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Is Associated with Clinicopathologic Features In CLINICAL RESEARCH e-ISSN 1643-3750 © Med Sci Monit, 2018; 24: 7303-7308 DOI: 10.12659/MSM.910956 Received: 2018.05.03 Accepted: 2018.06.05 Expression of B7 Homolog 1 (B7H1) Is Published: 2018.10.13 Associated with Clinicopathologic Features in Urothelial Bladder Cancer Authors’ Contribution: ACE 1 Qingyuan Li 1 Department of Urology, Yantai Affiliated Hospital of Binzhou Medical University, Study Design A ACFG 2 Feng Li Yantai, Shandong, P.R. China Data Collection B 2 Experiment Support Center, Binzhou Medical University, Yantai, Shandong, Statistical Analysis C BCF 1 Jizhong Che P.R. China Data Interpretation D CF 1 Yang Zhao 3 Traditional Chinese Medicine Hospital of Xiajin County, Dezhou, Shandong, Manuscript Preparation E DE 3 Chengjian Qiao P.R. China Literature Search F Funds Collection G Corresponding Author: Feng Li, e-mail: [email protected] Source of support: Departmental sources Background: B7 homolog 1 (B7H1) plays an important role in regulating tumor immunity. The purpose of this study was to probe the relationship between B7H1 expression and clinical outcomes in urothelial bladder cancer. Material/Methods: We investigated 110 urothelial bladder cancer cases. The expressions of B7H1 in tumors were analyzed by im- munohistochemistry and RT-PCR. The correlation between B7H1 expression and survival rate was analyzed by log-rank test. Results: B7H1 expression was significantly increased in cancerous tissues compared to normal tissues (p<0.05). B7H1 expression was not associated with sex, age, diameter, or the combination of these factors (p>0.05). The posi- tive expression of B7-H1 was positively correlated with grade, stage, recurrence, and metastasis (p<0.05). The RT-PCR results were consistent with the immunohistochemistry outcomes. Furthermore, the expression of B7H1 in tumors was highly correlated with the survival rate (p<0.05). Conclusions: Expression of B7H1 is correlated with clinicopathologic features in bladder cancer. Up-regulation of B7H1 can result in progression and recurrence of urothelial bladder cancer. MeSH Keywords: Antigens, CD274 • Survival Analysis • Urinary Bladder Neoplasms Full-text PDF: https://www.medscimonit.com/abstract/index/idArt/910956 1892 2 3 33 Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) 7303 [Chemical Abstracts/CAS] Li Q. et al.: CLINICAL RESEARCH Expression of B7H1 in bladder urothelial cancer © Med Sci Monit, 2018; 24: 7303-7308 Background Table 1. The clinicopathologic parameters of the 98 cases. Urothelial bladder cancer (UBC) is a common disease, account- n n ing for approximately 7% of all cancer cases [1,2]. Transurethral Age (y) Metastatic tumor resection combined with intravesical chemotherapy is the stan- dard treatment for Ta and T1 disease in urothelial bladder <65 46 Metastasis 11 cancer, but the outcomes remain poor [3]. Some studies have ³65 52 Non-metastasis 87 shown that the clinical outcome of UBC is associated with im- Tumor grade Tumor size (diameter) mune responses in cancer patients with anti-immune cells [4,5]. Response to therapy can be predicted by the combination of I 23 <3 cm 32 clinical outcomes and tissue-based molecular markers, which II 46 ³3 cm 66 could help in administering the optimal local and systemic III 29 Focus therapy in UBC patients. Tumor stage Solitary 45 B7 homolog 1 (B7-H1), also known as programmed death li- Ta–T1 25 Multiple 53 gand1 (PD-L1), is the third member of the B7 family [5]. T cells T2–T4 73 are immune cells that play a critical role in mediating anti-tumor immunity. A large numbers of T cells are present in bladder Tumor type cancer, and the cells positively control the local anti-tumor re- Primary 57 sponses, such as CD4+ T cells and CD86 [6]. B7-H1 can inhibit Recurrence 41 proliferation of T cells and is a negative stimulus in the pro- cess of activation of T cells through suppressing the secretion of some cytokines, for example, interferon-g (IFN-g), interleu- Material and Methods kin-10 (IL-10), IL-14, and IL-2 [7,8]. Expression of B7-H1 was reported to be higher in tumor tissues and the number of im- Clinical study mune cells in peripheral blood was found to be related with tumor escape [9]. Up-regulation of B7-H1 has been reported to We enrolled 98 patients (22 females and 76 males) who un- be correlated with progression of thyroid cancer, lung cancer, derwent bladder urothelial cancer surgery at Yantai Affiliated ovarian cancer, colon cancer, and melanoma [10–14]. A pre- Hospital of Binzhou Medical University from 1 September vious study showed that B7H1 expression is involved in the 2009 to 1 October 2011. We obtained cancerous tissue sam- mitogen-activated protein kinase (MAPK) pathway, especially ples from each patient, as well as 12 tissue samples adjacent the extracellular signal-regulated kinas (ERK) pathway in blad- to tumors. None of the patients in this study had received any der cancer [15]. In the in vitro lung cancer microenvironment, radiation therapy or chemotherapy before the operation. All B7H1 expression was up-regulated, with increasing trans- patients were diagnosed by the method of cystoscopy, ultra- forming growth factor b (TGF-b) and was associated with reg- sound, computerized tomography urography (CTU), and pa- ulatory T cell generation [16]. Moreover, overall survival was thology. Written permission was obtained from the patients correlated with B7H1 expression in patients with UBC. These prior to surgery. All patients were confirmed with pathological findings suggest that B7H1 plays an integral role in the im- examination. All tissues were stored in a liquid nitrogen tank munological escape mechanism in UBC. after surgical resection. Many studies showed that B7H1-positive expression was asso- The tumors were classified according to the tumor-node-metas- ciated with rapid cancer progression. The expression of B7H1 tasis (TNM) staging system of the International Union Against is significantly associated with the clinicopathological variables Cancer (1997) and the World Health Organization (WHO). All and postoperative survival in esophageal cancer [17]. Patients clinicopathologic parameters are summarized in Table 1. with high B7H1 expression show shorter survival than those with low B7H1 expression. This study was approved by Medical Ethics Committee of Binzhou Medical University. In this study, we investigated the B7H1 expression in UBC pa- tients and analyzed the relationship between expression of Immunohistochemistry B7H1 and clinicopathological features. The results may con- tribute to clinical treatment of UBC. Bladder cancer tissues and tissue adjacent to bladder cancer were fixed with 4% paraformaldehyde (Beijing North of Conde Clinical Reagent Co., Beijing, China), embedded in paraffin, and Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] This work is licensed under Creative Common Attribution- [ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) 7304 [Chemical Abstracts/CAS] Li Q. et al.: Expression of B7H1 in bladder urothelial cancer CLINICAL RESEARCH © Med Sci Monit, 2018; 24: 7303-7308 A B C Figure 1. B7H1 protein expression in normal bladder tissue, UBC tissue, and positive tissue. Representative slides (×400) of B7H1 protein staining in normal bladder tissue (A), UBC tissue (B), and positive tissue (C). then sliced into 4-μm sections. All sections were incubated Results for 4 h at room temperature with anti-B7H1 antibody (1: 50, Boster, China). Phosphate-buffered saline (PBS) was used as B7H1 overexpression in urothelial bladder cancer tissue a blank control to replace the first antibody. The second anti- body was diluted to 1: 500. Positivity of cell plasma or mem- The expression of B7-H1 protein was analyzed by immunohis- branes was indicated by tan color. Three randomly selected tochemistry (Figure 1). We found that the B7H1 expression was fields were observed at high magnification (*400). Greater negative in normal bladder tissue. However, the rate of positive than 10% positive cells was regarded as positive. The results expression in urothelial bladder cancer tissue was 57.14% (56/98). were analyzed using AlphaView SA software (Thermo Fisher Scientific, Waltham, MA, USA). The correlation between B7H1 expression and clinicopathological parameters of UBC cases RT-PCR We analyzed the correlation between the percentage of B7H1 Total RNA was extracted from fresh urothelial bladder cancer expression and clinicopathological parameters (Table 2). The tissue. The mRNA was reverse-transcribed into first-strand cDNA results showed that there was no association between B7H1- using M-MLV reverse transcriptase (Promega, USA). PCR reac- positive expression and either sex or age (p>0.05). Furthermore, tions were carried out using specific primers for B7H1 (sense: B7H1 expression was not found to be associated with tumor 5’-GCCGACTACAAGCGAATTAC-3’, antisense: 5’-TCTCAGTGTG metastasis, tumor size, or focus (p>0.05). According to the 2004 CTGGTCACAT-3’, product length 233 bp). b-actin served as the WHO criteria (low malignancy of urinary tract epithelial pap- internal control (sense: 5’-CGGGAAATCGTGCGTGAC-3’’, anti- illoma, low-grade Papillary urothelial carcinoma, high-grade sense: 5’-TAGAAGCATTT GCGGTGG-3’, product length511 bp). papillary urothelial carcinoma), 98 specimens were classified as grade group and stage group. Interestingly, we found that The cycling parameters were: 94°C for 3 min, 90°C for 30 s, and B7H1 expression was increased in the high-grade group and in 55°C for 30 s. The extension was 1 min at 72°C for 35 cycles. the stage group. In the grade groups, the percentages of B7H1 The PCR products were analyzed by Quantityone software expression were 34.78%, 50.00%, and 86.20%, respectively. (BioRad, USA). The change in mRNA expression was calculat- Similarly, the Ta-T1 group had 36% B7H1-positive expression ed by the 2–DDCT method.
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