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Prognostic Value of Caludin-4, nm23-H1 and MIB-1 in Undifferentiated Nasopharyngeal Carcinoma Nashwa M. Emara*, Ahlam A. Abd El-Maksoud*, Enas Ibrahim*, Ahmed M. Zeidan**, and Akram M. Nouh*** Pathology *& General Surgery** Departments; Faculty of Medicine, Benha University & National Cancer Institute; Cairo University*** Abstract: Nasopharyngeal cancer (NPC) is a tumor of epithelial origin with complex etiology, and it is one of the most confusing, commonly misdiagnosed and poorly understood diseases. Currently the standard treatment of NPC is radiotherapy, but therapy failure is quite common, making radioresistance an important issue. Claudin-4 is a major tight junction protein; that regulate the integrity and function of tight junctions. Aberrant expression of claudin-4 has been shown in various carcinomas with diverse prognostic implications. Ki67 as a maker of cell proliferation and nm23-H1as a metastasis marker have been widely used by many studies to indicate their close relationship with the progression and prognosis in many tumors. However, little is known about their prognostic values in undifferentiated NPC. Aim: The aim of this study is to evaluate the immunohistochemical staining of claudin-4, nm23-H1 protein and Ki-67 in human undifferentiated NPC, and the relationship between claudin-4, nm23-H1 and ki-67 expressions and both metastasis and prognosis of patients of undifferentiated NPC. Results: Results were correlated with sex, age, extent of tumor, lymph node status, the presence or absence of distant metastasis and patient survival. Low claudin-4 expression, low nm23-H1 and high ki-67 expression were associated with distant metastasis and poor survival. Conclusion: Claudin-4 may be a novel biomarker for prediction of distant metastasis and unfavorable prognosis in NPC, especially when combined with nm23-H1 and ki-67. Key Words: Ki67, nm23-H1, claudin-4 expression, NPC, prognosis. Introduction: NPC is an Epstein-Barr virus-related cancer with a high prevalence in Southeast Asia, Southern China, and North Africa. It has a high metastatic potential compares to other head and neck cancers. More than half of the patients present with detectable metastases in the regional lymph nodes or distant organs at diagnosis (Tsang et al; 2003, Jemal et al; 2011, Xuebing et al; 2012). NPC is notorious for its potential to metastasize at the early stages of the disease. For the NPC patients who have a distant metastasis, the outcome was generally ominous (Xuebing et al; 2012, Xu et al; 2015, Wang et al; 2015). Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma (Chan et al; 2005) and is subdivided into non-keratinizing and keratinizing squamous cell carcinomas. NPC accounts for 3.7% of upper aerodigestive tract carcinomas (Parkin et al; 2001). According to the tumor-node-metastasis staging system setted by the American Joint Committee on Cancer, patients are designated into stages 0, I, IIA, IIB, III, IVA, IVB and IVC (Cho; 2007). The etiology of NPC includes viral, genetic, and environmental factors. But the molecular mechanism of the development and progression of NPC remains poorly understood (Hsueh et al 2010). Identification of NPC metastasis-associated factors is helpful not only for understanding the mechanisms involved in NPC metastasis, but also for finding biomarkers for prognosis and potential therapeutic targets of NPC and hence may lead to more effective treatment of NPC patients (Liu et al; 2008 , Xuebing et al; 2012). Tight junctions (TJs) are specialized regions of cell-cell contact and have crucial roles in maintenance of cell polarity, adhesion, cellular arrangement, and paracellular permeability. Claudins have been recently identified as structural and functional components of the TJs in epithelial and endothelial cells and shown to play an important role in TJ function. They are a family of 24 proteins, and various claudins are expressed in different epithelial cells. Most tissues express multiple claudin proteins, which is thought to account for the selective variability of different tissue functions (Van Itallie & Anderson; 2006, Hewitt et al; 2006, Krause et al; 2007). Claudins have been of interest in cancer research. It has been hypothesized that changes and/or loss of claudin expression may play an important role in tumorigenesis and tumor progression, and altered expression of claudins has been reported in a variety of human neoplasms, including colorectal, breast, ovarian, pancreatic, prostate, tongue and esophageal carcinomas and in rectal well-differentiated endocrine neoplasms (Kojima et al; 2010). Although the expression pattern and prognostic value of claudins have been studied in many carcinomas, to the best of our knowledge, they have not been extensively investigated in NPC especially undifferentiated phenotype. Nucleoside diphosphate kinases, a highly conserved family in eukaryotes, are encoded by NM23 genes. NM23-H1 was identified by Steeg et al (1988a, b), based on its low expression in metastatic cells. Low expression or mutation of NM23-H1 has been implicated in cancer prognosis or metastasis in a variety of tumours and malignant transformation (Leone et al, 1991; Yih et al, 2002; Fan et al, 2003; Zhao and Li, 2004; Ma et al, 2005). Exogenous overexpression of the metastasis suppressor gene Nm23-H1 reduces the metastatic potential of multiple types of cancer cells and suppresses in vitro tumor cell motility and invasion (Horak et al; 2007). However, no definite data regarding NM23-H1 expression in the aggressive undifferentiated NPC phenotype. Ki-67 is a nuclear proliferation antigen that is expressed in proliferating cells during the G1, S, G2 and M phases of the cell cycle. Its expression is used as a marker of cell proliferation. Many studies have confirmed in a variety of human malignant tumors the relation between a high Ki-67 LI and poor prognosis. However, other studies, in prostate cancer and carcinomas of head and neck have shown no correlation between Ki-67 index and prognosis (őzel et al; 2004). Also, their results in nasopharyngeal carcinoma were debatable. The aim of this study is to evaluate the expression of claudin-4 protein in undifferentiated NPC patients and to correlate the results with clinicopathologic variables, other suggested markers; as nm23-H1 and Ki67 and disease outcome. Material and Methods: Human Tissue sampling and immunohistochemical analysis This retrospective study was carried upon 50 cases diagnosed as undifferentiated nasopharyngeal carcinoma during the years (2000-2010). Archival formalin-fixed, paraffin embedded blocks of tumor specimens, were collected from Pathology Department, Faculty of Medicine, Benha University and National Cancer Institute, Faculty of Medicine, Cairo University. The corresponding H&E slides and the clinicopathological data were retrieved from the patient archives and reviewed for confirmation of diagnosis and adequacy of materials. Follow up of only 22 patients was obtained for 5 years or until the time of death. From each block, 4 sections of 4 µm thickness were cut. One section was used for histopathological study by conventional H&E stain and the other 3 sections were cut on positively charged slides for immunehisochemical study. The histopathological classification of the tumor samples was based on Pathology and Genetics of Head and Neck Tumors" by World Health Organization in 2005 (Chan et al; 2005). Stage was defined according to American Joint Committee on Cancer Criteria ((Hsueh et al; 2010)). For immunohistochemical analyses, Nm23-H1 (Dako, USA), Claudin 4 (Neomarker, LABVISION, USA), and Ki67 (Dako, USA), prediluted ready to use, were applied for each case using the AvidinBiotin complex (APC) technique (Hsu et al., 1981). The procedure was as follow: Slides were dewaxed in xylen, and then treated with microwave heating at 90 C for 10 minutes in10 mmol/L citrate monohydrate (PH 6.0) for antigen retrieval. One to two drops of each of the primary monoclonal antibodies were applied to each section. Slides were incubated in humid chamber for one hour at room temperature. Then the sections were incubated for 20 min with a biotinylated secondary solution, and then rinsed with distilled water. This was followed by another 20 min of incubation with streptavidin solution. Freshly prepared chromogen diaminobenzine (DAB) was incubated with slides for 3-5 minutes then washed with distilled water. Slides were counterstained for 3 minutes with Mayer's hematoxylin then washed and covered with mounting media (DPX). Immunohistochemical Assessment: Nm23-H1 is expressed in the cytoplasm of tumor cells as yellow brown granules. A two point scale was used in its assessment as follows; Negative for no significant tumor cell staining & Positive for cytoplasmic staining of tumor cells (Xiang et al., 1998). The expression of Claudin 4 was assessed by semiquantative scoring of the extent and intensity of the staining. Only membranous staining was classified as positive. The staining extent was represented by the percentage of positively stained tumor cells and graded as less than 10% (1+), in between 10-50% (2+), or more than 50% (3+).The staining intensity was recorded as absent (0),weak (1+),moderate (2+), or strong (3+).The 2 scores were multiplied to give a final score of 0 to 9,which is grouped as low (final score 0-2) and high (final score 3-9) (Hsueh et al.,2010) MIB-1 immunoreactivity was assessed by counting the positive cells that show nuclear staining for MIB-1, under x400 magnification fields. Score for each tumor was reported as the means of the percentage of positive cells per high power field (proliferation index) (Xu et al., 2015).Using a cut off level of 20%, MIB-1 scores were ranked as high proliferation index (HPI), if ≥20% of tumor cells were positive, and low proliferation index (LPI), if <20% of tumor cells were positive (Guoqing & Xiaoyan; 2004). A negative control was used for each marker, by omitting the primary antibody and replacing it with normal rabbit serum IgG. Statistical analyses: All quantitative data are expressed as means ± standard deviation (SD). Statistical Package for the Social Sciences (SPSS) program, version 16.0 for windows (SPSS, Inc., Chicago, IL, USA) was used, on a personal computer. The Pearson correlation coefficient was used to correlate Nm23-H1, Claudin-4 and MIB-1 expression with clinicopathological data. The Kaplan-Meier method was used to calculate cumulative survival. Survival of patients was compared using the log rank test to compare the survival curves. Results Ages of the studied 50 cases of nasopharyngeal carcinoma ranged from 22 to 76 years with mean age (45.3± 9.26). Out of 50 cases, 35 cases (70%) were males and 15 cases (30%) were females. Table (1): Demographic and clinico-pathological characteristics of the 50 studied cases. Characteristics No. Of cases (%) Total patients 50 (100%) Age (Years) <45 21 (40%) >45 29 (60%) Gender Male 35 (70%) Female 15 (30%) T1 3 (6%) T2 18 (36%) T3 19 (38%) T4 10 (20%) N0 13 (26%) N1 12 (24%) N2 22 (44%) N3 3 (6%) M0 38 (76%) M1 12 (24%) I 2 (4%) II 7 (14%) III 18 (36%) IV 23(46%) Prognosis 5 year disease 10 (46%) (Of 22 cases only) free survival Primary Tumor Regional Lymph Nodes Distant Metastasis TNM Stage Recurrence 4 (18%) Death 8(36%) Immunohistochemical results: Nm23-H1 Expression Results (Figures 1,2): Staining of the product of Nm23-H1 gene was cytoplasmic. Positive cytoplasmic expression was seen in 18 cases (36%); while 32 cases (64%) showed negative expression (Table 2). The nm23-H1 expression in relation to age and sex is shown in Table 2. Nm23-H1 expression was insignificantly correlated with tumor size (P value >0.05) (Table 2). Nm23-H1 expression was significantly correlated to lymph node status (P value <0.01) (Table 2). Also the relation of nm23-H1 expression to distant metastasis was statistically significant (P value <0.05) (Table 2). Similarly the relation between nm23-H1 expression and TNM stage was statistically significant (P value <0.01) (Table 2). From previous results it was obvious that negative Nm23-H1 expression was associated with higher incidence of lymph node metastasis, distant dissemination and advanced clinical stage than those with positive Nm23-H1 expression. Claudin-4 Expression Results (Figures 3,4): Twenty cases (40%) showed high Claudin-4 expression, while 30 cases (60%) showed low expression for Claudin-4 (Table 2). The Claudin-4 expression in relation to age and sex is shown in Table 2. Claudin-4 expression was insignificantly correlated with tumor extent (P value >0.05) (Table 2). Similarly Claudin-4 expression was insignificantly correlated with lymph node status (P value >0.05) (Table 2). The relation of Claudin-4 expression to distant metastasis was statistically significant (P value <0.01) (Table 2). Also Claudin-4 expression was insignificantly correlated with TNM stage (P value >0.05) (Table 2). From previous results, it was found that low Claudin-4 expression was associated with distant metastasis. MIB-1 Expression Results (Figures 5,6): Regarding MIB-1 expression; it ranked from 1- 40%; with a mean of 16.08 ± 12.09, where 16 cases (32%) showed High Proliferation Index for MIB-1 expression (HPI), while 34 cases (68%) showed Low Proliferation Index (LPI) for MIB-1. The MIB-1 expression in relation to age and sex is shown in Table 2. MIB-1 expression was insignificantly correlated with tumor extent (P value >0.05) (Table 2). Similarly MIB-1 expression was insignificantly correlated with lymph node status (P value >0.05) (Table 2). The relation of MIB-1 expression to distant metastasis was statistically insignificant (P value >0.05) (Table 2). Also MIB-1 expression was insignificantly correlated with TNM stage (P value >0.05) (Table 2). Correlation of nm23-H1, MIB-1 and Claudin-4 Expression (Table 2): Regarding the relation between the expression results of the three used markers; the results were statistically insignificant, as out of 30 cases showing low expression for Claudin-4, 22 cases showed negative expression and 8 cases showed positive expression for nm23-H1; while 20 cases expressed LPI and 10 cases expressed HPI for MIB-1 respectively; whereas out of 20 cases showing high expression for Claudin-4, 10 cases showed negative expression and 10 cases showed positive expression for nm23-H1; while 14 cases expressed LPI and 6 cases expressed HPI for MIB-1 respectively. Also out of 34 cases showing LPI for MIB-1 expression; 20 cases were negative and 14 cases were positive for nm23-H1; while out of 16 cases showing HPI for MIB1, 12 cases were negative and 4 cases were positive for nm23-H1. Relation of Claudin-4, MIB-1 and nm23-H1 expressions to Prognosis (Table 3): Survival curves were plotted against nm23-H1, Claudin-4 and MIB-1 expressions (Diagrams 1, 2 &3). Log rank test showed significant correlations (P<0.05). It was found that 15 out of 22 followed-up cases were negative for nm23-H1; of which 3 cases recurred and 8 cases died; while 7 out of 22 cases were positive for nm23-H1; of which 6 cases showed disease free survival for 5 years. Nm23-H1 expression indicated favorable prognosis; with significant association (P value <0.05). Also 13 out of 22 followed-up cases showed low Claudin-4 expression; of which 2 cases recurred and 7 cases died; whereas 9 out of 22 cases showed high Claudin-4 expression; of which 6 cases showed disease free survival for 5 years. Low Claudin-4 expression correlated significantly with shorter recurrence free survival, hence, unfavorable prognosis (P value <0.05). Regarding MIB-1 expression; 14 out of 22 followed-up cases showed LPI for MIB-1; of which 8 cases showed disease free survival for 5 years; while 8 cases out of 22 followed-up cases showed HPI; of which 6 cases died. So; it can be suggested that High Proliferation Index was associated with poorer prognosis than LPI; with significant association (P value <0.05). Table (2): Relation between nm23-H, Claudin-4, MIB-1 expressions & clinico-pathological findings: Nm23-H1 Clinicopathological Data -ve +ve (No.&%) (No.&%) Claudin 4 P value Low High (No.&%) (No.&%) MIB-1 P value LPI HPI P (No.&%) (No.&%) value Age - <45 - >45 11 (34.4) 10 (55.6) >0.05 10 (33.3) 11 (55) 20 (66.7) 9 (45) 23 (76.7) 12 (60) >0.05 17 (50) 4 (25) 17 (50) 12 (75) 26 (76.5) 9 (56.3) 21 (65.6) 8(44.4) - Male 23 (71.9) 12 (66.7) - Female 9 (28.1) 6 (33.3) 7 (23.3) 8 (40) 8 (23.5) 7 (43.7) - T1 1 (3.1) 2 (11.1) 3 (10) 0 2 (5.9) 1 (6.2) - T2 12 (37.5) 6 (33.3) 12 (40) 6 (30) 15 (44.1) 3 (18.8) - T3 11 (34.4) 8 (44.4) 10 (33.3) 9 (45) 13 (38.2) 6 (37.5) - T4 8 (25) 2 (11.1) 5 (16.7) 5 (25) 4 (11.8) 6 (37.5) - N0 2 (6.2) 11 (61.1) 5 (16.7) 8 (40) 8 (23.5) 5 (31.2) - N1 11 (34.4) 1 (5.6) 9 (30) 3 (15) 12 (35.3) 0 - N2 16 (50) 6 (33.3) 13 (43.3) 9 (45) 13 (38.2) 9 (56.3) - N3 3 (9.4) 0 3 (10) 0 1 (2.9) 2 (12.5) - M0 21 (65.6) 17 (94.4) 19 (63.3) 19 (95) 26 (76.5) 12 (75) - M1 11 (34.4) 1 (5.6) 11 (36.7) 1 (5) 8 (23.5) 4 (25) -I 0 2 (11.1) 2 (6.6) 0 1 (2.9) 1 (6.2) - II 4 (12.5) 3 (16.7) 3 (10) 4 (20) 6 (17.6) 1 (6.2) - III 8 (25) 10 (55.5) 8 (26.7) 10 (50) 14 (41.2) 4 (25) - IV 20 (62.5) 3 (16.7) 17 (56.7) 6 (30) 13 (38.2) 10 (62.5) >0.05 Sex >0.05 >0.05 >0.05 1ry Tumor >0.05 >0.05 >0.05 LN <0.01** >0.05 >0.05 Distant Metastasis <0.05* <0.01** >0.05 TNM Stage <0.01** >0.05 >0.05 Nm23-H1 Clinicopathological Data -ve +ve (No.&%) (No.&%) Claudin 4 P value Low High (No.&%) (No.&%) MIB-1 P value LPI HPI P (No.&%) (No.&%) value NM23: - Negative 22 (73.3) 10 (50) 8 (26.7) 10 (50) >0.05 ---- ---- >0.05 20 (66.7) 10 (33.3) 14 (70) 6 (30) 30 20 ----- ---- ---- 22 (68.8) 10 (31.2) 8 (44.4) 10 (55.6) - High 20 (62.5) 12 (37.5) 14 (77.8) 4 (22.2) Total 32 18 - Positive Claudin 4: - Low - High >0.05 20 (58.8) 12 (75) >0.05 14 (41.2) 4 (25) ---- 20 (58.8) 14 (41.2) 10 (62.5) 6 (37.5) >0.05 >0.05 ---- ---- ---- 34 16 MIB-1: - Low N.B.: > 0.05= insignificant correlation, * <0.05= significant correlation, ** <0.01= highly significant correlation Table (3): Relation of nm23-H1, Claudin-4 and MIB-1 expressions to clinical outcome: Clinical outcome -Disease Nm23-H1 -ve +ve (No.&%) (No.&%) 4 (26.7) Claudin 4 P value 6 (85.7) Low High (No.&%) (No.&%) 4 (30.8) MIB-1 P value 6 (66.7) LPI HPI (No.&%) (No.&%) 8 (57.1) P value 2 (25) free survival -Recurrence <0.05* <0.05* <0.05* -Death 3 (20) 8 (53.3) 1 (14.3) 0 2 (15.4) 7 (53.8) 2 (22.2) 1 (11.1) 4 (28.6) 2 (14.3) 0 6 (75) Total 15 7 13 9 14 8 Diagram (1): Relation of nm23-H1 expression to the Survival. Diagram (2): Relation of claudin-4 expression to the Survival. Diagram (3): Relation of MIB-1 expression to the Survival. Figure (1) showing strong positive cytoplasmic expression for nm-23 in non-distant metastasizing undifferentiated NPC (Immunostaining, x400). Figure (2): showing negative cytoplasmic expression for nm-23 in distant metastasizing undifferentiated NPC (Immunostaining, x400). Figure (3): showing high Claudin-4 membranous expression in non-distant metastasizing undifferentiated NPC (Immunostaining, x400). Figure (4): showing low Claudin-4 membranous expression in distant metastasizing undifferentiated NPC (Immunostaining, x400). Figure (5): showing HPI for MIB-1 nuclear expression in distant metastasizing undifferentiated NPC (Immunostaining, x400). Figure (6): showing LPI for MIB-1 nuclear expression in non-distant metastasizing undifferentiated NPC (Immunostaining, x400). Discussion Primary NPC has unique pathological and clinical characteristics and radiotherapy with or without chemotherapy is the mainstream treatment. Although the 5-year survival of patients with NPC has steadily improved over the past three decades (Lee et al; 2005, Chan; 2011), overall 15‑58% of patients experienced recurrence after radical radiotherapy in the era of conventional radiotherapy (Chang et al; 2000), and 13‑22% have experienced recurrence in the era of intensitymodulated radiation therapy (Ng et al; 2011, Su et al; 2011). In most patients with recurrence after complete remission following radical radiotherapy, the cancer returns within an average of 1.5 years, with local recurrences accounting for 70% of such cases (Yang et al; 1996, Lee et al; 1999). Recurrent NPC may be local, regional, or distant and is usually treated with radiation therapy and/or chemotherapy and occasionally with surgery. Retreatment for recurrent NPC poses a critical challenge given its poor efficacy and serious toxicities (Xu et al; 2013). Improved identification of prognostic factors by means of molecular testing may be useful in the diagnosis of diseases and their exact subtypes and may aid physicians in selecting individualized treatment, increasing the likelihood of local salvage. Several prognostic factors have been identified in recent years including recurrent tumor T stage, histologic type, patient age and disease-free interval to recurrence (ITR). Of these factors, short-term ITR has been shown to correlate with poor outcome (Chou et al; 2008, Xu et al; 2015). Therefore, identification of molecular markers that may lead to an improved understanding of recurrent NPC and to individualized treatment is imperative. It is generally accepted that decreased expression of claudins leads to diminished cellular adhesion and differentiation with subsequent tumor cell dissociation and invasion. The notion is supported by the previous findings of reduced expression of claudins 1, 4, and 7 during progression of high-grade intraepithelial neoplasia to invasive carcinomas in urinary bladder (Boireau et al; 2007) and lost expression of claudins 1, 4, and 7 in diffuse type or poorly differentiated gastric adenocarcinoma (Resnick et al; 2005, Johnson et al; 2005). It has been demonstrated that small interfering RNA-mediated knockdown of claudin 7 in esophageal SCC cell lines decreased E-cadherin expression and increased tumor cell growth and invasion (Lioni et al; 2007). However other studies revealed that overexpression of claudin 4 has been reported in carcinomas of breast, prostate, stomach, uterus, ovary, pancreas, and kidney (Sato et al; 2004, Tokes et al; 2005, Resnick et al; 2005, Zhu et al; 2006, Sheehan et al; 2007, Santin et al; 2007, Lechpammer et al; 2008, Lanigan et al; 2009), with correlation to aggressive tumor behavior or decreased survival. Also Lee et al (2009) demonstrated that overexpression of claudin 1 confers resistance to cell death in NPC cell lines. In contrast, our study demonstrated the expression pattern of claudin-4 in undifferentiated NPC patients and its association with prognostic variables and survival and showed that there was a significant correlation of high claudin-4 expression with lower rate of distant metastasis and it was associated with improved distant metastasis free survival (DMFS). Consistent with our study results, it has been shown in pancreatic cancer cell lines that up-regulation of claudin 4 diminished the invasiveness and the metastatic potential of the cancer cells (Michl et al; 2003). This may be explained by the review held by Ding et al; 2013 who reported that downregulation of claudins contributes to epithelial transformation by increasing the paracellular permeability of nutreints and growth factors to cancerous cells. Possible explanation for studies revealing opposite opinion is through the interaction with Zona Occludens-1 (ZO-1) involved in tight junctions, to affect other cell signaling pathways involved in neoplastic transformation (Resnick et al; 2005). Few other plausible mechanisms have been proposed, one is through the suppression of apoptosis by increased expression of claudin 1 in NPC cell lines (Lee et al; 2009) and the other is through the activation of matrix metalloproteinase (MMP) proteins (Oku et al; 2006, Agarwal et al; 2005). Up-regulation of claudin 1 in oral SCC enhances invasion by the activation of MMP-2 and MMP1, and overexpression of claudins 3 and 4 in ovarian surface epithelial cell promotes invasion by increasing MMP-2 activity (Agarwal et al; 2005). It is recognized that claudins 3 and 4 can function as receptors for the Clostridium perfringens enterotoxin (CPE). CPE can cause cytolysis upon binding to its receptors through the effects on membrane permeability (Morin et al; 2005, Swisshelm et al; 2005, Santin et al; 2007). Therefore, cancer cells expressing claudins 3 and 4 can be the potential target for CPE toxin mediated therapy. Experiments have established that breast, ovarian, prostatic, and pancreatic cancer cells are sensitive to CPE treatment (Morin et al; 2005). NPC may also be a possible candidate for such therapy because positive immunoreactivity for claudin-4 was detected in 60 % of undifferentiated NPC patients in this study. The nm23 gene is one of the tumor suppressor genes. The expression of nm23-H1 has been reported to be inversely associated with metastatic potentiality in a number of human carcinomas, including breast, colorectal, gastric, hepatocellular and gall bladder carcinomas. Also exogenous overexpression of the metastasis suppressor gene Nm23H1 reduces the metastatic potential of multiple types of cancer cells and suppresses in vitro tumor cell motility and invasion (Xiang et al; 1998, Horak et al; 2007). This study revealed that reduced expression of nm23-H1 correlated with node and distant metastasis, advanced TNM stage, recurrence and poor survival and the results were statistically significant. In agreement with the results of this study, Liu et al (2008) reported that downregulation of nm23-H1 expression was associated with intracranial invasion in NPC and hence poor prognosis. Also Xuebing et al (2012) revealed that nm23-H1 downregulation is a biomarker for metastasis and poor prognosis in NPC. However, these findings cannot be generalized, and the biological functions of nm23 in cancer cells are perplexed. A positive correlation between nm23-H1 level and metastatic potential has also been observed in other human tumors including pancreatic carcinoma (Nakamori et al. 1993), osteosarcoma (Oda et al; 2000), neuroblastoma (Almegren et al; 2004), and esophageal squamous cell carcinoma (Wang et al; 2004). Given the complexity of metastasis, multiple pathways downstream of nm23-H1 presumably mediate its phenotypic effects. MIB-1 (Ki-67) is a maker of cell proliferation and has been widely used to assess cell proliferation in neoplastic tissues. Many studies indicate that MIB-1 has a close relationship with the replication, progression and prognosis of tumors (Guoqing & Xiaoyan; 2004). In this study, we detected the expression of MIB-1 in undifferentiated NPC in order to study the relationship between biological behavior and prognosis of NPC. Although this study revealed insignificant results with TNM stage, yet it revealed that HPI for MIB-1 protein was associated with poor survival. Small number of cases tested may contribute to this insignificance as regards TNM staging. In contrast to our results Masuda et al (1998) found no association with distant metastasis and survival and also zel et al (2004) reported LPI of Ki67 in undifferentiated NPC. Also Guoqing & Xiaoyan (2004) found that of 56 cases studied, 16 cases (28.6%) showed MIB-1 HPI and 40 cases (71.4%) showed MIB-1 LPI and also Xiaoyan et al (2005) reported that the patients having low expression of ki67 were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while tumors which highly expressed Ki67 (high proliferated tumors) were sensitive to radiotherapy and had a better prognosis. In our study, the percentage of Ki67 positive cells in NPC was up to 40% and the HPI accounted for 32% which was coincidence with the studies in the literature (Gabusi et al; 2001, Xiaoyan et al; 2005). Our statistical outcome indicated that the expression of Ki67 had no relationship to the stage of NPC (P<0.05), but significantly correlated with prognosis. A possible explanation could be that highly proliferating tumors confers an aggressive biological behavior. In agreement with our results, Raybaud et al (1997) found that patients (with poorly differentiated squamous cell carcinoma, in head and neck) who lowly expressed Ki67 would receive a better curative effect on radiotherapy, even if they had a late clinical stage. Meanwhile, according to the expression of MIB-1 before the treatment, an optimal method could be chosen and a series of measures can be taken to increase the cure rate of NPC patients. For example, we may add new remedy that can increase the radiosensitivity to the patients or use the adjuvant chemical treatment before or after the radiotherapy if the patients were highly expressed MIB-1 and were not sensitive to the radiotherapy. In conclusion, Claudin-4 may serve as a useful biomarker for the prediction of distant metastasis and patient survival in NPC. However, more cases need to be studied to confirm these findings. Further investigations are warranted to elucidate the role of claudins-4 in the pathogenesis of NPC especially undifferentiated type for the development of effective treatment. All the results of the used markers in relation to prognosis were statistically significant in a meaning of low Claudin-4 expression; Negative nm23-H1 expression and HPI for MIB-1 were associated with poor survival. References: Agarwal R, D'Souza T, Morin PJ: Claudin-3 and claudin-4 expression in ovarian epithelial cells enhances invasion and is associated with increased matrix metalloproteinase-2 activity. Cancer Res 65: 7378-85, 2005. Almgren MA, Henriksson KC, Fujimoto J, et al: Nucleoside diphosphate kinase A/nm23-H1 promotes metastasis of NB69-derived human neuroblastoma [J]. Mol Cancer Res 2(7): 387-394, 2004. 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