诊断

Review

Progress of tumor marker research in nasopharyngeal carcinoma

Yuan Zhu1, Weiguo Huang1,2,3, Sanyuan Tang1

 

1Center for Gastric Cancer Research of Human Province, The First Affiliated Hospital, University of South China, Hengyang 421001, Hunan, China

2Cancer Research Institute, University of South China, Hengyang 421001, Hunan Province, China

3Key Laboratory of Cancer Cellular and Molecular Pathology, University of South China, Hengyang 421001, Hunan, China

Corresponding author: Sanyuan Tang, Center for Gastric Cancer Research of Human, The First Affiliated Hospital, University of South China, Hengyang 421001, Hunan, China. E-mail address: tsy1123@163.com.

Weiguo Huang, Tel/fax: +86 734 8281547. Key Laboratory of Cancer Cellular and Molecular Pathology, University of South China, Hengyang 421001, Hunan Province, China. E-mail address: hwg_doctor@126.com


 

Citation: Zhu Y, Huang WG, Tang SY. Progress of tumor marker research in nasopharyngeal carcinoma. J Nasopharyng Carcinoma, 2014, 1(10): e10. doi:10.15383/jnpc.10.

Funding:This project were supported by Chinese National Science foundation (No. 81172210).

Competing interests:The authors have declared that no competing interests exist.

Conflict of interest: None.

Copyright:image001.gif2014 By the Editorial Department of Journal of Nasopharyngeal Carcinoma. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 

Abstract: Nasopharyngeal carcinoma (NPC) is a prevalent tumour in southern China and southeast Asia, particularly in the Cantonese population, where the incidence has remained high for decades. NPC is an Epstein-Barr virus (EBV) associated malignancy with a remarkable racial and geographical distribution. It is highly prevalent in southern Asia where the disease occurs at a prevalence about a 100-fold higher compared with other populations not at risk. We still observed the patients recur after primary treatment with radiotherapy or chemo-irradiation. Management of nasopharyngeal carcinoma remains one of the biggest clinical challenges. There have been many break-throughs in early detection, diagnosis, multi-modality treatment and also the disease monitoring for NPC. Systemic treatment is crucial to the management of locally advanced or metastatic NPC. With the advent of molecular targeted therapy and personalized medicine, novel therapies based on molecular targets of NPC have become the focus of research and development over the last decade, the etiology of NPC is thought to be associated with a complex interaction of genetic, viral, environmental and dietary factors. Thanks to the advancements in genomics, proteomics and bioinformatics in recent decades, more understandings of the disease etiology, carcinogenesis and progression have been gained. Research into these components may unravel the pathways in NPC development and potentially decipher the molecular characteristics of the malignancy in recent years. In order to the study of tumor markers associated with the NPC, this paper summarized the research progress of the NPC in recent years, a variety of related research progress of tumor markers and its early diagnosis and curative effect evaluation to the NPC, treatment and prognosis of great significance.

Keywords: NPC, tumor marker

 


Background

Nasopharyngeal carcinoma (NPC), is a highly metastatic epithelial malignancy showing high prevalence in Southeast Asia and North Africa[1]. It can be classified into three histological types, namely nonkeratinizing squamous cell carcinoma, keratinizing squamous cell carcinoma and undifferentiated carcinoma. At diagnosis, most NPC patients have locally advanced disease, which includes stages ranging from T2b N0 to T4 N3. Radiotherapy (RT) can effectively control the early stage of NPC, yielding an excellent 90%-95% of a 5-year local control rate in clinical trials. However, radiotherapy alone is not the optimal treatment for patients with locally advanced disease, which is the most frequent clinical presentation at diagnosis, since it yields an unsatisfactory 5-year survival rate of about 50%[2-3]. Failing to find an early nasopharyngeal carcinoma metastasis is the main reason of death. So, looking for early markers of specific patients with nasopharyngeal carcinoma will help early detection, early diagnosis and early treatment to improve survival rate of patients with nasopharyngeal carcinoma.

 

The Etiology of NPC Markers

NPC is characterized by its distinct racial and geographical distribution with a multifactorial etiology. It has been well accepted that, NPC is related to EBV infection, but environmental and genetics factors also play critical roles[4]. Among host genetic markers associated with NPC, the highly variable class I human leukocyte antigen (HLA) genes on chromosome 6 (6p21.3) have shown a strong and consistent association with NPC risk. It has been observed that, the frequency of some HLA alleles and/or haplotypes is increased (susceptible) or decreased (protective) in patients with NPC compared with normal individuals. In the HLA loci, A2, Bw46 were slightly higher than the non-B17 antigen in nasopharyngeal cancer family two times, which greatly increased (about 21 times) the risk of suffering from nasopharyngeal[5]. Nitrosamine has been identified as a carcinogen for NPC, cytochrome P450 2A6 (CYP2A6) played an important role in NPC development. Relationships between the disease and the CYP2A6 were studied on 74 NPC patients and 137 age-matched healthy controls by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to distinguish between a wide type allele. Overall, a significant association between CYP2A6 polymorphism and NPC development was observed (P<0.05) .This suggested that CYP2A6 polymorphism may play a crucial role in NPC susceptibility and it may be used as a risk marker for NPC[6]. Old et al[7] for the first time proved that EBV and nose using immunodiffusion serological relationship pharyngeal cancer. With the high incidence of nasopharyngeal carcinoma in southern China, EBV is closely related to the prevalence in Africa is dense with Burkitt lymphoma Cut relevant.

 

EBV-Associated Markers of Nasopharyngeal Carcinoma

EBV was the first identified human tumor virus associated with various malignancies. The fact that EBV genome is present in almost all NPC tissues renders is an ideal tumor marker for NPC. EBV gains access to the B-cell compartment, where it drives robust B-cell proliferation through expression of six EBV nuclear antigens, multiple non-coding RNAs, and two integral membrane proteins, latent membrane protein (LMP1) and latent membrane protein 2A (LMP2A). Assessment of immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies responses to various EBV antigen complexes, usually involving multiple serological assaysthe positive rates of EBV Rta-IgG, VCA-IgA, EA-IgA and EBV DNA in untreated NPC patient group were higher than those in other two groups. High-titer antibodies to VCA and early antigen, especially of high IgA class or high titers that persist after therapy, were found to be associated with a poorer prognosis [10].

A study found that patients with NPC serum EB virus DNA level and clinical staging and tumor progression was significantly correlated [11]. The γ-H2AX level was detected by Gou et al. in NPC cells CNE1 before and after EBV infection using Western blotting.γ-H2AX was expressed in most NPC specimens (94%), which was much higher than that in nasopharyngitis(NPI)(40%), and EBV was presented in 94% of NPC but only 30% in NPI. Finally, Western blotting showed that γ-H2AX level significantly increased in CNE1 cells after EBV infection. This study demonstrated that an intimate connection existed betweenγ-H2AX expression and EBV infection in NPC both in vivo and in vitro. EBV infection might induce DNA damage in CNE1 cells, which causes genome instability and initiates or promotes the tumorigenesis and development of NPC[12].

The EBV oncogene BARF1 is expressed in a high proportion of NPC. The structure of the secreted BARF1 glycoprotein expressed in a human cell line was solved by X-ray crystallography[13]. BARF1 was closely related to glycoprotein CD80 or B71. BARF1 mRNA detection of EBV DNA and molecular level can help the non-invasive diagnosis of NPC [14]. BARF1 was present in the serum and saliva from North African and Chinese patients with NPC. The secreted EBV oncoprotein showed a powerful mitogenic activity in B cells. BARF1 is a particularly promising marker for all ages of patients with NPC, its mitogenic activity suggests their implication in the oncogenic development of NPC[15].

LMP-1 played an important role in enhancing NPC cell response to arsenic trioxide (As2O3). The elongation of telomere length induced by LMP-1 might contribute to the mechanisms of As2O3 sensitivity[16]. Preclinical studies demonstrated that As2O3 could inhibit LMP-1 expression, dictate apoptosis and alterations of cell cycle distribution and growth retardation. LMP-1-positive NPC cells were more sensitive to As2O3 treatment than LMP-1-negative NPC cells [17]. Further study found that As2O3 could reduce metastatic potential of NPC cells, involving inhibition of MMP-9 expression. LMP-1 were reduced in this process and seemed to enhance anti-metastatic activity of As2O3 [18]. Of all the EBV-encoded product, latent membrane protein LMP-1 is considered to be an oncogene which playing an essential role in cell transformation and metastasis[19]. The EBV protein, latent membrane protein 2A (LMP2A), is expressed in NPC and can modulate epithelial proliferation, transformation and differentiation, and as such may promote malignancy[20].

A summary estimates for VCA-IgA in the diagnosis of NPC were: sensitivity 0.91 (95% confidence interval (CI): 0.90 - 0.92), specificity 0.92 (95%CI: 0.92 - 0.93) , positive likelihood ratio 31.65 ( 95%CI: 10.99 - 91.15 ), negative likelihood ratio 0.10 (95%CI: 0.07 - 0.13) and diagnostic odds ratio 414.59 (95%CI: 174.96 - 982.42). The area under the summary receiver operating characteristic curves was 0.98. The sensitivity and the specificity of serum VCA-IgA are very high, suggesting that the presence of VCA-IgA in peripheral blood is a valuable predictor for NPC, The sensitivity of VCA-IgA and the specificity of EA-IgA are the highest while detecting solely. Combined determination could improve the diagnostic sensitivity and accuracy for NPC[21].

A high-throughput approach shows that EBV microRNAs are generally more up-regulated than microRNAs of human origin. Authors found that their distinct presence in the serum of NPC patients positively correlated with cellular copy numbers of EBV microRNAs. Further investigation of potential EBV microRNA target genes revealed inhibition of tumor suppressor genes (e.g. phosphatase and tensin homolog deleted on chromosome ten, PTEN) and extensive deregulation of several pathways frequently involved in NPC (e.g. Wnt signaling), Profiling of EBV-encoded microRNAs in nasopharyngeal carcinoma reveals potential biomarkers and oncomirs[22].

 

Diagnostic Markers

Diagnosis was established by biopsy of the nasopharyngeal mass. Fused positron emission tomography/computed tomography is a valuable imaging tool in patients for staging diagnosis of NPC. However, NPC is commonly diagnosed late due to its deep location and vague symptoms [23]. By measuring the nuclear DNA content, DNA diploidy was found to occur earlier in the progression from premalignant to malignant head and neck squamous cell carcinomas (including NPC). This finding was promising to demonstrate methods that were readily applicable for routine diagnostic work [24]. It has been reported that the high sensitivity (81%) and specificity (0% false positives) of detecting aberrant methylation of CDH13 (encoded a cell adhesion molecule Hcadherin) from nasopharyngeal swabs suggested it could be utilized as a tool for early diagnosis [25].

Serum samples from NPC patients and healthy subjects with four specific VCA-IgA/EA-IgA profiles were tested with an anti-EBV Western blot test kit from EUROIMMUN AG, among the markers screened, EA-D p45-IgG showed a statistically significant difference (p < 0.05) between NPC and non-NPC subjects with VCA-IgA positivy. In the verification experiment, the specificity and sensitivity of EA-D p45-IgG were 75.0% and 90.6 %, respectively. EA-D p45-IgG might be a potential biomarker for NPC diagnosis[26]. Cf-DNA was extracted from serum collected from NPC patients and sex-matched healthy subjects. The promoter hypermethylation status of the five genes (RASSF1, CDKN2A, DLEC1, DAPK1 and UCHL1) was assessed by methylation-specific PCR after sodium bisulfite conversion. The combination of four-gene marker - CDKN2A, DLEC1, DAPK1 and UCHL1 - had the highest sensitivity and specificity in predicting NPC.Screening DNA hypermethylation of tumor suppressor genes in serum was a promising approach for the diagnosis of NPC[27]. The levels of CYFRA21-1 and Tumor Specific Growth Factor (TSGF) in NPC group were remarkably higher than those in benign nasopharyngeal disease and normal control groups (P < 0.01). The detection sensitivity is much higher than other tumor markers. Combined detection of two kinds of serum tumor markers increases the detective positive rate. CYFRA21-1 and TSGF can serve as the serum tumor marker in clinical diagnosis of NPC[28].

Tumor necrosis factor a (TNF-a) as another tumor immune cytokine, tumor necrosis with degradation effects, the content of TNF-a detected in NPC patients and normal control can be used as one of indicators of the early diagnosis of NPC[29]. More evidences indicated that inactivation of tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event during carcinogenesis, The researchers use the multiplex methylation specific-PCR (MMSP) assay which was designed to detect tumor-specific methylation status of several NPC-related genes, they collected paired nasopharyngeal (NP) swabs and NPC biopsies from 49 NPC patients and twenty noncancerous controls. The results showed that MMSP patterns of NPC swab were largely consistent with those of corresponding biopsies, MMSP assay is a reliable and potential diagnostic tool for NPC[30]. A study showed that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC[31]. miRNAs and EBV encoded miRNAs play key roles in almost all the steps of epithelia cell carcinogenesis, More importantly, some miRNAs could be secreted out and play a role in the microenvironments. The level of sera miRNAs is correlated with the copy numbers of host miRNAs in NPC. Promising results of gene therapy have also been achieved by lentiviral delivered miRNAs. so miRNAs would be potential biomarkers of early clinical diagnosis of NPC[32].

 

The Markers of Targeted Therapies

High-dose radiotherapy with adjunctive chemotherapy is the primary treatment of NPC[33]. Radiotherapy dose and field margins are individually tailored to the location and size of the primary tumour and lymph nodes. New types of treatment are being tested in clinical trials, which include biological therapy and intensitymodulated radiation therapy. It offer hope for better control of the disease [34].

A study found that the epidermal growth factor receptor (EGFR) was one of the most targeted receptors in the field of oncology. It has been highly expressed in nasopharyngeal , and its expression is often closely associated with a poor prognosis[35]. Tips for EGFR molecular targeted therapy may improve the prognosis of patients. Basic research has proved more EGFR antagonist used alone, in combination with radiotherapy or radiotherapy plus chemotherapy combined use could significantly inhibit the growth of nasopharyngeal carcinoma cell lines, proliferation and increased radiation and chemotherapy for nasopharyngeal carcinoma cells kill strains [36]. The recombinant plasmid hTERTp/tk/pGL3 was transfected into human NPC HNE1 cells and the expressions of TK and telomerase were investigated. The targeted killing effect induced by hTERTp/tk on HNE1 cells was assessed using RT-PCR and MTT assay. the tumor cell-killing effect of hTERTp/tk/pGL3 was slightly milder than that of the positive control CMV/tk/pGL3 that produced nonselective cell killing.it indicated that hTERTp/tk, a tumor-specific expression system, allows targeted tumor cell killing and reduces the activity of telomerase in NPC cells in vitro[37]. The antiapoptotic gene bcl-2antisense oligodeoxynucleotide, G3139, was found to have proapoptotic effects in C666-1 cell line. Combining with cisplatin, it was curative in C666-1 NPC xenograft tumours in vivo. The sequence-dependency of these effects was consistent with an antisense mechanism. The result suggested that bcl-2 might represent a biologically relevant target for the development of novel combinatorial therapies for NPC[38].

LMP2A is found to play a key role in the development of NPC. In this study, they inhibited LMP2A gene expression by lentivirus-mediated RNAi and construct a efficient and stable lentivirus vector, which efficiently downregulate the expression of LMP2A gene in infected cell line C666-1, which inhibits the proliferation and colony formation of C666-1 cells, the result shows that lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of NPC cell line C666-1 in vitro, and LMP2A may be a potential target for gene therapy in treatment of NPC[39]. A study has explored the effects of RNA interference (RNAi) targeting four different genes (VEGF, c-myc, survivin, hTERT) on the growth and proliferation of NPC CNE-2Z cells. The inhibitory effect of the plasmids on xenograft tumors were observed in nude mice.they found that RNA interference targeting multiple genes can effectively inhibit NPC proliferation and induce apoptosis, which provides an experiment basis for NPC gene therapy [40]. Pathway analyses by microarrays revealed that upregulation of NF-κB2 and survivin played central roles in increasing resistance to apoptosis and demonstrated significant inhibition of NPC cell proliferation, migration and invasion. These antitumor effects were associated with induction of G2/M cell cycle arrest and apoptosis, and downregulation of NF-kappaB target genes (EGFR, cyclin D1 and survivin). This first demonstration of therapeutic benefits of NF-kappaB targeting in NPC implicates the importance of targeting this pathway in NPC[41].

It has been reported that frequent down regulation of the microRNA miR-218 in primary NPC tissues and cell lines plays a critical role in NPC progression. Exogenous expression of miR-218 caused significant toxicity in NPC cells in vitro and delayed tumor growth in vivo. A negative feedback loop indicated wherein miR-218 regulates NPC cell migration via the SLIT-ROBO pathway. Our findings define an integrative tumor suppressor function for miR-218 in NPC and further suggest that restoring miR-218 expression in NPC might be useful for its clinical management[42]. To explore the inhibiting effect of siRNA transfection against Survivin on the growth of nasopharyngeal carcinoma cells. A study showed that the expression of survivin in xenograft tumor was significantly inhibited by pshRNA-survivin/shRNA, the apoptosis of tumor cells was accelerated and the growth speed of NPC cells in xenograft tumor was retarded. The high expression of nasopharyngeal carcinoma's gene could significantly be silenced by using technology of RNAi, the growth of tumors could be inhibited also. It’s a novel treatment that have a good prospect[43].

Some researchers detected reduced 14-3-3 sigma expression in 5/6 NPC tumor lines by quantitative reverse transcription PCR (RT-PCR) and Western blotting. By immunohistochemical staining, significant down-regulation of 14-3-3 sigma was also found in 26/72 (36.1%) primary tumors of NPC patients, who were treated with curative radiotherapy. Importantly, they demonstrated that 14-3-3sigma expression is significantly associated with both overall survival (OS) and cancer-specific survival (CSS), but not with the clinical staging of NPC patients. In conclusion, low expression of 14-3-3sigma appears to be a valuable marker for better survival in patient with NPC. These results provide the evidence that 14-3-3 sigma expression is a significant prognostic factor for NPC patients[44].

 

Molecular Biomarkers For Prognosis And Progression of Cancer

According to clinical data reported by conventional radiotherapy of nasopharyngeal carcinoma after chemotherapy, early 5-year survival rate of patients with nasopharyngeal carcinoma 70% -90%, but the 5-year survival in patients with advanced nasopharyngeal carcinoma is only 20% -30%. Therefore, early diagnosis huge impact on prognosis of nasopharyngeal carcinoma.It has been reported that Human leukocyte antigen G (HLA-G) has multiple immune regulatory functions including the induction of immune tolerance in malignancies. Western assays showed high HLA-G expression in NPC cell lines, but not in normal nasopharyngeal epithelium tissue, Moreover, high expression of HLA-G predicted poor survival of NPC patients. Multivariate analysis indicated that HLA-G was an independent and unfavorable prognostic factor[45]. Prohibitin-1 (PHB, also known as PHB1) is a pleiotropic protein in cells involved in the regulation of proliferation, apoptosis, transcription, and mitochondrial protein folding. The scholars examined PHB mRNA levels using 24 nasopharyngeal carcinoma (NPC) and eight normal nasopharyngeal epithelium (NPE) tissues. PHB mRNA and protein expression levels were significantly down regulated in NPC tissue specimens compared with the NPE samples (P<0.01). In addition, decreased PHB expression correlates significantly with a poor prognosis, whereas decreased PHB protein expression is closely associated with advanced clinical stage and metastasis in NPC lesions. Therefore, Prohibitin-1 is an important biomarker for nasopharyngeal carcinoma progression and prognosis[46]. DNA-binding protein inhibitor 2 (ID2) in NPC was significantly increased in NPC cell’s nucleus when compared with that in normal nasopharynx tissues. Furthermore, the higher expression level of nuclear ID2 was significantly associated with tumor size (T classification), lymph node metastasis (N classification), and clinical stage. The study demonstrated that over-expression of ID2 protein is an unfavorable prognostic factor which promotes cell proliferation in NPC[47].

HOTAIR, a cancer-related long non-coding RNA, evaluated its prognostic value for NPC. Quantified using real-time PCR, which was higher compared with non-cancer tissue samples. Most importantly, NPC patients with higher HOTAIR levels had a poor prognosis for overall survival using univariate and multivariate analysis. HOTAIR is a potential biomarker for the prognosis of NPC, and dysregulation of HOTAIR might play an important role in NPC progression[48]. The expressions of nm23-H1 and VEGF protein were examined by immunohistochemistry S-P staining in NPC tissues. The low level expression of nm23-H1 protein and the high level expression of VEGF protein might be associated with the development and poor prognosis of NPC[49]. The phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) by MAP kinase-interacting kinases (Mnk) on Ser-209 promotes cellular proliferation, survival, malignant transformation and metastasis. The results showed that the positive percentage of p-Mnk1 and p-eIF4E proteins expression in NPC (83.5% and 75.4%, respectively) was significantly higher than that in non-cancerous nasopharyngeal epithelium (40.0% and 32.9%, respectively). Increase of p-eIF4E and p-Mnk1 expression was significantly correlated inversely with overall survival.in summery,high expression of p-Mnk1 and p-eIF4E might be novel valuable biomarkers to predict poor prognosis of NPC[50].

Periostin, a stroma-associated protein, statistical analysis showed over-expression of periostin was significantly associated with advanced clinical stage (P < 0.001) and lymph node metastasis (P < 0.001) and decreased overall survival (P < 0.001) in NPC. The result showed that periostin was able to promote invasiveness of NPC cell. Cox regression analysis indicated over-expression of periostin was an independent prognostic factor. Periostin is a potential biomarker for the differentiation and prognosis of NPC, and it might play an important role in the progression of NPC[51]. A correlation analysis demonstrated that high expression of H3K27me3 was positively associated with tumor later T classification, tumor metastasis, advanced clinical stage and chemoradioresistance (P < 0.05). These findings provide evidence that H3K27me3 expression, as examined by IHC, has the potential to be used as an immunomarker to predict NPC chemoradiotherapy response and patient prognosis[52]. Secretory cells and tissue proteome transcriptome analysis of serum marker screening nasopharyngeal carcinoma, serum cystatin A levels found in patients with nasopharyngeal carcinoma was significantly higher than that in healthy controls [53]. Pre-treatment of nasopharyngeal carcinoma patients with high levels of cystatin A N the higher stages, the prognosis is poor. Tip cystatin A may be a potential prognostic marker of nasopharyngeal carcinoma.

 

Emerging Perspectives

Currently, these markers used existing in tumors that also existing in the normal population and non-cancer patient's blood and body fluids. In the diagnosis of malignancy, the sensitivity and specificity is not high, it is currently only used for secondary tumors diagnosis, these markers cannot be solely over the reference range for diagnosis. Therefore, these markers in the early diagnosis of NPC need requires a lot of research, but it makes sense in the monitoring of prognosis and treatment of NPC. With the development of molecular biology, there have appeared new detection methods, Genomics, proteomics, metabolomics and bioinformatics each plays a more and more important role for molecular biomarker discovery[54]. Make the specificity and sensitivity improved in the related markers of NPC, but still not ideal. We now have a better understanding of this disease, including its diagnosis, monitoring, treatment and prognosis. In the era of molecular targeted therapy, explore the best combined detection of new markers is still the direction of research.

 

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