Int J Clin Exp Med 2015;8(9):14782-14792 www.ijcem.com /ISSN:1940-5901/IJCEM0012703

Original Article Expression of Jagged1 predicts postoperative clinical outcome of patients with gastric cancer Hao Liu1*, Heng Zhang1*, Zhenbin Shen1*, Xuefei Wang1, Zhenglin Wang1, Jiejie Xu2, Yihong Sun1 Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China; 2Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China. *Equal contributors.

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Received July 10, 2015; Accepted September 10, 2015; Epub September 15, 2015; Published September 30, 2015 Abstract: Background: Clinical significance of Notch1 activation in gastric cancer has been elucidated in our previous study, but the role of its ligands remains obscure. This study aims to evaluate the prognostic value of Jagged1 expression in patients with gastric cancer. Methods: We examined Jagged1 expression in tumor and nontumor tissues in retrospectively enrolled 302 patients with gastric cancer undergoing gastrectomy at Zhongshan Hospital of Fudan University in 2008 by immunohistochemical staining. The Kaplan-Meier method and Cox regression models were used to evaluate the prognostic value of Jagged1 expression and its association with clinicopathological features. We created a predictive nomogram by integrating Jagged1 expression with the TNM staging system for overall survival of gastric cancer patients. Results: Jagged1 expression in gastric cancer was decreased compared with that in nontumor tissues. Low expression of Jagged1 in tumor and nontumor both predicted a dismal outcome. The Jagged1 risk derived from Jagged1 expression in tumor/nontumor tissue gave a further discrimination for the prognosis of gastric cancer patients. By Cox multivariate analysis, the Jagged1 risk was defined as an independent prognostic factor. The generated nomogram performed well in predicting the 3- and 5-year overall survival of gastric cancer patients. Conclusion: Jagged1 is a potential prognostic biomarker for overall survival, which could be integrated with TNM stage to give a better risk stratification for gastric cancer patients. Keywords: Gastric cancer, Jagged1, overall survival, prognosis, biomarker, nomogram

Introduction Although the incidence and mortality rates have been dramatically decreased since 1930s, gastric cancer remains the fourth most common malignant neoplasm worldwide and accounts for the second cancer-related death in both sex. The incidence of gastric cancer in China and some East Asia countries is much higher than that in America or Europe countries [1]. Furthermore, due to atypical symptoms at the early stage, over 80% of patients with gastric cancer were diagnosed at an advanced stage, which usually indicates a poor prognosis [2]. Thus, elucidation of key molecules in carcinogenesis of gastric cancer and targeting the underlying mechanisms may provide novel therapeutic targets for gastric cancer. Notch signaling is an evolutionally conserved cell interaction mechanism, which could play crucial roles in diverse cellular processes including cell-fate determination, regulation of

apoptosis and self-renewal of stem cells [3]. Four Notch molecules (Notch1 to Notch4) were identified as receptors for five ligands (Jagged1, Jagged2, DLL1, DLL3 and DLL4) in mammals. Aberrant expression of Notch1 has been identified correlated with the prognosis of patients in some malignant tumors. Being the main ligand for Notch1, aberrant expression of Jagged1 was found to be related to a poor prognosis in human renal clear cell carcinoma, breast cancer and papillary bladder transitional cell carcinoma [4-6]. Activated Notch1, accomplished by up-regulated Jagged1 was also found critical in development of colorectal cancer [7]. However, the expression of Jagged1 and its clinical significances in gastric cancer remain largely unknown and need to be further studied. Since Dufraine et al first discovered that activated Notch1 mutations in human T cell could lead to acute lymphoblastic leukemia, increasing studies have unraveled the role of Notch signaling in human malignant tumors [8-10].

Jagged1 in gastric cancer Given the critical role of Notch signal in carcinogenesis, it seems to be a potential therapeutic target for the treatment of malignant tumor. Previous studies have identified gamma-secretase inhibitors, which could prevent the generation of the intracellular domain of Notch molecules by targeting Notch receptors to suppress Notch activity [11]. However, targeting the binding of the ligands to Notch receptors could also have therapeutic value for blocking the activation of Notch signaling in malignant tumor. Previous studies have proved that the activation of Notch1 signaling in gastric cancer was related to rapid disease progression, and high expression of intracellular domain of Notch1 (NICD), which represented activated Notch1 in gastric tumor tissue, predicted a poor clinical outcome in patients with gastric cancer [12, 13]. Although DLL1 was reported to be involved in the activation of Notch1 [14], whether Jagged1 is involved in Notch1 activation in gastric cancer remains unknown. In this study, we sought to investigate the expression of Jagged1 in gastric cancer and determine whether Jagged1 is responsible for Notch1 activation. Furthermore, we also explored its relation with the clinicopathological characteristics and clinical outcomes. In addition, a nomogram integrated Jagged1 expression with TNM stage was developed to predict the 3- and 5-year overall survival in patients with gastric cancer after surgery. Materials and methods Patients A total of 302 patients who received gastrectomy from the same surgical team in Zhongshan Hospital of Fudan University (Shanghai, China) in 2008 were enrolled in the study. Six of these patients had distant metastasis, whose surgery was more focused on relieving symptoms while the others all received a radical (R0) resection. None of these patients received antitumor therapy before surgery. All the clinicopathological and baseline demographic characteristics were collected retrospectively, including age, gender, tumor size, tumor location, Lauren classification, tumor differentiation and tumor stage. Tumor stage and tumor differentiation were reassessed by two independent gastroenterology pathologists according to the 2010 International Union against Cancer TNM classification system. The average 14783

age of these patients is 59.5 years old with a range from 29 to 88, and 69.2% of these patients are male. Referred to Lauren classification, 63.9% are intestinal type, 22.8% are diffuse type, and the rest are mixed type. 63.9% of the patients have lymph nodes metastasis. All the patients were followed up until April 2014 with a median follow-up time of 45 months. Overall survival was defined as the time from the date of surgery to the date of death or last visit. Written informed consent from each patient was achieved. The use of human specimens was approved by the Clinical Research Ethics Committee of Zhongshan Hospital. Tissue microarray, immunohistochemistry and evaluation The construction of tissue microarray and the immunohistochemical protocols were as previously described [5, 12]. Primary antibody was rabbit polyclonal anti-Jagged1 antibody (1.00 mg/ml, ab7771, Abcam, Cambridge, MA, USA). The density of positive staining was measured using the computerized image system composed of an Olympus CCD camera connected to a Nikon eclipse Ti-s microscope. The stained sections were scanned at high-power magnification (×200) and captured by NIS-Elements F3.2 software to identify three independent microscopic fields with the strongest staining to ensure representativeness and homogeneity. Each photo used an identical setting. The density was counted by Image-Pro Plus version 6.0 software (Media Cybernetics Inc., Bethesda, MD, USA). Integrated optical density (IOD) of all the positive staining in each photograph was measured to give a quantitative assessment for the staining. The mean IOD of the three captured computerized microscopic fields was regarded as the density of Jagged1 expression in the represented tissue. The evaluation of immunostaining was performed by two independent gastroenterology pathologists who were blinded to the patient outcomes and clinicopathological characteristics. For immunohistochemical density, the cut-points for the definition of high/low expression subgroups were determined by X-tile software [15]. Statistical analysis Analyses were performed with SPSS 19.0 (SPSS Inc., Chicago, IL) and R software version 3.0.2 and the “rms” package (R Foundation for Int J Clin Exp Med 2015;8(9):14782-14792

Jagged1 in gastric cancer

Figure 1. Immunohistochemical analysis of Jagged1 expression in gastric cancer. A. High Jagged1 expression in nontumor tissue; B. Low Jagged1 expression in nontumor tissue; C. High Jagged1 expression in tumor tissue; D. Low Jagged1 expression in tumor tissue. Magnification 200×.

Statistical Computing, Vienna, Austria). Pearson χ2 test or Fisher’s exact test was used to compare categorical variables Continuous variables were analyzed by one-way ANOVA. KaplanMeier analysis was used to determine the survival and Log-rank test was used to compare the patient survival between subgroups. Cox proportional hazards model was used to perform multivariate analysis. Nomogram was created by R software using “rms” package. Calibration plots were generated to examine the performance characteristics of the predictive nomogram. The Harrell’s concordance indices (c-indices) were used to measure the prog14784

nostic accuracy. All statistical analyses were two-sided and P

Expression of Jagged1 predicts postoperative clinical outcome of patients with gastric cancer.

Clinical significance of Notch1 activation in gastric cancer has been elucidated in our previous study, but the role of its ligands remains obscure. T...
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