Archives of Medical Research 45 (2014) 229e236

ORIGINAL ARTICLE

Expression of Epstein-Barr Virus-encoded Latent Membrane Protein (LMP-1),p16 and p53 Proteins in Nonendemic Nasopharyngeal Carcinoma (NPC):

A Clinicopathological Study

Samuel Rosales-P�erez,a Ana M. Cano-Valdez,b Christian H. Flores-Balc�azar,c Ferran Guedea-Edo,d

Leonardo S. Lino-Silva,b Alicia Lozano-Borbalas,d Arturo Navarro-Mart�ın,d andAdela Poitevin-Chac�one

aRadiation Oncology Department, Oncology Hospital, Centro Medico Nacional Siglo XXI (IMSS), Mexico, D.F., MexicobPathology Department, cRadiation Oncology Department, National Cancer Institute of Mexico (INCan), Mexico, D.F., Mexico

dRadiation Oncology Department, Catalan Institute of Oncology (ICO-L’Hospitalet), Barcelona, SpaineRadiation Oncology Department, Medica Sur Hospital, Mexico, D.F., Mexico

Received for publication February 14, 2013; accepted January 31, 2014 (ARCMED-D-13-00096).

Address reprint

Oncology Departmen

pital de Oncolog�ıa, A

D.F., Mexico; Phone:

0400; E-mail: samuel

0188-4409/$ - see frohttp://dx.doi.org/10

Background and Aims. Although the latent membrane protein type 1 (LMP1) isfrequently expressed in Epstein-Barr virus (EBV) malignancies, its contribution to thepathogenesis of nasopharyngeal carcinoma (NPC) is not fully defined. LMP1 functionsas a viral mimic of the TNFR family member engaging a number of signaling pathwaysthat induce morphological and phenotypic alterations. This study aimed to investigate theLMP1 expression and EBV infection in relation to clinical outcome and survival in a se-ries of Mexican NPC patients. We also studied expression of p16 and p53 proteins.

Methods. We analyzed in 25 tumor specimens the expression of LMP1, p16 and p53 byimmunohistochemistry (IHC) and EBV presence by IHC/in situ hybridization. Differ-ences in clinical outcome and survival in relation to protein expression were correlatedthrough c2 statistics and Kaplan-Meier survival curves.

Results. Our results showed a rate of 92% (23/25) of EBV infection. The expressions ofLMP-1, p16 and p53 proteins were 40.0, 44.0 and 40.0%, respectively. LMP-1 immu-noexpression was more common in older patients (O50 vs. !50 years old, p 5 0.02)and with parapharyngeal space invasion ( p 5 0.02). The presence of metastatic diseaseat diagnosis ( p 5 0.03), distant recurrence disease ( p 5 0.006) and shorter distancerecurrence-free survival ( p 5 0.05) was associated with lack of p16.

Conclusions. In our series, EBV infection rates are particularly high for nonendemicNPC, although without a statistically significant difference in overall survival, LMP1and p16 expression was correlated with poorer clinical prognosis. Probably, LMP1 andp16 detection identify a worse clinical prognosis in NPC patient subgroup. � 2014IMSS. Published by Elsevier Inc.

Key Words: Nasopharyngeal carcinoma, EBV-LMP1, p16, p53, Immunohistochemistry.

Introduction

Nasopharyngeal carcinoma (NPC) is a squamous epithelialcancer originating from the lateral wall surface of the

requests to: Samuel Rosales-P�erez, Radiation

t, Centro M�edico Nacional Siglo XXI, UMAE Hos-

v. Cuauht�emoc #330 Col. Doctores, 06725 M�exico,

(52) 5556 28 0400 ext. 22624; FAX: (52) 5556 28

.rosales@me.com

nt matter. Copyright � 2014 IMSS. Published by Elsevier.1016/j.arcmed.2014.02.002

nasopharynx that usually arises around the ostium tubarius(1). NPC is an endemic head and neck malignancy in south-eastern Asia (25e50 cases/100,000 persons) and Alaskaand native Greenlanders, strongly linked to Epstein-Barr vi-rus infection (EBV). In the U.S. and other Western coun-tries it is an uncommon tumor where the incidence is|0.2e0.5 cases/100,000 persons (2,3). Other ethnic, ge-netic susceptibility, environmental factors and food con-sumption have been associated with NPC etiology (4).

Inc.

230 Rosales-P�erez et al./ Archives of Medical Research 45 (2014) 229e236

According to current WHO tumor classification, NPCcan be divided into three subtypes of histological patterns.Type I, keratinizing squamous cell carcinoma shows pre-dominant features of keratin productions; type II, differen-tiated nonkeratinizing carcinoma and type III,nonkeratinizing with less differentiation. Type II/III is anendemic neoplasm in the previously mentioned geographicareas and type I is more common in nonendemic areas.Although NPC types II/III are more associated with EBVinfection, almost all NPC cancer has a comorbid virus rela-tionship (5). In the U.S., the majority of NPCs have kerati-nizing squamous cell histologic findings, whereas in Asia,the undifferentiated histologic type is by far the mostcommon (6).

The prognosis depends mainly on the TNM stage classi-fication. Many patients are diagnosed in advanced stagesbecause the anatomic site of cancer growth is located inpainless areas and no other signs or symptoms are found.The 5-year survival rate of stages IeII ranges from72e90%. However, the 5-year overall survival (OS) rateof stages III and IV are 55% and 30%, respectively, duemainly to a high incidence of locoregional recurrences ordistance metastases (7).

Radiation therapy is the cornerstone of treatment with orwithout concurrent and adjuvant chemotherapy dependingon clinical stage. The M.D. Anderson Cancer Center(MDACC) series showed the long-term outcome in a largepopulation of patients treated with radiotherapy alone.Five-year local control rates for T1, T2, T3 and T4 cate-gories are 93, 79, 68, and 53%, respectively (8). The Inter-group 0099 explored concurrent chemo-RT followed byadjuvant chemo in NPC stage IIIeIV patients (2/70 Gy þcisplatin x 3 cycles plus adjuvant cisplatin/5-FU x 3 cycles).Chemo-RT improved 3-year overall survival (OS) in 31%and progression-free survival (PFS) in 45% (9).

Even though advances in treatment have achieved betterlocoregional control, it appears necessary to identify poten-tial biomarkers with better correlation to tumor growth,dissemination and/or treatment outcomes in patients withNPC, subsequently, to aid in stratification and developmentof molecular therapeutic targets (10).

LMP1, a complex 66-kDa integral membrane protein, isone of five key EBV-encoded viral proteins required forB-cell immortalization. In contrast to hematopoietic neo-plasms, LMP1 does exert a variety of growth-promoting ef-fects in human epithelial cells (11). In non-tumorigenic celllines, LMP1 expression can induce an epidermal hyperpla-sia (12) or a cellular phenotype similar to ‘‘wounded’’keratinocytes (13), but when expressed in NPC cells poten-tiates anchorage-independent growth and greatly enhancescell motility and invasion (14). Although showing no appre-ciable homology to known cellular proteins, LMP1 displaysfunctional similarities to the tumor necrosis factor receptor(TNFR) family members: TNRF1 and CD40. Unlike theseproteins, LMP1 signals in a constitutive ligand independent

manner (15). Taken as a whole, these findings demonstratethat LMP1 can induce profound effects in epithelial cells,many of which may account for its oncogenic properties.

Accordingly, we aimed to systematically analyze LMP1immunoexpression in patients with nonendemic NPC andmake a correlation with clinicopathological features andpatient survival. We also analyzed the expression of p16and p53 tumor suppressor genes (TSG) in these specimens.

Materials and Methods

Patients and Tumor Specimens

We retrospectively analyzed data from 29 patients withnonendemic primary NPC who were diagnosed and treatedat the National Cancer Institute in Mexico City (Table 1).Biopsy samples were collected from our biobank between1998 and 2008. Available paraffin-embedded tissue blockswere retrieved from 29 patients but only 25 analyzed withsufficient material. These patients were free of treatmentat initial presentation. The histological subtypes were reap-praised according to the current WHO classification (16).TNM staging was evaluated with the 6th American JointCommittee on Cancer system (17) by two pathologistsindependently. This study was conducted with the approvalof the Institutional Review Board.

Immunohistochemical Staining and Assessment of LMP1,p16 and p53 Proteins

Immunohistochemical staining was performed accordinglyto the protocol of our pathology department. Briefly, 3-mm-thick tissue sections were cut onto precoated slidesfrom paraffin-embedded tissue blocks and were thenroutinely deparaffinized with xylene and rehydrated withethanol washes. Slides were heated using the microwavewith citrate buffer (pH 6.0) for 7 min to retrieve antigens.Endogenous peroxidase was blocked with 3% H2O2. Slideswere next washed by Tris-buffered saline for 15e30 minand subsequently incubated with CS1-4 (anti-LMP1; dilu-tion 1:100, Dako, Carpinteria, CA) p16 (dilution 1:50;LabVision; Thermo Fisher Scientific, MA) y p53 (dilu-tion1:100; ImmunoVision; Springdale, AR). DAB (diamino-benzidine) was used as a chromogen. We also determinedlatent virus infection by EBV-encoded small RNAs (EBERs)in situ hybridization (ISH) using EBER PNA probes (Dako)according to the manufacturer’s protocol.

Immunohistochemical staining evaluation was evaluatedonly in the neoplasic cells (carcinoma cells) excluding lym-phocytes and was classified quantitatively as follows:0 (negative), 1þ (staining in !10% of the neoplasic cells)2þ (staining in 10e50% of the neoplasic cells) and 3þ(staining in O50% of the neoplasic cells). Staining inten-sity was evaluated semiquantitatively as follows: 0 (nega-tive), 1þ (weak) and 2þ (strong). We use positive and

Table 1. Clinical features of 29 patients with NPC

Variable n (%)

Gender

Male 20 (69.0)

Female 9 (31.0)

Age (years)

!50 15 (51.8)

$50 14 (48.2)

Performance status (ECOG)

0 18 (62.1)

1e2 8 (27.6)

O2 3 (10.3)

Smoking

Yes 8 (27.6)

No 21 (72.4)

Alcohol

Yes 7 (24.1)

No 22 (75.9)

WHO histological type

I (keratinizing) 4 (13.8)

II/III (nonkeratinizing) 25 (86.2)

Stagea

I 1 (3.5)

II 7 (24.1)

III 3 (10.3)

IV 18 (62.1)

Primary tumor (T)a

T1 10 (34.5)

T2 8 (27.6)

T3 3 (10.3)

T4 8 (27.6)

Nodal status (N)a

N0 6 (21.0)

N1 7 (24.0)

N2 4 (14.0)

N3 12 (41.0)

Metastasis (M)a

0 27 (93.1)

1 2 (6.9)

PSI

Yes 14 (48.3)

No 15 (51.7)

NPC, nasopharyngeal carcinoma; ECOG, Eastern Cooperative Oncology

Group; WHO, World Health Organization; PSI, parapharyngeal space

invasion.aAccording to the AJCC 6th edition.

Table 2. Treatment characteristics of 29 patients with NPC

Variable n (%)

Chemotherapy (n 5 22)

Neoadjuvant � chemoradiation 12 (54.5)

Adjuvant 10 (45.5)

Chemotherapy regimen (n 5 22)

CDDP 12 (54.5)

CDDP/5-FU 9 (41.0)

Gemcitabine 1 (4.5)

Radiotherapy (n 5 29)

Radiotherapy alone 17 (58.6)

Chemoradiation 12 (41.4)

Dose for primary site (Gy)

!60 9 (31.0)

$60 20 (69.0)

OTT (day)

!60 15 (51.7)

$60 14 (48.3)

NDPNN (n 5 28)

Yes 2 (7.1)

No 26 (92.9)

OTT, overall treatment time of radiotherapy; NDPNN, neck dissection for

persistent neck nodes.

231Epstein-Barr Virus Membrane Protein p16 and p53 in Nonendemic Nasopharyngeal Carcinoma

negative control tissues as recommend by the manufacturerfor all immunohistochemistry antibodies. For LMP-1 andEBER reactions, we used LCL (B-lymphoblastoid celllines) immortalized with Akata EBV as a positive control.A sense probe against EBER-1 was used for negative con-trol studies.

Treatment and Outcome Evaluation

All 29 patients with at least 18 months of follow-up wereretrospectively analyzed. Among the 29 patients, nonehad a past history suggestive of chronic active EBV-associated disease. All 29 patients with follow-up for

outcome received radiotherapy. The planned dose was 70Gy (2 Gy per day, five times per week for 7 weeks, meantotal dose 67.7 Gy). Twenty-two patients received chemo-therapy. Concurrent weekly cisplatin (CDDP) basedchemotherapy was administered in 12 patients (55.0%),nine patients (40.0%) received chemo-radiotherapyregimen of CDPP followed by adjuvant CDPP/5-FU andone patient (5.0%) received chemo-radiotherapy plus adju-vant gemcitabine.

Statistical Analysis

The influence of LMP1, p16 and p53 expression on clinico-pathological outcomes was evaluated by the Fisher exacttest. OS and PFS were measured and calculated from thestarting date of RT to the date of event developed. Survivalcurves were plotted using the Kaplan-Meier method, andstatistical significance was assessed by the long-rank test.All p values were 2-sided, and values !0.05 were consid-ered statistically significant. These analyses were conduct-ed with Statistical Product and Service Solutions software(SPSS) v.17.0.

Results

Clinical and Treatment Characteristics of Patients

A total of 29 patients with at least 18 months of follow-upwere retrospectively analyzed. Patient characteristics aresummarized (Tables 1 and 2). There were 20 males(69.0%) and nine females (31.0%) with a median age of

Figure 1. Immunostaining on tissue sections of NPC samples. (A) Low magnification of EBER expression by in situ hybridization (ISH) with hematoxylin

counterstain (magnification x25). (B) High magnification of EBER expression showing dark signal in the nuclei of neoplasic cells (ISH with hematoxylin

counterstain, x400). (C) Low magnification of P16 expression by immunohistochemistry with hematoxylin counterstain (magnification x25). (D) High

232 Rosales-P�erez et al./ Archives of Medical Research 45 (2014) 229e236

233Epstein-Barr Virus Membrane Protein p16 and p53 in Nonendemic Nasopharyngeal Carcinoma

50 years (range, 22e72 years). In most cases (23 patients,80.0%) the initial presenting symptom was uni- or bilateralcervical mass. Parapharyngeal space invasion was recordedin 14 patients (48.0%). Nasal cavity infiltration wasobserved in ten patients (34.0%). According to the 6th

American Joint Committee on Cancer (AJCC) system,one patient was classified as stage I (3.0%), seven as stageII (24.0%), three as stage III (11.0%) and 18 as stage IV(62.0%). Among the 18 stage IV cases, two patients showedmetastatic disease at diagnosis (M1).

All 29 patients with follow-up for outcome receivedradiotherapy. The planned dose was 70 Gy (2 Gy per day,five times per week for 7 weeks, mean total dose 67.7Gy). The plan design included two standard laterallyopposed fields and a bilateral supraclavicular field. Radio-therapy was delivered using linear accelerator (VarianC600, 2100; Palo Alto, CA) or a telecobalt unit. Although28 patients were classified as stage IIeIV disease, only 22received chemotherapy. Concurrent weekly cisplatin(CDDP)-based chemotherapy was administered in 12 pa-tients (55.0%), nine patients (40.0%) received chemo-radiotherapy regimen of CDPP followed by adjuvantCDPP/5-FU and one patient (5.0%) received chemo-radiotherapy plus adjuvant gemcitabine. All patients weremonitored every 4 months for the first 2 years and every6 months for next 3 years. After 5 years the evaluationwas once per year.

Morphologic and Immunophenotypic Features

Morphologic (29 patients) and immunophenotypic (25 pa-tients) features were evaluated. The neoplasic cells con-sisted of a mixture of small- to medium-sized cells with around-oval nuclear shape (Figure 1). Among the 29 cases,25 (86.0%) were classified as type II/III and four (14.0%)as type I according to WHO tumor classification.

Immunohistochemistry in 25 patients showed that 23specimens (92%) were positive for EBERs indicating thepresence of EBV-latent infected cells. LMP1 was detectedin ten cases (40%), p16 in 11 cases (44.0%) and p53 in10 cases (40.0%). Immunophenotypic features are listedin Table 3.

Prognostic Factor, Therapy and Impact of LMP1, p16 andp53 Expression on Clinical Outcome

Among 29 patients, OS was worse in male than females( p 5 0.03) and with non-keratinizing carcinoma variant(WHO types II/III, p 5 0.05). Chemo-radiotherapy

magnification of P16 expression showing brown signal in the nuclei and cytopla

x100). (E) Low magnification of P53 expression by immunohistochemistry with

stain, x25). (F) High magnification of P53 expression showing brown signal in th

stain, x100). (G) Low magnification of LMP-1 expression by immunohistochem

counterstain, x25). (H) High magnification of LMP-1 expression showing brown

with hematoxylin counterstain, x400). (A color figure can be found in the onlin

(chemo-RT) regimen of CDPP followed by adjuvantCDPP/5-FU was the best OS treatment vs. other therapeuticmodalities ( p 5 0.001). Treatment response is shown inTable 4. No statistically significant differences were foundbetween OS and other demographic, clinical or treatmentvariables. We attempted to find relationships of theLMP1, p16 y p53 score with various clinical parameters.

Prognostic Impact of LMP1 Expression in NPC

LMP1 staining was observed and successfully scored in 25cases. Immunoexpression was more common in olderpatients (O50 vs. !50 years old, p 5 0.02) and with para-pharyngeal space invasion (p 5 0.02). Complete treatmenttumor response was better in patients with negative LMP1expression ( p 5 0.06) with nonstatistically significant dif-ferences (NS). There was a trend suggesting of distantrecurrence disease with LMP1 positivity ( p 5 0.09). Wefound no statistically significant differences betweenLMP1 expression and OS or other clinicopathologicalfactors.

Prognostic Impact of p16 and p53 Expression in NPC

Immunoexpression of p16 protein was found in 11 cases(44.0%). The presence of metastasic disease at diagnosis( p 5 0.03) and development of distant recurrence disease( p 5 0.006) was significantly associated with lack of p16expression. Of note, p16 negative correlated with a moreaggressive clinical course with a shorter distance recurrencefree survival (DRFS, p 5 0.05). We found p53 expressionin ten cases (40.0%) with nonsignificant differences be-tween groups. There was also no relationship with p16 orp53 expression and OS.

Discussion

Nasopharyngeal carcinomas occur worldwide with variableincidence. In Western countries the tumor is rare (annualincidence, !0.5 cases/100,000), whereas in some parts ofSoutheast Asia, North Africa, Greenland, and Alaska it isa common human cancer (annual incidence in Hong Kong25/100,000). The association of nonkeratinizing NPC(WHO types II/III), particularly of the undifferentiated sub-type, with EBV is well established. EBV has been detectedin virtually all cases, irrespective of the geographic origin,and thus appears to be a rate-limiting step in the pathogen-esis of these tumors (18). In our series we found that almost

sm of neoplasic cells (immunoperoxidase with hematoxylin counterstain,

hematoxylin counterstain (immunoperoxidase with hematoxylin counter-

e nuclei of neoplasic cells (immunoperoxidase with hematoxylin counter-

istry with hematoxylin counterstain (immunoperoxidase with hematoxylin

signal in the cytoplasmic membrane of neoplasic cells (immunoperoxidase

e version of this article.)

Table 3. Clinical treatment response and incidence and site of

progression or recurrent disease

Variable n (%)

Treatment response (n 5 29)

Complete 20 (69.0)

Partial 6 (20.7)

Disease progression 3 (10.3)

Recurrent disease (n 5 29)

Local � regional 5 (17.2)

Distant only 5 (17.2)

No recurrence 19 (65.6)

Table 4. Expression level of EBER, LMP-1, p53 and p16 proteins

(n 5 25)

Variable Positivea n (%) Negativeb n (%)

EBER 23 (92.0) 2 (8.0)

LMP-1 10 (40.0) 15 (60.0)

p16 11 (44.0) 14 (56.0)

p53 10 (40.0) 15 (60.0)

aPositive (O10% tumor cells).bNegative (#10% tumor cells).

234 Rosales-P�erez et al./ Archives of Medical Research 45 (2014) 229e236

all cases (92%) were positive for EBERs indicating thepresence of EBV-latent infected cells and among the 29cases, 25 (86.0%) were classified as type II/III, accordingto WHO tumor classification. The relative percentage ofO85% of II and III WHO NPC types in Mexico are com-parable to those of countries where the incidence of NPC ishigher (Asia, Singapore, Malaysia, Greenland) and diversefrom that in Caucasian individuals of Western countries(19e21). Our results are consistent with published dataabout epidemiology of NPC-EBV associated in highendemic areas.

With regard to clinical outcome our findings show, sur-prisingly, that OS was worse in patients with undifferenti-ated vs. differentiated carcinomas ( p 5 0.05). Perhapsthe fact that this observation is different from others thathave been published (22,23) may be related to our smallsample size. EBER positivity, as a surrogate for EBV pres-ence, was associated with an improved survival and clinicaloutcome in some studies (24,25). However, we did not findthis relationship. We also observed a worse prognosis inmales than in females ( p 5 0.03).

The Intergroup 0099 established the role for chemo-RTin the treatment of NPC, and multiple randomized phaseIII trials (26) have shown the benefit of chemo-RT. In ourstudy we confirm the superiority in OS of chemo-RTregimen of CDPP followed by adjuvant CDPP/5-FU vs.other treatment modalities ( p 5 0.001).

LMP1 is a transmembrane protein encoded by the LMP1gene, one of the EBV latent genes (27). Although EBV isassociated with 85e90% of NPC, the reported detectableexpression rate of LMP1 determined by current techniquesvaries from 50e80% (28). We observed LMP1 staining inonly 10/25 cases (40%). Failure to detect the protein inmore specimens may reflect biological heterogeneity ormay be due to limitations of the paraffin-embedded immu-nohistochemical method as has been reported by others(29,30). Some researchers showed that the invasion andmetastasis of NPC was promoted by LMP1 expressionand was inhibited when LMP1 expression was knocked-down by RNA interference in vitro and in vivo (31). Afew clinical studies with a small sample size have showna worse prognosis and survival between LMP1 (þ)

compared to LMP1 (�) expression (32,33), whereas othersargued that LMP1 is not associated with NPC prognosis(34,35). A meta-analysis (36) concluded that LMP1 expres-sion is positively associated with metastasis in NPC. Ourresults showed that LMP1-immunoexpression was morecommon in older patients (O50 vs. !50 years old, p 50.02) and in those with parapharyngeal space invasion(p 5 0.02); this latter finding may be associated with amore aggressive behavior and hence a poorer prognosis(37). We also observed that complete treatment tumorresponse was better in patients with negative LMP1 expres-sion ( p 5 0.06; NS). This finding is probably in line withstudies (38,39) that demonstrated enhancing radiosensi-tivity in LMP1 downregulation intracellular signal path-ways by genetic manipulation. We found no otherdifferences between LMP1 expression and OS or other clin-icopathological variables.

The p16 tumor suppressor gene at 9p21 is an inhibitor ofcell cycling by blocking the G1-S phase of the cell cycle.Protein p16 is frequently deleted, mutated, or methylatedin squamous cell carcinoma of the head and neck (40)and this loss has a prediction for poor clinical outcome inseveral tumors (41). The importance of methylation of thepromoter region of the p16 gene in oral and pharyngeal-laryngeal cancers has been recently published (42). Amongthe 25 NPC cases in our study, 14 (56.0%) were negativefor p16 protein immunoexpression. These finding areconsistent with reports published by others (43,44). Thenonexpression rate of p16 protein also correlated with thepresence of metastatic disease at diagnosis ( p 5 0.03)and development of distant recurrence disease ( p 50.006). We also observed that nonexpression of p16 proteincorrelated to a more aggressive clinical course with a short-er distance recurrence-free survival (DRFS, p 5 0.05). Thelink between nonexpression of p16 and metastatic potentialbehavior has been reported previously (44). We did not findthat inactivation of p16 appears to be a significant predictorfor poor OS in NPC patients.

Regarding p53 protein, we found immunoexpression inten cases (40.0%), and the frequency of positive malignantcells differed from case to case with heterogeneous stainingintensity. There was no relationship with p53 expressionwith clinical variables and OS.

235Epstein-Barr Virus Membrane Protein p16 and p53 in Nonendemic Nasopharyngeal Carcinoma

There is now strong evidence supporting a role of EBVencoded LMP-1 protein in the pathogenesis of NPC, butthere are also other molecular pathology variables thatcan predict clinical outcome in these patients. The abovedata suggest that LMP1 expression could be correlated witha poorer clinical outcome and prognosis and p16 gene inac-tivation may thus play an important role in the pathogenesisof NPC, especially in terms of its metastatic potential. Toour knowledge, this series is the first one published innonendemic/non-white population of NPC and supportsthe importance, like others reports, to explore the molecularsignaling pathways to provide a substantial opportunity foridentification of novel diagnostic and prognostic bio-markers that could improve individual treatment in patientswith NPC.

AcknowledgmentsWe thank Evangelina Figueroa Medina, M.Sc. for her valuablehelp in statistical analyses.

Conflicts of Interest

The authors declare no conflict of interest.

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