Investigation of pd-l1 (cd274), pd-l2 (pdcd1lg2), and ctla-4 expressions in malignant pleural mesothelioma by immunohistochemistry and real-time polymerase chain reaction methods.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an aggressive malignant disease with a poor prognosis, which affects the surface mesothelium of the pleural cavity. Immune checkpoints are responsible for controlling the immune system to avoid autoimmunity and prevent tissue damage. In this study, we aimed to investigate the expression of cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2) immuno-control receptors in MPM patients and the relationship of the expression with tumour types and prognostic parameters. MATERIAL AND METHODS: In this study, we evaluated 50 MPM cases. Immunohistochemically CTLA-4, PD-L1, and PD-L2 were detected by using monoclonal anti-CTLA-4, anti-PD-L1, and anti-PD-L2. Real-time polymerase chain reaction (RT-PCR) analysis was performed with the primers CTLA-4, PD-L1, and PD-L2. RESULTS: Statistically, no significant relation was determined between the PD-L1, PD-L2, and CTLA-4 expressions (immunohistochemical and RT-PCR methods) and the MPM histological type. Interestingly significant correlation was observed between the mean survival time and immunohistochemical PD-L2 expression; thus, long-term survival was observed in cases with PD-L2 expression. CONCLUSIONS: Programmed death ligand 1, PD-L2, and CTLA-4 expression were observed in some MPM cases, suggesting that treatments targeting immune checkpoints may be effective. Because immunohistochemical expression of PD-L2 is associated with better prognosis, it may provide useful clues in the follow-up of patients.

Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions.

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell-mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell-mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1-triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.

Soluble programmed cell death protein 1 (sPD-1) and the soluble programmed cell death ligands 1 and 2 (sPD-L1 and sPD-L2) in lymphoid malignancies.

BACKGROUND: The programmed cell death protein 1 (PD-1) and its ligand 1 and 2 (PD-L1/PD-L2) regulate the immune system, and the checkpoint pathway can be exploited by malignant cells to evade anti-tumor immune response. Soluble forms (sPD-1/sPD-L1/sPD-L2) exist in the peripheral blood, but their biological and clinical significance is unclear. METHOD: Time-resolved immunofluorometric assay (TRIFMA) and enzyme-linked immunosorbent assay (ELISA) were used to measure sPD-1, sPD-L1, and sPD-L2 levels in serum from 131 lymphoma patients and 22 healthy individuals. RESULTS: Patients had higher sPD-1 and sPD-L2 levels than healthy individuals. In diffuse large B-cell lymphoma, patients with high International Prognostic Index score had higher sPD-1 levels and sPD-L2 levels correlated with subtype according to cell of origin. Compared to other lymphoma types, follicular lymphoma displayed higher sPD-1 and lower sPD-L1 levels along with lower ligand/receptor ratios. CONCLUSION: This is the first study to simultaneously characterize pretherapeutic sPD-1, sPD-L1, and sPD-L2 in a variety of lymphoma subtypes. The relation between higher sPD-1 levels and adverse prognostic factors suggests a possible biological role and potential clinical usefulness of sPD-1. Moreover, the reverse expression pattern in follicular lymphoma and T-cell lymphoma/leukemia may reflect biological information relevant for immunotherapy targeting the PD-1 pathway.

Programmed cell death-1 blockade in kidney carcinoma may induce eosinophilic granulomatosis with polyangiitis: a case report.

BACKGROUND: Immune checkpoint inhibitors have potential applications in treating various cancers but are associated with immune-related adverse events, such as inflammation, in a wide range of organs; however, allergic inflammation caused by these agents has not been extensively studied. CASE PRESENTATION: A 65-year-old man was diagnosed with a kidney neuroendocrine carcinoma. Three months after kidney resection surgery, the tumor cells had metastasized to his liver and lymph nodes. Subsequently, the patient started chemotherapy; however, regardless of treatment, the tumor grew, and the patient experienced a series of adverse effects, such as taste disorder, anorexia, and general fatigue. Finally, he was administered a programmed cell death (PD)-1 inhibitor, nivolumab (biweekly, toal 200 mg/body), which was effective against kidney carcinoma. However, the patient had a bronchial asthma attack at 22 cycles of nivolumab treatment and chest computed tomography (CT) revealed an abnormal bilateral shadow after 37 cycles of nivolumab treatment. Bronchoscopy findings revealed eosinophil infiltration in the lungs along with severe alveolar hemorrhage. Paranasal sinus CT scanning indicated sinusitis and nerve conduction analysis indicated a decrease in his right ulnar nerve conduction velocity. Based on these findings, the patient was diagnosed with eosinophilic granulomatosis with polyangiitis; he was treated with prednisolone, which alleviated his bronchial asthma. To restart nivolumab treatment, the dose of prednisolone was gradually tapered, and the patient was administered a monthly dose of mepolizumab and biweekly dose of nivolumab. To date, there have been no bronchial attacks or CT scan abnormalities upon follow up. CONCLUSIONS: We present a rare case in which a patient with cancer was diagnosed with eosinophilic granulomatosis with polyangiitis following treatment with a PD-1 inhibitor. Blockade of PD-1 and the programmed cell death ligand (PD-L) 1/PD-1 and PD-L2/PD-1 signaling cascade may cause allergic inflammation. Further studies are needed to identify the specific mechanisms underlying allergic inflammation after PD-1 blockade.

Cisplatin-induced programmed cell death ligand-2 expression is associated with metastasis ability in oral squamous cell carcinoma.

Programmed cell death ligands (PD-Ls) are expressed in tumor cells where they bind to programmed cell death-1, an immunocyte co-receptor, resulting in tumor cell evasion from the immune system. Chemotherapeutic drugs have been recently reported to induce the expression of PD-L, such as PD-L1, in some cancer cells. However, little is known regarding PD-L2 expression and its role in oral squamous cell carcinoma (OSCC). In this study, we examined the effect of cisplatin on the expression and regulation of PD-L2 in OSCC cell lines and analyzed malignant behavior in PD-L2-expressing cells using colony, transwell and transformation assays. In addition, we examined PD-L2 expression in the tumor tissues of OSCC patients using cytology and tissue microarray methods. In OSCC cell lines, cisplatin treatment upregulated PD-L2 expression, along with that of the drug efflux transporter ABCG2, via signal transducers and activator of transcription (STAT) 1/3 activation. Moreover, PD-L2-positive or PD-L2-overexpressing cells demonstrated upregulation in both invasion and transformation ability but not in proliferation compared with PD-L2-negative or PD-L2-silencing cells. PD-L2 expression was also observed in OSCC cells of cytology samples and tissue from OSCC patients. The intensity of PD-L2 expression was correlated with more malignant morphological features in the histological appearance and an invasive pattern. Our findings indicate that cisplatin-upregulated PD-L2 expression in OSCC via STAT1/3 activation and the expression of PD-L2 are likely to be associated with malignancy in OSCC. The PD-L2 expression in cisplatin-resistant OSCC cells may be a critical factor in prognosis of advanced OSCC patients.

Serum levels of soluble programmed cell death protein 1 and soluble programmed cell death protein ligand 2 are increased in systemic lupus erythematosus and associated with the disease activity.

OBJECTIVE: Programmed cell death protein 1 (PD-1) pathway plays important roles in systemic lupus erythematosus (SLE). We aimed to elucidate the association of serum soluble PD-1 (sPD-1) and related molecules with SLE and to explore their usefulness as biomarkers. METHODS: We retrospectively measured the serum levels of sPD-1, soluble PD-ligand 1 (sPD-L1), soluble PD-ligand 2 (sPD-L2) and interleukin (IL)-21 by ELISA in SLE patients, systemic sclerosis patients and healthy controls. Repeat sera samples were also obtained post treatment. RESULTS: The serum levels of sPD-1 and sPD-L2 in SLE patients with high disease activity were significantly higher than those in SLE patients with low disease activity, systemic sclerosis patients and healthy controls (n = 58, 15, 20 and 21, respectively; p < 0.001). However, the serum levels of sPD-L1 and IL-21 were not elevated in SLE patients. The serum levels of sPD-1 and sPD-L2 were higher among active SLE patients who tested positive for anti-dsDNA antibodies than in those who tested negative (p = 0.002 and <0.001, respectively). There were moderate correlations between the serum levels of sPD-1 and sPD-L2 and the SLE Disease Activity Index 2000 scores, the titres of anti-dsDNA antibodies and the serum levels of complements. Furthermore, the serum levels of sPD-1 and sPD-L2 decreased significantly in accordance with disease amelioration following treatment (p < 0.001). CONCLUSION: The present study demonstrated the association of serum sPD-1 and sPD-L2 with SLE and suggests their usefulness as disease activity biomarkers for SLE.

Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract.

Low molecular weight fucoidan extract (LMF), prepared by an abalone glycosidase digestion of a crude fucoidan extracted from Cladosiphon novae-caledoniae Kylin, exhibits various biological activities, including anticancer effect. Various cancers express programmed cell death-ligand 1 (PD-L1), which is known to play a significant role in evasion of the host immune surveillance system. PD-L1 is also expressed in many types of normal cells for self-protection. Previous research has revealed that selective inhibition of PD-L1 expressed in cancer cells is critical for successful cancer eradication. In the present study, we analyzed whether LMF could regulate PD-L1 expression in HT1080 fibrosarcoma cells. Our results demonstrated that LMF suppressed PD-L1/PD-L2 expression and the growth of HT1080 cancer cells and had no effect on the growth of normal TIG-1 cells. Thus, LMF differentially regulates PD-L1 expression in normal and cancer cells and could serve as an alternative complementary agent for treatment of cancers with high PD-L1 expression.

Combined approach for predicting the efficacy of nivolumab in head and neck carcinoma by tissue and soluble expressions of PD-L1 and PD-L2.

BACKGROUND: Predictive biomarkers for nivolumab in recurrent or metastatic head and neck squamous cell carcinoma (RMHNSCC) have not yet been established. METHODS: The tumor proportion score (TPS), combined positive score (CPS), and soluble forms of programmed cell death ligand-1 (PD-L1) and programmed cell death ligand-2 (PD-L2) were retrospectively analyzed in patients with RMHNSCC treated with nivolumab. RESULTS: The positivity rates for TPS (PD-L1), CPS (PD-L1), TPS (PD-L2), and CPS (PD-L2) were 73.8%, 78.2%, 56.4%, and 78.2%, respectively. Patients with high TPS (PD-L1), CPS (PD-L1), or CPS (PD-L1 and PD-L2) showed significantly prolonged progression-free survival. Favorable overall survival was associated with high CPS (PD-L1 and PD-L2) and low soluble PD-L1 and PD-L2 levels. The expressions of tissue and soluble PD-L1/2 were not correlated. CONCLUSIONS: Our study revealed that compared to PD-L1 expression alone, dual expression of PD-L1 and PD-L2 in tissue or soluble form could be feasible biomarkers in patients with RMHNSCC who received nivolumab.

The prognostic significance of PD-1 and its ligands in non-small cell lung cancer.

Background: In this study, we aimed to investigate the prognostic value of programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), and programmed cell death ligand 2 (PD-L2) expressions on immune and cancer cells in terms of survival in patients with lung adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Methods: Between January 2000 and December 2012, a total of 191 patients (172 males, 19 females; mean age: 60.3±8.4 years; range, 38 to 78 years) who were diagnosed with non-small cell lung cancer and underwent anatomic resection and mediastinal lymph node dissection were retrospectively analyzed. The patients were evaluated in three groups including lung squamous cell carcinoma (n=61), adenocarcinoma (n=66), and large-cell carcinoma (n=64). The survival rates of all three groups were compared in terms of immunohistochemical expression levels of PD-1, PD-L1, and PD-L2. Results: The mean follow-up was 71.8±47.9 months. In all histological subtypes, PD-1 expressions on tumor and immune cells were observed in 33% (61/191) and in 53.1% (102/191) of the patients, respectively. Higher expression levels of PD-L1 and PD-L2 at any intensity on tumor and immune cells were defined only in lung adenocarcinomas, and PD-L1 and PD-L2 values were detected in 36.4% (22/64) of these patients. The PD-L1 expressions on tumor and immune cells were observed in 41.7% (10/24) and 25% (6/24) of the patients, respectively. The PD-L2 expressions on tumor and immune cells were detected in 16.7% (4/24) and 8.4% (2/24) of the patients, respectively. Univariate and multivariate analyses revealed that PD-1 expression in tumor cells was an independent prognostic factor in all histological subtypes. Conclusion: Our study results suggest that PD-1 expression is a poor prognostic factor for overall survival in patients with completely resected adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

A Chromosome 9p24.1 Amplification in Colorectal Cancer with Metastases to the Kidney and Adrenal Gland: A Case Report.

Colorectal cancer (CRC) is the third leading cause of mortality worldwide. The Food and Drug Administration recently designated pembrolizumab, an immune checkpoint inhibitor (ICI) against a programmed death-1 receptor, as a breakthrough drug for the treatment of patients with mCRC whose tumors have deficient mismatch-repair gene expression (as evidenced by microsatellite instability-high) and patients with solid tumors with a high tumor mutational burden with ≥10 mutations/megabase. We present a patient with metastatic CRC having renal and adrenal gland metastases. Comprehensive molecular profiling performed on a site of metastatic CRC in the kidney revealed multiple genomic alterations characteristic of CRC and rare chromosome 9p24.1 amplification, resulting in a co-amplification of the PDL1, PDL2, and JAK2 genes. Although this genomic alteration may predict the response to ICI, the lack of pembrolizumab prevented the patient from receiving targeted treatment and succumbing to the disease.

Clinical significance of programmed cell death-ligand expression in small bowel adenocarcinoma is determined by the tumor microenvironment.

BACKGROUND: Comprehensive genomic analysis has shown that small bowel adenocarcinoma (SBA) has different genomic profiles from gastric and colorectal cancers. Hence, it is essential to establish chemotherapeutic regimens based on SBA characteristics. The expression of programmed cell death-ligand 1 (PD-L1) and programmed cell death-ligand 2 (PD-L2) in SBA is not fully understood. Anti-PD-L1/PD-1 therapy uses tumor-infiltrating lymphocytes (TILs); therefore, the status of TILs in the tumor microenvironment (TME) may influence their efficacy. The ratio of FoxP3+ to CD8+ T cells has been reported to be useful in predicting the prognosis of digestive system cancers. AIM: To investigate the clinicopathological significance of PD-L1/2 expression according to the status of TILs in SBA tissues. METHODS: We performed immunohistochemical analysis for PD-L1, PD-L2, CD8, FoxP3, and DNA mismatch repair (MMR) proteins using formalin-fixed, paraffin-embedded tissues from 50 patients diagnosed with primary SBA. The immunoreactivities of PD-L1 and PD-L2 were determined separately in tumor cells and tumor-infiltrating immune cells throughout the tumor center and invasive margins, and finally evaluated using the combined positive score (CPS). We assessed CD8+ and FoxP3+ T cells in the intratumoral and tumor-surrounding stroma. Subsequently, we calculated and summed the ratio of FoxP3 to CD8+ T cell counts. Immune-related cell densities were graded as low or high. Immunohistochemical results were compared with clinicopathological factors and patient prognosis. The distribution of cancer-specific survival (CSS) was estimated using the Kaplan-Meier method, and the log-rank test was used to test for significant differences in CSS. A Cox proportional hazard model was also used to assess the effect of tumor variables on CSS. RESULTS: PD-L1 expression was positive in 34% in tumor cells (T-PD-L1) and 54% in tumor-infiltrating immune cells (I-PD-L1) of the cases examined. T-PD-L2 was positive in 34% and I-PD-L2 was positive in 42% of the cases. PD-L1 CPS ≥ 10 and PD-L2 CPS ≥ 10 were observed in 50% and 56% of the cases, respectively. Deficient MMR (dMMR) was 14% of the cases. T-PD-L1, I-PD-L1 and PD-L1 CPS ≥ 10 were all significantly associated with dMMR (P = 0.037, P = 0.009, and P = 0.005, respectively). T-PD-L1, I-PD-L1, and PD-L1 CPS ≥ 10 were all associated with deeper depth of invasion (P = 0.001, P = 0.024, and P = 0.002, respectively). I-PD-L2 expression and PD-L2 CPS ≥ 10 were significantly higher in the differentiated histological type (P = 0.015 and P = 0.030, respectively). The I-PD-L1 and I-PD-L2 levels were significantly associated with better CSS (P = 0.037 and P = 0.015, respectively). CD8-high was significantly associated with less lymph node metastasis (P = 0.047), less distant metastasis (P = 0.024), less peritoneal dissemination (P = 0.034), and earlier TNM stage (P = 0.047). The CD8-high group had better prognosis than the CD8-low group (P = 0.018). FoxP3 expression was not associated with any clinicopathological factors or prognosis. We found that patients with PD-L2 CPS ≥ 10 tended to have worse prognosis in the FoxP3/CD8-low group (P = 0.088). CONCLUSION: The clinicopathological significance of PD-L1/2 expression may differ depending on the TME status. Immune checkpoint inhibitors may improve the prognosis of SBA patients with low FoxP3/CD8 ratio and PD-L2 expression.

Efficacy and Safety of PD-1/PD-L1 Inhibitor as Single-Agent Immunotherapy in Endometrial Cancer: A Systematic Review and Meta-Analysis.

The programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway plays a crucial role in the immune escape mechanism and growth of cancer cells in endometrial cancer (EC). Clinical trials investigating PD-1/PD-L1 inhibitor have shown promising results in other cancers, but their efficacy in EC still remains uncertain. Therefore, this meta-analysis aims to provide an updated and robust analysis of the effectiveness and safety of PD-1/PDL1 inhibitor as single-agent immunotherapy in EC, focusing on the objective response rate (ORR), disease control rate (DCR), and adverse events (AEs). This meta-analysis utilized STATA version 17 and RevMan version 5.4 software to pool the results of relevant studies. Five studies conducted between 2017 and 2022, comprising a total of 480 EC patients enrolled for PD-1/PD-L1 inhibitor immunotherapy met the inclusion criteria. The pooled proportion of EC patients who achieved ORR through PD-1/PD-L1 inhibitor treatment was 26.0% (95% CI: 16.0-36.0%; p < 0.05). Subgroup analysis based on mismatch repair (MMR) status showed an ORR of 44.0% (95% CI: 38.0-50.0%; p = 0.32) for the deficient mismatch repair (dMMR) group and 8.0% (95% CI: 0.0-16.0%; p = 0.07) for the proficient mismatch repair (pMMR) group. Pooled proportion analysis by DCR demonstrated an odds ratio (OR) of 41.0% (95% CI: 36.0-46.0%, p = 0.83) for patients undergoing PD-1/PD-L1 inhibitor treatment. Subgroup analysis based on MMR status revealed DCR of 54.0% (95% CI: 47.0-62.0%; p = 0.83) for the dMMR group, and 31.0% (95% CI: 25.0-39.0%; p = 0.14) for the pMMR group. The efficacy of PD-1/PD-L1 inhibitors was significantly higher in the dMMR group compared to the pMMR group, in terms of both ORR (OR = 6.30; 95% CI = 3.60-11.03; p < 0.05) and DCR (OR = 2.57; 95% CI = 1.66-3.99; p < 0.05). In terms of safety issues, the pooled proportion of patients experiencing at least one adverse event was 69.0% (95% CI: 65.0-73.0%; p > 0.05), with grade three or higher AEs occurring in 16.0% of cases (95% CI: 12.0-19.0%; p > 0.05). Based on the subgroup analysis of MMR status, PD-1/PD-L1 inhibitor immunotherapy showed significantly better efficacy among dMMR patients. These findings suggest that patients with dMMR status may be more suitable for this treatment approach. However, further research on PD-1/PD-L1 inhibitor immunotherapy strategies is needed to fully explore their potential and improve treatment outcomes in EC.

Programmed cell death 1 and programmed cell death ligand 1 expression in invasive breast carcinoma using CAL10 and NAT105 immunostaining.

Increased incidence of breast cancer has stimulated development of new diagnostic and therapeutic methods. The programmed cell death 1 (PD1) pathway and its inhibitors are promising avenues for investigation. PD1 includes PD ligands 1 (PDL1) and 2 (PDL2). We investigated the expression of PD1 and PDL1 in invasive breast carcinomas using immunohistochemical staining. We used 171 invasive breast carcinoma specimens from which tissue microarray blocks were created. Immunohistochemical staining of PD1 using NAT105, and PDL1 using CAL10 was performed on tissue microarray sections. NAT105 and CAL10 are useful clones for detecting expression of PD1 and PDL1. PD1 and PDL1 immunostaining was significantly stronger in carcinomas with basal-like phenotype compared to other molecular breast cancer types. PD1 and PDL1 expression also was associated with a high histologic grade and a high Ki-67 index. PD1 expression also was associated with lymphovascular invasion and axillary metastasis. PD1 and PDL1 expression is associated with aggressive tumor behavior and a basal-like phenotype in breast cancer. We suggest that inhibition of the PD1/PDL1 pathway, particularly in triple negative breast carcinomas with basal-like phenotype, might be useful for targeted immunotherapy.

PD-L2 Is Constitutively Expressed in Normal and Malignant Urothelium.

The use of immune checkpoint blockade, in particular PD-1 and PD-L1 inhibitors, is now commonplace in many clinical settings including the treatment of muscle-invasive bladder cancer (MIBC). Notwithstanding, little information exists regarding the expression of the alternative PD-1 ligand, PD-L2 in urothelial bladder cancer (UBC). We therefore set out to characterise the expression of PD-L2 in comparison to PD-L1. Firstly, we assessed PD-L2 expression by immunohistochemistry and found widespread expression of PD-L2 in UBC, albeit with reduced expression in MIBC. We further investigated these findings using RNA-seq data from a cohort of 575 patients demonstrating that PDCD1LG2 (PD-L2) is widely expressed in UBC and correlated with CD274 (PD-L1). However, in contrast to our immunohistochemistry findings, expression was significantly increased in advanced disease. We have also provided detailed evidence of constitutive PD-L2 expression in normal urothelium and propose a mechanism by which PD-L2 is cleaved from the cell surface in MIBC. These data provide a comprehensive assessment of PD-L2 in UBC, showing PD-L2 is abundant in UBC and, importantly, constitutively present in normal urothelium. These data have implications for future development of immune checkpoint blockade, and also the understanding of the function of the immune system in the normal urinary bladder.

The influence of programmed cell death ligand 2 (PD-L2) expression on survival outcome and tumor microenvironment in diffuse large B cell lymphoma.

The frequency and significance of programmed cell death ligand (PD-L) 2 expression in diffuse large B cell lymphoma (DLBCL) remain undefined. We described the expression pattern of PD-L/PD-1 in 88 DLBCL patients using immunohistochemistry. The association between PD-L expression and clinical characteristics/outcomes were analyzed. PD-L1 and PD-L2 were expressed in 14.8% and 68.2% of DLBCL patients with median positivity on tumor cells of 100% and 90%, respectively. PD-1 on tumor-infiltrating lymphocytes (TILs) was expressed in 12.5% of patients. Interestingly, 45.5% of patients had PD-L2 expressing TILs which were significantly associated with bulky disease (p = .046) and elevated lactate dehydrogenase (p = .048). PD-L1 and/or PD-L2 expression on lymphoma cells was associated with inferior progression-free survival (Hazard ratio [HR] 2.20; 95% Confidence Interval [CI] 1.004-4.84, p = .049) and overall survival (HR 2.27; 95%CI 1.03-4.98, p = .042), using multivariate analysis. In summary, PD-L2 expression on DLBCL is common and, together with PD-L1, were related to poor outcomes.

PD-L1 and PD-L2 Expression in Cervical Cancer: Regulation and Biomarker Potential.

PD-1/PD-L1 immune checkpoint inhibitors show potential for cervical cancer treatment. However, low response rates suggest that patient selection based on PD-L1 protein expression is not optimal. Here, we evaluated different PD-L1 detection methods and studied transcriptional regulation of PD-L1/PD-L2 expression by The Cancer Genome Atlas (TCGA) mRNAseq analysis. First, we determined the copy number of the PD-L1/PD-L2 locus by fluorescence in situ hybridization (FISH), PD-L1 mRNA expression by RNA in situ hybridization (RNAish), and PD-L1/PD-L2 protein expression by immunohistochemistry (IHC) on tissue microarrays containing a cohort of 60 patients. Additionally, distribution of PD-L1/PD-L2 was visualized based on flow cytometry analysis of single-cell suspensions (n = 10). PD-L1/PD-L2 locus amplification was rare (2%). PD-L1 mRNA expression in tumor cells was detected in 56% of cases, while 41% expressed PD-L1 protein. Discordant scores for PD-L1 protein expression on tumor cells between cores from one patient were observed in 27% of cases. Interestingly, with RNAish, PD-L1 heterogeneity was observed in only 11% of the cases. PD-L2 protein expression was found in 53%. PD-L1 mRNA and protein expression on tumor cells were strongly correlated (p < 0.001). PD-L1 and PD-L2 protein expression showed no correlation on tumor cells (p = 0.837), but a strong correlation on cells in stromal fields (p < 0.001). Co-expression of PD-L1 and PD-L2 on macrophage-like populations was also observed with flow cytometry analysis. Both PD-L1 and PD-L2 TCGA transcript levels strongly correlated in the TCGA data, and both PD-L1 and PD-L2 strongly correlated with interferon gamma (IFNG) expression/transcript levels (p < 0.0001). Importantly, patients with high PD-L1/PD-L2/IFNG transcript levels had a survival advantage over patients with high PD-L1/PD-L2 and low IFNG expression. Based on these findings, we conclude that PD-L1/PD-L2 expression in cervical cancer is mainly associated with interferon induction and not gene amplification, which makes FISH unsuitable as biomarker. The heterogeneous PD-L1 and PD-L2 expression patterns suggest IHC unreliable for patient selection. RNAish, in conjunction with interferon signaling evaluation, seems a promising technique for immune checkpoint detection. These results warrant further investigation into their prognostic and predictive potential.

Aberrant PD-1 ligand expression contributes to the myocardial inflammatory injury caused by Coxsackievirus B infection.

Coxsackievirus group B (CVB) is considered as one of the most common pathogens of human viral myocarditis. CVB-induced myocarditis is mainly characterized by the persistence of the virus infection and immune-mediated inflammatory injury. Costimulatory signals are crucial for the activation of adaptive immunity. Our data reveal that the CVB type 3 (CVB3) infection altered the expression profile of costimulatory molecules in host cells. CVB3 infection caused the decrease of PD-1 ligand expression, partially due to the cleavage of AU-rich element binding protein AUF1 by the viral protease 3Cpro, leading to the exacerbated inflammatory injury of the myocardium. Moreover, systemic PD-L1 treatment, which augmented the apoptosis of proliferating lymphocytes, alleviated myocardial inflammatory injury. Our findings suggest that PD1-pathway can be a potential immunologic therapeutic target for CVB-induced myocarditis.

A case of nivolumab-induced isolated adrenocorticotropic hormone (ACTH) deficiency.

One of the novel PD-1 antibodies/immune checkpoint inhibitors, nivolumab is reported to be associated with a wide range of immune-related adverse events (irAEs). We hereby report a case of isolated adrenocorticotropic hormone (ACTH) deficiency developing in a patient with squamous cell lung cancer (SCC) during nivolumab therapy. CASE: A 79-year-old man with SCC was started on nivolumab therapy as a fifth-line treatment after 4 lines of cytotoxic anticancer therapy. After 20 courses of nivolumab therapy, he had nausea, appetite loss, and difficulty walking. A close laboratory examination led to the diagnosis of isolated ACTH deficiency in this patient. Hydrocortisone replacement therapy led to amelioration of his symptoms and allowed him to continue with nivolumab therapy. The present case of isolated ACTH deficiency was characterized by a slowly progressive decline in the serum sodium level, which became manifest well before appearance of any clinical symptoms, suggesting that the serum sodium level may be used to predict progression to isolated ACTH deficiency.Thus, not only serum sodium levels need to be monitored in patients suspected of having isolated ACTH deficiency, but ACTH and cortisol levels need to be monitored in those exhibiting a decline in serum sodium levels. Again, nivolumab-induced isolated ACTH deficiency needs to be appropriately diagnosed and treated to ensure that patients continue with, and maximize survival benefit from, nivolumab therapy.

Intrinsic and Extrinsic Regulation of PD-L2 Expression in Oncogene-Driven Non-Small Cell Lung Cancer.

INTRODUCTION: The interaction of programmed cell death ligand 2 (PD-L2) with programmed cell death 1 is implicated in tumor immune escape. The regulation of PD-L2 expression in tumor cells has remained unclear, however. We here examined intrinsic and extrinsic regulation of PD-L2 expression in NSCLC. METHODS: PD-L2 expression was evaluated by reverse transcription and real-time polymerase chain reaction analysis and by flow cytometry. RESULTS: BEAS-2B cells stably expressing an activated mutant form of EGFR or the echinoderm microtubule associated protein like 4 (EML4)-ALK receptor tyrosine kinase fusion oncoprotein manifested increased expression of PD-L2 at both the mRNA and protein levels. Furthermore, treatment of NSCLC cell lines that harbor such driver oncogenes with corresponding EGFR or ALK tyrosine kinase inhibitors or depletion of EGFR or ALK by small interfering RNA transfection suppressed expression of PD-L2, demonstrating that activating EGFR mutations or echinoderm microtubule associated protein like 4 gene (EML4)-ALK receptor tyrosine kinase gene (ALK) fusion intrinsically induce PD-L2 expression. We also found that interferon gamma (IFN-γ) extrinsically induced expression of PD-L2 through signal transducer and activator of transcription 1 signaling in NSCLC cells. Oncogene-driven expression of PD-L2 in NSCLC cells was inhibited by knockdown of the transcription factors signal transducer and activator of transcription 3 (STAT3) or c-FOS. IFN-γ also activated STAT3 and c-FOS, suggesting that these proteins may also contribute to the extrinsic induction of PD-L2 expression. CONCLUSIONS: Expression of PD-L2 is induced intrinsically by activating EGFR mutations or EML4-ALK fusion and extrinsically by IFN-γ, with STAT3 and c-FOS possibly contributing to both intrinsic and extrinsic pathways. Our results thus provide insight into the complexity of tumor immune escape in NSCLC.

PD-L2 Expression as a Potential Predictive Biomarker for the Response to Anti-PD-1 Drugs in Patients with Non-small Cell Lung Cancer.

BACKGROUND/AIM: To investigate the role of programmed cell death-ligand 2 (PD-L2) expression as a predictive biomarker for response to anti-programmed cell death-1 (PD-1) drugs in patients with non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Ten patients who had undergone curative lung resection and received the anti-PD-1 drugs for the recurrence were enrolled. The cut-off value for PD-L2 (antibody clone 176611) expression on tumor cells was set at 50%. Tumor response was evaluated according to immune-related response criteria. RESULTS: Seven patients (70.0%) were positive for PD-L2. The response rates were 28.6% (2/7) and 33.3% (1/3) in patients with PD-L2-positive and PD-L2-negative NSCLC, respectively. Disease control was obtained in 2 patients despite the programmed cell death-ligand 1 (PD-L1)-negativity (antibody clone 22C3: 0%, antibody clone SP142: 0%), and these tumors expressed PD-L2 (≥1%). CONCLUSION: PD-L2 expression may be a target of immunotherapy in patients with PD-L1-negative NSCLC.