Investigation of CA9 expression in pulmonary metastatic lesions from patients with clear cell renal cell carcinoma

internalchildlikeInternet και Εφαρμογές Web

12 Νοε 2013 (πριν από 3 χρόνια και 8 μήνες)

74 εμφανίσεις

Investigation of CA9 expression in pulmonary metastatic lesions from patients with
clear cell renal cell carcinoma

Pierre Tennstedt
1
, Peter Schneider
1
, Egbert Oosterwijk
2
, Axel Rolle
4
, Susanne Fuessel
1
, Matthias Meinhardt
3
,
Marc
-
Oliver Grimm
1
, Manfred P. Wirth
1

1 Department of Urology, Technical University Dresden, Germany; 2 Experimental Urology, Radboud University Nijmegen,
Netherlands, 3 Institute of Pathology, Technical University Dresden, Germany, 4 Center for Pneumology and Thoracic Surgery,
Fachkrankenhaus Coswig, Germany

Introduction:


renal

cell

carcinoma

(RCC)

is

one

of

the

most

aggressive

tumors


30
%

of

patients

have

developed

metastases

at

time

of

diagnosis

and

up

to

40
%

develop

local

recurrence

or

metastatic

disease

(Lam

et

al
.
,

2005
)


2

year

survival

rate

after

development

of

metastases

is

10
-
20
%


Metastases

to

lung

are

most

frequent

with

prevalence

rates

as

high

as

72
%

(Weiss

et

al
.
,

1988
)

and

76
%

(Saitoh

et

al
.
,

1981
)

in

autopsy

studies
;



in

RCC,

expression

of

prognostic

marker

carbonic

anhydrase

9

(CA
9
)

was

found

in

95
%

of

the

clear

cell

subtype

(ccRCC)

(Liao

et

al
.
,

1997
)


high

expression

of

CA
9

was

associated

with

improved

prognosis

in

ccRCC

patients

(Bui

et

al
.
,

2003
)


controversially

high

expression

of

CA
9

was

associated

with

worse

prognosis

in

several

other

malignancies

e
.
g
.

cervix,

uterine

corpus

and

lung

and

breast

cancer

(Loncaster

et

al
.
,

2001
;

Giatromannolaki

et

al
.
,

2001
;

Chia

et

al
.
,

2001
)


aim

of

this

study

was

to

examine

the

expression

of

CA
9

in

pulmonary

metastases

from

ccRCC

patients

which

were

treated

by

laser
-
based

surgery

Patients:

Materials and Methods:

Tissue microarray construction:

Immunohistochemistry:

Results:

Table 1
: Patients clinical characteristics by CA9 expression in lung metastases of ccRCC

characteristics

overall

CA9

low

high

Patients

82

40

42

Gender






male

51

23

28

0.395


female

31

17

14

Age (year)



at time of nephrectomy




0.101



mean

59

61

58



median

60

62

59



range

40
-
75

40
-
75

42
-
70


at time of metastases surgery




0.622



mean

63

64

63



median

63

65

63



range

41
-
77

41
-
77

49
-
77

T stage primary tumor




0.394



T1+T2

40

19

21



T3+T4

33

19

14

Node metastases at primary tumor




0.451



no

52

25

27



yes

8

5

3

Metastasis at time of nephrectomy




0.243



No

52

24

28



Yes

23

14

9

Grading primary tumor




0.059


1+2

47

20

27




3


19

13

6

Staging primary tumor




0.278


I+II

30

14

16


III


19

9

11


IV


25

15

9

No. lung metastases




0.888



mean

8.2

8

8.4



median

3

3

3



range

1
-
64

1
-
49

1
-
64

Grading lung metastases




0.398


1+2

57

26

31


3


25

14

11

Node metastases at pulmonary surgery



0.054



No

58

24

34



Yes

23

15

8

Relapse after first pulmonary surgery



0.508


No


16

9

7



Yes

66

31

35

Deaths





0.098


No


30

11

19


Yes

52

29

23

p value

Fig
.

2

Kaplan
-
Meier

estimates

according

to

CA
9

expression

for

A

progression

free

survival

(PFS)

and

B

metastases

free

survival

(MFS)

for

patients

with

metastatic

ccRCC
.

Conclusion:


investigations

of

CA
9

expression

in

ccRCC

patients

with

pulmonary

metastases

demonstrates

that

CA
9

is

associated

with

survival


patients

with

high

CA
9

expression

in

lung

metastases

had

a

significant

higher

survival

than

patients

with

low

CA
9

expression


CA
9

expression

specifically

stratified

survival

for

clinical

variables

and

is

an

independent

predictor

of

survival


investigation

of

CA
9

expression

in

patients

with

ccRCC

could

be

an

important

factor

to

decide

adjuvant

therapy

corresponding should be address to:

Dr. rer. nat. Pierre Tennstedt


Technical University Dresden


Department of Urology


Fetscherstraße 74


01307 Dresden


Germany



Email: pierre.tennstedt@uniklinikum
-
dresden.de


study

cohort

consisted

of

82

ccRCC

Patients

(
51



and

31


)


all

underwent

radical

or

partial

nephrectomy

and

have

developed

lung

metastases,

which

were

resected

by

laser
-
based

surgery

from

1999

to

2004

in

Coswig

(Germany),

Center

for

Pneumology

and

Thoracic

surgery


at

lung

metastases

surgery

lymph

nodes

were

resected

and

reviewed

on

microscopic

slides

for

tumor

affection


median

age

63

years

(range

41

to

77
)


4

patients

died

not

by

a

cancer

specific

death


median

survival

after

metastases

surgery

for

48

patients

who

died

of

RCC

related

cause

was

25

months

(range

2

to

60
)

and

for

all

patients

41

months

(range

2

to

95
)


median

overall

survival

after

nephrectomy

was

45

months

(range

5

to

376
)

and

69

months

(range

5

to

376
),

respectively


median

progression

free

survival

was

19

months

(range

1

to

176
)

and

65

months

(range

0

to

351
),

respectively


primary

tumors

classified

after

the

2002

TNM

staging

and

classified

after

UICC

1997


lung

metastases

from

a

cohort

of

82

patients

were

obtained

from

Center

for

Pneumology

and

Thoracic

Surgery,

Coswig

Germany

and

embedded

in

paraffin


all

548

metastases

and

106

normal

lung

tissues

from

normal

appearing

region

of

the

metastases

of

82

patients

were

evaluated

after

hematoxylin
-
eosin

staining

by

a

skilled

pathologist

(MM)



two

punches

from

every

lung

metastases

were

taken

from

selected

morphologically,

representative

regions

and

one

punches

from

morphologically

normal

appearing

region

of

each

tumor


punches

were

precisely

arrayed

on

a

recipient

block

using

a

manual

tissue

array

instrument

as

described

by

Kononen

et

al
.

(Kononen

et

al
.
,

1998
)



Sections

(
4

µm)

from

each

tissue

array

block

were

transferred

to

silanized

slides

(Dako)


evaluation

of

histology

and

grade

after

Furman

criteria

(Fuhrman

et

al
.
,

1986
)

was

assessed

on

hematoxylin

and

eosin

stained

tissue

array

sections

by

a

pathologist

(MM)

blinded

to

clinicopathological

variables


all

lung

metastases

were

of

the

clear

cell

subtype

of

RCC


immunohistochemical

staining

of

tissue

sections

for

CA
9

was

done

using

a

peroxidase

technique

with

antigen

retrieval

using

heat

treatment

in

citrate

buffer

pH

6
.
0


CA
9

antibody

M
75

(kindly

provided

by

Dr
.

Oosterwijk)

was

used

in

a

dilution

of

1
:
200


evaluation

of

positively

stained

tumor

cells

was

based

on

scale

of

0

to

100

percent

and

staining

intensity

on

a

4

point

scale

of

0

to

3



CA
9

score

was

calculated

by

product

of

positively

stained

tumor

cells

and

staining

intensity

(low

<
300
,

high

=

300
)


Comparison

of

CA
9

expression

between

the

two

punches

of

all

metastases

showed

a

concordance

of

93
.
4
%

for

intensity

and

62
.
3
%

for

area


Statistical analysis:


analyses

were

performed

with

SPSS

software


outcome

of

interest

was

progression
-
free

survival

(PFS)

from

time

at

nephrectomy

to

first

pulmonary

metastases,

metastases
-
free

survival

(MFS)

from

time

at

pulmonary

surgery

to

metastases

relapse,

metastases

survival

(MS)

from

time

at

pulmonary

surgery

to

death

or

last

follow

up

and

overall

survival

(OS)

from

time

at

nephrectomy

to

death

or

last

follow
-
up


statistical

software

was

used

to

define

a

cutoff

to

classified

patients

according

to

CA
9

expression


Mann
-
Whitney
-
U

test

was

used

to

test

CA
9

association

with

clinicopathologic

variables



Kaplan
-
Meier

curves

were

used

to

visualize

association

of

CA
9

expression

with

survival


log
-
rank

test

was

used

to

test

difference

between

stratified

survival

functions

References:

Bui

MHT,

Visapaa

H,

Seligson

D,

Kim

H,

Han

K,

Huang

Y,

Horvath

S,

Stanbridge

EJ,

Palotie

A,

Figlin

RA,

Bellegrun

AS

(
2004
)

J
.

of

Urol
.

171
:
2461
-
2466
.

Chia SK, Wykoff CC, Watson PH (2001)
J Clin. Oncol.
19: 3660

8

Giatromanolaki A, Koukourakis MI, Sivridis E (2001)

Cancer Res.
61: 7992

8

Lam

JS,

Shvarts

O,

Leppert

JT,

Figlin

RA,

Bellegrun

AS

(
2005
)

J
.

Urol
.

173
:
1853
-
1862

Liao S
-
Y, Aurelio ON, Jan K, Zavada J, Stanbridge EJ (1997) Cancer Res. 57:2827
-
2831.

Loncaster JA, Harris AL, Davidson SE (2001)
Cancer Res.
61: 6394

9

Saitoh

H
.

(
1981
)

Cancer

48
:
1487

91
.

Weiss

L,

Harlos

JP,

Torhost

J

(
1988
)

J
.

Cancer

Res
.

Clin
.

Oncol
.

114
:
605

12
.

Fig
.

1

CA
9

staining

was

present

in

93
,
3

%

of

all

metastases

from

ccRCC

patients
.

Representative

punches

shows

A

low

CA
9

expression

and

B

high

CA
9

expression
.

C

In

normal

lung

tissue

no

CA
9

staining

was

observed

A

B

C

A

B

Fig
.

3

Kaplan
-
Meier

estimates

according

to

CA
9

expression

for

A

Metastases

survival

(MS)

and

B

overall

survival

(OS)

for

patients

with

metastatic

ccRCC
.

A

B

Table 4
. Stepwise Cox proportional multivariate hazard analysis for PFS

Parameter

Staging primary tumor

2.212

0.000

1.552
-
3.151

Grading primary tumor

2.732

0.000

1.661
-
4.492

HR

p value

95% CI

Table 5
. Stepwise Cox proportional multivariate hazard analysis for MS and OS

Parameter

HR

p value

95% CI

HR

p value

95% CI

MS

OS

No. lung metastases

1.750

0.015

1.116
-
2.743

1.780

0.044

1.015
-
3.122

Metastases relapse

4.225

0.007

1.477
-
12.086

5.630

0.021

1.296
-
24.467

Node metastases


At nephrectomy




3.779

0.006

1.456
-
9.804

Grading lung metastases

not significant


not significant

Table 2
. Univariate Cox proportional hazard analysis for PFS

Parameter

T stage primary tumor

1.680

0.040

1.023
-
2.758

Node metastases at nephrectomy

1.735

0.049

1.003
-
3.003

Gender

1.885

0.008

1.183
-
3.004

HR

p value

95% CI

Table 3
. Univariate Cox proportional hazard analysis for MS and OS

Parameter

HR

p value

95% CI

HR

p value

95% CI

MS

OS

No. lung metastases


not significant

2.050

0.021

1.117
-
3.765

Grading lung metastases


not significant

2.067

0.019

1.126
-
3.794

Metastases relapse

1.804

0.044

1.105
-
3.206

2.291

0.007

1.248
-
4.207

Node metastases


At nephrectomy


not significant

2.044

0.044

1.021
-
4.092

Gender


not significant

2.005

0.022

1.104
-
3.643

PSN
-
AUA08PS1
-
384