TUBITAK Grant No:SBAG-2567(102S045)

Asbestos Usage In Asbestos Usage In TurkeyTurkey

• Interior of house plastered with asbestos-containing ‘white soil’.

• Outdoors and floors of the same house plastered with asbestos-containing ‘white soil’.

Fairy Chimneys in Cappadocia

Erionite fibre

• Houses constructed with bricks containing erionite fibres

From A. Umran Dogan

Intrapleural inoculation of erionite displayed 300-800 and 100-500 times more carcinogenic activity than chrysotile and crocidolite respectivelyCarthew P, Hum Exp Toxicol 1992; 11: 530-4

Patients with erionite-induced MPM have significantly shorter life time. Hence, erionite induced cases have higher aneuploidy compared with asbestos induced cases (72.7% vs 13.8%) suggesting more aggressive tumor behavior. Emri S. Lung Cancer 2001; 33: 109-114

INTRODUCTION

Since erionite and asbestos induced MPM differs in biological behavior, their contributions of apoptotic pathways in tumor development could be different.

HYPOTHESIS

Mitochondrial Mitochondrial pathwaypathwayAnti-Anti-

apoptoticapoptoticPro-Pro-

apoptoticapoptoticHR3HR3

Homology OnlyHomology Only

Bcl-2Bcl-2 BaxBax BadBad

Bcl-XBcl-XLL BakBak BidBid

Bcl-WBcl-W Bcl-XBcl-XSS BikBik

Mcl-1Mcl-1 Bok/MtdBok/Mtd Bim/BodBim/Bod

Boo/DivaBoo/Diva

NR-13NR-13

HrkHrk

NixNix

Bcl-2 Bcl-2 Prevents The Cancer Prevents The Cancer Cell DeathCell Death

Bcl-2/bax: Worse prognosisAdvance tumor grade

↓Bcl-2/Bax :Lower tumor grade. Better prognosisBetter treatment response

Caspase Activation

Cell Death

The Final Common The Final Common PathwayPathway

Cytochrome c

Fas –Fas L

AIMAIM

• Bcl 2/Bax and Fas/Fas L pathways were studied in vivo in a comparative manner in tissue sections from patients with erionite and asbestos induced MPM.

• The association of expression pattern and survival has been evaluated.

• Sections from adenocarcinoma served as control group.

52 patients including 16 (30.7%) patients with erionite and 19 (36.5%) patients with asbestos induced MPM were enrolled in the study.

Tissue samples from patients with adenocarcinoma [n=17 (32.6%)] served as control groups.

MATERIAL AND METHOD

METHOD 2

•Immunohistochemically semiquantitative

the staining extensionexpressing the

percentages of positive cells/tissue section

the staining intensity0=null, 1=weak, 2=moderate

and 3=strong.

Statistical AnalysisStatistical AnalysisSPSS 10.01 for Windows (1999) was SPSS 10.01 for Windows (1999) was

used for statistical analysis. Chi used for statistical analysis. Chi square, Fisher’s exact chi square, square, Fisher’s exact chi square, Kruskal Wallis ANOVA and, Mann Kruskal Wallis ANOVA and, Mann Whitney U tests were performed for Whitney U tests were performed for comparisons . comparisons .

Survival analysis was done by Kaplan Survival analysis was done by Kaplan Meier Survival analysis Meier Survival analysis

Prognostic factors were analysed by Prognostic factors were analysed by Cox’s Multivariate Regression Model. Cox’s Multivariate Regression Model.

RESULTS

Patient Patient characteristicscharacteristics

ErionitErionitee MPMMPMN=16(%)N=16(%)

AsbestAsbestosos MPMMPMN=19 (%)N=19 (%)

AdenoAdenocarcicarcinomanomamamaN=17 (%)N=17 (%)

E/KE/K 11/511/5 10/910/9 14/314/3

Mean age Mean age (year)(year)

48.7348.7312.412.422

47.8947.8911.11.7373

53.8853.8811.2511.25

StageStage

Stage Stage II 2 (14.3)2 (14.3) 8 (42.1)8 (42.1) 3 (18.8)3 (18.8)

Stage Stage IIII 2 (14.3)2 (14.3) 1 (5.3)1 (5.3) 1 (6.3)1 (6.3)

StageStage III III 6 (42.9)6 (42.9) 7 (36.8)7 (36.8) 8 (50.0)8 (50.0)

StageStage IV IV 4 (28.6)4 (28.6) 3 (15.8)3 (15.8) 4 (25.0)4 (25.0)

Tumor typeTumor type

EpithelialEpithelial 10 (62.5)10 (62.5) 12 (63.2)12 (63.2)

SarcomatousSarcomatous 3 (18.8)3 (18.8) 2 (10.5)2 (10.5)

MixedMixed 3 (18.8)3 (18.8) 5 (26.3)5 (26.3)

Diagnostic Diagnostic methodmethod

Erionit MPMErionit MPM

N=16(%)N=16(%) Asbest Asbest MPMMPM

N=19 (%) N=19 (%)

AdenokarsinoAdenokarsinomama

N=17 (%) N=17 (%)

Toracotomy+opeToracotomy+open biopsyn biopsy

88 (50) (50) 16 (84.2)16 (84.2) 8 (47)8 (47)

TorachoscopyTorachoscopy 4 (25)4 (25) 3 (15.8)3 (15.8) 00

Closed pleural Closed pleural biopsybiopsyBronchoscopic Bronchoscopic biopsybiopsyTrucut BiopsyTrucut Biopsy

4 (25)4 (25) 00

00

00

3 (17.6)3 (17.6)

3 (17.6)3 (17.6)

3 (17.6)3 (17.6)

ErionitErionitee MPMMPMN=16 (%)N=16 (%)

AsbestAsbestosisosis MPMMPMN=19 (%)N=19 (%)

SurgerySurgery 9 (56.3)9 (56.3) 16 (84.2)16 (84.2)Type of SurgeryType of Surgery

Paliative tumor Paliative tumor reduction surgeryreduction surgery

Extrapleural Extrapleural pneumonectomypneumonectomy

00 1 (5.3)1 (5.3)

Pleurectomy+dePleurectomy+decorticationcortication

3 (18.8)3 (18.8) 99 ( (4747))

Paliative minor Paliative minor surgerysurgery

Toracoscopy+intToracoscopy+intracavitary racavitary cisplatincisplatin

6 (37.5)6 (37.5) 66 ( (3232))

Antibody Intensity Erionite MPM No (%)

Asbestos MPMNo (%)

AdenocarcinomaNo (%)

Bcl-2 Null 14 (88) 18 (95) 17 (100)

Weak 2 (13) 1 (5) 0

Fas Null 15 (94) 18 (95) 14 (82)

Weak 1 (6) 0 3 (18)

Moderate 0 1 (5) 0

Bax  

Null 10 (63) 12 (63) 8 (47)

Weak 2 (13) 5 (26) 6 (35)

Moderate 4 (25) 2 (11) 3 (18)

Fas Ligand  

Null 4 (25) 2 (11) 1 (6)

Weak 6 (38) 5 (26) 1 (6)

Moderate 6 (38) 10 (53) 15 (88)

Strong 0 2 (11) 0

BAX: 14 %, 5 %, 8 %

Fas L: 27 %, 47 %, 26 %

Fig. 1: Sections with antibody stainings (X400). a)  Skin epidermis. Positive control for Fas antibody. The image shows positive staining.

Fig. 1: Sections with antibody stainings (X400). Section from sarcomatous type MPM (asbestos). Moderately intense staining with Fas Ligand antibody.

Fig. 1: Sections with antibody stainings (X400). Section from mixed type MPM (erionite). Moderately intense staining with Bax antibody.

The median survival time in erionite induced MPM (14 months) was similar to time in asbestosis induced MPM (14 months).

Difference in median survival according to tumor stage, different treatment modalities and different histological subtypes was not statistically significant in whole MPM group.

Survival 1

In conjunction with antibody staining, Bax negative erionite patients had better survival (18 months) than bax positive erionite patients (14 months, OR: 18.83)

This was not true for the asbestosis group.

In Whole MPM group, Fas L positive patients (15 months) showed statistically better survival when compared to Fas L negative patients (12 months) (OR: 3.12, p=0.05) in whole MPM group

Survival 2

Survival time (months)

403020100

Pro

ba

bili

ty o

f S

urv

iva

l

1,2

1,0

,8

,6

,4

,2

0,0

-,2

BAX

Exp

Alive

No exp

Alive

Patients with no Bax expression showed better survival than patients with Bax expression in erionite group (p=0.06).

Survival time (months)

50403020100

Pro

ba

bili

ty o

f S

urv

iva

l

1,2

1,0

,8

,6

,4

,2

0,0

-,2

FAS LIGAND

Exp

Alive

No exp

Alive

Patients with Fas Ligand expression showed better survival than patients without expression in whole MPM group (p=0.05).

TARTIŞMA

This is the first study that compares This is the first study that compares common apoptotic protein expression in common apoptotic protein expression in erionite and asbestos induced MPM. erionite and asbestos induced MPM.

As similar to previous studies, Bcl-2 As similar to previous studies, Bcl-2 expression was not showed in the MPM expression was not showed in the MPM sections. This may imply that Bcl 2 does sections. This may imply that Bcl 2 does not take an important part in the not take an important part in the pathogenesis of MPM. pathogenesis of MPM. Narasimhan et al. Am J Physiol 1998.Narasimhan et al. Am J Physiol 1998.

Bax is a proapoptotic protein. We have Bax is a proapoptotic protein. We have shown that it expresses similarly in all shown that it expresses similarly in all groups.groups.

A previous study has shown that Bax had A previous study has shown that Bax had extensively expressed in MPM cell lines. extensively expressed in MPM cell lines. However, they concluded that Bax has However, they concluded that Bax has been disfunctional in MPM.been disfunctional in MPM.Narasimhan et al. Am J Physiol 1998.Narasimhan et al. Am J Physiol 1998.

•There is only one study that investigate the relation of apoptotic protein expression and survival.

•Accordingly, there is no relation of Bax and Bcl-2 and survival.

Soini Y et al. Clin Cancer Res 1999.

•In our study, in only erionite group, Bax had been inversely related with survival.

Bax is a proapoptotic protein Bax is a proapoptotic protein

RELATED WRELATED WIITH WORSE PROGNOSISTH WORSE PROGNOSIS Other antiapoptotic proteins may use this Other antiapoptotic proteins may use this

pathway to exert their activitypathway to exert their activity This finding indicates a possible different This finding indicates a possible different

mechanism in apoptotic pathways in mechanism in apoptotic pathways in erionite group. erionite group.

INDICATES FURTHER STUDIES IN CELL INDICATES FURTHER STUDIES IN CELL LINES LINES

•Stewart et al have shown that Fas/Fas L pathway may intact in MPM and may be enhanced by chemotherapeutics. Stewart et al. J Thorac Cardiovasc Surg 2002.

•In our study there is almost no expression of Fas expression. This was also true for adenocarcinoma. However, Fas L expressed heavily and showed better prognosis.

In Conclusion,

•Bcl-2 may not involve in the tumorigenesis of MPM.

Bax expression was associated with poor prognosis in only erionite induced MPM. This may imply a difference in apoptotic behaviour in erionite and asbestos induced MPM.

•Fas L pathway may be intact and may be associated with better survival in whole MPM group.