This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Leukemia Research 33 (2009) 1627–1635

Contents lists available at ScienceDirect

Leukemia Research

journa l homepage: www.e lsev ier .com/ locate / leukres

Pronostic significance of angiogenic/lymphangiogenic, anti-apoptotic,inflammatory and viral factors in 88 cases with diffuse large B celllymphoma and review of the literature�

Semra Paydas a,∗, Melek Ergin b, Gulsah Seydaoglu c, Seyda Erdogan b, Sinan Yavuz a

a Department of Oncology, Cukurova University Faculty of Medicine, 01330 Adana, Turkeyb Department of Pathology, Cukurova University Faculty of Medicine, 01330 Adana, Turkeyc Department of Biostatistics, Cukurova University Faculty of Medicine, 01330 Adana, Turkey

a r t i c l e i n f o

Article history:Received 11 January 2009Received in revised form 11 February 2009Accepted 14 February 2009Available online 16 March 2009

Keywords:LymphomaVEGF-AVEGF-CCox-2TSP-1SurvivinEBVPrognosisAngiogenesisLymphangiogenesis

a b s t r a c t

Background: Diffuse large B cell lymphoma (DLBCL) is the most common subtype of Non-Hodgkinslymphomas (NHL). The outcome of these patients shows a wide variation. We evaluated the effectof six biologic parameters including Cyclooxygenase-2 (Cox-2), Survivin, Epstein Barr Virus (EBV),Vascular Endothelial Growth Factor-A (VEGF-A), Vascular Endothelial Growth Factor-C (VEGF-C),Thrombospondin-1 (TSP-1) and clinical parameters.Patients and methods: A follow up study was conducted and 88 cases with DLBCL were included in thestudy. The data of 72 patients were eligible for the survival analyses. Immunohistochemistry was used todetect these parameters.Results: The ratio of positive cases for Cox-2, VEGF-A, Survivin, VEGF-C, EBV, TSP-1 were 71.6%, 64.8%,60.2%, 36.4%, 21.6%, 14.8%, respectively. Survivin (+) cases showed higher LDH levels and VEGF-A (+) casesshowed higher beta 2 microglobulin (B2M) levels compared with (−) cases. Mean survival rates werefound to be significantly shorter in cases expressing VEGF-A, VEGF-C, EBV and Survivin than cases notexpressing these. EBV expression (HR: 3.78; 95%CI: 1.47–9.74; p = 0.006), VEGF-C expression (HR: 3.22;95%CI: 1.07–9.68; p = 0.037) and extranodal involvement (HR: 3.02; 95%CI: 1.01–8.97; p = 0.047) werefound to be independent risk factors for prognosis according to the Cox regression analysis.Comment: Lymphangiogenesis (VEGF-C) and EBV related viral lymphomagenesis have been found to berelated with prognosis in DLBCL patients.

© 2009 Elsevier Ltd. All rights reserved.

1. Introduction

NHLs are a group of heterogeneous malignant disorders. DLBCLis the most common subtype and accounts for approximately35–40% of the cases. In this subgroup, there are different clin-ical entities showing different biology and clinical outcomes.Although characteristic morphological, immunophenotypic, cyto-genetic and clinical features have been recognized, DLBCL hasdifferent subgroups showing different outcome clinically, morpho-logically (centroblastic, immunoblastic, anaplastic, plasmablastic,primary mediastinal large B cell lymphoma, primary effusion lym-phoma, intravascular large B cell lymphoma etc) and genetically(Germinal center B cell like (GCB), activated B cell like (ABC) andtype III DLBCL). Classification of NHLs, especially micro array and

� This study has been presented at LLM meeting October 16–18, 2008 New York.∗ Corresponding author. Tel.: +90 322 3386060; fax: +90 322 3386153.

E-mail address: sepay@cu.edu.tr (S. Paydas).

other developing technologies will be more informative to deter-mine the biology of these entities [1]. There are many studiesdetermining the prognostic and predictive value of various biologicparameters in this heterogeneous entity. So far, several biologicmarkers have been reported, some of which give prognostic andsome predictive information about the biology of these differentsubgroups. In this paper we studied angiogenic, lymphangiogenic,anti-apoptotic, inflammatory and viral markers in DLBCL.

In this study VEGF-A and VEGF-C as angiogenic and lymphangio-genic, Survivin as anti-apoptotic, Cox-2 for inflammation and EBVfor viral lymphomagenesis and TSP-1 have been studied and theprognostic importance of these have been compared with classicalprognostic factors including age, stage, B symptom, internationalprognostic index (IPI), extranodal involvement, lactic dehydroge-nase (LDH), B2M and overall survival (OS).

2. Patients and methods

Patients were evaluated with standard methods including history, physicalexamination and biochemical–hematological tests. Ann Arbor Staging System was

0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.leukres.2009.02.015

Author's personal copy

1628 S. Paydas et al. / Leukemia Research 33 (2009) 1627–1635

Table 1Distributions of biological parameters.

n (%)

COX-2 Negative 25(28.4)Positive 63(71.6)

TSP-1 Negative 75(85.2)Positive 13(14.8)

EBV Negative 69(78.4)Positive 19(21.6)

SURVIVIN Negative 35(39.8)Positive 53(60.2)

VEGF-A Negative 31(35.2)Positive 57(64.8)

VEGF-C Negative 56(63.6)Positive 32(36.4)

Table 2Distributions of combination of biological parameters.

Parameter Both (+) Both (−) Either one +

EBV/Survivin 32 40 16EBV/VEGF-A 28 44 16EBV/VEGF-C 47 31 10EBV/TSP1 59 26 3EBV/Cox2 22 50 16VEGF-A/Cox2 13 30 45VEGF-A/Survivin 18 30 40VEGF-A/TSP1 28 50 10VEGF-A/VEGF-C 30 27 31VEGF-C/Survivin 25 41 22VEGF-C/Cox2 18 45 25VEGF-C/TSP1 51 29 8Survivin/Cox2 12 36 40Survivin/TSP1 32 46 10TSP-1/Cox2 21 58 9

used to determine the disease status of the patients. Biopsy samples were re-evaluated according to the WHO classification. VEGF-A, VEGF-C, Cox-2, TSP-1,EBV-LMP-1 and Survivin were studied by immunohistochemistry. Originally, 177cases with NHL including low and high grade lymphomas were evaluated for sixparameters; each parameter was published in whole group [2–5]. In this study,88 cases with DLBCL were evaluated for all of these six biologic parameters. Five-micron-thick sections were cut from formalin-fixed and paraffin-embedded tissueblocks, dewaxed and hydrated. Usual immunohistochemical staining procedureswere applied to the samples for every biological parameter. Sections were incu-bated with Survivin (Santa Cruz Biotechnology Inc. cat# sc-17779 Positive control:tonsil staining pattern: nuclear), TSP-1 (Thrombospondine-1: Novacastra Cat # NCL-TSP1 Positive control: placenta, staining pattern: cytoplasmic), VEGF-A (VEGF (A-20)Santa Cruz Biotechnology Inc. cat# sc-150 Positive control: tonsil, staining pat-tern: cytoplasmic), VEGF-C (VEGF-C Zymed Laboratories Inc. cat# 18-2255 Positivecontrol: colon carcinoma, staining pattern: cytoplasmic), EBV-LMP1 (Epstein-Barrvirus-latent membrane protein, Novacastra NCL-EBV-CS1- Positive control: Hodgkindisease, staining pattern: cytoplasmic), and Cox-2 (Cyclooxygenase-2 NovacastraNCL-COX-2 Positive control: Ulcerative colitis staining pattern: H score:). Cytoplas-mic staining for EBV, LMP1, VEGF-A, VEGF-C, TSP-1 and nuclear staining for Survivinat the tumor cells were considered as positive. Immunohistochemical Score (IHS)was determined by combining an estimate of the ratio (%) of immunoreactive cells(quantity score) with an estimate of the staining intensity (intensity score) for Cox-2. The details of staining steps and evaluation parameters were explained in ourprevious papers [2–5].

3. Statistics

The results are reported as mean ± SD or n and percentages (%)ranges. The categorical variables between the groups were analyzedby using the Chi square test or Fisher’s exact test Comparisons wereapplied using the student t test. The predictors of survival were ana-lyzed by the Kaplan–Meier method and compared by the Mantellog-rank test. Overall Survival (OS) was defined as the time intervalbetween diagnosis and death of any cause related to lymphoma orcensoring. Censoring was defined as being alive at the last follow- Ta

ble

3C

orre

lati

onbe

twee

ncl

inic

alan

dbi

olog

ical

par

amet

ers.

COX

-2TS

PEB

VSu

rviv

inV

EGF-

AV

EGF-

C

Neg

ativ

ePo

siti

veN

egat

ive

Posi

tive

Neg

ativ

ePo

siti

veN

egat

ive

Posi

tive

Neg

ativ

ePo

siti

vN

egat

ive

Posi

tive

Sex

(mal

e/fe

mal

e)15

/10

34/2

943

/32

6/7

38/3

111

/822

/13

27/2

617

/14

32/2

529

/27

20/1

2PS

(goo

d/p

oor)

19/6

40/1

852

/18

7/6

48/

1612

/826

/733

/17

22/8

37/1

638

/17

21/7

BSy

mpt

om(n

o/ye

s)5/

2014

/45

17/5

42/

1116

/49

3/16

6/28

13/3

77/

2312

/42

13/4

26/

23EN

inv

(no/

yes)

5/20

22/3

925

/48

2/11

23/4

44/

159/

2518

/34

9/22

18/3

721

/35

6/24

Hep

atom

egal

y(n

o/ye

s)21

/44

9/9

60/1

010

/353

/11

17/2

28/5

42/8

26/4

44/

94

8/7

22/6

Sple

nom

egal

y(n

o/ye

s)18

/745

/13

52/1

811

/247

/17

16/3

25/8

38/1

225

/238

/15

41/1

422

/6LA

P(n

o/ye

s)11

/14

20/3

826

/44

5/8

27/3

74/

1517

/16*

14/3

6*15

/15

16/3

722

/33

9/19

An

emia

(no/

yes)

10/1

425

/32

30/3

85/

828

/24

7/12

13/1

822

/28

12/1

623

/30

19/3

416

/12

Leu

kop

enia

(no/

yes)

23/1

51/6

61/7

13/0

58/4

16/3

30/1

44/

624

/450

/346

/7*

28/0

*

Thro

mbo

cyop

enia

(no/

yes)

22/2

55/2

65/3

12/1

60/2

17/2

29/2

48/

226

/251

/250

/327

/1LD

H(n

orm

al/h

igh

)4/

2015

/39

16/4

93/

1018

/41*

1/8*

8/22

11/3

76/

2113

/38

14/3

65/

23B

2M(n

orm

al/h

igh

)11

/921

/31

27/3

25/

827

/85/

1217

/13

15/2

717

/9*

15/3

1*23

/23

9/17

Alb

um

in(n

orm

al/l

ow)

16/7

37/1

943

/23

10/3

41/2

012

/618

/13

35/1

315

/13*

38/1

3*31

/21*

22/5

*

RF

(nor

mal

/abn

orm

al)

21/3

49/

759

/811

/251

/10

19/0

26/5

44/

525

/345

/743

/927

/1R

elap

se(n

o/ye

s)3/

710

/16

12/9

1/4

11/1

62/

78/

85/

156/

117/

1211

/62/

7IP

I(lo

w/h

igh

)15

/34

10/2

44

4/5

26/8

42/7

22/1

2*23

/26

10/2

420

/29

10/2

435

/14

20/1

4St

age

(Ear

ly/A

dva

nce

d)

15/1

033

/27

40/3

28/

539

/27

9/10

22/1

226

/25

20/1

028

/27

31/2

417

/13

Age

**51

.0±

17.5

54.8

±14

.753

.0±

15.4

57.3

±16

.852

.6±

16.3

57.5

±12

.24

8.3

±15

.457

.2±

14.8

*50

.6±

16.8

55.4

±14

.853

.2±

15.4

54.7

±16

.1

Abb

revi

atio

ns:

PS:

per

form

ance

stat

us

(goo

d:

1–2;

poo

r:3–

4),S

x:sy

mpt

om,E

N:

extr

anod

al(n

o:th

ere

isn

oEN

invo

lvem

ent,

yes:

ther

eis

ENin

volv

emen

t),L

AP:

lym

ph

aden

opat

hy,C

T:ch

emot

her

apy,

RF:

ren

alfu

nct

ion

,IPI

:in

tern

atio

nal

pro

gnos

tic

ind

ex(l

ow:

0–1–

2,h

igh

:3–

4)*

p<

0.05

,bet

wee

nn

egat

ive

and

pos

itiv

egr

oup

s.**

Dat

aex

pre

ssed

asm

ean

±SD

.

Author's personal copy

S. Paydas et al. / Leukemia Research 33 (2009) 1627–1635 1629

Fig. 1. (a) Survival curve according to the Cox-2 expression. (b) Survival curve according to the TSP-1 expression. (c) Survival curve according to the EBV expression. (d)Survival curve according to the Survivin expression. (e) Survival curve according to the VEGF-A expression. (d) Survival curve according to the VEGF-C expression.

Author's personal copy

1630 S. Paydas et al. / Leukemia Research 33 (2009) 1627–1635

up. A multivariate analysis was performed with the variables thathave a p value < 0.2 in the univariate analysis or that were consid-ered clinically or pathophysiologically important (age) with usingthe Cox regression model. A p value less than 0.05 was consideredto be significant. Age has been used as a continuous variable, notcategorized. The IPI 0 + 1 + 2 were categorized into low grade and3 + 4 were categorized into high grade for IPI.

4. Results

Eighty eight cases with DLBCL were included in this study.Female/male ratio was 39/49, mean age was 53.7 ± 15.6 (range20–82). 56.5% of the cases had early and 43.5% of the cases hadadvanced stage disease. Two thirds of the patients had extra-nodal involvement. Anemia, leucopenia and thrombocytopeniawere detected in 56.8%, 8.6% and 4.9% of the cases at diagnosis,respectively. LDH was high in 75% of the cases and 55.6% of thecases had high B2M level. Cox-2 was found to be positive in 71.6%,TSP-1 in 14.8%, EBV in 21.6%, Survivin in 60%, VEGF-A in 64.8% andVEGF-C in 36.4% of the cases. The expression of these biologicalparameters has been shown in Table 1. Distributions of combinationof these biological parameters have been shown in Table 2. Whenwe compared the clinical and biological parameters, there wasan association between Survivin expression and lymphadenopa-thy (0.031), EBV expression and high LDH (0.013), and VEGF-Aexpression and high B2M (0.007). Table 3 shows the correlationbetween clinical parameters and examined biological parametersin this study. Among these six parameters there was a correlationbetween VEGF-A and Cox-2 (kappa: 0.21, p: 0.038), VEGF-A and Sur-vivin (kappa: 0.27, p: 0.010) and VEGF-A and VEGF-C (kappa: 0.43,p: 0.0001).

Overall survival times were shorter in cases with EBV, Survivin,VEGF-A, and VEGF-C expression as compared to cases not express-ing these parameters (p values were 0.0001, 0.02, 0.03 and 0.001,respectively). The median follow up time was 22 months (min: 1max: 154). The 5-year OS was 49% for all cases (mean survival rate:88 months, median: 60 months). The shortest survival time wasseen in cases showing EBV expression: mean survival time was 21months in EBV positive cases while this time was 106 months incases without EBV expression. As expected, cases with advancedstage disease, having higher IPI and developing relapse had shortersurvival. Table 4 shows the overall survival times of the cases andFig. 1 shows the survival curves of the cases according to the Cox-2, TSP-1, EBV, Survivin, VEGF-A and VEGF-C expression. Double,either or no expression for these parameters and their overall sur-vival times has been shown in Table 5. In all cases showing doubleexpression for these biologic parameters showed shorter survivaltimes as compared with no expression.

When we evaluated these six biologic parameters and clinicalparameters including stage, age, IPI and extra-nodal involvementin multivariate analyses, the independent prognostic factors werefound to be EBV expression (HR: 3.78; 95%CI: 1.47–9.74; p = 0.006),VEGF-C expression (HR: 3.22; 95%CI: 1.07–9.68; p = 0.037) andextranodal involvement (HR: 3.02; 95%CI: 1.01–8.97; p = 0.047).

5. Discussion

DLBCL is the most frequent subtype of NHLs. DLBCL is het-erogeneous entity; clinically, morphologically and genetically.Classification of NHLs, especially DLBCL, is a continuous process.Micro array and other developing technologies will be more infor-mative to determine the biology of these entities [1]. There are manystudies determining the prognostic and predictive role of variousbiologic parameters in this heterogeneous entity. Here we studiedVEGF-A for angiogenesis, VEGFC for lymphangiogenesis, Survivin

Table 4Overall survival times of the cases according to the expression of the examinedbiologic parameter.

Overall survival (month)mean/median

Total/dead n p value

COX-2 Negative 113/– 22/5Positive 71/57 50/21 0.2

TSP-1 Negative 90/– 61/21Positive 39/30 11/5 0.4

EBV Negative 106/– 57/15Positive 21/13 15/11 0.0001

SURVIVIN Negative 118/– 37/7Positive 60/35 40/19 0.02

VEGF-A Negative 112/– 28/6Positive 66/35 44/20 0.03

VEGF-C Negative 109/– 48/11Positive 40/23 24/15 0.001

IPI grade Low 106/– 46/12High 35/31 26/14 0.006

Stage Early 107/– 44/11Advanced 56/31 28/15 0.01

EN inv No 110/– 24/5Yes 63/31 48/21 0.03

Relapse No 140/– 13/1Yes 43/30 22/13 0.001

for anti-apoptotic signal, Cox-2 for inflammatory and EBV for virallymphomagenesis in DLBCL.

5.1. Survivin

Survivin is a 165 kDa protein that belongs to the IAP family. Sur-vivin has multiple functions including cytoprotection, inhibition ofcell death and cell cycle regulation and it is important in mitoticprocess which is very important in malignant cell survival [6,7].Survivin is over-expressed in most of the malignant tumors whilethere is no expression it is not expressed in differentiated adult tis-sues. In lymphomas the frequency of Survivin expression changesaccording to the subtype of NHL and is between 55–100%. In DLBCLSurvivin has been shown in 55–82% of the cases [8–14]. Survivin hasbeen found to be a poor prognostic indicator in DLBCL: overexpres-sion of it has been shown to be correlated with reduced remissionrates and shorter survival times. Thus Survivin expression has beenfound in more aggressive forms of this entity: high IPI, the pres-ence of B symptom and/or extranodal disease, high LDH, shorter5-year OS, older age and advanced stage were found to be associ-ated with Survivin expression but Survivin expression has not beenfound to be related with all the poor prognostic indicators in allstudies [8–10,13,15–24]. In our cases, we found Survivin expressionin 60% of the cases and this expression was found to be associatedwith lymphadenopathy and high LDH which is associated with hightumor burden and shorter OS.

5.2. VEGF-A and VEGF-C

Angiogenesis and lymphangiogenesis are the essential stepsin carcinogenesis and VEGF-A and VEGF-C are the main play-ers of tumor angiogenesis/lymphangiogenesis [25–28]. There isan important association between the angiogenesis and lymphan-giogenesis and also with inflammatory signals ([29–31]. In DLBCLangiogenesis has been studied in a relatively higher number of thecases, however lymphangiogenesis has been studied in very lim-ited number of these cases. VEGF-A expression in DLBCL has beenfound in 60–80% of the cases and generally it has been found that

Author's personal copy

S. Paydas et al. / Leukemia Research 33 (2009) 1627–1635 1631

Table 5Overall survival times of the cases with or without one or more biologic parameter.

Overall survival (month) mean/median Total/dead n p

EBV SURVIVIN Both (−) 120/– 30/6 0.2*

Either one + 81/– 29/10 0.0001**

Both (+) 18/13 13/10 0.001***

EBV VEGF-A Both (−) 117/– 26/5 0.2*

Either one + 85/– 33/11 0.0001**

Both (+) 19/33 13/10 0.0006***

EBV VEGF-C Both (−) 125/– 41/6 0.0008*

Either one + 44/31 23/14 0.0001**

Both (+) 10/13 8/6 0.008***

EBV TSP-1 Both (−) 108/– 48/12 0.004*

Either one + 34/30 22/12 0.0004**

Both (+) 10/5 2/2 0.1***

EBV COX-2 Both (−) 1/8 20/4 0.5*

Either one + 87/– 39/12 0.002**

Both (+) 22/13 13/10 0.0005***

SURVIVIN COX-2 Both (−) 116/– 12/3 0.4*

Either one + 96/– 30/6 0.1**

Both (+) 50/30 30/17 0.005***

TSP-1 COX-2 Both (−) 108/– 20/5 0.6*

Either one + 78/60 43/16 0.1**

Both (+) 33/19 9/5 0.1***

VEGF-A COX-2 Both (−) 126/– 12/2 0.6*

Either one + 84/– 26/7 0.1**

Both (+) 61/30 34/17 0.05***

VEGF-C COX-2 Both (−) 125/– 17/3 0.6*

Either one + 90/– 36/10 0.02**

Both (+) 30/19 19/13 0.002***

VEGF-A SURVIVIN Both (−) 127/– 18/3 0.6*

Either one + 71/– 24/7 0.01**

Both (+) 44/27 30/16 0.008***

VEGF-C SURVIVIN Both (−) 134/– 24/3 0.05*

Either one + 77/60 32/12 0.0007**

Both (+) 25/23 16/11 0.03***

VEGF-A TSP-1 Both (−) 105/– 25/6 0.2*

Either one + 77/57 39/15 0.009**

Both (+) 23/19 8/5 0.09***

VEGF-C TSP-1 Both (−) 104/– 43/11 0.1*

Either one + 53/35 23/10 0.002**

Both (+) 21/14 6/5 0.1***

VEGF-C COX-2 Both (−) 121/– 22/4 0.3*

Either one + 84/– 32/10 0.006**

Both (+) 30/19 18/12 0.01***

VGF-A COX-2 Both (−) 118/– 15/3 0.6*

Either one + 83/– 26/7 0.06**

Both (+) 57/27 31/16 0.04***

VEGF-A VEGF-C Both (−) 111/– 27/6 0.6*

Either one + 93/– 22/5 0.004**

Both (+) 40/23 23/15 0.03***

SURVIVIN TSP-1 Both (−) 127/– 29/5 0.003*

Either one + 51/31 35/18 0.2**

Both (+) 46/– 8/3 0.4***

* p between both (−) and either one+ groups** p between either one + and both+ groups

*** p between both (−) and both (+) groups

lymphomas showing higher angiogenic potential are related withshortest disease free survival (DFS) and/or OS and poor prognos-tic indicators including aggressive histology and/or transformedmorphology [32–43]. Additionally, correlations between VEGF-Aand Cox-2, Survivin and VEGF-C were detected. These correlationsshow and confirm the strong prognostic value of VEGF-A and com-plex interaction with lymphangiogenesis, anti-apoptotic signalsand also inflammatory signals [44–52]. We found VEGF-A expres-

sion in 65% of the cases, this expression was found to be associatedwith high B2M and shorter survival.

VEGF-C expression has been studied in small number of thecases and in these studies. In one study covering 39 cases, VEGF-Cexpression has been found in almost all of the cases with DLBCL. Inone study VEGF-C was found to be associated with high LDH andIPI, in second study VEGF-C was found to be associated with angio-genesis, higher IPI and shorter OS [41,49,53]. We found VEGF-C

Author's personal copy

1632 S. Paydas et al. / Leukemia Research 33 (2009) 1627–1635

expression in only one third of the cases; however in our studyVEGF-C was one of the most important prognostic parametersamong the biologic and clinical prognostic indicators. We needmore studies covering more patients to solve this discrepancy tounderstand the biologic significance of VEGF-C in DLBCL.

5.3. TSP-1

TSP-1 is a 450 kDa extra cellular matrix protein with multi-functional capacity. Classically it is known that TSP-1 is a potentinhibitor of physiological angiogenesis. It may suppress the cellcycle, inhibits angiogenesis, metastasis and is important in suppres-sion of carcinogenesis [54–57]. Regulation of cell growth, motilityand apoptosis both in physiological and pathological conditions arevariable. The main function of TSP-1 is not clear and controversialresults have been shown in different studies. It can stimulate tumorgrowth, but also induce apoptosis. It blocks tumoral invasion andinhibits tumor angiogenesis. TSP-1 expression in tumors must becarefully evaluated because of its stimulatory and inhibitory effectson tumors. The function of TSP-1 may be dependent on the celltype and microenvironment. It has been shown that TSP-1 overex-pression in some cancers and cancer cell lines inhibits angiogenesisand suppresses tumor progression [58–61]. For example, elevatedTSP1/TSP2 levels have been found in bone marrow cultures fromMM patients as compared to healthy donors and an interactionbetween MM plasma cells and microenvironment. With these find-ings, it has been suggested that CD47 and TSP1/TSP2 may have arole in the pathogenesis of MM [62]. In our study we did not findan important data about the TSP-1 expression in lymphomas. Wefound TSP-1 expression in 15% of the cases, and TSP-1 expressingcases showed shorter survival (39 months vs. 90 months) but differ-ence was statistically non significant,. Also double positive cases forTSP-1 and other biologic markers such as Survivin, VEGF-A, VEGF-C, EBV and Cox-2 showed the shortest survival times as comparedcases without TSP-1 expression and other parameters. Although wedid not find an important prognostic importance of TSP-1 in DLBCL,shorter survival in cases with TSP-1 suggest that TSP-1 may be abad prognostic indicator in DLBCL and must be studied in furtherstudies.

5.4. EBV

EBV belongs to the herpes virus family and infects B cellsand epithelial cells. EBV-induced upregulation of angiogenic fac-tors may play a significant role in tumor progression. EBVassociated hemopoietic neoplasias are Hodgkin’s lymphoma andnon-Hodgkin’s lymphomas [63–66]. The highest prevalence hasbeen reported in HIV related lymphomas: nearly 100% of pri-mary central nervous system lymphomas, 80% of DLBCL withimmunoblastic features, 30–50% of Burkitt’s lymphomas, 60% ofplasmablastic lymphomas, 70% of primary effusion lymphoma, andnearly 100% of Hodgkin’s lymphomas. In one of the largest stud-ies about EBV and lymphoma (n: 520); EBV-encoded RNA-1 in situhybridization (EBER-ISH) positivity has been detected in 7.4% and31% of the cases with B-NHL and T-NHL, respectively [67–73]. InDLBCL, EBV has been found in 9–12% of the cases using EBER-ISH,in one of these latent membrane protein-1 (LMP-1) has been foundin only 3% of the cases [51,74,75]. In most of these studies, EBVwas found to be a bad prognostic indicator and has been foundto be associated with older age, advanced stage, more than oneextranodal involvement, presence of B symptom, poor responseto initial therapy, higher IPI and shorter disease free and overallsurvival [76]. EBV and older age is an important relation and inrecent times and the nosological category of senile or age-relatedEBV (+) B-lymphoproliferative disorder has been proposed for thesepatients [76]. In non-GCB subtype, in multivariate analysis, it has

been determined that EBER-ISH positivity is significant in non-GCBDLBCL patients; OS is poorer in EBER (+) cases [51]. In our studygroup, EBV-LMP1 expression was found in 22% of the cases andwas associated with high LDH and EBV was found to be the mostimportant poor prognostic indicator and cases with EBV expressionhad the shortest survival times.

5.5. Cox-2

The association between chronic inflammation and cancer is avery old history proposed by Rudolf Virchow [6,14]. Cyclooxyge-nase is a key enzyme required for the conversion of arachidonicacid to prostaglandins and two isoforms of this enzyme have beenidentified. Cox-2 induction is very important in sites of inflam-mation and cancer and Cox-2 overexpression reduces apoptosis,increases the invasive capacity of malignant cells and angiogenesis.With these properties Cox-2 gene overexpression has a critical rolein carcinogenesis in many cancers [77–83]. Cox-2 overexpressionwas found to be a bad prognostic indicator. In some studies, Cox-2 has been found to be associated with shorter survival, but wasnot found to be related with all poor prognostic indicators. On theother hand, selective inhibition of Cox-2 has been found to be asso-ciated with response in a variety of cancers cells or cancer cell lines[32,33,71,84]. Cox-2 expression in hemopoietic neoplasms has beenstudied in relatively limited number of the cases [85]. Cox-2 expres-sion by immunohistochemistry has been studied in 52 cases withmalignant lymphoma, and has been detected in 57% of the NHL.Cox-2 expression has been found to be correlated with advancedstage and poor response to chemotherapy [86]. In our study, wefound Cox-2 expression in 72% of the cases and did not show a cor-relation with clinical parameters. OS times were shorter in Cox-2expressors but statistically non significant.

It is very well known that carcinogenesis is a complex andmultistep process. Lymphomagenesis is a complex process andsequential and/or combined events are necessary for the develop-ment/progression of the lymphomas. Angiogenesis is one of the firststeps in the development of neoplasias. Inflammation is anotherimportant regulator of the tumorigenesis. Viral factors generallyplay a cofactor role in this complicated cascade. The inhibitors ofapoptotic cell death are the important players. The interactionsbetween endothelium and tumor cells and microenvironment havevery important roles in the development of cancer and we are learn-ing more clearly their roles in these complex processes. There ismuch evidence on the cross talk between angiogenesis and viralcarcinogenesis [87]. LMP1 increases the VEGF production and someother angiogenic factors such as Cox-2, MMP-9, IL-8 and NFKB. EBVand VEGF (+) cases had higher rate of recurrence, node positivityand lower survival times [3,4,88,89]. So there are many interactionsbetween these players in every step.

The net results of these findings are not clear enough but tailoredtreatments according to the responsible factors will be more usefulin some subgroups of these patients. For this reason we would liketo see the role of different pathways in lymphomagenesis and stud-ied angiogenic, lymphangiogenic, anti-apoptotic, viral, endothelialand inflammatory mediators. We found an important associationbetween EBV and VEGF-A expression and EBV and VEGF-A positivecases showed the shortest survival. This finding suggests the impor-tant role of angiogenesis in the pathogenesis of DLBCL and EBVcontribution to lymphomagenesis. It has been shown that LMP1must be a major target for gene therapy [54]. On the other handangiogenesis inhibitors are able to inhibit tumor growth in mousemodels [2,3,90]. VEGF anti-sense is an anti-sense oligonucleotidethat targets VEGF, by inhibiting angiogenesis and tumor cell prolif-eration. These findings suggest the anti- LMP1 and anti-angiogenicdrugs will be novel therapeutic strategies for lymphomas as shownin other malignant tumors. Poor survival rates in cases with double

Author's personal copy

S. Paydas et al. / Leukemia Research 33 (2009) 1627–1635 1633

VEGF-A and EBV expressors may suggest the importance of target-ing both the angiogenesis and EBV in the therapy of these caseswith unfavorable characteristics. Targeting both the angiogenesisand EBV may be important in the therapy of cases with NHL express-ing EBV and/or VEGF-A. For example drugs targeting angiogenesisand lymphangiogenesis may be more useful in such cases [44,91].

Survivin has been found to be associated with resistance tochemotherapy and/or radiation therapy, and associated with poorprognosis. Thus, inhibition of Survivin induces or enhances apopto-sis in tumor cells. It is known that some antitumor agents can reduceSurvivin expression. These findings suggest that Survivin may be apromising molecular target in DLBCL like other malignancies [6,16]Survivin is not expressed in normal tissues, for this reason it is anideal target for cancer therapy and Survivin antagonizing modalitieshave been developed. YM155, a small-molecule Survivin suppres-sant, and its efficacy has been shown in a phase II study. It wasproposed that these agents not only increase the objective responsewhen used with conventional chemotherapeutics, but also cir-cumvent drug resistance which is very important in malignantdiseases [19]. We found a significant association between Survivinand VEGF-A expression and this is compatible with the known asso-ciation between angiogenesis and inhibition of apoptosis.

This may suggest the better response in cases treated by thecombination of angiogenesis inhibitors and Survivin inhibitors.

Our results and literature data suggest that it is possible toincrease the therapeutic efficacy of conventional anti-neoplasticdrugs with the addition of anti-angiogenic, anti-inflammatory andanti-Survivin strategies. As an example in a multicenter phase IIstudy, low dose cyclophosphamide and high dose celecoxib havebeen successfully used in relapsed/refractory cases [92]. VEGFinhibitor bevacizumab has been used with R-CHOP and ORR andCR rates were found to be 85 and 38%, respectively [93].

6. Conclusion

(1) Anti-apoptotic factor Survivin and viral factor EBV positivitywere found to be associated with higher LDH.

(2) VEGF-A (+) cases showed higher B2M levels indicating highertumor burden.

(3) The correlation between angiogenic factor VEGF-A and inflam-mation marker Cox-2, and anti-apoptotic factor Survivin andlymphangiogenic factor VEGF-C suggest the crosstalk betweenangiogenic and inflammatory signals.

(4) EBV, VEGF-C and extranodal involvement were found to beindependent risk factors among all the biologic factors and clin-ical prognostic factors. This finding needs to be confirmed withlarger study groups. The shortest survival times were foundin EBV positive cases and this finding supports the prognosticvalue of EBV in DLBCL.

(5) Angiogenic/lymphangiogenic and anti-apoptotic signals areimportant in the pathogenesis of DLBCL. EBV related virallymphomagenesis is important biologic parameter in DLBCL.These findings suggest that different therapeutic approachesare needed for these cases.

Acknowledgement

We thank Terry Fox Foundation for support.

References

[1] Paepe PD, Wolf-Peeters CD. Diffuse large B-cell lymphoma: a heterogeneousgroup of non-Hodgkin’s lymphomas comprising several distinct clinicopatho-logical entities. Leukemia 2007;21:37–43.

[2] Paydas S, Ergin M, Erdogan S, Seydaoglu G, Yavuz S, Disel U. Trombospondin-1 (TSP-1) and survivin (S) expression in non-Hodgkin’s lymphomas. Leuk Res2008;32:243–50.

[3] Paydas S, Ergin M, Erdogan S, Seydaoglu G. Prognostic significance of EBV-LMP-1 and VEGF-A expressions in non-Hodgkin’s lymphomas. Leuk Res2008;32:1424–30.

[4] Paydas S, Ergin M, Erdogan S, Seydaoglu G. Cylooxygenase-2 expression in non-Hodgkin’s lymphomas. Leuk Lymphoma 2007;48:389–95.

[5] Paydas S, Seydaoglu G, Ergin M, Erdogan S. Prognostic significance of VEGF-Aand VEGF-C in non-Hodgkin’s lymphomaa and review of the literature. LeukLymphoma; in press.

[6] Yamamoto H, Ngan CY, Monden M. Cancer cells survive with surviving. CancerSci 2008;99:1709–14.

[7] Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin,expressed in cancer and lymphoma. Nat Med 1997;3:917–21.

[8] Xiang XJ, He YJ, Li YH, Huang H, Xu F. Clinical significance of survivin expressionin peripheral T-cell lymphoma. Ai Zheng 2006;25:758–61.

[9] Xiang XJ, He YJ. Clinical significance of survivin and caspase-3 expressionin diffuse large B-cell lymphoma. Zhonghua Zhong Liu Za Zhi 2006;28:298–301.

[10] Li JF, Li GD, Liu WP, Wang Y, Cheng JR, Chen Y, et al. Expression of anaplasticlymphoma kinase and survivin proteins in anaplastic large cell lymphoma andits significance. Zhonghua Bing Li Xue Za Zhi 2006;35:213–7.

[11] Schlette EJ, Mederios LJ, Goy A, Lai R, Rassidakis GZ. Survivin expressionpredicts poorer prognosis in anaplastic large cell lymphoma. J Clin Oncol2004;22:1682–8.

[12] Zuo XL, Zhou X, Meng J, Zhang KJ, Liu XH, Yang HQ. A study on the expres-sion of myeloid cell leukemia 1 proteins and survivin and their relationwith B cell apoptosis in non-Hodgkin’s lymphoma. Zhonghua Nei Ke Za Zhi2006;45:133–5.

[13] Adida C, Haioun C, Gaulard P, Lepage E, Morel P, Briere J, et al. Prognosticsignificance of survivin expression in diffuse large B-cell lymphomas. Blood2000;96:1921–5.

[14] Kuttler F, Valnet-Rabier MB, Angonin R, Ferrand C, Deconinck E, Mougin C, etal. Relationship between expression of genes involved in cell cycle control andapoptosis in diffuse large B cell lymphoma: a preferential survivin-cyclin B link.Leukemia 2002;16(4):726–35.

[15] Troeger A, Siepermann M, Escherich G, Meisel R, Willers R, Gudowius S, etal. Survivin and its prognostic significance in pediatric acute lymphoblasticleukemia. Haematologica 2007;92:1043–50.

[16] Romagnoli M, Trichet V, David C, Clement M, Moreau P, Batallie R, et al. Signif-icant impact of survivin on myeloma cell growth. Leukemia 2007;21:1070–8.

[17] Liu L, Zhang M, Zou P. Expression of PLK1 and survivin in diffuse large B-celllymphoma. Leuk Lymphoma 2007;48:2179–83.

[18] Caldas H, Fangusaro JR, Boue DR, Holloway MP, Altura RA. Dissecting the roleof endothelial survivin Ex3 in angiogenesis. Blood 2007;109:1479–89.

[19] Mita AC, Mita MM, Nawrocki ST, Giles FJ. Survivin: key regulator of mito-sis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res2008;14:5000–5.

[20] Kuttler F, Valnet-Rabier MB, Angonin R, Ferrand C, Deconinck E, Mougin C, etal. Relationship between expression of genes involved in cell cycle control andapoptosis in diffuse large B cell lymphoma: a preferential survivin-cyclin B link.Leukemia 2002;16:726–35.

[21] Watanuki-Miyauchi R, Kojima Y, Tsurumi H, Hara T, Goto N, Kasahara S, et al.Expression of antigen detected by a novel monoclonal antibody, T332, is asso-ciated with outcome of diffuse large B-cell lymphoma and its subtypes. PatholInt 2005;55:324–30.

[22] Mainou-Fowler T, Overman LM, Dignum H, Wood K, Crosier S, Angus B, et al.A new subtype-specific monoclonal antibody for IAP-survivin identifies high-risk patients with diffuse large B-cell lymphoma and improves the prognosticvalue of bcl-2. Int J Oncol 2008;32:59–68.

[23] Tracey L, Perez-Rosado A, Artiga MJ, Camacho F, Rodriguez A, Martinez N, etal. Expression of the NF-k B targets BCL2 and BIRC5/survivin characterizessmall B cell and aggressive B-cell lymphomas, respectively. J Pathol 2005;206:123–34.

[24] Martinez A, Bellesillo B, Bosch F, Ferrer A, Marce S, Villamor N, et al. Nuclear sur-vivin expression in mantle cell lymphoma is associated with cell proliferationand survival. Am J Pathol 2004;164:501–10.

[25] Folkman J. Angiogenesis in cancer, vascular, rheumatoid and molecular regula-tion. Nat Med 1995;1:27–31.

[26] Joukov V, Sorsa T, Kumar V, Jeltsch M, Claesson-Welsh I, Cao Y, et al. Prote-olytic processing regulates receptor specificity and activity of VEGF-C. EMBO J1997;16:3898–911.

[27] Ellis I, Filde I. Angiogenesis and metastasis. Eur J Cancer 1996;32A:2451–60.[28] Beck LJ, D’Amore P. Vascular development: cellular and molecular regulation.

FASEB J 1997;11:365–73.[29] Kerbel RS. Tumor angiogenesis. N Engl J Med 2008;358:2039–49.[30] Zhang XH, Huang DP, Guo GL, Chen GR, Zhang HX, Wan L, et al. Coexpression

of VEGF-C and COX-2 and its association with lymphangiogenesis in humanbreast cancer. BMC Cancer 2008;8:4.

[31] Mohammed RAA, Green A, El-Shikh S, Paish EC, Ellis IO, Martin SG. Prognosticsignificance of vascular endothelial cell growth factors-A, C, and–D in breastcancer and their relationship with angi- and lymphangiogenesis. Br J Cancer2007:1092–100.

[32] Salven P, Teerenhovi L, Joensun H. A high pretreatment serum vascularendothelial growth factor concentration is associated with poor outcome innon-Hodgkin’s lymphoma. Blood 1997;90:3167–72.

[33] Kini AR, Day NE, Peterson LC. Increased bone marrow angiogenesis in B-cellchronic lymphocytic leukemia. Leukemia 2000;14:1414–8.

Author's personal copy

1634 S. Paydas et al. / Leukemia Research 33 (2009) 1627–1635

[34] Giles FJ, Vose JM, Do KA, Johnson MM, Manshouri T, Bociek G, et al. Clini-cal relevance of circulating angiogenic factors in patients with non-Hodgkin’slymphoma or Hodgkin’s lymphoma. Leuk Res 2004;28:595–604.

[35] Ho CL, Sheu LF, Li CY. Immunohistochemical expression of basic fibrob-last growth factor, vascular endothelial growth factor, and their receptorsin stage IV non-Hodgkin lymphoma. Appl Immunohistochem Mol Morphol2002;10:316–21.

[36] Moehler TM, Ho AD, Goldschmidt H, Barlogie B. Angiogenesis in hematologicmalignancies. Crit Rev Oncol Hematol 2003;45:227–44.

[37] Jorgensen JM, Sorensen FB, Bendix K, Nielsen JL, Olsen ML, Funder AMD,et al. Angiogenesis in non-Hodgkin’s lymphoma: clinico-pathological cor-relations and prognostic significance in specific subtypes. Leuk Lymphoma2007;48:584–95.

[38] Korkolopoulou P, Thymara I, Kavantzas N, Vassilakopoulos TP, Kokoris SI,Dmitriadou EM, et al. Angiogenesis in Hodgkin’s lymphoma: a morpho-metric approach in 286 patients with prognostic implications. Leukemia2005;1:894–900.

[39] Hazar B, Paydas S, Zorludemir S, Sahin B, Tuncer I. Prognostic significance ofmicrovessel density and vascular endothelial growth factor (VEGF) expressionin non-Hodgkin’s lymphoma. Leuk Lymphoma 2003;44:2089–93.

[40] Gratzinger D, Zhao S, Marinelli RJ, Kapp AV, Tibshirani RJ, Hammer AS, etal. Microvessel density and expression of vascular endothelial growth fac-tor and its receptors in diffuse large B cell lymphoma subtypes. Am J Pathol2007;170:1362–9.

[41] Ganjoo KN, Moore AM, Orazi A, Sen JA, Johnson CS, An CS. The importanceof angiogenesis markers in the outcome of patients with diffuse large B celllymphoma: a retrospective study of 97 patients. J Cancer Res Clin Oncol2008;134:381–7.

[42] Jørgensen JM, Sørensen FB, Bendix K, Nielsen JL, Olsen ML, Funder AM,et al. Angiogenesis in non-Hodgkin’s lymphoma: clinico-pathological cor-relations and prognostic significance in specific subtypes. Leuk Lymphoma2007;48:584–95.

[43] Gratzinger D, Zhao S, Marinelli RJ, Kapp AV, Tibshirani RJ, Hammer AS, etal. Microvessel density and expression of vascular endothelial growth fac-tor and its receptors in diffuse large B-cell lymphoma subtypes. Am J Pathol2007;170:1362–9.

[44] Sundar SS, Ganesan TS. Role of lymphangiogenesis in cancer. J Clin Oncol2007;25:4299–307.

[45] Nagy JA, Vasile E, Feng D, Sundberg C, Brown LF, Detmar MJ, et al. Vascular per-meability factor/vascular endothelial growth factor induces lymphangiogenesisas well as angiogenesis. J Exp Med 2002;196:1497–506.

[46] Jeltsch M, Kaipinen A, Joukov V, Meng X, Lakso M, Rauvala H, et al. Hyperplasiaof lymphatic vessels in VEGF-C transgenic mice. Science 1997;276:1423–5.

[47] Oh SJ, Jeltsch MM, Birkenhager R, McCarthy JE, Weich HA, Christ B, et al.VEGF- and VEGF-C: specific induction of angiogenesis and lymphangiogene-sis in the differentiated avian chorioallantoic membrane. Dev Biol 1997;188:96–109.

[48] Hirakawa S, Kodama S, Kunstfeld R, Kajiya K, Brown LF, Detmar M. VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promoteslymphatic metastasis. J Exp Med 2005;201:1089–99.

[49] Pazgal I, Boycov O, Shpilberg O, Okon E, Bairey O. Expression of VEGF-C, VEGF-Dand their receptor VEGFR-3 in diffuse large B-cell lymphomas. Leuk Lymphoma2007;48:2213–20.

[50] Murono S, Inoue H, Tanabe T, Joab I, Yoshizaki T, Furukawa M, et al. Induction ofcyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involvedvascular endothelial growth factor production in nasopharyngeal carcinomacells. PNAS 2001;98:6905–10.

[51] Park S, Lee J, Ko YH, Han A, Jun HJ, Lee SC, et al. The impact of the Epstein-Barr virus status on clinical outcome in diffuse large B cell lymphoma. Blood2007;110:972–8.

[52] Tzankov A, Heiss S, Ebner S, Sterlacci W, Schaefer G, Augustin F, et al. Angio-genesis in nodal B cell lymphomas: a high throughput study. J Clin Pathol2007;60:476–82.

[53] Kudowaki I, Ichinohasama R, Harigae H, Ishizawa K, Okitsu Y, Kameoka J, etal. Accelerated lymphangiogenesis in malignant lymphoma: possible role ofVEGF-A and VEGF-C. Br J Haematol 2005;130:86–77.

[54] Yamauchi Mai, Imajoh-Ohmi Shinobu, Shibuya Masabumi. Novel antiangio-genic pathway of thrombospondin-1 mediated by suppression of the cell cycle.Cancer Sci 2007;98:1491–7.

[55] Merle B, Malaval L, Lawler J, Delmas P, Clezardin P. Decorin inhibits cell attach-ment to thrombospondin-1 by binding to a KKTRdependent cell adhesivesite present within the N-terminal domain of thrombospondin-1. J Biochem1997;67:75–83.

[56] Clezardin P, Lawler J, Amiral J, Quentin G, Delmas P. Identification of celladhesive sites in the N-terminal domain of thrombospondin-1. Biochem J1997;321:819–27.

[57] Lawler J, Ferro P, Duquette M. Expression and mutagenesis of thrombospondin.Biochemistry 1992;31:1173–80.

[58] Ambrosini G, Adida C, Sirugo G, Altieri DC. Induction of apoptosis and inhibitionof cell proliferation by survivin gene targeting. J Biol Chem 1998;273:11177–82.

[59] Bellon G, Martiny L, Robinet A. Matrix metalloproteinases and matrikines inangiogenesis. Crit Rev Oncol Hematol 2004;49:203–20.

[60] Scarpino S, Di Napoli A, Taraboletti G, Cancrini A, Ruco LP. Hepatocyte growthfactor (HGF) downregulates thrombospondin-1 (TSP-1) expression in thyroidpapillary carcinoma cells. J Pathol 2005;205:50–6.

[61] YeeKO, Streit M, Hawighorst T, Detmer M, Lawler J. Expression of the type-1 repeats of thrombospondin-1 inhibits tumor growth through activation oftransforming growth factor. Am J Pathol 2004;165:541–52.

[62] Rendtlew DJM, Knudsen LM, Dahl IM, Lodahl M, Rasmussen T. Dysregulationof CD47 and the ligands thrombospondin 1 and 2 in multiple myeloma. Br JHaematol 2007;138:756–60.

[63] Fukayama M, Ibuka T, Hayashi Y, Oaba T, Koike M, Mizutani S. Epstein-Barr virusin pyothorax-associated pleural lymphoma. Am J Pathol 1993;143:1044–9.

[64] Timms JM, Flavell JR, Murray PG, Rickinson AB, Traverse- Glehen A, Berger F,et al. Target cells of Epstein-Barr-virus (EBV)-positive posttransplant lympho-proliferative disease: similarities to EBV-positive-Hodgkin’s lymphoma. Lancet2003;361:217–23.

[65] Horenstein MG, Nador RG, Chadburn A, Hyjek EM, Inghirami G, Knowles DM, etal. Epstein-Barr virus latent gene expression in primary effusion lymphomascontaining Kaposi’s sarcoma-associated herpes virus/human herpesvirus-8.Blood 1997;90:1186–91.

[66] Siu LL, Chan JK, Kwang YL. Natural killer cell malignancies: clinicopathologicaland molecular features. Histol Histopathol 2002;17:539–54.

[67] Raphael M, Borisch B, Jaffe ES. Lymphomas associated with infection by thehuman immunodeficiency virus (HIV). In: Jaffe ES, Harris NL, Stein H, VardimanJW, editors. World Health Organization classification of tumors. Pathology andgenetics of tumors of hematopoietic and lymphoid tissues. Lyon: IARC Press;2001.

[68] Lee OJ, Kim KW, Lee GK. Epstein-Barr virus and human immunodeficiencyvirus–negative oral plasmablastic lymphoma. J Oral Pathol Med 2006;35:382–4.

[69] Trivedi P, Takazawa K, Zompetta C, et al. Infection of HHV-8+ primary effu-sion lymphoma cells with a recombinant Epstein-Barr virus leads to restrictedEBV latency, altered phenotype, and increased tumorigenicity without affectingTCL1 expression. Blood 2004;103:313–6.

[70] Hamilton-Dutoit SJ, Rea D, Raphael M, et al. Epstein-Barr virus–latentgene expression and tumor cell phenotype in acquired immunodeficiencysyndrome-related non-Hodgkin’s lymphoma. Correlation of lymphoma phe-notype with three distinct patterns of viral latency. Am J Pathol 1993;143:1072–85.

[71] d’Amore F, Johansen P, Houmand A, Weisenburger DD, Mortensen LS.Epstein-Barr virus genome in non-Hodgkin’s lymphomas occurring in immuno-competent patients: highest prevalence in nonlymphoblastic T-cell lymphomaand correlation with a poor prognosis. Blood 1996;87:1045–55.

[72] Glaser SL, Gulley ML, Borowitz MJ, Craig FE, Mann RB, Stewart SL, et al. Inter andintra-observer reliability of Epstein-Barr virus detection in Hodgkin lymphomausing histochemical procedures. Leuk Lymphoma 2004;45:489–547.

[73] Kwon JM, Park YH, Kang JH, Kim K, Ko YH, Ryoo BY, et al. The effect of Epstein-Barr virus status on clinical outcome in Hodgkin’s lymphoma. Ann Hematol2006;85:463–8.

[74] Park S, Lee, Ko YH, Han A, Jun HJ, Lee SC, et al. The impact of the Epstein-Barr virus status on clinical outcome in diffuse large B cell lymphoma. Blood2007;110:972–8.

[75] Kuze T, Nakamura N, Hashimoto Y, Sasaki Y, Abe M. The characteristics ofEpstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma: comparisonbetween EBV(+) and EBV(−) cases in Japanese population. Jpn J Cancer Res2000;91:1233–40.

[76] Shimoyama Y, Yamamoto K, Asano N, Oyama T, Kinoshita T, Nakamura S. Age-related Epstein–Barr virus-associated B-cell lymphoproliferative disorders:Special references to lymphomas surrounding this newly recognized clinico-pathologic disease. Cancer Sci 2008:1–7.

[77] Turini ME, DuBois RN. Cyclooxygenase-2A therapeutic target. Annu Rev Med2002;53:35–57.

[78] Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis inepithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell1995;83:493–501.

[79] Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expressionin human coloncancer cells increases metastatic potential. Proc Natl Acad Sci 1997;94:3336–40.

[80] Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. Cyclooxygenaseregulates angiogenesis induced by colon cancer cells. Cell 1998;93:705–16.

[81] Pathak SK, Sharma RA, Steward WP, Mellon JK, Griffiths TRL, Gescher AJ. Oxida-tive stress and cyclooxygenase activity in prostate carcinogenesis: targets forchemopreventive strategies. Eur J Cancer 2005;41:61–70.

[82] Tucker ON, Dannanberg AJ, Yang EK, Zhang F, Teng L, Daly JM, et al.Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer. Can-cer Res 1999;59:987–90.

[83] Ristimaki A, Honkanen N, Jankala H, Sipponen P, Harkonen M. Cyclooxygenase-2in human gastric carcinoma. Cancer Res 1997;57:1276–80.

[84] TsimberidouAM, Keating MJ, Bueso-Ramos CE, Kurzrock R. Epstein- Barr virusin patients with chronic lymphocytic leukemia: a pilot study. Leuk Lymphoma2006;47:827–36.

[85] Ladetto M, Vallet S, Trojan A, Dell’Aquilla M, Monitillo L, Rosato R, et al.Cyclooxygenase-2 is frequently expressed in multiple myeloma and is an inde-pendent predictor of poor outcome. Blood 2005;105:4784–91.

[86] Hazar B, Ergin M, Seyrek E, Erdogan S, Tuncer I, Hakverdi S. Cyclooxygenase-2expression in lymphomas. Leuk Lymphoma 2004;45:1395–9.

[87] Yoshizaki T, Horikawa T, Quing-Chun R, Wakisaka H, Takeshita H, Sheen TS, etal. Induction of interleukin-8 by Epstein-Barr virus latent membrane protein-1and its correlation to angiogenesis in nasopharyngeal carcinoma. Clin CancerRes 2001;7:1946–51.

Author's personal copy

S. Paydas et al. / Leukemia Research 33 (2009) 1627–1635 1635

[88] Wakisaka N, Kondo S, Yoshizaki T, Murono S, Furukawa M, Pagano JS. Epstein-Barr virus latent membrane protein 1 induces synthesis of hypoxia induciblefactor 1 alpha. Mol Cell Biol 2004;24:5223–34.

[89] Mainou-Fowler T, Overman LM, Dignum H, Wood K, Crosier S, Angus B, et al.A new subtype-specific monoclonal antibody for IAP-survivin identifies high-risk patients with diffuse large B-cell lymphoma and improves the prognosticvalue of bcl-2. Int J Oncol 2008 Jan;32(1):59–68.

[90] Kuttler F, Valnet-Rabier MB, Angonin R, Ferrand C, Deconinck E, Mougin C, etal. Relationship between expression of genes involved in cell cycle control andapoptosis in diffuse large B cell lymphoma: a preferential survivin-cyclin B link.Leukemia 2002 Apr;16(4):726–35.

[91] Dudek AZ, Bodempudi V, Welsh BW, Jasinski P, Griffin RJ, Milbauer L, et al. Sys-temic inhibition of tumour angiogenesis by endothelial cell-based gene therapy.Br J Cancer 2007;97:513–22.

[92] Buckstein R, Kerbel RS, Shaked Y, Nayar R, Foden C, Turner R, et al. High-dose celecoxib and metronomic Low-dose cyclophosphamide is an effectiveand safe therapy in patients with relapsed and refractory aggressive histologynon-Hodgkin’s lymphoma. Clin Cancer Res 2006;12:5190–8.

[93] Ganjoo KN, An CS, Robertson MJ, Gordon LI, Sen JA, Weisenbach J, et al. Rit-uximab, bevacizumab and CHOP (RA-CHOP) in untreated diffuse large B-celllymphoma: safety, biomarker and pharmacokinetic analysis. Leuk Lymphoma2006;47:998–1005.