High B7-H3 Expression Research Articles

Abstract 5461: B7-H3 regulates NF-kB signaling in cancer cells by modulating the degradation and re-synthesis of IkBα

Abstract B7-H3, a B7 homologue, is highly expressed on tumors and tumor vasculature. Although its role as a T cell inhibitor or co-stimulator is still under investigation, high expression of membranous B7-H3 in the tumor compartment is correlated with poor overall survival. NF-kB activation, standing at the intersection between inflammation and cancer, can lead to both the production of cytokines that engage the innate immune system, as well as pro-proliferative signals in cancer. B7-H3 has been shown to enhance and suppress inflammatory signaling by modulating NF-kB. However, the mechanism by which this occurs in cancer cells remains unknown. In this study, we aim to understand how B7-H3 modulates the NF-kB pathway in order to regulate incoming inflammatory signals in cancer cells. The human colon cancer cell line, HCT116, was stably transfected with a transcriptionally-coupled post-translational NF-kB luciferase reporter comprising of the inhibitor of NF-kB, IkBα, fused to firefly luciferase and placed downstream of an NF-kB responsive promoter, kB5. This construct allows for the real-time readout of the degradation of IkBα and NF-kB driven transcriptional re-synthesis of IkBα in response to a stimulus. The reporter cell line was treated with two stimulants, TNFα and flagellin, that activate NF-kB through different pathways and to different degrees with or without treatment with human B7-H3. Treatment of the reporter cell line with B7-H3 alone did not induce IkBα degradation or re-synthesis. However, stimulation of the reporter cell line with TNFα or flagellin in combination with B7-H3 attenuated the degradation of IkBα and its re-synthesis compared to stimulation with TNFα or flagellin alone. Maximal re-synthesis of IkBα when stimulated with TNFα was 8.25 ± 1.51 fold (S.D.) and significantly increased compared to cells additionally treated with B7-H3 (5.83 ± 1.23 fold, p = .01). In addition, peak degradation time of IkBα when cells were stimulated by TNFα was 45 ± 5 min (S.D.), and significantly shorter than when cells were also treated with B7-H3 (61.67 ± 2.89 min, p = .01). Similar trends were observed when cells were treated with both flagellin and B7-H3 compared to stimulation with flagellin alone. These studies show that B7-H3 suppresses the response of colon cancer cells to incoming inflammatory stimuli and attenuates NF-kB signaling by tempering the kinetics and dynamics of IkBα. Citation Format: Sarah Glazer, Seth Gammon, David Piwnica-Worms. B7-H3 regulates NF-kB signaling in cancer cells by modulating the degradation and re-synthesis of IkBα [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5461.

Abstract 3686: RAIDs: Future prospects for innovative targeted treatments in cervical cancer

Abstract Routine molecular diagnostic tests have yet to be introduced to guide personalized cervical cancer treatment in contrast to other solid cancers. To assess cancer functional events (CFEs), the RAIDs consortium (Rational Molecular Assessment and Innovative Drug Selection, www.raids-fp7.eu) accrued consecutive tumour tissues, whole blood and sera from 419 cervical cancer patients in 18 centers from 7 European Union countries, between 2013-2016 (BioRAIDs: NCT02428842). Whole exome sequencing (WES) is presently available for the first 98 patients, for 20 CC cell lines (list available in supplementary data) and Reverse Phase Protein Array (RPPA) data for 154 patients with a common core set of 91 patients. By comparing the proportion of mutated tumours for each gene and after correction for multiple testing, we detected no significant difference in the frequency of significantly mutated genes (SMG) between the TCGA and RAIDs datasets. However, alterations in epigenetic regulator genes, which are of potential high clinical interest, are not discussed in the TCGA paper. Mutations, frameshift deletions, insertions or stop codons in MLL2 and MLL3 genes, are present in 17% and 19 % respectively (total: 30%) of the first 98 RAIDs tumours, as well as in all 20 cervical cancer cell lines analyzed. Similar to the TCGA results, frequent mutations in genes coding for acetyltransferases such as EP300/CREBBP (9%) were detected in RAIDs tumours and cell lines (15%/25%). With the exception of SHKBP1, all other novel and previously confirmed genes by TCGA were equally detected in the RAIDs dataset, such as PIK3CA (33%), FBXW7 (14%), NFE2L2 (7%), KRAS (4%), ERBB2 (4%), PTEN (5%), CASP8 (3%). Similarly to TCGA, RAIDs RPPA data defined 3 stable clusters. TCGA and RAIDs cluster 1 regrouped proteins frequently associated with EMT (epithelial mesenchymal transition). Significant features of RAIDs cluster 1 were high phospho-YAP (p= 3.11e-06) and phospho-Met (p=4.74e-03), high Met (p=2.42e-11), high Notch (p=4.14e-17), low E-cadherin (p=2.41e-06) as well as significant levels of the phosphorylated forms of EGFR, HER2-3 and AKT and high PD-L1 and B7-H4 expression. While TCGA cluster 2 had been associated with “Hormone” signaling, RAIDs cluster 2 was associated with “DNA damage” signaling. Antibody selection was not identical between TCGA and RAIDs and further comparisons are warranted. In RAIDs, pre-treatment activation of DNA repair proteins (Rad50, Phospho-Chk2, Phospho-DNA-PK, Phospho-FANCD2, Phospho-53BP1) together with low levels of Ki67 (proliferation) appeared predictive for a complete response to standard radio-chemotherapy. This needs validation on an independent population. RAIDs and TCGA cluster 3 were associated with p38 MAPK and PI3K signaling. The RAIDs consortium's future objectives are to better define actionable CFE in relation to outcome and patient's quality of life, allowing the focus on relevant CFEs for patients at high risk. Citation Format: Maud Kamal, Els Berns, Windy Luscap Rondof, Leanne De koning, Gemma Kenter, Attila Kerezst, Marina Popovic, Choumouss Kamoun, Balazs Balint, Suzy Scholl, RAIDs Consortium. RAIDs: Future prospects for innovative targeted treatments in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3686.

Expression of B7-H3 in muscle infiltrating bladder cancer and its clinical significance

Objective To investigate the expression of B7-H3 in muscle infiltration bladder transitional cell carcinoma and its clinical significance. Methods The clinical and pathological data of 115 patients with muscle infiltration bladder transitional cell carcinoma who underwent radical surgery were retrospectively analyzed. The expression of B7-H3 in bladder cancer tissues and corresponding paracancerous tissues was detected by immunohistochemical staining, and the relationship between B7-H3 and clinicopathological characteristics was analyzed. Results The positive expression of B7-H3 was mainly in the cytoplasm, presenting brownish yellow granules. B7-H3 had positive expression in all bladder cancer tissues but no expression in paracancerous tissues. The high positive rate of B7-H3 expression in bladder cancer was 68.7%. The abnormal expression of B7-H3 was correlated with the distant metastasis and intravascular tumor thrombi (all P 0.05). The survival analysis result showed that the survival time of the patients with high expression of B7-H3 in the bladder cancer tissues was significantly lower than that of patients with low expression of B7-H3, and the difference was statistically significant (P<0.05). Conclusion The expression of B7-H3 in muscle infiltration bladder cancer tissues is up-regulated and is positively associated with the distant metastasis of bladder cancer and intravascular tumor thrombus. The expression of B7-H3 may indicate poor prognosis of the patients with bladder cancer. Key words: Bladder cancer; Immunohistochemistry; B7-H3; Prognosis

Correlation of IDH1 and B7[sbnd]H3 expression with prognosis of CRC patients

BackgroundB7H3 is an immuno-stimulatory glycoprotein that is overexpressed in cancer. However, its functional contributions to cancer development and progression are not well understood. In several reports, it was demonstrated that B7H3 reprograms lipid metabolism and regulates glucose metabolism. Isocitrate dehydrogenase 1 (IDH1), a metabolic enzyme in the TCA cycle, its reaction product is involved in lipid synthesis. Thus, we aimed to identify a novel marker to predict the prognosis of CRC patients and to investigate the relationship between IDH1 and B7H3. MethodsWe analyzed IDH1 and B7H3 expression levels in 225 CRC specimens by immunochemistry. Moreover, in vitro studies were performed to demonstrate the correlation between IDH1 and B7H3. ResultsAmong 225 tissues, the positive rates of IDH1 and B7H3 were 37.8% (85/225) and 87.6% (197/225), respectively. In CRC samples, IDH1 significantly correlated with B7H3 expression (P = 0.044). Moreover, multivariate analyses revealed that high expression of both B7H3 and IDH1 and a high tumor grade were related to the prognosis of CRC patients. Kaplan-Meier survival analysis revealed that patients with co-expression of IDH1 and B7H3 had a poor overall survival. In SW480B7H3-EGFP cells, which highly express B7H3, IDH1 was up-regulated. Similarly, knockdown of B7H3 expression in Caco-2-shB7-H3 contributed to reduced IDH1 levels. ConclusionsAlthough IDH1 and B7H3 cannot be used as independent prognostic factors, co-expression of IDH1 and B7H3 significantly correlated with the prognosis of CRC patients and may serve as a combined predictive marker. Thus, the correlation between IDH1 and B7H3 has been proven in vivo and in vitro.

Immunohistochemical and Image Analysis-Based Study Shows That Several Immune Checkpoints are Co-expressed in Non-Small Cell Lung Carcinoma Tumors.

The understanding of immune checkpoint molecules' co-expression in non-small cell lung carcinoma (NCLC) is important to potentially design combinatorial immunotherapy approaches. We studied 225 formalin-fixed, paraffin-embedded tumor tissues from stage I-III NCLCs - 142 adenocarcinomas (ADCs) and 83 squamous cell carcinomas (SCCs) - placed in tissue microarrays. Nine immune checkpoint markers were evaluated; four (programmed death ligand 1 [PD-L1], B7-H3, B7-H4, and indoleamine 2,3-dioxygenase 1 [IDO-1]) expressed predominantly in malignant cells (MCs) and five (inducible T cell costimulator, V-set immunoregulatory receptor, T-cell immunoglobulin mucin family member 3, lymphocyte activating 3, and OX40) expressed mostly in stromal tumor-associated inflammatory cells (TAICs). All markers were examined using a quantitative image analysis and correlated with clinicopathologic features, TAICs, and molecular characteristics. Using above the median value as positive expression in MCs and high density of TAICs expressing those markers, we identified higher expression of immune checkpoints in SCC than ADC. Common simultaneous expression by MCs was PD-L1+ B7-H3+ IDO-1 in ADC and PD-L1+ B7-H3, or B7-H3+ B7-H4, in SCC. TAICs expressing checkpoint were significantly higher in current smokers than in never smokers. Almost all the immune checkpoint markers showed positive correlation with TAICs expressing inflammatory cell markers. KRAS-mutant ADC specimens showed higher expression of PD-L1 in MCs and of B7-H3, T-cell immunoglobulin mucin family member 3, and IDO-1 in TAICs than wild type. Kaplan-Meier survival curves showed worse prognosis in ADC patients with higher B7-H4 expression by MCs. We found frequent immunohistochemical co-expression of immune checkpoints in surgically resected NCLC tumors and correlated with tumor histology, smoking history, tumor size, and the density of inflammatory cells and tumor mutational status.

High expression of B7-H3 and CD163 in cancer tissues indicates malignant clinicopathological status and poor prognosis of patients with urothelial cell carcinoma of the bladder.

The objective of the present study was to investigate the association of B7-H3 expression and cluster of differentiation (CD)163+ tumor-associated macrophage (TAM) infiltration with clinicopathological parameters in urothelial cell carcinoma of the bladder (UCB), and to investigate their potential conjoint effects on progression of UCB. B7-H3 expression and CD163+ TAM infiltration in tumor specimens from 134 consecutive patients that underwent radical cystectomy for UCB were tested using immunohistochemistry, followed by statistical analysis. In these 134 patients, B7-H3 expression and CD163+ TAM infiltration in the bladder carcinoma tissues were significantly associated with an increased ratio of vascular invasion (P=0.009; P=0.012) and distant metastasis (P=0.015; P=0.038); however, they were not associated with gender, age, pathologic grade, tumor stage, recurrence or lymphatic metastasis. The results of χ2 test analysis indicated that CD163+ TAM infiltration and B7-H3 expression were positively correlated (χ2=20.714; P<0.001). Overall survival (OS) and progression-free survival (PFS) rates were significantly worsened by high B7-H3 expression (P=0.002; P=0.020). However, CD163+ TAM infiltration was not associated with OS or PFS rate. Notably, the OS and PFS rates in patients with high B7-H3 expression or high CD163+ TAM infiltration were significantly poorer than the patients with low B7-H3 expression (P<0.001; P<0.001) or low CD163+ TAM infiltration (P=0.022; P=0.017) in the subgroup of 115 patients with muscle-invasive bladder cancer. The results of the present study indicate that B7-H3 expression level could be used as an independent prognostic indicator following radical cystectomy for UCB and patients with high B7-H3 expression and high CD163+ TAM infiltration experience a poorer prognosis.

The prognostic value of B7-H4 in pancreatic cancer

Background:There were many reports suggesting that different kinds of tumors can express B7-H4; however, the prognostic value in cancer was still unclearly. Therefore, we conducted a meta-analysis to investigate the relationship between overexpression of B7-H4 with the prognostic value in pancreatic cancer patients.Materials and Methods:The Pubmed, Embase, Cochrane Library, Ovid, Web of Science, and Chinese research database (including CBM, CNKI, and WAN FANG) were searched for related literature published until October 12, 2017. The pooled odds ratios (ORs) and/or pooled hazard ratios (HRs) for clinical pathological factors and overall survival (OS) were calculated and analyzed using Stata software. To assess whether an individual study had an impact on the result, sensitivity analysis was performed for all included individual studies using the fixed-effects model. Publication bias was evaluated using Egger's and Begg's tests.Results:Data from 6 observational studies including 442 patients were summarized in this meta-analysis, and each study was eligible for inclusion based on included and exclude criteria. The pooled results indicated that the B7-H4 overexpression could predict the presentation of lymph node metastasis (OR = 3.94, 95% CI: 1.22–12.66, P = .022), advanced TNM stage (T = the extent of the primary tumor, N = regional lymph nodes, M = distant metastases) (III+IV vs I+II; OR = 7.63, 95% CI: 2.46–23.66, P < .001), and the poor OS (HR = 3.00, 95%CI = 2.20–4.10, P < .001) in PC patients.Conclusions:This study reveals that high expression of B7-H4 is an unfavorable prognostic factor for patients with pancreatic cancer. These results may guide the clinical management of this patient population.

PD-L1, B7-H3, and PD-1 expression in immunocompetent vs. immunosuppressed patients with cutaneous squamous cell carcinoma.

To characterize the expression of co-signaling molecules PD-L1, PD-1, and B7-H3 in cutaneous squamous cell carcinoma (cSCC) by immune status. We retrospectively analyzed 66 cases of cSCC treated with surgical resection from 2012 to 2015. Immunostained tumor sections were analyzed for percent of tumor cells expressing PD-L1 (Tum-PD-L1%), B7-H3 (Tum-B7-H3%), density of peri and intratumoral CD8 T cells (CD8 density), proportion of CD8 T cells expressing PD-1 (CD8-PD-1%) and of tumor-infiltrating immune cells (TII) expressing PD-L1 (TII-PD-L1%). Of 66 cases, 42 were immunocompetent, 24 immunosuppressed (13 organ transplant, 8 HIV+, 3 other). Defining positive expression at > 5%, 26% of tumors were positive for PD-L1, 85% for B7-H3, 80% had CD8 T cells that expressed PD-1 and 55% had TII that expressed PD-L1. Tum-B7-H3% was significantly higher (median 60 vs. 28%, p = 0.025) in immunocompetent vs. immunosuppressed patients, including when factoring in cause of immunosuppression. No significant difference in Tum-PD-L1%, TII-PD-L1%, CD8 density, or CD8-PD-1% was observed. Tumors from HIV+ patients lacked PD-L1 expression, and had lower B7-H3% (median 2.5 vs. 60%, p = 0.007), and higher CD8 density (median 75% vs. 40%, p = 0.04) compared to immunocompetent patients. Higher tumor grade (Rs = 0.34, p = 0.006) and LVI (Rs = 0.61, p < 0.001) were both associated with higher Tum-PD-L1%. cSCC showed expression of PD-L1 on tumor in 26% of cases, and high tumor B7-H3 expression (85%) and PD-1 expression on CD8 TILs (80%). Tumor B7-H3 expression was significantly higher in immunocompetent vs. immunosuppressed patients, largely driven by very low expression in HIV+ patients.

Silencing of B7-H4 suppresses the tumorigenicity of the MGC-803 human gastric cancer cell line and promotes cell apoptosis via the mitochondrial signaling pathway.

B7-H4 is a transmembrane protein which is a member of the B7 superfamily. It is overexpressed in various types of cancer, including gastric cancer. However, the effects of B7-H4 on the tumorigenicity of gastric cancer and the underlying mechanisms have not yet been fully explored. Thus, the aim of this study was to examine the effects of B7-H4 on the tumorigenicity of gastric cancer cells and to elucidate the underlying mechanisms. For this purpose, B7-H4 expression in gastric cancer tissues was detected by immunohistochemical staining. The effects of B7-H4 on the biological behavior of the MGC-803 human gastric cancer cell line were examined by Cell Counting kit-8 (CCK-8) assay, cell cycle analysis, wound healing assay, AnnexinV/propidium iodide staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Moreover, the expression levels of apoptotic markers, such as cleaved caspase‑3, cleaved caspase‑9, Bcl-2 and Bax were examined by western blot analysis. Immunohistochemical staining revealed that a high expression of B7-H4 was found in about 41.8% of tissues obtained from patients with gastric cancer. Comparative analysis revealed that B7-H4 expression significantly correlated with lymph node metastasis and the TNM stage. The results of CCK-8 assay, cell cycle analysis, wound healing assay, AnnexinV/propidium iodide staining assay and TUNEL assay all demonstrated that the silencing of B7-H4 by small interfering RNA decreased cell proliferation, suppressed cell motility, and induced cell cycle arrest and the apoptosis of MGC-803 human gastric cancer cells. Furthermore, the results of western blot analysis indicated that the downregulation of B7-H4 induced the apoptosis of the MGC-803 cells via the mitochondrial signaling pathway through the activation of caspase‑3 and caspase‑9, and by altering the Bax/Bcl-2 ratio in a manner that favored apoptosis. Based on the findings on human gastric cancer cell line MGC-803, the findings of this study suggested that B7-H4 may have the potential to be a valuable prognostic marker and a target for individualized therapies for gastric cancer. However, further investigations are required in order to confirm our findings on a larger scale.

Upregulation of B7-H4 promotes tumor progression of intrahepatic cholangiocarcinoma

Recent reports show that B7-H4 is highly expressed in a variety of tumor cells, functions as a negative regulator of T cells and then promotes tumor progression. However, its expression and role in intrahepatic cholangiocarcinoma (ICC) remain unclear. In present study, B7-H4 expression in ICC and peritumoral tissues was determined at the level of mRNA and protein, and its bioactivity in ICC cells was studied after modification of B7-H4 expression. Then, the mechanism related to tumor progression induced by B7-H4 expression in ICC cells was explored. Finally, clinical significance of B7-H4 expression in ICC patients was further analyzed. The results showed that B7-H4 expression in ICC was much higher than that in peritumoral tissues at the level of both mRNA and protein. The high level of B7-H4 in ICC cells induced epithelial-to-mesenchymal transitions and promoted invasion and metastasis of tumor cells through activation of ERK1/2 signaling. The elevated B7-H4 expression was associated with the downregulated Bax, upregulated Bcl-2 expression, and activation of caspase-3. Clinically, high B7-H4 expression in tumor samples was significantly related to malignant phenotype, such as lymph node metastasis, high tumor stage, and poor differentiation. ICC patients with high expression of B7-H4 had shorter overall survival (OS) and disease-free survival. Moreover, the B7-H4 expression was an independent prognostic factor for predicting OS and tumor recurrence of ICC patients after operation. In conclusion, high expression of B7-H4 promotes tumor progression of ICC and may be a novel therapeutic target for ICC patients.

Expression of CD24 and B7-H3 in breast cancer and the clinical significance.

This study aimed to investigate the correlation between the expression of CD24 and B7-H3 in breast cancer tissues and the clinical significance. Expression of CD24 and B7-H3 in breast cancer and adjacent tissues were detected by immunohistochemistry. Quantitative PCR was used to detect the expression of CD24 and B7-H3 mRNA in breast cancer and adjacent tissues. The expression of CD24 and B7-H3 protein in breast cancer and adjacent tissues was detected by immunoblotting. The correlation between the expression levels of the two proteins was analyzed and the relationship between the expression of two proteins and the 5-year survival of breast cancer patients was investigated. CD24 and B7-H3 were positively expressed in breast cancer and adjacent tissues, the CD24-positive rate was 75.7 and 25.7%, respectively, and the B7-H3-positive rate was 56.8 and 43.2%, respectively, and the differences were statistically significant (P<0.05). The expression of CD24 was positively correlated with the expression of B7-H3 (Spearman's correlation coefficient r, 0.297; p=0.036). The positive and negative expression of CD24 and B7-H3 significantly affected the 5-year survival of breast cancer patients (P<0.05). Quantitative PCR results showed that the expression levels of CD24 and B7-H3 mRNA in breast cancer tissues were significantly higher than those in adjacent tissues (P<0.05). The expression levels of CD24 and B7-H3 protein in breast cancer tissues were also significantly higher than those in adjacent tissues (P<0.05). CD24 and B7-H3 were highly expressed in breast cancer, suggesting that both CD24 and B7-H3 were related to the development of breast cancer. Five-year survival analysis of breast cancer patients showed that the high expression of CD24 and B7-H3 were correlated with the poor prognosis of patients. Thus, CD24 and B7-H3 may become new targets for the treatment of breast cancer.

Immune Surveillance Plays a Role in Locally Aggressive Giant Cell Lesions of Bone.

Giant cell lesions are locally aggressive intraosseous neoplasms with capacity to metastasize. The role of immune surveillance in the pathophysiology of giant cell lesions is poorly understood, and understanding what role the immune system plays in giant cell lesions may lead to the development of more effective treatment. The aim of this study was to explore the role of immune surveillance in giant cell lesions by examining the expression of the HLA class I and class II antigens and tumor infiltrating lymphocytes. In addition, we examined the role of the immune modulating surface antigen B7-H3, which belongs to the B7 superfamily, a group of molecules that modulates T-cell responses. (1) Is an immune response elicited by giant cell lesions? (2) Do clinically relevant human leukocyte antigen (HLA) defects exist in giant cell lesions? (3) Is B7-H3 a clinically relevant immune modulator? The study sample was derived from the population of patients presenting to the Massachusetts General Hospital for evaluation and management of giant cell lesions from 1993 to 2008. We included patients with histologically confirmed giant cell lesions with a minimum followup of 6months. Patients with systemic diseases (n = 4 [3%]), syndromes associated with giant cell lesions (n = 4 [3%]), and those without sufficient followup (n = 26 [19%]), inadequate records (n = 7 [5%]), or inadequate tissue available (n = 2 [1%]) were excluded. Tissue microarray, containing 288 tissue cores for 93 patients, was carefully constructed. This contained tissue from 45 patients with maxillofacial lesions, 38 with aggressive and seven with nonaggressive lesions, and 48 patients with axial and appendicular lesions, 30 with aggressive lesions and 18 with nonaggressive lesions. The population mean age was 28 ± 12years and the duration of followup was 4 ± 3years. The tissue microarray was immunohistochemically stained with monoclonal antibodies specific for HLA classes I and II and B7-H3 antigens and analyzed for tumor infiltrating lymphocytes. Antigen expression was examined in multinucleated giant cells and mononuclear stromal cells. The results were correlated with local invasion and tumor aggressiveness, which is based on accepted staging criteria. Tumor infiltrating lymphocytes were detected in all the tumors. The mean number of CD8+ T cell infiltration was lower in aggressive tumors (median, 4.8; interquartile range [IQR], 0.4-13.4), when compared with nonaggressive tumors (median, 15.8; IQR, 4.3-46.3; p = 0.007). HLA class I antigens were highly expressed by multinucleated giant cells in all tumors, but were lightly expressed on mononuclear stromal cells in 53% (45 of 84) to 73% (56 of 77) of tumors. HLA class I antigen low expression in mononuclear stromal cells was associated with tumor aggressiveness (odds ratio [OR], 4.3; p = 0.005). Low HLA class I expression combined with low CD8+ T cell infiltration was most highly associated with tumor aggressiveness (OR, 7.81; p = 0.011). B7-H3 antigen was expressed in 36.9% mononuclear stroma cells and also was associated with local tumor invasion (OR, 1.36; p < 0.001). Similarly, giant cell lesions with high B7-H3 expression and low CD8+ tumor infiltrating lymphocytes were associated with increased tumor aggressiveness (OR, 8.89; p = 0.0491). Locally aggressive giant cell lesions are associated with low HLA class 1 antigen expression, low CD8+T cell infiltration, and high expression of the immune modulator B7-H3. Failure of immune surveillance implies that there may be an opportunity to target aspects of the immune surveillance machinery to treat giant cell lesions.

B7-H4 as an independent prognostic indicator of cancer patients: a meta-analysis.

The expression of B7-H4 was observed in a variety of tumors, however the prognostic value in cancer was still controversial. Therefore, we conducted this meta-analysis to explore the potential role of B7-H4 in cancer prognostic prediction. Twenty-seven studies including 3771 patients were brought into the analysis according to the inclusion and exclusion criteria. The pooled results demonstrated that elevated B7-H4 predicted a poor OS (HR = 1.93, 95% CI 1.71-2.18, P < 0.001) and DFS (HR = 1.84, 95% CI 1.46-2.33, P < 0.001). Subgroup analysis showed that races, tumor types, sample sources, analysis types, sources of HR and sample sizes exhibited non-significant distinctions with OS (PS = 0.878, PS = 0.143, PS = 0.613, PS = 0.639, PS = 0.48 and PS = 0.528, respectively). PubMed, Embase and the Cochrane Library were searched up to April 7, 2017, to recognize the available studies for assessing the association between B7-H4 and cancer patients’ outcome. We extracted the hazard ratio (HR), relative ratio (RR), odds ratio (OR) with their 95% confidence interval (CI) for overall survival (OS) or disease-free survival (DFS) as the effect size (ES) for the analysis. This meta-analysis demonstrates high expression of B7-H4 is a negative correlation with the outcome of cancer patients.

A phase 1, open-label, dose escalation study of enoblituzumab (MGA271) in pediatric patients with B7-H3-expressing relapsed or refractory solid tumors.

TPS2596 Background: Enoblituzumab, is an Fc optimized humanized IgG1 monoclonal antibody that binds to B7-H3 (CD276), a member of the B7 family. It is Fc-engineered to enhance effector function including antibody dependent cellular cytotoxicity (ADCC). IHC analyses with the parental anti-B7H3 mAb specificity incorporated in enoblituzmab revealed limited B7-H3 expression in normal tissues but high expression in many cancers (Loo et al., 2012). Among pediatric solid tumors, high expression of B7-H3 has been reported in neuroblastoma, rhabdomyosarcoma, osteosarcoma, Wilms tumor, Ewing’s sarcoma and desmoplastic small round cell tumor. B7-H3 overexpression correlates with poor prognosis in a broad range of cancers in adults suggesting a potential role in enabling tumor immune escape. ADCC and potential modulation of T cell function resulting in enhanced antitumor immune response are presumed mechanisms of action of enoblituzumab. Methods: This is an open-label, dose escalation / cohort expansion phase 1 study (NCT02982941) designed to characterize the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of enoblituzumab in children and young adults with B7-H3-expressing relapsed or refractory malignant solid tumors. A 3+3 design is used in escalating dose cohorts of weekly intravenous (IV) enoblituzumab starting at 10 mg/kg. Response is first determined at 8 weeks. irRECIST is used for response assessment for patient management. Enoblituzumab may continue up to 2 years based on response. Cohort expansion phase, to further define the safety and initial antitumor activity of enoblituzumab, will start after maximum tolerated dose is determined. The patients are assigned to 1 of 5 cohorts based on disease type as follows: 1) neuroblastoma - measurable disease, 2) neuroblastoma - non-measurable disease, 3) rhabdomyosarcoma, 4) osteosarcoma, and 5) Ewing’s sarcoma, Wilms’ tumor and desmoplastic small round cell tumors. Enrollment is ongoing. Ref : Development of an Fc-enhanced anti-B7-H3 monoclonal antibody with potent antitumor activity. Loo D, Alderson RF, Chen FZ, Huang L et al. Clin Cancer Res. 2012; 18:3834-45. Clinical trial information: NCT02982941.

Case-control study of PD-1, PD-L1, and B7-H3 expression in human immunodeficiency virus infected (HIV+) and uninfected patients with head and neck cancers.

e23194 Background: HIV+patients are typically excluded from immunotherapy trials but there are limited data on co-signaling molecule expression in these patients, including in head and neck squamous cell carcinoma (HNSCC). Methods: A case control study was conducted to evaluate tumor tissue sections for PD-L1, PD-1, and B7-H3 expression in HIV+HNSCC patients (n = 12) and HNSCC controls (n = 12). Cases and controls were matched for age at diagnosis, race, gender, TNM stage, and primary site. Smoking status, HIV RNA viral load (VL), and CD4 count were also recorded. Immunostained tumor sections were analyzed for percent of tumor cells expressing PD-L1 (PD-L1%) and B7-H3(B7-H3%) (Abcam), and percent of tumor infiltrating lymphocytes (TIL) expressing PD-1 (TIL-PD-1%) and PD-L1 (TIL-PD-L1%) (Abcam). Statistical analysis used the non-parametric Mann-Whitney test, chi-square test and Spearman’s rank correlation, Rs. Results: The 12 HIV+ HNSCC cases were predominantly male (67%), black race (67%) and cigarette smokers (100%), with a median age of 50.4 years, median viral load (VL) of 52399 copies/mL, median CD4 count (CD4) of 236cells/uL, predominantly locally advanced (75% Stage III/IVa/IVb), and oropharyngeal primary site (42%). Defining positive expression as &gt; 5%, 42% of HIV+ HNSCC patients tumors were positive for PD-L1, 100% for B7-H3, and 92% had TILs that expressed PD-1 and PD-L1. HIV+ patients had significantly higher B7-H3% (Median 60% vs. 20%, p = 0.03) and TIL-PD-L1% (Median 15% vs. 10%, p = 0.045) compared to controls. There were no differences in PD-L1% or TIL-PD-1%. In HIV+ cases, increased VL correlated with increased TIL-PD-1% (Rs = 0.73, p = 0.011) and increased CD4 count correlated with increased tumor PD-L1% (Rs = 0.62, p = 0.04). There were no other significant correlations between CD4 count or VL and co-signaling molecule expression. Conclusions: HIV+ HNSCC patients had significantly higher tumor B7-H3 and TIL PD-L1 expression, with similar tumor PD-L1 and TIL PD-1 expression, compared to HIV negative HNSCC control patients. These findings support inclusion of HIV+ HNSCC patients in immunotherapy trials with checkpoint inhibitors.

Clinical significance of the B7-H4 as a novel prognostic marker in breast cancer

Increasing evidence has suggested that B7-H4 plays an important role in cancer development not only in immune field. However, the correlation between B7-H4 protein expression and clinical outcome in primary breast cancer has not been investigated. We examined the expression of B7-H4 by Immunohistochemistry in 147 cases of invasive breast carcinomas. Correlation of B7-H4 protein levels with pathological tumor stages and overall survival rates of breast cancer patients were also analyzed. Uni-and multivariate analyses methods were performed to evaluate the prognostic value of B7-H4 expression levels. We found that different expression levels of B7-H4 on breast cancer cells were significantly correlated with patient's pTNM (pathological staging, T: primary tumor; N: regional lymph node; M: distant metastasis) stage (P<0.001), overall survival (P=0.000), p53 (P=0.041). Furthermore, univariate analyses showed that the overall survival rate of the patients with higher B7-H4 expression was significantly worse than that of the patients with lower B7-H4 expression (P<0.001). Multivariate analyses showed that B7-H4 was a significant independent prognostic indicator (P=0.000). Our results suggest that B7-H4 might be closely associated with breast cancer progression. Its high expression may serve as a potential prognostic biomarker in human breast cancer.

Analysis of the newly defined immune checkpoints in pancreatic cancer.

109 Background: Pancreatic cancer has not been improved with the five-year survival rate for more than 10 years, which is a refractory cancer of poor prognosis. Development of novel therapeutic agents has been desired, because the standard chemotherapy has not been satisfied yet. Anti-tumor immunotherapy is expected for less burden on the patient in recent years. However, it is limited by multiple tolerogenic mechanisms related to the tumor, the host immune system, and the tumor microenvironment. Therefore satisfactory results are not obtained in the clinical trials for several vaccinations. In this experiment, we focused on negative co-signaling molecules, especially immune checkpoints. The purpose of the present study was to investigate the relationship between the expression of immune checkpoints and clinico-pathological findings, including prognosis in pancreatic cancer patients. Methods: We enrolled 81 patients (52 men and 29 women; age range: 36-84 years) with pancreatic cancer who underwent curative resection with lymphadenectomy at Gunma University Hospital and Saiseikai Maebashi Hospital (Maebashi, Japan) between 2000 and 2011. Patients who had undergone palliative resection, preoperative chemotherapy and radiation therapy were excluded from this study. Furthermore, none of the patients enrolled in this study had synchronous or metachronous cancer in other organs. Results: Immunohistochemical staining of immune checkpoints in primary pancreatic tissue was performed. Patients with high B7-H4 expression had a significantly poorer prognosis than those with low B7-H4 expression. It was also seen in B7-H3 expression in this experiment. Furthermore, in analysis for the expression of CD8, IFN-γ and FOXP3, patients with high FOXP3 expression had a significantly poorer prognosis than those with low FOXP3 expression. Conclusions: The B7-h3 and B7-H4 are negative co-stimulatory molecules and a novel prognostic marker related to the T-cell mediated immune response. It’s pathway may be a molecular target for controlling tumor progression in patients with pancreatic cancer.

B7-H3 promotes the proliferation, migration and invasiveness of cervical cancer cells and is an indicator of poor prognosis.

B7-H3 is an immune regulatory molecule whose aberrant expression in tumors is associated with adverse outcomes. Upregulation of B7-H3 may promote tumor cell proliferation and metastasis invitro, but the role of B7-H3 in cervical cancer has not yet been investigated. We measured B7-H3 expression in 90cervical cancer patient and 20non‑cervical lesion patient tissues using immunohistochemistry and in 30cervical cancer patient and 30healthy donor blood samples using ELISA. The association of B7-H3 expression and the prognosis of cervical cancer patients was investigated. B7-H3 knockdown in CaSki and SiHa cell lines was performed using small hairpin (sh)RNA lentiviral transfection and B7-H3 overexpression in CaSki and HeLa cell lines was performed using plasmid-vector lentivirus transduction. Cell proliferation, invasion and migration were then measured using MTT and Transwell assays invitro. B7-H3 expression was significantly higher in the cervical cancer tissues compared to that noted in the normal cervical tissues (mean 72.22 vs.15.00%; p<0.001). Using Kaplan‑Meier and Cox analyses, our data revealed that patients with strong intensity staining were significantly more likely to have a worse prognosis. The B7-H3 level in cervical cancer patient blood was significantly higher than that in the normal donors (13.41±6.12 vs. 9.90±3.16ng/ml; p=0.007). MTT assay revealed that high expression of B7-H3 promoted cervical cancer cell proliferation. Transwell assay data revealed that high expression of B7-H3 enhanced cervical cancer cell migration and invasion (CaSki,p=0.003; HeLa,p=0.03). In conclusion, expression of B7-H3 was significantly higher in cervical cancer tissues compared to normal cervical tissues, and this high expression was associated with worse prognosis for cervical cancer patients. In addition, B7-H3 promoted proliferation, invasion and migration of cervical cancer and may be a potential target for treating cervical cancer.

Tumor B7-H3 (CD276) expression and smoking history in relation to lung adenocarcinoma prognosis

ObjectivesCompared with non-smoking counterparts, smoking-associated lung cancers have a higher mutational load, resulting in the creation of more tumor neoantigens and increased immunogenicity. B7-H3 (also known as CD276) belongs to a family of immune modulators that includes PD-1 and PD-L1 (also known as B7-H1 or CD274). Considering the evidence that PD-L1 inhibitors have been shown to be more effective against lung cancer in smokers, we herein examined the prognostic interaction of tumor B7-H3 expression level with smoking history in lung adenocarcinoma patients. Materials and methodsUsing tissue microarrays comprising 270 consecutive cases of lung adenocarcinoma, we evaluated tumor B7-H3 expression by immunohistochemistry. We examined the prognostic association between B7-H3 expression levels and smoking history, using Cox proportional hazards regression analysis and the log-rank test. Additionally, we used logistic regression analysis to examine the correlations between B7-H3 expression levels and clinicopathological/molecular features of lung adenocarcinoma. ResultsThe association of B7-H3 expression with survival differed by smoking history (Pinteraction=0.014); high B7-H3 expression was associated with decreased lung cancer-specific survival in moderate/heavy-smoking patients (smoking index [SI]≥400) (hazard ratio [HR]=3.07, 95% confidence interval [CI]=1.74–5.49, P=0.0001; log-rank: P<0.0001), but not in non/light-smoking patients (SI<400) (HR=1.14, 95% CI=0.63–1.96, P=0.64; log-rank: P=0.64). Interestingly, in moderate/heavy-smoking patients, high B7-H3 expression was associated with decreased survival in stage I cancer (log-rank; P=0.0005), whereas it showed no significant difference of survival in stage II–IV cancer (P=0.37). High B7-H3 expression was associated with smokers (univariable odds ratio [OR]=2.63, 95% CI=1.51–4.65; P=0.0005) and independently associated with EGFR wild-type status (multivariable OR=2.80, 95% CI=1.38–5.84; P=0.0042). ConclusionsWe demonstrated that the prognostic association of B7-H3 expression indeed differed according to smoking history. Our study also showed the significant association of high B7-H3 expression with EGFR wild-type and smoking patients, indicating the potential effectiveness of anti-B7-H3 therapy for EGFR wild-type or smokers’ lung adenocarcinoma.

Correlation of B7-H3 with androgen receptor, immune pathways and poor outcome in prostate cancer: an expression-based analysis.

B7-H3 (CD276), part of the B7 superfamily of immune checkpoint molecules, has been shown to have an immunomodulatory role. Its regulation, receptor and mechanism of action remain unclear. B7-H3 protein expression correlates with prostate cancer outcomes, and humanized monoclonal antibodies (that is, enoblituzumab) are currently being investigated for therapeutic use. Here we used genomic expression data to examine the relationship between B7-H3 mRNA expression and prostate cancer. Prostatectomy tissue from 2781 patients were profiled using the Affymetrix HuEx 1.0 ST microarray. Pairwise comparisons were used to identify significant associations between B7-H3 expression and clinicopathologic variables, and survival analyses were used to evaluate the prognostic significance of B7-H3. Pearson's correlation analyses were performed to assess the relationship of B7-H3 expression with molecular subtypes and individual transcripts. Androgen receptor (AR) occupancy at the B7-H3 locus was determined using chromatin immunoprecipitation (ChIP), and androgen-dependent expression changes in B7-H3 was evaluated by quantitative reverse transcription PCR in LNCaP cell lines. Oncomine was queried to evaluate B7-H3 expression in metastatic disease. B7-H3 mRNA expression was positively associated with higher Gleason score (P<0.001), tumor stage (P<0.001), and castrate resistant metastatic disease (P<0.0001). High B7-H3 expression correlated with the development of metastasis and prostate cancer specific mortality, but this was not significant on multi-variable analysis. B7-H3 expression correlated with ERG-positive disease (r=0.99) and AR expression (r=0.36). ChIP revealed an AR-binding site upstream of B7-H3, and the presence of androgens decreased B7-H3 expression in LNCaP suggesting potential direct AR regulation. Gene set enrichment analysis demonstrated an association of B7-H3 with androgen signaling as well as immune regulatory pathways. Higher B7-H3 expression correlates with Gleason grade, prostate cancer stage and poor oncologic outcomes in prostatectomy cohorts. B7-H3 expression appears to be related to androgen signaling as well as the immune reactome.