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 metastasis 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 metastasis 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; expression of carbonic anhydrase 9 (CA9) was found in 95% of the clear cell subtype of renal cell carcinoma (ccRCC) (Liao et al., 1997) high expression of CA9 is associated with improved prognosis in ccRCC patients (Bui et al., 2004) controversially high expression of CA9 was associated with worst 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: Results: Table 1: Patients clinical characteristics by CA9 expression in lung metastases of ccRCC characteristicsoverall CA9 lowhigh patients sex men52 women31 age at time of nephrectomy mean median range at time of metastases surgery mean median range metastasis at time of nephrectomy no yes T stage primary tumor node status primary tumor no yes grading (Fuhrman) primary tumor staging (UICC 1997) primary tumor I+II III+IV lung metastases mean834 median range grading lung metastases relapse after first metastases surgery no yes node metastases after first metastases surgery no yes deaths p value (ANOVA) AB C progression free survival overall survival Fig. 2 Kaplan-Meier estimates according to CA9 expression for A progression free survival, B metastases relapse free survival and C overall survival for patients with metastatic ccRCC. months following nephrectomy months following metastases surgery metastases survival group 1 with low CA9 expression group 2 with high CA9 expression log-rank p = log-rank p = log-rank p = Conclusion:  investigations of CA9 expression in patients with metastatic ccRCC demonstrates that CA9 is associated with survival  patients with high CA9 expression in lung metastases had a significant higher survival than patients with low CA9 expression  CA9 expression was significantly associated with age at time of nephrectomy and with grading of lung metastases  CA9 expression also influenced by metastases at time of nephrectomy and lymph node metastases at time of lung surgery, but not significantly  interestingly number of metastases is not associated with survival between CA9 groups  CA9 is an independent predictor of survival for metastatic ccRCC patients corresponding should be address to:Dr. rer. nat. Pierre Tennstedt Technical University Dresden Department of Urology Fetscherstraße Dresden Germany study cohort consisted of 83 ccRCC Patients (52 ♂ 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 metastasis surgery lymph nodes were resected and reviewed on microscopic slides for tumor affection median age 60 years (range 40 to 75) median survival after metastases surgery for 45 patients who died of RCC related cause was 25 months (range 2 to 56) and for all patients 40 months (range 1 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 17 months (range 1 to 351) and 27 months (range 1 to 351), respectively primary tumors classified after the 2002 TNM staging and classified after UICC 1997 lung metastases from cohort of 83 patients were obtained from Center for Pneumology and Thoracic Surgery, Coswig Germany two punches from every lung metastasis were taken from select morphologically, representative regions of each paraffin embedded metastasis and homolog normal lung tissue from every patient 557 metastases and 109 normal lung tissue were arrayed on 9 paraffin blocks Sections (4 µm) from each tissue array block were transferred to silanized slides (Dako) evaluation of histology and grade (after Fuhrman) 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 expression of CA9 was analyzed by product of positively stained tumors cells and staining intensity evaluation of positively stained tumor cell was based on scale of 0 to 100 percent and staining intensity on a 4 point scale of 0 to 3 (low <300, high = 300) CA9 staining was highly reproducible indicated by a high level of concordance of both punches from the same metastases Statistical analysis: analyses were performed with SPSS software outcome of interest was overall survival from time after nephrectomy to death or last follow-up and survival from first lung metastases surgery to death or last follow-up statistical software was used to define a cutoff to classified patients according CA9 expression Kaplan-Meier curves were used to visualize association of CA9 expression with overall survival log-rank test was used to test difference between stratified survival functions analysis of variance (ANOVA) was used to test stratified CA9 groups 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: 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: Liao S-Y, Aurelio ON, Jan K, Zavada J, Stanbridge EJ (1997) Cancer Res. 57: 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. median group 1: 21 median group 2: 53 median group 1: 36 median group 2: 55 median group 1: 60 median group 2: 101 AM Fig. 1 CA9 staining was present in 93,3 % of all metastases from ccRCC patients. Representative punches shows A low CA9 expression and B high CA9 expression. C In normal lung tissue no CA9 staining was observed ABC