Elevated PD-L1 Expression Research Articles

317. PD-L1 DYNAMICS DURING THE CISPLATIN-INDUCED ACQUIRED RESISTANCE PROCESS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Immunotherapy combined with chemotherapy has been the first-line treatment and the most widely-used neoadjuvant regimen in esophageal squamous cell carcinoma (ESCC). Resistance to chemotherapy is an inevitable clinical problem, however, the impact of chemoresistance on immunotherapy efficacy remains unclear. This study aimed to investigate the impact of chemoresistance formation on PD-L1 dynamic changes both in vivo and in vitro during the construction of cisplatin-resistant ESCC models, with the goal of exploring the potential influence of chemoresistance on immunotherapy. Methods For in vivo model, fluorescent-labeled ESCC cells (AKR) were implanted into C57 mice, followed by cisplatin administration for six cycles. Surviving tumor cells were isolated using flow cytometry, and reinjected to establish cisplatin-resistant model. Tumors were harvested to evaluate PD-L1 expression by immunohistochemistry. For in vitro model, ESCC cell lines (TE-1, KYSE150) received low-dose cisplatin for 48h. After three cycles, resistance index (RI, RI=IC50 of drug-expose cell/IC50 of parental cell) was determined by CCK8 assay. The process was repeated with escalating cisplatin doses until resistant cell lines were established (RI>5). PD-L1 expression was measured using RT-qPCR/Western-Blot after each generation. Results In vivo (Figure A), cisplatin-resistant tumor exhibited elevated PD-L1 expression compared to control group (H-score: 22.17 vs 2.09, p<0.001). In vitro (Figure B), PD-L1 expression exhibited an initial increase, followed by a gradual decrease during the process of cisplatin-induced resistance. Specifically, in early stages of cisplatin-resistance in TE-1 cells, PD-L1 mRNA expression rose 4.9 times, consistently paralleled by an elevation in protein level. Subsequently, PD-L1 expression gradually decreased in both mRNA and protein levels. KYSE150 cells exhibited a trend of upregulation in PD-L1 expression in the early stages of cisplatin resistance but later decreased, returning to parental cell levels. Conclusion Under in vivo conditions, acquired cisplatin-resistance in ESCC cells significantly upregulated PD-L1 expression, indicating a potential mechanism for PD-L1-mediated immune escape and its impact on immunotherapy efficacy. However, under in vitro conditions, no consistent trend in PD-L1 expression was observed during the acquisition of cisplatin resistance. This suggests a potentially crucial role of the immune system involved in the chemoresistance regulatory mechanism. Further investigations are needed to explore the impact of chemoresistance on tumor immunology.

SUV3 knockdown inhibits proliferation, migration, and invasion of hepatocellular carcinoma cells and induces PD-L1 expression

Objective: To study the SUV3 gene role during the process of occurrence and advancement of hepatocellular carcinom. Methods: The The differences in SUV3 expression between hepatocellular carcinoma tissues and normal liver tissues were compared by analyzing transcriptome sequencing data from TCGA and GTEx databases. SUV3 knockdown in different hepatocellular carcinoma cells was performed using RNA interference technology. Overexpression vectors were constructed to overexpress SUV3 in different hepatocellular carcinoma cells. The SUV3 regulatory effect was studied on proliferation, migration, and invasion of hepatocellular carcinoma cells. A subcellular fraction isolation approach was used to investigate whether SUV3 knockdown resulted in the release of mitochondrial DNA into the cytoplasm. Quantitative reverse transcription PCR was applied to investigate whether SUV3 knockdown affected PD-L1 expression. The two groups were compared using a two-tailed t-test. Results: The TCGA database analysis revealed that SUV3 expression was higher in hepatocellular carcinoma tissues than in normal liver tissues, and the prognosis of patients with high SUV3 expression in hepatocellular carcinoma tissues was poor. The quantitative RT-PCR results showed that SUV3 expression was higher in hepatocellular carcinoma tissues than that in paracancerous liver tissue. The MTS assay showed that with SUV3 knockdown, the proliferation rate was significantly lower in hepatocellular carcinoma cells than that of the control hepatocellular carcinoma cells (P<0.01). The proliferation rate was significantly higher in SUV3-overexpressed hepatocellular carcinoma cells than that of control hepatocellular carcinoma cells (P<0.01). Cell scratch assay and cell migration and invasion assay showed that SUV3 knockdown inhibited the migration and invasion of hepatocellular carcinoma cells (P<0.01), while SUV3 overexpression promoted the migration and invasion of hepatocellular carcinoma cells (P<0.05). SUV3 Knockdown led to a decrease in the overall level of mtDNA (P<0.01) in accompanied by an increase in mtDNA level in the cytoplasm (P<0.01), indicating that SUV3 knockdown led to mitochondrial DNA leakage into the cytoplasm. SUV3 knockdown resulted in elevated PD-L1 expression (P<0.001), and overexpression of TREX1 in SUV3 knockdown cells decreased mtDNA levels in the cytoplasm and inhibited SUV3 knockdown, resulting in elevated PD-L1 expression, indicating that SUV3 knockdown induced PD-L1 expression by increasing cytoplasmic DNA levels. Conclusions: The SUV3 gene may play an oncogenic function in hepatocellular carcinoma cells.

PGM5-AS1 Promotes Progression of Diffuse Large B-Cell Lymphoma and Immune Escape by Regulating miR-503-5p.

Diffuse large B-cell lymphoma (DLBCL) is a prevalent malignant condition with a dismal prognosis. LncRNA PGM5 antisense RNA 1 (PGM5-AS1) appears to be intricately involved in the progression of DLBCL, yet the modulatory mechanism remains unclear. The purpose of this study was to explore the expression of lncRNA PGM5-AS1 in DLBCL and its effect on the disease progression of DLBCL, as well as to explore its mechanisms. A total of 35 patients were included in the study. The expression levels of PGM5-AS1 and miR-503-5p in DLBCL tumor tissues and cell lines were detected by RT-qPCR. Cell proliferation was assessed using CCK8. Apoptosis rate was determined by flow cytometry. Cell invasion was examined by transwell assays. The specific interaction between PGM5-AS1 and miR-503-5p was verified through dual luciferase reporter gene assays. The immune related factors were detected by ELASA kits. The CD8+ T cells cytotoxicity was evaluated by LDH cytotoxicity kit. In DLBCL tumor tissues and cells, upregulated PGM5-AS1 expression, downregulated miR-503-5p expression, and elevated PD-L1 expression were observed. PGM5-AS1 functioned as a regulator in controlling DLBCL cell proliferation, apoptosis, and invasion by downregulating miR-503-5p expression. When CD8+ T cells were co-cultured with cells transfected with si-PGM5-AS1, the secretion of immunoregulatory factors increased, and the cytotoxicity of CD8+ T cells increased. These effects were mitigated by miR-503-5p inhibitors. PGM5-AS1 accelerated DLBCL development and facilitated tumor immune escape through the miR-503-5p. Our discoveries offered an insight into lncRNA PGM5-AS1 serving as a prospective therapeutic target for DLBCL.

The frequency of EGFR gene mutations in a cohort of Romanian patients with non-small cell lung cancer and their association with PD-L1 expression level and ALK rearrangements

Abstract Background The mortality rate linked to non-small cell lung cancer (NSCLC) has notably decreased in recent years, primarily due to refined diagnostic techniques. This retrospective study aims to offer new insights into the frequency of EGFR gene mutations in Romanian NSCLC patients, examining potential associations or exclusions with ALK rearrangements and elevated PD-L1 expression level and seeks to contribute crucial insights into molecular marker alterations associated with NSCLC, advancing our understanding of targeted therapy prospects for oncology patients diagnosed with NSCLC in Romania. Methods DNA was extracted from the FFPET sections using the DNA Sample Preparation kit from Roche Diagnostics while the EGFR mutation detection test was performed using Real-Time PCR methods. PD-L1 expression levels and ALK rearrangements were immunohistochemically assessed. Results Among the 453 patients, 42 displayed EGFR gene mutations. The most prevalent mutation was Ex19Del, observed in 3.5% of cases, followed by the L858R substitution (2.9%). A noticeable elevation of PD-L1 expression level was observed on average when comparing patients EGFR Wild-Type with patients with EGFR gene mutations (40.37% versus 26.13%). The association of the L858R mutation and positive ALK was observed in one patient in our study cohort. Conclusions The study reveals a significantly higher prevalence of EGFR gene mutations among females and non-smokers. EGFR mutations were exclusively identified in patients with lung adenocarcinoma. This study data act as a catalyst for future investigations into resistance mechanisms to anti-EGFR TKIs in NSCLC patients in Romania and the prevalence of EGFR gene mutations associated with this phenomenon.

Whole exome sequencing analysis of intratumoral heterogeneity, tumor neoantigen burden and human leukocyte antigen loss of heterozygosity in HCC.

e16003 Background: Hepatocellular Carcinoma (HCC) patients has a high postoperative recurrence within 2 years and a low 5-year survival rate. However, molecular mechanisms for early recurrence are poorly understood. Immune checkpoint blockades (ICBs) show clinical benefits in HCC, with the immune microenvironment influencing ICBs therapeutic effects. Exploring the relationship between intratumoral heterogeneity (ITH), tumor neoantigen burden (TNB), and human leukocyte antigen loss of heterozygosity (HLA LOH) can deepen our understanding of the immune evasion mechanisms. Methods: In this study, whole exome sequencing (WES) was employed to analyze tumor specimens and matched peripheral blood controls derived from a cohort of 235 HCC patients. PD-L1 expression levels were assessed in the samples using immunohistochemistry. NetMHCpan was applied to predict neoantigen and quantified the peptide affinity of MHC-I genes measured by IC50. Neoantigen-MHC affinity was categorized as strong (IC50≤50nM), weak (50nM &lt; IC50≤500nM), and negative (IC50 &gt; 500nM). TNB was defined by the number of high affinity neoantigens per megabase in WES. Analysis of LOH was performed on HLA-I class genes and categorized as strong positive, weak positive, and negative groups. We used PyClone to conduct ITH analysis to assess the distribution of multiple subclones within a tumor. The average Cancer Cell Fraction (CCF) for each somatic mutation cluster was calculated, and these clusters were classified into major clones and subclones. Results: In 235 HCC samples, median TMB and TNB were 2.48 Muts/Mb and 0.30 Neos/Mb. Only 6.81% exhibited TMB-H (TMB≥10 Muts/Mb). TMB and TNB are significantly positively correlated (p &lt; 0.0001). HLA LOH occurred in 19.57% HCC patients, with high LOH samples having more high-affinity neoantigens (mean TNB 1.65 Neos/Mb). ITH analysis showed that 83.83% samples harbored subclonal clusters. HCC samples were divided into high (ITH≥0.908) and low (ITH &lt; 0.908) groups according to the median ITH of 0.908. High ITH group had higher TNB (median 0.43 vs 0.20 Neos/Mb) , more HLA LOH (9.24% vs 5.17% strong positive LOH). The high ITH groups exhibited a lower percentage of samples with elevated PD-L1 expression (14.29% vs 25.86%). The Combined Positive Score (CPS) revealed a significant difference between the groups (Fisher's test p = 0.03). Conclusions: In HCC, the high ITH group exhibited a higher proportion of HLA LOH compared to the low ITH group. Moreover, a decreased proportion of PD-L1 positive samples was observed in the high ITH group, suggesting that high ITH may be a key factor influencing treatment failure in immunotherapy. ITH analysis demonstrated a potential multi-subclonal origin of recurrent tumor cells, which is associated with immune escape, tumor malignancy,as well as the development of resistance.

Prognostic and Clinical Significance of PD-L1, EGFR and Androgen Receptor (AR) Expression in Triple-Negative Breast Cancer (TNBC) Patients.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is associated with high recurrence rates, a high incidence of distant metastases and poor overall survival. The aim of this study was to investigate the role of PD-L1, EGFR and AR expression in TNBC promotion and progression. To that end, we analyzed the immunohistochemical expression of these genes in 125 TNBC patients and their relation to clinicopathological parameters and survival. An elevated expression of PD-L1 was significantly correlated with higher tumor and nuclear grade, while a low expression was correlated with loco-regional recurrence without any influence on survival. Contrary to this, the expression of AR showed a positive impact on the DFI and a negative association with tumor grade. Furthermore, PD-L1 and AR demonstrated simultaneous expression, and further co-expression analysis revealed that a positive expression of PD-L1/AR notably correlates with tumor and nuclear grade and has a significant impact on a longer DFI and OS, while a negative PD-L1/AR expression is significantly associated with metastases. Therefore, our results suggest that positive PD-L1/AR expression is beneficial for TNBC patients. In addition, an elevated expression of EGFR contributes to metastases and a worse DFI and OS. In conclusion, we think that low PD-L1/low AR/high EGFR expression followed by high Ki67 expression constitutes a 'high risk' profile of TNBC.

Loss of heterozygosity impacts MHC expression on the immune microenvironment in CDK12-mutated prostate cancer

BackgroundIn prostate cancer (PCa), well-established biomarkers such as MSI status, TMB high, and PDL1 expression serve as reliable indicators for favorable responses to immunotherapy. Recent studies have suggested a potential association between CDK12 mutations and immunotherapy response; however, the precise mechanisms through which CDK12 mutation may influence immune response remain unclear. A plausible explanation for immune evasion in this subset of CDK12-mutated PCa may be reduced MHC expression.ResultsUsing genomic data of CDK12-mutated PCa from 48 primary and 10 metastatic public domain samples and a retrospective cohort of 53 low-intermediate risk primary PCa, we investigated how variation in the expression of the MHC genes affected associated downstream pathways. We classified the patients based on gene expression quartiles of MHC-related genes and categorized the tumors into “High” and “Low” expression levels. CDK12-mutated tumors with higher MHC-expressed pathways were associated with the immune system and elevated PD-L1, IDO1, and TIM3 expression. Consistent with an inflamed tumor microenvironment (TME) phenotype, digital cytometric analyses identified increased CD8 + T cells, B cells, γδ T cells, and M1 Macrophages in this group. In contrast, CDK12-mutated tumors with lower MHC expression exhibited features consistent with an immune cold TME phenotype and immunoediting. Significantly, low MHC expression was also associated with chromosome 6 loss of heterozygosity (LOH) affecting the entire HLA gene cluster. These LOH events were observed in both major clonal and minor subclonal populations of tumor cells. In our retrospective study of 53 primary PCa cases from this Institute, we found a 4% (2/53) prevalence of CDK12 mutations, with the confirmation of this defect in one tumor through Sanger sequencing. In keeping with our analysis of public domain data this tumor exhibited low MHC expression at the RNA level. More extensive studies will be required to determine whether reduced HLA expression is generally associated with primary tumors or is a specific feature of CDK12 mutated PCa.ConclusionsThese data show that analysis of CDK12 alteration, in the context of MHC expression levels, and LOH status may offer improved predictive value for outcomes in this potentially actionable genomic subgroup of PCa. In addition, these findings highlight the need to explore novel therapeutic strategies to enhance MHC expression in CDK12-defective PCa to improve immunotherapy responses.

Abstract PO5-06-09: Metastatic triple negative breast cancer has distinct tumor immune landscape

Abstract Background: Triple negative breast cancer (TNBC) is considered the most immunogenic breast cancer subtype due to higher levels of tumor infiltrating immune cells (TILS), elevated tumor mutational burden and PD-L1 expression, providing a rationale for immunotherapy. Here we aimed to investigate the differences in the immune microenvironment of primary vs. metastatic sites in TNBC vs. non-TNBC and their impact on survival. Methods: Comprehensive immune profiling, including PD-L1 IHC and the expression of 395 immune genes, was performed on 147 real-world FFPE breast cancer samples (32 primary, 115 metastatic). 37 samples were, by definition, triple negative for ER, PR, and HER2 overexpression. PD-L1 (CPS positive ≥1% and CPS positive ≥10%) was determined using immunohistochemistry (22C3). mRNA expression signatures of tumor inflammation (TIGS, weak/moderate/strong) and cell proliferation (CP, poor/moderate/high) were determined by RNA-sequencing. Demographic and clinicopathologic variables were collected. Statistical comparisons of biomarkers between groups were calculated using the Wilcoxon Rank-Sum test for continuous variables and the proportions test for categorical variables (p≤0.05 for significance). Survival differences were quantified by Cox proportional hazards analysis (p≤0.05 for significance). Results: Triple negative status was associated with current use of alcohol [p=0.03]. Black patients were also more likely to have TNBC, constituting 24% of TNBC patients while only making up 14% of the total cohort [p=0.04]. Conversely, white patients were less likely to have TNBC, constituting 73% of TNBC patients while making up 84% of the total cohort [p=0.04]. Comparing TNBC and non-TNBC cases, TNBC cases were observed to have significantly higher TIGS [p=0.014] and PD-L1 expression [p=4.4 × 10−7]. Additionally, TNBC cases trended towards exhibiting greater cell proliferation [p=0.069]. These differences were found to be driven primarily by metastatic tumors, among which TNBC tumors showed significantly higher TIGS [p=0.015], cell proliferation [p=0.021], and PD-L1 expression [p=2.9 × 10−7] than non-TNBC tumors, while none of these significant associations were observed among primary tumors. Triple negative status was significantly associated with PD-L1 expression (assessed by IHC), with a majority of PD-L1 positive cases determined to be triple negative [70% of CPS≥1% cases, p=5 × 10-5; 59% of CPS≥10% cases, p=6 × 10−4]. Several immune-related genes and immune checkpoint molecules were over-expressed in triple negative breast cancer, including CD8, GZMB, PRF1, CCL5, IFNG, TIGIT and CXCL10. A number of genes were also significantly overexpressed in metastatic tumors: KRT5 [p=0.02], TFRC [p=0.04], ABCF1 [p=0.04], FCRLA [p=0.05], LAMP3 [p=0.05], and underexpressed in metastatic tumors: ENTPD1 [p=0.05], IGSF6 [p=0.03], and ITGA1 [p=0.03]. Conclusions: Our comprehensive biomarker analyses showed that metastatic TNBC has a more inflamed tumor microenvironment and higher checkpoint target expression compared to non-TNBC. Further analysis of immune expression by site-specific metastases and correlation with immunotherapy outcomes is warranted to guide clinicians in selection of the ideal metastatic site to biopsy for therapeutic decision making. However, in a collective TNBC context, these data support the use of immunotherapy in metastatic TNBC. Citation Format: Robert Seager, Heidi Ko, Sarabjot Pabla, Maria-Fernanda Senosain, Erik Van Roey, Shuang Gao, Kyle Strickland, Rebecca Previs, Mary Nesline, Stephanie Hastings, Shengle Zhang, Jeffrey Conroy, Taylor Jensen, Marcia Eisenberg, Brian Caveney, Eric Severson, Shakti Ramkissoon, Shipra Gandhi. Metastatic triple negative breast cancer has distinct tumor immune landscape [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-09.

Stat5b/Ezh2 axis governs high PD-L1 expressing tolerogenic dendritic cell subset in autoimmune diabetes

Dendritic cells (DCs) are specialized antigen-presenting cells that play an important role in inducing and maintaining immune tolerance. The altered distribution and/or function of DCs contributes to defective tolerance in autoimmune diseases such as type 1 diabetes (T1D). In human T1D and in NOD mouse models, DCs share some defects and are often described as less tolerogenic and excessively immunogenic. In the NOD mouse model, the autoimmune response is associated with a defect in the Stat5b signaling pathway. We have reported that expressing a constitutively active form of Stat5b in DCs of transgenic NOD mice (NOD.Stat5b-CA), re-established their tolerogenic function, restored autoimmune tolerance and conferred protection from diabetes. However, the role and molecular mechanisms of Stat5b signaling in regulating splenic conventional DCs tolerogenic signature remained unclear. In this study, we reported that, compared to immunogenic splenic DCs of NOD, splenic DCs of NOD.Stat5b-CA mice exhibited a tolerogenic profile marked by elevated PD-L1 and PD-L2 expression, reduced pro-inflammatory cytokine production, increased frequency of the cDC2 subset and decreased frequency of the cDC1 subset. This tolerogenic profile was associated with increased Ezh2 and IRF4 but decreased IRF8 expression. We also found an upregulation of PD-L1 in the cDC1 subset and high PD-L1 and PD-L2 expression in cDC2 of NOD.Stat5b-CA mice. Mechanistically, we demonstrated that Ezh2 plays an important role in the maintenance of high PD-L1 expression in cDC1 and cDC2 subsets and that Ezh2 inhibition resulted in PD-L1 but not PD-L2 downregulation which was more drastic in the cDC2 subset. Additionally, Ezh2 inhibition severely reduced the cDC2 subset and increased the cDC1 subset and Stat5b-CA.DC pro-inflammatory cytokine production. Together our data suggest that the Stat5b-Ezh2 axis is critical for the maintenance of tolerogenic high PD-L1-expressing cDC2 and autoimmune tolerance in NOD.Stat5b-CA mice.

The clinical association of programmed death-1/PD-L1 axis, myeloid derived suppressor cells subsets and regulatory T cells in peripheral blood of stable COPD patients.

Myeloid-derived suppressor cells (MDSCs) have crucial immunosuppressive role in T cell dysfunction in various disease processes. However, the role of MDSCs and their impact on Tregs in COPD have not been fully understood. The aim of the present study is to investigate the immunomodulatory role of MDSCs and their potential impact on the expansion and function of Tregs in COPD patients. Peripheral blood samples were collected to analyze circulating MDSCs, Tregs, PD-1/PD-L1 expression to assess the immunomodulatory role of MDSC and their potential impact on the expansion and function of Treg in COPD. A total of 54 COPD patients and 24 healthy individuals were enrolled in our study. Flow cytometric analyses were performed to identify granulocytic MDSCs (G-MDSCs), monocytic MDSCs (M-MDSCs), Tregs, and the expression of PD-1/PD-L1(L2) on MDSCs and Tregs in peripheral blood. Our results revealed a significantly higher percentage of G-MDSCs and M-MDSCs (p < 0.001) in COPD patients compared to the healthy controls. Additionally, a significantly higher proportion of peripheral blood Tregs was observed in COPD patients. Furthermore, an increased expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on Tregs (p < 0.01) was detected in COPD patients. The expression of PD-1 on CD4+ Tcells and Tregs, but not CD8+Tcells, was found to be increased in patients with COPD compared to controls. Furthermore, an elevated expression of PD-L1 on M-MDSCs (p < 0.01) was also observed in COPD patients. A positive correlation was observed between the accumulation of M-MDSCs and Tregs in COPD patients. Additionally, the percentage of circulating M-MDSCs is positively associated with the level of PD-1 (r = 0.51, p < 0.0001) and CTLA-4 (r = 0.42, p = 0.0014) on Tregs in COPD. The recruitment of MDSCs, accumulation of Tregs, and up-regulation of CTLA-4 on Treg in COPD, accompanied by an increased level of PD-1/PD-L1, suggest PD-1/PD-L1 axis may be potentially involved in MDSCs-induced the expansion and activation of Treg at least partially in COPD.

Abstract 7488: Detection of PD-L1 mRNA expression in circulating tumor cells (CTCs) from patients with metastatic NSCLC, HNSCC and melanoma using a novel highly sensitive commercially available CE-IVD kit

Abstract Background: We have previously reported the development of an RT-qPCR assay for PD-L1 expression in CTCs (Strati et al, Ann Oncol, 2017). This assay is now commercially available as Oncolipsy PD-L1 kit (CE-IVD, Pharmassist, Greece) and we report here it’s application in size-based enriched CTCs from patients with metastatic NSCLC, HNSCC and melanoma, before and during PD-1/PD-L1 blockade therapies. Patients and Methods: The Oncolipsy PD-L1 kit was analytically validated using Mini-Genes as positive controls (IDT, USA). The analytical sensitivity was tested by spiking known numbers of H1975 cells in peripheral blood (PB) samples from healthy donors (HD). In total 95 patients were enrolled in the study: 69 patients with metastatic NSCLC, 19 patients with metastatic HNSCC and 7 patients with metastatic melanoma. 40 PB samples from HD were used as a control group. 10 ml of PB was collected at baseline (V0), two months after immunotherapy (V1) and at the time of disease progression (PD) or 18 months after initiation of immunotherapy (V2). PD-L1 and B2M transcripts were quantified in cDNAs obtained from CTCs isolated with the size-based PARSORTIX (ANGLE, UK) device. A sample was considered as CTC-positive when cDNAs were positive for CK-8, CK-18 or CK-19 expression. Results: Spiking experiments have shown that 10 H1975cells/10ml PB can be detected using the kit. In CTC-positive samples, overexpression of PD-L1 was detected in 15/58(25.8%) patients at V0, in 6/34(17.6%) patients at V1 and in 3/11(27.3%) at V2. Patients with elevated expression of PD-L1 at V1 in respect to V0 and detectable CTCs had longer PFS (18.5mo vs. 13.2mo, p=0.033) and longer OS (24.8mo vs. 12.8mo, p = 0.071) after initiation of immunotherapy. Patients with elevated PD-L1 at V1 compared to V0 benefited from immunotherapy, with a longer DFS (17.8mo vs. 13.4mo) and OS (29.4mo vs. 26.9mo). Conclusions: The commercially available Oncolipsy PD-L1 kit can be used for the quantification of PD-L1 transcripts in CTCs. Expression of PD-L1 in size-based enriched CTCs can predict response to PD-1/PD-L1 blockade therapy. Acknowledgements: This study has been financially supported by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T1RCI-02935). Trial registration: ClinicalTrials.gov Identifier: NCT04490564 Citation Format: Areti Strati, Martha Zavridou, Stavroula Smilkou, Victoria Tserpeli, Eleni Tzanikou, Dimitra Stergiopoulou, Eleni Efthimiadou, Emily Tsaroucha, Amanda Psyrri, Aggeliki Sfika, Evangelos Bournakis, Ioanna Balgkouranidou, Stylianos Kakolyris, Ioannis Boukovinas, Christos Papadimitriou, Loukas Kaklamanis, Ioanna Koukli, Evi Lianidou. Detection of PD-L1 mRNA expression in circulating tumor cells (CTCs) from patients with metastatic NSCLC, HNSCC and melanoma using a novel highly sensitive commercially available CE-IVD kit [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 7488.

Abstract 5667: Intratumoral heterogeneity in gastric cancer related with survival and tumor microenvironment

Abstract Gastric cancer (GC), often diagnosed at an advanced stage, poses a significant therapeutic challenge due to its resistance to treatment. The understanding of intratumoral heterogeneity, crucial for tumor cell survival, remains limited in GC. We conducted a comprehensive analysis of three patient groups (intraperitoneal metastasis [IM], hematogenous metastasis [HM], and both metastasis [HIM]) undergoing immunotherapy (IO) and chemotherapy. Utilizing single-cell RNA sequencing and TCR sequencing, we examined 11 adjacent normal, 10 HM, 8 IM, and 5 HIM primary GC samples. In epithelial cells, three distinct tumor clusters (Tumor_1, Tumor_2, and Tumor_3) were identified. Trajectory analysis revealed the differentiation of chief cells into Tumor_1, subsequently dividing into more malignant cell types, Tumor_2 and Tumor_3. Tumor_2 was enriched in IM, while Tumor_1 was enriched in HM and HIM. Patients grouped by Tumor_1, 2, 3 levels showed varying survival outcomes. Surprisingly, high Tumor_2 levels correlated with increased survival, attributed to elevated HLA and PDL1 expression, promoting immune cell recruitment and robust response to IO therapy. Conversely, Tumor_3 high patients exhibited worse survival, with enrichment in metabolic pathways associated with glycolysis and hypoxia, linking to metastasis. We observed the proportion of T cells that increased presence of CD8 effector cells among patients exhibiting high levels of Tumor_1 and Tumor_2. Through TCR analysis, we found heightened clonality in both CD8 effector cells and exhausted T cells in patients with elevated Tumor_1 and Tumor_2 levels. This suggests that the activated CD8 effector T cells in these patients may enhance their ability to effectively target and attack cancer cells. In summary, this study provides a comprehensive picture of intratumoral heterogeneity in GC can impact the survival and tumor microenvironment. The unexpected high survival in Tumor_2 patients emphasizes the potential of targeting specific pathways for improved therapeutic outcomes. Additionally, the observed T cell dynamics and clonality in Tumor_1 and 2 underscore the importance of understanding the immune microenvironment for effective cancer attack. Citation Format: Do-eon Gu, Woo Sun Kwon, Sun Young Rha, Woong-Yang Park. Intratumoral heterogeneity in gastric cancer related with survival and 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 5667.

Integrating molecular subtype and CD8+ T cells infiltration to predict treatment response and survival in muscle-invasive bladder cancer

BackgroundLuminal and Basal are the primary intrinsic subtypes of muscle-invasive bladder cancer (MIBC). The presence of CD8+ T cells infiltration holds significant immunological relevance, potentially influencing the efficacy of antitumor responses. This study aims to synergize the influence of molecular subtypes and CD8+ T cells infiltration in MIBC.MethodsThis study included 889 patients with MIBC from Zhongshan Hospital, The Cancer Genome Atlas, IMvigor210 and NCT03179943 cohorts. We classified the patients into four distinct groups, based on the interplay of molecular subtypes and CD8+ T cells and probed into the clinical implications of these subgroups in MIBC.ResultsAmong patients with Luminal-CD8+Thigh tumors, the confluence of elevated tumor mutational burden and PD-L1 expression correlated with a heightened potential for positive responses to immunotherapy. In contrast, patients featured by Luminal-CD8+Tlow displayed a proclivity for deriving clinical advantages from innovative targeted interventions. The Basal-CD8+Tlow subgroup exhibited the least favorable three-year overall survival outcome, whereas their Basal-CD8+Thigh counterparts exhibited a heightened responsiveness to chemotherapy.ConclusionsWe emphasized the significant role of immune-molecular subtypes in shaping therapeutic approaches for MIBC. This insight establishes a foundation to refine the process of selecting subtype-specific treatments, thereby advancing personalized interventions for patients.

Clinical significance of blocking novel immune checkpoint B7-H4 in urothelial carcinoma of bladder as a potential therapeutic target.

Urothelial Carcinoma of Bladder is complex disease with high mortality and recurrence rates. Current standard regimes have exhibited anti-tumor activity but still, a proportion of patients are non-responsive or in-eligible to receive such treatments. Immune checkpoints have emerged as potential class of therapeutics to be tested in UCB patients. Clinical trials targeting PD-1/PD-L1 axis have been tested in UCB but still a proportion of patients are non-responsive to it which stresses upon identifying new targets. New immune checkpoint B7-H4 has been shown to negatively regulate T cell activity in cancer and is a poor prognostic factor in various solid tumors. In this study we assessed the novel immune checkpoint B7-H4 status in UCB patients. We observed elevated expression of B7-H4 and PD-L1 on CD8+ T cells in circulation of UCB patients. Relative mRNA expression and immunohistochemistry displayed upregulation in bladder tumor tissue. Increased expression of B7-H4 along with PD-L1 in periphery and tumor of UCB patients highlights involvement of B7-H4 in disease progression. Combinatorial blocking of B7-H4 and PD-L1 enhanced IFN-γ and granzyme B in CD8+T cells functional T cell immune response in UCB patients. Also, B7-H4 was significantly associated with clinico-pathological parameters. Our findings highlight B7-H4 as potential therapeutic target for treatment of UCB patients in future after further validation.

Tumor immune microenvironment of KRAS G12C mutated pancreatic cancer as compared to other mutations and non-cancer pancreata.

704 Background: KRAS G12C mutation occurs in approximately 1% of pancreatic ductal adenocarcinoma (PDA). KRAS G12C inhibitors have a reported response rate of 21-33% in patients with previously treated advanced PDA. KRAS is a known modulator of the tumor immune microenvironment (TME) and a greater understanding of the TME in KRAS G12C as compared to other mutations is crucial for developing rational therapeutic combination strategies in PDA. Methods: We performed multiplex fluorescent immunohistochemistry (mfIHC) on tissue obtained from patients with histologically confirmed PDA (KRAS G12C n=8; other mutations n=108) and normal pancreas (n=38). Serial staining was performed using antibodies against CD3, CD8, CD163, FoxP3 and PanCK for simple and complex phenotyping as well as spatial analyses in the TME. After tyramide based signal amplification, the slides were imaged at 20x magnification using the Mantra Quantitative Pathology workstation. Images were analyzed using inForm Cell Analysis software (Akoya Biosciences). All statistical analyses were performed using JMP Pro 13.2.0. Differences in phenotype, distances, and engagement were evaluated by 2-tailed Student’s t test or ANOVA. Results: Patients with KRAS G12C mutation had a median age of 70 (45-73) years of which majority (n=5, 62.5%) were men with advanced stage at diagnosis (n=6; 75%). The median overall survival is not yet reached (n=5, alive) after a median follow-up time of 14.4 months through reverse censoring methodology. When examining cellular infiltration, CD8+ cytotoxic T lymphocyte cells were significantly more abundant in the G12C TME compared to the cancer and non-cancer controls (p &lt;0.01). Additionally, the relative frequency of CD4+ and Tregs (CD3+/CD8-/FoxP3+) were also significantly higher in G12C (p&lt;0.01). The percentage of CD163+ antigen presenting cells (APCs) out of all cells was significantly higher in G12C (p&lt;0.01) while the percentage of non-functional APCs (PDL1+/CD163+) of all APCs was significantly higher in G12C than cancer and non-cancer controls (p=0.03). In comparison, the PDL1+ epithelial cells were much lower in G12C TME (p&lt;0.01). The cellular engagement and interaction data from the spatial analysis in the TME will be presented at the meeting. Conclusions: We identified increased infiltration of cytotoxic T lymphocytes and APCs in PDA patients with KRAS G12C mutations relative to other mutations and non-cancerous pancreata. Furthermore, while all PDAs had elevated PD-L1 expression on APCs, KRAS G12C tumors had minimal expression on epithelial cells suggesting a mutation specific impact and a potential role of immune checkpoint inhibitors in combination with G12C inhibitors.

Tumor-associated macrophages and PD-L1 in prostate cancer: a possible key to unlocking immunotherapy efficacy.

Prostate cancer (PCa) is often considered as a "cold" tumor with low responsiveness to immunotherapy. Recent evidence suggests the activation of specific immune cells, such as tumor-associated macrophages (TAMs), could potentially influence the efficacy of immunotherapy in PCa. However, the relationship between TAMs and PD-L1, a significant regulator in immunotherapy, within PCa remains unexplored. In this study, we assessed TAM infiltration and PD-L1 expression levels in a local cohort of 95 PCa tissue samples and two publicly available PCa datasets. We employed a combination of bioinformatics and experimental techniques, including gene set enrichment analysis, CIBERSORTx, tissue microarray, immunohistochemistry staining, and analysis of single-cell sequencing datasets, to provide a comprehensive understanding of the association between PD-L1 and TAMs in the PCa microenvironment. The study showed that CD68+ TAMs and CD163+ TAMs (M2-TAMs) were more abundant in the tumor microenvironment than in non-cancerous surrounding tissues. The infiltration of CD163+ TAMs was significantly associated with the Gleason score and risk stratification of PCa. Importantly, elevated PD-L1 expression correlated significantly with high infiltration of CD163+ TAMs. Furthermore, patients displaying high levels of CD163+ TAMs and PD-L1 expression exhibited shorter times to biochemical recurrence-free survival. Our study suggests that CD163+ TAMs are closely associated with PD-L1 expression and can act as a valuable prognostic indicator for PCa. The high infiltration of M2-TAMs, coupled with the overexpression of PD-L1, may contribute to immune escape mechanisms in PCa, thereby influencing disease prognosis.

PD-1 signaling in neonates restrains CD8+ T cell function and protects against respiratory viral immunopathology

Respiratory viral infections, including human metapneumovirus (HMPV), remain a leading cause of morbidity and mortality in neonates and infants. However, the mechanisms behind the increased sensitivity to those respiratory viral infections in neonates are poorly understood. Neonates, unlike adults, have several anti-inflammatory mechanisms in the lung, including elevated baseline expression of programmed death ligand 1 (PD-L1), a ligand for the inhibitory receptor programmed cell death protein 1 (PD-1). We thus hypothesized that neonates would rely on PD-1:PD-L1 signaling to restrain antiviral CD8 responses. To test this, we developed a neonatal primary HMPV infection model using wild-type C57BL/6 (B6) and Pdcd1-/- (lacking PD-1) mice. HMPV-infected neonatal mice had increased PD-L1/PD-L2 co-expression on innate immune cells but a similar number of antigen-specific CD8+ T cells and upregulation of PD-1 to that of adult B6 mice. Neonatal CD8+ T cells had reduced interferon-gamma (IFN-γ), granzyme B, and interleukin-2 production compared with B6 adults. Pdcd1-/- neonatal CD8+ T cells had markedly increased production of IFN-γ and granzyme B compared with B6 neonates. Pdcd1-/- neonates had increased acute pathology with HMPV or influenza. Pdcd1-/- neonates infected with HMPV had long-term changes in pulmonary physiology with evidence of immunopathology and a persistent CD8+ T-cell response with increased granzyme B production. Using single-cell ribonucleic acid sequencing from a child lacking PD-1 signaling, a similar activated CD8+ T-cell signature with increased granzyme B expression was observed. These data indicate that PD-1 signaling critically limits CD8+ T-cell effector functions and prevents immunopathology in response to neonatal respiratory viral infections.

Immune checkpoint inhibitors targeting PD-1/PD-L1 in the treatment of human lymphomas.

Non-Hodgkin lymphomas (NHLs) encompass a diverse group of malignancies arising from B cells, T cells, and natural killer (NK) cells at various stages of differentiation. Conversely, classical Hodgkin lymphomas (cHLs) primarily feature Reed-Sternberg cells (RSCs) amid a background of reactive immune cells. Immunomodulatory pathways, notably the PD-1/PD-L1 axis, play pivotal roles in tumor immune evasion across both NHLs and cHLs. Elevated expression of PD-1 and PD-L1 is observed in a spectrum of lymphomas, influencing prognosis and treatment response. Therapeutically, immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 have revolutionized lymphoma management, particularly in relapsed/refractory cases. Nivolumab and pembrolizumab, among others, have demonstrated efficacy in various B-cell lymphomas, with promising outcomes in cHL. Combination strategies incorporating ICIs with conventional chemotherapy or targeted agents show enhanced efficacy and are being explored extensively. In this review we discuss the most important features of the tumor microenvironment of NHLs and cHLs, address the therapeutic approaches with ICIs and try to outline future perspectives.

CD58 Alterations Govern Antitumor Immune Responses by Inducing PDL1 and IDO in Diffuse Large B-Cell Lymphoma.

Recurrent abnormalities in immune surveillance-related genes affect the progression of diffuse large B-cell lymphoma (DLBCL) and modulate the response to therapeutic interventions. CD58 interacts with the CD2 receptor on T cells and NK cells and is recurrently mutated and deleted in DLBCL, suggesting that it may play a role in regulating antitumor immunity. In this study, we comprehensively analyzed the genomic characteristics of CD58 through targeted next-generation sequencing, RNA sequencing (RNA-seq), whole-exome sequencing, and single-cell RNA-seq in patients with newly diagnosed DLBCL. The CD58 mutation rate was 9.1%, and the copy number loss rate was 44.7% among all enrolled patients with DLBCL. Notably, CD58 genetic alterations, along with low CD58 expression, significantly correlated with reduced rates of response to R-CHOP therapy and inferior progression-free survival and overall survival. Single-cell RNA-seq revealed that CD58 expression in tumor cells was negatively correlated with CD8+ T-cell exhaustion/dysfunction status. Insufficient T-cell activation resulting from CD58 alterations could not be attributed solely to CD2 signaling. CD58 inhibited the activity of the JAK2/STAT1 pathway by activating the LYN/CD22/SH2 domain-containing phosphatase 1 (SHP1) axis, thereby limiting PDL1 and IDO expression. Elevated PDL1 and IDO expression in CD58-deficient DLBCL cells led to immune evasion and tumor-intrinsic resistance to chimeric antigen receptor T-cell therapy. Direct activation of CD58-CD2 costimulatory signaling in combination with anti-PDL1 blockade or IDO inhibitor sensitized CD58-deficient DLBCL to chimeric antigen receptor T-cell therapy. Collectively, this work identified the multiple roles of CD58 in regulating antitumor immune responses in DLBCL. Significance: Loss of CD58 mediates immune evasion and therapy resistance in diffuse large B-cell lymphoma by upregulating PDL1 and IDO through LYN/CD22/SHP1 signaling, providing potential targets and therapeutic strategies to improve patient treatment.

P53 deficient breast cancer cells reprogram preadipocytes toward tumor-protective immunomodulatory cells.

The TP53 gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients.