PD-L1 Expression In Tumor Tissue Research Articles

PD-L1 as a Urine Biomarker in Renal Cell Carcinoma-A Case Series and Proof-of-Concept Study.

Renal cell carcinoma (RCC) is among the most lethal urologic malignancies once metastatic. Current treatment approaches for metastatic RCC (mRCC) involve immune checkpoint inhibitors (ICIs) that target the PD-L1/PD-1 axis. High PD-L1 expression in tumor tissue has been identified as a negative prognostic factor in RCC. However, the role of PD-L1 as a liquid biomarker has not yet been fully explored. Herein, we analyze urine levels of PD-L1 in mRCC patients before and after either ICI therapy or surgical intervention, as well as in a series of patients with treatment-naïve RCC. The mid-stream urine of patients with mRCC (n = 4) or treatment-naïve RCC, i.e., prior to surgery from two centers (cohort I, n = 49: cohort II, n = 29) was analyzed for PD-L1 by ELISA. The results from cohort I were compared to a control group consisting of patients treated for non-malignant urologic diseases (n = 31). In the mRCC group, urine PD-L1 levels were measured before and after tumor nephrectomy (n = 1) or before and after ICI therapy (n = 3). Exosomal PD-L1 in the urine was analyzed in selected patients by immunoblotting. A strong decrease in urine PD-L1 levels was found after tumor nephrectomy or following systemic treatment with ICIs. In patients with treatment-naïve RCC (cohort I), urine PD-L1 levels were significantly elevated in the RCC group in comparison to the control group (median 59 pg/mL vs. 25.7 pg/mL, p = 0.011). PD-L1 urine levels were found to be elevated, in particular, in low-grade RCCs in cohorts I and II. Exosomal PD-L1 was detected in the urine of a subset of patients. In this proof-of-concept study, we show that PD-L1 can be detected in the urine of RCC patients. Urine PD-L1 levels were found to correlate with the treatment response in mRCC patients and were significantly elevated in treatment-naïve RCC patients.

Effects of immunogenic cell death-inducing chemotherapeutics on the immune cell activation and tertiary lymphoid structure formation in melanoma.

The infiltration and activation of immune cells in the tumor microenvironment (TIME) affect the prognosis of patients with cancer. Tertiary lymphoid structure (TLS) formation favors tumour- infiltrating-lymphocyte (TIL) recruitment and is regarded as an important indicator of good prognosis associated with immunotherapy in patients with tumors. Chemotherapy is currently one of the most commonly used clinical treatment methods. However, there have been no clear report to explore the effects of different types of chemotherapy on TLS formation in the TIME. This study examined the effects of immunogenic cell death (ICD)-inducing chemotherapeutics on immune cells, high-endothelial venules (HEV), and TLSs in mouse melanomas. Doxorubicin (an ICD inducer), gemcitabine (non-ICD inducer), and a combination of the two drugs was delivered intra-peritoneally to B16F1-loaded C57BL/6 mice. The infiltration of immune cells into tumor tissues was evaluated using flow cytometry. HEV and TLS formation was assessed using immunohistochemistry and multiple fluorescent immunohistochemical staining. Doxorubicin alone, gemcitabine alone, and the two-drug combination all slowed tumor growth, with the combined treatment demonstrating a more pronounced effect. Compared with the control group, the doxorubicin group showed a higher infiltration of CD8+ T cells and tissue-resident memory T cells (TRM) and an increase in the secretion of interferon-γ, granzyme B, and perforin in CD8+ T subsets and activation of B cells and dendritic cells. Doxorubicin alone and in combination with gemcitabine decreased regulatory T cells in the TIME. Moreover, doxorubicin treatment promoted the formation of HEV and TLS. Doxorubicin treatment also upregulated the expression of programmed cell death protein (PD)-1 in CD8+ T cells and programmed cell death protein ligand (PD-L)1 in tumor cells. These results indicate that doxorubicin with an ICD reaction promotes TLS formation and increases PD-1/PD-L1 expression in tumor tissues. The results demonstrate the development of a therapeutic avenue using combined immune checkpoint therapy.

Anti-PD-1 Antibody (Tislelizumab) Combined with R-CHOP for the Treatment of Previously Untreated Primary Mediastinal B-Cell Lymphoma: A Prospective Phase II Study

Background: Primary mediastinal B-cell lymphoma (PMBCL) is a rare aggressive B-cell lymphoma of thymic origin. Due to the low incidence rate of PMBCL, there is currently no internationally accepted standard first-line treatment regimen. Dose-adjusted (DA) EPOCH-R which is the more commonly used first-line therapy plan is not so effective because patients usually cannot tolerate it. The R-CHOP regimen is comparable to the R-DA-EPOCH for the first-line treatment of PMBCL in terms of survival and has a better safety profile (Bhatt, V.R. et al, Cancer Treat Rev 2015; Iwanicka el al., Curr Hematol Malig Rep 2014). Meanwhile, patients with PMBCL usually have alterations of 9p24.1, leading to overexpression of PDL-1/2. Previous studies have found that PD-1 monoclonal antibody shows good efficacy in recurrent and refractory PMBCL (Steidl et al., Blood 2011; Zinzani et al., Blood 2017). Tislelizumab, a humanized IgG4 antibody that binds PD-1 with high affinity, minimizes binding to Fcγ receptor 1 (FcγRI) on macrophages by the genetic engineering of Fc segment, abrogating antibody-dependent phagocytosis (ADCP)-induced T-cell clearance. The efficacy and safety of its combination with R-CHOP in previously untreated PMBCL is not yet clear. We speculate that the combination of both may be a new option for the first-line therapy for PMBCL patients with good efficacy and low toxicity. Methods: This is a single-arm, multi-center, phase II clinical study (ChiCTR2100044313). Patients aged 18 years or older with a diagnosis of PMBCL and previously untreated were eligible. Eligible patients received 6 cycles of Tislelizumab (200 mg D1), Rituximab (375 mg/m 2 D1), Cyclophosphamide (750 mg/m 2 D2), Pirarubicin (50 mg/m 2 D2), Vincristine (1.4 mg/m 2, maximum dose 2 mg, D2), Prednisone (100 mg/d D2-6), every 21 days for 1 cycle. Disease response assessments were performed by positron emission tomography and computed tomography (PET-CT) or enhanced CT every two cycles, meanwhile peripheral blood ctDNA was monitored, and treatment-related adverse effects were assessed and recorded. The primary endpoint was objective remission rate (ORR). The secondary endpoints included treatment safety, progression-free survival (PFS) and overall survival (OS), and the exploratory analyses of the correlation between PD-L1 expression in tumor tissues, tumor-associated macrophages (TAM) phenotype in tumor microenvironment and efficacy. Results: From June 2020 to June 2023, 12 patients were enrolled and 9 patients had evaluable efficacy with the median age of 34 years, 3 males and 6 females, all patients had large mediastinal mass at initial diagnosis. 7 patients had PD-L1 expression (VENTANA SP263) above 80% and 2 cases could not be further tested due to insufficient tissue specimens. The ORR was 100%, and the CR rate was 77.8% (7/9) after 6 cycles of treatment. The 5 th and 9 th patient reached PR after 6 cycles of treatment due to irregular hospitalization. And CR was achieved after 2 cycles of regular treatment for the 5 th patient. NGS from formalin-fixed paraffin-embedded (FFPE) and circulating tumor DNA (ctDNA) evaluation from blood were performed across all patients before treatment. 5 patients underwent end of treatment-ctDNA test. Median follow-up 15m (6.8m-26.3m), the third patient was lost to follow-up at 9m. The other patients who achieved CR are still at complete remission. Moreover, we found SOCS1, TNFAIP3 are the most frequently mutated genes in biopsies of PMBCL and only the 5th patient had RHOA mutation which is highly related to PMBCL. The PET-CT and ctDNA assessments were negative in three patients at the end of 6 cycles with 60% concordance, and the 5 th and 9 th patient achieved PR on PET-CT, but the ctDNA assessments were negative. The most common adverse events (AEs) were gastrointestinal reactions, 2 patients experienced grade IV myelosuppression and 1 patient had grade I hyperthyroidism. No treatment-related deaths occurred. Conclusion: Tislelizumab combined with R-CHOP for the treatment of previously untreated PMBCL had good efficacy and sustained response with low toxicity. Longer follow-up is required to access the survival benefit of this regimen. Figure 1: Disease Remission in the Included Patients

A Study on the Clinical Significance of Blood Exosomal PD-L1 in Non-Small Cell Lung Cancer Patients and its Correlation with PD-L1 in Tumor Tissues.

Exosomal programmed cell-death ligand 1 (ePD-L1) can influence immune inhibition and dysfunction. We were dedicated to unearthing the relation between ePD-L1 in blood and pathological characteristics as well as PD-L1 in tumor tissues. We recruited 65 non-small cell lung cancer (NSCLC) patients for exosome extraction and detected the blood ePD-L1 expression in these patients by enzyme-linked immunosorbent assay (ELISA) method. Besides, the correlation between blood ePD-L1 and patients' pathological characteristics was also analyzed. The expression of PD-L1 in tumor tissues was tested by immunohistochemistry (IHC) and its correlation with blood ePD-L1 expression level was analyzed by Spearman correlation coefficient. No significant correlation was observed in PD-L1 expression levels between blood-derived exosome and tumor tissue. Altogether, high blood ePD-L1 expression was relevant to NSCLC progression, while no such relevance to PD-L1 expression in tumor tissue.

Dynamic change in Siglec-15 expression in peritumoral macrophages confers an immunosuppressive microenvironment and poor outcome in glioma.

Sialic acid-binding immunoglobulin-like lectin-15 (Siglec-15) was reported to be a novel immune checkpoint molecule comparable to programmed cell death 1 ligand 1 (PD-L1). However, its expression profile and immunosuppressive mechanisms in the glioma tumor microenvironment have not yet been fully explored. To identify the expression profile and potential function of Siglec-15 in glioma tumor microenvironment. We investigated Siglec-15 and PD-L1 expression in tumor tissues from 60 human glioma patients and GL261 tumor models. Next, Siglec-15 knockout macrophages and mice were used to elucidate the immunosuppressive mechanism of Siglec-15 impacting macrophage function. Our results demonstrated that high levels of Siglec-15 in tumor tissues was positively correlated with poor survival in glioma patients. Siglec-15 was predominantly expressed on peritumoral CD68+ tumor-associated macrophages, which accumulated to the highest level in grade II glioma and then declined as grade increased. The Siglec-15 expression pattern was mutually exclusive with that of PD-L1 in glioma tissues, and the number of Siglec-15+PD-L1- samples (n = 45) was greater than the number of Siglec-15-PD-L1+ samples (n = 4). The dynamic change in and tissue localization of Siglec-15 expression were confirmed in GL261 tumor models. Importantly, after Siglec15 gene knockout, macrophages exhibited enhanced capacities for phagocytosis, antigen cross-presentation and initiation of antigen-specific CD8+ T-lymphocyte responses. Our findings suggested that Siglec-15 could be a valuable prognostic factor and potential target for glioma patients. In addition, our data first identified dynamic changes in Siglec-15 expression and distribution in human glioma tissues, indicating that the timing of Siglec-15 blockade is critical to achieve an effective combination with other immune checkpoint inhibitors in clinical practice.

Clinical significance of serum-derived exosomal PD-L1 expression in patients with advanced pancreatic cancer

BackgroundInteractions between the programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) lead to immune evasion in various tumors and are associated with poor prognosis in patients with pancreatic cancer; however, the roles of PD-L1-containing exosomes in pancreatic cancer is poorly understood. Here, we investigated the correlation between circulating exosomal PD-L1 (exoPD-L1) and PD-L1 expression in tumor tissue, and survival outcomes in patients with advanced PDAC.MethodsExosomes were derived from pre-treatment serum samples isolated using ExoQuick kit from 77 patients with advanced pancreatic cancer. Exosomal PD-L1 (exoPD-L1) was detected by enzyme-linked immunosorbent assay, and matched tumor tissues PD-L1 expression were evaluated by PD-L1 immunohistochemistry (22C3) assay, described with combined positive score. Cutoff value of exoPD-L1 for survival was assessed with receiver operating characteristic curve analysis. Kaplan-Meier analysis was performed to obtain median overall survival (OS), and hazard ratio was estimated using a stratified Cox regression model.ResultsThe median exoPD-L1 serum concentration was 0.16 pg/mg, with undetected levels in seven patients. ExoPD-L1 levels were significantly higher in patients with systemic disease than in those with locally advanced disease (p = 0.023). There was a significantly higher proportion of elevated exoPD-L1 levels in patients with positive PD-L1 expression compared to patients with negative PD-L1 expression (p = 0.001). Patients were classified into groups with low and high exoPD-L1 levels using ROC curve-derived cutoffs (0.165 pg/mg; area under the curve, 0.617; p = 0.078). At a median follow-up of 8.39 months, the median OS was 13.2 (95% CI, 8.17–18.3) and 6.36 months (95% CI, 3.27–9.45) in the low and high exoPD-L1 groups, respectively (HR = 0.61; 95% CI, 0.35–1.04; p = 0.059). ExoPD-L1 levels did not affect the proportion of CD8+CD69+ effector cytotoxic T cells in either of the groups (p = 0.166).ConclusionsThe serum-derived exoPD-L1 levels were higher in metastatic pancreatic cancer than locally advanced disease. Collectively, higher serum exoPD-L1 levels in patients with advanced pancreatic cancer suggested worse survival outcomes and may have clinical implications.

Abstract 235: Establishing clinical utility for genomic profiling with plasma cell free DNA in urothelial carcinoma

Abstract Purpose: Genomic profiling in urothelial carcinoma (UC) conventionally uses tissue DNA. However, tissue biopsy is so invasive that it can be difficult in some cases. Cell-free DNA (cfDNA) assay is a minimally invasive tool obtaining a comprehensive genomic information reflecting a tumor heterogeneity. Recently, the investigation of cfDNA assay in UC is widely conducted, but its clinical utility remains largely unknown. We aimed to establish plasma cfDNA assay and clarify its clinical role in UC. Method: Blood and matched tissue were obtained from 48 cases with advanced UC before 1st line systemic therapy or radical surgery. For genomic profiling of all samples, we applied a customed targeted DNA-sequencing strategy capturing 54 UC-relevant genes. Somatic mutations were required to be supported by a minimum variant allele frequency (VAF) of 0.5% in plasma cfDNA and 5% in tissue DNA. All mutation calls were filtered against matched leukocyte DNA. The result of mutational analysis was validated with digital PCR. The proportion of tumor-derived cfDNA (circulating tumor DNA (ctDNA) fraction) was estimated. We investigated the association between genomic profile and molecular subtyping with RNA sequencing and immunohistochemistry (IHC). Result: Fifty-five somatic mutations were detected in 22 cfDNA samples (22/48, 45.8%). Twenty-six of all somatic mutations identified in cfDNA (26/55, 47.3%) were concurrently present in tumor tissues. In 14 mutations randomly selected from all of those detected in cfDNA, VAF of digital PCR was statistically consisted with the result of NGS (p<0.001, R2=0.95). Furthermore, digital PCR showed that mutations detected in cfDNA alone were cleared via radical surgery. And those were found in another tissue sample from the same patient. Of the 22 cases, 6 had a ctDNA fraction greater than 2% of total cfDNA. High ctDNA fraction was associated with advanced clinical tumor stage, high PD-L1 expression in tumor tissue by IHC and basal molecular subtype (p = 0.04, 0.02, <0.01, respectively). Conclusions: We established cfDNA assay for genome profiling in advanced UC. The result of validation using digital PCR suggested that cfDNA assay can identify the mutation which cannot be detected in tissue sample as a tumor heterogeneity. Additionally, cfDNA assay generate the information for classifying UC cases by molecular subtypes and predicting the expression of immune checkpoint molecules. Further investigation is needed as a promising minimally invasive assay to predict an immunotherapy responsiveness against metastatic UC. Citation Format: Yuki Kita, Takayuki Sumiyoshi, Takeshi Sano, Akihiro Hamada, Toru Sakatani, Kenji Nakamura, Hideaki Takada, Ryousuke Ikeuchi, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. Establishing clinical utility for genomic profiling with plasma cell free DNA in urothelial carcinoma [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 235.

Outcomes of programmed death protein-1 inhibitors treatment of chronic active Epstein Barr virus infection: A single center retrospective analysis.

Chronic active Epstein-Barr virus (CAEBV) disease is a high-mortality disease, which is characterized by persistent infectious mononucleosis-like symptoms. There is no standard treatment for CAEBV and allogeneic hematopoietic stem cell transplantation (HSCT) was considered the only potentially therapeutic approach. PD-1 inhibitors have achieved high response in many Epstein-Barr virus-related diseases. In this single-center retrospective analysis, we report the outcomes of PD-1 inhibitors treatment of CAEBV. All CAEBV patients without hemophagocytic lymphohistiocytosis (HLH), who were treated with PD-1 inhibitors in our center between 6/1/2017 and 12/31/2021, were retrospectively analyzed. The efficacy and safety of the PD-1 inhibitors were evaluated. Among the sixteen patients with a median age at onset of 33 years (range, 11-67 years), twelve patients responded to PD-1 inhibitors and the median progression-free survival (PFS) was 11.1 months (range, 4.9-54.8 months). Three achieved clinical complete response (clinical CR), as well as molecular CR. Five patients achieved and remained partial response (PR), and four converted from PR to no response (NR). For three CR patients, the median time and cycles from the first application of PD-1 inhibitor to clinical CR were 6 weeks (range, 4-10 weeks) and 3 cycles (range, 2-4 cycles), and molecular CR was achieved after a median of 16.7 weeks (range, 6.1-18.4 weeks) and 5 cycles (range, 3-6 cycles) of PD-1 inhibitor infusion. No immune-related adverse events have been observed except for one patient who suffered immune-related pancreatitis. There was no correlation of treatment outcome with blood count, liver function, LDH, cytokine or ferritin levels. NK cell function, PD-L1 expression in tumor tissue and gene mutation possibly correlated with treatment response. In patients with CAEBV, PD-1 inhibitors have tolerable toxicity and comparable outcomes while improving quality of life and financial toxicity. Larger prospective studies and longer follow-up time is needed to be conducted.

Prognostic importance of PD-L1 expression in bladder cancer patients.

PD-L1/PD-1 molecules are known as important mediators in immune-escape mechanisms of tumors. PD-L1 is highly expressed in various malignancies, including bladder cancer. However, the prognostic value of PD-L1 in bladder cancer patients remains controversial. To investigate the prognostic significance of PD-L1 expression in tumor tissues of bladder cancer patients. RNA was isolated from FFPE tumor tissues of 48 bladder cancer patients using the monophasic phenol and guanidine isothiocyanate method. Total RNA was converted to cDNA and gene expression levels were analyzed by qRT-PCR. The differential expression levels of the PD-L1 gene between tumor grade and cancer stage groups were analyzed by independent student's t-test and one-way ANOVA. Statistically significantly increased PD-L1 expression was observed in the high-grade tumor group (p < 0.05). No significant difference in PD-L1 expression was found among pTa, pT1, and pT2 groups. In addition, the difference in overall survival was not significantly different between groups. The results showed that high PD-L1 expression in bladder cancer was associated with tumor aggressiveness and grade. Despite the inability of the qRT-PCR to show the PD-L1 expression at different locations of tumor tissue, evaluation of PD-L1 mRNA expression by qRT-PCR, which is a highly sensitive and specific assay, appears to be a robust approach. Furthermore, these findings may contribute to a rationale for recommending anti-PD-L1 immunotherapy as an alternative to standard therapy for bladder cancer patients who are most likely to benefit from it.

Soluble Programmed Cell Death Ligand-1 (sPD-L1) Levels in Various Cancer Types and Normal Populations.

To date, PD-L1 expression in tumor tissue, assessed by immunohistochemical stain, is clinically applicable as a predictive companion biomarker for PD-1/PD-L1 inhibitor which has been highlighted over the past several years. Before blood-based sPD-L1 enters clinical use, it is critical to establish the reference range. This study was designed to investigate soluble sPD-L1 levels in various cancer patients and normal population. For the detection of sPD-L1, 4 cancer groups (hepatocellular carcinoma, lung cancer, bladder cancer, gastric cancer) and healthy volunteers' samples were analyzed using an ELISA kit. Using a receiver operating characteristic curve, optimal sPD-L1 cutoff levels were determined. The mean serum sPD-L1 level of the normal population was 59.97 pg/mL (range; 23.780 - 115.2 pg/mL). In various cancer types, serum sPD-L1 levels ranged from 38.696 pg/mL to 228.77 pg/mL, and cutoff values under AUC ranged from 60.307 pg/mL to 64.371 pg/mL. sPD-L1 can be used as a screening biomarker in various cancer patients referring to optimal cutoff levels suggested.

Assessment of PD-L1 mRNA expression in gastrointestinal tumors and the response to immunotherapy.

Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) has been proposed as a predictive biomarker to predict response to immunotherapy. Given the limitations of IHC test in PD-L1 detection, this study aimed to investigate the technical feasibility of using quantitative RT-PCR (qRT-PCR) to replace IHC in PD-L1 detection in gastrointestinal tumors. The Cancer Genome Atlas database was used to evaluate the relationship between PD-L1 expression in tumor tissue and the patient prognosis. In addition, 52 patients with gastrointestinal cancer were enrolled and divided into the stomach (STAD), colon (COAD), and rectum (READ) adenocarcinoma cohorts. IHC test was used to determine the PD-L1 level of the tissue specimens, and the qRT-PCR test was used to analyze the mRNA expression in both blood and tissue specimens. Moreover, the correlation between blood PD-L1 mRNA expression and immunotherapy efficacy was investigated in additional 15 patients with gastric cancer that further enrolled. The expression level of PD-L1 in tumor tissue is related to the tumor stage of COAD (p-value = 0.001) and primary therapy outcomes in patients with READ (p-value = 0.003) but not significantly correlated to the overall survival (OS) time of patients with gastrointestinal cancer. Moreover, the concordance of PD-L1 mRNA expression level of tissue and paired blood samples is low, despite a weak linear relationship that was found in the STAD cohort (r = 0.43, p-value = 0.049). We further demonstrated that qRT-PCR results in both tissue and blood specimens were numerically but not statistically significant consistent with IHC results (corresponding to a p-value of 0.84 and 0.55, respectively). Remarkably, high PD-L1 expression in blood of patients with STAD shows a better response to immunotherapy (p-value = 0.04), which could be well identified at the relative expression cutoff of 1.5 (sensitivity of 85.7%, specificity of 75.0%, and AUC of 0.82). Our study established a novel strategy for rapidly distinguishing patients with gastrointestinal cancer with the response to immunotherapy and has potential clinical benefits.

Outcomes of PD-1 Inhibitors Treatment of Chronic Active Epstein Barr Virus Infection : A Single Center Retrospective Analysis

INTRODUCTION Chronic active Epstein-Barr virus (CAEBV) disease is a high-mortality disease, which is characterized by persistent infectious mononucleosis-like symptoms. There is no standard treatment of CAEBV and allogeneic hematopoietic stem cell transplantation (HSCT) was considered as an only potentially therapeutic approach. PD-1 inhibitors have achieved high response in many Epstein-Barr virus-related diseases. In this single-center retrospective analysis, we report the outcomes of PD-1 inhibitors treatment of CAEBV. METHODS Sixteen CAEBV patients were treated with PD-1 inhibitors between 6/1/2017 and 12/31/2021. Hemophagocytic lymphohistiocytosis (HLH) was excluded. Patients received Nivolumab or Sindilizumab therapy every 3 to 4 weeks, 100 to 200 mg intravenously. Clinical and laboratory assessments were performed on patients before each infusion during treatment. Adverse events were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0. RESULTS Among the sixteen patients with a median age at onset of 33 years (range, 11-67 years), twelve patients responded to PD-1 inhibitors, and their median duration of relapse-free survival (RFS) was 10.3 months (range, 1.0-54.8 months). Three of them achieved clinical complete response (clinical CR), as well as molecular CR, and nine patients achieved partial response (PR) (Figure 1). Four of the nine PR patients converted from PR to no response (NR), and five remained in PR. The remaining four patients did not respond to PD-1 inhibitor therapy. One patient eventually died from a severe lung infection and one patient died of severe liver failure. The median time and cycles from the first application of PD-1 inhibitor to clinical CR in three patients with clinical CR were 43 days (range, 30-72 days) and 3 cycles (range, 2-4 cycles). Patient 14 suffered immune-related pancreatitis (Grade 2, CTCAE 5.0) after 6 cycles of PD-1 inhibitor therapy. Four patients developed persistent or intermittent high fever after the first infusion of PD-1 inhibitors. Other than that, no treatment-related adverse events have been observed. There was no correlation of treatment outcome with blood count, aspartate aminotransferase (AST) level, alanine aminotransferase (ALT) level, LDH level, cytokine level or ferritin level. Comparing the NK cell function of responders and non-responders, the test results showed higher numbers of patients with defective NK cell function among non-responders. The immunohistochemical analysis showed that patients with high PD-L1 expression in tumor tissue may have a more significant response to PD-1 inhibitors. Genetic testing indicated that, except for 1 patient without gene sequencing and 8 patients with no related gene mutation detected, the other 7 patients were detected with potentially pathogenic or susceptibility mutations. CONCLUSION In patients with CAEBV, PD-1 inhibitors have tolerable toxicity and comparable outcomes while improving quality of life and financial toxicity. Larger prospective studies and longer follow-up time is needed to be conducted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High Serum sPD-L1 Level Predicts Poor Outcome in Hepatocellular Carcinoma Patients Treated with Radiotherapy

<h3>Purpose/Objective(s)</h3> To determine the clinical significance of serum soluble (s)PD-1 and sPD-L1 in hepatocellular carcinoma (HCC) received radiotherapy (RT). <h3>Materials/Methods</h3> In cohort 1, 144 HCC patients treated with liver RT were obtained from a prospectively collected database. Blood samples were collected 1 day before and 2 weeks after RT. In cohort 2, formalin-fixed paraffin-embedded samples were obtained from 46 patients with HCC, among whom 23 received preoperative RT and the other 23 received direct surgery. The relationship of sPD-1/sPD-L1 in serum or PD-L1 expression in tumor tissues and clinicopathological features were evaluated. <h3>Results</h3> In cohort 1, patients with tumor thrombosis (p = 0.01) and advanced stage (p = 0.00) had significantly higher serum sPD-L1 concentrations. Higher sPD-L1 level was negative predictor of progression-free survival (PFS) (HR = 1.53, p=0.042). In addition, RT significantly increased the expression of sPD-L1 (p = 0.000), which negatively associated with content of serum CD8+ T cells (R = -0.24, p = 0.033). Moreover, no significant associations were found between clinicopathological features and sPD-1. In cohort 2, we confirmed the phenomenon that RT could increase the expression of PD-L1 in tumor tissues (p = 0.001) and the expression level of PD-L1 in tumor tissues was related to decreased cytotoxic T-cell infiltration and a trend of poor prognosis. <h3>Conclusion</h3> High PD-L1 expression was associated with poor prognoses in HCC treated with RT. The increases in PD-L1 induced by RT indicated the combined treatment with RT and immune therapy may be promising strategy for HCC.

Higher serum sPD-L1 levels after radiotherapy indicate poor outcome in hepatocellular carcinoma patients

BackgroundOur preclinical research reveals that radiotherapy (RT) promoted PD-L1 upregulation in tumor tissues and that higher PD-L1 after RT worsened the prognosis through immunosuppression. We sought to validate our experimental results in clinical cohorts and promote clinical application. Patients and methodsIn cohort 1, formalin-fixed paraffin-embedded samples were obtained from 46 HCC patients, 23 of whom received preoperative RT and the other 23 received direct surgery. A prospectively collected database contained 122 HCC patients treated with liver RT were enrolled in cohort 2. Blood samples were taken a day before and two weeks after RT. Patients in cohort 2 were further divided into two groups, exploration (73 patients) and validation (49 patients) groups. ResultsIn cohort 1, RT increased the expression of PD-L1 in tumor tissues (p = 0.001), and PD-L1 levels were associated with decreased cytotoxic T-cell infiltration and a trend toward poor prognosis (p = 0.14). Moreover, PD-L1 expression in tumor tissue positively correlated with soluble (s) PD-L1 in serum (R = 0.421, p = 0.046). Then, in cohort 2, we revealed RT increased sPD-L1 in serum (p < 0.001), which was associated with the number of circulating CD8+ T cells (R = -0.24, p = 0.036), indicating poor survival. Furthermore, patients with higher rate of sPD-L1 increase after RT have better treatment response (p < 0.001), PFS (p = 0.032) and OS (p = 0.045). ConclusionHigher post-RT serum sPD-L1, which may potentiate immune suppression effects, indicates a poor prognosis for HCC patients treated with RT.

Could We Predict the Response of Immune Checkpoint Inhibitor Treatment in Hepatocellular Carcinoma?

Simple SummaryThe use of anti-programmed cell-death protein (ligand)-1 (PD-[L]1) is now a standard of care for treating hepatocellular carcinoma (HCC). However, the treatment only benefits 10–20% of patients when used as a monotherapy. The unique environments of hepatitis and/or cirrhosis, which continuously interact with the hosts’ immune systems, make it difficult to find appropriate biomarkers to predict the response or lack of response of anti-PD-1/PD-L1 treatment in HCC. The current review aimed to present both clinical and translational biomarkers for anti-PD-1/PD-L1 treatment in HCC.The use of anti-programmed cell-death protein (ligand)-1 (PD-[L]1) is an important strategy for treating hepatocellular carcinoma (HCC). However, the treatment only benefits 10–20% of patients when used as a monotherapy. Therefore, the selection of patients for anti-PD-1/PD-L1 treatment is crucial for both patients and clinicians. This review aimed to explore the existing literature on tissue or circulating markers for the identification of responders or non-responders to anti-PD-1/PD-L1 in HCC. For the clinically available markers, both etiological factors (viral versus non-viral) and disease extent (intra-hepatic vs. extrahepatic) impact the responses to anti-PD-1/PD-L1, warranting further studies. Preliminary data suggested that inflammatory indices (e.g., neutrophil-lymphocyte ratio) may be associated with clinical outcomes of HCC during the anti-PD-1/PD-L1 treatment. Finally, although PD-L1 expression in tumor tissues is a predictive marker for multiple cancer types, its clinical application is less clear in HCC due to the lack of a clear-cut association with responders to anti-PD-1/PD-L1 treatment. Although all translational markers are not routinely measured in HCC, recent data suggest their potential roles in selecting patients for anti-PD-1/PD-L1 treatment. Such markers, including the immune classification of HCC, selected signaling pathways, tumor-infiltrating lymphocytes, and auto-antibodies, were discussed in this review.

Biomarkers predicting the effect of immunotherapy: what you need to know in practice?

Immune checkpoint inhibitors (ICT) therapy is a successful immunotherapy strategy that is quite effective in a 20–40% of patients with solid tumors. Nevertheless, there is a large group of patients, the proportion of which do not have a clinical response to such therapy and demonstrate adverse event. This explains the importance of personification of immunotherapy and the search for predictive markers. The article is devoted to the review of clinically significant biomarkers of the effectiveness of immunotherapy, such as PD-L1 expression in tumor tissue, microsatellite instability, tumor mutation burden and tumor infiltrating lymphocytes.

Abstract 1349: Hypomethylating agents modulate the PD-1/PD-L1 signaling in a murine breast cancer model and show additive effects in the combination with PD-1 blockage in vitro and in vivo

Abstract With epigenetic therapy emerging as a potential therapy for solid tumors and recent reports that indicate an influence of hypomethylating agents on the PD-1/PD-L1 expression in tumor tissue, epigenetic therapy in combination with immune-modulatory compounds might open-up new avenues for cancer treatment. To investigate the influence of hypomethylating agents on the PD-1/PD-L1 signaling cascade in breast cancer and its implication on possible treatment options, we determined the effect of 5-Azacytidine (5-AZA), decitabine (DAC) and anti-PD1 in mono- and combined therapy on the mouse breast cancer model 4T1 in vitro and in vivo. In a life cell imaging based 2D system we evaluated the influence of the different treatment arms on tumor growth, methylation status and PD-L1 expression. Furthermore, we determined the effect of 5-AZA and DAC on autologous T cells in the presence and absence of 4T1 cancer cells. The combination of 5-AZA and anti-PD1 treatment was further evaluated in an in vivo setting. 24 4T1 bearing balb/c mice were treated with either an isotype control, 5-AZA, anti-PD1 or a combination of the latter. Tumor growth was monitored by caliper measurement. At the end of the experiment tumor and hematopoietic organs were analyzed by flow cytometry. A panel of 36 murine cytokines was determined in the murine serum at different time points. Low dose 5-AZA (IC10) and DAC (IC2.5) induced a down regulation of PD-L1 on 4T1 cells in vitro. Interestingly, this effect was most prominent at the dose level and time point when the hypomethylation effect of the respective compound was most pronounced (day 7). 5-AZA and DAC induced an upregulation of PD-1 on autologous CD4+ as well as CD8+ T cells. Of note, 4T1 cells reduced the expression of PD-1 on autologous CD4+ and CD8+ T cells. Antitumoral activity in vitro was determined in all treatment arms. An additive effect of the combination was specifically determined when combining 5-AZA and anti PD-1. Hence, 5-AZA and anti PD-1 were investigated in vivo more in detail. 4T1 is characterized by an immune suppressive tumor microenvironment as indicated by the in vitro results: we determined a reduced CD4/CD8 ratio and a high percentage of MDSC. These features were reverted by the applied treatments. Along the same line, 5-AZA in mono- and in combination therapy with anti-PDL1 specifically increased the G-CSF levels in murine serum. In addition, the PDL-1 downregulation on the tumor cells was reproduced in vivo. Finally, the combination arm displayed the most prominent antitumoral activity. In summary, we have strong evidence that low dose hypomethylating agents influence the immune landscape of breast cancer via the modulation of PD-1/PD-L1 signaling. The combination with checkpoint inhibitors was superior to the monotherapy and might lead to new treatment options for breast cancer patients. Citation Format: Julia Schüler, Agon Kolica, Hung Ngyuen, Anna Edinger, Kanstantsin Lashuk, Eva Oswald. Hypomethylating agents modulate the PD-1/PD-L1 signaling in a murine breast cancer model and show additive effects in the combination with PD-1 blockage in vitro and in vivo [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 1349.

Nivolumab with chemotherapy as neoadjuvant treatment for inflammatory breast cancer.

e12633 Background: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer with poor prognosis and is often resistant to neoadjuvant systemic therapy with early recurrence and metastases. PD-L1 expression in IBC is moderate to high, and blockade of the PD-1/PD-L1 axis with checkpoint inhibitors has emerged as a promising treatment to enhance clinical response. We examined the efficacy of neoadjuvant nivolumab (anti-PD-1 antibody) with chemotherapy in IBC. Methods: This is an open-label multicohort multicenter study of nivolumab with neoadjuvant chemotherapy in patients with newly diagnosed non-metastatic IBC (n=52). All breast cancer subtypes (ER/PR/HER2) were allowed. Patients received nivolumab 360 mg on day 1 (21-day cycle) x 4 cycles with standard chemotherapy. Cohort 1 (HER2-negative) received nivolumab with paclitaxel (80 mg/m2)x12 weeks followed by doxorubicin (60 mg/m2) and cyclophosphamide (600 mg/m2) (AC) x 4 cycles. Cohort 2 (HER2-positive) received nivolumab with taxane (paclitaxel 80 mg/m2, docetaxel 75 mg/m2, or nab-paclitaxel 100 mg/m2), trastuzumab (8 mg/kg then 6 mg/kg), and pertuzumab (840 mg then 420 mg) x 4 cycles followed by AC x 4 cycles. All patients underwent modified radical mastectomy (MRM) followed by radiation and adjuvant therapy per institutional standard of care. Primary objective was pathologic complete response (pCR) (ypT0/Tis ypN0). Residual Cancer Burden (RCB) was assessed. Secondary objectives were safety/tolerability and invasive recurrence-free interval at 2 years. Breast biopsies, residual tumor tissue, and peripheral blood samples were collected for correlative biomarker testing. PD-L1 expression in tumor tissue will be assessed as a predictive marker. Study was closed after 8 patients were enrolled due to slow accrual. Results: 8 patients were enrolled from June 2019-December 2020. All completed neoadjuvant systemic therapy with nivolumab and none had disease progression. They underwent MRM between January 2020-June 2021. Mean age was 57 years (range 43-74). 4 were HER2-positive, 3 were TNBC, and 1 was HR-positive/HER2-negative. 3 were Caucasian, 2 were Latino, 2 were Black, and 1 was Asian. There was no grade 4 toxicity. Most common grade 3 toxicity was neutropenia (n=3). Immune-related events were diarrhea/colitis (n=2) and elevated liver transaminases (n=1). At time of MRM, 4 patients had pCR, 1 had RCB-I, 2 had RCB-II, and 1 had RCB-III. They remain with no evidence of disease and are in follow-up. Tumor biological correlatives are being performed. Conclusions: Addition of nivolumab to neoadjuvant therapy was tolerable and safe and demonstrated anti-tumor activity in IBC with high pCR rate in this pilot study. This supports further investigation of the role of checkpoint inhibitors in treatment of IBC. Clinical trial information: NCT03742986. [Table: see text]

Relationship and prognostic significance of IL-33, PD-1/PD-L1, and tertiary lymphoid structures in cervical cancer.

IL-33, an epithelial-derived cytokine, functions as an alarmin for the immune system in the tumor microenvironment (TME). However, the expression and role of IL-33 on cervical cancer remain unclear. The aim of this study was to investigate the expression of IL-33 and its relationship with clinicopathologic features, tertiary lymphoid structures (TLS), and programmed cell death 1 (PD-1)/programmed cell death 1 ligand (PD-L1) immune checkpoints by immunohistochemistry in 93 cervical cancer patient specimens. Down-regulation of IL-33 expression was observed in tumor tissues compared with adjacent tissues. More importantly, IL-33 was detected in the cytoplasm of tumor fraction. IL-33 expression in tumor cytoplasm was associated with tumor size and the invasive depth of tumors (p<0.05). Meanwhile, IL-33 expression in tumor cytoplasm was positively correlated with infiltration of CD3+ T cells, CD8+ T cells, and PD-L1 expression in tumor tissues (p<0.05). The number of TLS strongly correlated with the depth of tumor invasion, preoperative chemotherapy, human papillomavirus infection, and high level of PD-1 (p<0.05). However, there was no significant relationship between IL-33 and TLS. Kaplan-Meier survival curves showed that the formation of TLS was associated with a better prognosis (p=0.008). In multivariable Cox regression modeling, high expression of PD-L1 in tumor tissues was correlated with poor prognosis (HR=0.128; 95% CI: 0.026-0.646; p=0.013), whereas the high expression of IL-33 in tumor tissues was associated with better prognosis (HR=5.097; 95% CI:1.050-24.755; p=0.043). These results indicate that IL-33, TLS, and PD-L1 are potentially valuable prognostic predictor for cervical cancer. IL-33 has potential for combination with PD-L1-related antitumor therapy.

Mucin-type sialyl-Tn antigen is associated with PD-L1 expression and predicts poor clinical prognosis in breast cancer.

A recent study showed that mucin-type sialylated O-linked glycans could induce the increased expression of PD-L1 via binding to Siglec receptors. However, the relationship between the expression of the mucin-type sialyl-Tn antigen (sTn) and PD-L1 remains unclear in breast cancer (BC). Therefore, we investigate the clinicopathological and prognostic effects of sTn expression and its relationship with PD-L1 expression in BC tissues. We retrospectively analyzed the clinical data of 380 invasive BC patients between January 2011 and January 2014. The last follow-up time was January 31, 2019 with a median follow-up of 62 months. The expression of the sTn antigen and PD-L1 in 380 tumor specimens was assessed by immunohistochemistry. Correlations between sTn/PD-L1 expression and clinicopathological features and prognoses were analyzed. In BC tissues, the positive expression rate of PD-L1 (20.5%) was much lower than that of sTn (41.8%). Pearson's contingency analysis showed that sTn and PD-L1 expression in tumor tissues demonstrated a high correlation (P<0.001). High sTn expression was associated with negative ER expression (P<0.001), positive HER-2 status (P<0.001), advanced tumor stage (P<0.001), high density of CD8+ tumor-infiltrating lymphocytes (TILs) (P=0.028), and positive lymph node metastasis (P=0.002). Moreover, patients with concomitant high expression of both markers had the highest risk of relapse (P<0.001) and mortality (P<0.001). The multivariate Cox regression model revealed that positive sTn expression (HRos: 1.941, 95% CI: 1.168, 3.223, Pos=0.028; HRpfs: 1.739, 95% CI: 1.063, 2.847, Ppfs=0.010) and positive PD-L1 expression (HRos: 1.912, 95% CI: 1.138, 3.212, Pos=0.017; HRpfs: 1.863, 95% CI: 1.116, 3.110, Ppfs=0.014) were independent indicators for poor overall survival (OS) and progression-free survival (PFS), respectively. BC patients who expressed both sTn and PD-L1 had poorer survival. Therefore, combinational therapy with dual blockade might benefit BC patients with sTn(+)/PD-L1(+) expression, which requires further examination in future clinical trials.