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

renal cell carcinomaPierre Tennstedt1, Peter Schneider1, Egbert Oosterwijk2, Axel Rolle4, Susanne Fuessel1, Matthias Meinhardt3, Marc-Oliver Grimm1, Manfred P. Wirth1

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 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 (CA9) was found in 95% of the clear cell subtype (ccRCC)

(Liao et al., 1997) high expression of CA9 in primary tumors was associated with improved prognosis in ccRCC patients (Bui et al., 2003) controversially, high expression of CA9 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 CA9 in pulmonary metastases from ccRCC patients which were treated

by laser-based surgery

Patients

Materials and Methods

Tissue microarray construction

Immunohistochemistry

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 median age at pulmonary surgery was 63 (41 - 77) years at lung metastases surgery, lymph nodes were resected and reviewed on microscopic slides for tumor affection 48/82 patients died of RCC, 4 patients did not die by a cancer-specific death median survival after metastases surgery was 25 (2 – 60) months for the 48 patients who died of RCC-related cause

and 41 (2 - 95) months for all patients median overall survival after nephrectomy was 45 (5 - 376) months and 69 (5 - 376) months, respectively median progression-free survival (PFS) was 19 (1 – 176) months and 65 (0 – 351) months, respectively median follow-up after pulmonary surgery was 40 (2 – 95) months, after nephrectomy 71 (5 – 376) months primary tumors were classified according to the 2002 TNM staging and the UICC criteria of 19974

lung metastases were obtained from Center for Pneumology and Thoracic Surgery, Coswig Germany and embedded in paraffin

548 metastases of 82 patients were evaluated by a skilled pathologist (MM) after hematoxylin-eosin staining two punches from every lung metastases and one punch from morphologically normal appearing lung tissue were taken

from selected morphologically representative regions of each tissue block 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 Thoenes criteria 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 CA9 was done using a peroxidase technique with antigen retrieval

using heat treatment in citrate buffer pH 6.0 CA9 antibody M75 (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 (3 = strongest staining) CA9 score was calculated by product of positively stained tumor cells and staining intensity (low <300, high = 300) comparison of CA9 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 classify patients according to the CA9 expression Mann-Whitney-U test was used to test association of CA9 expression with clinicopathologic variables Kaplan-Meier method was used to visualize association of CA9 expression with survival log-rank test was used to test difference between stratified survival functions Cox proportional hazard model was used to test the statistical independence and significance of CA9 expression for the

prediction of survival based on clinical variables

Results

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

characteristics overallCA9

low high

Patients82 40 42Gender

male 51 23 28 0.395female 31 17 14

Age (year) at time of nephrectomy 0.101

mean 59 61 58median 60 62 59range 40-75 40-75 42-70

at time of metastases surgery 0.622mean 63 64 63median 63 65 63range 41-77 41-77 49-77

T stage primary tumor 0.394T1+T2 40 19 21T3+T4 33 19 14

Node metastases at primary tumor 0.451no 52 25 27yes 8 5 3

Metastasis at time of nephrectomy 0.243No 52 24 28Yes 23 14 9

Grading primary tumor 0.0591+2 47 20 273 19 13 6

Staging primary tumor 0.278I+II 30 14 16III 19 9 11IV 25 15 9

No. lung metastases 0.888mean 8.2 8 8.4median 3 3 3range 1-64 1-49 1-64

Grading lung metastases 0.3981+2 57 26 313 25 14 11

Node metastases at pulmonary surgery 0.054No 58 24 34Yes 23 15 8

Relapse after first pulmonary surgeryNo 16 9 7Yes 66 31 35

DeathsNo 30 11 19Yes 52 29 23

p value

Fig. 1: CA9 staining was present in 93,3 % of all metastases from ccRCC patients. Representative punches show A low CA9 expression and B high CA9 expression. C In normal lung tissue no CA9 staining was observed.

A B C

Fig. 2: Kaplan-Meier estimates according to CA9 expression for A progression-free survival (PFS) and B metastases-free survival (MFS) for patients with metastatic ccRCC

A

median PFS: 53 vs. 21 months

PF

S

B

median MFS: 21 vs. 9 months

(N=42)(N=40)

Conclusion

CA9 expression in 93,3% of all pulmonary metastases (Fig. 1) CA9 expression in pulmonary metastases of ccRCC demonstrates its association

with survival (Fig. 2 & 3):

significant longer overall survival after nephrectomy (OS) and metastasectomy

(MS) as well as longer progression-free survival (PFS) and time to

metastases relapse (MFS) for patients with high CA9 expression CA9 expression specifically stratified OS and MS for clinical variables (primary pN0,

primary low grade, low metastases´ grade; examples in Fig. 4)

CA9 is an independent predictor of survival, particularly for stratification of

survival for patients with favorable clinical parameters investigation of CA9 expression in patients with ccRCC could be an important factor

to decide on adjuvant therapy

corresponding should be address to:

Dr. rer. nat. Pierre TennstedtTechnical University DresdenDepartment of UrologyFetscherstraße 7401307 DresdenGermanye-mail: pierre.tennstedt@uniklinikum-dresden.de

A B

A B

Fig. 4: Kaplan-Meier estimates for A overall survaval (OS) and B metastases survival (MS) according to low primary tumor grade substratified by CA9 expression.

median MS: 144 vs. 58 months

(N=40)

(N=37)

median MS: 56 vs. 35 months

(N=40)

(N=37)

A B

Fig. 3: Kaplan-Meier estimates according to CA9 expression for A overall survival (OS) and B metastases survival (MS) for patients with metastatic ccRCC.

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