Despite progress in understanding the biology of adrenocortical carcinoma (ACC), treatment options have not dramatically changed in the last three decades, nor have we learned how to avoid some of its long-term side effects. Our goal was to improve the understanding of immune pathways that may include druggable targets to enhance immune responses of patients with ACC, focusing on immune evasion and the activation of immune cells against ACC. Our strategy was aimed at improving insight regarding gene expression without steroid interference. Using approaches based on high and low steroid phenotypes (HSP and LSP, respectively), we characterized immune pathways using The Cancer Genome Atlas (TCGA) ACC cohort data. Although previous studies have suggested that patients with ACC receive minimal benefit from immunotherapy, high expression of immune modulators was noted in patients with LSP, suggesting the activation of these biomarkers may be an important adjuvant therapy target after clearance of excess glucocorticoids. In addition, patients with LSP ACC had higher immune cell infiltration than patients with HSP ACC and other cancer subtypes. Our findings can be summarized as follows (1): we confirmed and improved the definition of two immune response pathways to ACC (HSP and LSP) based on in silico transcriptome analysis (2), we demonstrated the steroid profile should be considered, otherwise analyses of ACC immune characteristics can generate confounding results (3), among the overexpressed immunotherapy targets, we demonstrated that LSP was rich in PDCD1LG2 (PD-L2) and both HSP and LSP overexpressed CD276 (B7-H3), which was associated with resistance to anti-PD1 therapy and may have accounted for the modest results of previous clinical trials, and (4) identification of patients with LSP or HSP ACC can be used to help determine whether immunotherapy should be used. In conclusion, we highlighted the differences between LSP and HSP, drawing attention to potential therapeutic targets (CD276, PDCD1, and PDCD1LG2). Treatments to reduce immune evasion, as well as the use of other natural and pharmacological immune activators, should include prior pharmacological inhibition of steroidogenesis. Attempts to combine these with tumor cell proliferation inhibitors, if they do not affect cells of the immune system, may produce interesting results.

Keywords: B7-H3; CD276; CD8+ T lymphocytes; PD-1/PD-L1/PD-L2; PDCD1/CD274/PDCD1LG2; adrenocortical carcinoma; steroidogenesis.

Background: The immune mechanism was shown to be involved in the development of adenomyosis. The aim of the current study was to evaluate the expression of the immune checkpoints B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and to explore the effect of mifepristone on the expression of these immune checkpoints.

Methods: The expression of B7-H2, B7-H3, B7-H4 and PD-L2 in normal endometria and adenomyosis patient samples treated with or without mifepristone was determined by immunohistochemistry analysis.

Results: In adenomyosis patient samples, the expression of B7-H2, B7-H3 and B7-H4 was increased in the eutopic and ectopic endometria compared with normal endometria, both in the proliferative and secretory phases. Moreover, the expression of B7-H2 and B7-H3 was higher in adenomyotic lesions than in the corresponding eutopic endometria, both in the proliferative and secretory phases. The expression of PD-L2 was higher in adenomyotic lesions than in normal endometria in both the proliferative and secretory phases. In the secretory phase but not the proliferative phase, the expression of B7-H4 and PD-L2 in adenomyotic lesions was significantly higher than that in the corresponding eutopic endometria. In normal endometria and eutopic endometria, the expression of B7-H4 was elevated in the proliferative phase compared with that in the secretory phase, while in the ectopic endometria, B7-H4 expression was decreased in the proliferative phase compared with the secretory phase. In addition, the expression of B7-H2, B7-H3, B7-H4 and PD-L2 was significantly decreased in adenomyosis tissues after treatment with mifepristone.

Conclusions: The expression of the immune checkpoint proteins B7-H2, B7-H3, B7-H4 and PD-L2 is upregulated in adenomyosis tissues and is downregulated with mifepristone treatment. The data suggest that B7 immunomodulatory molecules are involved in the pathophysiology of adenomyosis.

Keywords: Adenomyosis; B7-H2; B7-H3; B7-H4; Mifepristone; PD-L2.

Purpose: Accurate identification and assessment of sentinel lymph node (SLN) using noninvasive imaging methods can play a vital role in tumor staging, surgical planning, and prognostic evaluation. In this study, we assessed the efficacy of B7-H3-targeted molecular-ultrasound imaging for the early SLN detection, and characterization in a mouse model of orthotopic breast cancer.

Procedures: We established a mouse breast cancer model with lymph node metastasis by injecting MAD-MB 231 cells which were engineered to express firefly luciferase reporter gene into the fat pad of the right 4th mammary gland in female BALB/c nude mice. The sole lymph node (LN) close to the tumor was regarded as the SLN for imaging investigation, which included metastatic and non-metastatic SLNs. The LN in the right 4th mammary gland from normal mice was used as normal control (normal mice LN). The commercially available preclinical streptavidin-coated, perfluorocarbon-containing lipid-shelled microbubbles (VisualSonics, Toronto, Canada) were used to generate B7-H3-targeted microbubbles (MB_B7-H3) and control microbubbles (MB_Control). Then, ultrasound molecular imaging (USMI) was performed using a high-resolution transducer (MS250; center frequency, 21 MHz; Vevo 2100; VisualSonics, Toronto, Canada) after intravenous injection of microbubbles.

Results: The SLN was clearly detected and located under conventional (B-mode) and contrast-enhanced ultrasonography with microbubble injection. The metastatic SLNs showed a markedly higher signal from B7-H3-targeted microbubbles (MB_B7-H3) compared to the non-metastatic SLNs and normal LNs. The metastatic SLN was further confirmed by ex vivo bioluminescence imaging and eventually verified by histological analysis.

Conclusions: Our findings suggest the potential value of USMI using B7-H3 targeted microbubbles in breast cancer and establish an effective imaging method for the non-invasive detection and characterization of SLN.

Keywords: B7-H3; Breast cancer; Microbubbles; Molecular imaging; Sentinel lymph node; Ultrasound.

<i><em>B7</em>-<em>H3</em></i> isoforms, <i>4Ig<em>B7</em>-<em>H3</em></i> (<i>4Ig</i>) and <i>2Ig<em>B7</em>-<em>H3</em></i> (<i>2Ig</i>) expression characteristics in acute myeloid leukemia (AML) remain unknown. This study investigated mRNA and membrane protein expression of two isoforms in AML cell lines and de novo patients by using RT-PCR and flow cytometry, and analyzed the <i><em>B7</em>-<em>H3</em></i> promoter methylation state by utilizing RQ-MSP. <i>4Ig</i> was the dominant <i><em>B7</em>-<em>H3</em></i> isoform. <i>2Ig</i> mRNA expression rate and abundance were elevated in AML in comparison with controls (<i>P</i>=0.000 and 0.000), while no significant difference in <i>4Ig</i> (<i>P</i>=0.802, <i>P</i>=0.398). Membrane protein levels of <i><em>B7</em>-<em>H3</em></i> isoforms in AML was higher than controls, detected by total <i><em>B7</em>-<em>H3</em></i> expression rate (<i>P</i>=0.002), total <i><em>B7</em>-<em>H3</em></i> mean fluorescence intensity (MFI) ratio of blast cells and lymphocytes (MFI ratio) (<i>P</i>=0.000), and <i>4Ig <em>B7</em>-<em>H3</em></i> MFI ratio (<i>P</i>=0.005). Compared with <i>2Ig</i>^low group, <i>2Ig</i>^high patients had older age, lower <i>NPM1</i> mutation, higher <i>FLT3-ITD</i> mutation, and declining complete remission (CR) rates (<i>P</i>=0.026, 0.012, 0.047, and 0.028). In <i><em>B7</em>-<em>H3</em></i>^high group, there was a trend towards older age, -M4 and M5, worse karyotype, and lower CR rates, although with no marked difference (<i>P</i>>0.05). The overall survival (OS) of <i>2Ig</i>^high and <i><em>B7</em>-<em>H3</em></i>^high groups were shorter than that of <i>2Ig</i>^low and <i><em>B7</em>-<em>H3</em></i>^low groups in the whole and non-M3 AML cohorts (<i>P</i>=0.006 and 0.046; <i>P</i>=0.003 and 0.032). Besides, an unmethylated <i><em>B7</em>-<em>H3</em></i> promoter was identified. In conclusion, <i>2Ig</i> mRNA and total <i><em>B7</em>-<em>H3</em></i> isoforms membrane protein tended to have potential diagnostic value for AML. Specifically, high <i>2Ig</i> mRNA expression and total <i><em>B7</em>-<em>H3</em></i> membrane protein expression indicate worse OS. <i>4Ig</i> and <i>2Ig</i> expression are methylation-independent.

Keywords: 2Ig B7-H3; 4Ig B7-H3; acute myeloid leukemia; prognosis.

Background: B7 family molecules affect both immune responses and cancer progression via immunological and non-immunological pathways. However, the specific expression and prognostic value of B7 members in acute myeloid leukemia (AML) remains unclear; hence, an investigation using online bioinformatics databases is required.

Methods: In this study, we explored the expression of B7 molecules using the ONCOMINE, Gene Expression Profiling Interactive Analysis 2 (GEPIA2), and UALCAN databases, while the prognostic value of B7 molecules in AML was analyzed using the LinkedOmics, GEPIA2, UALCAN, and TCGAportal databases. Additionally, genetic alteration and gene co-expression analysis of the B7 family was performed via the cBioPortal and LinkedOmics databases, while functional and pathway enrichment analyses were conducted using the Metascape databases for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses.

Results: The message RNA (mRNA) levels of B7 family members varied in AML patients, and aberrant highly expressed B7 members were correlated with poor prognosis in AML, including B7.1, B7-DC, B7-H3, B7-H5, and B7-H7. B7-H6 acted as a protective molecule for overall survival (OS), while B7-H1 overexpression was inclined to predict poor prognosis. B7 family gene alteration occurred frequently in AML, and the altered B7 group seemed to exhibit a trend towards worse OS. The co-expression genes and relative signaling pathways of the B7 family might be involved in oncogenesis and be associated with prognosis in AML.

Conclusions: Our study showed that aberrantly expressed B7 family molecules affected the prognosis of AML patients, and thus, could be promising prognostic biomarkers and new therapeutic targets.

Keywords: B7; Co-stimulatory molecules; acute myeloid leukemia (AML); bioinformatics integration analysis; prognosis.

Immature dendritic cells (iDCs) and tolerogenic DCs are essential for the induction and maintenance of peripheral tolerance. Tumors produce immuno-modulatory factors which imprint a pro-tolerogenic, maturation-resistant state in DCs. Here we asked for common markers of differentially tolerized human monocyte-derived DC populations. For this, PBMC-derived monocytes were differentiated to DCs in the presence of established immuno-modulators as released by tumors (IL-6, IL-10, TGF-β, glucocorticoid [GC], prostaglandin E2 [PGE2]). Most unstimulated pro-tolerogenic DC populations commonly over-expressed some tolerance-associated markers (ILT-4, IL-10, HO-1) as compared with iDCs. These markers may contribute to imprint a pro-tolerogenic state in DCs. Furthermore, some tolerance markers were overexpressed in an immuno-modulator specific manner in DCs differentiated in the presence of TGF-β (overexpressed tolerance markers: B7-H3, CD103, TGF-β1), IL-10 (B7-DC, ILT-3) and PGE2 (IDO). Upon stimulation, matured control DCs (mDCs) down-regulated most pro-tolerogenic markers monitored, while some were upregulated (IkBα, IDO, B7-H1, B7-DC). In contrast, the different groups of tolerized DCs largely retained expression of pro-tolerogenic markers after stimulation. In contrast to mDCs, most groups of tolerized DCs showed impaired upregulation of CD80, and all groups retained IL-10 cytokine production after stimulation. All tolerized DC populations commonly exerted an attenuated allogenic T cell stimulatory capacity as compared with mDCs.

For the developing field of gene therapy the successful address of the basic requirement effective gene delivery has remained a critical barrier. In this regard, the "Holy Grail" vector envisioned by the field's pioneers embodied the ability to achieve efficient and specific in vivo gene delivery. Functional linkage of antibody selectivity with viral vector efficiency represented a logical strategy but has been elusive. Here we have addressed this key issue by developing the technical means to pair antibody-based targeting with adenoviral-mediated gene transfer. Our novel method allows efficient and specific gene delivery. Importantly, our studies validated the achievement of this key vectorology mandate in the context of in vivo gene delivery. Vectors capable of effective in vivo delivery embody the potential to dramatically expand the range of successful gene therapy cures.

Keywords: Adenoviral vectors (Ad); CD276 [B7-H3]; Camelid single domain antibody (sdAb); Gene delivery; Human epithelial ovarian Cancer cell (SKOV3.ip1); Ovarian Cancer (OvCa) xenograft mouse model.

Objective: To investigate the expression and clinical significance of costimulatory molecule B7-H3 and microRNA-145 (miR-145) in lung cancer patients with malignant pleural effusion.

Patients and methods: A total of 100 cases of patients with lung cancer who admitted to our hospital for treatment from March 2017 to September 2018 were selected. Forty-nine cases of patients diagnosed with malignant pleural effusion were included in the study group, and 51 cases with benign pleural effusion in the control group. The content of B7-H3 in pleural effusion of the two groups was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of miR-145 in pleural effusion of the two groups was analyzed by Real-time quantitative PCR (qRT-PCR). The relationships between the expressions of B7-H3 and miR-145 and the clinicopathological characteristics were analyzed. The diagnostic value of B7-H3 and miR-145 in lung cancer was analyzed.

Results: The expression level of B7-H3 in the study group was significantly higher than that in the control group (p<0.050), while the expression level of miR-145 was significantly lower than that in the control group (p<0.050). The expression levels of B7-H3 and miR-145 in the study group were correlated with lymph node metastasis, differentiation degree of lung cancer and TNM stage (p<0.001). The sensitivity and specificity of miR-145 in single diagnosis of lung cancer were 64.71% and 79.59%, respectively. The sensitivity and specificity of B7-H3 in single diagnosis of lung cancer were 80.39% and 61.22%, respectively.

Conclusions: B7-H3 and miR-145 are abnormally expressed in lung cancer, and are closely related to the lymphatic metastasis, differentiation degree and TNM stage of lung cancer. They may be potential markers for the diagnosis of malignant pleural effusion in lung cancer in the future.

A number of sequencing studies identified the prognostic impact of somatic mutations in myelodysplastic syndrome (MDS). However the majority of them focused on methylation regulation, apoptosis and proliferation genes. Despite the number of experimental studies published on the role of micro-RNA processing and checkpoint genes in the development of MDS, the clinical data about mutational landscape in these genes is limited. We performed a pilot study which evaluated mutational burden in these genes and their association with common MDS mutations. High prevalence of mutations was observed in the genes studied: 54% had mutations in DICER1, 46% had mutations in LAG3, 20% in CTLA4, 23% in B7-H3, 17% in DROSHA, 14% in PD-1 and 3% in PD-1L. Cluster analysis that included these mutations along with mutations in ASXL1, DNMT3A, EZH2, IDH1, RUNX1, SF3B1, SRSF2, TET2 and TP53 effectively predicted overall survival in the study group (HR 4.2, 95%CI 1.3-13.6, p = 0.016). The study results create the rational for incorporating micro-RNA processing and checkpoint genes in the sequencing panels for MDS and evaluate their role in the multicenter studies.

Yeast surface display is a proven tool for the selection and evolution of ligands with novel binding activity. Selections from yeast surface display libraries against transmembrane targets are generally carried out using recombinant soluble extracellular domains. Unfortunately, these molecules may not be good models of their true, membrane-bound form for a variety of reasons. Such selection campaigns often yield ligands that bind a recombinant target but not target-expressing cells or tissues. Advances in cell-based selections with yeast surface display may aid the frequency of evolving ligands that do bind true, membrane-bound antigens. This study aims to evaluate ligand selection strategies using both soluble target-driven and cellular selection techniques to determine which methods yield translatable ligands most efficiently and generate novel binders against CD276 (B7-H3) and Thy1, two promising tumor vasculature targets. Out of four ligand selection campaigns carried out using only soluble extracellular domains, only an affibody library sorted against CD276 yielded translatable binders. In contrast, fibronectin domains against CD276 and affibodies against CD276 were discovered in campaigns that either combined soluble target and cellular selection methods or used cellular selection methods alone. A high frequency of non target-specific ligands discovered from the use of cellular selection methods alone motivated the development of a depletion scheme using disadhered, antigen-negative mammalian cells as a blocking agent. Affinity maturation of CD276-binding affibodies by error-prone PCR and helix walking resulted in strong, specific cellular CD276 affinity ( K_d = 0.9 ± 0.6 nM). Collectively, these results motivate the use of cellular selections in tandem with recombinant selections and introduce promising affibody molecules specific to CD276 for further applications.

Tumour angiogenesis is a crucial factor associated with tumour growth, progression, and metastasis. The whole process is the result of an interaction between a wide range of different molecules, influencing each other. Herein we summarize novel discoveries related to the less known angiogenic molecules such as galectins, pentraxin-3, Ral-interacting protein of 76 kDa (RLIP76), long non-coding RNAs (lncRNAs), B7-H3, and delta-like ligand-4 (DLL-4) and their role in the process of tumour angiogenesis. These molecules influence the most important molecular pathways involved in the formation of blood vessels in cancer, including the vascular endothelial growth factor (VEGF)-vascular endothelial growth factor receptor interaction (VEGFR), HIF1-a activation, or PI3K/Akt/mTOR and JAK-STAT signalling pathways. Increased expression of galectins, RLIP76, and B7H3 has been proven in several malignancies. Pentraxin-3, which appears to inhibit tumour angiogenesis, shows reduced expression in tumour tissues. Anti-angiogenic treatment based mainly on VEGF inhibition has proved to be of limited effectiveness, leading to the development of drug resistance. The newly discovered molecules are of great interest as a potential source of new anti-cancer therapies. Their role as targets for new drugs and as prognostic markers in neoplasms is discussed in this review.

Keywords: angiogenesis; carcinogenesis; galectins.

Purpose: The aim of this study was to characterize chondrosarcoma tumor infiltration by immune cells and the expression of immunologically relevant molecules. This information may contribute to our understanding of the role of immunological events in the pathogenesis of chondrosarcoma and to the rational design of immunotherapeutic strategies.

Patients and methods: A tissue microarray (TMA) containing 52 conventional and 24 dedifferentiated chondrosarcoma specimens was analyzed by immunohistochemical staining for the expression of parameters associated with tumor antigen-specific immune responses, namely, CD4⁺ and CD8⁺ tumor infiltrating lymphocytes (TILs) and the expression of HLA class I heavy chain, beta-2 microglobulin (β2m), HLA class II and immune checkpoint molecules, B7-H3 and PD-1/PD-L1. The results were correlated with histopathological characteristics and the clinical course of the disease.

Results: CD8⁺ TILs were present in 21% of the conventional and 90% of the dedifferentiated chondrosarcoma tumors tested. B7-H3 was expressed in 69% of the conventional and 96% of the dedifferentiated chondrosarcoma tumors tested. PD-1 and PD-L1 were expressed 53% and 33% respectively of the dedifferentiated tumors tested. PD-L1 expression was associated with shorter time to metastasis.

Conclusion: The tumor infiltration by lymphocytes suggests that chondrosarcoma is immunogenic. Defects in HLA class I antigen and expression of the checkpoint molecules B7-H3 and PD-1/PD-L1 suggest that tumor cells utilize escape mechanisms to avoid immune recognition and destruction. This data implies that chondrosarcoma will benefit from strategies that enhance the immunogenicity of tumor antigens and/or counteract the escape mechanisms.

Keywords: B7-H3; HLA class I; PD-L1; T cell infiltration; chondrosarcoma.

The families of miR-34 and miR-449 share the same seed region. However, the members showed differential effects on the expression of B7-H3 and PD-L1 in HCT-116 cells. Using miR-34a as a template, the non-seed region was modified by nucleotide alteration, yielding four synthetic microRNA (miRNA) analogs. Among those, NS-MX3, with a base alteration from G to C at the 18th locus of miR-34a, showed the most potent inhibition on both B7-H3 and PD-L1 expression. Subsequent investigations demonstrated that NS-MX3 had a broad anti-proliferation activity against several colorectal tumor cell lines and its antitumor effect was consistently reflected by tumor growth inhibition (TGI) in the HCT-116 xenograft model. In addition, NS-MX3 displayed a synergistic effect on TGI when combined with bevacizumab or regorafenib. Further analysis revealed that the superior antitumor activity of NS-MX3 was correlated to concomitant suppression of both B7-H3 and PD-L1 expression in tumor tissues. Taken together, the present study indicates that the non-seed region of miRNAs plays an important role in the regulation of checkpoint genes, thus showcasing single nucleotide alteration of the non-seed region as a promising approach to discover and develop novel immunotherapies.

Keywords: Antitumor activity; B7-H3; Combination; MicroRNA; Single nucleotide alteration; Synthetic analogs.

B7-H3 is an immunomodulatory member of the B7-superfamily with limited expression in normal tissues, but overexpression in several types of cancer. Therefore it is currently being explored as a potential target for cancer immunotherapy. The biological relevance of B7-H3 expression in pancreatic cancer is unclear, while there are no data on B7-H3 expression in ampullary cancer. We aimed to compare intra-tumoral B7-H3 expression between these two closely related cancer types and analyze its association with post-surgical disease course. B7-H3 expression levels were determined by immunohistochemistry in tissue microarrays of resected tumors of 137 pancreatic cancer patients and 83 patients with ampullary cancer of the pancreato-biliary subtype. B7-H3 was more frequently expressed in cancer cells of ampullary cancer patients compared to pancreatic cancer patients (51% versus 21%; p< 0.001). In ampullary cancer patients, but not in pancreatic cancer patients, B7-H3 cancer cell expression was associated with longer disease-free survival and patient survival. However, the prognostic value of B7-H3 was lost upon adjustment for CA19-9 levels. The frequencies of B7-H3 expression in tumor stroma did not differ between the two types of cancer (66% versus 63%). In both cancer types, stromal B7-H3 expression was not associated with post-surgical disease course. Compared to pancreatic cancer, B7-H3 is more frequently expressed in cancer cells of patients with the pancreato-biliary subtype of ampullary cancer. These data suggest that B7-H3 may represent an interesting potential target for immunotherapy in ampullary cancer rather than in pancreatic cancer.

Keywords: B7H3; CD276; cancer immunotherapy; immune checkpoint; pancreas.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a high mortality rate. Epithelial-to-mesenchymal transition (EMT) confers cancer cells with immune evasive ability by modulating the expression of immune checkpoints in many cancers. Thus, the aim of our study is to examine the interplay between EMT and immune checkpoint molecules in HCC. A reversible EMT model was utilised with transforming growth factor (TGF)-<i>β</i>1 as an EMT inducer for HCC cell lines Hep3B and PLC/PRF/5. HCC cells were treated with TGF-<i>β</i>1 for 72 h and the EMT status and immune checkpoint expression were examined. In addition, the migratory ability of HCC cells were examined using wound healing and transwell migration assays in the reversible EMT model. siRNA-mediated knockdown of immune checkpoint molecule, <em>B7</em>-<em>H3</em>, was further utilised to validate the association between TGF-<i>β</i>1-mediated EMT and immune checkpoint expression in HCC. In addition, a web-based platform, SurvExpress, was utilised to evaluate the association between expression of TGF-<i>β</i>1 in combination with immune checkpoint molecules and overall survival in HCC patients. We observed induction of EMT upon treatment of HCC cells with TGF-<i>β</i>1 revealed by reduced expression of epithelial markers along with increased expression of mesenchymal markers. Withdrawal of TGF-<i>β</i>1 reversed the process of EMT with elevated expression of epithelial markers and reduced expression of mesenchymal markers. TGF-<i>β</i>1 treatment elevated the migratory potential of HCC cells which was reversed following reversal assay. Notably, during TGF-<i>β</i>1-induced EMT, there was upregulation of immune checkpoint molecules PD-L1 and <em>B7</em>-<em>H3</em>. However, the reversal of EMT decreased the expression of PD-L1 and <em>B7</em>-<em>H3</em>. In addition, TGF-<i>β</i>1 driven EMT was reversed following knockdown of <em>B7</em>-<em>H3</em> in both HCC cells further validating the interplay between TGF-<i>β</i>1-mediated EMT and immune checkpoint expression in HCC. Furthermore, the coordinate expression of TGF-<i>β</i>1 with PD-L1 (<i>p=0.01487</i>) and <em>B7</em>-<em>H3</em> (<i>p=0.009687</i>) was correlated with poor overall survival in 422 HCC patients. Our study has demonstrated a close association between TGF-<i>β</i>1-mediated EMT and regulation of immune checkpoints in HCC.

Keywords: epithelial-to-mesenchymal transition; hepatocellular carcinoma; immune checkpoint molecules; transforming growth factor-β1.

Introduction: Glioblastoma (GBM) is the most common primary adult brain tumour with a median overall survival (OS) of 12-15 months. Molecular characterization of multiple immunooncology targets in GBM may help target novel immunotherapeutic strategies. We used NanoString GeoMx® Digital Spatial Profiling (DSP) to assess multiple immunooncology protein targets in methylated versus unmethylated IDH-wild-type glioblastoma.

Methods: NanoString GeoMx® DSP technology uses multiple primary antibodies conjugated to indexing DNA oligos with a UV photocleavable linker. Tissue regions of interest (ROIs) are selected with bound fluorescent antibodies; oligos are released via a UV-mediated linker and quantitated. We used DSP multiplex analysis of 31 immunooncology proteins and controls (CD4, CD14, CD68, CD8A, B7-H3, PD-L1, CD19, FOXP3, CD44, STAT3 (phospho Y705), CD45, Pan Cytokeratin, MS4A1/CD20, CD45RO, PD1, CD3, beta-2 microglobulin, VISTA, Bcl2, GZMB, PTEN, beta-catenin, CD56, Ki-67, STAT3, AKT, p-Akt, S6, Histone H3, IgG Rabbit control, and Mouse IgG control) from ROIs in a cohort of 10 IDH-wild-type glioblastomas (5 methylated and 5 unmethylated). An nCounter platform allowed quantitative comparisons of antibodies between ROIs in MGMT methylated and unmethylated tumours. Mean protein expression counts between methylated and unmethylated GBM were compared using technical and biological replicates.

Results: The analysis showed 10/27 immunooncology target proteins were significantly increased in methylated versus unmethylated IDH-wild-type glioblastoma tumour core (false discovery rate (FDR) <0.1 by Benjamini-Hochberg procedure).

Conclusions: NanoString GeoMx® DSP was used to analyse multiple immunooncology protein target expression in methylated versus unmethylated IDH-wild-type glioblastoma. In this small study, there was a statistical increase in CD4, CD14, CD68, CD8A, B7-H3, PDL-1, CD19, FOXP3, CD44, and STAT3 protein expression in methylated versus unmethylated GBM tumour core; however, this requires larger cohort validation. Advanced multiplex immunooncological biomarker analysis may be useful in identifying biomarkers for novel immunotherapeutic agents in GBMs.

Background: Novel immune checkpoint-based immunotherapies may benefit specific groups of prostate cancer patients who are resistant to other treatments.

Methods: We analyzed by immunohistochemistry the expression of B7-H3, PD-L1/B7-H1, and androgen receptor (AR) in tissue samples from 120 prostate adenocarcinoma patients treated with radical prostatectomy in Spain, and from 206 prostate adenocarcinoma patients treated with radical prostatectomy in Norway.

Results: B7-H3 expression correlated positively with AR expression and was associated with biochemical recurrence in the Spanish cohort, but PD-L1 expression correlated with neither of them. Findings for B7-H3 were validated in the Norwegian cohort, where B7-H3 expression correlated positively with Gleason grade, surgical margins, seminal vesicle invasion, and CAPRA-S risk group, and was associated with clinical recurrence. High B7-H3 expression in the Norwegian cohort was also consistent with positive AR expression.

Conclusion: These results suggest distinct clinical relevance of the two immune checkpoint proteins PD-L1 and B7-H3 in prostate cancer. Our findings highlight B7-H3 as an actionable novel immune checkpoint protein in prostate cancer.

Keywords: B7-H3/CD276; PD-L1/CD274; androgen receptor (AR); immune checkpoint protein; prostate cancer.

Osteosarcoma is the most common bone tumor in children and young adults. Metastatic and relapsed disease confer poor prognosis, and there have been no improvements in outcomes for several decades. The disease's biological complexity, lack of drugs developed specifically for osteosarcoma, imperfect preclinical models, and limits of existing clinical trial designs have contributed to lack of progress. The Children's Oncology Group Bone Tumor Committee established the New Agents for Osteosarcoma Task Force to identify and prioritize agents for inclusion in clinical trials. The group identified multitargeted tyrosine kinase inhibitors, immunotherapies targeting B7-H3, CD47-SIRPα inhibitors, telaglenastat, and epigenetic modifiers as the top agents of interest. Only multitargeted tyrosine kinase inhibitors met all criteria for frontline evaluation and have already been incorporated into an upcoming phase III study concept. The task force will continue to reassess identified agents of interest as new data become available and evaluate novel agents using this method.

Keywords: chemotherapy; clinical trials; metastasis; osteosarcoma; relapsed; therapeutics.

Objective: chordomas are rare bone tumors with few therapeutic options. Skull base and sacrum are the two most common origin sites. Immunotherapies are emerging as the most promising approaches to fight various cancers. This study tends to identify new cell surface targets for immunotherapeutic options of skull base chordomas.

Methods: we profiled 45 skull base chordoma clinical samples by immunohistochemistry for the expression of six CAR-Targets (PD-L1, B7-H3, B7-H4, VISTA, HER2 and HER3). In addition, we generated B7-H3 targeted CAR-T-cells and evaluated their antitumor activities in vitro.

Results: We found that B7-H3 was positively stained in 7 out of 45 (16%) chordoma samples and established an expression hierarchy for these antigens (B7-H3 > HER3 > PD-L1 > HER2 = VISTA = B7-H4). We then generated a B7-H3 targeted CAR vector and demonstrated that B7-H3-CAR-T-cells recognized antigen positive cells and exhibited significant antitumor effects, including suppression of tumor spheroid formation, CAR-T-cell activation and cytokine secretion.

Conclusions: Our results support B7-H3 might serve as a promising target for CAR-T-cell therapies against chordomas.

Keywords: B7-H3; CAR-T cells; chordoma; immunotherapy; skull base chordoma.

B7 family proteins serve as checkpoint molecules that protect tumors from T cell mediated lysis. Tryptophan degrading enzymes indoleamine 2,3 dioxygenase (IDO) and tryptophan 2,3 dioxygenase (TDO) also induce T cell immune tolerance. However, little is known about the relative contribution of B7 molecules, tryptophan degrading enzymes, as well as the impact of tumor and stromal cell interactions to the development of immunosuppressive tumor microenvironment. To investigate such interactions, we used a tripartite model of human hepatocellular carcinoma cell line (HepG2) and mesenchymal stromal cells (MSCs) co-cultured with peripheral blood mononuclear cells (PBMCs). Co-culture of HepG2 cells and activated PBMCs demonstrate that HepG2 cells undergo PBMC mediated cytolysis, despite constitutive expression of B7-H3 and upregulation of PD-L1 by IFNγ. Knockdown of B7-H3, PD-L1 or IDO does not modulate PBMC mediated lysis of HepG2 cells. However, TNFα preactivation enhances lysis of HepG2 cells, and blocking of TNFα production from PBMCs protects HepG2 cells. On the other hand, MSCs protect HepG2 cells from PBMC mediated lysis, even in the presence of TNFα. Further investigation showed that MSC mediated protection is associated with the unique secretome profile of upregulated and downregulated cytokines and chemokines. IFNγ activated MSCs are superior to TNFα activated or control MSCs in protecting HepG2 cells. Blockade of IFNγ driven IDO activity completely abolishes the ability of MSCs to protect HepG2 cells from cytolysis by PBMCs. These results suggest that inhibition of IFNγ activation of IDO induction in stromal cells, combined with usage of TNFα, could be a novel immunotherapeutic strategy to induce regression of hepatocellular carcinoma.

Keywords: B7 family checkpoints; hepatocellular carcinoma; immunotherapy; indoleamine 2,3 dioxygenase (IDO); mesenchymal stromal cells.

[This corrects the article DOI: 10.2147/OTT.S318082.].

The immune checkpoint molecule, B7-H3, which belongs to the B7 family, has been shown to be overexpressed in various cancers. Its role in tumors is not well defined, and many studies suggest that it is associated with poor clinical outcomes. The effect of B7-H3 on laryngeal cancer has not been reported. This study investigated the expression of B7-H3 in laryngeal squamous cell carcinoma (LSCC), and its relationship with clinicopathological factors and prognosis of LSCC patients. The gene expression quantification data and clinical data of LSCC retrieved from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database were analyzed to determine the diagnostic and prognostic roles of B7-H3. Quantitative real-time polymerase chain reaction (qRT-PCR) was then performed to determine the gene expression level of B7-H3 between LSCC tissues and paired normal adjacent tissues. In addition, TCGA RNA-seq data was analyzed to evaluate the expression level of B7 family genes. Next, the protein expression of B7-H3 and CD8 in LSCC was determined using immunohistochemistry and immunofluorescence. qRT-PCR results showed that the expression level of B7-H3 mRNA was significantly higher in LSCC tissues than in adjacent normal tissues. Similar results were obtained from the TCGA analysis. The expression of B7-H3 was significantly associated with T stage, lymph node metastasis, and pathological tumor node metastasis (TNM) stage, and it was also an independent factor influencing the overall survival time (OS) of patients with LSCC. In addition, B7-H3 was negatively correlated with CD8⁺T cells. These results show that B7-H3 is upregulated in LSCC. Therefore, B7-H3 may serve as a biomarker of poor prognosis and a promising therapeutic target in LSCC.

Keywords: B7-H3; LSCC - laryngeal squamous cell carcinoma; biomarker; diagnosis; prognosis.

Immunotherapy has been a milestone for muscle-invasive bladder cancer (MIBC), but only a small portion of patients can benefit from it. Therefore, it is crucial to develop a robust individualized immune-related signature of MIBC to identify patients potentially benefiting from immunotherapy. The current study identified patients from the Cancer Genome Atlas (TCGA) and immune genes from the ImmPort database, and used improved data analytical methods to build up a 45 immune-related gene pair signature, which could classify patients into high-risk and low-risk groups. The signature was then independently validated by a Gene Expression Omnibus (GEO) dataset and IMvigor210 data. The subsequent analysis confirmed the worse survival outcomes of the high-risk group in both training (p < 0.001) and validation cohorts (p = 0.018). A signature-based risk score was proven to be an independent risk factor of overall survival (p < 0.001) and could predict superior clinical net benefit compared to other clinical factors. The CIBERSORT algorithm revealed the low-risk group had increased CD8⁺ T cells plus memory-activated CD4⁺ T-cell infiltration. The low-risk group also had higher expression of PDCD1 (PD-1), CD40, and CD27, and lower expression of CD276 (B7-H3) and PDCD1LG2 (PD-L2). Importantly, IMvigor210 data indicated that the low-risk group had higher percentage of "inflamed" phenotype plus less "desert" phenotype, and the survival outcomes were significantly better for low-risk patients after immunotherapy (p = 0.014). In conclusion, we proposed a novel and promising prognostic immune-related gene pair (IRGP) signature of MIBC, which could provide us a panoramic view of the tumor immune microenvironment of MIBC and independently identify MIBC patients who might benefit from immunotherapy.

Keywords: immune-related gene pair signature; immunotherapy; muscle-invasive bladder cancer; prognosis; tumor microenvironment.