Epstein-Barr Virus Infection as Significant Predisposing Factor and Post-Radiotherapy Prognostic Indicator among Libyan Patients with Nasopharyngeal Cancer

Background and Objectives: Libya is one of the North African countries which constitute an area of nasopharyngeal carcinoma endemicty, particularly at the north part of Libya as compared to its south portion. Although from the clinical as well as histopathological presentation, there is no uniquely specific pattern of appearance, but there is high deficiency in the data that confirm the possible predisposing factors which may play a role in the development of this common variety of head and neck cancer at this country, and whether these factors affecting the patients response to treatment. Therefore this study was conducted to elucidate the suggestion of the role of Epstein-Barr Virus (EBV) in the risk of nasopharyngeal cancer at Libyan population; the results were correlated with the radiotherapy response and the improvement in the post-therapy prognostic value.

Patients and Methods: Sixteen patients aged 9-80 years presented at ENT department -Althowra central teaching hospital-elbyda-Libya at period from September- 2005 up to January-2014 with variable patterns of clinical presentation suggestive nasopharyngeal carcinoma. All patients were evaluated radio- logically as well as endoscopically, the diagnosis was confirmed by the biopsy and further histo-pathological assessment. For all patients the serological elucidation for IgG and IgM anti-EBV antibodies was done, and the results were correlated with the age of the incidence, sex of the incidence, the pattern of clinical presentation, the pattern of histo-pathological presentation, the response to the radiotherapy, the rate of the recurrence, and five years survival.

Results: 88% of the patients showed significant increase of serum IgG against Viral Capsid Antigen (VCA) of EBV. 83% of the cases presented with cervical masses and unilateral otitis media with effusion. 86% of cases showed lympho-epithelioma as histo-pathological pattern. On the other hand among all patients, those patients with lympho-epithelioma showed significant response to the concomitant radio- chemotherapy (100%) with high survival rate exceed five years.

Conclusion: The EBV infection can be considered as one of main predisposing factors of nasopharyngeal carcinoma at Libyan population. As it was noted from this presenting study that the induction of the cancer by EBV is mainly due to chronic infection rather than acute infection and this was confirmed by significant elevation of serum IgG rather than IgM. In addition it was postulated that the EBV infection most likely to be associated with lympho-epithelioma variety of histo-pathology rather than well-differentiated type, and as it was elucidated via this study that the association of the cancer with EBV infection increased the sensitivity of the tumor to concomitant radio-chemotherpy, thus this will improve the five years survival after the treatment.

Nasopharyngeal Carcinoma (NPC) is a tumor arising from the epithelial cells that cover the surface and line the nasopharynx. NPC was first described as a separate entity by Regaud and Schmincke in 1921 [1,2]. NPC is 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 males. Also Chinese men in the fifth decade were at highest risk. The incidence of the disease at southern China was 15:100,000 and at Hong Kong was 30:100,000. On the other hand the annual incidence of NPC in the UK is 0.25 per million (age standardized, age 0-14 years), 0.1 per million at age 0-9 years and 0.8 per 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 nasopharyngeal cancers at age 15-19 years are carcinomas. This suggests an incidence of 1 to 2 per million for NPC at age 15-19 years [4].

At the northern Africa, the NPC is mainly located with higher incidence at Libya, and Tunisia. The eastern part of Libya showed higher incidence of NPC as compared to the western part [5]. On the basis of some Libyan researches, it was found that the higher incidence was at age group 40-49 years (32%) and least cases were registered at age group 10-19 years (14%) [6]. As it is well-known that, there is a pathogenesis triad which constitutes the main three combined predisposing factors for NPC namely genetic, environmental, and viral infection. Regarding the viral infection, it is well-established that, EBV is considered as one of the significant and strongly associated predisposing etiological factor with the pathogenesis of NPC [7]. The EBV infection doesn’t only play a significant role in the pathogenesis of the NPC but it acts as one of highly valuable determinants for the histopathological pattern of this cancer as well as its response manner to the radiotherapy [8].

Epstein and his group discovered novel viral particles within cultured cells from patients with Burkitts’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 approximately 172 kilobases (kb) in length. Approximately 90% of the adult population throughout the world is EBV-positive by serology. Elevated titers of IgA antibody to EBV Viral Capsid Antigen (VCA) are usually found in patients with NPC, therefore this method of measuring patients’ EBV-specific IgA antibodies is useful in screening for early detection of NPC [1-8]. EBNA1 and EBERS are expressed in all EBV-positive cases of NPC and LMP1 is present in up to approximately 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 [1-8]. Epstein-Barr virus 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 (CR2), or (CD21), the putative EBV receptor. Hence, EBV appears to home to the oropharynx, and more specifically, the B-cells within the oropharynx. The strain B95-8 can be found in EBV-positive cell lines such as Raji, Namalwa, and CA46. These cell lines are all of B-lymphocyte lineage. This strain has been used as a benchmark in order to check for EBV positively in NPC. The EBV latent proteins include the 6 nuclear antigens (EBNAs 1, 2, 3A, 3B and 3C, and EBNA-LP) and the 3 latent membrane proteins (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 (TR) region, which is formed during the circularization of the linear DNA to produce the viral episome. EBER1 and EBER2 are highly transcribed non-poly-adenylated RNAs and their transcription is a consistent feature of latent EBV infection [1-8].

It was in 1966 that Old et al first discovered the relationship between Epstein Barr virus and NPC, using in situ hybridization and the Anti-Complement Immune-Fluorescent (ACIF) assay. Subsequent studies by others demonstrated the expression of EBV latent genes-Epstein-Barr Virus Nuclear Antigen (EBNA), latent membrane protein-1 (LMP1-), LMP-2 and Epstein-Barr virus encoded small RNAs (EBER)-in NPC cells confirming the infection of tumour cells by EBV [1-11]. Intriguingly, expression of EBV Early Antigen (EA) is positively correlated with the consumption of salted and preserved food, suggesting that development of EBV-positive NPC could be related to dietary habits 21 and provides another link to the epidemiological studies with NPC. Future studies should consider the effects of dietary risk factors on the risk of specific histologic subsets of NPC, and not assume that the disease is a etiologically homogeneous [1-8,12-15]. Since EBV infection indeed precedes clonal expansion of malignant cells. EBV is thought to contribute, at least in part, to the overall pathogenesis of NPC. Many studies have shown that Undifferentiated Nasopharyngeal Carcinomas (UNPC) are invariably EBV-positive, regardless of geographical origin. The process of EBV entry into keratinocytes and NPC cells is more complex, as both keratinocytes and NPC cells express only low levels of CR2 receptor. In addition, the relevance of serological tests for EBV infection in predicting the occurrence of NPC is presently still unclear. Specifically, a positive serological test for EBV EA may be found in greater than 80% of patients with NPC in Singapore, yet many normal individuals also express a positive EBV EA serological test, and never develop NPC. The procedures that researchers have carried out, such as PCR, revealed expression of the LMP2A and LMP2B genes and of latent transcripts running through the BamHI A region of the EBV genome in the opposite direction to the conventional lytic cycle mRNAs transcribed over this region [1-8].

It is well known that the EBV contributes directly to tumourigenesis in nasopharyngeal carcinoma NPC, primarily in the undifferentiated 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-positive (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 finding 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. In order to better understand and treat NPC, there is an urgent need to develop more EBV+ human NPC cell lines. Histoimmunochemically speaking, EBV acts to induce cell proliferation pathways in NPC via the activation of anti-apoptotic mechanism, i.e. NPC is consistently associated with EBV infection. EBV-encoded LMP1, expressed in most 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 2 C-terminal activating regions, interact with TNF Receptor Associated Factors (TRAFs) and TNF Receptor Associated Death Domain protein (TRADD). In vitro, LMP1 expression in epithelia cells can up regulate the expression of intercellular adhesion molecule 1 (ICAM-1), CD40, and cytokines such as interleukin 6 (IL-6) and IL-8. LMP1 also can 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 C-terminal activating regions 1 and 2 (CTAR-1 and CTAR-2), an effect blocked by overexpression of inhibitor of nuclear factor kappa B (IκB). LMP1 encoded by EBV is a membrane protein that activates multiple signaling pathways and transcription factors, including Nuclear Factor-kappaB (NF-κB). NF-κB activation is necessary for Hodgkin/Reed-Sternberg (HRS) cells to proliferate and inhibit apoptosis. Furthermore, activation of NF-κB is essential for B-cell immortalisation by EBV and LMP1-mediated transformation of fibroblasts. EBER-negative Hodgkin Lymphoma (HL) also displays constitutive NF-κB activation, indicating that without LMP1, other mechanisms exist to activate NF-κB. LMP1 has been shown to activate the mitogen activated protein kinase pathways and Janus kinase (JAK) signal transducers and activators (STATs) of transcription pathway in epithelia cells, as well as in B cells. LMP1 and 2A also activate the phosphatidylinositol3-OH kinase (P13K)/Akt pathway, which is commonly activated inappropriately in malignancy [1-8,13-15]. A recent study showed that P13K/Akt pathway was of importance in NPC pathogenesis. P13K/Akt pathway activation with subsequent phosphorylation and inactivation of GSK-3β and nuclear β-catenin accumulation were characteristic of primary NPC specimens. Thus it is clear that NPC uses its viral proteins to activate numerous cellular pathways in the NPC tumour that results in proliferation and prevents the transformed cells from dying [1,3,4,7].

On the other hand there are a lot of difficulties for performance and elucidation of proper research activity to confirm all these concepts and to correlate them with the clinical presentations, hitopathological patterns, and radiotherapy response of NPC, these difficulties come from the point of limitation of numbers of authentic EBV + NPC cell lines in existence as well as there are many inherent limitations to culturing NPC cells In vitro. Typically, nasopharyngeal biopsies provide only small tissue fragments. In order to increase the amount of available tissue, subcutaneous tumors may be first transferred to euthymic mice for initial in vivo expansion before seeding into tissue culture flasks 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 difficulties, at our area which is considered as one of most important regions for endemic NPC there were leak of the sufficient data regarding NPC as a general and from the pathogenesis point of view particular. For this reason this presenting original study was planned and decided to be conducted to achieve these specific aims:

a) To confirm whether there is any significant relationship between NPC and EBV infection among Libyan population.

b) To postulate the possible correlation between the EBV infection and clinical presentation of NPC among Libyan population.

c) To elucidate the possible correlation between the EBV infection and histopathological pattern of NPC among Libyan population.

d) To demonstrate the possible correlation between the EBV infection and the response of NPC to concomitant radio-chemotherapy among Libyan population.

Sixteen patients aged from nine years to eighty years presented at ENT department- Althowra central teaching hospital-Albyda city-Libya at period from September- 2005 up to January-2014 as cases of variable clinical presentations suggestive nasopharyngeal growths. Patients underwent further radiological as well as endoscopic evaluations and biopsies taken for histopathological confirmation of NPC diagnosis.

On the other hand, all patients were assessed immunologically for EBV antibodies Viral Capsid Antigen (VCA) in form of VCA-IgG as well as VCA-IgM. All patients were decided for concomitant radio-chemotherapy course after confirmation of the diagnosis and full staging. The immunological results were correlated with the clinical manifestations, histopathological patterns, and patients' response to the treatment.

Data were expressed by using descriptive analysis as means ±, standard error of mean (s.e.m) and per­centages. Test of significance was carried out; using chi-square test and two-way analysis of variance ~.A probability less than 0.05 was consid­ered as significant, the degree of signifi­cance was determined by using level of standard deviation test. Student — t — test was used for dependent sample, as well as contingency coefficient was calculated as measurement of association between nominal variables.

As shown in tables 1 & 2 the NPC incidence was correlated with patients demographic factors namely age and sex. The males were representing higher percentage (57%) among the total presented cases as compared to females who are representing (43%) of total cases, On the other hand the highest percentage of patients (58%) were at age 39- 48 years, followed by (14%) at age 29-38 years. As illustrated in table 3 that there was a significant elevation of VCA-IgG among (88%) of NPC cases as compared to VCA-IgM. As demonstrated in table 4 the commonest clinical presentation of NPC among Libyan patients at Albyda city was neck mass (81%), followed by halitosis (64%), weight loss as well as metabolic changes (56%) and unilateral otitis media with effusion (53%).As can be seen in table 5 the commonest radiological presentation of NPC among Libyan patients at Albyda city was in form of obliteration of unilateral Rosen- mullar fossa (64%) followed by obliteration of bilateral Rosen- mullar fossae, localized bone destruction, base of skull invasion, parapharngeal space invasion, and intracranial invasion, (36%), (18%), (18%), (9%), and (9%) consecutively. From the other view, table 6 shown that the different varieties of histo-pathological presentations among NPC Libyan patients at Albyda city. As can be seen the Lympho-Epithelioma (LE) constituted (86%) of NPC cases who were presented at ENT department-Althowra central teaching hospital-Albyda city-Libya.

Table 7 demonstrated the relationship between the variety of the histo-pathological pattern of NPC in one side and the response to the concomitant radio-chemotherapy with five years survival rate in the other side. The (LE) presented significantly (p < 0,001) the highest response to the treatment with the maximum five years survival rate and no any evidences of the recurrence throughout seven years of follow up.

The Incidence of nasopharyngeal carcinoma 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 with precision, confirmation is dependent on histology. Epstein-Barr Virus (EBV)-encoded RNA signal is present in all nasopharyngeal carcinoma cells, and early diagnosis of the disease is possible through the detection of raised antibodies against EBV. The quantity of EBV DNA detected in blood indicates the stage and prognosis of the disease. Radiotherapy with concomitant chemotherapy has increased survival, and improved techniques (such as intensity-modulated radiotherapy), early detection of recurrence, and application of appropriate surgical salvage procedures have contributed to improved therapeutic results. Screening of high-risk individuals in endemic regions together with developments in gene therapy and immunotherapy might further improve outcome [1-5,10].

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 well-defined populations, including natives of southern China, Southeast Asia, the Arctic, and the Middle East/North Africa. The distinctive racial/ethnic and geographic distribution of NPC worldwide suggests that both environmental factors and genetic traits contribute to its development [7,9-18].

Convincing evidence implicates dietary factors as the primary cause of NPC among Chinese. A series of case-control studies conducted in various Chinese populations with distinct risks of NPC, ranging from the very high-risk Cantonese populations to the relatively low-risk northern Chinese, have suggested that ingestion of salted fish 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 suggests that ingestion of preserved foods by these population groups may be responsible for their raised incidence of NPC [7,9-18].

A recent study suggests that among occupational hazards such as exposure to smoke, construction, metal, wood dust, motor fuel and oil, paint and varnishes etc… only wood dust was statistically significant for the development of NPC. Therefore, the development of NPC disease is multi-factorial with genetics, diet, and environmental exposure all playing large roles [9,10,12].

The positive IgA/VCA and intake of salted fish were associated with a strong excess risk of NPC. The association persisted after adjustment for other factors. The combination of salted fish and EBV was strongly associated with NPC, and more so than EBV or salted fish per se. Multivariate analyses showed that IgA/VCA was the most important predictor of NPC, and salted fish the second most important. These results suggest that EBV has a strong effect on the development of NPC. The exclusion of EBV and genetic factors in earlier epidemiological studies may have resulted in an overestimation of salted fish as important etiological factor causing NPC [19-28].

As it was confirmed by serial local studies which done at different regions of Libya that NPC can remain silent for a long time causing few primary symptoms, so it is difficult to make an early diagnosis. It has an early tendency to cervical lymph node involvement regardless of the size of the primary. Regional Lymph node enlargement, headache, nasal, and ear symptoms were common presenting complaints. Cranial nerves palsies and trismus, were found in the late stages [29].

Thus by the correlation of all previously mentioned reviews with the results of this presenting original study we can note that, the males have higher prevalence for NPC as compared to females and the peak age incidence was ranged from 39-48, this comes in agreement with other many national and inter-national studies. These figures can be explained by the higher chance for the exposure of the males particularly at these middle adult age groups to environmental predisposing factors in for of industrial as well as occupational hazards as compared to females [9,10,13-15].

On the other hand the commonest clinical presenting pattern of NPC was in form of cervical metastatic lymph node enlargement. This can be discussed in relation to three patho-anatomical facts that: a) the nasopharyngeal cavity is considered as wide cavity with sufficient space that makes the small sized tumors asymptomatic at early stage in relation to oto-rhino-pharyngeal obstructive symptoms and signs, b) the nasopharynx is rich in the lymphatic drainage with much decussated distributions resulting in early lymphatic spreading to sentinel lymph node groups and subsequently to other draining lymph nodes station groups, and c) the lateral wall of nasopharyngeal cavity is directly communicated with parapharyngeal space via sinus of Morgagni, this resulting in the facilitation of local, and rapid invasion of tumor into this space. In addition the histo-pathological picture of the tumor may determine its clinical as well as the radiological presentation. I.e. the tumors which are from the epithelial origin, toward the non-differentiation manner and those from mesenchymal origin as sarcomas have higher tendency for more aggressive and rapidly invasive behavior [13-15,27,29].

In accordance the detection of nuclear antigen associated with Epstein-Barr virus (EBNA) and viral DNA in NPC type- 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 significant role in the predisposition for NPC particularly LE (type-III). The serum VCA-IgG was significantly elevated as compared to VCA-IgM. This may indicate the association of risk of the cancer with longer duration histo-immunological activity of the virus which resulting in the stimulation of production of IgG rather than IgM, because immunologically speaking, as it is well-known that high titer of IgG almost always associated with chronic and prolonged- subclinical stage of infection rather than the acute and sever stage [1-8,12,15,17,19,22].

From the other view, as it was shown via this presenting study that the commonest histo-pathological pattern of NPC is LE which considered as one of the undifferentiated categories of NPC (type-III). This may determine the response to concomitant radio-chemotherapy and further improvement in the five years survival rate, because as it is well-established that LE is classified as highly sensitive variety of NPC to radiotherapy. In the same manner, the LE is strongly associated with EBV infection as a significant etiological predisposing factor. In agreement with all previously mentioned concepts, our results postulated that the LE which is the commonest pattern recognized among Libyan patients with NPC had been confirmed with high titer of VCA-IgG of EBV, on the other hand those patients elucidated sufficient response to concomitant radio-chemotherapy, and thus high rate of five years survival.

Therefore from this original descriptive study we can conclude to that NPC at Libyan population is commonly presented clinically with cervical metastatic lymph node enlargement, halitosis and unilateral otitis media with effusion. From the histo-pathological point of view, the commonest pattern is LE, which had significant association with EBV infection. Thus it had shown a significant response to the treatment and subsequent significant elevation of five years survival rate.

Libya is considered as one of North African countries which has endemic epidemiological presentation of NPC, this may stimulate for continuous and wider spectrum of research activity regarding this type of malignancies. For this reason as a recommendation, further confirmatory studies are required for more assessment by elucidation and estimation of antibodies against other more specific viral products at the cellular level. This may need more advanced histo-immuno-chemical techniques just to find out those viral related cell lines.

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