PDCD1LG2 Expression Research Articles

Genomic Profiling of Rare Undifferentiated Sarcomatoid Subtypes of Pancreatic Carcinomas: In Search of Therapeutic Targets

PURPOSE The highly aggressive undifferentiated sarcomatoid carcinoma (USC) subtype of pancreatic ductal adenocarcinoma (PDAC) remains poorly characterized because of its rarity. Previous case reports suggest that immune checkpoint inhibitors could be a promising treatment strategy, but the prevalence of established predictive biomarkers of response is largely unknown. The objective of this study was to leverage comprehensive genomic profiling of USC PDAC tumors to determine the prevalence of biomarkers associated with potential response to targeted therapies. METHODS USC tumors (n = 20) underwent central pathology review by a board-certified gastrointestinal pathologist to confirm the diagnosis. These samples were compared with non-USC PDAC tumors (N = 5,562). Retrospective analysis of DNA and RNA next-generation sequencing data was performed. RESULTS USC PDACs were more frequently PD-L1+ by immunohistochemistry than non-USC PDAC (63% v 16%, respectively, P < .001). Furthermore, USC PDAC had an increase in neutrophils (8.99% v 5.55%, P = .005) and dendritic cells (1.08% v 0.00%, q = 0.022) and an increased expression of PDCD1LG2 (4.6% v 1.3%, q = 0.001), PDCD1 (2.0% v 0.8%, q = 0.060), and HAVCR2 (45.9% v 21.7%, q = 0.107) than non-USC PDAC. Similar to non-USC PDAC, KRAS was the most commonly mutated gene (86% v 90%, respectively, P = 1). CONCLUSION To our knowledge, this work represents the largest molecular analysis of USC tumors to date and showed an increased expression of immune checkpoint genes in USC tumors. These findings provide evidence for further investigation into immune checkpoint inhibitors in USC tumors.

Diagnostic Values of METTL1-Related Genes and Immune Characteristics in Systemic Lupus Erythematosus.

Methyltransferase like 1 (METTL1) regulates epitranscriptomes via the m7G modification in mammalian mRNA and microRNA. Systemic lupus erythematosus (SLE) is caused by abnormal immune reactivity and has diverse clinical manifestations. RNA methylation as a mechanism to regulate gene expression is widely implicated in immune regulation. However, the role of m7G in immune response of SLE has not been extensively studied. Expression of METTL1 was identified in the public dataset GSE122459 and validated in an independent cohort of SLE patients. We investigated the association between METTL1-expression and clinical manifestations of SLE. Subsequently, differentially expressed genes (DEG) that were correlated with METTL1-expression in GSE122459 were used for functional enrichment analysis. The correlation between infiltrating immune cells and METTL1, as well as candidate biomarkers identified to be correlated with either METTL1 or immune cell infiltration were assessed by single-sample GSEA. Potential mechanisms were explored with Gene ontology and KEGG pathway enrichment. Diagnostic performances of candidate biomarkers in SLE were analyzed. The mRNA and protein expression of METTL1 in SLE patients were significantly decreased in both datasets. METTL1-coexpressed DEGs were enriched in several key immune-related pathways. Activated CD8 T cells, activated CD4 T cells, memory B cells and type 2 helper T cells were different between patients with high and low METTL1 expression. Further, activated CD8 T-cells, activated CD4 T-cells, memory B-cells were correlated with METTL1. The genes of LAMP3, CD83, PDCD1LG2, IGKVD3D-20, IGKV5-2, IGKV2D-30, IGLV3-19 and IGLV4-60 were identified as candidate targets that were correlated with immune cell proportion. Moreover, LAMP3, CD83, and PDCD1LG2 expression were of diagnostic value in SLE as indicated by ROC analysis. Our findings suggested that METTL1 and its candidate targets LAMP3, CD83, PDCD1LG2 may be used for diagnosing SLE and could be explored for developing targeted molecular therapy for SLE.

PD-L1 and PD-L2 immune checkpoint protein induction by type III interferon in non-small cell lung cancer cells

IntroductionDespite the clinical success of PD-1/PD-1-ligand immunotherapy in non-small cell lung cancer (NSCLC), the appearance of primary and acquired therapy resistance is a major challenge reflecting that the mechanisms regulating the expression of the PD-1-ligands PD-L1 and PD-L2 are not fully explored. Type I and II interferons (IFNs) induce PD-L1 and PD-L2 expression. Here, we examined if PD-L1 and PD-L2 expression also can be induced by type III IFN, IFN-λ, which is peculiarly important for airway epithelial surfaces. MethodsIn silico mRNA expression analysis of PD-L1 (CD274), PD-L2 (PDCD1LG2), and IFN- λ signaling signature genes in NSCLC tumors and cell lines was performed using RNA sequencing expression data from TCGA, OncoSG, and DepMap portals. IFN-λ-mediated induction of PD-L1 and PD-L2 expression in NSCLC cell lines was examined by real-time quantitative polymerase chain reaction and flow cytometry. ResultsIFNL genes encoding IFN- λ variants are expressed in the majority of NSCLC tumors and cell lines along with the IFNLR1 and IL10R2 genes encoding the IFN-λ receptor subunits. The expression of PD-L1 and PD-L2 mRNA is higher in NSCLC tumors with IFNL mRNA expression compared to tumors without IFNL expression. In the NSCLC cell line HCC827, stimulation with IFN-λ induced both an increase in PD-L1 and PD-L2 mRNA expression and cell surface abundance of the corresponding proteins. In the NSCLC cell line A427, displaying a low basal expression of PD-L1 and PD-L2 mRNA and corresponding proteins, stimulation with IFN-λ resulted in an induction of the former. ConclusionThe type III IFN, IFN- λ, is capable of inducing PD-L1 and PD-L2 expression, at least in some NSCLC cells, and this regulation will need acknowledgment in the development of new diagnostic procedures, such as gene expression signature profiles, to improve PD-1/PD-1-ligand immunotherapy in NSCLC.

Comparative analysis of the molecular profile and tumor immune microenvironment (TIME) of human epidermal growth factor receptor 2 (HER2) low (L)- versus high (H)-expressing gastroesophageal cancers (GEC).

287 Background: Addition of immune checkpoint blockade to anti-HER2 therapy has improved outcomes in HER2-positive GEC. Anti-HER2 antibody-drug conjugates have shown activity in some HER2-L tumors in other tumor types. We aimed to compare the molecular profile and TIME of HER2-L and HER-H GEC. Methods: 8678 GEC (gastric, GE junction, and esophageal) adenocarcinoma and squamous cell carcinoma samples were analyzed by next-generation sequencing (NGS) of RNA (whole transcriptome, NovaSeq), DNA (592 genes, NextSeq, or whole exome sequencing, NovaSeq), and immunohistochemistry (IHC, Caris Life Sciences, Phoenix, AZ). Cohorts were stratified by IHC HER2 values of 0 (non-expressors), 1-2+ (HER2-L), or 3+ (HER2-H) and compared using X2 or Fisher-Exact. Statistical significance was determined as P-value adjusted for multiple comparisons (q < 0.05). Microenvironment cell population (MCP) counter was used to quantify immune cell infiltration. Results: Tumor were grouped into HER2 non-expressors (N = 5217), L (N = 2660), and H (N = 801). Mutations of TP53 (72% vs 92%) and amplification of MYC (4% vs 7%), CCNE1 (7% vs 12%), CCND3 (2% vs 4%), CDK6 (3% vs 6%), SMARCE1 (2% vs 25%) and RARA (3% vs 24%) were significantly lower in HER2-L compared to HER2-H (q < 0.05). ARID1A (14% vs 9%), PIK3CA (8% vs 3%), KRAS (9% vs 2%), GNAS (2% vs 0.3%), KMT2D (6% vs 1%), CDH1 (5% vs 1%), and ATM (4% vs 1%) mutations were significantly higher in HER2-L compared to HER2-H (q < 0.05). HER2-L was associated with more TMB-H (9.3% vs. 5%; q<0.05), MSI-H (4.7% vs. 0.6%; q<0.05), and a trend towards higher PD-L1 expression than in HER2-H (80.8% vs 76.1%, p < 0.05 but q > 0.05). Immuno-oncology (IO)-related gene expression inversely correlated with HER2 expression with lowest expression of PDCD1LG2, CD274, CTLA4, PDCD1, HAVCR2, CD80, IFNG, LAG3, and CD86 in HER2-H (q < 0.05). HER2-L had significantly higher median immune infiltration of B cells (fold change [FC]: 1.22), T cells (FC: 1.16), CD8+ T cells (FC: 1.56), NK cells (FC: 1.12), neutrophils (FC: 1.10), cytotoxic lymphocytes (FC: 1.36), and myeloid dendritic cells (FC: 1.34), compared to HER2-H (q < 0.05). HER2 non-expressors showed similar immune cell infiltrates compared to HER2-H. HER2-H was associated with lower T-cell inflamed scores and IFN gamma signature when compared to HER2-L and non-expressors (q < 0.05). Conclusions: To our knowledge, this study is the first and largest comparison of molecular profile and TIME in HER2-expressing GEC. We demonstrated distinct molecular and TIME profiles with higher immunogenic profiles in HER-L as compared to HER2-H. IO-related gene expression and TIME cell distribution differences in HER2-H GEC suggest that response to IO and HER2 therapy combinations is likely related to HER2-targeted treatment effect on TIME rather than baseline immunogenicity of the tumor.

Integrative study reveals the prognostic and immunotherapeutic value of CD274 and PDCD1LG2 in pan-cancer.

Background: Disorders of CD274 and PDCD1LG2 contribute to immune escape in human cancers, and treatment with anti-programmed death receptor 1 (PD-1) has been widely used in recurrent or metastatic tumors. However, integrated studies considering CD274 and PDCD1LG2 across cancers remain limited. Materials and Methods: Differences in expression levels of CD274 and PDCD1LG2 were analyzed in diverse cancer types using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. The clinical information and matched expression profiles of TCGA patients were obtained to determine the prognostic value of CD274 and PDCD1LG2. Moreover, correlations between CD274 and PDCD1LG2 and the immune signature were analyzed by exploring the TIMER2 and TISIDB databases. We also investigated correlations between CD274 and PDCD1LG2 and immunotherapeutic biomarkers, including mismatch repair (MMR), tumor mutation burden (TMB), microsatellite instability (MSI), and DNA methylation. Results: Expression levels of CD274 and PDCD1LG2 varied across multiple cancer types. CD274 and PDCD1LG2 not only impacted the prognosis of patients with cancer but were associated with clinical characteristics (lymph node metastasis, tumor stage, and sex) in kidney renal papillary cell carcinoma, thyroid carcinoma, and some other cancer types. Typically, CD274 and PDCD1LG2 could be strongly correlated with macrophages, dendritic cells, neutrophils, and CD8+ T-cells. Furthermore, CD274 and PDCD1LG2 expression were associated with various immunosuppressive biomarkers, such as CTLA4, TIGIT, and LAG3. In addition, CD274 and PDCD1LG2 were significantly associated with MMR, TMB, MSI, and DNA methylation. Finally, enrichment analysis confirmed that CD274 and PDCD1LG2 were associated with numerous biological pathways, such as: “Activation of Immune Reactions” and “Epithelial-Mesenchymal Transition,” suggesting that CD274 and PDCD1LG2 play crucial roles in cancer immunity and tumor metastasis. Conclusion: CD274 and PDCD1LG2 play critical roles in cancer progression and immune response and could serve as effective biomarkers to predict the prognosis and immune signature of cancer.

Abstract 761: Pan-cancer landscape and impact of PD-L1 and PD-L2 structural variation

Abstract Purpose PD-1 receptor and ligand interactions are the target of immunotherapies across 16 cancer types. Biomarkers that predict response to immunotherapy are microsatellite instability, tumor mutational burden and PD-L1 immunohistochemistry. Structural variations in PD-L1 (CD274) and PD-L2 (PDCD1LG2) have been observed across cancer, but the full landscape is unknown. Here we describe the genomic landscape of PD-L1 and PD-L2 structural variation, their potential impact on the tumor microenvironment and evidence that patients with these alterations can benefit from immunotherapy. Methods We analyzed sequencing data from cancer cases with PD-L1 (CD274) and PD-L2 (PDCD1LG2) structural variations across published data, The Cancer Genome Atlas (TCGA) and the Oncology Research Information Exchange Network. From TCGA we obtained copy number status through cBioPortal, gene expression using TCGABiolinks and PD-L1 structural variations identified by Kataoka et al. Gene fusions were detected in ORIEN using STAR-Fusion and Arriba. To evaluate immune signature enrichment we ran software ImSig on gene expression data, using Mann-Whitney tests to determine significant results. We curated literature of patients with structural variations in PD-L1 or PD-L2 receiving PD-1 immunotherapy. Findings From 18 studies and datasets we curated 319 cases with structural variations in PD-L1 and PD-L2. We observed breakpoint ‘hotspots’ in the untranslated regions (UTRs) of both genes including 70 duplications, 48 deletions, 78 inversions and 106 translocations. Leveraging TCGA, we found that PD-L1 amplified tumors had significantly upregulated PD-L1 expression and signatures for interferon signaling and immune cell proliferation, compared to PD-L1 copy neutral tumors, each p < 0.001. Similarly, in PD-L1 rearranged tumors we observed upregulation in PD-L1 expression and signatures for interferon signaling, macrophages, monocytes, T cells and immune cell proliferation, all p < 0.001 compared to PD-L1 copy neutral tumors. Further, retrospective review of 7 studies including patients with structural variations in PD-L1 or PD-L2 revealed >50% (43/77) response rate to immunotherapy. Implications Our evaluation of PD-L1 and PD-L2 structural variations show that the 3’ UTR is affected in hotspots involving a variety of structural variations. Our findings from TCGA suggest PD-L1 structural variation may play a role in driving expression of PD-L1 and immune dysregulation. Enriched interferon signaling in PD-L1 rearranged tumors is of interest as interferon exposure is known to drive PD-L1 and PD-L2 expression. Retrospective evidence from curated studies suggest that this genomic alteration could help identify candidates for PD-1 inhibitors. Based on these findings we propose further study to optimize detection of PD-L1 and PD-L2 structural variation in cancer and design of a pan-cancer prospective clinical trial to target these alterations. Citation Format: Emily L. Hoskins, Eric Samorodnitsky, Michele R. Wing, Julie W. Reeser, Julia Hopkins, Karthikeyan Murugesan, Zheng Kuang, Leah Stein, Zach Risch, Raven Vella, Serifat Adebola, Lianbo Yu, Anoosha Paruchuri, Richard S. Huang, Lee A. Albacker, Sameek Roychowdhury. Pan-cancer landscape and impact of PD-L1 and PD-L2 structural variation [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 761.

Pan-cancer landscape of CD274 (PD-L1) and PDCD1LG2 (PD-L2) structural variations.

3133 Background: PD-1 receptor and PD-L1 ligand interactions are the target of immunotherapies across multiple tumor types. Established biomarkers that predict response to immunotherapy are microsatellite instability, tumor mutational burden, and PD-L1 immunohistochemistry. Structural variations in CD274 (PD-L1) and PDCD1LG2 (PD-L2) have been observed in cancer, but the comprehensive landscape is unknown. Here we describe the genomic landscape of CD274 and PDCD1LG2 structural variations, their potential impact on the tumor microenvironment, and evidence that patients with these alterations can benefit from immunotherapy. Methods: We analyzed sequencing data from 514 cancer cases with CD274 and PDCD1LG2 structural variations across 25 publications and data sources, including large pan-cancer sources: Foundation Medicine, Inc (FMI), The Cancer Genome Atlas (TCGA), and Oncology Research Information Exchange Network. To evaluate immune signature enrichment, we ran the software ImSig on gene expression data. We curated literature reporting clinical outcomes of patients harboring structural variations in CD274 and PDCD1LG2. Results: From 25 studies and datasets, we curated 514 cancer cases with structural variations in PD-L1 and PD-L2, including 158 duplications, 126 deletions, 97 inversions, 178 translocations, and 96 unclassified structural variations, totaling to 655 events. We observed breakpoint ‘hotspots’ in the 3’-untranslated regions (UTRs) of PD-L1 and PD-L2. Leveraging TCGA data, we observed, in CD274-rearranged tumors, significant upregulation in PD-L1 and PD-L2 expression and signatures for interferon signaling, macrophages, monocytes, T cells, and immune cell proliferation (each p < 0.001, compared to CD274 non-rearranged, copy neutral tumors). Furthermore, retrospective review of 12 studies that included patients with structural variations in CD274 or PDCD1LG2, including duplications, inversions and copy number amplifications, revealed a 73% (52/71) response rate to PD-1 immunotherapy with durable responses. Conclusions: Our evaluation of CD274 and PDCD1LG2 structural variations shows that the 3’-UTR is frequently affected and is associated with increased expression of ligands and immune signatures. Enriched interferon signaling in CD274-rearranged tumors is of particular interest, as interferon exposure is known to drive PD-L1 and PD-L2 expression. Retrospective evidence from curated studies suggests that these genomic alterations could identify candidates for PD-1 or PD-L1 immunotherapy. We expect that these findings will better define CD274 and PDCD1LG2 structural variations in cancer and support our pan-cancer prospective clinical trial to target these alterations.

The Aging-Related Prognostic Signature Reveals the Landscape of the Tumor Immune Microenvironment in Head and Neck Squamous Cell Carcinoma.

BackgroundNumerous studies have shown that the aging microenvironment played a huge impact on tumor progression. However, the clinical prognostic value of aging-related risk signatures and their effects on the tumor immune microenvironment (TIME) in head and neck squamous cell carcinoma (HNSCC) remains largely unclear. This study aimed to identify novel prognostic signatures based on aging-related genes (AGs) and reveal the landscape of the TIME in HNSCC.MethodsDifferentially expressed AGs were identified using the gene set enrichment analysis (GSEA). The prognostic risk model of AGs was established by univariate and multivariate Cox regression and least absolute shrinkage and selection operator (LASSO) regression analyses. The independent prognostic value of the risk model and the correlations of the prognostic signature with immune score, tumor immune cell infiltration, and immune checkpoints were systematically analyzed.ResultsA prognostic risk model of four AGs (BAK1, DKK1, CDKN2A, and MIF) was constructed and validated in the training and testing datasets. Kaplan–Meier curves and time-dependent receiver operating characteristic (ROC) curve analysis confirmed that the four-AG risk signature possessed an accurate predictive value for the prognosis of patients with HNSCC. Correlation analysis revealed that the risk score was negatively associated with immune score and immune cell infiltration level while positively correlated with immune checkpoint blockade (ICB) response score. Patients of the high-risk subtype contained higher infiltration levels of resting natural killer (NK) cells, M0 macrophages, M2 macrophages, and resting mast cells while having lower infiltration levels of memory B cells, CD8+ T cells, follicular helper T cells, regulatory T cells (Tregs), and activated mast cells than did those of the low-risk subtype. The expressions of CTLA4, PDCD1, and TIGIT were downregulated while the PDCD1LG2 expression was upregulated in the high-risk subtype compared to those in the low-risk subtype. Furthermore, the four selected AGs in the risk model were demonstrated to possess important functions in immune cell infiltration and ICB response of HNSCC.ConclusionsThe aging-related risk signature is a reliable prognostic model for predicting the survival of HNSCC patients and provides potential targets for improving outcomes of immunotherapy.

Comparative molecular profiling of pancreatic ductal adenocarcinoma (PDAC) of the head (H) versus body/tail (B/T) and the tumor immune microenvironment (TIME).

598 Background: PDAC of the H and B/T differ in embryonic origin, cell composition, blood supply, lymphatic and venous drainage, and innervation. H tumors tend cause symptoms earlier and to present at earlier stages compared to B/T cancers. The impact of PDAC tumor location on patient presentation and survival has been shown in large national data-based analyses, although with conflicting results. We aimed to compare the molecular and tumor immune microenvironment (TIME) profiles of PDAC of the H vs. B/T. Methods: A total of 3499 PDAC samples were analyzed via next-generation sequencing (NGS) of RNA (whole transcriptome, NovaSeq), DNA (NextSeq, 592 genes or NovaSeq, whole exome sequencing) and immunohistochemistry (IHC, Caris Life Sciences, Phoenix, AZ). RNA deconvolution was performed using QuantiSeq (Finotello 2019, Genome Medicine) to quantify the immune cell infiltration. Pathway gene enrichment analyses were done using Gene Set Enrichment Analysis (GSEA, Subramaniam 2015, PNAS). Significance was determined as p values adjusted for multiple correction (q) of < 0.05. Results: Anatomic subsites of PDAC tumors were grouped by primary tumor sites into H (N = 2058) or B/T (N = 1384). There were significantly more metastatic tumors profiled from H vs. B/T (57% vs. 44%, p < 0.001). KRAS mutations (93.8% vs. 90.2%), genomic loss of heterozygosity (12.7% vs. 9.1%), and several copy number alterations ( FGF3, FGF4, FGF19, CCND1, ZNF703, FLT4, MUTYH, TNFRS14) trended higher in B/T when compared to H (p < 0.05 but q > 0.05). GNAS mutations (2.2% vs. 0.7%) trended higher in H vs. B/T (p < 0.05). No significant difference in immuno-oncology (IO) markers (TMB, PD-L1, MSI-H) were observed, but expression analysis of IO-related genes showed significantly higher expression of CTLA4 and PDCD1 in H (q < 0.05, fold change 1.2 and 1.3) and IDO1 and PDCD1LG2 expression trended higher in B/T (p < 0.05, fold change 0.95). When comparing median cell abundance values as part of TIME analysis, H had increased immune infiltration of B cells (0.045 vs. 0.043), M2 macrophages (0.035 vs. 0.032), neutrophils (0.056 vs. 0.052), NK cells (0.027 vs. 0.026), CD8+ T cells (% > 0: 48.2% vs. 43.2%), while B/T had increased infiltration of M1 macrophages (0.035 vs. 0.032) (all q < 0.05). GSEA showed enrichment of CTLA4 (normalized enrichment score (NES) 1.6, false discovery rate (FDR) 0.19) and primary immunodeficiency pathway enrichment (NES 1.7, FDR 0.11) in H. Conclusions: To our knowledge, this is one of the largest cohort of PDAC tumors subjected to broad molecular profiling. Differences in IO-related gene expression and TIME cell distribution suggest that response to IO therapies may differ in PDAC arising from H vs B/T. Subtle differences in the genomic profliles of H vs. B/T tumors were also observed.

Identification of PDCD1 and PDCD1LG2 as Prognostic Biomarkers and Associated with Immune Infiltration in Hepatocellular Carcinoma.

BackgroundHepatocellular carcinoma has been identified to be among the most prevalent malignancies in the world and has an unfavorable prognosis. Immune checkpoints perform an essential function in many biological processes and are associated with the survival of cancer patients. The function of immune checkpoints remains unknown.MethodsWe used bioinformatic methods to examine the prognostic value of immune checkpoints and the corresponding link to immune infiltration in HCC. qRT-PCR was used to validate the expression of immune checkpoints and their prognostic significance in HCC.ResultsThe level of mRNA of SIGLEC15, PDCD1LG2, LAG3, PDCD1, CTLA4 as well as PDCD1LG2 was increased in HCC tissues as opposed to liver tissues. Immune checkpoints were shown to participate in the activation of the apoptotic pathway in HCC patients. The elevated expression of PDCD1 and PDCD1LG2 were shown to have a favorable recurrence-free survival (RFS), progression-free survival (PFS), disease-specific survival (DSS), and overall survival (OS). PDCD1, PDCD1LG2, and pT stage were independent variables that affect the HCC patients’ prognoses as revealed by the multivariate and univariate analyses. A prediction nomogram indicated that the calibration plots for OS rates over three and five years had a stronger predictive performance in the TCGA HCC cohort in contrast with an ideal model. Positive correlations were observed between the PDCD1 and PDCD1LG2 expression and immune biomarkers, immune cells, chemokine receptors, as well as chemokines.ConclusionThe present research performed a thorough examination of the prognostic significance of immune checkpoints in HCC and its correlation with immune infiltration, which suggested that PDCD1 and PDCD1LG2 were prognostic biomarkers in HCC and related to the immune infiltration.

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 Blood Microenvironment Influences the Molecular Phenotypes of Circulating Tumor Cells in Head and Neck Squamous Cell Carcinoma.

Circulating tumor cells (CTCs) may be affected by the environment encountered during blood circulation. We aimed to explore the association between the molecular phenotype of CTCs and systemic inflammatory markers. CTCs isolated from patients with recurrent/metastatic head and neck squamous cell carcinoma by CD45-negative selection were analyzed for the expression of multiple genes. The correlations between gene expression levels in CTCs and systemic inflammation markers were examined. Thirty-five (83.3%) of the 42 patients were positive for CTCs. No significant differences in systemic inflammatory markers were observed between CTC-positive and CTC-negative patients. Notably, VIM or ZEB2 expression was strongly correlated with that of CD44 or ALDH1. PIK3CA, CD44, ALDH1A1, and PDCD1LG2 expression in CTCs was correlated with lymphocyte- and/or albumin-related systemic inflammatory markers. CTCs acquire a survival advantage through phenotypic alterations in the hostile blood environment, and evade circulatory immune surveillance.

Prognostic relevance of programmed cell death 1 ligand 2 (PDCD1LG2/PD-L2) in patients with advanced stage colon carcinoma treated with chemotherapy

Colorectal cancer (CRC) is the leading cause of cancer-related mortality worldwide. Although the role of tumor programmed cell death 1 ligand 1 (PD-L1) in suppressing antitumor immunity has been validated in various malignances, the impact of PD-L2 (PD-L2/PDCD1LG2) within tumors remains elusive. Here, we examined tumor PD-L2 expression by immunohistochemical analysis and assessed its association with clinicopathological characteristics and the infiltration of intratumoral T lymphocytes in colon carcinoma patients (n = 1264). We found that tumor PD-L2 status was correlated with perineural invasion (PNI) and associated with survival outcome in colon carcinoma patients. The level of tumor PD-L2 was positively associated with tumor PD-L1 expression but inversely associated with the density of CD8+ tumor-infiltrating lymphocytes (TILs). Patients with elevated tumor PD-L2 levels had a favorable 5-year overall survival (OS) compared to patients with low PD-L2 levels (57% vs 40%, p < 0.001), especially in advanced stage colon carcinoma patients. Low tumor PD-L2 expression was associated with an increased 5-year OS risk among advanced stage colon carcinoma patients by univariate analysis [hazard ratio (HR) = 1.69, 95% CI 1.324–2.161, p < 0.001] and multivariate analysis [HR = 1.594, 95% CI 1.206–2.106, p = 0.001]. Moreover, tumor PD-L2 expression was inversely associated with the lymphocytic reaction in advanced stage colon carcinoma, suggesting that PD-L2 may be upregulated by a compensatory mechanism to inhibit T cell-mediated anticancer immunity. Taken together, these results show that tumor PD-L2 expression may be an independent prognostic factor for survival outcome in patients with advanced stage colon carcinoma.

CBX3 Promotes Gastric Cancer Progression and Affects Factors Related to Immunotherapeutic Responses.

BackgroundChromobox 3 (CBX3) is a member of the chromobox family proteins, which plays a critical role in tumor progression, but the exact function of CBX3 in gastric cancer remains unknown. The current research mainly investigates the underlying mechanisms and clinical value of CBX3 in gastric cancer.MethodsGene expression cohorts from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were analyzed to assess the effect of CBX3 in gastric cancer. CBX3 expression was further determined by immunohistochemistry (IHC). The function of CBX3 on proliferation, migration and the cell cycle was explored via colony-forming, cell cycle and transwell assays, respectively. Moreover, RNA sequencing (RNA-seq) in AGS cells and two cohorts was utilized to explore the specific mechanism of CBX3.ResultsCBX3 expression was upregulated in human gastric cancer tissues and the expression level was closely associated with adverse signs. Knockdown of CBX3 in gastric cancer cells significantly inhibited the malignant phenotype. In addition, RNA-seq analysis revealed that CBX3 regulates genes related to the cell cycle, mismatch repair and immune-related pathways. Furthermore, the expression of CBX3 was significantly and inversely related to the abundance of tumor-infiltrating lymphocytes (TILs), PDCD1 and PDCD1LG2 expression and immunotherapy responses. Moreover, CBX3 influences the effectiveness of chemotherapy, thereby impacting the prognosis of gastric cancer patients.ConclusionCBX3 contributes to gastric cancer progression and is associated with chemotherapy and immunotherapy response. CBX3 may serve as a new diagnostic biomarker and potential target for immunotherapy and chemotherapy in gastric cancer.

Integrated Genetic and Topological Analysis Reveals a Hodgkin-like Mechanism of Immune Escape in T-Cell/Histiocyte-Rich Large B-Cell Lymphoma

Introduction:T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive large B cell lymphoma that typically presents with disseminated disease. In contrast to diffuse large B-cell lymphoma, not otherwise specified (DLBCL), TCRLBCL is characterized histologically by rare malignant B-cells within a robust but ineffective inflammatory background composed of numerous T cells and macrophages. TCRLBCL shows a “tolerogenic” immune signature by gene expression profiling, as well as frequent upregulation of PD-L1 (Van Loo et al. PMID: 19797726; Chen et al. PMID: 23674495). Although these features suggest that active immune evasion is central to TCRLBCL pathogenesis, its mechanistic basis is poorly understood. Accordingly, we performed an integrated analysis of tumor genetics and cell-cell interactions within the tumor microenvironment to comprehensively study PD-1:PD-L1 interactions in a multi-institutional cohort of TCRLBCL.Methods:34 cases of TCRLBCL were identified from the pathology archives of four academic medical centers. Control cohorts containing 21 cases of DLBCL and 106 cases of classic Hodgkin Lymphoma (CHL) were used as comparators. An established fluorescence in situ hybridization (FISH) assay was used to identify copy number changes and structural rearrangements of CD274 (PD-L1) and PDCD1LG2 (PD-L2) on chromosome 9p24.1, which represents the primary genetic mechanism of PD-L1/L2 expression in CHL (Roemer et al. PMID: 27069084). Tumor-specific expression of PD-L1 and PD-L2 protein was assessed by immunohistochemistry (IHC) and scoring by two pathologists using a modified H-score (percentage of positive tumor cells [0-100%] multiplied by the mean staining intensity [0-3+]). The topology of PD-L1/PD-1 expression and cell-cell interactions in the tumor microenvironment was determined by multispectral immunofluorescence (IF) microscopy and spatial image analysis, as previously performed for CHL (Carey et al. PMID: 28893733).Results:By FISH, copy gain or amplification of PD-L1 and PD-L2 was identified in 22/34 (64.7%) cases of TCRLBCL (Figure 1A) and was associated with a 4.9-fold increase in tumor PD-L1 expression relative to cases with disomy or polysomy (mean PD-L1 H-score 72 vs 14.7, p = 0.02). A rearrangement of PD-L2 was identified in one case and associated with diffuse expression of PD-L2. These findings contrasted with those observed in the DLBCL cohort, which showed a low overall frequency of 9p24.1 copy gain/amplification (5/21 cases, 23.8%) and only minimal tumor PD-L1 expression (mean PD-L1 H-score 15.6), and were intermediate to those observed in CHL, which shows near universal copy gain/amplification of 9p24.1 (98/106 cases, 92%) and extensive tumor PD-L1 expression (mean PD-L1 H-score 143.7; Figure 1B).By multispectral IF, TCRLBCL showed prominent infiltration by PD-L1+ tumor-associated macrophages (TAM) (Figure 1C), which were 5.5-fold increased relative to DLBCL and 6.6-fold increased relative to CHL (p < 0.001). TCRLBCL also showed marked infiltration by PD-1+ T cells, which were 12.3-fold increased relative to DLBCL and 3.4-fold increased relative to CHL (p < 0.001). By spatial analysis, PD-L1+ TAMs in TCRLBCL were located in closer proximity to tumor cells than PD-L1- TAMs (p < 0.001, Figure 1D-E) and also showed frequent direct interactions with PD-1+ T cells. These findings contrasted with those in DLBCL, where no local enrichment of PD-L1+ TAMs or PD-1+ T cells was identified, and were similar but more prominent than those observed in CHL.Conclusion:TCRLBCL is characterized by recurrent gains of PD-L1 and PD-L2 on chromosome 9p24.1 in association with tumor-specific expression of PD-1 ligands, as well as prominent infiltration by PD-L1+ TAMs and PD-1+ T cells. PD-L1+ TAMs in TCRLBCL are enriched around individual tumors cells and also show frequent direct interactions with PD-1+ T cells, consistent with the establishment of an immunoevasive-niche. These findings contrast with those observed in DLBCL and are most similar to those identified in CHL. Relative to CHL, however, TCRLBCL shows less frequent gains of 9p24.1 and tumor cell expression of PD-L1, and a greater degree of infiltration by PD-L1+ TAMs and PD-1+ T cells. These findings suggest that the PD-1:PD-L1 pathway is central to immune evasion in TCRLBCL and highlight the need to test the clinical efficacy of PD-1 blockade in this patient population. [Display omitted] DisclosuresGriffin:Moderna Therapeutics: Consultancy. Freeman:Novartis: Patents & Royalties; AstraZeneca: Patents & Royalties; Dako: Patents & Royalties; Roche: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Patents & Royalties; Merck: Patents & Royalties; EMD-Serono: Patents & Royalties; Roche: Patents & Royalties; Xios: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Patents & Royalties; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees; Origimed: Membership on an entity's Board of Directors or advisory committees. Hodi:Merck: Consultancy. Shipp:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Bayer: Research Funding; AstraZeneca: Honoraria. Rodig:KITE: Research Funding; Affimed: Research Funding; Merck: Research Funding; Bristol Myers Squibb: Research Funding.

Characterization of the Neuroendocrine Tumor Immune Microenvironment.

The immune environment and the potential for neuroendocrine tumors (NETs) to respond to immune checkpoint inhibitors remain largely unexplored. We assessed immune checkpoint marker expression, lymphocytic infiltrate, and associated mutational profiles in a cohort of small intestine and pancreatic NETs. We assessed expression of PDCD1 (PD-1), CD274 (PD-L1), and PDCD1LG2 (PD-L2) in archival tissue from 64 small intestine (SINETs) and 31 pancreatic NETs (pNET). We additionally assessed T-cell infiltrates, categorizing T-cell subsets based on expression of the T-cell markers CD3, CD8, CD45RO (PTPRC), or FOXP3. Finally, we explored associations between immune checkpoint marker expression, lymphocytic infiltrate, and tumor mutational profiles. Expression of PD-1 or PD-L1 in small intestine or pancreatic NET was rare, whereas expression of PD-L2 was common in both NET subtypes. T-cell infiltrates were more abundant in pNET than in SINET. We found no clear associations between immune checkpoint marker expression, immune infiltrates, and specific mutational profile within each tumor type. Our findings provide an initial assessment of the immune environment of well-differentiated NETs. Further studies to define the immunologic differences between pNET and SINET, as well as the role of PD-L2 in these tumors, are warranted.

Topological analysis reveals a PD-L1-associated microenvironmental niche for Reed-Sternberg cells in Hodgkin lymphoma

AbstractSignaling between programmed cell death protein 1 (PD-1) and the PD-1 ligands (PD-L1, PD-L2) is essential for malignant Hodgkin Reed-Sternberg (HRS) cells to evade antitumor immunity in classical Hodgkin lymphoma (cHL). Copy number alterations of 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) contribute to robust PD-L1 and PD-L2 expression by HRS cells. PD-L1 is also expressed by nonmalignant tumor-associated macrophages (TAMs), but the relationships among PD-L1+ HRS cells, PD-L1+ TAMs, and PD-1+ T cells remain undefined. We used multiplex immunofluorescence and digital image analysis to examine the topography of PD-L1+ and PD-1+ cells in the tumor microenvironment (TME) of cHL. We find that the majority of PD-L1 in the TME is expressed by the abundant PD-L1+ TAMs, which physically colocalize with PD-L1+ HRS cells in a microenvironmental niche. PD-L1+ TAMs are enriched for contacts with T cells, and PD-L1+ HRS cells are enriched for contacts with CD4+ T cells, a subset of which are PD-1+. Our data define a unique topology of cHL in which PD-L1+ TAMs surround HRS cells and implicate CD4+ T cells as a target of PD-1 blockade.

Tumor PDCD1LG2 (PD-L2) Expression and the Lymphocytic Reaction to Colorectal Cancer.

Expression of the immune checkpoint ligand CD274 (programmed cell death 1 ligand 1, PD-L1, from gene CD274) contributes to suppression of antitumor T cell-mediated immune response in various tumor types. However, the role of PDCD1LG2 (PD-L2, CD273, from gene PDCD1LG2) in the tumor microenvironment remains unclear. We hypothesized that tumor PDCD1LG2 expression might be inversely associated with lymphocytic reactions to colorectal cancer. We examined tumor PDCD1LG2 expression by IHC in 823 colon and rectal carcinoma cases within two U.S.-nationwide cohort studies and categorized tumors into quartiles according to the percentage of PDCD1LG2-expressing carcinoma cells. We conducted multivariable ordinal logistic regression analysis to assess the associations of tumor PDCD1LG2 expression with Crohn-like lymphoid reaction, peritumoral lymphocytic reaction, intratumoral periglandular reaction, or tumor-infiltrating lymphocytes, controlling for potential confounders, including microsatellite instability, CpG island methylator phenotype, long-interspersed nucleotide element-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Tumor PDCD1LG2 expression was inversely associated with Crohn-like lymphoid reaction (Ptrend = 0.0003). For a unit increase in the three-tiered ordinal categories of Crohn-like lymphoid reaction, a multivariable OR in the highest (vs. lowest) quartile of the percentage of PDCD1LG2-expressing tumor cells was 0.38 (95% confidence interval, 0.22-0.67). Tumor PDCD1LG2 expression was not associated with peritumoral lymphocytic reaction, intratumoral periglandular reaction, tumor-infiltrating lymphocytes, or patient survival (Ptrend > 0.13). Thus, tumor PDCD1LG2 expression is inversely associated with Crohn-like lymphoid reaction to colorectal cancer, suggesting a possible role of PDCD1LG2-expressing tumor cells in inhibiting the development of tertiary lymphoid tissues during colorectal carcinogenesis. Cancer Immunol Res; 5(11); 1046-55. ©2017 AACR.

Immunotherapy in lymphoma.

Immunotherapy in lymphoma.