE120575 indicated significant enrichment of TREM2+ TAMs in melanoma patients (n=48) with poor immunotherapy response, which had an identical gene signature with TREM2+ TAMs from ESCC. Analysis of 29 bulk-RNA melanoma samples from dataset GSE78220 revealed that a gene signature of 40 genes associated with TREM2+ TAMs was upregulated in the transcriptome of melanomas that did not respond to anti-PD1 therapy. Validation in the TCGA ESCC cohort (n=80) showed that a high enrichment score of the TREM2+ TAM was associated with poor prognosis. In addition, 10 ESCC patients treated with anti-PD1 therapy suggested that patients who are not sensitive to immunotherapy have higher density of TREM2+TAMs infiltration. Overall, TREM2+ TAM infiltration in ESCC is associated with poor prognosis and may serve as a biomarker for predicting outcomes and immunotherapy modulation in this patient population. modulation; single-cell RNA sequencing.

Expression-based subtypes define pathologic response to neoadjuvant immune-checkpoint inhibitors in muscle-invasive bladder cancer

Checkpoint immunotherapy (CPI) has increased survival for some patients with advanced-stage bladder cancer (BCa). However, most patients do not respond. Here, we characterized the tumor and immune microenvironment in pre- and post-treatment tumors from the PURE01 neoadjuvant pembrolizumab immunotherapy trial, using a consolidative approach that combined transcriptional and genetic profiling with digital spatial profiling. We identify five distinctive genetic and transcriptomic programs and validate these in an independent neoadjuvant CPI trial to identify the features of response or resistance to CPI. By modeling the regulatory network, we identify the histone demethylase KDM5B as a repressor of tumor immune signaling pathways in one resistant subtype (S1, Luminal-excluded) and demonstrate that inhibition of KDM5B enhances immunogenicity in FGFR3-mutated BCa cells. Our study identifies signatures associated with response to CPI that can be used to molecularly stratify patients and suggests therapeutic alternatives for subtypes with poor response to neoadjuvant immunotherapy.

Apatinib remodels the immunosuppressive tumor ecosystem of gastric cancer enhancing anti-PD-1 immunotherapy

Apatinib has been shown to clinically enhance anti-PD-1 immunotherapy for advanced gastric cancer (GC). However, the complexity of GC immunosuppression remains a challenge for precision immunotherapy. Here, we profile the transcriptomes of 34,182 single cells from GC patient-derived xenografts of humanized mouse models treated with vehicle, nivolumab, or nivolumab plus apatinib. Notably, excessive expression of CXCL5 in the CellCycle malignant epithelium, induced by anti-PD-1 immunotherapy and blocked by combined apatinib treatment, is found to be a key driver of tumor-associated neutrophil (TAN) recruitment in the tumor microenvironment through the CXCL5/CXCR2 axis. We further show that the protumor TAN signature is associated with anti-PD-1 immunotherapy-related progressive disease and poor cancer prognosis. Molecular and functional analyses in cell-derived xenograft models confirm the positive invivo therapeutic effect of targeting the CXCL5/CXCR2 axis during anti-PD-1 immunotherapy. Altogether, our study elucidates the GC immunosuppressive landscape in anti-PD-1 immunotherapy and highlights potential targets for overcoming checkpoint immunotherapy resistance.

1243 Longer recurrence free survival (RFS) in germline pathogenic variant (gPV) positive patients with melanoma on adjuvant checkpoint immunotherapy (IO)

1243 Longer recurrence free survival (RFS) in germline pathogenic variant (gPV) positive patients with melanoma on adjuvant checkpoint immunotherapy (IO)

The pathological significance and potential mechanism of ARHGEF6 in lung adenocarcinoma

BackgroundEmerging evidences suggest that ARHGEF6 is involved in cancers but the exact significance and underlying mechanism are unclear. This study aimed to elucidate the pathological significance and potential mechanism of ARHGEF6 in lung adenocarcinoma (LUAD). MethodsBioinformatics and experimental methods were used to analyze the expression, the clinical significance, the cellular function and potential mechanisms of ARHGEF6 in LUAD. ResultsARHGEF6 was downregulated in LUAD tumor tissues and correlated negatively with poor prognosis and tumor stemness, positively with the Stromal score, the Immune score and the ESTIMATE score. The expression level of ARHGEF6 was also associated with drug sensitivity, the abundance of immune cells, the expression levels of Immune checkpoint genes and immunotherapy response. Mast cells, T cells and NK cells were the first three cells with the highest expression of ARHGEF6 in LUAD tissues. Overexpression of ARHGEF6 reduced proliferation and migration of LUAD cells and the growth of xenografted tumors, which could be reversed by re-knockdown of ARHGEF6. Results of RNA sequencing revealed that ARHGEF6 overexpression induced significant changes in the expression profile of LUAD cells, and genes encoding uridine 5′-diphosphate-glucuronic acid transferases (UGTs) and extracellular matrix (ECM) components were downregulated. ConclusionsARHGEF6 functions as a tumor suppressor in LUAD and may serve as a new prognostic marker and potential therapeutic target. Regulating tumor microenvironment and immunity, inhibiting the expression of UGTs and ECM components in the cancer cells, and decreasing the stemness of the tumors may among the mechanisms underlying the function of ARHGEF6 in LUAD.

Abstract 2332: VSTM2A modulation of immune response in colorectal cancer through abrogating PD-L1 and PD-1 interaction

Abstract Background: Checkpoint immunotherapy using humanized antibody is a new therapeutic strategy against advanced colorectal cancer (CRC). Nonetheless, it shows low tumor penetration due to the large molecule size. Therefore, small immune regulatory molecules are actively sought. Our previous study showed that a small secretory protein VSTM2A suppressed Wnt signalling pathway through inducing LRP6 lysosome dependent degradation. However, its role in immune modulation is largely unknown. Methods: Clinicopathological features were analyzed using CRC patient cohort. Functional biology of VSTM2A was investigated using T cell-antigen presenting cell (APC) co-culture models and knockout mice model. Liquid chromatography with tandem mass spectrometry (LC-MS) was carried out for mechanistic investigation. Results: Gene ontology analysis of colon RNA-seq dataset from TCGA/GTEx revealed significant enrichment of adaptive immune response and lymphocyte activation with high VSTM2A expression. CRC patients with inferior VSTM2A expressions were associated with lower immunoscores in both early and late stages of CRC. In addition, our immunohistochemical examinations of 208 in-house CRC cases further pinpoints to a considerable fraction of VSTM2A-negative cases showing avoidance of spontaneous CD8a+ cells infiltration (Pearson's chi-squared test P &amp;lt; 0.001, φ = 0.24) and worse clinical outcome. LC-MS assay followed by reciprocal immunoprecipitation revealed that VSTM2A interacted with the IgV motif of immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1). We further found that VSTM2A recognized PD-L1 with a dissociation constant (Kd) of 2.6 nM. Recombinant VSTM2A dose dependently suppressed the interaction of PD-L1 and PD-1 with IC50 of 4.51 nM, indicating VSTM2A is a potential intrinsic PD-L1 antagonist. Moreover, our in-vitro mechanistic studies revealed that VSTM2A protein could block the interaction of PD-1/PD-L1, and induced NFAT-RE luciferase activity dose-dependently using a bioluminescent cell-based PD-1/PD-L1 blockade bioassay. VSTM2A could dramatically activate the interleukin-2 production from DO11.10 T cell in the presence non-T cell spherocyte isolated from BALB/c mice and OVA323-339 peptide. Furthermore, our in-vivo study revealed that VSTM2A triggered a distinct increase in CD45+CD3+ T cells infiltration in the syngeneic MC38 mouse model. Vstm2a+/- knockout mice challenged with azoxymethane and dextran sodium sulfate showed significantly high CRC incidence and tumor burden in comparison to wild-type littermates. Tumor from Vstm2a+/- mice showed significantly less CD8+ T cell infiltration. Conclusions: our study demonstrate the tumor extrinsic role of VSTM2A in modulation of microenvironment through abrogating PD-L1/PD-1 interaction. Silencing of VSTM2A in CRC could suppress T cell activation and infiltration in the tumor sites. Citation Format: Yujuan Dong, Yunfei Zhou, Nathalie Wong, Jun Yu, Siu Man Simon Ng. VSTM2A modulation of immune response in colorectal cancer through abrogating PD-L1 and PD-1 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2332.

Abstract 4619: Spatial analysis of the immunosuppressive tumor microenvironment in brain cancer applying a novel multiplex immunofluorescence panel

Abstract Glioblastoma (GBM) and brain metastases (BM) account for &amp;gt;70% of all brain cancers. The annual incidence averages to 10-17 cases per 100.000 person (~ 3 cases for GBM and 7-14 for BM), resulting in 45.000-77.000 new cases in the European Union alone. The standard of care for GBM includes maximal surgical resection followed by radiation and chemotherapy with temozolomide, whilst whole brain irradiation or surgical removal with or without chemotherapy are used for BM. In spite of therapy, the median overall survival remains &amp;lt; 6 months for BM, and 16 months for GBM, suggesting brain cancers as one of the most therapy-resistant tumors. Despite the emergence of novel therapeutic strategies, such as anti-angiogenic therapy, targeting myeloid cells or checkpoint immunotherapy, no significant improvement in overall survival of brain cancer patients was observed. Several studies have suggested the resistance to therapy in brain cancers is largely attributed by the presence of an immunosuppressive brain tumor microenvironment. To investigate the spatial architecture of the tumor microenvironment in BM derived from lung and breast cancers, we employed the Lunaphore 13-plex immune-oncology (IO) Core Panel, consisting of clinically relevant IO biomarkers (CD3, CD4, CD8, CD45, FoxP3, PD1, PD-L1, CD11c, CD20, CD56, CD68, aSMA and Ki-67) for multiplex immunofluorescence (mIF) assays. These findings were then compared to isocitrate dehydrogenase-wildtype glioblastoma, which is a T-cell-poor cancer that is also characterized by an immunosuppressive tumor microenvironment. Further, the IO Core Panel was expanded with previously established antibodies targeting myeloid, blood-brain barrier and tumor cell markers in a proteomic workflow on COMET™, a fully automated, sequential immunofluorescence (seqIF™) platform. Comprehensive mapping of the GBM microenvironment and BM confirmed previous observations obtained by bulk and single cell RNA sequencing analysis that described profound differences in the tumor microenvironment of BM versus GBM. In contrast to GBM, anti-tumor immunity in BM was increased, whereas immunosuppressive cells remained unchanged between the two. Of note, T- and B-cells were not evenly distributed within the tumor tissue, providing an additional level of spatial information that would have been missed by bulk RNA or proteomic analyses. Our study is the first to demonstrate a successful application of Lunaphore’s IO core panel on GBM and BM samples. This highlights potential future use in the development of tools for personalized medicine for GBM and other tumor types. Citation Format: Yvonne Reiss, Jadranka Macas, François Rivest, Victor de Gautard, Saska Brajkovic, Bastian Nicolai, Tatjana Starzetz, Kathleen Sommer, Pinar Cakmak, Jonathan Schup, Jennifer H. Lun, Karl H. Plate. Spatial analysis of the immunosuppressive tumor microenvironment in brain cancer applying a novel multiplex immunofluorescence panel. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4619.

Abstract 4525: YAP1 in relapsed pulmonary high-grade neuroendocrine carcinomas (NEC) is associated with CDKN2A loss, intact RB1, EMT and therapeutic vulnerability to MEK1 and CDK4/6 inhibition

Abstract Neuroendocrine carcinomas (NECs) are clinically aggressive carcinomas commonly arising from the respiratory and gastrointestinal tracts, typically categorized as large-cell neuroendocrine carcinomas (LCNECs) or small cell carcinomas (most commonly small cell lung cancer (SCLC)). Clinically, pulmonary LCNECs (pLCNECs) mirror the course common to SCLC - initial response followed by rapid and insurmountable resistance to one-size-fits-all approaches. Recently, SCLC has been subdivided into four subtypes with unique vulnerabilities, three of which are defined by the transcription factors ASCL1, NEUROD1, and POU2F3, while a fourth group exhibits an inflamed signature. We hypothesize that pLCNEC may be similarly classified into molecularly distinct subsets with unique therapeutic vulnerabilities - a fundamental step toward personalized medicine. We applied our SCLC 1300 gene signature to pLCNEC patient tumors and, as in SCLC, found three distinct subtypes determined by differential expression of ASCL1, NEUROD1, and POU2F3, but with a unique fourth subtype marked by expression of the transcription factor YAP1. Unlike in treatment-naïve SCLC, where YAP1 is absent, YAP1 expression clearly defines pLCNEC as two, roughly equal subsets with the YAP1-low tumors encompassing tumors expressing the other three transcription factors. Conversely, YAP1-high pLCNEC is more mesenchymal and inflamed, and less neuroendocrine (NE), reminiscent of inflamed SCLC. Additionally, YAP1-high status is associated with smoking exposure (P&amp;lt;0.001, FC=81), high frequency of CDKN2A homozygous deletion and SMARCA4 mutations, as well as intact RB1. These features are distinct from SCLC, wherein transcriptional subtypes lack distinct genomic characteristics. Consistent with CDKN2A deletion, YAP1-high pLCNEC cell lines have increased sensitivity to MEK1 and CDK4/6 inhibition. We also demonstrate that RB1 loss downregulates YAP1 expression, which may account for the absence of YAP1 in treatment-naïve SCLC due to ubiquitous loss of RB1. In contrast to treatment-naïve SCLC, where our group and others have been unable to detect YAP1, single-cell RNAseq analysis of biopsies from patients with relapsed SCLC identified emerging YAP1-positive cancer cell populations, which are similarly associated with increased EMT, immune cell infiltration (CD8+ T-cells), and loss of NE gene expression. This suggests that the ability for cancer cells to acquire YAP1 expression and, perhaps, pLCNEC-like features, may be a resistance mechanism in relapsed SCLC, contributing to the abundant intratumoral heterogeneity and highlighting potential vulnerabilities to overcome resistance. In summary, YAP1 may be a predictive biomarker of intact RB1 and response to cellular and checkpoint immunotherapy and MEK1/CDK4/6 inhibition in pLCNEC and relapsed SCLC. Citation Format: C. Allison Stewart, Lixia Diao, Yuanxin Xi, Runsheng Wang, Kavya Ramkumar, B. Leticia Rodriguez, Benjamin B. Morris, Li Shen, Bingnan Zhang, Yan Yang, Azusa Tanimoto, Veronica Y. Novegil, Luisa M. Solis Soto, Pedro F. Simoes da Rocha, Natalie Vokes, Don L. Gibbons, Michael Frumovitz, Junya Fujimoto, Jing Wang, Bonnie Glisson, Lauren A. Byers, Carl M. Gay. YAP1 in relapsed pulmonary high-grade neuroendocrine carcinomas (NEC) is associated with CDKN2A loss, intact RB1, EMT and therapeutic vulnerability to MEK1 and CDK4/6 inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4525.

Abstract 5963: Germline variant positive melanoma patients exhibit an enhanced inflammatory microenvironment

Abstract Germline pathogenic variants (gPV) in cancer susceptibility genes are present in roughly 15% of all melanoma diagnoses. Prior studies note improvements in checkpoint immunotherapy responsiveness in patients with a gPV, which is associated with an increased neo-antigen load in gPV patients. We have also previously reported improvements in melanoma specific survival (MSS) in gPV patients vs non-gPV. No studies to date have examined the immune environment in gPV patients which may account for improvements in survival. In our study, melanoma patients with a personal or family history of multiple cancers were offered germline testing utilizing a commercial germline cancer sequencing panel. Enrolled patients also received a blood draw with subsequent flow cytometry to analyze the immune phenotype. In a cohort of 540 melanoma patients, 80 had a gPV. Multivariable linear regression indicated that significantly fewer polymorphonuclear myeloid derived suppressor cells (PMN-MDSC’s) were noted relative to non-gPV patients when adjusting for melanoma stage at germline testing [β = -.45, 95% CI (-.76, -.15), p = .0036]. This difference was most significant in cutaneous, later stage (IIB-IV) patients, primarily stage III. We also interrogated patterns in gene signature between gPV and non-gPV patients in the TCGA PanCancer cutaneous melanoma cohort. We identified reductions in CCL27 (p = .007) and CCL1 (p = .06), genes associated with MDSC recruitment, across all stages in gPV patients, as well as reductions in ARG1 (p= .05) and S100A7 (p = .08), genes associated with PMN-MDSC’s, in later stage gPV patients. Pathway differences were analyzed using gene set enrichment analysis (GSEA). Analysis of Hallmark gene sets identified significantly upregulated signatures in inflammatory response, TNF-α signaling, IFN-γ signaling, and IL-2 STAT5 signaling in late stage gPV patients. KEGG pathway analysis further identified NK cell mediated cytotoxicity and TCR signaling as significantly upregulated in late stage gPV patients. Notably, fatty acid metabolism pathways, which have been implicated in immunosuppression through MDSC suppressive function, were downregulated in gPV patients. These results suggest that gPV melanoma is associated with both a depletion of PMN-MDSC’s and an upregulation of inflammatory microenvironmental genes in later stages which may explain improved outcomes. Citation Format: Alan Shen, Roshan Lodha, Brandon Bungo, Michelle Arbesman, Nikhil Pramod, Jennifer Ko, Brian Gastman, C. Marcela Diaz, Timothy Chan, Joshua Arbesman, Pauline Funchain. Germline variant positive melanoma patients exhibit an enhanced inflammatory microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5963.