Anaplastic thyroid cancer (ATC) and advanced differentiated thyroid cancers (DTCs) show fatal outcomes, unlike DTCs. Here, we demonstrate mutational landscape of 27 ATCs and 86 advanced DTCs by massively-parallel DNA sequencing, and transcriptome of 13 ATCs and 12 advanced DTCs were profiled by RNA sequencing. TERT, AKT1, PIK3CA, and EIF1AX were frequently co-mutated with driver genes (BRAF

Gastric cancer is diagnosed in nearly one million new patients each year and it remains the second leading cause of cancer-related deaths worldwide. Although gastric cancer represents a heterogeneous group of diseases, chronic inflammation has been shown to play a role in tumorigenesis. Cancer development is a multistep process characterized by genetic and epigenetic alterations during tumour initiation and progression. The stromal microenvironment is important in maintaining normal tissue homeostasis or promoting tumour development. A plethora of immune cells (i.e., lymphocytes, macrophages, mast cells, monocytes, myeloid-derived suppressor cells, Treg cells, dendritic cells, neutrophils, eosinophils, natural killer (NK) and natural killer T (NKT) cells) are components of gastric cancer microenvironment. Mast cell density is increased in gastric cancer and there is a correlation with angiogenesis, the number of metastatic lymph nodes and the survival of these patients. Mast cells exert a protumorigenic role in gastric cancer through the release of angiogenic (VEGF-A, CXCL8, MMP-9) and lymphangiogenic factors (VEGF-C and VEGF-F). Gastric mast cells express the programmed death ligands (PD-L1 and PD-L2) which are relevant as immune checkpoints in cancer. Several clinical undergoing trials targeting immune checkpoints could be an innovative therapeutic strategy in gastric cancer. Elucidation of the role of subsets of mast cells in different human gastric cancers will demand studies of increasing complexity beyond those assessing merely mast cell density and microlocalization.

BACKGROUND: In clinical practice, the detection of biomarkers is mostly based on primary tumors for its convenience in acquisition. However, immune checkpoints may express differently between primary and metastatic tumor. Therefore, we aimed to compare the differential expressions of PD-1, PD-L1 and PD-L2 between the primary and metastatic sites of renal cell carcinoma (RCC).
METHODS: Patients diagnosed with RCC by resection or fine needle aspiration of metastasis were included. Immunohistochemistry (IHC) was applied to detect PD-1, PD-L1 and PD-L2 expressions. SPSS 22.0 was applied to conduct Chi-square, consistency tests and Cox's proportional hazards regression models. GraphPad Prism 6 was used to plot survival curves and R software was used to calculate Predictive accuracy (PA).
RESULTS: In the whole cohort (N = 163), IHC results suggested a higher detection rate of PD-L1 in the metastasis than that of the primary site (χ2 = 4.66, p = 0.03), with a low consistent rate of 32.5%. Among different metastatic tumors, PD-1 was highly expressed in the lung/lymph node (65.3%) and poorly expressed in the brain (10.5%) and visceral metastases (12.5%). PD-L1 was highly expressed in lung/lymph node (37.5%) and the bone metastases (12.2%) on the contrary. In terms of survival analysis, patients with PD-1 expression either in the primary or metastasis had a shorter overall survival (OS) (HR: 1.59, 95% CI 1.08-2.36, p = 0.02). Also, PD-L1 expression in the primary was associated with a shorter OS (HR 2.55, 95% CI 1.06-6.15, p = 0.04). In the multivariate analysis, the predictive accuracy of the whole model for PFS was increased from 0.683 to 0.699 after adding PD-1.
CONCLUSION: PD-1, PD-L1 and PD-L2 were differentially expressed between primary and metastatic tumors. Histopathological examination of these immune check points in metastatic lesions of mRCC should be noticed, and its accurate diagnosis may be one of the effective ways to realize the individualized treatment.

Immune infiltration of tumors is closely associated with clinical outcome in renal cell carcinoma (RCC). Tumor-infiltrating immune cells (TIICs) regulate cancer progression and are appealing therapeutic targets. The purpose of this study was to determine the composition of TIICs in RCC and further reveal the independent prognostic values of TIICs. CIBERSORT, an established algorithm, was applied to estimate the proportions of 22 immune cell types based on gene expression profiles of 891 tumors. Cox regression was used to evaluate the association of TIICs and immune checkpoint modulators with overall survival (OS). We found that CD8+ T cells were associated with prolonged OS (hazard ratio [HR] = 0.09, 95% confidence interval [CI].01-.53; P = 0.03) in chromophobe carcinoma (KICH). A higher proportion of regulatory T cells was associated with a worse outcome (HR = 1.59, 95% CI 1.23-.06; P < 0.01) in renal clear cell carcinoma (KIRC). In renal papillary cell carcinoma (KIRP), M1 macrophages were associated with a favorable outcome (HR = .43, 95% CI .25-.72; P < 0.01), while M2 macrophages indicated a worse outcome (HR = 2.55, 95% CI 1.45-4.47; P < 0.01). Moreover, the immunomodulator molecules CTLA4 and LAG3 were associated with a poor prognosis in KIRC, and IDO1 and PD-L2 were associated with a poor prognosis in KIRP. This study indicates TIICs are important determinants of prognosis in RCC meanwhile reveals potential targets and biomarkers for immunotherapy development.

Cell lines are essential tools to standardize and compare experimental findings in basic and translational cancer research. The current dogma states that cancer stem cells feature an increased tumor initiation capacity and are also chemoresistant. Here, we identified and comprehensively characterized three morphologically distinct cellular subtypes in the non-small cell lung cancer cell line A549 and challenge the current cancer stem cell dogma. Subtype-specific cellular morphology is maintained during short-term culturing, resulting in the formation of holoclonal, meroclonal, and paraclonal colonies. A549 holoclone cells were characterized by an epithelial and stem-like phenotype, paraclone cells featured a mesenchymal phenotype, whereas meroclone cells were phenotypically intermediate. Cell-surface marker expression of subpopulations changed over time, indicating an active epithelial-to-mesenchymal transition (EMT), in vitro and in vivo. EMT has been associated with the overexpression of the immunomodulators PD-L1 and PD-L2, which were 37- and 235-fold overexpressed in para- versus holoclone cells, respectively. We found that DNA methylation is involved in epigenetic regulation of marker expression. Holoclone cells were extremely sensitive to cisplatin and radiotherapy in vitro, whereas paraclone cells were highly resistant. However, inhibition of the receptor tyrosine kinase AXL, whose expression is associated with an EMT, specifically targeted the otherwise highly resistant paraclone cells. Xenograft tumor formation capacity was 24- and 269-fold higher in holo- than mero- and paraclone cells, respectively. Our results show that A549 subpopulations might serve as a unique system to explore the network of stemness, cellular plasticity, tumor initiation capacity, invasive and metastatic potential, and chemo/radiotherapy resistance.

Esophageal and gastric cancers represent tumors with poor prognosis. Unfortunately, radiotherapy, chemotherapy, and targeted therapy have made only limited progress in recent years in improving the generally disappointing outcome. Immunotherapy with checkpoint inhibitors is a novel treatment approach that quickly entered clinical practice in malignant melanoma and renal cell cancer, but the role in esophageal and gastric cancer is still poorly defined. The principal prognostic/predictive biomarkers for immunotherapy efficacy currently considered are PD-L1 expression along with defects in mismatch repair genes resulting in microsatellite instability (MSI-H) phenotype. The new molecular classification of gastric cancer also takes these factors into consideration. Available reports regarding PD-1, PD-L1, PD-L2 expression and MSI status in gastric and esophageal cancer are reviewed to summarize the clinical prognostic and predictive role together with potential clinical implications. The most important recently published clinical trials evaluating checkpoint inhibitor efficacy in these tumors are also summarized.

AIM: Programmed cell death-ligand 1 and 2 (PD-L1 and PD-L2) are ligands of the programmed cell death-1 (PD1) receptor. PD1/PD-L1 inhibitors have shown clinical efficacy in non-small cell lung cancer (NSCLC). However, relatively little is known about the expression of PD-L2, or its association with the clinicopathological features of NSCLC. Here, the radiological features of PD-L2-positive lung adenocarcinoma were evaluated.
MATERIALS AND METHODS: PD-L1 and PD-L2 expression were evaluated by immunohistochemical staining of surgically-resected specimens from 393 patients with primary lung adenocarcinoma who underwent preoperative thin-section computed tomography (CT), 222 of whom also underwent
RESULTS: Among the 393 specimens, 132 (33.6%) and 266 (67.7%) were positive for PD-L1 and PD-L2 expression, respectively. Multivariate analysis showed that the absence of surrounding ground glass opacity and the presence of air bronchogram were significantly associated with PD-L2 expression; however, there was no significant association between PD-L2 expression and the consolidation/tumor ratio. In 222
CONCLUSION: PD-L2-positive lung adenocarcinomas are less radiologically malignant and invasive than their PD-L1-positive counterparts.

The immune status of tumors critically influences their responsiveness to PD1 blockades and other immune-based therapies. Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is a clinically validated predictive biomarker of response to checkpoint-inhibitor therapy in a limited number of clinical settings but is poorly predictive in most. With emerging evidence that multiple pathways and immune-checkpoint proteins may coordinately contribute to the adaptive immune resistance, the identification and quantitation of multiple immune markers in tumor tissue could help identify the controlling pathways in a given patient, guide the selection of optimal therapy, and monitor response to treatment. We developed and validated a sensitive and robust immuno-liquid chromatography-parallel reaction monitoring assay to simultaneously quantify the expression levels of six immune markers (CD8A, CD4, LAG3, PD1, PD-L1, and PD-L2) using as little as 1-2 mg of fresh frozen tissue. The lower limit of quantitation ranged from 0.07 ng/mg protein for PD1 to 1.0 ng/mg protein for CD4. The intrabatch accuracy was within -16.6% to 15.0% for all proteins at all concentrations, and the variation ranged from 0.8% to 14.7%, while interbatch accuracy was within -6.3% to 8.6%, and the variation ranged from 1.3% to 12.8%. The validated assay was then applied to quantify all six biomarkers in different tissues and was confirmed to have sufficient sensitivity (0.07-1.00 ng/mg protein) and reproducibility (variation ranged from 4.3 to 12.0%). In an analysis of 26 cervical tumors, CD8A and CD4 were detected in all tumors, followed by PD-L1 in 85%, LAG-3 in 65%, PD1 in 50%, and PD-L2 in 35%. The strongest correlations were observed between CD8A and CD4 ( r = 0.88) and CD8A and LAG-3 ( r = 0.86). PD1 was not significantly correlated with any of the other proteins tested. This method can be applied to survey the immune signatures across tumor types and tailored to incorporate additional markers as needed.

Aging is the single most significant risk factor for cancer development. However, the potential impact of aging on cancer microenvironment remains poorly understood. Here, we performed a pan-cancer transcriptome analysis to identify aging-specific molecular patterns across 18 cancer types. Strikingly, aging-specific molecular features define human cancers into two types, including the strong and weak aging-effect groups. Significant aging associated molecular signature was observed in 16 cancer types (strong aging-effect group) such as breast invasive carcinoma and acute myeloid leukemia. In such 16 cancer types, old patients showed obvious poor survival compared to young patients, but this observation was not found in the weak aging-effect cancers. Aging-associated cancer-relevant molecules significantly enriched in 23 pathways including EMT and KRAS signaling. More interestingly, in cancer microenvironment, aging significantly restrains adaptive immunity, but strikingly, increases the number of infiltrated innate immune cells. Further analysis shows that the expression of immune checkpoints including PD-1, PD-L1, PD-L2 and CTLA-4 are mostly correlated with age. In general, cancer cells in elderly patients show a more aggressive phenotype and their surrounding microenvironment is under a more immune suppression status compared to young patients. Our study provides a systematic understanding of aging-associated molecular features in pan-cancer and indicates a clinical requirement to develop aging-specific therapeutic strategies in a majority of cancer types. Furthermore, aging-altered immune cells and immune checkpoints should be considered in cancer immunotherapy.

Peripheral T-cell lymphoma (PTCL), not otherwise specified (PTCL-NOS) is among the most common disease subtypes of PTCL, one that exhibits heterogeneous clinicopathological features. Although multiple disease-stratification models, including the cell-of-origin or gene-expression profiling methods, have been proposed for this condition, their clinical significance remains unclear. To establish a clinically meaningful stratification model, we analyzed gene-expression signatures of tumors and tumor-infiltrating immune cells using the nCounter system, which enables accurate quantification of low abundance and/or highly fragmented transcripts. To do so, we assessed transcripts of 120 genes related to cancer or immune cells using tumor samples from 68 newly diagnosed PTCL-NOS patients and validated findings by immunofluorescence in tumor sections. We show that gene-expression signatures representing tumor-infiltrating immune cells, but not those of cancerous T cells, dictate patient clinical outcomes. Cases exhibiting both B-cell and dendritic cell (DC) signatures (BD subgroup) showed favorable clinical outcomes, whereas those exhibiting neither B-cell nor DC signatures (non-BD subgroup) showed extremely poor prognosis. Notably, half of the non-BD cases exhibited a macrophage signature, and macrophage infiltration was evident in those cases, as revealed by immunofluorescence. Importantly, tumor-infiltrating macrophages expressed the immune-checkpoint molecules programmed death ligand 1/2 and indoleamine 2, 3-dioxygenase 1 at high levels, suggesting that checkpoint inhibitors could serve as therapeutic options for patients in this subgroup. Our study identifies clinically distinct subgroups of PTCL-NOS and suggests a novel therapeutic strategy for 1 subgroup associated with a poor prognosis. Our data also suggest functional interactions between cancerous T cells and tumor-infiltrating immune cells potentially relevant to PTCL-NOS pathogenesis.

Chromosome 9 harbors several relevant oncogenes related to hematolymphoid malignancies and one specific region, 9p24, has come into the focus of attention in the last years as it contains recurrently mutant genes of therapeutic interest. The most prominent genes of this locus are programmed death ligands 1 and 2 (PDL1/PDL2), with the amplification of PDL1 being a hallmark of both classical Hodgkin and primary mediastinal B cell lymphoma, and Janus kinase 2 (JAK2), which is point-mutated in myeloproliferative neoplasms and other myeloid malignancies, and rearranged in PCM1-JAK2-positive myeloid/lymphoid neoplasms with eosinophila. Finally, this locus contains the lysine (K)-specific demethylase 4C (KDM4C/JMJD2C), which is also relevant for oncogenesis. Activation of these genes is effectuated, as exemplified, by multiple mechanisms, which is rather unique to oncogenes, since they are usually affected by just one type of mutation, and points towards the central role of these genes in tumor initiation and growth. Amplifications and, less frequently, translocations are the most common findings for PDL1/PDL2 and JAK2 in lymphomas. In this review, we describe the role of genes located on chromosome 9p24 and their derived proteins in diverse subtypes of lymphomas, with a special focus on PDL1 and PDL2, which are becoming a central target of immunotherapy, not only in classical Hodgkin lymphoma but also in various types of solid cancers. We also elucidate the role of the surgical pathologists in this setting - concerning what they can contribute - both diagnostically and predictively.

Although immune checkpoint molecules regulate the progression of certain cancers, their significance in malignant development of Waldenstrom macroglobulinemia (WM), an incurable low-grade B-cell lymphoma, remains unknown. Recently, cytokines in the bone marrow (BM) microenvironment are shown to contribute to the pathobiology of WM. Here, we investigated the impact of cytokines, including interleukin-6 (IL-6) and IL-21, on immune regulation and particularly on the programmed death-1 (PD-1) and its ligands PD-L1 and PD-L2. We showed that IL-21, interferon γ, and IL-6 significantly induced PD-L1 and PD-L2 gene expression in WM cell lines. Increased PD-L1 and PD-L2 messenger RNA was also detected in patients' BM cells. Patients' nonmalignant BM cells, including T cells and monocytes, showed increased PD-L1, but minimal or undetectable PD-L2 surface expression. There was also very modest PD-L1 and PD-L2 surface expression by malignant WM cells, suggesting that ligands are cleaved from the cell surface. Levels of soluble ligands were higher in patients' BM plasma and blood serum than controls. Furthermore, IL-21 and IL-6 increased secreted PD-L1 in the culture media of WM cell lines, implying that elevated levels of soluble PD-1 ligands are cytokine mediated. Soluble PD-1 ligands reduced T-cell proliferation, phosphorylated extracellular signal-regulated kinase and cyclin A levels, mitochondrial adenosine triphosphate production, and spare respiratory capacity. In conclusion, we identify that soluble PD-1 ligands are elevated in WM patients and, in addition to surface-bound ligands in WM BM, could regulate T-cell function. Given the capability of secreted forms to be bioactive at distant sites, soluble PD-1 ligands have the potential to promote disease progression in WM.

BACKGROUND: The Cancer Genome Atlas (TCGA) is an important data resource for cancer biologists and oncologists. However, a lack of bioinformatics expertise often hinders experimental cancer biologists and oncologists from exploring the TCGA resource. Although a number of tools have been developed for facilitating cancer researchers to utilize the TCGA data, these existing tools cannot fully satisfy the large community of experimental cancer biologists and oncologists without bioinformatics expertise.
METHODS: We developed a new web-based tool The Cancer Omics Atlas (TCOA, http://tcoa.cpu.edu.cn ) for fast and straightforward querying of TCGA "omics" data.
RESULTS: TCOA provides the querying of gene expression, somatic mutations, microRNA (miRNA) expression, protein expression data based on a single molecule or cancer type. TCOA also provides the querying of expression correlation between gene pairs, miRNA pairs, gene and miRNA, and gene and protein. Moreover, TCOA provides the querying of the associations between gene, miRNA, or protein expression and survival prognosis in cancers. In addition, TCOA displays transcriptional profiles across various human cancer types based on the pan-cancer analysis. Finally, TCOA provides the querying of molecular profiles for 2877 immune-related genes in human cancers. These immune-related genes include those that are established or promising targets for cancer immunotherapy such as CTLA4, PD1, PD-L1, PD-L2, IDO1, LAG3, and TIGIT.
CONCLUSIONS: TCOA is a useful tool that supplies a number of unique and new functions complementary to the existing tools to facilitate exploration of the TCGA resource.

BACKGROUND: Gene expression of specific therapeutic targets in non-malignant lung tissue might play an important role in optimizing targeted therapies. This study aims to identify different expression patterns of fifteen genes important for targeted therapy in non-small cell lung cancer (NSCLC).
METHODS: We prospectively collected tissue of NSCLC and non-malignant lung tissue from 25 primary resected patients. RNA-sequencing and 450 K methylation array profiling was applied to both NSCLC and non-malignant lung tissue and data were analyzed for 14 target genes. We analyzed differential expression and methylation as well as expression according to patient characteristics like smoking status, histology, age, chronic obstructive pulmonary disease, C-reactive protein (CRP) and gender. TCGA data served as a validation set.
RESULTS: Nineteen men and 6 women were included. Important targets like PD-L2 (p = 0.035), VEGFR2 (p < 0.001) and VEGFR3 (p < 0.001) were downregulated (respective fold changes = 1.8, 3.1, 2.7, 3.5) in tumor compared to non-malignant lung tissue. The TCGA set confirmed these findings almost universally. PD-L1 (p < 0.001) became also significantly downregulated in the TCGA set. In NSCLC, MUC1 (p = 0.003) showed a higher expression in patients with a CRP < 5 mg/L compared to > 5 mg/L. In the TCGA data but not in our primary data, PD-L1 & 2 were both borderline more expressed in tumors of active smokers vs. tumors of ex-smokers (p = 0.044 and 0.052).
CONCLUSIONS: Our results suggest a lower PD-L1 & 2 and VEGFR expression in NSCLC vs. non-malignant lung tissue. Specific patient characteristics did not seem to change the overall expression differences as they were in line with the overall results. This information may contribute to the optimization of targeted treatments.

Recent progress in anti-tumor immunotherapy has focused on the significance of the tumor microenvironment in tumor progression and resistance to chemo/radio-therapy. Myeloid cells such as macrophages are predominant stromal components in hematological malignancies. In the present study, we investigated the regulation of programmed death-1 (PD-1) ligand expression in primary central nervous system lymphoma (PCNSL) using PCNSL cell lines and human monocyte-derived macrophages. TK PCNSL cell line-derived soluble factors induced overexpression of PD-1 ligands, indoleamine 2,3-dioxygenase (IDO1), and several other cytokines in macrophages. The expression of PD-1 ligands was dependent on the activation of signal transducer and activator of transcription 3. PD-L1 and IDO1 were overexpressed by macrophage/microglia in PCNSL tissues, and gene expression profiling indicated that IDO1 expression was positively correlated with the expression of macrophage and lymphocyte markers. Macrophage-derived factors did not influence the proliferation or chemo-sensitivity of cell lines. These data suggest that the expression of immunosuppressive molecules, including PD-1 ligands and IDO1, by macrophage/microglia may be involved in immune evasion of lymphoma cells.

PURPOSE: Programmed death-1 (PD-1) inhibitors are approved for the treatment of patients with recurrent and metastatic squamous cell carcinoma of the head and neck (SCCHN). Ongoing and planned randomized phase 3 trials are testing the benefit of combining PD-1/programmed death-ligand 1 (PD-L1) inhibitors with chemoradiation for patients with locoregionally confined SCCHN. Few studies have investigated relationships among potential predictive pathologic biomarkers such as PD-L1, PD-L2, and PD-1 in this population and associations between these markers and clinical characteristics.
METHODS AND MATERIALS: We retrospectively reviewed records and pathology from 81 patients with locoregional oropharynx SCCHN treated with curative intent. Samples were analyzed for PD-L1, PD-L2, PD-1, CD8, and CD56 expression using immunohistochemistry. Human papilloma virus (HPV) status was determined by p16-immunohistochemistry and confirmed by in situ hybridization or polymerase chain reaction-based HPV typing. Correlations between HPV status, clinical features, and recurrence status with immune markers in both tumor and tumor-associated stroma were determined. Hazard ratios were estimated via Cox proportional hazards model.
RESULTS: Tumor PD-L1 expression was inversely associated with age (P = .01) and the highest levels of expression (>30% of tumor cells) were observed in HPV-associated tumors. There was a correlation between tumor and stromal PD-L1 expression (P = < .0001). PD-1 and CD8 expression within tumor deposits was associated with HPV status (P = 0.003 and P = .008, respectively) and decreased local recurrence (P = .001 and P < .001, respectively). In addition to the association between tumor and stromal PD-1 (P < .0001), PD-1 was also correlated with tumor PD-L1 expression (P < .001). CD56+ natural killer cell infiltrates correlated with PD-L1 expression.
CONCLUSIONS: In patients with untreated oropharyngeal SCCHN, HPV-associated tumors displayed the highest levels of PD-L1 expression and PD-1+ and CD8+ immune cells. Locally recurrent tumors had lower levels of PD-L1, PD-1, and CD-8 positivity. Whereas almost all SCCHN tumors had CD56+ infiltrating natural killer cells, most tumors didn't have PD-L2 expression. These associations may help predict which patients may benefit most from immunotherapeutic approaches.

BACKGROUND: Activation of the JAK/STAT pathway is common in triple-negative breast cancer (TNBC) and affects the expression of genes controlling immune signaling. A subset of TNBC cases will have somatic amplification of chromosome 9p24.1, encoding PD-L1, PD-L2, and JAK2, which has been associated with decreased survival.
MATERIALS AND METHODS: Eleven TNBC cell lines were evaluated using array comparative genomic hybridization. A copy number gain was defined as an array comparative genomic hybridization log
RESULTS: The cell line HCC70 had 9p24.1 copy number amplification that was associated with both increased JAK2 and pSTAT3; however, knockdown of JAK2 inhibited cell growth independently of 9p24.1 copy number status. In TNBC cell lines with 9p24.1 gain or amplification, PD-L1 expression rapidly and strikingly increased 5- to 38-fold with interferon-γ (P < .05), and inducible PD-L1 expression was completely blocked by JAK2 knockdown and the JAK1/2 inhibitor ruxolitinib. In tumor tissue, expression of interferon-γ-related genes correlated with 9p24.1 copy number status.
CONCLUSION: These data suggest that the JAK2/STAT1 pathway in TNBC might regulate the dynamic expression of PD-L1 that is induced in the setting of an inflammatory response. Inhibition of JAK2 might provide a synergistic therapy when combined with other immunotherapies in the subset of TNBC with 9p24.1 amplification.

Soluble PD-1 (sPD1) can bind with ligands PD-L1/PD-L2 on the surface of dendritic cells (DCs). Therefore, a sPD1 vaccine fused with an immunogen can increase T cell activation against cancer. Here, we constructed a MUC1 and survivin (MS) combination gene tumor vaccine expressing MS fused with soluble PD-1 (sPD1/MS). To investigate whether the sPD1/MS fusion vaccine could enhance tumor-specific immune responses, its immunogenicity and anti-tumor activity were examined after intramuscular immunization in mice. Compared with the MS DNA vaccine, the specific cytolysis rate of the sPD1/MS fusion DNA vaccine was increased from 21.64% to 34.77%. Moreover, the sPD1/MS vaccine increased the tumor suppression rate from 17.18% to 30.96% and prolonged survival from 6.96% to 19.44% in a murine colorectal cancer model. Combining the sPD1/MS vaccine with oxaliplatin improved the tumor suppression rate to 74.71% in the murine colorectal cancer model. The sPD1/MS vaccine could also exert a good anti-tumor effect, increasing the tumor infiltrated CD8

BACKGROUND & AIMS: Immune checkpoint inhibition may affect growth or progression of highly aggressive cancers, such as esophageal adenocarcinoma (EAC). We investigated the regulation of expression of major histocompatibility complex, class 1 (MHC-I) proteins (encoded by HLA-A, HLA-B, and HLA-C) and the immune response to EACs in patient samples.
METHODS: We performed quantitative polymerase chain reaction array analyses of OE33 cells and OE19 cells, which express different levels of the ATP binding cassette subfamily B member 1 (TAP1) and TAP2, required for antigen presentation by MHC-I, to identify microRNAs (miRNAs) that regulate their expression. We performed luciferase assays to validate interactions between miRNAs and potential targets. We overexpressed candidate miRNAs in OE33, FLO-1, and OACP4 C cell lines and performed quantitative polymerase chain reaction, immunoblot, and flow cytometry analyses to identify changes in messenger RNA (mRNA) and protein expression; we studied the effects of cytotoxic T cells. We performed miRNA in situ hybridization, RNA-sequencing, and immunohistochemical analyses of tumor tissues from 51 untreated patients with EAC in the Netherlands. Clinical and survival data were collected for patients, and EAC subtypes were determined.
RESULTS: We found OE19 cells to have increased levels of 7 miRNAs. Of these, we found binding sites for miRNA 125a (MIR125a)-5p in the 3' untranslated region of the TAP2 mRNA and binding sites for MIR148a-3p in 3' untranslated regions of HLA-A, HLA-B, and HLA-C mRNAs. Overexpression of these miRNAs reduced expression of TAP2 in OE33, FLO-1, and OACP4 C cells, and reduced cell-surface levels of MHC-I. OE33 cells that expressed the viral peptide BZLF1 were killed by cytotoxic T cells, whereas OE33 that overexpressed MIR125a-5p or MIR 148a along with BZLF1 were not. In EAC and nontumor tissues, levels of MIR125a-5p correlated inversely with levels of TAP2 protein. High expression of TAP1 by EAC correlated with significantly shorter overall survival times of patients. EACs that expressed high levels of TAP1 and genes involved in antigen presentation also expressed high levels of genes that regulate the adaptive immune response, PD-L1, PD-L2, and IDO1; these EACs had a poor response to neoadjuvant chemoradiotherapy and associated with shorter overall survival times of patients.
CONCLUSIONS: In studies of EAC cell lines and tumor tissues, we found increased levels of MIR125a-5p and MIR148a-3p to reduce levels of TAP2 and MHC-I, required for antigen presentation. High expression of MHC-I molecules by EAC correlated with markers of an adaptive immune response and significantly shorter overall survival times of patients.

BACKGROUND: Salivary duct carcinoma (SDC) is a rare tumor occurring in the salivary gland. SDC is a highly aggressive tumor and its prognosis is extremely poor. Effective treatments in advanced SDC have not yet been established. Recently, immune checkpoint inhibitors have paved the way for the treatment of various malignancies. We examined the expressions of programed death ligand (PD-L) 1/PD-L2 and programed death (PD-1), and the correlation of clinicopathological findings.
METHODS: We examined 18 cases of SDC and conducted immunohistochemical staining using formalin-fixed paraffin-embedded full-face sections.
RESULTS: The expression of PD-L1 and PD-L2 in tumor cells was observed in nine cases (50%) and 14 cases (78%), respectively. Cases with a high expression of PD-L1 and PD-L2 were found in four (22%) and seven cases (39%), respectively. The cases with a high expression of PD-L1 showed significantly shorter overall survival compared to those with low PD-L1 expression and null expression. We also examined the expression of PD-L1/PD-L2 and PD-1 of tumor-infiltrating mononuclear cells (TIMC) in stroma. The expressions of PD-L1 in tumor cells and stroma had a significant correlation. Association between the expressions of PD-L1 in tumor cells and those of PD-1 in stroma was significant. However, PD-L2 expression in the tumor had no significant correlation with expression in TIMCs. PD-L1, PD-L2 and PD-1 expressions in stroma were not associated with patient prognosis.
CONCLUSIONS: High PD-L1 expression in SDC was strongly associated with unfavorable prognosis, indicating that PD-1/PD-L1 inhibitors could be effective in SDC.

The prognosis for patients with malignant glioma is very poor and thus the identification of new potential therapeutic targets is critically important. In this work, we report a previously unknown role for the homeobox transcription factor HOXC10 in regulating immunosuppressive gene expression in glioma cell lines and their proliferative and invasive capacities. Although HOXC10 expression is dysregulated in several types of tumors, its potential function in glioma was not known. We found that HOXC10 expression was upregulated in glioma compared with normal tissue, and that HOXC10 expression positively associated with high grading of glioma. In three independent datasets (REMBRANDT glioma, The Cancer Genome Atlas glioblastoma multiforme and GSE4412), HOXC10 upregulation was associated with short overall survival. In two glioma cell lines, HOXC10 knock-down inhibited cell proliferation, colony formation, migration and invasion, and promoted apoptosis. In addition, HOXC10 knock-down suppressed the expression of genes that are involved in tumor immunosuppression, including those for transforming growth factor-β 2, PD-L2, CCL2 and TDO2. A ChIP assay showed that HOXC10 directly bound to the PD-L2 and TDO2 promoter regions. In summary, our results suggest that HOXC10 upregulation in glioma promotes an aggressive phenotype and induces immunosuppressive gene expression, supporting further investigation of the potential of HOXC10 as a therapeutic target in glioma.

A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1β), M2-associated (CCL13, CD206), Mreg-associated (IL-10, TGF-β), and angiogenesis associated (MMP9, VEGF) genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1β, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

BACKGROUND: Cancer of unknown primary (CUP) accounts for approximately 3% of all malignancies. Avoiding immune destruction is a major cancer characteristic and therapies aimed at immune checkpoint blockade are in use for several specific cancer types. A comprehensive survey of predictive biomarkers to immune checkpoint blockade in CUP were explored in this study.
METHODS: About 389 cases of CUP were analysed for mutations in 592 genes and 52 gene fusions using a massively parallel DNA sequencing platform (next-generation sequencing [NGS]). Total mutational load (TML) and microsatellite instability (MSI) were calculated from NGS data. PD-L1 expression was explored using immunohistochemistry (with 5% cutoff value).
RESULTS: High TML was seen in 11.8% (46/389) of tumours. MSI-high (MSI-H) was detected in 7/384 (1.8%) of tumours. Tumour PD-L1 expression was detected in 80/362 CUP (22%). A small proportion of CUP cases harboured genetic alterations of negative predictive biomarkers to immune checkpoint inhibitors (predictors to hyperprogression) including MDM2 gene amplification (2%) and loss of function JAK2 gene mutations (1%). Amplifications of CD274 (PD-L1) and PDCD1LG2 (PD-L2) genes were also rare (1.4% and 0.8%, respectively). The most frequently mutated genes were TP53 (54%), KRAS (22%), ARID1A (13%), PIK3CA (9%), CDKN2A (8%), SMARCA4 (7%) and PBRM1, STK11, APC, RB1 (5%, respectively).
CONCLUSIONS: Using a multiplex testing approach, 28% of CUP carried one or more predictive biomarkers (MSI-H, PD-L1 and/or TML-H) to the immune checkpoint blockade, providing a novel option for treatment in patients with CUP.

BACKGROUND/AIM: The squamous cell carcinoma antigen (SCCA), encoded by the genes SERPINB3/B4, is a tumour marker produced by head and neck squamous cell carcinoma (HNSCC). We aimed to examine SERPINB3/B4 mRNA levels and its clinical significance in the therapeutic context.
MATERIALS AND METHODS: We retrieved mRNA expression levels, clinical, pathological and genomic data for 520 HNSCC from The Cancer Genome Atlas (TCGA).
RESULTS: HNSCC tumours express high levels of SERPINB3/B4 mRNA. SERPINB3 expression differs depending on Human papillomavirus (HPV) infection status, primary tumour location, grade and differentiation, extension to lymph nodes and extracapsular spread. Interestingly, we observed an association between SERPINB3/B4 and the presence of tumour immune infiltrate as well as the expression of the immune checkpoint regulators PD-L1/PD-L2 that depended on HPV status.
CONCLUSION: Our findings point to potential interest of SERPINB3/B4 for the stratification of HNSCC patients in the therapeutic context.

Much focus has been on the interaction of programmed cell death ligand 1 (PD-L1) on malignant B cells with programmed cell death 1 (PD-1) on effector T cells in inhibiting antilymphoma immunity. We sought to establish the contribution of natural killer (NK) cells and inhibitory CD163

Immune checkpoints are important targets for immunotherapies. However, knowledge on the epigenetic modification of immune checkpoint genes is sparse. In the present study, we investigated promoter methylation of CTLA4, PD-L1, PD-L2, and PD-1 in diffuse lower-grade gliomas (LGG) harboring isocitrate dehydrogenase (IDH) mutations with regard to mRNA expression levels, clinicopathological parameters, previously established methylation subtypes, immune cell infiltrates, and survival in a cohort of 419 patients with IDH-mutated LGG provided by The Cancer Genome Atlas. PD-L1, PD-L2, and CTLA-4 mRNA expression levels showed a significant inverse correlation with promoter methylation (PD-L1: p=0.005; PD-L2: p<0.001; CTLA-4: p<0.001). Furthermore, immune checkpoint methylation was significantly associated with age (PD-L2: p=0.003; PD-1: p=0.015), molecular alterations, i.e. MGMT methylation (PD-L1: p<0.001; PD-L2: p<0.001), ATRX mutations (PD-L2: p<0.001, PD-1: p=0.001), and TERT mutations (PD-L1: p=0.035, PD-L2: p<0.001, PD-1: p<0.001, CTLA4: p<0.001) as well as methylation subgroups and immune cell infiltrates. In multivariate Cox proportional hazard analysis, PD-1 methylation qualified as strong prognostic factor (HR=0.51 [0.34-0.76], p=0.001). Our findings suggest an epigenetic regulation of immune checkpoint genes via DNA methylation in LGG. PD-1 methylation may assist the identification of patients that might benefit from an alternative treatment, particularly in the context of emerging immunotherapies.

OBJECTIVES: We aimed at investigating the relationship between classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBL), and gray zone lymphoma (GZL) with intermediate characteristics between cHL and PMBL, from the perspective of the aberration in programed cell death 1 and the programed death ligands (PDLs) network.
METHODS: We explored the expression levels of PDLs and chromosomal anomalies in 67 cases: 34 cases with cHL, 20 with PMBL, and 13 with GZL, using immunohistochemical analyses and Fluorescence In Situ Hybridization (FISH).
RESULTS: Twenty-one cHL (62%), 3 PMBL (15%), and 6 GZL (46%) cases showed staining to PD-L1 antibodies in more than 70% of tumor cells. Two cHL (6%), 10 PMBL (50%), and 3 GZL (23%) cases were not stained by PD-L1 antibodies. Patients over 40 years old manifest more frequent expression of PD-L1 in cHL. Proportion of tumors stained by PD-L2 antibody was increased in PMBL. FISH analyses with a PD-L1/PD-L2 probe detected 5 amplification, 1 gain, and 7 polysomy cases in cHL, 1 amplification and 1 polysomy case in GZL, and amplification in 1 PMBL case.
CONCLUSION: We identified increased staining of PD-L1 in cHL and that of PD-L2 in PMBL. GZL had a pattern similar to that of cHL.

Renal cell carcinoma exome-derived, V(D)J recombination reads had an elevated presence and variability, for both TcR-α and -β, when compared to marginal tissue, reflecting an opportunity to assess tumor immunogenicity by comparison with marginal tissue T cells. PD-1, PD-L2, CTLA4 and FOXP3, all of which are implicated in the evasion of an anti-tumor immune response, had a significantly higher expression for samples representing co-detection of productive TcR-α and -β recombination reads. Samples representing tumors with productive TcR-α recombination reads but no detectable, productive TcR-β recombination reads, reflected a 20% survival advantage, and RNASeq data indicated an intermediate level of immune checkpoint gene expression for those samples. These results raise the question of whether relatively high levels of detection of productive TcR-α recombination reads, in comparison with detection of reads representing the TcR-β gene, identify a microenvironment that has not yet entered a T-cell exhaustion phase and may thereby represent conditions for immune enhancements that do not require anti-immune checkpoint therapies.

Head and neck squamous cell carcinoma (HNSCC) comprises a heterogeneous group of tumors that arise from the squamous epithelium of the oral cavity, oropharynx, larynx and hypopharynx. While many HNSCCs are related to classical etiologic factors of smoking and alcohol, a clinically, genomically, and immunologically distinct subgroup of tumors arise from the epithelium of the tonsil and the base of tongue as a result of infection with Human Papilloma Virus (HPV). In this review we describe the genomic and immunologic landscape of HNSCC, highlighting differences between HPV-positive and HPV-negative HNSCC. While HPV-negative tumors are characterized by tobacco-associated mutations in genes including TP53 and CDKN2A, in HPV-positive HNSCC integration of viral genome from HPV into the host cellular genome results in expression of the E6 and E7 viral oncoproteins, with consequent degradation of p53 and functional inactivation of Rb. The immune microenvironment of HNSCC is characterized by changes in immune cell populations, immune checkpoints, as well as tumor or microenvironmental factors that alter the balance of the immune milieu in favor of immunosuppression, allowing tumor evasion and escape from immune surveillance. Immune therapies, in particular those targeting the PD1 receptor or its ligand PD-L1, including nivolumab, pembrolizumab, durvalumab, and atezolizumab have shown significant efficacy in subsets of patients with HNSCC. Current trials are evaluating the efficacy of these agents in combination with chemotherapy, radiotherapy and other immune therapies including CTLA-4 and IDO-1 inhibitors. While biomarkers including PD-L1 expression, PD-L2 expression and the interferon-gamma gene signature show potential to predict benefit from checkpoint inhibitor therapy - it is hoped that improved understanding of the genomic and immune landscape will lead to ways to improved strategies to stratify patients and to select which HNSCC are most likely to benefit from these therapies.

Primary testicular lymphomas (PTL) are the most prevalent type of testicular cancer arising in men over the age of 60. PTL accounts for approximately 1-2% of all non-Hodgkin lymphomas and most present with localized disease but despite this, outcome is poor. The majority of cases represent an extranodal manifestation of diffuse large B-cell lymphoma (DLBCL), known as primary testicular DLBCL (PT-DLBCL). Gene expression profiling has established that over 75% of PT-DLBCLs resemble the activated B-cell-like (ABC) or non-germinal center subtype of nodal DLBCL. In distilling the specific mutational landscape and immunophenotypic profiles, immune-escape and sustained signalling emerge as prominent features of PT-DLBCL. These include genomic alterations arising within the core components of antigen presentation (CIITA, B2M, and HLA loci) and structural rearrangements of programmed death ligands 1 (CD274) and 2 (PDCD1LG2). Enrichment for somatic mutations within NF-κB pathway genes (MYD88, CD79B, NFKBIZ, BCL10, and MALT1) also feature prominently in PT-DLBCL. Taken together, the unique molecular and clinical characteristics of PT-DLBCL have informed on aspects of the distinct disease biology of this organotypic lymphoma that may guide rational therapeutic strategies.