1

The relation between latent membrane protein 1 expressions in NPC

tumor tissue with survival

Cyprianus Murtono*, Sofia Mubarika**, Soeripto*** * Pathological Anatomy Department Faculty of Medicine, Atmajaya Catholic University, Jakarta

** Histology Department Faculty of Medicine, Gadjah Mada University, Yogyakarta

*** Pathological Anatomy Department Faculty of Medicine, Gadjah Mada University, Yogyakarta

ABSTRACT

Background: Latent membrane protein 1 (LMP1) is a product of oncogene, expressed by

EBV associated NPC. LMP1 was reported to have a role on the immune response through

activation of MHC class I and II. Purpose: To know the role of LMP1 expression on the survival

that take place sequentially through its relation with the molecules MHC class I (HLA A10,

HLA2A, beta 2-microglobuline) and II (HLA DR) and with the density of mononuclear cells

CD3, CD4, CD8, CD68, S100 in the tumor tissue. Methods: Thirty seven NPC cases were used

as research sample. The exclusive criterium was the absence of EBV in the tumor tissue. RISH

EBER was used to detect the EBV infection. Results: There were significance correlations

between the expression of LMP1 with the expressions of MHC class I HLA2A (T value: 2.28),

beta 2-microglobuline (T value: 1.88) and MHC class II HLA DR (T value: 3.00). The expression

of LMP1 in the tumor tissue was significancely correlated with the density of S100+ cells (T

value: 2.08). Conclusion: There was a sequential process between LMP1 expression, expression

of MHC class I (HLA2A, beta 2-microglobulin) and MHC class II HLA DR in tumor tissue, the

density of CD3+ and the survival. There was a direct correlation between LMP1 and the survival

and S100 density. Also between CD8 and survival.

Key words: LMP1, nasopharyngeal carcinoma, cytokines, proteases, survival

ABSTRAK

Latar belakang: LMP1 adalah satu produk gen yang memiliki sifat onkogenik yang

diekspresi oleh KNF yang terkait dengan infeksi EBV. LMP1 dilaporkan berperan dalam respons

imun melalui aktivasi MHC kelas I dan II. Tujuan: Untuk mengetahui pengaruh ekspresi LMP1

terhadap ketahanan hidup yang terjadi secara bertahap melalui hubungannya dengan molekul-

molekul MHC class I (HLA A10, HLA2A, beta 2-microglobuline) and II (HLA DR) dan melalui

kepadatan sel radang CD3, CD4, CD8, CD68 dan S100 di dalam jaringan tumor. Metode: Tiga

Research Report

2

puluh tujuh kasus NPC digunakan sebagai sampel penelitian. Kriteria eksklusif adalah tidak

adanya EBV dalam jaringan tumor. RISH EBER digunakan untuk pemeriksaan adanya infeksi

EBV. Pemeriksaan adanya protein di jaringan menggunakan metode immunohistokimiawi

dengan memakai antibodi monoklonal yang sesuai. Hasil: Ada korelasi yang signifikan antara

ekspresi LMP1 dengan ekspresi MHC kelas I HLA2A (T: 2,28), beta 2-mikroglobulin (T: 1,88),

dan MHC kelas II HLA DR (T: 3,00) juga antara MHC kelas I HLA2A dengan kepadatan sel

CD3+ (T: 2,24), antara kepadatan sel CD8+ dengan ketahanan hidup (T: 2,01) dan antara

ekspresi LMP1 dengan kepadatan sel S100+ (T: 2,08). Kesimpulan: Terdapat proses sekuensial

antara ekspresi LMP1 dengan ekspresi MHC kelas I (HLA2A, beta 2-mikroglobulin) dan MHC

kelas II HLA DR di jaringan tumor juga antara MHC kelas I HLA2A dengan kepadatan sel

CD3+. Terdapat korelasi langsung antara ekspresi LMP1 dengan ketahanan hidup dan dengan

kepadatan S100. Juga antara CD8 dengan ketahanan hidup.

Kata kunci: LMP1, karsinoma nasofaring, sitokin, enzim protease, ketahanan hidup

Alamat korespondensi: Cyprianus Murtono, Departemen Patologi Anatomi FK Universitas

Katolik Atmajaya, Jakarta. E-mail: murtono@hotmail.com

INTRODUCTION

Nasopharyngeal carcinoma (NPC)

is a malignancy of squamous epithelial

cell of the nasopharynx. On the basis of

the differentiation WHO classified NPC

in three types, namely type I

(keratinizing squamous carcinoma), type

II (non-keratinizing carcinoma) and type

III (undifferentiated carcinoma).1 NPC is

most frequent tumor in Yogyakarta. The

incidence of NPC is 4.95 per 100.000

population in man and 2.09 in women.2

Factors as race, genetic, geographic and

environment have roles on the

pathogenesis of NPC.1 Epstein Barr

virus (EBV) has a role on oncogenesis of

NPC since EBV genome is consistently

found in the tumor cells. The gene

products such as EBERs, EBNA1,

BARF 1, LMP2 and LMP1 expressed by

EBV associated NPC.3 Except LMP1,

the function of the gene products on

oncogenesis is not clear.

Tumor progression is a sequential

process whereby many factors are

involved. LMP1, which has an

oncogenic property has been suggested

to be associated with tumor progression,

since there was an orderly progression

from pre-neoplastic lesion to invasive

3

cancer.4 Latent membrane protein 1

(LMP1) was reported to induce factors

of invasion and metastasis by inducing

growth factors, proteases and adhesion

molecules.5 Reports about the interaction

between these factors were still

controversial.6,7

Some antigens mutated or lost and

genetically tumor is not stable. Certain

cancers lost molecules, especially the

MHC class I. CTL did not recognize

cells if the cells lost the MHC class I

expression, even though NK cells did it

well. IFN-gamma enhanced T cell to

increase MHC class I production but

MHC class II did not increased,8

although another study found inversely.9

Reports about the role of expression of

MHC class I beta 2-microglobulin in

NPC tissue were still varies. All NPC

expressed beta 2-microglobulin,10

but

there is no correlation between EBER or

LMP1 expression in tumor tissue with

the expression of beta 2-microglobulin.

There is increase of beta 2-

microglobulin in serum level compared

with the control.11

The level decreased

after radiotherapy.

NPC is characterized by dense

infiltration of mononuclear cells as

immune reaction against tumor tissue.

The proteins are found on the cell

surface and recognized by immune

system cells and enhance immune

response. Tumor infiltrating

lymphocytes (TIL) in NPC type 3 was

reported as in activated status and this

response occurred due IFN-gamma

stimulation against the tumor cell.12

Contrary CD8 cell infiltration is

predominant in the LMP1 expressing

tumor cells but this CD8 cells express

very low CD25 and TIA-1, indicating

that these cells neither activated nor

having cytotoxic properties since the

apoptotic index is very low.13

There were no unanimity reports

about the role of Granzyme B and IFN

gamma expression on the tumor

progression. The density of Granzyme B

(+) cells in Granzyme B(+) gastric

cancer is significancely lower than in

Granzyme B (-) gastric cancer.14

In lung

and breast cancer the percentage of

positive perforine and Granzyme B

expression tumor cells is inversely

correlated with regional metastasis so

that both molecules seemed has role on

suppression to nodal metastasis.15 On the

other side the existence of many

Granzyme B+ lymphocytes is a strong

parameter for fatal clinical outcome of

4

NPC that independent from stadium.16

The function of IFN gamma is to

enhance proliferation and activity of B

cell, T cell, NK cell and macrophages.

IFN-gamma is a strong macrophage

activator and caused increased of

microbicid and cytotoxic of macrophage

that also enhance cytokine production.17

The present study shall uncover the

relation of LMP1 expression on the

survival that take place sequentially

through its relation with the molecules

MHC class I (HLA A10, HLA2A, beta

2-microglobuline) and II (HLA DR) and

with the density of mononuclear cells

CD3, CD4, CD8, CD68, S100 in the

tumor tissue.

The aims of the research are to find

out: 1) Is there a correlation between the

expression of LMP1 of the tumor tissue

and the survival of the NPC patients? 2)

Is there a correlation between the

expression of LMP1 in the tumor tissue

and the survival that take place

sequentially through the expression of

MHC class I namely HLA 10, HLA2A,

HLA beta 2-microglobulin and through

MHC class II (HLA DR) in the tumor

tissue? 3) Is there correlation between

the expression of LMP1 in the tumor

tissue with the survival that take place

sequentially through the expression of

MHC class I, namely HLA 10, HLA2A,

HLA beta 2-microglobulin and MHC

class II (HLA DR) and through the dens

mononuclear inflammatory cells CD3+,

CD4+, CD8+, CD68+ and S100+ in the

tumor tissue? 4). Is there a correlation

between the expression of LMP1 in the

tumor tissue with the survival that take

place sequentially through the density of

the mononuclear CD3+, CD4+, CD8+,

CD68+ and S100+ in the tumor tissue?

METHODS

The population as subject of the

study was the NPC patients from the

region of Yogyakarta and nearby. Thirty

seven NPC patients from Sardjito

Hospital were used as research sample.

Hematoxyllin Eosin was used

confirm the diagnosis. To know is there

association between tumor and EBV

infection RISH study for EBER was

performed using PNA probe with

protocol of Pathological Anatomy of

Vrij Universiteit Medical Center,

Amsterdam. The protocols from the

same Medical Center were used to do

other IHC staining. For the study the

expression of EBNA1 ARS was done

with microwave oven in citric buffer

5

solution (pH: 6.0); the duration is 4.5

minutes in 100% wattage and later on 10

minutes in 30% wattage. OT1X was

used as monoclonal antibody. The

duration of incubation for EBNA1 was 1

hour with concentration 1:100. Envision

was used in this staining. The expression

of LMP1 in the tumor tissue was

detected with monoclonal antibody S-12

(dilution 1:400) after ARS with TRIS

buffer (pH 9.0) in autoclave for 50

minutes. IHC staining was performed by

automatic staining machine Ventana.

To recognize T cell, T helper,

Cytotoxic T cell, Macrophage, Natural

Killer cells, Dendritic Reticular cells,

Granzyme B positive cells and IFN

gamma positive cells. IHC stainings

were performed using monoclonal

antibodies against CD3, CD4, CD8,

CD56, CD68, S100, Granzyme B and

IFN gamma. The density of each cell

determined with the number of cells with

positive IHC staining using relevant

monoclonal antibody in 10 separated

fields and each field is 1 mm2. The value

is the average number of the 10

microscopic fields.18

The correlation between the

expression of LMP1 and the survival

through the relations between the

expression of LMP1 with the

expressions of MHC class I (HLA 10,

HLA2A, and beta 2-microglobuline) and

II (HLA DR) and through the density of

the CD3, CD4, CD8, CD68, S100 cells

was determined with LISREL 8.50

program. T value >1.96 was declared as

significance. The segment in the path

diagram with significance correlation

determined a causal relationship between

the variables.

RESULT

The genes product of EBV (EBER,

EBNA1, LMP1)

The result of

immunohistochemistry study of EBER,

EBNA1 and LMP1 on the research

sample (37 cases) is shown in table 1.

EBER was expressed in all samples

which revealed that NPC is consistently

associated with EBV infection. EBER

expression was very intense and

homogenous while in the rest the

expression was heterogenous or weak.

The strong EBER expression is shown in

picture 1. EBNA1 was positive in 29 of

37 samples (74%). LMP1 expression

was detected in 20 of 37 specimens

(54.5%). Picture 2 showed the

expression of LMP1.

6

Tabel 1. Gene product of EBV (EBER, EBNA1 and LMP1) of the NPC patients

________________________________________________________________________

Gene product number of cases of % of positivity Number of cases

Expression strength

0 1 2

EBER 0 17 20 100% 37

EBNA1 8 11 18 74% 37

LMP1 17 16 4 54.5% 37

Immunohistochemistry staining

for CD3, CD4, CD8, D68, S100,

Granzyme B and IFN gamma. The

result of the IHC staining for these cells

can be seen in table 2. It has been shown

the number of specimen with tumor

tissue. Also the maximal, minimal and

mean numbers of positive cells per mm2.

The study showed that the most infiltrate

cells was CD3+.

Table 2. The result of IHC staining for CD3, CD4, CD8, CD68 and S100

Cell type N Minimum Maximum Mean Std.deviasi

CD3+ 32 0 120 25.09 32.09

CD4+ 25 0 24 5.12 5.48

CD8+ 28 0 80 16.54 24.55

CD68+ 31 1 43 18.03 13.56

S100+ 32 0 50 11.25 13.01

Picture 1. Strong EBER expression. The arrow

head shows the nuclei of tumor cells with

homogenous, very intense EBER expression.

Picture 2. LMP1 expression of the NPC tissue.

The arrow head shows the cytoplasm and cell

membran of tumor cells with homogenous, very

intense LMP1 expression.

7

The relation between LMP1

expression with the expression of

MHC class I (HLA10, HLA2A, beta 2-

microglobulin), MHC class II (HLA

DR) and the density of mononuclear

cells (CD3, CD4, CD8, CD68, S 100) in

the tumor tissue.

Statistical analysis of the relation

between the LMP1 expression in the

tumor tissue with the survival either

directly or through the expression of

MHC class I (HLA10, HLA2A, beta 2-

microglobulin) and II (HLA DR) and the

density of CD3, CD4, CD8, CD68, S100

cells in the tumor tissue can be seen in

table 3 and 4. In table 3 could be seen

that there was a significance correlation

between the expression of LMP1 of the

tumor tissue with the survival (T value:

2.61), with the expression of HLA2A of

the tumor (T value: 2.28) and HLA DR

(T value: 3.00). There was also a

significance correlation between the

expression of LMP1 with the density of

S100 (T value: 2.08).

There were non-significance

correlations between LMP1 with the

expression of HLA beta 2-microglobulin

(T value: 1.88) and between HLA2-

microglobulin with the density of CD4

(T value: 1.81). However these

correlations are not significance. It is not

impossible that the correlation should be

significance if the number of samples is

higher. The density of CD8 correlated

significancely with the survival (T value:

2.01). It was shown in table 4 that the

density of CD3 significancely correlated

with HLA2A (T value: 2.24).

Table 3. T value from the path analysis LMP1-MHC class I and II mononuclear cell

density- the survival

LMP1 survival

Path coef T-value Path coeff T-value

HLA10 0.38 12.90 1.60

HLA2A 2.28 5.55 0.67

Beta 2-micro 1.88 -0.30 -0.036

HLA DR 3.00 -24.55* -2.65

CD3+density -1.16* -0.13 -0.19* -1.03

CD 4+ density -2.41 -1.65 -0.14* 0.098

CD8+ density -4.04 -0.52 +0.45* 2.01

CD68+ density -0.94 -0.22 -0.46 -1.13

8

S100+ density 7.32 2.08 +0.15 0.31

LMP1 25.51 2.61

Note: T value < 1.96: Not significance

> 1.96: Significance (bold)

Table 4. Path coefficients and T value between MHC class I and II with the density of

mononuclear cell

HLA10 HLA2A Beta 2-micro HLA DR

I II I II I II I II

CD3+ density 1.67 0.29 16.56 2.24 1.69 0.22 18.41 -2.37

CD4+ density -3.95 -4.03 1.51 1.23 2.35 1.81 1.17 0.90

CD8+ density 1.13 0.22 3.23 0.48 -0.0062 -0.00089 -6.93* -1.00

CD68+ density -1.53 -0.53 1.06 0.28 2.98 0.78 0.44 0.12

S100+ density 0.028 0.012 -2.94 -0.99 1.42 0.45 -8.05* -2.55

Note: 1. Column I: Path coefficient

Column II: T value

2. T value <1.96: Not significance

>1.96: Significance (bold)

Expression of LMP1 2.61

survival

2.08

2.28 2.01

3.00

Density of mononucl.cells

HLA10, beta 2-micro CD8+

HLA2A

HLADR 2.24 S100+

Sel CD3+ Note: : Not significance

: Significance

9

Picture 3. Paths that have a significance role in the Path diagram LMP1 expression with survival through

the expression of MHC class I and II and the density of mononuclear CD3+, CD4+, CD8+, CD68+ and

S100 cells.

DISCUSSION

LMP1 is an oncogene product that

has a role on cytokine regulation in NPC

which is characterized with dens

mononuclear inflammatory cells. LMP1

and MHC class I and II was reported are

able to influence the recruitment and

function of these inflammatory cells in

the tumor tissue and lateron influence

the survival.9,11,19

Since the dense

mononuclear inflammatory cell is

characteristic in NPC and its mechanism

takes place in the tumor progression so it

rise a question whether LMP1 as an

oncogene product has a role in the

survival of the NPC patients through its

influence on the expression of MHC

class I and II in the tumor tissue that

influence the recruitment of dens

mononuclear inflammatory cells.8,12

It

was shown by this study that there was a

significance correlation between the

LMP1 expression with the survival (T

value: 2.61) and that means there is a

significance role LMP1 on the survival

of the NPC patient. Literature studies on

the role of LMP1 on the survival are still

a controversial one.

On one side LMP1 has a

imunosupresif property against the

lymphocytes in the tumor tissue.20

On

the other side LMP1 (+) NPC growth is

faster, more expansif with a better

prognosis.21

The outcome of patients

LMP1 (-) NPC and weak TCR is poor.22

Statement of Hu (1995) was based on

the facts that NPC grows on an

immunocompetent individu that means

the tumor cells escape from

immunosurveillance system. NPC

patients showed an increased titer of

LMP1 compared with a normal EBV

carrier and this increase took place

before any symptoms appear. LMP1

could change the non-immunogenic S6C

mammary tumor cell line into an

immunogenic one with rejection

capability. It was shown that immune

surveillance could be hindered by

mutation immunogenic LMP1 gene

epitope into non-immunogenic one in

LMP1 (+) NPC. Another method is to

down regulate the immunogenic LMP1

by promoter methylation. If mutation

and down regulation could hinder LMP1

dependent immunogenecity it will be

10

analogically relevant to compare

between LMP1 (+) isolate from NPC

with LMP1 (-) one. It appeared that

LMP1 (-) isolate mutated in lower

frequency but more immunogenic than

LMP1 (+) NPC which is highly mutated

but less immunogenic.

This study showed that the density

of CD8 correlated with the survival (T

value: 2.01). Literature study showed

controversies about the role and function

of CD8 cells in tumor tissue. It was

noted that CD8+ cells are activated

lymphocytes and work on the tumor

cells due the increase of IFN-gamma+

and CD25+ cells.12

TIL of NPC can

lyses C15 NPC tumor cells. If the

activation path paralysed the TIL

cytolytic potency is still exist.23

This

finding is conform with prior report that

lymphocytes infiltration was denser in

metastasized tumor and that the cell

density correlated with the survival.19

Contrary CTL was not activated cells

and the dens CTL infiltration in

LMP1(+) NPC was not cytotoxic against

the tumor since the apoptosis grade is

low.13 A support finding is that IFN-

gamma level in NPC was very low

compared with healthy or in remission

people.24

Other reported that a poor

outcome correlates with the density of

CD8+16

and the increase of CD8+ had

no influence on the survival.25 In was

shown in this study that there was

increase of IFN-gamma that has a role

on the survival, so that the increase of

IFN-gamma has a significance role on

this process.

This study noted that there was a

significance correlation between the

LMP1 expression in the tumor tissue

with the expression of MHC class I

HLA2A (T value: 2.28) and MHC class

II HLA DR (T value: 3.00). The relation

between the LMP1 expression in the

tumor tissue with MHC class 1 beta 2-

microglobulin was high but not

significance (T value: 1.88). This value

could be higher with more cases

involved in this study. The T-values of

the correlations showed a significance

correlation between the LMP1

expression with MHC class I and II.

There was an increase of CD8+ cells in

tumor tissue and a significance

correlation between the densities of

CD8+ cells with the survival of NPC

patients. The increase of expression of

MHC class I and II in virus infected cells

increases the antigen recognizing and

cell destruction. This can explain how

11

the LMP1 expression correlated with the

survival in accordance the increase of

CD8 cells and correlation between CD8

cells and the survival of the NPC

patients.

One finding of the study was a

significance correlation between the

expression of HLA2A with the density

of CD3+ cells (T value: 2.24). This

study also showed CD3 had highest

density compared with the other

inflammatory cells which is conform

with previous report, since the function

of CD3 that is associated with TCR and

is needed for superficial TCR expression

and its signal transduction.23

This study showed which path is

responsible in the path diagram LMP1

expression- the survival through the

expression of MHC class I and II and

through the density of mononuclear cells

could be seen in picture 3. This picture

explains summarize that LMP1

significancely corelated with the survival

of the NPC patients. This correlation

was caused by immune escape

mechanism of the tumor cells of the

immunosurveilance system that take

place during tumor progression either

due to selective down regulation of

LMP1 expression, methylation of LMP1

promoter sekuens or LMP1 mutation of

LMP1+ tumor. Beside this, LMP1

mutation decrease immunogenic

property of tumor cells and improved

tumor growth. This study also confirmed

the significance correlation between

CD8 expressions with the survival.

Cytotoxic role of CD8 still active CD8 is

activated lymphocytes that directed to

the tumor cells and this fact is based on

the increase of IFN-gamma.12

Considering that TIL from NPC is able

to lysis NPC tumor cells (C15) revealed

that even though the activation path is

paralyzed the cytolitic potency of TIL

that proliferate in the tumor tissue still

active.23

In conclusion of this study, there

was a significance correlation between

the LMP1 expression of the tumor tissue

with the survival, so that the higher

LMP1 expression the better the survival.

There was also a significance correlation

between LMP1 expression with the

survival that take place sequentially

through the expression of HLA2A, beta

2-microglobulin and HLA DR of the

tumor tissue since there was a

significance correlations between LMP1

expression of the tumor tissue with the

expression of either HLA2A, beta 2-

12

microglobulin or MHC class II HLA DR

of the tumor tissue. The relations

between the expression of LMP1 and

survival did not take place through the

density of CD4+, CD8+ and CD68+

cells in the tumor tissue. There was a

correlation between the expression of

LMP1 of the tumor cells with either the

density of S100+ cells or the expression

of MHC class I HLA2A in the tissue

which was correlated with the density of

CD3+ cells. This meant that there was a

sequential relationship between the

expression of LMP1 with the density of

CD3+ cells in the tumor tissue through

MHC class I and density of S100+ cells

but the density of CD3+ and S100+ were

not correlated with the survival. The

study also showed that the relation

between LMP1 with the survival

sequentially was not caused by the

density of CD4+, CD8+ and CD68+

cells in the tumor tissue. This study

confirmed the significance correlation

between the density of CD8+ cells with

the survival that mean CD8+ cells

influence the survival but it could not be

shown that sequentially caused by

LMP1.

REFERENCES

1. Shanmugaratnam K, Sobin LH.

Nasopharyngeal carcinoma.

International histological classification

of tumors No.19. Geneva: WHO; 1978.

p. 13-15.

2. Soeripto, Dachlan Z, Pradjatmo H,

Tirtoprodjo P, Widyo K. Study on

population based cancer registry in the

special region of Yogyakarta. Research

report. Ministry of Health of Republic of

Indonesia, Jakarta.1988.

3. Brooks L, Yao QY, Rickinson AB,

Young LS. Epstein Barr virus latent

genaes transcription in nasopharyngeal

carcinoma cells: coexpression of

EBNA1, LMP1 and LMP2 transcript. J

Virol 1992; 66:2689-97.

4. Pathmanathan R., Prasad U, Sadler R,

Flynn K, Raab-Traub N. Clonal

proliferations of cells infected with

Epstein-Barr virus in preinvasive lesions

related to nasopharyngeal carcinoma. N

Engl J Med 1995; 333(11):693-8.

5. Yoshizaki T. Promotion of metastasis in

nasopharyngeal carcinoma by Epstein

Barr virus latent membrane protein-1.

Histol Histopathol 2002; 17:845-50.

6. Murono S, Inoue H, Tanabe T, Joab I,

Yoshizaki T, Furukawa M, et al.

Induction of cyclooxygenase-2 by

Epstein-Barr virus latent membrane

protein-1 is involved in vascular

endothelial growth factor production in

13

nasopharyngeal carcinoma cells. Proc

Natl Acad Sci 2001; 98(12):6905-10.

7. Yoshizaki T, Horikawa T, Qing-Chun

R, Wakisaka N, Takeshita H, Sheen TS,

et al. Induction of interleukin 8 by

Epstein Barr virus latent protein-1 and

its correlation to angiogenaesis in

nasopharyngeal carcinoma. Clin Cancer

Res 2001; 7(7):1946-51.

8. Tizard IA. Immunology an introduction

4th. Philadelphia: WB Saunders Co;

1996.

9. Oppenheim JJ, Ruscetti FW. Medical

immunology. In: Parslow TG, Stites DP,

Terr AI, Imboden JB, eds. Lange

medical. 10th ed. New York: McGraw-

Hill; 2001.

10. Kouvidou C, Rontogianni D, Tzardi M,

Datseris G, Panayiotides I, Darivianaki

K, et al. Beta 2-microglobulin and HLA-

DR expression in relation to the

presence of Epstein-Barr virus in

nasopharyngeal carcinomas. Patholbiol

1995; 63(6):320-7.

11. Lee JK, Tsai SC, Hsieh JF, Ho YJ, Sun

SS, Kao CH. Beta 2-microglobulin beta

2M) as a tumor marker in

nasopharyngeal carcinoma. Anticancer

Res 2000; 20(6C):4765-8.

12. Tang KF, Chan SH, Loh KS, Chong

SM, Wang D, Yeoh KH, et al. Increased

production of interferon-gamma by

tumor infiltrating T lymphocytes in

nasopharyngeal carcinoma: indicative of

an activated status. Cancer Lett 1999;

140(1-2):93-8.

13. Shao JY, Ernberg I, Biberfeld P, Heiden

T, Zeng YX, Hu LF. Epstein Barr virus

LMP1 status in relation to apoptosis,

p53 expression and leucocytes

infiltration in nasopharyngeal

carcinoma. Anticancer Res 2004;

24(4):2309-18.

14. Zhou Y, Shi D. Effect of GrB- positive

expression in benign and malignant

gastric epithelial tissue on local immune

response. Zhonghua Yi Xue Za Zhi

2002; 82(14):966-9.

15. Kontani K, Sawai S, Hanaoka J, Tezuka

N, Inoue S, Fujino S. Involvement of

granzyme B and perforin in suppressing

nodal metastasis of cancer cells in breast

and lung cancers. Eur J Surg Oncol

2001; 27(2):180-6.

16. Oudejans JJ, Harijadi H, Kummer JA,

Tan IB, Bloemena E, Middeldorp JM, et

al. High numbers of granzyme B/CD8-

positive tumor-infiltrating lymphocytes

in nasopharyngeal carcinoma biopsies

predict rapid fatal outcome in patients

treated with curative intent. J Pathol

2002; 198(4):468-75.

17. Oppenheim JJ, Ruscetti FW, Taltynek

C. Cytokine. In: Stites DP, Terr AI,

Parslow TG, eds. Basic and clinical

immunology. 8th ed. Connecticut,

Stamford: Lange medical; 1994. p. 116.

14

18. Cassanova JF, Torres A, Martinez-

Garcia, Hardisson D, Nistal M,

Regadera J. Statistical consideration in

the methodology of quantifying

immunocompetent cellos in tumors. Ann

Quant Cytol Histol 1999; 21:227-34.

19. Jayasurya A, Bay BH, Yap WM, Tan

NG. Lymphocytic infiltration in

undefferentiated nasopharyngeal

carcinoma. Arch Otolaryngol Head

Neck Surg 2000; 126(11):1329-32.

20. Dukers DF, Meij P, Vervoort MB, Vos

W, Scheper RJ, Meijer CJ, et al. Direct

immunosuppressive effects of EBV-

encoded latent membrane protein 1. J

Immunol 2000; 165(2):663-70.

21. Hu LF, Chen F, Zhen QF, Zhang YW,

Luo Y, Zheng X, et al. Differences in

the growth pattern and clinical course of

EBV-LMP1 expressing and non-

expressing nasopharyngeal carcinomas.

Eur J Cancer 1995; 31A(5):658-60.

22. Laytragoon-Lewin N, Porwit-

MacDonald A, Mellstedt H, Lewin F.

Alteration of cellular mediated

cytotoxicity, T cell receptor zeta (TcR

zeta) and apoptosis related genae

expression in nasopharyngeal carcinoma

(NPC) patients: possible clinical

relevance. Anticancer Res 2000;

20(2B):1093-100.

23. Ferradini L, Miescher S, Stoeck M,

Busson P, Barras C, Cerf-Bensussan N,

et al. Cytotoxic potential despite

impaired activation pathways in T

lymphocytes infiltrating nasopharyngeal

carcinoma. Int J Cancer 1991;

47(3):362-70.

24. Zheng Y, Cao KY, Chua DT, Sham JS,

Kwong DL, Ng MH, et al.

Complementary activation of peripheral

natural killer cell immunity in

nasopharyngeal carcinoma. Cancer Sci

2006; 97(9):912-9.

25. Gallo O, Bianchi S, Giannini A, Gallina

E, Libonati GA, Fini-Storchi O.

Correlations between histopathological

and biological findings in

nasopharyngeal carcinoma and its

prognostic significance. Laryngoscope

1991; 101(5):487-93.