PD-L1 Expression In Tumor Cells Research Articles

PD-L1 expression in tumor and inflammatory cells is associated with favorable tumor features and favorable prognosis in muscle-invasive urothelial carcinoma of the bladder not treated by immune checkpoint inhibitors

BackgroundA high level of PD-L1 expression is the most relevant predictive parameter for response to immune checkpoint inhibitor (CPI) therapy in urinary bladder cancer. Existing data on the relationship between PD-L1 expression and the natural course of disease are controversial and sparse.MethodsTo expand our understanding of the relationship between PD-L1 expression and parameters of cancer aggressiveness, PD-L1 was analyzed on tissue microarrays containing 2710 urothelial bladder carcinomas including 512 patients with follow-up data who underwent radical cystectomy and follow-up therapies in the pre-immune checkpoint inhibitor therapy era.ResultsTumor cell positivity in ≥10% of cells were seen in 513 (20%) and an immune cell positivity occurred in 872 (34%) of 2566 interpretable cancers. PD-L1 positivity in tumor cells increased from pTaG2 low grade (0.9% positive) to pTaG3 high grade (4.1%; p = 0.0255) and was even higher in muscle-invasive (pT2–4) carcinomas (29.3%; p < 0.0001). However, within pT2–4 carcinomas, PD-L1 positivity was linked to low pT stage (p = 0.0028), pN0 (p < 0.0001), L0 status (p = 0.0005), and a better prognosis within 512 patients with cystectomy who never received CPIs (p = 0.0073 for tumor cells and p = 0.0086 for inflammatory cells). PD-L1 staining in inflammatory cells was significantly linked to PD-L1 staining in tumor cells (p < 0.0001) and both were linked to a positive p53 immunostaining (p < 0.0001).ConclusionIt cannot be fully excluded that the strong statistical link between PD-L1 status and favorable histological tumor features as well as better prognosis could influence the outcome of studies evaluating CPIs in muscle-invasive urothelial carcinoma.

Tumor-associated macrophage subtypes on cancer immunity along with prognostic analysis and SPP1-mediated interactions between tumor cells and macrophages.

Tumor-associated macrophages (TAM) subtypes have been shown to impact cancer prognosis and resistance to immunotherapy. However, there is still a lack of systematic investigation into their molecular characteristics and clinical relevance in different cancer types. Single-cell RNA sequencing data from three different tumor types were used to cluster and type macrophages. Functional analysis and communication of TAM subpopulations were performed by Gene Ontology-Biological Process and CellChat respectively. Differential expression of characteristic genes in subpopulations was calculated using zscore as well as edgeR and Wilcoxon rank sum tests, and subsequently gene enrichment analysis of characteristic genes and anti-PD-1 resistance was performed by the REACTOME database. We revealed the heterogeneity of TAM, and identified eleven subtypes and their impact on prognosis. These subtypes expressed different molecular functions respectively, such as being involved in T cell activation, apoptosis and differentiation, or regulating viral bioprocesses or responses to viruses. The SPP1 pathway was identified as a critical mediator of communication between TAM subpopulations, as well as between TAM and epithelial cells. Macrophages with high expression of SPP1 resulted in poorer survival. By in vitro study, we showed SPP1 mediated the interactions between TAM clusters and between TAM and tumor cells. SPP1 promoted the tumor-promoting ability of TAM, and increased PDL1 expression and stemness of tumor cells. Inhibition of SPP1 attenuated N-cadherin and β-catenin expression and the activation of AKT and STAT3 pathway in tumor cells. Additionally, we found that several subpopulations could decrease the sensitivity of anti-PD-1 therapy in melanoma. SPP1 signal was a critical pathway of communication between macrophage subtypes. Some specific macrophage subtypes were associated with immunotherapy resistance and prognosis in some cancer types.

Abstract CT191: First-in-human, dose escalation and expansion study of MT-6402, a novel engineered toxin body (ETB) targeting PD-L1, in patients with PD-L1 expressing relapsed/refractory advanced solid tumors: Interim data

Abstract MT-6402 is a PD-L1-targeted ETB capable of directly killing PD-L1 expressing cells by internalization of a de-immunized Shiga-like toxin A subunit (SLTA), resulting in ribosomal destruction. Targeting PD-L1 expressing tumor cells may directly drive tumor regression, whereas targeting PD-L1 expressing immune cells may release immunosuppression and drive tumor immune recognition. MT-6402 also delivers an HLA-A*02 restricted cytomegalovirus (CMV) class I antigen into PD-L1 expressing cells (antigen seeding) that can be recognized by existing CMV-specific cytotoxic T cells. A first-in-human dose escalation (Part A) and expansion (Part B) study with a weekly intravenous administration of MT-6402 in 4-week cycles was initiated in 2021. Safety and tolerability are primary objectives. Efficacy, pharmacokinetics, and pharmacodynamic data are also collected. As of 10Dec2023, 53 total patients (pts) with PD-L1-expressing advanced solid tumors received ≥ 1 dose of MT-6402: 48 pts in Part A ranging in dose 16 to 100 µg/kg and 5 pts in Part B at 63 µg/kg, just below the maximum tolerated dose (MTD) of 83 µg/kg. MT-6402 was well tolerated in Part A, completed Sept2023. 5 dose limiting toxicities have been reported: a Grade (G)3 maculopapular rash at 24 µg/kg, a G3 infusion related reaction at 63 µg/kg, a G1 cardiac troponin T increase leading to dosing hold for ≥2 weeks at 83 µg/kg, a G3 maculopapular rash at 100 µg/kg, and a G1 cardiac troponin T increase at 100 µg/kg. There were no G4 or G5 drug-related adverse events. In Part B, pts with PD-L1 scores of TPS ≥ 50% will be treated at MTD or lower and 5 pts have been treated at 63 µg/kg. Signs of antitumor activity have been reported including a long-lasting PR with 70% tumor reduction have been observed in a pt with CPI-refractory nasopharyngeal carcinoma on treatment 14+ cycles at 63 µg/kg. This pt has a PD-L1 TPS score of 2% indicating immune cell clearance may have driven the response. The most common cancer in Part A is Squamous Cell Carcinoma of the Head and Neck (10 pts). Pharmacodynamic effects include a significant reduction in peripheral PD-L1+ immune cells, indicating a PD-L1 target-specific effect. Substantial alterations in serum cytokines associated with the tumor microenvironment were noted after each dose in most pts in a dose dependent manner. Most pts display increased CD8/CD4 ratios with IL-2 and TNFα cytokine release, indicative of immune activation due to elimination of immune cells. CMV-specific CD8 T-cells exhibited a pattern of extravasation from the periphery in HLA-A*02+/CMV+ pts suggesting antigen seeding. These results describe a novel approach to checkpoint modulation by MT-6402 with targeted cell killing and altered tumor immunophenotype, particularly in HLA-A*02/CMV+ pts. These results provide rationale for continued development of MT-6402. Citation Format: Brian A. Van Tine, Eugene Ahn, Rebecca A. Redman, Minal A. Barve, William J. Edenfield, Julio Peguero, Victoria M. Villaflor, Drew W. Rasco, Steven F. Powell, Admasu T. Mamuye, Chris B. Moore, Joshua M. Pelham, Soratree Charoenthongtrakul, David R. Spigel. First-in-human, dose escalation and expansion study of MT-6402, a novel engineered toxin body (ETB) targeting PD-L1, in patients with PD-L1 expressing relapsed/refractory advanced solid tumors: Interim data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT191.

Abstract 1149: Spatial transcriptomic analysis of tumor microenvironment in patients with triple negative breast cancer with residual disease after neoadjuvant chemotherapy with immune checkpoint inhibitor

Abstract Recent advance of treatment strategy for triple negative breast cancer (TNBC) including immune checkpoint inhibitor improved survival outcome. Especially, adding pembrolizumab on neoadjuvant chemotherapy (NAC) increased pathologic complete response (pCR) rate at the time of curative surgery. Previous studies suggested that PD-L1 expression in both tumor and immune cells was associated with the response of pembrolizumab in TNBC, but PD-L1 expression state were not associated with pembrolizumab response in NAC setting. In addition, neoadjuvant pembrolizumab increased event-free survival (DFS) rate after NAC even though they had not achieved pCR. However, there were no biomarker for EFS increasement of using pembrolizumab. We designed prospective translational research for evaluating tumor microenvironment (TME) of residual TNBC which had treated with NAC followed by curative surgery. We collected formalin fixed paraffin embedded (FFPE) surgical specimens after NAC with or without pembrolizumab. TNBC samples were used for spatial transcriptomics (ST) analysis, with H&amp;E staining and inspection by an experienced pathologist. Libraries were prepared using Visium Spatial Gene Expression Reagent Kit protocol for FFPE (10x Genomics). Sequencing was conducted on the NovaSeq 6000 System (Illumina). From February 2023 to October 2023, 23 TNBC specimens from 22 patients were collected. Twenty specimens were breast tissues and three were lymph nodes, 18 TNBCs had been treated with NAC with pembrolizumab and four with only NAC, and five had residual cancer burden(RCB) class I, 11 of RCB class II and one of RCB class III. We collected BC and LN samples from BC with class III. Of 23 samples, we performed spatial transcriptomic analysis in 12 samples. In intrinsic subtyping, eight specimens were categorized into normal like, two of basal like and two of luminal A BC subtypes. In TNBC subtyping, mesenchymal stem cell like (MSL) were in six specimens, three of stem cell like (SL), two of luminal androgen receptor (LAR) and one of immunomodulatory (IM). We also performed cell type annotation with unsupervised method and categorized into 11 cell types; three cancer cell types, two macrophages, B and T cells, adipocytes, normal epithelium and perivascular-like and cancer associated fibroblasts. Among these 11 cell types, increasement of immune cells including B cells, T cells and macrophage proportions was associated with pembrolizumab treatment in NAC setting. In our study, we evaluated TME state of TNBC after NAC with or without pembrolizumab. We suggested that MSL or SL TNBC subtypes were associated with residual disease after NAC regardless of pembrolizumab use and pembrolizumab may recruit more immune cells in TME of TNBC. For concreating our suggestion, further analyses would be warranted. Citation Format: Ji-Yeon Kim, Kyunghee Park, Yoon La Choi, Jeong Eon Lee, Byung Joo Chae, Jonghan Yu, Seok Won Kim, Seok Jin Nam, Jin Seok Ahn, Woong-Yang Park, Yeon Hee Park. Spatial transcriptomic analysis of tumor microenvironment in patients with triple negative breast cancer with residual disease after neoadjuvant chemotherapy with immune checkpoint inhibitor [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 1149.

Cabozantinib Plus Atezolizumab or Cabozantinib Alone in Patients With Advanced NSCLC Previously Treated With an Immune Checkpoint Inhibitor: Results From the Phase 1b COSMIC-021 Study

IntroductionWe evaluated efficacy and safety of cabozantinib plus atezolizumab or cabozantinib alone in advanced NSCLC previously treated with an immune checkpoint inhibitor (ICI). MethodsCOSMIC-021 (NCT03170960) is a phase 1b, multicenter study in advanced solid tumors. This analysis included patients with stage IV non-squamous NSCLC without actionable genomic aberrations in EGFR, ALK, ROS1, or BRAF-V600E who progressed on one prior ICI and less than or equal to two prior lines of systemic anticancer therapy. Patients received cabozantinib 40 mg orally/day plus atezolizumab 1200 mg intravenously every three weeks (combination cohort) or cabozantinib 60 mg orally/day (single-agent cabozantinib cohort). Primary end point of the combination cohort was objective response rate per Response Evaluation Criteria in Solid Tumors v1.1 by investigator. Outcomes in the single-agent cabozantinib cohort were exploratory. ResultsEighty-one patients assigned to combination therapy and 31 assigned to single-agent cabozantinib received greater than or equal to one dose of study treatment. Median (range) follow-up was 26.1 months (12.1–44.2) and 22.4 months (1.5–29.0), respectively. Objective response rate was 20% (95% confidence interval: 11.7%–30.1%) in combination cohort and 6% (95% confidence interval: 0.8%–21.4%) in single-agent cabozantinib cohort. Treatment-related adverse events (TRAEs) occurred in 86% of patients in the combination cohort and 90% in the single-agent cabozantinib cohort; grade 3/4 TRAEs were 44% and 48%, respectively. There were two grade 5 TRAEs: pneumonitis (n = 1, combination) and gastric ulcer hemorrhage (n = 1, single-agent). Neither PD-L1 expression in tumor cells nor tumor mutation burden correlated with outcomes. ConclusionsCabozantinib plus atezolizumab demonstrated modest clinical activity and manageable toxicity in advanced NSCLC after progression on prior ICI.

Targeting two radiation-induced immunosuppressive pathways to improve the efficacy of normofractionated radiation therapy in a preclinical colorectal cancer model

Purpose We have previously demonstrated in a murine colorectal cancer model that normofractionated RT (normoRT: 18 × 2 Gy) induced MDSC infiltration and PD-L1 expression, while hypofractionated RT (hypoRT: 3 × 8 Gy) induced Treg. Here, we wanted to assess whether the association of normoRT with treatments that target two radiation-induced immunosuppressive pathways (MDSC and PD-L1) could improve tumor control. Materials and methods Subcutaneous tumors were induced using colon tumor cells (CT26) in immunocompetent mice (BALB/c) and were treated with RT alone (18 × 2 Gy or 3 × 8 Gy), or concomitantly with 5-Fluorouracil (5FU) (10 mg/kg) to deplete MDSC, and/or anti-PD-L1 (10 mg/kg). We assessed the impact of these combinations on tumor growth and immune cells infiltration by flow cytometry. In addition, we performed tumor rechallenge experiments and IFN-γ ELISpots to study the long-term memory response. Results Even though tumor growth was significantly delayed in the RT + 5FU compared to 5FU and untreated groups (p < .05), there was no significant difference between RT + 5FU (CRT) and RT alone. The rate of MDSC increased significantly 1 week after the end of normoRT (8.09% ± 1.03%, p < .05) and decreased with the addition of 5FU (3.39% ± 0.69%, p < .05). PD-L1 expressing tumor cells were increased after treatment. Adding anti-PD-L1 significantly delayed tumor growth, achieved the highest complete response rate, and induced a long-lasting protective specific anti-tumor immunity. Conclusions These results tend to demonstrate the interest of inhibiting two radiation-induced immunosuppressive mechanisms. In patients, the combination of normoRT with 5FU is already the standard of care in locally advanced rectal cancer. Adding an anti-PD-L1 to this treatment could show promising results.

Tumor-targeted delivery of copper-manganese biomineralized oncolytic adenovirus for colorectal cancer immunotherapy

Oncolytic viral therapy (OVT) is a novel anti-tumor immunotherapy approach, specifically replicating within tumor cells. Currently, oncolytic viruses are mainly administered by intratumoral injection. However, achieving good results for distant metastatic tumors is challenging. In this study, a multifunctional oncolytic adenovirus, OA@CuMnCs, was developed using bimetallic ions copper and manganese. These metal cations form a biomineralized coating on the virus's surface, reducing immune clearance. It is known that viruses upregulate the expression of PD-L1. Copper ions in OA@CuMnCs can decrease the PD-L1 expression of tumor cells, thereby promoting immune cell-related factor release. This process involves antigen presentation and the combination of immature dendritic cells, transforming them into mature dendritic cells. It changes "cold" tumors into "hot" tumors, further inducing immunogenic cell death. While oncolytic virus replication requires oxygen, manganese ions in OA@CuMnCs can react with endogenous hydrogen peroxide. This reaction produces oxygen, enhancing the virus's replication ability and the tumor lysis effect. Thus, this multifunctionally coated OA@CuMnCs demonstrates potent amplification in immunotherapy efficacy, and shows great potential for further clinical OVT. Statement of significanceOncolytic virus therapy (OVs) is a new anti-tumor immunotherapy method that can specifically replicate in tumor cells. Although the oncolytic virus can achieve a therapeutic effect on some non-metastatic tumors through direct intratumoral injection, there are still three major defects in the treatment of metastatic tumors: immune response, hypoxia effect, and administration route. Various studies have shown that the immune response in vivo can be overcome by modifying or wrapping the surface protein of the oncolytic virus. In this paper, a multifunctional coating of copper and manganese was prepared by combining the advantages of copper and manganese ions. The coating has a simple preparation method and mild conditions, and can effectively enhance tumor immunotherapy.

Assessing the genomic feature of Chinese patients with ampullary adenocarcinoma: potential therapeutic targets

BackgroundsAmpullary adenocarcinoma (AMPAC) is a rare malignancy, treated as pancreatic or intestinal cancer based on its histologic subtype. Little is known about the genomic features of Chinese patients with AMPAC.Materials and methodsWe enrolled 145 Chinese AMPAC patients in our local cohort and performed a compressive somatic and germline genetic testing using a 156 gene panel. Expression of PD-L1 (clone 28 − 8) was also assessed in tumor specimens from 64 patients.ResultsThe frequency of genetic alterations (GAs) in Chinese patients with AMPAC was found to be distinctive, with TP53, KRAS, SMAD4, APC, CTNNB1, ARID1A, and CDKN2A emerged as the most frequently mutated genes. Comparing with Western patients, significant differences were observed in the prevalence of PIK3CA and ARID2. Furthermore, the incidence of MSI-H was lower in the Chinese cohort, with only two patients identified as MSI-H. Conversely, 11 patients (8.27%) had pathogenic/likely pathogenic germline alterations, all of which were in the DNA damage response (DDR) pathway. In our cohort, 34.48% (22/64) of patients exhibited positive PD-L1 expression in tumor cells, and this expression was associated with GAs in CTNNB1 and BLM. Importantly, over three-fourths of Chinese AMPAC patients in our study had at least one actionable GA, with more than one-fifth of them having actionable GAs classified as Level 3. These actionable GAs were primarily involved in the DDR and PI3K pathways. Notably, GAs in the DDR pathway were detected in both Chinese and Western patients, and regardless of their functional impact, these alterations demonstrated enhanced overall survival rates and higher tumor mutational burden (TMB) levels.ConclusionThese findings underscore the distinct genomic landscape of Chinese AMPAC patients and highlight the potential for targeted therapies based on the identified GAs.

79 Biomarker of response to immunotherapy

79 Biomarker of response to immunotherapy

Lingual Denervation Improves the Efficacy of Anti-PD-1 Immunotherapy in Oral Squamous Cell Carcinomas by Downregulating TGFβ Signaling.

Intratumoral nerve infiltration relates to tumor progression and poor survival in oral squamous cell carcinoma (OSCC). How neural involvement regulates antitumor immunity has not been well characterized. This study aims to investigate molecular mechanisms of regulating tumor aggressiveness and impairing antitumor immunity by nerve-derived factors. We performed the surgical lingual denervation in an immunocompetent mouse OSCC model to investigate its effect on tumor growth and the efficacy of anti-PD-1 immunotherapy. A trigeminal ganglion neuron and OSCC cell coculture system was established to investigate the proliferation, migration, and invasion of tumor cells and the PD-L1 expression. Both the neuron-tumor cell coculture in vitro model and the OSCC animal model were explored. Lingual denervation slowed down tumor growth and improved the efficacy of anti-PD-1 treatment in the OSCC model. Coculturing with neurons not only enhanced the proliferation, migration, and invasion but also upregulated TGFβ-SMAD2 signaling and PD-L1 expression of tumor cells. Treatment with the TGFβ signaling inhibitor galunisertib reversed nerve-derived tumor aggressiveness and downregulated PD-L1 on tumor cells. Similarly, lingual denervation in vivo decreased TGFβ and PD-L1 expression and increased CD8+ T-cell infiltration and the expression of IFNγ and TNFα within tumor. Neural involvement enhanced tumor aggressiveness through upregulating TGFβ signaling and PD-L1 expression in OSCC, while denervation of OSCC inhibited tumor growth, downregulated TGFβ signaling, enhanced activities of CD8+ T cells, and improved the efficacy of anti-PD-1 immunotherapy. This study will encourage further research focusing on denervation as a potential adjuvant therapeutic approach in OSCC. This study revealed the specific mechanisms for nerve-derived cancer progression and impaired antitumor immunity in OSCC, providing a novel insight into the cancer-neuron-immune network as well as pointing the way for new strategies targeting nerve-cancer cross-talk as a potential adjuvant therapeutic approach for OSCC.

Methylthioadenosine Phosphorylase Genomic Loss in Advanced Gastrointestinal Cancers.

One of the most common sporadic homozygous deletions in cancers is 9p21 loss, which includes the genes methylthioadenosine phosphorylase (MTAP), CDKN2A, and CDKN2B, and has been correlated with worsened outcomes and immunotherapy resistance. MTAP-loss is a developing drug target through synthetic lethality with MAT2A and PMRT5 inhibitors. The purpose of this study is to investigate the prevalence and genomic landscape of MTAP-loss in advanced gastrointestinal (GI) tumors and investigate its role as a prognostic biomarker. We performed next-generation sequencing and comparative genomic and clinical analysis on an extensive cohort of 64 860 tumors comprising 5 GI cancers. We compared the clinical outcomes of patients with GI cancer harboring MTAP-loss and MTAP-intact tumors in a retrospective study. The prevalence of MTAP-loss in GI cancers is 8.30%. MTAP-loss was most prevalent in pancreatic ductal adenocarcinoma (PDAC) at 21.7% and least in colorectal carcinoma (CRC) at 1.1%. MTAP-loss tumors were more prevalent in East Asian patients with PDAC (4.4% vs 3.2%, P = .005) or intrahepatic cholangiocarcinoma (IHCC; 6.4% vs 4.3%, P = .036). Significant differences in the prevalence of potentially targetable genomic alterations (ATM, BRAF, BRCA2, ERBB2, IDH1, PIK3CA, and PTEN) were observed in MTAP-loss tumors and varied according to tumor type. MTAP-loss PDAC, IHCC, and CRC had a lower prevalence of microsatellite instability or elevated tumor mutational burden. Positive PD-L1 tumor cell expression was less frequent among MTAP-loss versus MTAP-intact IHCC tumors (23.2% vs 31.2%, P = .017). In GI cancers, MTAP-loss occurs as part of 9p21 loss and has an overall prevalence of 8%. MTAP-loss occurs in 22% of PDAC, 15% of IHCC, 8.7% of gastroesophageal adenocarcinoma, 2.4% of hepatocellular carcinoma, and 1.1% of CRC and is not mutually exclusive with other targetable mutations.

CXCL13 Positive Cells Localization Predict Response to Anti-PD-1/PD-L1 in Pulmonary Non-Small Cell Carcinoma.

Background: Immune checkpoint inhibitors (ICIs) have revolutionized non-small cell lung cancers (NSCLCs) treatment, but only 20-30% of patients benefit from these treatments. Currently, PD-L1 expression in tumor cells is the only clinically approved predictor of ICI response in lung cancer, but concerns arise due to its low negative and positive predictive value. Recent studies suggest that CXCL13+ T cells in the tumor microenvironment (TME) may be a good predictor of response. We aimed to assess if CXCL13+ cell localization within the TME can predict ICI response in advanced NSCLC patients. Methods: This retrospective study included 65 advanced NSCLC patients treated with Nivolumab/Pembrolizumab at IUCPQ or CHUM and for whom a pretreatment surgical specimen was available. Good responders were defined as having a complete radiologic response at 1 year, and bad responders were defined as showing cancer progression at 1 year. IHC staining for CXCL13 was carried out on a representative slide from a resection specimen, and CXCL13+ cell density was evaluated in tumor (T), invasive margin (IM), non-tumor (NT), and tertiary lymphoid structure (TLS) compartments. Cox models were used to analyze progression-free survival (PFS) and overall survival (OS) probability, while the Mann-Whitney test was used to compare CXCL13+ cell density between responders and non-responders. Results: We showed that CXCL13+ cell density localization within the TME is associated with ICI efficacy. An increased density of CXCL13+ cells across all compartments was associated with a poorer prognostic (OS; HR = 1.22; 95%CI = 1.04-1.42; p = 0.01, PFS; HR = 1.16; p = 0.02), or a better prognostic when colocalized within TLSs (PFS; HR = 0.84, p = 0.03). Conclusion: Our results support the role of CXCL13+ cells in advanced NSCLC patients, with favorable prognosis when localized within TLSs and unfavorable prognosis when present elsewhere. The concomitant proximity of CXCL13+ and CD20+ cells within TLSs may favor antigen presentation to T cells, thus enhancing the effect of PD-1/PD-L1 axis inhibition. Further validation is warranted to confirm the potential relevance of this biomarker in a clinical setting.

A NRF2 Regulated and the Immunosuppressive Microenvironment Reversed Nanoplatform for Cholangiocarcinoma Photodynamic-Gas Therapy.

Photodynamic therapy (PDT) is a minimally invasive and controllable local cancer treatment for cholangiocarcinoma (CCA). However, the efficacy of PDT is hindered by intratumoral hypoxia and the presence of an antioxidant microenvironment. To address these limitations, combining PDT with gas therapy may be a promising strategy to enhance tumor oxygenation. Moreover, the augmentation of oxidative damage induced by PDT and gas therapy can be achieved by inhibiting NRF2, a core regulatory molecule involved in the antioxidant response. In this study, an integrated nanotherapeutic platform called CMArg@Lip, incorporating PDT and gas therapies using ROS-responsive liposomes encapsulating the photosensitizer Ce6, the NO gas-generating agent L-arginine, and the NRF2 inhibitor ML385, is successfully developed. The utilization of CMArg@Lip effectively deals with challenges posed by tumor hypoxia and antioxidant microenvironment, resulting in elevated levels of oxidative damage and subsequent induction of ferroptosis in CCA. Additionally, these findings suggest that CMArg@Lip exhibits notable immunomodulatory effects, including the promotion of immunogenic cell death and facilitation of dendritic cell maturation. Furthermore, it contributes to the anti-tumor function of cytotoxic T lymphocytes through the downregulation of PD-L1 expression in tumor cells and the activation of the STING signaling pathway in myeloid-derived suppressor cells, thereby reprogramming the immunosuppressive microenvironment via various mechanisms.

HER2 and PD-L1 immunohistochemistry (IHC) expression, and HER2 genomic alterations: Associations and clinical outcomes for advanced bladder cancer.

538 Background: Bladder cancer (BC) has a relatively high rate of human epidermal growth factor receptor 2 (HER2) alterations. The association between HER2 mutation/amplification, HER2, and PD-L1 immunohistochemistry (IHC) expression and associations with clinical outcomes for advanced bladder cancer (BC) has not been studied. Methods: We retrospectively analyzed BC samples for PD-L1 and HER2 IHC expression and compared HER2 alterations from genomic profiling with the MSK IMPACT platform. HER2 IHC expression was defined as 0, 1+, 2+, 3+, and PD-L1 IHC was the combined tumor and immune cell PD-L1 expression score (CPS). HER2 alteration was defined as either pathogenic mutation and/or amplification. We studied pairwise associations between HER2 alteration, PD-L1, and HER2 IHC expression in all patients and their associations with progression-free survival (PFS) and overall survival (OS) for muscle-invasive bladder cancer (MIBC) pts. Association analyses were performed using the Wilcoxon rank-sum test or Fisher’s exact test. Kaplan-Meier method and Cox proportional hazard models were used for time-to-event analyses. Results: Among 202 pts with HER2 IHC, 188 had MSK IMPACT, and 168 had PD-L1 CPS. The overall incidence of HER2 alteration was 22.3%; 48.2% had CPS ³10, and HER2 IHC distribution was 0:18.8%, 1+:29.7%, 2+:33.7%, and 3+:17.8%. The CPS score was inversely associated with HER2 IHC expression (p&lt;0.001). No association was noted between CPS score and HER2 alteration (p=0.735). HER2 altered tumors were strongly correlated with high-level HER2 IHC expression (p&lt;0.001). However, 41% (n=14/34) of HER2 IHC 3+ samples did not have HER2 alterations, and 17% (n=7/36) of HER2 altered samples had HER2 IHC expression of 0. In patients with MIBC, HER2 alteration and HER2 IHC expression (0/1+ vs. 2+/3+) were not associated with PFS (p=0.5 and p=0.4, respectively) or OS for MIBC pts (p=0.84 and p=0.94, respectively). A higher PD-L1 CPS score (&gt;=10 vs &lt;10) was associated with improved PFS for MIBC pts (p=0.03). Conclusions: Our study is the first to describe an inverse correlation between HER2 IHC expression and PD-L1 CPS score. Furthermore, HER2 IHC overexpression is strongly associated with HER2 amplification, but a subset of patients with high HER2 protein expression are potentially missed by genomic profiling alone. HER2 expression by IHC or HER2 genomic alteration is not a prognostic marker for MIBC pts in this cohort. This data provides the foundation for further HER2-directed advanced BC studies. [Table: see text]

Potential biomarkers for treatment response in advanced non-pancreatic neuroendocrine tumors.

599 Background: Well-differentiated NETs are highly vascular and hence, inhibition of angiogenesis is of interest. VEGF, a key molecule in promoting angiogenesis, has also been shown to promote an immunosuppressive tumor microenvironment. Nintedanib, an oral inhibitor of FGFR1–3, VEGFR1–3 and PDGFRA, has previously been evaluated in a phase II clinical trial in well-differentiated, non-pancreatic NETs, where 83% of the evaluable patients were progression-free at 16 weeks and median PFS was 11 months. In preclinical studies, nintedanib has been shown to increase PD-L1 expression in tumor cells, which then results in improved outcomes with immune checkpoint inhibition. There are multiple indicators of T-cell exhaustion, including PD-L1 and LAG-3. LAG-3+ T-cells have been found to correlate with poor response to treatment in neuroendocrine neoplasms. Given these findings, this study evaluates serum VEGF and LAG-3 expression to determine potential biomarkers of response in patients treated with nintedanib. Methods: 27 sets of paired samples collected at baseline and 8-weeks post treatment with nintedanib, from clinical trial NCT02399215 were used for this study. With IRB approval, we tested for serum VEGFa and serum LAG-3 using Luminex test kits: HCKP1-11K-01 (LAG-3) and HCYTA-60K-01 (VEGFa). We compared the baseline, 8-week, and percentage change in serum VEGF and serum LAG-3 levels with Rd Cd8+ pAkt+ T-cells (activated cytotoxic T-cells) as a marker of immune activation, Rd CD25+ pAkt+ T-cells (regulatory T-cells) as a marker of immune suppression, and PFS. Results: The median VEGF levels at baseline and 8-weeks were 10.50 pg/mL and 11.90 pg/mL respectively. The median LAG-3 level at baseline and 8-weeks were 59000 pg/mL and 62900 pg/mL respectively. Higher VEGFa levels in the post-treatment samples were associated with poorer progression free survival (HR= 1.009, 95% HR CI: [0.999, 1.019], with P= 0.0760). Higher levels of VEGF at baseline were also associated with lower numbers of Rd CD8+ pAkt + cells (Pearson coefficient = -0.42, p-value 0.04) at baseline. Increased baseline CD25+ pAkt+ levels were positively correlated with percent change in serum LAG-3 levels between baseline and post-treatment samples. (Pearson Co-efficient: 0.45; p=value: 0.02). Conclusions: Elevated serum VEGFa levels at 8-weeks post treatment correlated with lower PFS. Serum VEGFa level is a potential indicator of effector T-cell suppression, as evidenced by low number of activated cytotoxic T-cells in patients with higher VEGFa levels at baseline. Higher baseline regulatory T-cells were associated with a greater increase in serum LAG-3 levels, suggesting that serum LAG-3 may be an indicator of an immunosuppressive tumor microenvironment.

Pharmaceutical targeting of OTUB2 sensitizes tumors to cytotoxic T cells via degradation of PD-L1

PD-1 is a co-inhibitory receptor expressed by CD8+ T cells which limits their cytotoxicity. PD-L1 expression on cancer cells contributes to immune evasion by cancers, thus, understanding the mechanisms that regulate PD-L1 protein levels in cancers is important. Here we identify tumor-cell-expressed otubain-2 (OTUB2) as a negative regulator of antitumor immunity, acting through the PD-1/PD-L1 axis in various human cancers. Mechanistically, OTUB2 directly interacts with PD-L1 to disrupt the ubiquitination and degradation of PD-L1 in the endoplasmic reticulum. Genetic deletion of OTUB2 markedly decreases the expression of PD-L1 proteins on the tumor cell surface, resulting in increased tumor cell sensitivity to CD8+ T-cell-mediated cytotoxicity. To underscore relevance in human patients, we observe a significant correlation between OTUB2 expression and PD-L1 abundance in human non-small cell lung cancer. An inhibitor of OTUB2, interfering with its deubiquitinase activity without disrupting the OTUB2-PD-L1 interaction, successfully reduces PD-L1 expression in tumor cells and suppressed tumor growth. Together, these results reveal the roles of OTUB2 in PD-L1 regulation and tumor evasion and lays down the proof of principle for OTUB2 targeting as therapeutic strategy for cancer treatment.

A study on expression of programmed death ligand-1 in small cell lung carcinoma and correlation with clinicopathological parameters.

Small cell lung carcinoma (SCLC) is characterized by rapid growth and an aggressive clinical course. Standard therapy regimes have limited effects on disease course; therefore theprognosis ofSCLC is poor. In thecurrent study, thefrequency ofprogrammed death ligand 1 (PD-L1) expression in SCLC and its correlation with clinico-pathological features were evaluated. Thestudy included 100 cases ofSCLC wherein testing for PD-L1 was done with theSP263 clone on theVentana benchmark XT system. Cases with > 1% PD-L1 expression in tumour cells or immune cells were categorized as positive. PD-L1 expression was identified in 14% ofcases using thecut-off of≥ 1%. Thetumour proportion score was 10% and theimmune proportion score was 9.78% using acut-off of≥ 1%. PD-L1 positive expression was more frequent in themale population with age > 40 years. All thepatients with positive PD-L1 expression were smokers. In thePD-L1 positive group, presence ofnecrosis was identified in 71.4% ofcases and when compared with thePD-L1 negative subgroup this finding was statistically significant (p = 0.010). Personalized targeted therapy for cases ofSCLC is still under evaluation. Theuse ofimmunotherapeutic targets, such as PD-L1, may help to define anew treatment strategy for SCLC. Development ofnew treatment strategies may improve prognosis and survival.

IRGD-mediated liposomal nanoplatforms for improving hepatocellular carcinoma targeted combination immunotherapy and monitoring tumor response via IVIM-MRI.

The combination therapy of targeted treatments and immune checkpoint blockade (ICB) holds great promise for hepatocellular carcinoma (HCC) treatment. However, challenges such as immunogenicity, off-target toxicity of ICB antibodies, low drug co-delivery efficiency, and lack of effective biomarkers to monitor treatment response limit the efficacy of existing targeted immunotherapies. Herein, we synthesized iRGD-modified pH-sensitive liposomal nanoparticles co-encapsulating lenvatinib (Len) and the small molecule PD-1/PD-L1 inhibitor BMS-202 (iRGD-lip@Len/BMS-202) to address issues related to inadequate tumor enrichment and distinct pharmacokinetics of these drugs. Furthermore, intravoxel incoherent motion-magnetic resonance imaging (IVIM-MRI), which is calculated using a biexponential model, can simultaneously reflect both the diffusion of water molecules within the tissue and the microcirculatory perfusion of capillaries. Consequently, we further assessed the feasibility of using IVIM-MRI to monitor the cancer treatment response in nanodrug therapy. These results demonstrated that the iRGD-targeted liposomal nanodrug effectively accumulated in tumors and released in acidic microenvironments. The sustained release of Len facilitated tumor vascular normalization, decreased the presence of Tregs and MDSCs and activated the IFN-γ signaling pathway. This led to increased PD-L1 expression in tumor cells, enhancing the sensitivity of BMS-202. Consequently, there was a synergistic amplification of antitumor immune therapy, resulting in the shrinkage of subcutaneous and orthotopic HCC and inhibition of lung metastasis. Furthermore, IVIM-MRI technology facilitated the non-invasive monitoring of the tumor microenvironment (TME), revealing critical therapeutic response indicators such as the normalization of tumor blood vessels and the degree of hypoxia. Collectively, the combination of Food and Drug Administration (FDA)-approved drugs with iRGD-modified liposomes presents a promising strategy for HCC treatment. Simultaneously, IVIM-MRI provides a non-invasive method to accurately predict the response to this nanodrug.

Inhibition of α4β1 Integrin Activity by Small Tellurium Compounds Regulates PD-L1 Expression and Enhances Antitumor Effects.

Various cancer treatment approaches that inhibit the activity of the programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) axis, a key player in tumor immune evasion, have been developed. We show that the immunomodulatory small tellurium complexes AS101 (ammonium trichloro(dioxoethylene-o,o')tellurate) and SAS (octa-O-bis(R,R)-tartarate ditellurane) suppress PD-L1 expression in a variety of human and mouse malignant cells via the modulation of α4β1 very late antigen-4 (VLA-4) integrin activity. Consequently, the expression of pAkt and its downstream effector pNFκB are inhibited. Additionally, SAS promotes the death of mouse malignant cells by activated syngeneic splenocytes or CD8+ T cells, preventing the development of chemoresistance in malignant cells. Moreover, AS101 and SAS may increase, at least in part, chemosensitivity through inhibition of the VLA-4/IL-10/PD-L1 pathway. Additionally, AS101 or SAS treatment of B16/F10 melanoma-bearing mice decreased tumor cell PD-L1 expression, leading to increased CD8+ T-cell infiltration into the tumors and tumor shrinkage. Combination treatment with an αPD-1 antibody and either tellurium compound significantly increased the antitumor efficacy of immunotherapy. Overall, VLA-4 integrin signaling is critical for tumor immune evasion and is a potential target for cancer treatment. Finally, AS101 or SAS, biologically active tellurium compounds, can effectively enhance the therapeutic efficacy of αPD-1-based cancer immunotherapy.

Development, Characterization, and Radiation Dosimetry Studies of 18F-BMS-986229, a 18F-Labeled PD-L1 Macrocyclic Peptide PET Tracer

PurposeIn cancer immunotherapy, the blockade of the interaction between programmed death-1 and its ligand (PD-1:PD-L1) has proven to be one of the most promising strategies. However, as mechanisms of resistance to PD-1/PD-L1 inhibition include variability in tumor cell PD-L1 expression in addition to standard tumor biopsy PD-L1 immunohistochemistry (IHC), a comprehensive and quantitative approach for measuring PD-L1 expression is required. Herein, we report the development and characterization of an 18F-PD-L1-binding macrocyclic peptide as a PET tracer for the comprehensive evaluation of tumor PD-L1 expression in cancer patients.Procedures18F-BMS-986229 was characterized for PD-L1 expression assessment by autoradiography or PET imaging. 18F-BMS-986229 was utilized to evaluate tumor PD-L1 target engagement in competition with a macrocyclic peptide inhibitor of PD-L1 (BMS-986189) over a range of doses using PET imaging. A whole-body radiation dosimetry study of 18F-BMS-986229 in healthy non-human primates (NHPs) was performed.ResultsIn vitro autoradiography showed an 8:1 binding ratio in L2987(PD-L1 +) vs. HT-29 (PD-L1-) tumors, more than 90% of which could be blocked with 1 nM of BMS-986189. Ex vivo autoradiography showed that 18F-BMS-986229 detection was penetrant over a series of sections spanning the entire L2987 tumor. In vivo PET imaging in mice demonstrated a 5:1 tracer uptake ratio (at 90–100 min after tracer administration) in L2987 vs. HT-29 tumors and demonstrated 83%-93% specific binding of BMS-986189 within those dose ranges. In a healthy NHP dosimetry study, the resultant whole-body effective dose was 0.025 mSv/MBq.Conclusion18F-BMS-986229 has been preclinically characterized and exhibits high target specificity, low background uptake, and a short blood half-life supportive of same day imaging in the clinic. As the PET tracer, 18F-BMS-986229 shows promise in the quantification of PD-L1 expression, and its use in monitoring longitudinal changes in patients may provide insights into PD-1:PD-L1 immuno-therapy treatment outcomes.