PD-L1 Levels Research Articles

Abstract 3065: Targeting of metabolic reprogramming in lung cancer by utilizing patient-derived organoid tissue for precision medicine

Abstract In a preliminary pilot study, we reported elevated activity of indoleamine 2,3-dioxygenase 1 (IDO1) in serum from cisplatin-resistant (CR) lung cancer patients compared to cisplatin-sensitive (CS) patients. This heightened IDO1 activity was associated with increased tryptophan (TRP) catabolism, resulting in the upregulation of kynurenine (KYN) production. The detection of KYN can now be readily achieved through immunohistochemistry (IHC). We demonstrated that this upregulation contributed to increased Treg (regulatory T cell) and MDSC (myeloid-derived suppressor cell) populations resulting in an immunosuppressive tumor microenvironment. To investigate this effect, we used dual inhibition of IDO1/TDO2 (tryptophan2,3-dioxygenase 2) in an orthotopic mouse model of CR-Lewis lung carcinoma. This resulted in a significant reduction in tumor volume compared to LLC-CS (n=7, p<0.01), confirming the role of TRP catabolism in CR tumorigenesis. To determine the relevance of the data derived from this mouse model to clinical settings, we established 50 patient-derived organoid tissues (PDOT) from patients with NSCLC. PDOT is an experimental ex vivo tissue model developed to recapitulate the intricate features of lung architecture, including the lung acinus. Upon optimizing our collection and growth protocols, each PDOT successfully developed into a spherical body within 14 days. Furthermore, through bulk RNA-seq analysis, we showed that PDOT closely mirrors the expression of key molecular markers (PD-L1 and mutations), as observed in the original tumor tissues. PDOT from patients possessing high levels of PD-L1 and KYN were exquisitely sensitive to dual inhibition with 2-3-fold greater sensitivity compared to patients with undetectable KYN expression in growth inhibition assays (GI50=20±5.5μM vs. 65±7.5μM). Increased lipid peroxidation was detected via flow cytometry by accumulation of the C11-BODIPY reporter dye in the dual inhibition treatment group. Moreover, we integrated PDOT with a novel 3D microfluidic culture that can be supplied with patient-matched peripheral blood mononuclear cells (PBMC) to assess immune response. Using CyTOF, we showed that dual inhibitors with Atezolizumab (anti-PD-L1) enhanced immune effector (CD8+, NK) and suppressed immunosuppressive (Treg, MDSC) populations. We believe that our swift progress in PDOT development may bridge the divide between cancer biomarker expression and patient trials by timely complementing conventional drug studies based on cell lines and xenografts to facilitate the design of personalized therapies. As a result, the extensive reporting that we have contributed for KYN expression profiling in CR has the potential to serve as a valuable companion diagnostic tool in the future. This can assist in applying precision oncology by finding the most appropriate patients for immunotherapies. Citation Format: Chunjing Wu, Jonathan D. Nguyen, George Theodore, Estelamari Rodriguez, Sydney Spector, Emily Kim, Taranatee Khan, Pablo Puente, Yujie Wang, Dao M. Nguyen, Cheng-Bang Chen, Diane Lim, Lynn G. Feun, Niramol Savaraj, Medhi Wangpaichitr. Targeting of metabolic reprogramming in lung cancer by utilizing patient-derived organoid tissue for precision medicine [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 3065.

Abstract 6182: Artificial intelligence (AI)-powered quantification of glypican-3 (GPC3) expression facilitates patient selection for GPC3-targeted therapy in solid tumors

Abstract Introduction: GPC3 is a glycosyl-phosphatidylinositol-anchored cell surface glycoprotein that functions to control cell growth and differentiation in early development. Membrane GPC3 is highly expressed in hepatocellular carcinoma (HCC) and moderately in squamous non-small cell lung cancer (SQ-NSCLC) with limited expression in normal tissues which makes it an attractive target for immune cell engagers. This study aims to develop an AI-powered algorithm to evaluate GPC3 expression and compare it with manual immunohistochemistry (IHC) scoring in HCC, adeno-NSCLC and SQ-NSCLC. Methods: 147 NSCLC (94 SQ-NSCLC and 53 adeno-NSCLC) and 113 HCC FFPE tumor blocks were obtained from commercial sources. GPC3 protein level was quantified by a qualified IHC assay for tumor cell membrane and cytoplasm individually. PD-L1 expression is reported as tumor cell proportion score (TPS), combined positive score (CPS) and immune cell proportion score (IPS/IC) TPS, regardless of staining pattern and intensity. FFPE slides were stained for GPC3+ (GC33, Ventana), PD-L1+ (22C3, Dako) cells and then scanned at 20x using the Aperio Versa8 scanner. Machine learning (ML) models were developed using the digitized whole slide images to identify GPC3+ tumor area. Data-driven cutoffs were applied to model-generated human-interpretable features of GPC3+ percentage of tumor area to classify samples as positive and negative. As an orthogonal approach to the GPC3+ percentage cutoff method, tissue and cell annotations were used to train a convolutional neural network (CNN) to classify GPC3 positivity. Results: The epidemiology profiling results revealed that GPC3 protein is highly expressed in HCC (57.5% cases with > 1% positive cells), followed by SQ-NSCLC (52.1%) and adeno-NSCLC (5.7%). GPC3 expression was only detected in tumor cells with two staining patterns observed: membrane + cytoplasm and cytoplasm-only. There was no significant correlation between GPC3 and PD-L1 expression across tumor types, although a trend of lower level of PD-L1 was observed in the samples with GPC3 ≥50%. ML model quantification of GPC3+ and GPC3- showed high concordance with the consensus score of 2 independent pathologists on a test set of held-out samples not used in training. This concordance was comparable to that of individual annotators to consensus. Instead, GPC3 positivity results using both cutoff and CNN methods showed moderate concordance with 2-way pathologist consensus. Conclusions: Here we profiled GPC3 expression across tumor types and showed high level of expression in HCC followed by SQ-NSCLC. We have established an AI-powered digital pathology platform that can provide a standardized, scalable, and reproducible method of characterizing GPC3 positivity to support further patient selection in clinical study. Citation Format: Ellen Meng, Robin Meng, Ying Tong, Yuchen Li, Hong Wang, Paola Fiorentini, Elham Attieh, Qi Tang, Asma Kefsi, Serena Masciari, Giovanni Abbadessa, Cecile Combeau, Lei Tang, Benoit Pasquier, Rui Wang. Artificial intelligence (AI)-powered quantification of glypican-3 (GPC3) expression facilitates patient selection for GPC3-targeted therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6182.

Abstract 5617: Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts the tumor microenvironment

Abstract Ataxia telangiectasia and Rad3-related protein (ATR) and ataxia telangiectasia mutated (ATM) are two of the main kinases of the DNA damage response (DDR). Recent work has demonstrated that the addition of ATM inhibitors enhances the cytotoxicity and in vivo anti-tumor efficacy of ATR inhibitors by abrogating the ATR inhibitor mediated G1 cell cycle arrest and enhancing chromosomal damage.1 Prior to causing cell death, damaged chromosome fragments can end up in the cytosol where they are detected by cytosolic nucleic acid sensors such as cGAS, resulting in the induction of inflammatory signaling pathways and an anti-tumor immune response. This suggests that the mode of action of DDR inhibitors may involve the tumor microenvironment (TME). We therefore aimed to assess the influence of combined pharmacological ATR and ATM inhibition on the TME to better understand how this could be exploited therapeutically. In line with published results in human tumor models, the treatment of murine colon cancer MC-38 cells in vitro with a combination of ATR inhibitor tuvusertib and ATM inhibitor lartesertib led to cell death. Further analysis revealed an activation of the cGAS-STING signaling pathway, an upregulation of PD-L1 and the release of inflammatory cytokines. These results confirmed the activation of immune signaling pathways upon combined inhibition of ATR and ATM. To analyze the stimulation of an anti-tumor immune response upon tuvusertib + lartesertib treatment in vivo, MC-38 cells were transplanted into immunocompetent C57BL/6J mice. Immunoprofiling by flow cytometry and immunohistochemistry staining confirmed that treatment with tuvusertib + lartesertib indeed altered the TME in this setting. In line with the in vitro findings, a significant upregulation of PD-L1 on immune and tumor cells could be observed after 24, 48, and 72 h of treatment even though MC-38 cells expressed high levels of PD-L1 already. Immunoprofiling did not reveal any changes in the TME immune cell content at these early time points. Interestingly, PD-L1 expression as well as tumor immune population changed after prolonged (23 days) treatment. PD-L1 was still expressed on tumor cells but to a significantly lesser extent and a depletion of CD8+ T-cells was observed. However, a significant increase in NK cells could be detected. In summary, we show that inhibition of the DDR through a combination of ATR and ATM inhibitors influences the TME in the MC-38 tumor model. These findings suggest an additional mode of action beyond direct cancer cell cytotoxicity. Further studies on how this may be leveraged therapeutically, for example through a combination with immune checkpoint inhibitors, are warranted. 1. Turchick A, Zimmermann A, et al. Mol Cancer Ther 2023;22(7):859-872. Citation Format: Karin Laaber, Audrey Turchick, Julia Jabs, Brian Elenbaas, Lyubomir T. Vassilev, Astrid Zimmermann. Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts the tumor microenvironment [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 5617.

Abstract 1208: Plasma proteomics-based models for predicting therapeutic benefit and immune-related adverse events in non-small cell lung cancer patients treated with immunotherapy

Abstract Background: Immune checkpoint inhibitors (ICIs) have significantly improved outcomes for patients with metastatic non-small cell lung cancer (NSCLC). However, clinical management is challenging. The PD-L1 biomarker does not optimally guide the choice between ICI monotherapy and combination ICI-chemotherapy, and there are currently no biomarkers to predict immune-related adverse events (irAEs). Here, we describe a versatile platform based on plasma proteomics and machine learning for generating predictive tools to support treatment decisions. Methods: Pre-treatment plasma samples were collected from 616 NSCLC patients receiving ICI-based therapy (NCT04056247). Clinical benefit (CB) data and irAEs were recorded. Proteomic profiling of plasma samples was performed using the SomaScan® assay, covering ~7000 proteins per sample. Proteins associated with Clinical Benefit (progression-free survival at 12 months) were classified as resistance-associated proteins (RAPs). Proteins associated with grade ≥3 irAEs occurring within the first 100 days of treatment were classified as toxicity-associated proteins (TAPs). RAPs and TAPs were integrated into two separate machine learning-based models, yielding CB and irAE probabilities, respectively. The CB model used a CB probability threshold to assign patients a PROphet-positive or PROphet-negative result. Bioinformatic analysis was performed on RAPs and TAPs. Results: CB and irAE models displayed strong predictive performance with a high correlation between predicted probability and observed CB or irAE rate (CB model: R2=0.98; p-value<0.0001; irAE model: R2= 0.92; p-value <0.0001). In the CB model, patients classified as PROphet-positive achieved significantly longer overall survival (OS) than PROphet-negative patients, with a median OS of 25.9 vs. 10.8 months (hazard ratio, HR = 0.51; p-value<0.001). Furthermore, patients with PD-L1 ≥50% tumors and a PROphet-negative result had significantly longer OS with ICI-chemotherapy in comparison to ICI monotherapy (HR=0.23, p-value = 0.0003), suggesting that combination therapy is preferable for such patients despite high PD-L1 levels. Bioinformatic analysis showed that RAPs related to immune regulation, angiogenesis, chemo-resistance, and other potential resistance mechanisms displayed higher expression levels in patients who did not benefit from treatment. Neutrophil- and inflammation-related proteins were significantly enriched in patients who experienced irAEs. Conclusions: We describe two machine-learning based predictive tools that may be used separately or in combination to inform treatment decisions based on the patient’s likelihood to benefit from ICI therapy and to develop serious irAEs. Our findings also provide biological insights related to treatment resistance and immune-related toxicity. Citation Format: Michal Harel, Petros Christopoulos, Igor Puzanov, Jair Bar, Iris Kamer, Niels Reinmuth, Ina Koch, Mor Moskovitz, Adva Levy-Barda, Alona Zer, Michal Lotem, David Farrugia, Rivka Katzenelson, Gillian Price, Abed Agbarya, Helen Cheley, Adam Hassani, Mahmoud Abu-Amna, Tom Geldart, Anirban Chatterjee, Andreas Polychronis, Maya Gottfried, Yanyan Lou, Tatiana Harkovsky, Alison Brewster, Ido Wolf, Ella Tepper, Ben Yellin, Itamar Sela, Coren Lahav, Yehonatan Elon, Raya Leibowitz, Adam P. Dicker, Young Kwang Chae, Ryan J. Sullivan, David Carbone, David Gandara, Jarushka Naidoo. Plasma proteomics-based models for predicting therapeutic benefit and immune-related adverse events in non-small cell lung cancer patients treated with immunotherapy [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 1208.

Abstract 5282: Sulindac modulates the response of triple negative breast cancer to anti-PD-L1 immunotherapy

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous tumor, and there is a lack of effective therapies. Immune checkpoint inhibitors (ICIs) therapy has been widely used to treat a variety of human cancers including TNBC. Despite the FDA's approval of therapies like Atezolizumab (anti-PD-L1) and Pembrolizumab (anti-PD-1) for certain TNBC cases, more than half of all TNBC patients, especially those with low PD-L1 levels and in advanced stages, remain unresponsive. Therefore, there is urgent clinical need to find other innovative combination therapy strategy for these TNBC patients. In this study, we examined the efficacy of sulindac to enhance the response of TNBC to anti-PD-L1 immunotherapy. We utilized a 4T1 syngeneic mouse tumor model to compare the inhibitory effects of PD-L1 antibody, sulindac, and their combination on 4T1 tumor growth. We found that mice treated with combination therapy showed a significant reduction in tumor volume, along with increased infiltration of activated T lymphocytes (Granzyme B+/CD8+ T cells) in the tumor tissues. We also established a PBMC humanized mouse model to further confirmed that combination therapy could significantly reduce the tumor size of TNBC patient-derived xenografts (56S) and the combination group were found to have the most infiltrating activated human T lymphocytes (Granzyme B+/CD8+ T cells) in the tumor tissues. Immunofluorescent staining of organoids also confirmed that organoids from combination group have more CD8+ T cell and the results of organoid cell viability assay in vitro indicated that combination of sulindac with PD-L1 antibody together with activated human PBMC significantly reduce 56S organoid cell viability, which is consistent with in vivo study. When investigating the mechanism of action, first we found sulindac could downregulate exosomal PD-L1 by decreasing expression of nsMase2 which is a major regulator in production of exosomal PD-L1. Second, we demonstrated that sulindac could downregulate PD-L1 by blocking Stat3 signaling and enhancing the expression of miR-570-3p which can potentially target PD-L1, which in turn led to a further decrease in exosomal PD-L1. Previous study showed that PD-L1 antibody could be bound and consumed by exosomal PD-L1 in the blood circulation. Therefore, in combination therapy, sulindac downregulating exosomal PD-L1 leads to increased availability of PD-L1 Ab, which potentially improves the overall efficacy of anti-PD-L1 therapy. In conclusion, our findings provide unique insights into the mechanism of action and efficacy for sulindac as an immunomodulatory agent in combination with anti-PD-L1 therapy for the treatment of TNBC. Citation Format: Bin Yi, Ruixia Ma, Yaguang Xi. Sulindac modulates the response of triple negative breast cancer to anti-PD-L1 immunotherapy [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 5282.

Heat-killed Prevotella intermedia promotes the progression of oral squamous cell carcinoma by inhibiting the expression of tumor suppressors and affecting the tumor microenvironment

BackgroundOral microbial dysbiosis contributes to the development of oral squamous cell carcinoma (OSCC). Our previous study showed that Prevotella intermedia (P. intermedia) were enriched in the oral mucosal surface, plaque, and saliva of patients with OSCC. Intratumoral microbiome could reshape the immune system and influence the development of various tumors. However, the invasion status of human OSCC tissues by P. intermedia and the pathway through which intratumoral P. intermedia potentiates tumor progression remain unexplored.MethodsP. intermedia in human OSCC or normal tissues was detected by FISH. A mouse OSCC cell line SCC7 was adopted to investigate the effects of heat-killed P. intermedia treatment on cell proliferation, invasion, and cytokine release by using CCK-8 assay, transwell invasion assay and ELISA. Moreover, we established a mouse transplanted tumor model by using SCC7 cells, injected heat-killed P. intermedia into tumor tissues, and investigated the effects of heat-killed P. intermedia on tumor growth, invasion, cytokine levels, immune cell infiltrations, and expression levels by using gross observation, H&E staining, ELISA, immunohistochemistry, mRNA sequencing, and transcriptomic analysis.ResultsOur results indicated that P. intermedia were abundant in OSCC and surrounding muscle tissues. Heat-killed P. intermedia promoted SCC7 cell proliferation, invasion and proinflammatory cytokine secretions, accelerated transplanted tumor growth in mice, exacerbate muscle and perineural invasion of OSCC, elevated the serum levels of IL-17A, IL-6, TNF-α, IFN-γ, and PD-L1, induced Treg cells M2 type macrophages in mouse transplanted tumors. The data of transcriptomic analysis revealed that heat-killed P. intermedia increased the expression levels of inflammatory cytokines and chemokines while reduced the expression levels of some tumor suppressor genes in mouse transplanted tumors. Additionally, IL-17 signaling pathway was upregulated whereas GABAergic system was downregulated by heat-killed P. intermedia treatment.ConclusionsTaken together, our results suggest that P. intermedia could inhibit the expression of tumor suppressors, alter the tumor microenvironment, and promote the progression of OSCC.

Nivolumab plus ipilimumab versus the EXTREME regimen in recurrent/metastatic squamous cell carcinoma of the head and neck: a cost-effectiveness analysis

In the CheckMate 651 study, nivolumab plus ipilimumab versus EXTREME (cisplatin/carboplatin + cetuximab + fluorouracil) regimen was compared for effectiveness. It is not known whether these immunotherapy agents are cost-effective for recurrent or metastatic squamous cell carcinomas of the head and neck (R/M SCCHN). The purpose of this study was to compare the cost-effectiveness of nivolumab plus ipilimumab with EXTREME in the first-line setting from the standpoint of third-party payers in the United States. The projecting of costs and outcomes over 15 years was done using a three-state partitioned survival model discounted by 3% per year. Long-term extrapolation of CheckMate 651 was used to model progression-free survival and overall survival (OS). The incremental net health benefit (INHB), incremental net monetary benefit (INMB), quality-adjusted life years (QALYs), and incremental cost-effectiveness ratio (ICER) were calculated. The uncertainty and stability of the model were accounted for via one-way and probabilistic sensitivity analyses. As compared with nivolumab plus ipilimumab, EXTREME was associated with an increase of 0.154 life-years and 0.076 QALYs, as well as a cost increase of $572 per patient. The corresponding ICERs were $7545/QALY along with the values of INMB and INHB were $113,267 and 0.076 QALYs, respectively, at a willingness to pay (WTP) threshold of $150,000/QALY. The probability of nivolumab plus ipilimumab being cost-effective was > 99% in patients with combined positive score (CPS) ≥ 1, CPS 1–19, or CPS ≥ 20. Moreover, hazard ratio for OS and body weight were the most sensitive parameters for the model. According to sensitivity analyses, these results were generally robust. In overall populations with R/M SCCHN, the EXTREME regimen is cost-effective compared with nivolumab plus ipilimumab. Given a WTP threshold of $150,000 per QALY, the probability of the EXTREME regiment being cost-effective compared with nivolumab and ipilimumab, was 64%. Importantly, there was heterogeneity in the cost-effectiveness probabilities, based on primary sites and expression levels of PD-L1. Therefore, tailored treatment based on individual patient and clinical characteristics, remains important, and may impact the cost-effectiveness of the regimens under study.

Stigmasterol Exerts an Anti-Melanoma Property through Down-Regulation of Reactive Oxygen Species and Programmed Cell Death Ligand 1 in Melanoma Cells.

Cancer immunotherapy as a promising anti-cancer strategy has been widely studied in recent years. Stigmasterol (STIG), a phytosterol, is known to have various pharmacological effects, including anti-inflammatory effects. However, the pharmacological role of STIG on melanoma immunotherapy has not been investigated. The present study demonstrates the anti-melanoma potency of STIG through the regulation of PD-L1 levels. The results reveal that STIG reduces reactive oxygen species (ROS) levels induced by hydrogen peroxide and increases glutathione levels decreased by α-MSH in B16F10 cells. Moreover, STIG significantly decreases melanin content and tyrosinase activities elevated by α-MSH. It also suppresses nitric oxide production induced by α-MSH. Additionally, STIG induces apoptosis with the up-regulation of PARP activation. STIG inhibits IFN-γ-induced PD-L1 expression and STAT1 phosphorylation levels. STIG also reverses the up-regulation of PD-L1 and phosphorylated STAT1 levels augmented by cisplatin, and STIG enhances CD8(+) T-cell-mediated cell death against B16F10 cells. These findings represent the first evidence of pro-apoptotic activity of STIG on melanoma cells through the down-regulation of ROS and PD-L1 pathways. Therefore, STIG may be an effective candidate for melanoma immunotherapy.

Neoadjuvant Chemoimmunotherapy for NSCLC

To date, no meta-analyses have comprehensively assessed the association of neoadjuvant chemoimmunotherapy with clinical outcomes in non-small cell lung cancer (NSCLC) in randomized and nonrandomized settings. In addition, there exists controversy concerning the efficacy of neoadjuvant chemoimmunotherapy for patients with NSCLC with programmed cell death 1 ligand 1 (PD-L1) levels less than 1%. To compare neoadjuvant chemoimmunotherapy with chemotherapy by adverse events and surgical, pathological, and efficacy outcomes using recently published randomized clinical trials and nonrandomized trials. MEDLINE and Embase were systematically searched from January 1, 2013, to October 25, 2023, for all clinical trials of neoadjuvant chemoimmunotherapy and chemotherapy that included at least 10 patients. Observational studies and trials reporting the use of neoadjuvant radiotherapy, including chemoradiotherapy, molecular targeted therapy, or immunotherapy monotherapy, were excluded. Surgical, pathological, and efficacy end points and adverse events were pooled using a random-effects meta-analysis. Among 43 eligible trials comprising 5431 patients (4020 males [74.0%]; median age range, 55-70 years), there were 8 randomized clinical trials with 3387 patients. For randomized clinical trials, pooled overall survival (hazard ratio, 0.65; 95% CI, 0.54-0.79; I2 = 0%), event-free survival (hazard ratio, 0.59; 95% CI, 0.52-0.67; I2 = 14.9%), major pathological response (risk ratio, 3.42; 95% CI, 2.83-4.15; I2 = 31.2%), and complete pathological response (risk ratio, 5.52; 95% CI, 4.25-7.15; I2 = 27.4%) favored neoadjuvant chemoimmunotherapy over neoadjuvant chemotherapy. For patients with baseline tumor PD-L1 levels less than 1%, there was a significant benefit in event-free survival for neoadjuvant chemoimmunotherapy compared with chemotherapy (hazard ratio, 0.74; 95% CI, 0.62-0.89; I2 = 0%). This study found that neoadjuvant chemoimmunotherapy was superior to neoadjuvant chemotherapy across surgical, pathological, and efficacy outcomes. These findings suggest that patients with resectable NSCLC with tumor PD-L1 levels less than 1% may have an event-free survival benefit with neoadjuvant chemoimmunotherapy.

Dynamic MicroRNA-Expression In Plasma of Melanoma Patients Correlates With Progression, PD-L1 Status and Overall Survival

Melanoma treatment has improved significantly with the development of immune checkpoint inhibition (ICI), which has greatly enhanced the survival rates of patients with metastatic melanoma. However, a significant number of patients do not respond well to ICI treatment and experience progression. This highlights the critical need for practical means to track melanoma patients' response to ICI. To address this issue, the patterns of circulating miRNAs were studied in liquid biopsies of melanoma patients. These miRNAs have the potential to provide essential information regarding the cancer stage, progression, and the presence of PD-L1 in tumor tissue. A sophisticated flow cytometric test was used to measure up to 63 different miRNAs at once. The study identified a combination of nine miRNAs that are capable of distinguishing between different stages of melanoma, particularly stage IV. Additionally, five miRNAs were pinpointed which are downregulated in patients who do not respond to ICI treatment. Furthermore, two miRNAs were found that correlate to the level of PD-L1 in tumor tissue, and low levels of miR-150-5p were linked to poorer overall survival. These findings suggest that circulating miRNAs could serve as valuable markers to predict the effectiveness of ICI, provide insights into the cancer's stage and PD-L1 status, and ultimately help physicians make better treatment decisions in the future. However, further research is needed to confirm these findings and establish their clinical usefulness.

Enhancing Tumor Immunotherapy by Multivalent Anti-PD-L1 Nanobody Assembled via Ferritin Nanocage.

Increasing immunotherapy response rate and durability can lead to significant improvements in cancer care. To address this challenge, a novel multivalent immune checkpoint therapeutic platform is constructed through site-specific ligation of anti-PD-L1 nanobody (Nb) on ferritin (Ftn) nanocage. Nb-Ftn blocks PD-1/PD-L1 interaction and downregulates PD-L1 levels via endocytosis-induced degradation. In addition, the cage structure of Ftn allows encapsulation of indocyanine green (ICG), an FDA-approved dye. Photothermal treatment with Nb-Ftn@ICG induces immunogenic death of tumor cells, which improves systemic immune response via maturation of dendritic cells and enhanced infiltration of T cells. Moreover, Nb-Ftn encapsulation significantly enhances cellular uptake, tumor accumulation and retention of ICG. In vivo assays showed that this nanoplatform ablates the primary tumor, suppresses abscopal tumors and inhibits tumor metastasis, leading to a prolonged survival rate. This work presents a novel strategy for improving cancer immunotherapy using multivalent nanobody-ferritin conjugates as immunological targeting and enhancing carriers.

Association of lymphocyte subsets with the efficacy and prognosis of PD‑1 inhibitor therapy in advanced gastric cancer: results from a monocentric retrospective study

PurposeThis retrospective study aimed to investigate the changes in peripheral blood lymphocyte subsets before and after immunotherapy in patients with advanced gastric cancer and their relationship n with the therapeutic efficacy and clinical prognosis.MethodsPeripheral blood lymphocyte subsets, including CD4 + T cells, CD8 + T cells, CD4+/CD8 + ratio, NK cells, Treg cells, and B cells, were collected from 195 patients with advanced gastric cancer who were admitted to the First Hospital of Shanxi Medical University with immunotherapy from January 2020 to October 2021, at the time of diagnosis of advanced gastric cancer, before immunotherapy and after 3 cycles of immunotherapy. T-tests were used to examine the factors influencing the patients’ peripheral blood lymphocyte subsets and the changes after immunotherapy. To examine the relationship between lymphocyte subsets and treatment outcomes, ROC curves were plotted using a logistic regression. Kaplan–Meier curve was drawn, and the Log Rank test was carried out to compare the differences in PFS between the different groups. Cox proportional hazards regression model was used to analyze the factors affecting PFS after calibration of other variables.ResultsThe proportion of peripheral blood lymphocyte subsets in patients with advanced gastric cancer was affected by age and PD-L1 level. Compared to the baseline, the treatment effective group had higher proportions of CD4 + T cells, a higher CD4+/CD8 + ratio, NK cells and Treg cells, and lower proportions of CD8 + T cells and B cells in the peripheral blood after three cycles of immunotherapy. In the treatment-naive group, there were no significant differences in the lymphocyte subsets. With cut-off values of 30.60% and 18.00%, baseline CD4 + T cell and NK cell ratios were independent predictors of immunotherapy efficacy and PFS. Treg cell ratio, gender, PD-L1 levels, and MMR status all predicted PFS independently.ConclusionThe proportion of peripheral blood lymphocyte subsets was modified in patients who responded to PD-1 inhibitors. Different lymphocyte subpopulation levels can be used as biomarkers to predict immunotherapy efficacy and clinical prognosis in patients with advanced gastric cancer.

USP24-i-101 targeting of USP24 activates autophagy to inhibit drug resistance acquired during cancer therapy

Drug resistance in cancer therapy is the major reason for poor prognosis. Addressing this clinically unmet issue is important and urgent. In this study, we found that targeting USP24 by the specific USP24 inhibitors, USP24-i and its analogues, dramatically activated autophagy in the interphase and mitotic periods of lung cancer cells by inhibiting E2F4 and TRAF6, respectively. USP24 functional knockout, USP24C1695A, or targeting USP24 by USP24-i-101 inhibited drug resistance and activated autophagy in gefitinib-induced drug-resistant mice with doxycycline-induced EGFRL858R lung cancer, but this effect was abolished after inhibition of autophagy, indicating that targeting USP24-mediated induction of autophagy is required for inhibition of drug resistance. Genomic instability and PD-L1 levels were increased in drug resistant lung cancer cells and were inhibited by USP24-i-101 treatment or knockdown of USP24. In addition, inhibition of autophagy by bafilomycin-A1 significantly abolished the effect of USP24-i-101 on maintaining genomic integrity, decreasing PD-L1 and inhibiting drug resistance acquired in chemotherapy or targeted therapy. In summary, an increase in the expression of USP24 in cancer cells is beneficial for the induction of drug resistance and targeting USP24 by USP24-i-101 optimized from USP24-i inhibits drug resistance acquired during cancer therapy by increasing PD-L1 protein degradation and genomic stability in an autophagy induction-dependent manner.

TMEM160 promotes tumor immune evasion and radiotherapy resistance via PD-L1 binding in colorectal cancer

BackgroundThe effectiveness of anti-programmed cell death protein 1(PD-1)/programmed cell death 1 ligand 1(PD-L1) therapy in treating certain types of cancer is associated with the level of PD-L1. However, this relationship has not been observed in colorectal cancer (CRC), and the underlying regulatory mechanism of PD-L1 in CRC remains unclear.MethodsBinding of TMEM160 to PD-L1 was determined by co-immunoprecipitation (Co-IP) and GST pull-down assay.The ubiquitination levels of PD-L1 were verified using the ubiquitination assay. Phenotypic experiments were conducted to assess the role of TMEM160 in CRC cells. Animal models were employed to investigate how TMEM160 contributes to tumor growth.The expression and clinical significance of TMEM160 and PD-L1 in CRC tissues were evaluated by immunohistochemistry(IHC).ResultsIn our study, we made a discovery that TMEM160 interacts with PD-L1 and plays a role in stabilizing its expression within a CRC model. Furthermore, we demonstrated that TMEM160 hinders the ubiquitination-dependent degradation of PD-L1 by competing with SPOP for binding to PD-L1 in CRC cells. Regarding functionality, the absence of TMEM160 significantly inhibited the proliferation, invasion, metastasis, clonogenicity, and radioresistance of CRC cells, while simultaneously enhancing the cytotoxic effect of CD8 + T cells on tumor cells. Conversely, the upregulation of TMEM160 substantially increased these capabilities. In severely immunodeficient mice, tumor growth derived from lentiviral vector shTMEM160 cells was lower compared with that derived from shNC control cells. Furthermore, the downregulation of TMEM160 significantly restricted tumor growth in immune-competent BALB/c mice. In clinical samples from patients with CRC, we observed a strong positive correlation between TMEM160 expression and PD-L1 expression, as well as a negative correlation with CD8A expression. Importantly, patients with high TMEM160 expression exhibited a worse prognosis compared with those with low or no TMEM160 expression.ConclusionsOur study reveals that TMEM160 inhibits the ubiquitination-dependent degradation of PD-L1 that is mediated by SPOP, thereby stabilizing PD-L1 expression to foster the malignant progress, radioresistance, and immune evasion of CRC cells. These findings suggest that TMEM160 holds potential as a target for the treatment of patients with CRC.

Plasma Proteome-Based Test for First-Line Treatment Selection in Metastatic Non-Small Cell Lung Cancer.

Current guidelines for the management of metastatic non-small cell lung cancer (NSCLC) without driver mutations recommend checkpoint immunotherapy with PD-1/PD-L1 inhibitors, either alone or in combination with chemotherapy. This approach fails to account for individual patient variability and host immune factors and often results in less-than-ideal outcomes. To address the limitations of the current guidelines, we developed and subsequently blindly validated a machine learning algorithm using pretreatment plasma proteomic profiles for personalized treatment decisions. We conducted a multicenter observational trial (ClinicalTrials.gov identifier: NCT04056247) of patients undergoing PD-1/PD-L1 inhibitor-based therapy (n = 540) and an additional patient cohort receiving chemotherapy (n = 85) who consented to pretreatment plasma and clinical data collection. Plasma proteome profiling was performed using SomaScan Assay v4.1. Our test demonstrates a strong association between model output and clinical benefit (CB) from PD-1/PD-L1 inhibitor-based treatments, evidenced by high concordance between predicted and observed CB (R2 = 0.98, P < .001). The test categorizes patients as either PROphet-positive or PROphet-negative and further stratifies patient outcomes beyond PD-L1 expression levels. The test successfully differentiates between PROphet-negative patients exhibiting high tumor PD-L1 levels (≥50%) who have enhanced overall survival when treated with a combination of immunotherapy and chemotherapy compared with immunotherapy alone (hazard ratio [HR], 0.23 [95% CI, 0.1 to 0.51], P = .0003). By contrast, PROphet-positive patients show comparable outcomes when treated with immunotherapy alone or in combination with chemotherapy (HR, 0.78 [95% CI, 0.42 to 1.44], P = .424). Plasma proteome-based testing of individual patients, in combination with standard PD-L1 testing, distinguishes patient subsets with distinct differences in outcomes from PD-1/PD-L1 inhibitor-based therapies. These data suggest that this approach can improve the precision of first-line treatment for metastatic NSCLC.

High-risk histopathologic features in local advanced conjunctival melanoma.

To identify high-risk histopathologic and molecular features of local recurrence, nodal metastasis, distant metastasis (DM) and disease-specific death (DSD) in conjunctival melanoma (CoM). Ninety patients with pathologically diagnosed CoM between 2008 and 2023 were enrolled. Immunohistochemistry staining of BRAFV600E, NRASQ61R, CD117, PD-1 and PD-L1 was performed in 65 and 45 patients, respectively. Cox regression and Kaplan-Meier survival analysis were conducted to identify risk factors for local recurrence, nodal metastasis, DM and DSD. Pathologically, ulceration (hazard ratio [HR]: 3.170; 95% CI: 1.312-7.659; p = 0.01) and regression (HR: 3.196; 95% CI: 1.094-9.335; p = 0.034) were risk factors for DM. Tumour thickness ≥ 4 mm (HR: 4.889; 95% CI: 1.846-12.946; p = 0.001) and regression (HR: 4.011; 95% CI: 1.464-10.991; p = 0.007) were risk factors for DSD. For patients with tumour thickness < 4 mm, the presence of ulceration indicated a higher risk of nodal metastasis (log-rank p = 0.0011), DM (log-rank p = 0.00051) and DSD (log-rank p = 0.02). Patients with regression (+)/tumour-infiltrating lymphocytes (TILs) (+) had a higher risk for DM (log-rank p = 0.011) and DSD (log-rank p = 0.0032). Molecularly, the positive rate of BRAFV600E, NRASQ61R, CD117, PD-1 and PD-L1 was 40.00% (26/65), 43.08% (28/65), 70.77% (46/65), 46.67% (21/45) and 28.89% (13/45), respectively. Positive BRAFV600E was identified as an independent risk factor for DM (HR: 2.533; 95% CI: 1.046-6.136, p = 0.039). The expression level of BRAFV600E was positively correlated with vascular invasion (p = 0.01), as well as the expression levels of PD-1 (p = 0.038) and PD-L1 (p = 0.049). Tumour thickness ≥ 4 mm, ulceration, the coexistence of regression and TILs, and positive BRAFV600E were risk factors for poor prognosis of CoM patients. Besides, expression level of BRAFV600E was positively correlated with the expression levels of PD-1 and PD-L1.

Correlation between PD-1 and sPD-L1 expression levels in peripheral blood of DLBCL patients and their clinicopathological characteristics.

Molecular pathology and clinical characteristics play a crucial role in guiding treatment selection and predicting the prognosis of diffuse large B-cell lymphoma (DLBCL). The programmed cell death protein 1 (PD-1) and its ligand (PD-L1), have emerged as pivotal regulators of immune checkpoints in cancer. The objectives of this study are to investigate the correlation between the expression levels of PD-1 and soluble PD-L1 (sPD-L1) in the peripheral blood of DLBCL patients, analyze their clinicopathological characteristics, and identify the optimal beneficiary group for PD-1/PD-L1 blockade. Peripheral blood samples were collected from 36 DLBCL patients before their initial treatment at Shandong Cancer Hospital between December 2018 and July 2019. The expression levels of PD-1 and sPD-L1 were measured using flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The clinicopathological characteristics, including age, sex, Ann Arbor stage, International Prognostic Index (IPI) score, response to treatment, etc., were recorded for each patient. The surface expression of PD-1 on peripheral blood T cells was significantly higher in DLBCL patients compared to healthy controls. There was a significant association between elevated PD-1 expression levels and the advanced Ann Arbor stage (P=0.0153) as well as the B group (P=0.0184). Higher sPD-L1 levels were associated with the GCB subtype according to Hans's classification (P=0.0435). The expression levels of PD-1 and sPD-L1 in the peripheral blood of DLBCL patients are significantly correlated with advanced disease stage, B group, and GCB subtype according to Hans's classification. This suggests that the PD-1/PD-L1 axis play a critical role in specific subgroups of DLBCL. Targeting this axis could serve as a potential therapeutic strategy to enhance the clinical outcomes of DLBCL patients. Further studies are necessary to explore the prognostic implications of PD-1 and sPD-L1 expression levels in DLBCL patients.

Comprehensive Co-Inhibitory Receptor (Co-IR) Expression on T Cells and Soluble Proteins in Rheumatoid Arthritis.

Co-IRs T cells were immunophenotyped employing multicolor flow cytometry, and ELISA was utilized for measuring soluble PD-1, PDL-2, and TIM3. Correlations have been detected across the percentage of T cells expressing Co-IRs (MFI) and different indicators in the blood, including ESR, high-sensitivity CRP (hsCRP), 28 joint disease activity scores (DAS28), and soluble PD-1/PDL-2/TIM3. In RA patients, we recognized elevated levels of PD-1 (CD279), CTLA-4, and TIGIT in CD4+ T cells; TIGIT, HLA-DR, TIM3, and LAG3 in CD8+ T cells; and CD8+CD279+TIM3+, CD8+HLA-DR+CD38+ T cells. The following tests were revealed to be correlated with hsCRP: CD4/CD279 MFI, CD4/CD279%, CD4/TIM3%, CD8/TIM3%, CD8/TIM3 MFI, CD8/LAG3%, and CD8+HLA-DR+CD38+%. CD8/LAG3 and CD8/TIM3 MFIs are linked to ESR. DAS28-ESR and DAS28-CRP exhibited relationships with CD4/CD127 MFI, CD8/CD279%, and CD8/CD127 MFI, respectively. CD4+CD279+TIM3+% was correlated with DAS28-ESR (p = 0.0084, N = 46), DAS28-CRP (p = 0.007, N = 47), and hsCRP (p = 0.002, N = 56), respectively. In the serum of patients with RA, levels of soluble PD-1, PDL-2, and Tim3 were extremely elevated. CD4+ TIM3+% (p = 0.0089, N = 46) and CD8+ TIM3+% (p = 0.0305, N = 46) were correlated with sTIM3 levels; sPD1 levels were correlated with CD4+CD279+% (p < 0.0001, N = 31) and CD3+CD279+% (p = 0.0084, N = 30). Co-IR expressions on CD4+ and CD8+ T cells, as well as soluble PD-1, PDL-2, and TIM3 levels, could function as indicators of disease activity and potentially play crucial roles in the pathogenesis of RA.

Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142−/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142−/low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Outcomes in patients treated with frontline immune checkpoint inhibition (ICI) for advanced NSCLC with KRAS mutations and STK11/KEAP1 comutations across PD-L1 levels

IntroductionIn patients with advanced NSCLC (aNSCLC), the impact of KRAS mutations (m) and comutations with STK11 and KEAP1 on outcomes across different PD-L1 levels remains incompletely understood. We aimed to investigate the frequency of KRAS mutations and comutations across PD-L1 levels, and the association between these mutations and survival, stratified by PD-L1 expression. MethodsWe conducted a nationwide cohort study of patients diagnosed with aNSCLC between 2016 and 2021 treated with frontline (chemo)immunotherapy, who underwent molecular genotyping including KRAS, STK11, and KEAP1. Real-world overall survival (OS) and progression-free survival (rwPFS) were estimated using Kaplan-Meier methodology. Cox multivariable regressions were used to evaluate the association between KRASm and survival across different PD-L1 strata, and to assess whether the association between KRASm and survival differed by PD-L1 level. Finally, within subgroups defined by PD-L1 expression, we used interaction terms to assess whether co-mutations with STK11 and KEAP1 moderated the association between KRAS mutation and survival. ResultsOf our 2593-patient cohort, 982 (37.9 %) were KRASm and 1611 (62.1 %) KRASwt. KRASm were enriched in the PD-L1 ≥50 % cohort (334/743, 45 %), but within patients with KRASm, co-mutations with STK11 and KEAP1 were enriched in the PD-L1 0 % cohort. KRASm was associated with significantly worse OS in the PD-L1 0 % cohort compared to the PD-L1 ≥50 % cohort (P for interaction = 0.008). On adjusted analyses stratified by PD-L1, KRASm was associated with worse survival only in the PD-L1 0 % group (OS HR 1.46, p = 0.001). KEAP1 and STK11 comutations were most strongly associated with worse OS in the PD-L1 0 % subgroup; patients with triple KRASm/KEAPm/STK11m PD-L1 0 % NSCLC experienced the worst outcomes. ConclusionsKRASm are associated with worse overall survival in PD-L1 negative NSCLC; however, this association is largely driven by comutations with STK11 and KEAP1, which are enriched in PD-L1 negative tumors.