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Original Article the Expression Status of ZIC2 As a Prognostic Marker for Nasopharyngeal Carcinoma

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Original Article the Expression Status of ZIC2 As a Prognostic Marker for Nasopharyngeal Carcinoma Int J Clin Exp Pathol 2018;11(9):4446-4460 www.ijcep.com /ISSN:1936-2625/IJCEP0081196 Original Article The expression status of ZIC2 as a prognostic marker for nasopharyngeal carcinoma Wei Yi1*, Jiani Wang4*, Zhicheng Yao5, Qinglei Kong6, Nana Zhang7, Wei Mo1, Lihua Xu2, Xian Li3 Departments of 1Radiotherapy, 2Hematology, 3Radiology, The First Affiliated Hospital of Guangzhou Medical Uni- versity, Guangzhou, Guangdong Province, P. R. China; 4Breast Cancer Center, Departments of 5General Surgery, 6Emergency, 7Pathology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, P. R. China. *Equal contributors. Received June 12, 2018; Accepted July 19, 2018; Epub September 1, 2018; Published September 15, 2018 Abstract: Zinc finger protein ZIC2 is a transcription factor encoded by the ZIC2 gene, which can interact with vari- ous DNAs and proteins. ZIC2 expression may promote cell proliferation and inhibit apoptosis. Recent studies have reported that ZIC2 acts as an oncogene in various cancers. The expression and distinct prognostic value of ZIC2 in NPC is not well established. The aim of this study was to investigate ZIC2 expression and its prognostic significance in nasopharyngeal carcinoma (NPC). The ZIC2 expressions at the mRNA levels in NPC tissues and normal tissues were investigated using Oncomine analysis. ZIC2 protein expression was analyzed in paraffin-embedded NPC tis- sues by immunohistochemistry. Statistical analyses were performed to evaluate the clinicopathological significance of ZIC2 expression. The result shows the expression of ZIC2 mRNA is significantly elevated in NPC tissue compared with normal nasopharynx tissues. In paraffin-embedded tissue samples, the immunoreactivity of ZIC2 was primarily seen in the nuclei and cytoplasms within tumor cells. High ZIC2 expression was obviously related to poor OS and DFS compared to low ZIC2 expression. In a multivariate analysis, the expression of ZIC2 was an independent prog- nostic factor for OS and DFS. ZIC2 is up-regulated in NPC and associated with histology and survival. ZIC2 may serve as a prognostic indicator for patients with NPC. Keywords: NPC, ZIC2, overexpression, prognosis, nasopharyngeal carcinoma (NPC), Epstein-Barr virus Introduction Currently, radiotherapy and concurrent chemo- radiotherapy (CCRT) are the preferred treat- Nasopharyngeal carcinoma (NPC), an Epstein- ment strategies for NPC. Despite a great im- Barr virus-associated malignancy, arises from provement in diagnosis and treatment, the the nasopharynx and has an unbalanced distri- 5-year overall survival for locally-advanced NPC bution of incidence and morbidity in different has remained poor [7]. Therefore, a novel target regions of the world. It is well known that NPC is is in urgent need for early diagnosis and an effi- endemic in southern China, Hong Kong, North cient therapeutic option for NPC. Zinc finger Africa [1], and Southeast Asia with an approxi- protein ZIC2 (ZIC2) is a transcription factor mate incidence of 30 per 100,000, yet NPC is encoded by the ZIC2 gene, which contains relatively rare among white populations [2-4]. C2H2 zinc fingers and can interact with various What causes these variations in the incidence DNA and proteins [8]. of NPC is unclear. Multiple factors, such as EBV infection, environmental factors, and genetic ZIC2 expression can also promote cell prolifera- susceptibility are believed to account for NPC’s tion and inhibit apoptosis during the develop- geographical distribution [5]. Since EBV infec- ment of pancreatic ductal adenocarcinoma [9]. tion is ubiquitous in the B lymphocytes but rare Recent studies have reported that ZIC2 acts as in epithelial cells [6], it is generally accepted an oncogene in various cancers, such as ovari- that other environmental and genetic factors an cancer [10], liver cancer [11], and pancreatic are also important determinants for NPC risk. cancer [9]. However, the clinical significance of ZIC2 is up-regulated in NPC Table 1. Correlation of ZIC2 expression with clinicopathological features Total ZIC2 expression Characteristics P value (n=108) Low (n=99) High (n=9) Age (years) 0. 305 ≤ 46 44 (40.7%) 42 (95.5%) 2 (4.5%) > 46 64 (59.3%) 57 (89.1%) 7 (10.9%) Gender 0.112 Male 78 (72.2%) 74 (94.9%) 4 (5.1%) Female 30 (27.8%) 25 (83.3%) 5 (16.7%) Histology 0.478 WHO I 0 (0%) 0 (0%) 0 (0%) WHO II 41 (38.0%) 39 (95.1%) 2 (6.9%) WHO III 67 (62.0%) 60 (89.6%) 7 (10.4%) Clinical stage (UICC) 0.683 I 3 (2.9%) 3 (100.0%) 0 (0.0%) II 23 (22.5%) 22 (95.7%) 1 (4.3%) III 47 (46.1%) 43 (91.5%) 4 (8.5%) IV 25 (24.5%) 21 (84.0%) 4 (16.0%) V 4 (3.9%) 4 (100.0%) 0 (0.0%) T classification 0.463 T1 11 (10.8%) 10 (90.9%) 1 (9.1%) T2 40 (39.2%) 36 (90.0%) 4 (10.0%) T3 31 (30.4%) 30 (96.8%) 1 (3.2%) T4 20 (19.6%) 17 (85.0%) 3 (15.0%) N classification 0.233 N0 29 (28.4%) 25 (86.2%) 4 (13.8%) N1 25 (24.5%) 25 (100.0%) 0 (0.0%) N2 38 (37.3%) 34 (89.5%) 4 (10.5%) N3 10 (9.8%) 9 (90.0%) 1 (10.0%) Carotid sheath involvement 0.617 No 15 (14.7%) 13 (86.7%) 2 (13.3%) Yes 87 (85.3%) 80 (92.0%) 7 (8.0%) Nasal cavity involvement 0.055 No 72 (70.6%) 63 (87.5%) 9 (12.5%) Yes 30 (29.4%) 30 (100.0%) 0 (0.0%) Maxillary sinus involvement 0.680 No 80 (78.4%) 72 (90.0%) 8 (10.0%) Yes 22 (21.6%) 21 (95.5%) 1 (4.5%) Neck lymph node level involvement 0.827 No 29 (28.4%) 25 (86.2%) 4 (13.8%) Level I-III 63 (61.8%) 59 (93.7%) 4 (6.3%) Level IV 10 (9.8%) 9 (90.0%) 1 (10.0%) Maximum neck lymph node diameter 0.488 <20 mm 52 (51.0%) 46 (88.5%) 6 (11.5%) ≥20 mm 50 (49.0%) 47 (94.0%) 3 (6.0%) WHO, World Health Organization. ZIC2 in NPC remains unclear. In the present tissue samples and revealed its significance in study, we examined the ZIC2 expression in NPC NPC prognosis and clinical pathology. 4447 Int J Clin Exp Pathol 2018;11(9):4446-4460 ZIC2 is up-regulated in NPC 48 years (range, 16 to 80 years old). The median follow- up time was 46 months (ran- ge, 3.0 to 89 months). All the patients were followed from diagnosis until death, or re- lapse, or the last census date. The clinicopathologic charac- teristics for these patients including age, gender, histolo- gy, clinical stage T classifica- tion, N classification, carotid sheath involvement, nasal ca- vity involvement, maxillary si- nus involvement, neck lymph node level involvement, and maximum neck lymph node Figure 1. ZIC2 expression in NPC and normal nasopharynx tissues. The fig- diameter are summarized in ure was derived from gene expression data in the Oncomine database com- Table 1. All the NPC patients paring expression levels of ZIC2 in normal (left plot) and cancer tissue (right were treated with standard plot). radical radiotherapy, except 4 patients with distant metasta- Material and methods sis at the time of diagnosis (with or without che- motherapy). The TNM stage was defined on the Oncomine analysis basis of the 8 th AJCC (American Joint Committee on Cancer) Staging System. Based on the pri- Oncomine (www.oncomine.org) is an online mary tumor sites and lymph nodes, there were database of cancer microarrays that aims at 3, 23, 47, 25 and 4 patients belonging to the collecting, standardizing, and analyzing cancer categories of stage I, II, III, IVa, and IVb respec- transcriptome data to the biomedical research tively. For the use of these clinical materials on community and providing gene expression as research purposes, prior consent was obtained well as clinical and pathological profiles for the from each of the patients, and the protocol of major types of cancer contrasted with respec- this study was approved by the ethics commit- tive normal tissues [12]. It contains 65 gene tee of the First Affiliated Hospital of Guangzhou expression datasets covering a wide variety of Medical University. human malignant cancers [12]. In this study, Immunohistochemistry (IHC) staining we took advantage of this high-throughput database to mine ZIC2 expression in nasopha- Formalin-fixed and paraffin-embedded NPC ryngeal carcinoma tissues and normal tissues. samples were cut into sequential sections, The threshold value of this study was a 2.0 fold each 4 μm thick. Then the slides were baked for change, a cut-off p-value <0.05, and top 10% 3 hours at 60°C. After being deparaffinized in gene rank. xylenes and rehydrated through graded alcohol to distilled water, these slides were immersed Patients and tissue specimens into 3% hydrogen peroxide to block endogenous peroxidase activity at room temperature for 20 All clinical samples used for the expression of min, and then we boiled the slides in an ethyl- ZIC2 studies by immunohistochemistry (IHC) enediamine tetraacetic acid (EDTA) buffer assay were collected from the First Affiliated (pH=8.0) for 3 min in a high-pressure cooker for Hospital of Guangzhou Medical University, antigen retrieval. After the retrieval solution Guangzhou, China, between April 2010 and returned to room temperature, the slides were December 2017. The original site of NPC was incubated with diluted rabbit polyclonal anti- involved in selecting retrievable biopsy sam- ZIC2 antibody (1:200 dilutions; Affinity, USA) in ples. These cases included 78 (72.2%) men a moist chamber at 4°C overnight. Subsequently, and 30 (27.8%) women, with a median age of after we washed the samples 3 times in ph- 4448 Int J Clin Exp Pathol 2018;11(9):4446-4460 ZIC2 is up-regulated in NPC (40×40) from necrotic areas were randomly selected for counting.
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