Effect of Epstein–Barr virus infection on predisposition and postradiotherapeutic prognostic value among Libyan patients with nasopharyngeal cancer

Introduction Nasopharyngeal carcinoma ( NPC) is a tumor arising from the epithelial cells that cover the surface and line the nasopharynx. NPC was fi rst described as a separate entity by Regaud and Schmincke in 1921 [ 1,2]. NPC is an uncommon disease; it is endemic in southern China, South-East Asia, and northern Africa [3]. In Malaysia, it is the second most common cancer in the male population. Also Chinese men in the fi fth decade were at the highest risk. Th e incidence of the disease in southern China was 15 : 100 000 and in Hong Kong was 30 : 100 000. In contrast, the annual incidence of NPC in the UK is 0.25/million (age standardized) at age 0–14 years, 0.1/million at age 0–9 years, and 0.8/ million at age 10–14 years. It seems reasonable to assume, on the basis of England and Wales’s cancer registry data, that at least 80% of NPCs at age 15–19 years are carcinomas. Th is suggests an incidence of 1–2/million for NPC at age 15–19 years [4].


Introduction
Nasopharyngeal carcinoma ( NPC) is a tumor arising from the epithelial cells that cover the surface and line the nasopharynx. NPC was fi rst described as a separate entity by Regaud and Schmincke in 1921 [ 1,2]. NPC is an uncommon disease; it is endemic in southern China, South-East Asia, and northern Africa [3]. In Malaysia, it is the second most common cancer in the male population. Also Chinese men in the fi fth decade were at the highest risk. Th e incidence of the disease in southern China was 15 : 100 000 and in Hong Kong was 30 : 100 000. In contrast, the annual incidence of NPC in the UK is 0.25/million (age standardized) at age 0-14 years, 0.1/million at age 0-9 years, and 0.8/ million at age 10-14 years. It seems reasonable to assume, on the basis of England and Wales's cancer registry data, that at least 80% of NPCs at age [15][16][17][18][19] years are carcinomas. Th is suggests an incidence of 1-2/million for NPC at age 15-19 years [4].
In northern Africa, NPC occurs with a higher incidence in Libya and Tunisia. Th e eastern part of Libya showed higher incidence of NPC as compared with the western part [5]. Some Libyan studies found that the highest

Effect of Epstein-Barr virus infection on predisposition and postradiotherapeutic prognostic value among Libyan patients with nasopharyngeal cancer
Khaled M. Bofares

Background and objective
Libya is one of the North African countries with an endemic of nasopharyngeal carcinoma, particularly in the northern part of Libya as compared with its southern part. The clinical and histopathological presentation reveals no uniquely speci c pattern of appearance; however, there is high de ciency in the data that con rm the possible predisposing factors that may play a role in the development of this common variety of head and neck cancer in this country, and whether these factors affect the patients' response to treatment. This study was conducted to determine the role of the Epstein-Barr virus ( EBV) in the risk for nasopharyngeal cancer in the Libyan population; the results were correlated with radiotherapy response and improvement in the post-therapeutic prognostic value.

Patients and methods
Sixteen patients aged 9-80 years presented at the ENT Department, Althowra Central Teaching Hospital, Albyda, Libya, from September 2005 to January 2014 with variable patterns of clinical presentation suggestive of nasopharyngeal carcinoma. All patients were evaluated radiologically and endoscopically. The diagnosis was con rmed by biopsy and further histopathological assessment. For all patients, serological elucidation for IgG and IgM anti-EBV antibodies was carried out, and the results were correlated with age at incidence, sex of the patient, pattern of clinical presentation, pattern of histopathological presentation, response to radiotherapy, rate of recurrence, and 5-year survival.

Results
In all, 88% of patients showed a signi cant increase in serum IgG against viral capsid antigen of EBV; 83% of the cases presented with cervical masses and unilateral otitis media with effusion; 86% of cases showed lymphoepithelioma as histopathological pattern. All patients with lymphoepithelioma showed signi cant response to concomitant radiochemotherapy (100%) with high survival rate exceeding 5 years.

Conclusion
The EBV infection can be considered one of the main predisposing factors to nasopharyngeal carcinoma in the Libyan population. It was noted from this study that the induction of cancer by EBV is mainly by chronic infection rather than by acute infection; this was con rmed by signi cant elevation in serum IgG rather than IgM. In addition, it was postulated that the EBV infection is most likely associated with the lymphoepithelioma variety of histopathology rather than with the well-differentiated type, and as it was elucidated through this study that the incidence was in the age group 40-49 years (32%) and the least in the age group 10-19 years (14%) [6]. It is well known that there is a pathogenesis triad that comprises the main three predisposing factors to NPC -namely, genetic, environmental, and viral infection. Regarding viral infection, it is well established that Epstein-Barr virus (EBV) is considered one of the most signifi cant and strongly associated predisposing etiological factors to the pathogenesis of NPC [7]. EBV infection not only plays a signifi cant role in the pathogenesis of NPC but also acts as one of the most highly valuable determinants of the histopathological pattern of this cancer as well as its response to radiotherapy [8].
Epstein and his group discovered novel viral particles within cultured cells from patients with Burkitt's lymphoma in 1964, 15 and 4 years later; this new virus was named EBV. EBV is a relatively large gamma herpes virus, and its DNA is double stranded and 172 kb in length. Approximately 90% of the adult population throughout the world is EBV+ by serology. Elevated titers of IgA antibody to EBV viral capsid antigen ( VCA) are usually found in patients with NPC. Th erefore, this method of measuring patients' EBVspecifi c IgA antibodies is useful in screening for early detection of NPC [1][2][3][4][5][6][7][8]. Epstein-Barr virus nuclear antigen 1 ( EBNA1) and Epstein-Barr virus-encoded small RNAs (EBERs) are expressed in all EBV+ cases of NPC, and latent membrane protein 1 ( LMP1) is present in up to 65% of cases. In almost all cases of EBV infection the oropharynx is the primary site of infection, as well as the site of viral replication [1][2][3][4][5][6][7][8]. Th e EBV is known to target primarily B lymphocytes. In-vitro studies demonstrate that EBV infects and potentially activates B cells by binding to the type-2 complement receptor, or to CD21, the putative EBV receptor. Hence, EBV appears to home to the oropharynx, and more specifi cally to the B cells within the oropharynx. Th e strain B95-8 can be found in EBV+ cell lines such as Raji, Namalwa, and CA46. Th ese cell lines are all of B-lymphocyte lineage. Th is strain has been used as a benchmark to check for EBV positivity in NPC. Th e EBV latent proteins include the six nuclear antigens (EBNAs 1, 2, 3A, 3B, and 3C, and LP) and the three LMPs (1, 2A, 2B). EBNA-LP is transcribed from variable numbers of repetitive exons. LMP2A and LMP2B are composed of multiple exons located on either side of the terminal repeats region, which is formed during the circularization of the linear DNA to produce the viral episome. EBER1 and EBER2 are highly transcribed nonpolyadenylated RNAs and their transcription is a consistent feature of latent EBV infection [1][2][3][4][5][6][7][8].
It was in 1966 that Old and colleagues fi rst discovered the relationship between EBV and NPC using in-situ hybridization and the anticomplement immunofl uorescent assay. Subsequent studies by others demonstrated the expression of EBV latent genes -EBNA, LMP1, LMP2, and EBERs -in NPC cells confi rming the infection of tumor cells by EBV [1][2][3][4][5][6][7][8][9][10]. Intriguingly, expression of EBV early antigen (EA) is positively correlated with the consumption of salted and preserved food, suggesting that development of EBV+ NPC could be related to dietary habits [21], providing another link to epidemiological studies on NPC. Future studies should consider the eff ects of dietary risk factors on the risk for specifi c histologic subsets of NPC and not assume that the disease is etiologically homogeneous [1][2][3][4][5][6][7][8][11][12][13][14]. EBV infection indeed precedes clonal expansion of malignant cells and is thought to contribute, at least in part, to the overall pathogenesis of NPC. Many studies have shown that undiff erentiated NPCs are invariably EBV+, regardless of geographical origin. Th e process of EBV entry into keratinocytes and NPC cells is more complex, as both keratinocytes and NPC cells express only low levels of type-2 complement receptor. In addition, the relevance of serological tests for EBV infection in predicting the occurrence of NPC is presently still unclear. Specifi cally, a positive serological test for EBV EA may be found in more than 80% of patients with NPC in Singapore, yet many normal individuals also express a positive EBV EA serological test and never develop NPC. Th e procedures that researchers have carried out, such as PCR, revealed expression of the LMP2A and LMP2B genes and of latent transcripts running through the Bam HI A region of the EBV genome in the opposite direction to the conventional lytic cycle mRNAs transcribed over this region [1][2][3][4][5][6][7][8].
It is well known that EBV contributes directly to tumorigenesis in NPC, primarily in the undiff erentiated form of NPC, which is commonly found in South-East Asia. Unfortunately, research in NPC has been severely hampered by the lack of authentic EBV+ human NPC cell lines for study. Since 1975, there have been more than 20 reported NPC cell lines. However, many of these NPC-derived cell lines do not express EBV transcripts in long-term culture, and therefore that fi nding may dispute the fundamental theory of NPC carcinogenesis. In fact, only one EBV+ human NPC cell line (C-666) in long-term culture has been reported. Hence, most of the NPC cell lines may not be representative of the disease itself. To better understand and treat NPC, there is an urgent need to develop more EBV+ human NPC cell lines. Immunohistochemically, EBV acts to induce cell proliferation pathways in NPC through the activation of an antiapoptotic mechanism; that is, NPC is consistently associated with EBV infection. EBV-encoded LMP1, expressed in most cases of NPC, has been suggested to have an important role in the pathogenesis and development of NPC, and its expression correlates with poor prognosis. LMP1 molecules aggregate in the cell membrane, and through two C-terminal activating regions (CTARs) they interact with tumor necrosis factor (TNF) receptor-associated factors and TNF receptor-associated death domain protein. In vitro, LMP1 expression in epithelia cells can upregulate the expression of intercellular adhesion molecule 1, CD40, and cytokines such as interleukin 6 and interleukin 8. LMP1 can also induce the expression of CD70 antigen, a member of the TNF family, in epithelial cells in vitro. LMP1 can induce a matrix metalloproteinase, MM-9, through CTAR-1 and CTAR-2, an eff ect blocked by overexpression of the inhibitor of nuclear factor B (I B). LMP1 encoded by EBV is a membrane protein that activates multiple signaling pathways and transcription factors, including nuclear factor B (NF-B). NF-B activation is necessary for Hodgkin/Reed-Sternberg cells to proliferate and inhibit apoptosis. Furthermore, activation of NF-B is essential for B-cell immortalization by EBV and LMP1-mediated transformation of fi broblasts. EBER-negative Hodgkin's lymphoma also displays constitutive NF-B activation, indicating that without LMP1 other mechanisms exist to activate NF-B. LMP1 has been shown to activate the mitogen-activated protein kinase pathways and Janus kinase ( JAK) signal transducers and activators of transcription pathway in epithelial cells and B cells. LMP1 and 2A also activate the phosphatidylinositol-3-OH kinase ( P13K)/Akt pathway, which is commonly activated inappropriately in malignancy [1][2][3][4][5][6][7][8][12][13][14]. A recent study showed that the P13K/Akt pathway is important in NPC pathogenesis. P13K/Akt pathway activation with subsequent phosphorylation and inactivation of GSK-3 and nuclear -catenin accumulation was characteristic of primary NPC specimens. Th us, it is clear that NPC uses its viral proteins to activate numerous cellular pathways in the NPC tumor, which results in proliferation and prevents the transformed cells from dying [1,3,4,7].
Th ere are a lot of diffi culties in the performance and elucidation of proper research activity to confi rm all these concepts and correlate them with clinical presentations, histopathological patterns, and radiotherapy response of NPC. Th ese diffi culties stem from the limited number of authentic EBV+ NPC cell lines in existence and from the inherent limitations to culturing NPC cells in vitro. Typically, nasopharyngeal biopsies provide only small tissue fragments. To increase the amount of available tissue, subcutaneous tumors may be fi rst transferred to euthymic mice for initial in-vivo expansion before seeding into tissue culture fl asks for in-vitro culture. Besides the potential introduction of murine cells, this method is likely to result in changes to the primary human biopsy tissue sample. In addition to these previously mentioned diffi culties, in our area, which is considered one of the most important regions for endemic NPC, there was lack of suffi cient data regarding nasopharyngeal carcinoma (NPC) as general and from pathogenesis point of view particular, for this reason this study was planned to achieve these specifi c aims:

Patients and methods
Sixteen patients aged 9-80 years presented at the ENT Department, Althowra Central Teaching Hospital, Albyda, Libya, from September 2005 to January 2014 with variable clinical presentations suggestive of nasopharyngeal growth were included in this study. Th e patients underwent further radiological and endoscopic evaluations and biopsies were taken for histopathological confi rmation of NPC diagnosis.
All patients were assessed immunologically for EBV antibody VCA in the form of VCA-IgG and VCA-IgM. All patients were indicated for concomitant radiochemotherapy after confi rmation of diagnosis and full staging. Th e immunological results were correlated with the clinical manifestations, histopathological patterns, and patients' response to the treatment.

Statistical analysis
Data were expressed using descriptive analysis as mean ± SEM and percentages. Test of signifi cance was carried out usi ng the 2 -test and two-way analysis of variance. A probability value of less than 0.05 was considered signifi cant; the degree of signifi cance was determined using the level of SD test. Student's t-test was used for dependent variables, and the contingency coeffi cient was calculated as measurement of association between nominal variables. Tables 1 and 2 the NPC incidence was correlated with patients' demographic factors -namely, age and sex. Th e percentage of male patients was higher (57%) than that of female patients (43%). Th e highest percentage of patients (58%) was found in the age group 39-48 years, followed by age 29-38 years (14%). As illustrated in Table 3 there was a signifi cant elevation in VCA-IgG in 88% of NPC cases as compared with VCA-IgM. As demonstrated in Table 4 the most common clinical presentation of NPC among Libyan patients at Albyda city was neck mass (81%), followed by halitosis (64%), weight loss and metabolic changes (56%), and unilateral otitis media with eff usion (53%). As can be seen in Table 5 the most common radiological presentation of NPC among Libyan patients at Albyda city was obliteration of unilateral Rosenmullar fossae (64%), followed by obliteration of bilateral Rosenmullar fossae, localized bone destruction, base of skull invasion, parapharyngeal space invasion,

Table 1 Sex distribution in relation to the incidence of nasopharyngeal carcinoma in the Libyan population at Albyda city
Types of sex % Male (n = 9) 57 Female (n = 7) 43 Total (n = 16) 100 P < 0.05.

Discussion
Th e incidence of NPC has remained high in endemic regions. Diagnosing the disease in the early stages requires a high index of clinical acumen and, although most cross-sectional imaging investigations show the tumors precisely, confi rmation is dependent on histology. Th e EBV-encoded RNA signal is present in all NPC cells, and early diagnosis of the disease is possible through the detection of raised antibodies against EBV. Th e quantity of EBV DNA detected in blood indicates the stage and prognosis of the disease. Radiotherapy with concomitant chemotherapy has increased the probability of survival and led to the development of improved techniques (such as intensity-modulated radiotherapy), aiding in the early detection of recurrence. Further, application of appropriate surgical salvage procedures has contributed to improved therapeutic results. Screening of highrisk individuals in endemic regions together with developments in gene therapy and immunotherapy might further improve outcome [1][2][3][4][5][6].
NPC has a unique and complex etiology that is not completely understood. Although NPC is rare in most populations, it is a leading form of cancer in a few welldefi ned populations, including natives of southern China, South-East Asia, the Arctic, the Middle East, and North Africa. Th e distinctive racial/ethnic and geographic distribution of NPC worldwide suggests that both environmental factors and genetic traits contribute to its development [6,7,[9][10][11][12][13][14][15][16][17].
Convincing evidence implicates dietary factors as the primary cause of NPC among the Chinese. A series of case-control studies conducted in various Chinese populations with distinct risk for NPC, ranging from the very high-risk Cantonese populations to the relatively low-risk northern Chinese, have suggested that ingestion of salted fi sh and other kinds of preserved foods constitutes the most important cause of NPC among these people. Preliminary data on Malays in South-East Asia, Eskimos in Alaska, and Arabs of North Africa also suggest that ingestion of preserved foods may be responsible for their raised incidence of NPC [6,7,[9][10][11][12][13][14][15][16][17].
A recent study suggests that, among occupational hazards such as exposure to smoke, construction sites, metal, wood dust, motor fuel and oil, paint and varnishes, etc., only wood dust was statistically signifi cant for the development of NPC. Th erefore, the development of NPC disease is multifactorial, with genetics, diet, and environmental exposure all playing large roles [6,9,11].
Th e positive VCA-IgA and intake of salted fi sh were associated with a strong excess risk for NPC. Th e association persisted after adjustment for other factors. Th e combination of salted fi sh and EBV was strongly associated with NPC, and more so than EBV or salted fi sh per se. Multivariate analyses showed that VCA-IgA was the most important predictor of NPC, and salted fi sh the second most important. Th ese results suggest that EBV has a strong eff ect on the development of NPC. Th e exclusion of EBV and genetic factors in earlier epidemiological studies may have resulted in an overestimation of salted fi sh as an important etiological factor causing NPC [18][19][20][21][22][23][24][25][26][27].
As confi rmed by a series of local studies conducted at diff erent regions of Libya, NPC can remain dormant for a long time, causing few primary symptoms; hence, it is diffi cult to make an early diagnosis. It has an early tendency to cervical lymph node involvement regardless of the size of the primary tumor. Regional lymph node enlargement, headache, and nasal and ear symptoms were common presenting complaints. Cranial nerve palsies and trismus were found in the late stages [28].
Th us, on the basis of the correlation between all previously mentioned reviews and the results of this study we can note that male patients have a higher incidence of NPC compared with female patients and the peak age at incidence ranged from 39 to 48 years. Th ese results are in agreement with those of many other national and international studies. Th ese fi gures can be explained by the higher probability for exposure of the male population, particularly the middle-aged group, to predisposing environmental factors from industrial and occupational hazards compared with females [6,9,[12][13][14].
Th e most commonly presenting clinical pattern of NPC was cervical metastatic lymph node enlargement. Th is can be discussed in relation to three pathoanatomical facts: (a) Th e nasopharyngeal cavity is considered a wide cavity with suffi cient space that makes the Libya is considered one of the North African countries with an endemic epidemiological presentation of NPC, encouraging a continuous and wider spectrum of research into this type of malignancy. Further studies are required for elucidation and estimation of antibodies against other more specifi c viral products at the cellular level, which may require more advanced immunohistochemical techniques.
small-sized tumors asymptomatic at early stage in relation to otorhinopharyngeal obstructive symptoms and signs; (b) Th e nasopharynx is rich in lymphatic drainage with much decussated distributions resulting in early lymphatic spread to sentinel lymph node groups and subsequently to other draining lymph node station groups; and (c) Th e lateral wall of the nasopharyngeal cavity is directly connected to the parapharyngeal space through the sinus of Morgagni, resulting in the facilitation of local and rapid invasion of tumor into this space.
In addition, the histopathological profi le of the tumor may determine its clinical and radiological presentation; that is, nondiff erentiated tumors of epithelial origin and those of mesenchymal origin, such as sarcomas, have higher tendency for more aggressive and rapidly invasive behavior [12][13][14]26,28].
In accordance, the detection of EBNA and viral DNA in NPC types II and III has revealed that EBV can infect epithelial cells and is associated with their transformation. It was illustrated from the results of this presenting study that EBV infection has a signifi cant role in the predisposition for NPC, particularly LE (type III). Th e serum VCA-IgG was signifi cantly elevated as compared with VCA-IgM. Th is may indicate the association of the risk for cancer with longer duration of the histoimmunological activity of the virus, resulting in the stimulation of production of IgG rather than IgM, because, immunologically, it is well known that a high titer of IgG is almost always associated with chronic and prolonged subclinical stage of infection rather than the acute and severe stage [1][2][3][4][5][6][7][8]11,14,16,18,21].
In contrast, it was shown through this presenting study that the most common histopathological pattern of NPC is LE, which is considered one of the undiff erentiated categories of NPC (type III). Th is may determine the response to concomitant radiochemotherapy and further improvement in the 5-year survival rate, because it is well established that LE is classifi ed as a highly sensitive variety of NPC to radiotherapy. In the same manner, LE is strongly associated with EBV infection as a signifi cant etiological predisposing factor. In agreement with all previously mentioned concepts, our results postulated that LE, which is the most common pattern recognized among Libyan patients with NPC, had been confi rmed with a high titer of VCA-IgG of EBV. Th ose patients showed suffi cient response to concomitant radiochemotherapy, and thus a high rate of 5-year survival.