High B7-H3 Expression Research Articles

High expression of B7-H3 on stromal cells defines tumor and stromal compartments in epithelial ovarian cancer and is associated with limited immune activation

BackgroundB7-H3 and B7-H4 are highly expressed by many human malignancies making them attractive immunotherapeutic targets. However, their expression patterns and immune contexts in epithelial ovarian cancer have not been well characterized.MethodsWe used flow cytometry, immunohistochemistry, and genomic analyses to determine the patterns of B7-H3, B7-H4, and PD-L1 expression by tumor, stromal, and immune cells in the ovarian tumor microenvironment (TME). We analyzed immune cell frequency and expression of PD-1, TIM3, LAG3, ICOS, TIA-1, granzyme B, 2B4, CD107a, and GITR on T cells; CD20, CD22, IgD, BTLA, and CD27 on B cells; CD16 on monocytes; and B7-H3, B7-H4, PD-L1, PD-L2, ICOSL, CD40, CD86, and CLEC9a on antigen-presenting cells by flow cytometry. We determined intratumoral cellular location of immune cells using immunohistochemistry. We compared differences in immune infiltration in tumors with low or high tumor-to-stroma ratio and in tumors from the same or unrelated patients.ResultsOn non-immune cells, B7-H4 expression was restricted to tumor cells whereas B7-H3 was expressed by both tumor and stromal cells. Stromal cells of the ovarian TME expressed high levels of B7-H3 compared to tumor cells. We used this differential expression to assess the tumor-to-stroma ratio of ovarian tumors and found that high tumor-to-stroma ratio was associated with increased expression of CD16 by monocytes, increased frequencies of PD-1high CD8+ T cells, increased PD-L1 expression by APCs, and decreased CLEC9a expression by APCs. We found that expression of PD-L1 or CD86 on APCs and the proportion of PD-1high CD4+ T cells were strongly correlated on immune cells from tumors within the same patient, whereas expression of CD40 and ICOSL on APCs and the proportion of PD-1high CD8+ T cells were not.ConclusionsThis study provides insight into the expression patterns of B7-H3 and B7-H4 in the ovarian TME. Further, we demonstrate an association between the tumor-to-stroma ratio and the phenotype of tumor-infiltrating immune cells. We also find that some but not all immune parameters show consistency between peritoneal metastatic sites. These data have implications for the design of immunotherapies targeting these B7 molecules in epithelial ovarian cancer.

TMIC-11. ENHANCED B7-H4 EXPRESSION IN GLIOMAS WITH LOW PD-L1 EXPRESSION IDENTIFIES COLD TUMORS

Abstract The expression profiles of different immune checkpoint molecules are promising for triaging personalized targeted immunotherapy. Gliomas have been shown as potential targets for immune checkpoint inhibitors. Our study was performed to determine co-expression levels of two major B7 immune molecules PD-L1 and B7-H4 in gliomas in which both have demonstrated to inhibit anti-tumor host immunity. We assessed tumor issues from primary gliomas stage II–IV(n=505) by immunohistochemistry (IHC) for protein levels of both PD-L1 and B7-H4. Gene co-expression analysis assessing clusters based on extent of PD-L1/B7-H4 classifier genes expression were investigated in two transcriptome datasets (TCGA and CGGA) to validate IHC expression profiles. Here, we found that 61% and 54% of patient samples were positive for PD-L1 and B7-H4 respectively, whereby high-expression of either protein was limited to 23% and 20% respectively. Co-expression of PD-L1 and B7-H4 in high levels was limited to 2%. Comparable results were seen in RNA-seq datasets when PD-L1 mRNA expression level corelated negatively with B7-H4. Gene co-expression modules clustered in each grade gliomas without Double-High modules (glioma cluster with high mRNA expression of both PD-L1 and B7-H4 classifier genes) also verified restricted co-expression pattern. B7-H4 mRNA expression level had negative correlation with extent of immune cell infiltration, including tumor-infiltrating lymphocytes (TILs), and High-B7-H4 module gliomas (high B7-H4 but low PD-L1 classifier genes expression) were related to a cold tumor with less TILs. The majority of gliomas express PD-L1 or B7-H4, however, co-expression of both at high levels is minimal. The High-B7-H4 module was significantly lacking in TILs, suggesting that B7-H4 might inhibit T cell trafficking into the central nervous system (CNS). This study demonstrates that PD-L1 expression alone is not fully informative in gliomas for immune targeted or active-specific immunotherapy, and PD-L1 and B7-H4 probably inhibit different aspects of the T cell functions.

Associations of B7-H3 and B7-H4 Expression in Ductal Carcinoma In Situ of the Breast With Clinicopathologic Features and T-Cell Infiltration.

B7-H3 and B7-H4 play an inhibitory role in T-cell function by limiting proliferation and cytokine production. Information about B7-H3 and B7-H4 expression in ductal carcinoma in situ (DCIS) remains uncertain. The objective of this study was to evaluate the expression levels of B7-H3 and B7-H4 in DCIS and their associations with clinicopathologic features and T-cell infiltration. B7-H3 and B7-H4 mRNA and protein expression levels in 8 pairs of DCIS tissues and matched normal adjacent tissues were examined by RNAscope in situ hybridization and immunohistochemistry analysis. Immunohistochemical staining of B7-H3, B7-H4, CD3, and CD8 was performed for 79 DCIS samples using tissue microarray. RNAscope in situ hybridization and immunohistochemistry analysis revealed that expression levels of B7-H3 and B7-H4 in DCIS tissues were higher than those in corresponding normal tissues. B7-H3 and B7-H4 mRNA and protein appeared to be mainly expressed in DCIS carcinoma cells. High B7-H3 and B7-H4 expression was observed in 58 (73.4%) and 62 (78.5%) cases with DCIS, respectively. High B7-H3 expression was significantly associated with high-nuclear grade and presence of comedo-type necrosis (both P<0.05). B7-H3 expression in HR/HER2 subtype was higher than that in HR/HER2 subtype (P<0.05). B7-H3 and B7-H4 expression levels were negatively related to the density of CD3 and CD8 T-cell infiltrates. B7-H3 and B7-H4 may play an important role in immune surveillance mechanisms of DCIS. They might be useful targets to develop immune-based therapy to alter or prevent DCIS progression.

The prognostic value of B7H1 and B7H4 expression in pancreatic cancer: A meta-analysis.

The clinical implications of B7H1 and B7H4 in pancreatic cancer have been described however, the prognostic significance of these genes in pancreatic cancer patients remains inconclusive. The aim of the present study was to evaluate the prognostic role of B7H1 and B7H4 in pancreatic cancer patients. Electronic databases (PubMed, EMBASE, and the Cochrane Library) were searched for relevant articles published before May 2019. Meta-analyses were performed by pooling the hazard ratios (HRs) between overall survival or cancer-specific survival and high or low expression of B7H1/B7H4 in pancreatic cancer patients. Subgroup and sensitivity analyses were performed, and sources of variabilities were explored by performing meta-regression. Sixteen studies (1434 patients' data) were included. Compared with low expression, high expression of B7H1 was associated with significantly poor overall survival (HR 1.92 (95% confidence interval (CI) 1.35, 2.74); P<0.001) and cancer-specific survival (HR 2.46 (95% CI 1.55, 3.90); P<0.001). High expression of B7H4 also predicted poor overall survival (HR 2.38 (95% CI 1.89, 3.00); P<0.001). In subgroup analyses, a significant association between B7H1 and overall survival was observed for trials conducted in China (HR 2.08 (95% CI 1.29, 3.34)) but not in Japan (HR 1.98 (95% CI 1.33, 2.96)); or in studies with <50% patients having high expression (HR 2.02 (95% CI 1.40, 2.91)) but not in studies with >50% patients with high expression (HR 1.40 (95% CI 0.87, 2.26)). The current study suggests that high B7H1 and B7H4 expression is associated with a poor prognosis in pancreatic cancer patients.

High expression of BTLA and B7-H4 on the surface of myeloid dendritic cells has a negative regulatory effect on their anti-tuberculosis immunity activity in pleural tuberculosis patients

To investigate the effects of the surface markers B- and T-lymphocyte attenuator (BTLA) and B7 homologous body 4 (B7-H4) on expression of CD83, and Human Leukocyte Antigen–DR isotype (HLA-DR) that can activate dendritic cells (DCs). Flow cytometry was used to detect the co-expression of BTLA and B7-H4 on myeloid DCs (mDCs) in peripheral blood (PB) and pleural effusions (PE) in 15 volunteers and 20 tuberculous pleurisy (TP) patients. Co-expression of BTLA and B7-H4 (double positive (DP)) mDCs in PB and PE of TP patients were enhanced. The proportion of DP mDC in PB decreased markedly after 2 weeks treatment, but was still greater than in controls. Expression of CD83 and HLA-DR on DP mDCs was higher than on BTLA and B7-H4 double negative (DN) expressing mDCs in PB of different TP groups. Expression of CD83 on DP mDCs in PB and PE of TP patients was greater than that of controls. Expression of HLA-DR on DP mDCs in TP patient PB was lower than in TP PE and controls. In pleural tuberculosis (TB) patients, high expression of BTLA and B7-H4 promoted a high level of CD83 and HLA-DR, which had a negative regulatory effect on mDCs on anti-TB immunity.

Tumor cell expression of B7-H4 correlates with higher frequencies of tumor-infiltrating APCs and higher CXCL17 expression in human epithelial ovarian cancer

ABSTRACTB7-H4, an immune suppressive member of the B7 family, is highly expressed in a wide variety of human malignancies making it an attractive immunotherapeutic target. However, the association between B7-H4 expression in the tumor microenvironment and the immune infiltrate has not been comprehensively examined. To evaluate the immune tumor microenvironment, we analyzed epithelial ovarian tumors from 28 patients using flow cytometry, immunohistochemistry, functional, and genomic analyses. We determined B7-H4 expression patterns and compared the immune infiltrates of tumors with high and low surface expression of B7-H4. Frequencies and phenotypes of tumor and immune cells were determined using multiple flow cytometry panels. Immunohistochemistry was used to analyze cellular infiltration and location. Publicly available datasets were interrogated to determine intratumoral cytokine and chemokine expression. We found that B7-H4 was predominantly expressed by tumor cells in the epithelial ovarian tumor microenvironment. Surface expression of B7-H4 on tumor cells was correlated with higher levels of infiltrating mature antigen-presenting cells. Further, expression of CXCL17, a monocyte and dendritic cell chemoattractant, correlated strongly with B7-H4 expression. T cells expressed activation markers, but T cells expressing a combination of markers associated with T cell activation/exhaustion phenotype were not prevalent. Overall, our data suggest that B7-H4 is associated with a pro-inflammatory tumor microenvironment.

B7-H3-redirected chimeric antigen receptor T cells target glioblastoma and neurospheres

BackgroundThe dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM.MethodsWe compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models.FindingsB7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines.InterpretationWe demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM.FundAlex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.

Enhanced B7-H4 Expression in Gliomas With Low PD-L1 Expression Identifies Cold Tumors

Abstract INTRODUCTION The expression profiles of different immune checkpoint molecules are promising for triaging personalized targeted immunotherapy. Our study was performed to determine co-expression levels of 2 major B7 immune molecules, PD-L1 and B7-H4, in gliomas where both have demonstrated to inhibit antitumor host immunity. METHODS We assessed tumor issues from primary gliomas stage II to IV (n = 505) by immunohistochemistry (IHC) for protein levels of both PD-L1 and B7-H4. Gene co-expression analysis assessing clusters based on extent of PD-L1/B7-H4 classifier genes expression were investigated in 2 transcriptome datasets (TCGA and CGGA) to validate IHC expression profiles and explore properties of the glioma immune microenvironment among specific co-expression PD-L1/B7-H4 cluster groups. RESULTS PD-L1 was detected in 61% of patients whereby 23% expressed high levels. B7-H4 was expressed in 54% whereby 20% were identified as high expression. Co-expression of PD-L1 and B7-H4 in high levels was limited to 2% cases. Comparable results were seen in RNA-sequencing datasets when PD-L1 mRNA expression level corelated negatively with B7-H4. Gene co-expression modules clustered in each grade gliomas without double-high modules (gliomas cluster with high mRNA expression of both PD-L1 and B7-H4 classifier genes) also verified restricted coexpression pattern. B7-H4 mRNA expression level had negative correlation with extent of immune cell infiltration, including tumor-infiltrating lymphocytes (TILs), and high-B7-H4 module gliomas (high B7-H4 but low PD-L1 classifier genes expression) was related to a cold tumor with less TILs. CONCLUSION The majority of gliomas express PD-L1 or B7-H4, however, co-expression of both at high levels is minimal. The high-B7-H4 module was significantly lacking in TILs, suggesting that B7-H4 might inhibit T cell trafficking into the central nervous system (CNS). This study demonstrates that PD-L1 expression alone is not fully informative in gliomas for immune targeted or active-specific immunotherapy, and PD-L1 and B7-H4 probably inhibit different aspects of the T cell functions.

B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

Glioblastoma (GBM) remains one of the most malignant primary tumors in adults, with a 5-year survival rate less than 10% because of lacking effective treatment. Here, we aimed to explore whether B7-H3 could serve as a novel therapeutic target for GBM in chimeric antigen receptor (CAR) T cell therapy. In this study, a CAR targeting B7-H3 was constructed and transduced into T cells by lentivirus. Antitumor effects of B7-H3-specific CAR-T cells were assessed with primary and GBM cell lines both in vitro and in vivo. Our results indicated that B7-H3 was positively stained in most of the clinical glioma samples, and its expression levels were correlated to the malignancy grade and poor survival in both low-grade glioma (LGG) and GBM patients. Specific antitumor functions of CAR-T cells were confirmed by cytotoxic and ELISA assay both in primary glioblastoma cells and GBM cell lines. In the orthotropic GBM models, the median survival of the CAR-T-cell-treated group was significantly longer than that of the control group. In conclusion, B7-H3 is frequently overexpressed in GBM patients and may serve as a therapeutic target in CAR-T therapy.

PD-L1, B7-H3 and VISTA are highly expressed in gestational trophoblastic neoplasia.

The B7 family check-point molecules are potential therapeutic targets in cancer immunotherapy. However, their expression status in human gestational trophoblastic neoplasia (GTN) remains unknown. We investigated the expression profiles of the B7 family check-point proteins PD-L1, PD-L2, B7-H3, B7-H4, VISTA and B7-H6 in GTN and their clinical significance. We identified 112 patients with GTN, including 68 with choriocarcinoma, 33 with placental-site trophoblastic tumour (PSTT) and 11 with epithelioid trophoblastic tumour (ETT). Immunohistochemical staining was performed on whole-tissue GTN sections using anti-B7 family antibodies. VISTA expression was immunohistochemically analysed using microarrays of normal human tissues and of 20 common cancers. PD-L1 and B7-H3 were highly expressed in all GTN tumours, while PD-L2 was expressed in 87.5% of the samples. B7-H4 and B7-H6 were negative in 100% and 98.2% of the samples, respectively. PD-L1, B7-H3 and VISTA levels were significantly higher in choriocarcinomas and PSTTs than in ETTs. There was no association between B7 family check-point expression in tumour cells and disease stage, prognostic score or patient outcomes (complete remission versus death). VISTA protein was widely overexpressed in 98.2% of all the GTN, but its expression varied in other cancer types and was negative in normal adult and fetal tissues except placental trophoblasts and splenic lymphocytes. The GTN trophoblast cells show high expression of PD-L1, B7-H3 and VISTA in a manner that is independent of clinical outcomes. These proteins may be potential immunotherapeutic targets when treating GTN.

Abstract 3248: B7-H3, an immune checkpoint protein is overexpressed in AML and the blocking monoclonal antibodies enhance NK cell-mediated apoptosis in AML cells

Abstract Acute myeloid leukemia (AML) is the most common and aggressive acute leukemia found in adults. Immune checkpoint inhibition has led to important clinical advances in cancer therapy in recent years due to superior cure rates compared with standard therapy. We hypothesize that B7-H3 (CD276) an immune checkpoint protein is overexpressed in AML cells and targeting B7-H3 activates immune cells against AML cells. We analyzed B7-H3 expression in peripheral blood (PB) and bone marrow (BM) mononuclear cells from AML patients (n=65) and healthy donors (n=10) at MD Anderson Cancer Center. Cell surface expression analysis by flow cytometry revealed that the cells of ~60% of the patients were positive for B7-H3 and its expression was 2- to 3-fold higher in AML cells than in healthy donor cells. B7-H3 expression is relatively higher in CD34+ AML cells than in CD34- AML cells (p&amp;lt;0.01). In contrast, no difference was observed between CD34+ and CD34- cells from healthy donors. The Cancer Genome Atlas RNA sequencing data revealed that patients with high B7-H3 expression had significantly lower overall and disease-free survival durations than did patients with low B7-H3 expression (p=0.024). To investigate the role of B7-H3 in immunomodulation, we stably knocked down B7-H3 in OCI-AML3 and co-cultured them with or without human PB-derived NK cells at a 2:1 ratio and measured apoptosis induction in AML cells by annexin-v binding approach. We found that knockdown of B7-H3 induced NK cell-mediated apoptosis in AML cells 3-fold compared to control AML cells. These data indicate that inhibition of B7-H3 in AML cells enhances NK cell-mediated apoptosis in AML cells. To target B7-H3, we have generated four monoclonal antibodies: B1, B2, B3 and B4 (codenamed to protect IP). To investigate whether these novel anti-B7-H3 monoclonal antibodies are able to block B7-H3 immunomodulatory function and activate NK cells, we performed a co-culture experiment with GFP-expressing OCI-AML3 cells and PB-derived NK cells in the presence or absence of anti-B7-H3 antibodies. Apoptosis induction was measured by real-time annexin-v binding using IncuCyte live cell imaging system. The addition of anti-B7-H3 monoclonal antibodies, B1, B2, B3 and B4 at 25μg/ml enhanced NK cell-induced apoptosis 3-fold in OCI-AML3 cells. These data indicate that anti-B7-H3 antibodies block the immunomodulatory function of B7-H3 and induce NK cell-mediated apoptosis in AML cells. In vivo testing of these antibodies against AML-PDX models is currently ongoing. In conclusion, we found that B7-H3 is overexpressed in AML cells and its expression is associated with bad prognosis in AML patients. Knockdown or antibody-mediated blocking of B7-H3 enhanced NK cell-induced apoptosis in AML cells. These data indicated that B7-H3 is a novel immune-checkpoint protein in AML and patients could potentially benefit from anti-B7-H3 therapies. Citation Format: Stanley Ly, Bin Yuan, Sabrina Grimm, Michael Andreeff, Hans-Jörg Bühring, Venkata Lokesh Battula. B7-H3, an immune checkpoint protein is overexpressed in AML and the blocking monoclonal antibodies enhance NK cell-mediated apoptosis in AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3248.

Abstract P4-06-25: B7-H3 and B7-H4 expression in ductal carcinoma in situ of the breast: Associations with clinicopathologic features and T-cell infiltration

Abstract Background: With the clinical success of immune check point blockades in treating malignant tumors, there are intense investigations in identifying new pathways to activate immune system by targeting immunoregulatory molecules. B7-H3 and B7-H4 play an inhibitory role in T cell function by limiting proliferation and cytokine production. Although several studies have investigated the expression of B7-H3 and B7-H4 in invasive breast cancers, the information of the B7-H3 and B7-H4 molecules in ductal carcinoma in situ (DCIS) remains uncertain. The present work was undertaken to evaluate the expression of B7-H3 and B7-H4 in DCIS and its association with clinicopathological features in patients with DCIS. In addition, the association of B7-H3 and B7-H4 expression with the T-cell infiltration was also assessed to investigate its roles in the regulation of tumor immune surveillance. Materials and methods: B7-H3 and B7-H4 expression was examined in 8 pairs of DCIS tissues and matched normal adjacent tissues at mRNA and protein levels by RNAscope in situ hybridization (ISH) and immunohistochemistry. Immunohistochemical staining of B7-H3 and B7-H4 was done in 79 DCIS samples with known hormone receptor (HR) and human epidermal growth factor 2 (HER2) expression using tissue microarray. In addition, immunohistochemical staining was also performed for the T cell lineage markers CD3 and CD8 in DCIS. Results: RNAscope ISH and immunohistochemistry of B7-H3 and B7-H4 confirmed their increases in DCIS tissues compared with their corresponding normal tissues. B7-H3 and B7-H4 mRNA and protein expression appeared to be concentrated mainly in the DCIS carcinoma cells. High B7-H3 and B7-H4 expression was observed in 58 (73.4%) and 62 (78.5%) cases with DCIS, respectively. High B7-H3 expression was significantly associated with high nuclear grade and presence of comedo-type necrosis (P &amp;lt; 0.05 and P &amp;lt; 0.05, respectively). B7-H3 expression was higher in the HR–/HER2+ subtypes than in the HR+/HER2– subtype (P &amp;lt; 0.05). B7-H3 and B7-H4 expression tended to be associated with low stromal tumor infiltrating lymphocytes density and was negatively related to the CD3+ and CD8+ T cell infiltrates density. Conclusions: These results suggest that B7-H3 and B7-H4 may play an important role in immune surveillance mechanisms of DCIS and may be useful targets for immune-based therapy to alter or prevent DCIS progression. Citation Format: Lee JS, Kim G-E, Park MH, Yoon JH. B7-H3 and B7-H4 expression in ductal carcinoma in situ of the breast: Associations with clinicopathologic features and T-cell infiltration [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-25.

Association of increased B7 protein expression by infiltrating immune cells with progression of gastric carcinogenesis.

B7 negative costimulatory molecules are a group of molecules associated with the occurrence, development, and therapy of cancers. Here, we aimed to determine the clinical significance of PD-L1, B7-H3, and B7-H4 and their expression in CD8 and CD68 positive cells at different stages of gastric carcinogenesis.We detected PD-L1, B7-H3, B7-H4, CD8, and CD68 expression in samples by immunohistochemical staining of 62 chronic superficial gastritis (CSG) samples, 72 chronic atrophic gastritis (CAG) samples, 68 low-grade intraepithelial neoplasia (LIN) samples, 65 high-grade intraepithelial neoplasia (HIN) samples obtained from gastroscopic biopsies and 50 gastric adenocarcinoma (GA) samples obtained from surgical resections. Then we statistically analyzed the expression differences and correlations.Our results indicated that B7 and CD68 expression on infiltrating immune cells was associated with disease progression. However, infiltration of CD8+ cells decreased with disease progression. B7-H3 expression was markedly enhanced at neoplasia and GA stages. B7-H3 in tumor cells was negatively correlated with CD8-expressing cells. Conversely, B7-H3 expression in tumor-infiltrating immune cells was positively correlated with CD68-expressing cells. B7-H4 expression was found in the cell membrane at the stages of gastritis and low-grade neoplasia and was gradually expressed in the cytoplasm at high-grade neoplasia and GA stages. High B7-H4 expression in infiltrating immune cells was also significantly associated with lower CD8-positive and higher CD68-positive cell densities.Increased B7 protein expression by infiltrating immune cells was associated with disease progression, and specifically, the level of B7-H3 expression and localization of B7-H4 expression differed significantly among different stages of gastric carcinogenesis.

High expression of B7-H2 or B7-H3 is associated with poor prognosis in hepatocellular carcinoma

High expression of B7-H2 or B7-H3 is associated with poor prognosis in hepatocellular carcinoma

Does B7-H4 expression correlate with clinicopathologic characteristics and survival in ovarian cancer?: A systematic review and PRISMA-compliant meta-analysis.

Background:Several studies have shown that B7-H4 expression is significantly increased in ovarian cancer. However, the role of B7-H4 expression in ovarian cancer remains unclear, and some studies reporting conflicting results. A systematic review of the literature and meta-analysis were conducted to assess the clinicopathologic characteristics and prognostic significance of B7-H4 in ovarian cancer.Methods:Eligible studies were searched in the PubMed, MEDLINE, Cochrane Library, and the China National Knowledge Infrastructure databases. The included studies assessed the relationship between B7-H4 expression and clinicopathologic features or prognosis in patients with ovarian cancer through September 2017. A total of 1045 patients in 10 studies were included in the meta-analysis. Stata software version 12.0 was used to analyze the data. We used an odds ratio (OR) or hazard ratio (HR) with a 95% confidence interval (CI) to assess the risk or hazard association.Results:B7-H4 expression in ovarian cancer patients was significantly increased (OR: 4.20, 95% CI: 2.85–6.18, Z = 6.91, P < .05), and heterogeneity was low between studies (I2 = 8.2%, P = .366). With respect to the clinicopathologic features, no relation was detected between B7-H4 expression and International Federation of Gynaecology and Obstetricsstages stages (OR: 0.81, 95% CI: 0.64–1.03, Z = 1.70, P = .09), pathologic grade (OR: 0.91, 95% CI: 0.72–1.16, Z = 0.76, P = .45), tumor metastasis (OR: 1.25, 95% CI: 0.90–1.74, Z = 1.34, P = .18), or histologic type (OR: 1.17, 95% CI: 0.85–1.60, Z = 0.96, P = .34) in ovarian cancer. Furthermore, B7-H4 expression was significantly associated with a worse progression-free survival (PFS) (HR: 1.30, 95% CI: 1.17–1.45, Z = 4.79, P < .05).Conclusion:B7-H4 expression was related to ovarian cancer, but not to patients’ clinicopathologic characteristics. High B7-H4 expression was negatively correlated with survival outcome, suggesting that B7-H4 plays an essential role in poor prognosis in ovarian cancer patients.

Abstract B31: Tumor-intrinsic B7-H3 regulates drug resistance, metabolism, and pathogenesis in ovarian cancer

Abstract B7-H3 (CD276) is a cell surface glycoprotein of B7 family of checkpoint blockade proteins that has been considered a valuable novel biomarker and a promising target for cancer treatment. B7-H3 is overexpressed in a wide variety of human cancers and is associated with tumor progression, metastasis, and poor patient outcome. In addition to its function in cancer immunity, B7-H3 also has tumor-intrinsic functions related to drug resistance, metabolic reprogramming, migration, and invasion of cancer cells. B7-H3 overexpression in the ovarian tumors and tumor vasculature has been reported and B7-H3 monoclonal antibodies have shown inhibitory effects in ovarian cancer xenograft studies. Nonetheless, the molecular pathways regulated by B7-H3 in ovarian cancer remain largely unknown. Here we explored the role of tumor intrinsic B7-H3 in ovarian cancer cells by genetic modulation of B7-H3 expression levels in ovarian cancer cells, using CRISPR/Cas9-mediated B7-H3 knockout and B7-H3 overexpression using retrovirus system for cell transduction. Knockout of B7-H3 in OV-90 cells, which showed high endogenous expression of B7-H3, resulted in decreased glycolysis and oxidative phosphorylation through downregulation of HIF1α, and its downstream target glycolytic proteins LDHA and PDK1. Furthermore, OV-90 B7-H3 KO cells showed decreased phosphorylation of the prosurvival protein STAT3, reduced clonogenic and migratory capability, and increased sensitivity to chemotherapeutic agent carboplatin. Overexpression of B7-H3 in TOV112D cells, which has low endogenous expression of B7-H3, resulted in the opposite effects, leading to increased glycolysis and mitochondrial respiration, increased STAT3 phosphorylation, increased migration, and increased resistance to carboplatin. We also performed RNA-seq analysis of OV-90 B7-H3 WT and KO clones and found several signaling pathways involved in cell migration, adhesion, and inflammatory response. Finally, we found that the growth of OV-90 B7-H3 KO cells was dramatically defective in xenograft models using both athymic nude and SCID immunocompromised mice. These results suggest that tumor-intrinsic B7-H3 plays a critical role in the growth of ovarian cancer and warrants further investigation of therapeutic approaches blocking B7-H3 functions for the treatment of this deadly malignancy in women. Citation Format: Holly Taylor, Jaroslav Slamecka, Alla Musiyenko, Elaine Gavin, Tiffany S. Norton, Ileana Aragon, Taylor Young, Jennifer Scalici, Rodney P. Rocconi, Ming Tan, Luciana Madeira da Silva. Tumor-intrinsic B7-H3 regulates drug resistance, metabolism, and pathogenesis in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B31.

Monitoring Immune Checkpoint Regulators as Predictive Biomarkers in Hepatocellular Carcinoma

The global burden of hepatocellular carcinoma (HCC), one of the frequent causes of cancer-related deaths worldwide, is rapidly increasing partly due to the limited treatment options available for this disease and recurrence due to therapy resistance. Immune checkpoint inhibitors that are proved to be beneficial in the treatment of advanced melanoma and other cancer types are currently in clinical trials in HCC. These ongoing trials are testing the efficacy and safety of a few select checkpoints in HCC. Similar to observations in other cancers, these immune checkpoint blockade treatments as monotherapy may benefit only a fraction of HCC patients. Studies that assess the prevalence and distribution of other immune checkpoints/modulatory molecules in HCC have been limited. Moreover, robust predictors to identify which HCC patients will respond to immunotherapy are currently lacking. The objective of this study is to perform a comprehensive evaluation on different immune modulators as predictive biomarkers to monitor HCC patients at high risk for poor prognosis. We screened publically available HCC patient databases for the expression of previously well described immune checkpoint regulators and evaluated the usefulness of these immune modulators to predict high risk, patient overall survival and recurrence. We also identified the immune modulators that synergized with known immune evasion molecules programmed death receptor ligand-1 (PD-L1), programmed cell death protein-1 (PD-1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and correlated with worse patient outcomes. We evaluated the association between the expression of epithelial-to-mesenchymal transition (EMT) markers and PD-L1 in HCC patient tumors. We also examined the relationship of tumor mutational burden with HCC patient survival. Notably, expression of immune modulators B7-H4, PD-L2, TIM-3, and VISTA were independently associated with worse prognosis, while B7-H4, CD73, and VISTA predicted low recurrence-free survival. Moreover, the prognosis of patients expressing high PD-L1 with high B7-H4, TIM-3, VISTA, CD73, and PD-L2 expression was significantly worse. Interestingly, PD-L1 expression in HCC patients in the high-risk group was closely associated with EMT marker expression and prognosticates poor survival. In HCC patients, high tumor mutational burden (TMB) predicted worse patient outcomes than those with low TMB.

Tumor B7-H3 (CD276) Expression and Survival in Pancreatic Cancer.

B7-H3 (CD276), a member of the family of immune modulators, orchestrates antitumor immunity. To date, only small-sized studies have examined the association of B7-H3 expression with survival in pancreatic cancer, yielding inconclusive results. We evaluated tumor B7-H3 expression in 150 consecutive patients with pancreatic ductal adenocarcinoma using immunohistochemistry. B7-H3 expression was positive (≥10% tumor cells) in 99 of 150 (66%) cases of pancreatic cancer. We classified the tumors into four groups depending on B7-H3 expression (negative, low, intermediate, and high) and found that higher B7-H3 expression was independently associated with lower disease-free survival (DFS; for high vs. negative B7-H3 expression: multivariable hazard ratio (HR) = 3.12; 95% confidence interval (CI) = 1.48–6.15; Ptrend = 0.0026). Furthermore, the association of B7-H3 expression with survival differed according to the pathological stage (p-stage) (Pinteraction = 0.048, between p-stages I–II and III–IV). The association of B7-H3 positivity with lower DFS was stronger in tumors with p-stage I–II (multivariable HR = 3.10, 95% CI = 1.75–5.69; P < 0.0001) than in those with p-stage III–IV (multivariable HR = 1.20, 95% CI = 0.67–2.28; P = 0.55). We demonstrated that tumor high B7-H3 expression is independently associated with poor survival in patients with pancreatic cancer and that this association is stronger in tumors with p-stage I–II than in those with p-stage III–IV. B7-H3 expression may be a useful prognostic biomarker for identifying aggressive early-stage pancreatic cancer.

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.