Percentage Of PD-L1 Research Articles

Dendrobine Suppresses Tumor Growth by Regulating the PD-1/PD-L1 Checkpoint Pathway in Lung Cancer

Background: Dendrobine is a bioactive alkaloid isolated from Dendrobium nobile. Studies have evaluated the anti-tumor effect of dendrobine in cancers, including lung cancer. However, the mechanism of dendrobine inhibiting tumors requires further study. Methods: Bioinformatics was performed to screen the potential targets of dendrobine. The in-tersection of dendrobine and lung cancer targets was performed for KEGG analysis. CCK-8 was used to detect cell viability after dendrobine treatment. A xenograft mouse model was es-tablished to explore the effect of dendrobine on lung cancer. The percentages of PD-L1+, CD4+, CD8+, CD11b+, CD25+FOXP3+ cells, the expression of Ki-67 and caspase-3, the ex-pression of pathway-related proteins, the levels of IL-2, IFN-γ, and TGF-β, and the changes of indicators of liver and renal function were measured. Results: Dendrobine regulated the PD1/PD-L1 checkpoint signaling pathway and affected the occurrence and development of lung cancer. Dendrobine decreased the cell viability of lung cancer. Dendrobine and anti-PD-L1 decreased tumor growth, increased caspase-3 expression, and reduced Ki-67 expression in tumor tissues. Dendrobine and anti-PD-L1 suppressed pro-tein expression of PD-L1, p-JAK1/JAK1, and p-JAK2/JAK2 in tumor tissues. Greatly, den-drobine and anti-PD-L1 decreased the percentages of PD-L1+, CD11b+, and CD25+FOXP3+ cells, increased the percentages of CD4+ and CD8+cells, and enhanced the levels of IL-2, IFN-γ, and TGF-β in tumor tissues. Dendrobine demonstrated no hepatorenal toxicity to the tumor mice. The combination of dendrobine and anti-PD-L1 greatly strengthened the effects of dendrobine on tumors. Conclusion: Dendrobine inhibited tumor immune escape by suppressing the PD-1/PD-L1 checkpoint pathway, thus restricting tumor growth of lung cancer. conclusion: Dendrobiine inhibited tumor immune escape by suppressing PD-1/PD-L1 checkpoint pathway, thus restricting tumor growth of lung cancer. other: N/A

Platinum-Based Chemotherapy Induces Opposing Effects on Immunotherapy Response-Related Spatial and Stromal Biomarkers in the Bladder Cancer Microenvironment.

Platinum-based chemotherapy and immune checkpoint inhibitors are key components of systemic treatment for muscle-invasive and advanced urothelial cancer. The ideal integration of these two treatment modalities remains unclear as clinical trials have led to inconsistent results. Modulation of the tumor-immune microenvironment by chemotherapy is poorly characterized. We aimed to investigate this modulation, focusing on potential clinical implications for immune checkpoint inhibitor response. We assessed immune cell densities, spatial relations, and tumor/stromal components from 116 patients with urothelial bladder cancer (paired data for 95 patients) before and after platinum-based chemotherapy. Several published biomarkers for immunotherapy response changed upon chemotherapy treatment. The intratumoral CD8+ T-cell percentage increased after treatment and was associated with increased TNFα-via-NF-κB signaling. The percentage of PDL1+ immune cells was higher after chemotherapy. An increase in chemo-induced changes that potentially inhibit an antitumor immune response was also observed, including increased fibroblast-based TGFβ signaling and distances from immune cells to the nearest cancer cell. The latter two parameters correlated significantly in posttreatment samples, suggesting that TGFβ signaling in fibroblasts may play a role in spatially separating immune cells from cancer cells. We examined specific chemotherapy regimens and found that treatment with methotrexate, vinblastine, doxorubicin, and cisplatin was associated with an increase in the macrophage cell percentage. Gemcitabine-containing chemotherapy was associated with upregulation of fibroblast TGFβ signaling. The opposing effects of platinum-based chemotherapy on the immune cell composition and stromal context of the tumor-immune microenvironment may explain the inconsistent results of clinical trials investigating chemotherapy and immune checkpoint inhibitor combinations in bladder cancer.

Potential Role of Circulating PD-L1+ Leukocytes as a Predictor of Response to Anti-PD-(L)1 Therapy in NSCLC Patients.

PD-(L)1 inhibitors are part of the treatment strategy for non-small cell lung cancer (NSCLC) although its efficacy is limited to certain patients. Our study aimed to identify patients who might benefit from anti-PD-(L)1 inhibitors by analyzing the PD-L1 expression on circulating leukocytes and its evolution during treatment. One hundred thirteen NSCLC patients, according to their radiological response after 10-12 weeks of treatment, were classified into responders, stable, and progressive disease. Percentages of circulating PD-L1+ leukocytes, PD-L1+ platelets (PLTs), and leukocyte-PLT complexes were assessed using flow cytometry, and plasma concentrations of soluble immunomodulatory factors were quantified by ELISA. Responders exhibited significantly higher pre-treatment percentages of PD-L1+ neutrophils, PD-L1+ CD14+ cells, and PD-L1+ PLTs than progressors. The percentages of these populations decreased in responders post-treatment, contrasting with stables and progressors. PLTs notably contributed to PD-L1 expression in CD14+ cells and neutrophils. Plasma cytokine analysis revealed baseline differences only in IL-17 concentration among groups, whereas network analyses highlighted distinct association patterns between plasma molecules and PD-L1+ leukocytes after 10-12 weeks of treatment. Our findings suggest that pre-treatment assessment of circulating PD-L1+ neutrophils, PD-L1+ CD14+ cells, and PD-L1+ PLTs may be helpful in identifying NSCLC patients who are potential candidates for anti-PD-(L)1 therapy.

First-line immune checkpoint inhibitors in low programmed death-ligand 1-expressing population.

Introduction: Inhibitors of programmed cell death 1 (PD1) and its ligand (PDL1) have exhibited favorable long-term survival in many types of advanced-stage cancer and current approvals have to date been granted in certain tumour types irrespective of PD-L1 status. Methods: We extracted the following information: study sample size, trial period, cancer types, intervention of treatment, type of PD-L1 antibody, immunohistochemistry (IHC) scoring method, number and percentage of PD-L1 < 1% population, and median follow- up time. PD-L1 expression was defined as percentage of number of PD-L1-stained tumor cells (TPS), area of tumor infiltrated by PD-L1-stained immune cells (IPS), number of PD-L1-stained cells (tumor cells, lymphocytes and macrophages; CPS). Different trials used distinct method to define low PD-L1 expression. The risk of bias of the included trials was assessed by using the Cochrane risk of bias tool for RCTs. Results: Here, a total of 34 trials were included to extract individual patient data (IPD) to evaluate the survival benefit of first line PD1/PDL1 inhibitors vs. standard-of-care (SOC) in patients with PDL1 < 1%. In term of anti-PD-1/PD-L1 monotherapy, OS (HR = 0.90, 0.81-1.01) and PFS (HR = 1.11, 0.97-1.27) between PD-1/PD-L1 inhibitor group and SOC group were comparable. In term of anti-PD-1/PD-L1 combination therapy, PD-1/PD-L1 inhibitor group exhibited longer OS (median 19.5 months vs. 16.3 months; HR = 0.83, 0.79-0.88, p < 0.001) and PFS than those of SOC group (median 8.11 months vs. 6.96 months; HR = 0.82, 0.77-0.87, p < 0.001).Subgroup analysis showed that survival benefit was mainly observed in non-small cell lung cancer (NSCLC) (HROS = 0.74; HRPFS = 0.69; p < 0.001), small-cell lung cancer (SCLC) (HROS = 0.58, p < 0.001; HRPFS = 0.55, p = 0.030), esophageal squamous cell carcinoma (ESCC) (HROS = 0.62, p = 0.005; HRPFS = 0.79, p < 0.001), melanoma (HROS = 0.53, p < 0.001) and nasopharyngeal carcinoma (NPC) (HRPFS = 0.35, p = 0.013). Conclusion: Anti-PD-1/PD-L1 combinational therapy rather than monotherapy exhibit survival benefit in the low PD-L1 population in the first-line setting, and the survival benefit was mainly observed in specific tumor types.

Exosomes released from PD-L1+ tumor associated macrophages promote peritoneal metastasis of epithelial ovarian cancer by up-regulating T cell lipid metabolism

Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneum, and the prognosis of patients is poor. In the peritoneum of patients with EOC, TAMs (tumor associated macrophages) regulate the imbalance of T cell ratio and promote the progression and metastasis of EOC. However, the mechanism of peritoneal metastasis in EOC patients remains unclear. Here, we confirmed that the percentages of PD-L1+ TAMs in EOC tissues increased significantly, and TAMs-derived PD-L1+ exosomes affected the transcription factor PPARα to up-regulate the expression of CPT1A in CD8+ T cells, promote fatty acid oxidation, and increase reactive oxygen species to cause cell damage. The apoptosis of CD8+ T cells was increased, and the expressions of their exhaustion markers LAG3, TIM-3, and PD-1 were also up-regulated. TAMs affect T cell function through lipid metabolism, leading to peritoneal immune imbalance and promoting peritoneal metastasis of EOC. This study reveals the mechanism by which TAMs in the peritoneal microenvironment regulate T cell lipid metabolism through exosome delivery of PD-L1, and the effect of lipid metabolism on T cell function, reveals the molecular mechanism of tumor immune microenvironment affecting EOC metastasis, and further explores related pathways whether molecular blockade can be used as a means to intervene in disease progression is expected to establish a new strategy for the diagnosis and treatment of EOC.

Lacking Immunotherapy Biomarkers for Biliary Tract Cancer: A Comprehensive Systematic Literature Review and Meta-Analysis.

Immunotherapy has recently been incorporated into the spectrum of biliary tract cancer (BTC) treatment. The identification of predictive response biomarkers is essential in order to identify those patients who may benefit most from this novel treatment option. Here, we propose a systematic literature review and a meta-analysis of PD-1, PD-L1, and other immune-related biomarker expression levels in patients with BTC. Prisma guidelines were followed for this systematic review and meta-analysis. Eligible studies were searched on PubMed. Studies published between 2017 and 2022, reporting data on PD-1/PD-L1 expression and other immune-related biomarkers in patients with BTC, were considered eligible. A total of 61 eligible studies were identified. Despite the great heterogeneity between 39 studies reporting data on PD-L1 expression, we found a mean PD-L1 expression percentage (by choosing the lowest cut-off per study) of 25.6% (95% CI 21.0 to 30.3) in BTCs. The mean expression percentages of PD-L1 were 27.3%, 21.3%, and 27.4% in intrahepatic cholangiocarcinomas (iCCAs-15 studies), perihilar-distal CCAs (p/dCCAs-7 studies), and gallbladder cancer (GBC-5 studies), respectively. Furthermore, 4.6% (95% CI 2.38 to 6.97) and 2.5% (95% CI 1.75 to 3.34) of BTCs could be classified as TMB-H and MSI/MMRd tumors, respectively. From our analysis, PD-L1 expression was found to occur approximately in 26% of BTC patients, with minimal differences based on anatomical location. TMB-H and MSI molecular phenotypes occurred less frequently. We still lack a reliable biomarker, especially in patients with mismatch-proficient tumors, and we must need to make an effort to conceive new prospective biomarker discovery studies.

Association between the Immunophenotype of Peripheral Blood from mCRPC Patients and the Outcomes of Radium-223 Treatment

(1) Background: Prostate cancer is the second most common cancer in men, with androgen suppression as the standard treatment. Despite initially responding to castration, most metastatic prostate cancer patients eventually experience progression. In these cases, Radium-223 is the chosen treatment. We hypothesized that the immunophenotype of circulating leukocytes conditions the response to Radium-223 treatment. (2) Material and Methods: In this prospective study, we collected peripheral blood from twelve mCRPC patients and nine healthy donors before (baseline) and during treatment with Radium-223. Immunophenotyping and the percentages of leukocyte–platelet complexes were determined by flow cytometry. The increments or decrements of leukocyte subsets between the baseline and the second Radium-223 injection were also calculated. (3) Results: At baseline, the mCRPC patients had a lower percentages of CD4+ T cells and B cells and higher percentages of NK and neutrophils than the HDs. In addition, they had more OX40+ CD4+ T cells, PD-L1+ CD8low cells, PD-L1+ B cells, PD-L1+ NK cells, and monocyte–platelet complexes than the HDs. Moreover, patients with slow and fast progression had different percentages of PD-L1+ CD8+ T cells. In particular, slow progression patients underwent an increment of PD-L1+ CD8+ T cells after two cycles of Radium-223. (4) Conclusions: The characterization of circulating immune cells before initiating Radium-223 treatment could become a non-invasive indicator of the response.

Molecular characterization of nectin-4 in non-small cell lung cancer subtypes.

e21119 Background: Nectin-4 (Nectin cell adhesion molecule-4) is a tumor associated antigen overexpressed in multiple solid tumor types. Studies show that over 60% of NSCLC are nectin-4 overexpressed. As enfortumab vedotin, an antibody-drug conjugate directed against nectin-4, has been FDA approved for advanced urothelial carcinoma, this holds treatment implications. Here, we investigated nectin-4 expression in NSCLC subtypes. Methods: 27,607 NSCLC tumors were tested for whole transcriptome sequencing (WTS, NovaSeq) and NextGen DNA sequencing (592 gene panel or whole exome sequencing, WES) at Caris Life Sciences (Phoenix, AZ). Tumors were analyzed overall in quartiles by nectin-4 expression (Q4-greatest, Q1-least) and further analyzed in the adenocarcinoma (AD) and squamous carcinoma (SQ) cohorts. PD-L1 expression was tested by IHC (Dako 22C3). Immune cell populations were estimated by QuanTISeq. Statistical significance was determined using Chi-square/Fisher-Exact/Mann-Whitney U tests and adjusted for multiple comparisons (q &lt; 0.05). Results: We saw significantly greater expression of nectin-4 in SQ (median TPM- 15.68) versus AD tumors (median TPM - 9.88) (p &lt; 0.05). We saw a greater prevalence of NFE2L2, STK11, ERBB2 mutations and genomic loss of heterozygosity (LOH) but a lower percentage of PD-L1+ tumors in Q4 vs Q1 tumors (all q-value &lt; 0.05, Table 1). mRNA expression of immune checkpoints PDL1, CTLA4, CD80, CD86, HAVCR2, and IDO1 was increased in Q4 tumors (1.1-1.3-fold, all q &lt; 0.05). Q4 tumors had reduced estimates of M1 macrophages, regulatory T cells (Tregs), and CD8+ T cells and increased estimates of neutrophils and NK cells. The association of nectin-4 expression with the immune landscapes of AD and SQ was similar. Q4 tumors in AD and SQ displayed increased expression of the above-mentioned immune checkpoints (q &lt; 0.05). In AD and SQ, estimates of M1 macrophages, Tregs, and CD8+ T cells were reduced, but neutrophils and NK cells were increased in Q4 tumors (q &lt; 0.05). The pattern of molecular alterations was different in AD and SQ. While KEAP1, STK11 and ERBB2 mutations were significantly enriched in AD Q4 tumors, NFE2L2 mutations were significantly enriched in SQ Q4 tumors (Table 1). Prevalence of LOH was higher and PD-L1+ tumors was lower in Q4 tumors in both AD and SQ (Table 1). Conclusions: NFE2L2 alterations in SQ and STK11, KEAP1 mutations in AD, which are associated with poor immunotherapy (IO) response, were enriched in Q4 tumors. This suggests that the immune landscape associated with high nectin-4 expression in NSCLC may be driven by the dysregulated KEAP1-NFE2L2 pathway. Targeted therapy towards nectin-4 may benefit these NSCLC subtypes that do not respond well to IO. [Table: see text]

Abstract 6363: Short-term exposure of cancer cells to ultra-high concentrations nitric oxide induces PDL-1 upregulation

Abstract Background: We have previously shown that treating mouse colon carcinoma (CT26) tumor-bearing mice with ultra-high concentrations of nitric oxide (UNO) upregulates innate and adaptive immune cells both locally and systemically. As immune-checkpoint molecules, such as the PD-L1 receptor PD-1, are expressed on immune cells, our group assessed the potential synergy of UNO and an anti-PD-1 antibody for treating tumors in vivo. Indeed, primary tumors regressed in 53% of the CT26 tumor-bearing mice treated with UNO and anti-PD-1. Moreover, these mice were primary and distant tumor-free and survived 100 days post-treatment. In the current study, we assessed PD-L1 expression levels on cancer cells in vitro following short-term exposure to UNO. Methods: CT26 cells were exposed to 25,000 - 100,000 ppm NO for 10 - 60 seconds. The percentage of PD-L1-expressing cells was assessed by flow cytometry analysis. Results: 1. Exposing CT26 cells to 100,000 ppm NO for 10 seconds upregulated PD-L1 expression. 85.1% of cells, which are still viable or at early apoptosis, expressed PD-L1 compared to 71% of untreated cells (p&amp;lt;0.0001). Exposure of CT26 cells to 25,000 ppm or 50,000 ppm NO for 10 seconds did not change the level of PD-L1 expression significantly. 2. Exposure to 100,000 ppm NO for 30 seconds further increased the percentage of cells expressing PD-L1 to 96.7% (p&amp;lt;0.0001 compared to untreated cells). For this duration, lower concentrations of NO also increased PD-L1-expression in viable early apoptotic CT26 cells to 82.8% - 92.3% when treated with 25,000 ppm or 50,000 ppm NO, respectively. 3. Finally, a 60 second exposure of CT26 cells to 25,000 ppm, 50,000 ppm, or 100,000 ppm NO resulted in the highest percentage of PD-L1 expressing cells. Approximately 95% of all viable or early apoptotic CT26 cells express PD-L1 under all NO conditions. This finding is significant compared to 71% of cells in the control arm that express PD-L1 (p&amp;lt;0.0001). Conclusion: Short-term exposure (10 - 60 seconds) to UNO upregulates PD-L1 expression on cancer cells in an NO concentration-dependent and exposure-dependent process. A 10-second exposure to 100,000 ppm NO is sufficient to significantly induce PDL-1 expression. Therefore, NO therapy can potentially be combined with anti-PD-L1 antibody treatment to improve therapeutic outcomes. Citation Format: Yana Epshtein, Matan Goldshtein, Selena Chaisson, Jedidiah Monson, Matt Johnson, Amir Avniel, Steve Lisi, Hila Confino. Short-term exposure of cancer cells to ultra-high concentrations nitric oxide induces PDL-1 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6363.

Coexpression of PD-L1/PD-1 with CXCR3/CD36 and IL-19 Increase in Extranodal Lymphoma.

Many studies have demonstrated that PD-L1/PD-1 signaling is an immune evasion mechanism in tumors. PD-L1/PD-1 coexpression with CXCR3/CD36 in peripheral lymphocytes in lymphoma still needs to be clarified. The current study investigated PD-L1/PD-1 coexpression with CXCR3/CD36 in circulating lymphocytes, serum IL-19 levels, and their correlation with clinical outcome and extranodal involvement in lymphoma. Subjects and Methods. The coexpression of PD-L1/PD-1 with CXCR3/CD36 on circulating lymphocytes was analyzed by flow cytometry in 78 lymphoma patients before and after therapy and in 50 healthy controls. The concentration levels of IL-19 in serum were assessed by an ELISA. Results. PD-L1 and PD-1 were expressed on circulating CXCR3+ and CD36+ lymphocytes in lymphoma and were significantly higher in patients with extranodal involvement than in lymphoma patients without extranodal involvement (P < 0.001). Elevated IL-19 levels were observed in lymphoma patients and increased significantly in extranodal involvement (P < 0.001). High percentages of PD-L1+CXCR3+ and PD-1+CXCR3+ lymphocytes were associated with high LDH levels, hepatomegaly, lymphedema, advanced tumor stage, and recurrence. Furthermore, patients with splenomegaly and generalized lymphadenopathy had high percentages of PD-L1+CXCR3+ lymphocytes. In addition, levels of PD-L1/PD-1 coexpression with CXCR3 and IL-19 were significantly associated with bone marrow, lung, and lymph vessel involvement. Further analysis revealed that high percentages of PD-L1+CD36+ and PD-1+CD36+ lymphocytes were associated with lung and bone marrow involvement. Patients with high levels of PD-L1/PD-1 coexpression with CXCR3 and IL-19 had inferior event-free survival (EFS) compared with that in lymphoma patients with low levels. EFS was decreased in patients with high percentages of PD-L1+CD36+ and PD-1+CD36+ lymphocytes. When using the receiver operating characteristic (ROC) curve, the superiority of IL-19 (area under the curve (AUC): 0.993) and PD-L1+CXCR3+% (AUC: 0.961) to PD-1+CXCR3+% (AUC: 0.805), PD-L1+CD36+% (AUC: 0.694), and PD-1+CD36+% (AUC 0.769) was evident in the diagnosis of extranodal involvement, identifying lymphoma patients with extranodal involvement from patients without extranodal involvement. Conclusions. Coexpression of PD-L1/PD-1 with CXCR3/CD36 in circulating lymphocytes and serum IL-19 levels contributes to poor prognosis and might be potential markers for extranodal involvement in lymphoma.

Artificial intelligence-based immunoprofiling serves as a potentially predictive biomarker of nivolumab treatment for advanced hepatocellular carcinoma.

Immune checkpoint inhibitors (ICI) have been applied in treating advanced hepatocellular carcinoma (aHCC) patients, but few patients exhibit stable and lasting responses. Moreover, identifying aHCC patients suitable for ICI treatment is still challenged. This study aimed to evaluate whether dissecting peripheral immune cell subsets by Mann-Whitney U test and artificial intelligence (AI) algorithms could serve as predictive biomarkers of nivolumab treatment for aHCC. Disease control group carried significantly increased percentages of PD-L1+ monocytes, PD-L1+ CD8 T cells, PD-L1+ CD8 NKT cells, and decreased percentages of PD-L1+ CD8 NKT cells via Mann-Whitney U test. By recursive feature elimination method, five featured subsets (CD4 NKTreg, PD-1+ CD8 T cells, PD-1+ CD8 NKT cells, PD-L1+ CD8 T cells and PD-L1+ monocytes) were selected for AI training. The featured subsets were highly overlapping with ones identified via Mann-Whitney U test. Trained AI algorithms committed valuable AUC from 0.8417 to 0.875 to significantly separate disease control group from disease progression group, and SHAP value ranking also revealed PD-L1+ monocytes and PD-L1+ CD8 T cells exclusively and significantly contributed to this discrimination. In summary, the current study demonstrated that integrally analyzing immune cell profiling with AI algorithms could serve as predictive biomarkers of ICI treatment.

Visible- and near-infrared hyperspectral imaging for the quantitative analysis of PD-L1+ cells in human lymphomas: Comparison with fluorescent multiplex immunohistochemistry

IntroductionWe analyzed the expression of PD-L1 in human lymphomas using hyperspectral imaging (HSI) compared to visual assessment (VA) and conventional digital image analysis (DIA) to strengthen further the value of HSI as a tool for the evaluation of brightfield-based immunohistochemistry (IHC). In addition, fluorescent multiplex immunohistochemistry (mIHC) was used as a second detection method to analyze the impact of a different detection method. Material and methods18 cases (6 follicular lymphomas and 12 diffuse large B-cell lymphomas) were stained for PD-L1 by IHC and for PD-L1, CD3, and CD8 by fluorescent mIHC. The percentage of positively stained cells was evaluated with VA, HSI, and DIA for IHC and VA and DIA for mIHC. Results were compared between the different methods of detection and analysis. ResultsAn overall high concordance was found between VA, HSI, and DIA in IHC (Cohens Kappa = 0.810VA/HSI, 0.710 VA/DIA, and 0.516 HSI/DIA) and for VAmIHCversus DIAmIHC (Cohens Kappa = 0.894). Comparing IHC and mIHC general agreement differed depending on the methods compared but reached at most a moderate agreement (Coheńs Kappa between 0.250 and 0.483). This is reflected by the significantly higher percentage of PD-L1+ cells found with mIHC (pFriedman = 0.014). ConclusionOur study shows a good concordance for the different analysis methods. Compared to VA and DIA, HSI proved to be a reliable tool for assessing IHC. Understanding the regulation of PD-L1 expression will further enlighten the role of PD-L1 as a biomarker. Therefore it is necessary to develop an instrument, such as HSI, which can offer a reliable and objective evaluation of PD-L1 expression.

Correlation of the expression of PD-L1 and Cyclin and Histopathological Grading in Colorectal Adenocarcinoma

One of the prognosis factors of colorectal adenocarcinoma is histopathological grading. The expression of programmed death-ligand (PD-L1) and cyclin D1 has the potential to become one of the predictive and prognostic factors for tumor cells in colorectal cancer. This study aimed to identify the relationship between the expression of PD-L1 and cyclin D1 and histopathological grading in colorectal adenocarcinoma. It is an analytic observational study with a cross-sectional approach using a sample of 34 paraffin blocks of colorectal adenocarcinoma preparations at the Anatomical Pathology Laboratory, RSUD Dr. Soetomo Surabaya for the period January 2016 to December 2020. The parameter of the assessment was the percentage of PD-L1 and cyclin D1 antibodies that were positively stained on the membrane and nucleus of tumor cells which was subjected to histopathological grading assessment based on hematoxylin-eosin (HE) staining. The data were analyzed using Spearman’s correlation. PD-L1 expression was positively correlated with histopathological grading in colorectal adenocarcinoma (p=0.006). Cyclin D1 expression did not correlate with histopathological grading in colorectal adenocarcinoma (p=0.891). Statistical analysis showed no significant correlation between PD-L1 and cyclin D1 expressions (r = 0.188; p = 0.286).

Abstract 6179: Increased circulating PD-L1 expressing CD14 high CD16 negative classical monocytes correlate with poor prognosis in cancer patients treated with PD-1 blockade therapies

Abstract Background: Immune checkpoint inhibitors (ICIs), like PD-1 and PD-L1 blockade therapies, are currently considered as one of the most significant breakthroughs in potent cancer immunotherapy. However, many patients are non-responders or develop resistance following an initial response to ICIs. ICIs have no accurate predictive biomarkers. Previously, we showed that increased expression of PD-L1 molecules on CD14+ monocytes was significantly correlated with shorter overall survival (OS) for patients with several cancer types on PD-1 blockade therapies. As monocytes can be further classified into subsets (classical, intermediate, non-classical), we herein investigated the prognostic factors in peripheral blood mononuclear cell (PBMC) subsets and assessed the clinical significance for the PD-L1-expressing subsets of each of these cells including classical, intermediate and non-classical monocyte subsets in patients with various cancer types, along with their clinical implications in PD-1 blockade therapies. Material and Methods: We evaluated 44 patients (20 with gastric cancer, 17 with non-small cell lung carcinoma, and 7 with esophageal cancer) undergoing PD-L1 blockade therapy (pembrolizumab or nivolumab) for PD-L1 expression levels in classical (CD14high, CD16-), intermediate (CD14high, CD16+), and non-classical (CD14low, CD16+) monocytes measured via flow cytometry before ICI treatment. The percentages of PD-L1+ cells in respective monocyte subsets were compared with respect to different clinicopathological conditions and the association with survival time was assessed. Results: The number of PD-1 positive cells in CD14+ monocytes was very low (almost &amp;lt;1%). Also, the monocyte subset expressing PD-L1 had a high percentage of classical monocytes, whereas non-classical monocytes expressed less PD-L1. Higher levels of classical monocytes were correlated with shorter OS (P = 0.044), whereas higher levels of non-classical monocytes were correlated with longer OS (P = 0.027). Focusing on non-small cell lung cancer population (N = 17), higher levels of PD-L1-expressing CD14+ monocytes were statistically correlated with shorter OS (P = 0.025). Furthermore, analysis of a subset of monocytes showed that the higher PD-L1-expressing classical monocytes correlated with the shorter OS, suggesting that they predict poor prognosis (P = 0.0095). Conclusion: Circulating PD-L1-expressing classical monocytes in the peripheral bloods were associated with poor prognosis in patients treated with PD-1 blockade therapies. Among the monocyte subsets, classical monocyte, especially PD-L1-expressing classical monocytes can be a potential biomarker for prognosis in these patients. Citation Format: Ryotaro Ohkuma, Katsuaki Ieguchi, Makoto Watanabe, Tsubasa Goshima, Rie Onoue, Kazuyuki Hamada, Yutaro Kubota, Atsushi Horiike, Toshiaki Tsurui, Risako Suzuki, Nana Iriguchi, Tomoyuki Ishiguro, Yuya Hirasawa, Hirotsugu Ariizumi, Kiyoshi Yoshimura, Mayumi Tsuji, Yuji Kiuchi, Shinichi Kobayashi, Takuya Tsunoda, Satoshi Wada. Increased circulating PD-L1 expressing CD14 high CD16 negative classical monocytes correlate with poor prognosis in cancer patients treated with PD-1 blockade therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6179.

Abstract 597: BLEACH&amp;STAIN 15 marker multiplexed imaging in 3098 human carcinomas revealed six major PD-L1 driven immune phenotypes with distinct spatial orchestration

Abstract Multiplex fluorescence immunohistochemistry (mfIHC) approaches were yet either limited to 6 markers or limited to a small (1.5cmx1.5cm) tissue size that hampers translational studies on large tissue microarray (TMA) cohorts. To be able to assess more marker in a large patient cohort, we have developed a BLEACH&amp;STAIN multiplex fluorescence immunohistochemistry approach that enabled the simultaneous analysis of 15 biomarkers. To study the relationship of PD-L1 expression on multiple different cell types and the relationship with various lymphocyte subtypes, PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, and CD31 were analyzed in 3098 tumor samples from 44 different tumor types. An artificial intelligence-based framework - incorporating three different deep learning systems - for automated marker quantification on tumor as well as immune cells was further established to study the spatial interplay between PD-L1 expression and the composition of tumor infiltrating leucocytes (TILs). Comparing the automated deep learning-based PD-L1 quantification with conventional brightfield PD-L1 data revealed a high concordance in tumor cells (p&amp;lt;0.0001) as well as immune cells (p&amp;lt;0.0001) and an accuracy of the automated PD-L1 quantification ranging from 90% to 95.2%. Unsupervised clustering showed that a major proportion of the three PD-L1 phenotypes (i.e., PD-L1+ tumor and immune cells [G1], PD-L1+ immune cells [G2], PD-L1 negative [G3]) were either inflamed (G1.1, G2.1, G3.1) or non-inflamed (G1.2, G2.2, G3.2). In the inflamed PD-L1+ patients (G.1.1), spatial analysis revealed that an elevated intratumoral CD68+CD163+ M2 macrophages as well as CD11c+ dendritic cell infiltration (p&amp;lt;0.001 each) was associated with a high (CD3+CD4±CD8±FOXP3±) T-cell exclusion and a high PD-1 expression on T-cells (p&amp;lt;0.001 each). In breast cancer, a particular poor prognosis for the non-inflamed PD-L1+ breast cancer patients (G1.2, G2.2) was found and the PD-L1 fluorescence intensity on tumor cells showed a significantly higher predictive performance for overall survival with an area under receiver operating curves (AUC) of 0.72 (p&amp;lt;0.001) than the percentage of PD-L1+ tumor cells (AUC: 0.54). In conclusion, BLEACH&amp;STAIN mfIHC in combination with a deep learning-based framework for automated PD-L1 assessment on tumor and immune cells enabled a rapid and comprehensive assessment of PD-L1 expression in different cell types and their interrelation with inflammatory cells. Our approach enabled the identification of six major PD-L1 phenotypes ranging from an PD-L1+ tumor cell inflamed phenotype (G1.1) with a spatial T-cell exclusion to a non-inflamed PD-L1+ immune cell phenotype showing a particular poor prognosis (G2.2) to a non-inflamed PD-L1 negative phenotype (G3.2). Citation Format: Elena Bady, Katharina Möller, Tim Mandelkow, Ronald Simon, Maximilian Lennartz, Claudia Hube-Magg, Guido Sauter, Niclas C. Blessin. BLEACH&amp;STAIN 15 marker multiplexed imaging in 3098 human carcinomas revealed six major PD-L1 driven immune phenotypes with distinct spatial orchestration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 597.

Immunological correlatives of durable responses and survival benefit for patients with cervical cancer in a trial of combined chemotherapy and immune therapy.

5537 Background: Recurrent, metastatic, or persistent cervical cancer is largely an incurable disease due to lack of effective therapies. New treatment strategies are needed to provide long-term anti-tumor responses. We sought to gain insight into the immune pathways contributing to treatment response to immunotherapy. Methods: Fourteen patients with recurrent, persistent, or metastatic cervical cancer were enrolled in a single-arm phase II clinical trial (NCT03367871) with the combination of chemotherapy, bevacizumab and pembrolizumab regardless of tumor PD-L1 status. We designed a 40-color full spectrum flow cytometry panel to profile the protein markers of immune cells in peripheral blood mononuclear cells (PBMCs) at the single cell level to assess immunological correlates in blood with patients' anti-tumor responses. Results: In this trial with a cohort of predominantly Black and Hispanic patients (93%), 50% had durable responses to a combination therapy, and no difference was observed in the combined PD-L1 score (CPS) between responders and non-responders. With 40-color flow cytometry multiplex assays, we detected several differences between the non-responders and responders within the B and T cell populations. Within the B cell population, we observed a significant increase in the HLA+CD200hi B cells, a potentially suppressive cell subset, within the non-responders while among the responders we observed an increase in the number of HLA+CD200lo B cells. This HLA+CD200lo subset was mainly composed of IgD-CD27- memory B cells, a population suggested to have effector function. Moreover, the HLA+CD200hi B cell population contained a higher percentage of PD-L1 expressing cells than the HLA+CD200lo population, further supporting the suggestion that the HLA+CD200hi population may be an immunosuppressive subset. Within the T cell compartment, we observed an increase in the number of CD4+CD69+ T cells, which represent an activated CD4 T cell population, in the responder group compared to the non-responder group. The number of activated CD4+ T cells was observed to correlate (r = 0.5) with the HLA+CD200hi B cell subset. Conclusions: This study identified novel immunological correlates in peripheral blood that were associated with durable antitumor responses and survival benefit for cervical cancer patients. These results suggest a role for T-B collaboration in effective antitumor immunity induced by combination of chemotherapy and immune therapy against cervical cancer. Clinical trial information: NCT03367871.

A comprehensive profiling of the immune microenvironment of breast cancer brain metastases.

Despite potential clinical implications, the complexity of breast cancer (BC) brain metastases (BM) immune microenvironment is poorly understood. Through multiplex immunofluorescence, we here describe the main features of BCBM immune microenvironment (density and spatial distribution) and evaluate its prognostic impact. Sixty BCBM from patients undergoing neurosurgery at three institutions (2003-2018) were comprehensively assessed using two multiplex immunofluorescence panels (CD4, CD8, Granzyme B, FoxP3, CD68, pan-cytokeratin, DAPI; CD3, PD-1, PD-L1, LAG-3, TIM-3, CD163, pan-cytokeratin, DAPI). The prognostic impact of immune subpopulations and cell-to-cell spatial interactions was evaluated. Subtype-related differences in BCBM immune microenvironment and its prognostic impact were observed. While in HR-/HER2- BM and HER2+ BM, higher densities of intra-tumoral CD8+ lymphocytes were associated with significantly longer OS (HR 0.16 and 0.20, respectively), in HR+/HER2- BCBMs a higher CD4+FoxP3+/CD8+ cell ratio in the stroma was associated with worse OS (HR 5.4). Moreover, a higher density of intra-tumoral CD163+ M2-polarized microglia/macrophages in BCBMs was significantly associated with worse OS in HR-/HER2- and HR+/HER2- BCBMs (HR 6.56 and 4.68, respectively), but not in HER2+ BCBMs. In HER2+ BCBMs, multiplex immunofluorescence highlighted a negative prognostic role of PD-1/PD-L1 interaction: patients with a higher percentage of PD-L1+ cells spatially interacting with (within a 20 µm radius) PD-1+ cells presented a significantly worse OS (HR 4.60). Our results highlight subtype-related differences in BCBM immune microenvironment and identify two potential therapeutic targets, M2 microglia/macrophage polarization in HER2- and PD-1/PD-L1 interaction in HER2+ BCBMs, which warrant future exploration in clinical trials.

Diagnostic Performance of PD-L1 versus PD-1 Expression in Circulating CD20 Cells in Diffuse Large B-Cell Lymphoma.

This study aimed to investigate PD-L1 and PD-1 expression in circulating CD20+ cells in diffuse larger B-cell lymphoma (DLBCL) and to evaluate the predictive and diagnostic performance of PD-L1 versus PD-1 expression in circulating CD20+ cells in DLBCL. Percentages of CD20+, PD-L1+CD20+, and PD-1+CD20+ cells were measured by flow cytometry in 40 DLBCL blood samples and 19 healthy controls. The DLBCL patient group was subdivided into 20 newly diagnosed patients with no treatment yet and 20 patients that had finished six cycles of CHOP therapy. Percentages of PD-L1+CD20+ and PD-1+CD20+ cells were highly significantly increased in pre-therapy patients in comparison to healthy volunteers (p < 0.001). Meanwhile, a significant decrease in percentages of PD-L1+CD20+ and PD-1+CD20+ was observed in post-CHOP therapy patients in comparison to pre-therapy patients (p < 0.001). PD-L1+CD20+ cells were significantly decreased in post-therapy patients when compared to normal controls (p < 0.001), while not for PD-1+CD20+ cells. A strong significant positive correlation between percentages of PD-L1+CD20+ and PD-1+CD20+ was detected in DLBCL patients (p < 0.001). In the pre-therapy group, high PD-L1+CD20+ and PD-1+CD20+ percentages were correlated with serum LDH levels (p = 0.021, p < 0.001). High percentages of PD-1+CD20+ were found in DLBCL patients with splenomegaly (p = 0.027). The results revealed that patients with advanced tumor stages, poor ECOG performance, and non-GCB DLBCL type had increased percentages of PD-L1+CD20+ and PD-1+CD20+ cells. Moreover, PD-L1+CD20+ % and PD-1+CD20+ % were significantly increased in DLBCL patients with bone marrow involvement or B symptoms. The superiority of PD-L1+CD20+ over PD-1+CD20+ was more profound in DLBCL prediction [AUC: 1.0] and in discriminating newly diagnosed patients [AUC: 1.0]. The findings suggest that increased PD-L1/PD-1 expression in peripheral CD20 cells may serve as a companion diagnostic marker for DLBCL. Moreover, percentages of PD-L1+CD20+ cells have better diagnostic performance with higher sensitivity and specificity than PD-1+CD20+ %.

The integration of systemic and tumor PD-L1 as a predictive biomarker of clinical outcomes in patients with advanced NSCLC treated with PD-(L)1blockade agents.

Tumor PD-L1 expression is a predictive biomarker for patients with NSCLC receiving PD-(L)1 blockade agents. However, although increased tumor PD-L1 expression predicts responsiveness, clinical benefit has been observed regardless of tumor PD-L1 expression, suggesting the existence of other PD-L1 sources. Theaim of our study was to analyze whether integrating systemic and tumor PD-L1 is more predictive of efficacy in patients with advanced NSCLC receiving PD-(L)1 blockade agents. Twenty-nine healthy donors and 119 consecutive patients with advanced NSCLC treated with PD-(L)1 drug were prospectively included. Pretreatment blood samples were collected to evaluate PD-L1 levels on circulating immune cells, platelets (PLTs), platelet microparticles (PMPs), and the plasma soluble PD-L1 concentration (sPD-L1). Tumor PD-L1 status was assessed by immunohistochemistry. The percentages of circulating PD-L1 + leukocytes, sPD-L1 levels, and tumor PD-L1 were correlated with efficacy. No differences in the percentages of circulating PD-L1 + leukocytes were observed according to tumor PD-L1 expression. Significantly longer progression-free survival was observed in patients with higher percentages of PD-L1 + CD14 + , PD-L1 + neutrophils, PD-L1 + PLTs, and PD-L1 + PMPs and significantly longer overall survival was observed in patients with higher percentages of PD-L1 + CD14 + and high tumor PD-L1 expression. Integrating the PD-L1 data of circulating and tumor PD-L1 results significantly stratified patients according to the efficacy of PD-(L1) blockade agents. Our results suggest that integrating circulating PD-L1 + leukocytes, PLT, PMPs, and sPD-L1 and tumor PD-L1 expression may be helpful to decide on the best treatment strategy in patients with advanced NSCLC who are candidates for PD-(L)1 blockade agents.

P57.02 Integration of Systemic / Tumor PD-L1 as a Predictive Biomarker of Clinical Outcome in Advanced NSCLC Patients Treated With Anti-PD-(L)1 Agents

Tumor PD-L1 expression is a predictive biomarker for NSCLC patients (pts) receiving anti-PD-(L)1 agents. However, clinical benefit has been observed regardless of tumor PD-L1 expression, suggesting the existence of other PD-L1 sources. The aim of our study was to analyze whether integrating systemic and tumor PD-L1 is more predictive of outcome in advanced NSCLC pts receiving PD-(L)1 blockade agents. Pretreatment blood samples were collected to evaluate PD-L1 levels on circulating immune cells, platelets (PLTs), platelet microparticles (PMPs) and plasmatic concentrations of soluble PD-L1 (sPD-L1). Tumor PD-L1 status was assessed by immunohistochemistry. The percentages of circulating PD-L1+ leukocytes, PLTs, sPD-L1 levels and tumor PD-L1 expression were correlated with clinical outcome. 29 healthy donors and 119 advanced NSCLC pts treated with anti-PD-(L)1 were prospectively included. Median follow-up: 10.97 months [IQR: 6.06-21.87]. Median age of NSCLC patients was 65 (36-84), 78.1% were male and the most common histology was non-squamous (61.3%). Tumor PD-L1 expression (IHC 22C3 pharmDx) : <1% 30 pts (25.2%), 1-49% 35 (29.4%), ≥50% 37 (31.1%), not evaluable 17 (14.3%). PD-(L)1 inhibitors were given in 1st line in 37 pts (31.1%) and in ≥2nd line in 82 (68.9%), and given as monotherapy in 104 pts (87.4%), combination with chemotherapy in 11 pts (9.2%), or with other immunotherapy in 4 pts (3.4%) pts. Significantly longer progression free survival was observed in pts with higher percentage of PD-L1+ CD14+, PD-L1+ neutrophils, PD-L1+ PLTs and PD-L1+ PMPs and significantly longer overall survival was observed in pts with higher percentages of PD-L1+ CD14+ and high tumor PD-L1 expression (Table 1). Higher levels of integrated PD-L1 data of circulating and tumor PD-L1 results significantly stratified patients according to efficacy of PD-(L1) blockade agents (PFS: HR 0.29; 95% CI: 0.16-0.54, p<0.0001; OS: HR 0.28; 95% CI: 0.14-0.54, p=0.0002), even when tumor PD-L1 expression was excluded.Table 1PFSOSVariablesHR95% CIPHR95% CIP% PD-L1+ CD4+0.760.44-1.320.330.790.45-1.390.42% PD-L1+ CD8+0.690.41-1.160.160.810.47 – 1.390.45% PD-L1+ NK1.170.69-1.990.551.10.63 – 1.90.72% PD-L1+ CD14+0.360.22-0.58<0.0010.580.37- 0.930.02% PD-L1+ Neutrophils0.510.32-0.8<0.010.680.43–1.070.09% PD-L1+ PLTs0.480.26-0.880.020.970.51-1.960.93% PD-L1+ PMPs0.490.28-0.870.020.570.29-1.10.09pg/ml sPD-L11.260.81-1.970.291.240.78-1.970.34% TPS ≥50% vs <1% ≥50% vs 1-49%0.65 0.610.36-1.18 0.32 - 1.150.23 0.230.47 0.590.24-0.91 0.31-1.120.02 0.02 Open table in a new tab Our results suggest that the integration of circulating PD-L1+ leukocytes, PLT, PMPs and sPD-L1 and tumor PD-L1 expression could be helpful to decide the best treatment strategy in advanced NSCLC pts candidates for anti-PD-(L)1 agents.