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Prognostic value of claudin-4, nm23-H1, and MIB-1 in undifferentiated nasopharyngeal carcinoma

Emara, Nashwa M.; Abd El-Maksoud, Ahlam A.; Ibrahim, Enas; Zeidan, Ahmed M.; Nouh, Akram M.

doi: 10.1097/01.XEJ.0000504533.36954.32

Introduction 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 for NPC is radiotherapy, but therapy failure is quite common, making radioresistance an important issue. Claudin-4 is a major tight junction protein that regulates the integrity and function of tight junctions. Aberrant expression of claudin-4 has been shown in various carcinomas with diverse prognostic implications. Ki-67 as a maker of cell proliferation and nm23-H1 as 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 was 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 with undifferentiated NPC.

Results The 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 the prediction of distant metastasis and unfavorable prognosis in NPC, especially when combined with nm23-H1 and Ki-67.

Departments of aPathology

bGeneral Surgery, Faculty of Medicine, Benha University, Benha

cNational Cancer Institute, Cairo University, Cairo, Egypt

Correspondence to Nashwa M. Emara, MD, PhD, Department of Pathology, Faculty of Medicine, Benha University, 13111 Benha, Egypt Tel: +201063346960; fax: +0133227518; e-mail:

Received July 14, 2016

Accepted August 14, 2016

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Nasopharyngeal cancer (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 compared with 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; Li 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 (Li et al., 2012; Wang et al., 2015; Xu et al., 2015).

NPC is a squamous cell carcinoma (Chan et al., 2005) and is subdivided into nonkeratinizing 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 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. However, 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 finding potential therapeutic targets of NPC, and hence may lead to a more effective treatment of NPC patients (Liu et al., 2008; Li et al., 2012).

Tight junctions (TJs) are specialized regions of cell–cell contact and have crucial roles in the 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 (Hewitt et al., 2006; Van Itallie and Anderson, 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, 1988b), 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 tumors 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, there is no definite data on 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 (Özel et al., 2004). Moreover, their results in NPC were debatable.

The aim of this study was to evaluate the expression of claudin-4 protein in undifferentiated NPC patients and to correlate the results with clinicopathologic variables, other suggested markers such as nm23-H1 and Ki-67, and disease outcome.

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Materials and methods

Human tissue sampling and immunohistochemical analysis

This retrospective study was conducted on 50 cases diagnosed as undifferentiated NPC during the years 2000–2010. Archival formalin-fixed, paraffin-embedded blocks of tumor specimens were collected from the 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, four sections of 4 µm thickness were prepared. One section was used for histopathological study using conventional H&E stain and the other three sections were prepared on positively charged slides for immunohistochemical study.

The histopathological classification of the tumor samples was based on the Pathology and Genetics of Head and Neck Tumors’ by WHO in 2005 (Chan et al., 2005). The stage was defined according to the American Joint Committee on Cancer Criteria (Hsueh et al., 2010).

For immunohistochemical analyses, nm23-H1 (Dako, USA), claudin-4 (Lab Vision; NeoMarkers, Fremont, California, USA), and Ki-67 (Dako, Carpinteria, California, USA), prediluted ready to use, were applied for each case using the Avidin–Biotin complex (APC) technique (Hsu et al., 1981). The procedure was as follows: the slides were dewaxed in xylene, and then treated with microwave heating at 90 C for 10 min 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. The slides were incubated in a humid chamber for 1 h at room temperature. Thereafter, 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 was incubated with slides for 3–5 min and then washed with distilled water. The slides were counterstained for 3 min with Mayer’s hematoxylin and then washed and covered with mounting media (DPX).

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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, no significant tumor cell staining; and positive, cytoplasmic staining of tumor cells (Xiang et al., 1998).

The expression of claudin-4 was assessed with semiquantitative 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 follows: less than 10%, 1+; between 10 and 50%, 2+; or more than 50%, 3+. The staining intensity was recorded as absent (0), weak (1+), moderate (2+), or strong (3+). The two scores were multiplied to give a final score of 0–9, 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 ×400 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 cutoff level of 20%, MIB-1 scores were ranked as high proliferation index (HPI) if greater than or equal to 20% of tumor cells were positive, and as low proliferation index (LPI) if less than 20% of tumor cells were positive (Guoqing and Xiaoyan, 2004).

A negative control was used for each marker, by omitting the primary antibody and replacing it with normal rabbit serum IgG.

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Statistical analyses

All quantitative data are expressed as mean±SD. Statistical package for the social sciences program, version 16.0 for windows (SPSS Inc., Chicago, Illinois, 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.

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Ages of the 50 cases of NPC ranged from 22 to 76 years, with a mean age of 45.3±9.26. Of the 50 cases, 35 (70%) cases were male and 15 (30%) cases were female (Table 1).

Table 1

Table 1

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Immunohistochemical results

Nm23-H1 expression results

Staining of the product of nm23-H1 gene was cytoplasmic. Positive cytoplasmic expression was seen in 18 (36%) cases, whereas 32 (64%) cases showed negative expression (Table 2). The nm23-H1 expression in relation to age and sex is shown in Table 2 (Figs 1 and 2).

Table 2

Table 2

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Nm23-H1 expression was nonsignificantly correlated with tumor size (P>0.05) (Table 2).

Nm23-H1 expression was significantly correlated with lymph node status (P<0.01) (Table 2).

Moreover, the relation of nm23-H1 expression with distant metastasis was statistically significant (P<0.05) (Table 2).

Similarly, the relation between nm23-H1 expression and TNM stage was statistically significant (P<0.01) (Table 2).

From previous results it was obvious that negative nm23-H1 expression was associated with a higher incidence of lymph node metastasis, distant dissemination, and advanced clinical stage compared with positive nm23-H1 expression.

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Claudin-4 expression results

Twenty (40%) cases showed high claudin-4 expression, whereas 30 cases (60%) showed low expression for claudin-4 (Table 2). Claudin-4 expression in relation to age and sex is shown in Table 2 (Figs 3 and 4).

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Claudin-4 expression was nonsignificantly correlated with tumor extent (P>0.05) (Table 2).

Similarly, claudin-4 expression was nonsignificantly correlated with lymph node status (P>0.05) (Table 2).

The relation of claudin-4 expression with distant metastasis was statistically significant (P<0.01) (Table 2).

Moreover, claudin-4 expression was nonsignificantly correlated with TNM stage (P>0.05) (Table 2).

From previous results, it was found that low claudin-4 expression was associated with distant metastasis.

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MIB-1 expression results

As regards MIB-1 expression, it ranged from 1 to 40%, with a mean of 16.08±12.09; 16 (32%) cases showed HPI for MIB-1 expression, whereas 34 (68%) cases showed LPI for MIB-1. The MIB-1 expression in relation to age and sex is shown in Table 2 (Figs 5 and 6).

Fig. 5

Fig. 5

Fig. 6

Fig. 6

MIB-1 expression was nonsignificantly correlated with tumor extent (P>0.05) (Table 2).

Similarly, MIB-1 expression was nonsignificantly correlated with lymph node status (P>0.05) (Table 2).

The relation of MIB-1 expression with distant metastasis was statistically nonsignificant (P>0.05) (Table 2).

Moreover, MIB-1 expression was nonsignificantly correlated with TNM stage (P>0.05) (Table 2).

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Correlation of nm23-H1, MIB-1 and Claudin-4 Expression

As regards the relation between the expression results of the three used markers, the results were statistically nonsignificant. Of 30 cases showing low expression for claudin-4, 22 cases showed negative expression and eight cases showed positive expression for nm23-H1; 20 cases expressed LPI and 10 cases expressed HPI for MIB-1. Of the 20 cases showing high expression for claudin-4, 10 cases showed negative expression and 10 cases showed positive expression for nm23-H1; 14 cases expressed LPI and six cases expressed HPI for MIB-1. Moreover, of the 34 cases showing LPI for MIB-1 expression, 20 cases were negative and 14 cases were positive for nm23-H1; of 16 cases showing HPI for MIB-1, 12 cases were negative and 4 cases were positive for nm23-H1 (Table 2).

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Relation of claudin-4, MIB-1, and nm23-H1 expressions with prognosis

Survival curves were plotted against nm23-H1, claudin-4, and MIB-1 expressions (Diagrams 1–3). Log-rank test showed significant correlations (P<0.05) (Table 3).

Diagram 1

Diagram 1

Diagram 2

Diagram 2

Diagram 3

Diagram 3

Table 3

Table 3

It was found that 15 of 22 followed-up cases were negative for nm23-H1, of which three cases recurred and eight cases died. 7 of 22 cases were positive for nm23-H1, of which six cases showed disease-free survival for 5 years. Nm23-H1 expression indicated favorable prognosis with significant association (P<0.05).

Moreover, 13 of 22 followed-up cases showed low claudin-4 expression, of which two cases recurred and seven cases died; however, nine of 22 cases showed high claudin-4 expression, of which six cases showed disease-free survival for 5 years. Low claudin-4 expression correlated significantly with shorter recurrence-free survival, and hence unfavorable prognosis (P<0.05).

As regards MIB-1 expression, 14 of 22 followed-up cases showed LPI for MIB-1, of which eight cases showed disease-free survival for 5 years. eight cases of 22 followed-up cases showed HPI, of which six cases died. Hence, it can be suggested that High Proliferation Index was associated with poorer prognosis compared with LPI, with significant association (P<0.05).

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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 intensity-modulated 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. Of these factors, short-term interval to recurrence 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 the urinary bladder (Boireau et al., 2007) and lost expression of claudins 1, 4, and 7 in diffuse type or poorly differentiated gastric adenocarcinomas (Johnson et al., 2005; Resnick 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 the breast, prostate, stomach, uterus, ovary, pancreas, and kidney (Sato et al., 2004; Resnick et al., 2005; Tokes et al., 2005; Zhu et al., 2006; Santin et al., 2007; Sheehan et al., 2007; Lechpammer et al., 2008; Lanigan et al., 2009), with correlation to aggressive tumor behavior or decreased survival. Lee et al. (2009) also 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 a lower rate of distant metastasis, and that it was associated with improved distant metastasis-free survival.

Consistent with our study results, it has been shown in pancreatic cancer cell lines that upregulation 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 the downregulation of claudins contributes to epithelial transformation by increasing the paracellular permeability of nutrients and growth factors to cancerous cells.

Possible explanation for studies revealing opposite opinion is through the interaction with zona occludens-1 involved in TJs, 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 through the 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 (Agarwal et al., 2005; Ku et al., 2006). Upregulation of claudin-1 in oral SCC enhances invasion through the activation of MMP-2 and MMP-1, 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, 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, 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. Moreover, 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 (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 the downregulation of nm23-H1 expression was associated with intracranial invasion in NPC and hence poor prognosis. Moreover, Li 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 (Almgren 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 marker 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 and Xiaoyan, 2004). In this study, we detected the expression of MIB-1 in undifferentiated NPC to study the relationship between biological behavior and prognosis of NPC.

Although this study revealed nonsignificant results with TNM stage, it revealed that HPI for MIB-1 protein was associated with poor survival. Small number of cases tested may contribute to this nonsignificance as regards TNM staging.

In contrast to our results, Masuda et al. (1998) found no association with distant metastasis and survival and also Özel et al. (2004) reported LPI of Ki-67 in undifferentiated NPC.

Moreover, Guoqing and Xiaoyan (2004) found that of the 56 cases studied, 16 (28.6%) cases showed MIB-1 HPI and 40 (71.4%) cases showed MIB-1 LPI. Shi et al. (2005) also reported that the patients having a low expression of Ki-67 were not sensitive to chemotherapy, liable to metastasis to distant organs, and had a poor prognosis, whereas tumors that highly expressed Ki-67 (high proliferated tumors) were sensitive to radiotherapy and had a better prognosis.

In our study, the percentage of Ki-67-positive cells in NPC was up to 40% and the HPI accounted for 32%, which is in agreement with the studies in the literature (Gabusi et al., 2001; Shi et al., 2005). Our statistical outcome indicated that the expression of Ki-67 had no relationship with the stage of NPC (P<0.05), but significantly correlated with prognosis. A possible explanation could be that highly proliferating tumors confer an aggressive biological behavior.

In agreement with our results, Raybaud-Diogene et al. (1997) found that patients (with poorly differentiated squamous cell carcinoma, in head and neck) who mildly expressed Ki-67 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 a new remedy that can increase the radiosensitivity to the patients or use the adjuvant chemical treatment before or after the radiotherapy if the patients highly expressed MIB-1 and were not sensitive to the radiotherapy.

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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 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.

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Conflicts of interest

There are no conflicts of interest.

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