EXTERNAL VALIDATION OF IMP3 EXPRESSION AS AN INDEPENDENT PROGNOSTIC MARKER FOR METASTATIC PROGRESSION AND DEATH FOR PATIENTS WITH CLEAR CELL RENAL CELL CARCINOMA

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CONCLUSIONS: These results demonstrate the exclusive activation of P2X1 receptors with no involvement of P2X3, muscarinic RU 3< UHFHSWRUV LQ Įȕ0H$73LQGXFHG EODGGHU FRQWUDFWLRQV LQ DQHVWKHWL]HGPLFH7KLVPRXVHPRGHOUHSUHVHQWVWKHUHIRUHDUHOLDEOH tool for in vivo screening of new P2X1 receptors antagonists for the treatment of overactive bladder. Source of Funding: None

Kidney Cancer: Basic Research (III) Moderated Poster Session 13 Sunday, May 18, 2008

1:00 - 3:00 pm

373 TYPE OF VHL MUTATION AND TUMOR SIZE AFFECT GROWTH RATES OF RENAL MASSES IN VHL PATIENTS Kiranpreet Khurana*, Paul Albert, Rabindra Gautam, Peter A Pinto, W Marston Linehan, Gennady Bratslavsky. Bethesda, MD. INTRODUCTION AND OBJECTIVE: Renal carcinogenesis LVNQRZQWREHLQÀXHQFHGE\VPRNLQJK\SHUWHQWLRQERG\PDVVLQGH[ (BMI), and mutation of the von Hippel Lindau (VHL) gene. Additionally, the frequency of multiple solid renal tumors in VHL patients is greater with germline mutations leading to a truncated VHL protein. With many patients undergoing active surveillance for small renal masses we HYDOXDWHWKHLQÀXHQFHRIWKHVHIDFWRUVRQJURZWKUDWHVRIUHQDOPDVVHV in VHL population. METHODS: We retrospectively reviewed radiographic studies (CT or MRI) of VHL patients managed at the National Cancer Institute within the past 24 months. Patients with at least one solid enhancing renal lesion greater than 1.8 cm during the last follow up who had at least two prior interval tumor measurements were included in the study. In each patient the largest solid lesion was selected and the volume of tumor calculated using two largest dimensions. The slope of the growth rate for the largest solid renal lesion was estimated using the least-squares regression. Wilcoxon rank sum test was used to compare the growth rates of renal masses in patients with and without smoking, hypertension, and truncating VHL mutations, while Pearson’s correlation was used to evaluate for relationship between BMI and the growth rates. 5(68/76:HLGHQWL¿HGSDWLHQWVWKDWIXO¿OOHGRXULQFOXVLRQ criteria. The median age was 44 years (range 23 to 69) and 56% were males. The median follow up was 3.0 years (range 0.5-6.3). The median increase in tumor volume was 54% per year (range -45% to 285%). There ZDVDQLQYHUVHUHODWLRQVKLSEHWZHHQWKHWXPRUVL]HDQGWKHJURZWKUDWH (p<0.0001). The growth rates of tumors with germline mutations leading WRWUXQFDWHG9+/SURWHLQZHUHVLJQL¿FDQWO\JUHDWHUWKDQWKHJURZWKUDWHV of tumors with non-truncating mutations with 74% and 41% increase in tumor volume per year, respectively (p<0.01). Smoking, hypertension, DQG%0,GLGQRWLQÀXHQFHWKHJURZWKUDWHV CONCLUSIONS: This study examines the growth rates of largest solid clear cell renal tumors in VHL patients. There is an inverse UHODWLRQVKLSEHWZHHQWKHWXPRUVL]HDQGWKHJURZWKUDWH$GGLWLRQDOO\ UHQDOPDVVHVZLWKWKHWUXQFDWLQJ9+/PXWDWLRQVJURZVLJQL¿FDQWO\IDVWHU WKDQWKHWXPRUVZLWKQRQWUXQFDWLQJPXWDWLRQV7KHVH¿QGLQJVPD\EH useful in determining patient’s follow up and designing active surveillance studies and clinical protocols. Source of Funding: NIH Intramural Research.

CHROMOSOMAL ABNORMALITIES OF TUMOR ENDOTHELIAL CELLS IN HUMAN RENAL CELL CARCINOMA Tomoshige Akino*, Kyoko Hida, Kunihiko Tsuchiya, Yasuhiro Hida, Michael Klagsbrun, Kohei Matsuda, Noritaka Ohga, Takashige Abe, Ataru Sazawa, Toru Harabayashi, Nobuo Shinohara, Masanobu Shindoh, Katsuya Nonomura. Sapporo, Japan, and Boston, MA. INTRODUCTION AND OBJECTIVE: An important concept in tumor angiogenesis is that tumor blood vessels contain genetically normal and stable endothelial cells unlike tumor cells, which typically display genetic instability. We have reported that endothelial cells in solid tumors were cytogenetically abnormal (Hida K. et al., Can Res 2004). In WKHPRXVH[HQRJUDIWPRGHOVÀXRUHVFHQFHLQVLWXK\EULGL]DWLRQ),6+  analysis showed that freshly isolated uncultured tumor were aneuploid. We herein tested the hypothesis that human tumor endothelial cells have chromosomal aberrations. METHODS: Twenty specimens of clinically obtained from UHQDO FHOO FDUFLQRPD DJHV  WR  JHQGHU  ZHUH PDOH 7 VWDJH777KLVWRORJLFDOW\SHVFOHDUFHOOSDSLOODU\ chromophobe, 1 clear cell with sarcmatoid change) were studied. Tumor endothelial cells were isolated from tumor specimens immediately after surgery using magnetic cell sorting system as previously described (Hida K. et al., Can Res 2004). As a normal counterpart, normal renal endothelial cells were isolated far from tumor in the same specimen when it was possible. The degree of aneuploidy in uncultured, freshly LVRODWHGDQGF\WRVSXQWXPRUHQGRWKHOLDOFHOOVZHUHDQDO\]HGE\),6+ :HDOVRSHUIRUPHGDQDO\VLVRISORLG\E\),6+LQWKHIUR]HQVHFWLRQVRI human renal cell carcinoma and normal renal tissue samples. RESULTS: Aneuploid endothelial cells were observed in all 20 of human renal cell carcinomas. We observed that more than 35% of uncultured renal tumor endothelial cells were aneuploid. However normal endothelial cells were mostly diploid, with only 5% aneuploid degree which is within normal range. In tissue sections, aneuploid endothelial cells were detected in renal cell carcinoma vessels, whereas there was no aneuploid endothelial cell detected in normal renal vessels. &21&/86,216 7KLV LV WKH ¿UVW UHSRUW RI FKURPRVRPDO abnormalities of tumor endothelial cells in human renal cell carcinoma. The possibility that cytogenetically abnormal tumor endothelial cells is chemotherapy resistant warrants further investigation. Source of Funding: None

375 EXTERNAL VALIDATION OF IMP3 EXPRESSION AS AN INDEPENDENT PROGNOSTIC MARKER FOR METASTATIC PROGRESSION AND DEATH FOR PATIENTS WITH CLEAR CELL RENAL CELL CARCINOMA Nathan E Hoffmann*, Yuri Sheinin, Christine M Lohse, Alexander S Parker, Bradley C Leibovich, Zhong Jiang, Eugene D Kwon. Rochester, MN, Jacksonville, FL, and Boston, MA. INTRODUCTION AND OBJECTIVE: High quality, external validation studies have recently been highlighted to be of paramount importance for proper translation of prognostic markers into the clinical setting. To that end, we examined associations of the insulin-like growth factor-II mRNA binding protein, IMP3, with clinical and pathologic features of clear cell renal cell carcinoma (ccRCC) in an independent cohort of patients to validate recent work showing IMP3 as a prognostic marker for RCC progression and death. METHODS: We studied 716 consecutive tumor specimens from patients treated with surgery at our institution for unilateral, sporadic, non-cystic ccRCC between 1990 and 1999. Tissues were stained and scored for IMP3 expression, and these expression levels were correlated with clinical and pathologic features as well as clinical outcomes including progression to distant metastases and death from RCC. RESULTS: We observed that 213 (29.8%) ccRCC specimens expressed tumor cell IMP3. IMP3 expression was associated with advanced stage and grade of primary tumors as well as other adverse features including coagulative tumor necrosis and sarcomatoid differentiation. After multivariate adjustment for ccRCC prognostic features, positive IMP3 expression was still associated with a 42% LQFUHDVHLQWKHULVNRIGHDWKIURP5&&ULVNUDWLRS  $PRQJ WKH VXEVHW RI SDWLHQWV ZLWK FOLQLFDOO\ ORFDOL]HG GLVHDVH SRVLWLYH ,03

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expression was associated with a nearly 5-fold increased risk of distant PHWDVWDVHVULVNUDWLRS  CONCLUSIONS: Using a large and independent cohort RI FF5&& SDWLHQWV ZH FRQ¿UPHG WKDW WXPRU FHOO ,03 H[SUHVVLRQ represents an independent predictor of aggressive ccRCC tumor behavior. Source of Funding:7KH5LFKDUG06FKXO]H)DPLO\ Foundation, The Commonwealth Foundation for Cancer Research, and The Helen and Martin Kimmel Foundation.

376 HEPARANASE EXPRESSION IN RENAL CELL CARCINOMAS AND ITS ASSOCIATION WITH TUMOR INVASION Shuji Mikami*, Mototsugu Oya, Masayuki Shimoda, Ryuichi Mizuno, Makio Mukai, Motowo Nakajima, Yasunori Okada. Tokyo, Japan. INTRODUCTION AND OBJECTIVE: Heparan sulfate proteoglycans (HSPGs) are one of the major components of extracellular matrix (ECM) and basement membrane (BM), and they play roles in the insolubility and stability of BM and ECM. Heparanase (HPSE) activity, which degrades HSPGs, had been detected in metastatic tumor cells, showing a positive correlation with metastatic potential. HPSE gene was recently cloned, and its expression is observed in malignant tumors. However, to our knowledge, there has been no report on HPSE expression in renal cell carcinomas (RCCs). The purpose of this study was to investigate HPSE expression in RCCs and its association with tumor invasion. METHODS: Seventeen RCCs and 6 non-neoplastic renal WLVVXHV ZHUH DQDO\]HG IRU +36( P51$ H[SUHVVLRQ E\ UHDOWLPH 3&5 )RUW\¿YH 5&&V DQG WKHLU QRQQHRSODVWLF UHQDO WLVVXHV ZHUH immunostained with anti-HPSE antibody and the data were semiquantitatively evaluated. Involvement of HPSE in the invasiveness of RCC cell lines, 786-O and Caki-2 cells was investigated by downregulating the gene expression with small interfering RNA (siRNA) using Matrigel invasion assay. RESULTS: Expression levels of HPSE mRNA and protein ZHUH VLJQL¿FDQWO\ KLJKHU LQ FOHDU FHOO 5&&V WKDQ LQ SDSLOODU\ 5&&V chromophobe RCCs and non-neoplastic renal tissues. HPSE protein expression levels were positively correlated with progression of primary tumor, distant metastasis, venous invasion and pathological stage. siRNA targeting HPSE down-regulated HPSE mRNA and protein expression, and resulted in inhibition of the invasion of 786-O and Caki-2 cells, whereas non-targeting siRNA showed no effect. CONCLUSIONS: Our study demonstrated the predominant HPSE expression in clear cell RCCs with positive correlation to malignant SKHQRW\SH:HKDYHDOVRSURYLGHGWKH¿QGLQJVWKDWWKHGRZQUHJXODWLRQ of HPSE expression effectively inhibits the invasion of RCC-derived cancer cells through Matrigel in vitro. The data suggest that HPSE plays a role in invasion and metastasis, and silencing of the gene may be a potential target for clear cell RCCs. Source of Funding:*UDQWLQ$LGIRU6FLHQWL¿F5HVHDUFK from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

377 KAI1 IS A NOVEL BIOMARKER FOR CHROMOPHOBE RENAL CELL CARCINOMA Eric C Kauffman*, Daniel A Barocas, Ximing J Yang, Huixian Liu, Douglas S Scherr, Jiangling J Tu. New York, NY, and Nashville, TN. INTRODUCTION AND OBJECTIVE: Identification of PROHFXODU PDUNHUV VSHFL¿F IRU UHQDO FHOO FDUFLQRPD 5&&  KLVWRORJLF subtypes and oncocytomas is an ongoing challenge. Chromophobe 5&& DQG RQFRF\WRPDV LQ SDUWLFXODU FDQ EH GLI¿FXOW WR GLVWLQJXLVK E\ convential light microscopy, and individual molecular markers have thus far been unreliable. KAI1 is a metastasis suppressor gene whose expression is lost in a wide range of high-grade cancer types and WKHLUPHWDVWDVHVEXWKDV\HWWREHFKDUDFWHUL]HGLQUHQDOWXPRUV:H investigated whether KAI1 is differentially expressed in RCC histologic subtypes and oncocytomas. METHODS: Immunohistochemical staining for KAI1 was performed using tissue microarrays with a total of 151 patient

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kidney specimens, including 47 clear cell RCC, 33 papillary RCC, 32 chromophobe RCC, 29 oncocytomas and 10 normal kidneys. KAI1 expression was assessed by an experienced genitourinary pathologist based on the percentage of cells staining and the degree of staining, and was scored as 0 (none), 1+ (low), 2+ (moderate), or 3+ (high). RESULTS: In normal kidney, KAI1 showed low to moderate membranous staining of the distal tubule cells but not cells in proximal tubules or glomeruli. Absence of KAI1 expression was observed in 94% (44/47) of clear cell cancers, 97% (32/33) of papillary cancers, and 97% (28/29) of oncocytomas, and when expressed in these tumor types, levels were typically low (1+). In contrast, 88% (28/32) of chromophobe cancers expressed KAI1, with most (66%) expressing moderate to high levels (2-3+). The positive and negative predictive values for KAI1 staining in distinguishing chromophobe RCC from oncocytoma were 97% and 88%, respectively. CONCLUSIONS: KAI1 is a novel biomarker for chromophobe 5&& DQG WR RXU NQRZOHGJH WKH ¿UVW UHSRUWHG FKURPRSKREH 5&& protein that is largely absent in oncocytoma. KAI1 staining may thus be clinically useful in the diagnostic differentiation of chromophobe RCC and oncocytoma. It remains to be determined whether KAI1 H[SUHVVLRQLQNLGQH\KDVDUROHLQFDQFHUUHJXODWLRQRUVLPSO\UHÀHFWV chromophobe RCC lineage from the distal nephron, where expression was also detected in this study KAI1 Expression in Renal Tumors KAI1 staining positive negative

Chromophobe Clear cell RCC Papillary RCC RCC (N=47) (n=33) (n=32) 3 (6.3%) 1 (3%) 28 (87.5%) 44 (92.7%) 32 (97%) 4 (12.5%)

Oncocytoma (n=29) 1 (3.4%) 28 (96.6%)

Source of Funding: None

378 COINHIBITORY MOLECULE EXPRESSION AND LYMPHOCYTIC INFILTRATION IN NON-CLEAR CELL RENAL CELL CARCINOMA Timothy J Roth*, Nathan E Hoffmann, R Houston Thompson, Christine M Lohse, Susan M Kuntz, Bradley C Leibovich, Michael L Blute, Yuri Sheinin, Eugene D Kwon. Rochester, MN, and New York, NY. INTRODUCTION AND OBJECTIVE: Introduction: Expression of the coinhibitory molecules B7-H1 and B7-H3 as well as lymphocytic invasion in clear cell RCC tumors is associated with aggressive forms of 5&&DVZHOODVLQFUHDVHGULVNRIFDQFHUVSHFL¿FGHDWK7KHJRDORIWKLV study is to evaluate these features in non-clear cell RCC tumors. 0(7+2'6:HHYDOXDWHGO\PSKRF\WLFLQ¿OWUDWLRQDQGWXPRU expression of B7-H1 and B7-H3 in 866 specimens from patients who underwent nephrectomy at our institution between 1990 and 1999 for sporadic, unilateral RCC. RESULTS: There were 661 (76.3%) non-cystic clear cell RCC specimens, 158 (18.2%) papillary RCC, and 47 (5.4%) chromophobe 5&&/\PSKRF\WLFLQ¿OWUDWLRQZDVHYLGHQWLQ FOHDUFHOO (41.1%) papillary, and 18 (38.3%) chromophobe RCC tumors (p<0.001). The proportion of tumors expressing B7-H1 or B7-H3 was also associated with histologic subtype: 128 (19.4%) clear cell, 13 (8.2%) papillary, and 7 (14.9%) chromophobe tumors expressed B7-H1 (p=0.004). B7-H3 H[SUHVVLRQZDVLGHQWL¿HGLQ FOHDUFHOO SDSLOODU\ and 21 (44.7%) chromophobe RCC tumors (p<0.001). CONCLUSIONS: Conclusion: RCC is considered an immunogenic tumor and accordingly we identified lymphocytic LQ¿OWUDWLRQ LQ QHDUO\  RI FOHDU FHOO 5&& WXPRUV DQG  RI QRQ FOHDUFHOOVXEW\SHV$VO\PSKRF\WLFLQ¿OWUDWLRQLQFOHDUFHOO5&&WXPRUV LVDVVRFLDWHGZLWKDQLQFUHDVHGULVNRIFDQFHUVSHFL¿FGHDWKWKHORZHU UDWHRIO\PSKRF\WLFLQ¿OWUDWLRQLQSDSLOODU\DQGFKURPRSKREHWXPRUVLV consistent with the improved outcomes observed in patients with these non-clear cell subtypes of RCC. Aberrant expression of the coinhibitory molecules B7-H1 or B7-H3 on RCC tumor cells is believed to result LQ 7 FHOO LQKLELWLRQ DOORZLQJ 5&& WXPRU SURJUHVVLRQ LQ D ORFDOL]HG environment of immunosuppression. We observed that both papillary and chromophobe subtypes of RCC can aberrantly express these molecules, as well as clear cell RCC tumors. Expression of either B7-H1 or B7-H3 is not restricted to a particular RCC subtype. Strikingly, B7-H3 expression is more than twice as prevalent in chromophobe RCC