S170 POSTERS 445 INHIBITION OF HYPOXIA-INDUCIBLE CARBONIC ANHYDRASE IX ENHANCES HEXOKINASE II INHIBITOR- INDUCED HEPATOCELLULAR CARCINOMA CELL APOPTOSIS J.H. Yoon 1 , S.J. Myung 1 , G.Y. Gwak 1 , S.J. Yu 1 , Y.J. Jung 1 , H.S. Lee 1 , J.S. Lee 2 , Y.J. Lee 3 . 1 Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, 2 Department of Internal Medicine, Ilsanpaik Hospital, Inje University, Goyang, 3 Department of Internal Medicine, Paik Hospital, Inje University, Pusan, South Korea E-mail: yoonjh@snu.ac.kr Background and Aims: Cancer cells may adapt to pH changes and thereby, grow at low pHs. In acidic environments, many signals are acti- vated to augment cancer cell growth and invasion. Carbonic anhydrase-9 (CA-IX), an enzyme involved in lowering pH, is known to be over- expressed in many cancer cells. Hypoxic condition, which large or infiltra- tive hypovascular tumor may encounter, also produces acidic environments. This study was to examine if CA-IX is hypoxia-inducibly expressed in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients. Methods: Human HCC cell line (Huh-7) was used in this study. CA- IX expression was evaluated using immunoblot analysis in cells grown either in a normoxic or hypoxic condition. Cells were treated with CA-IX inhibitor, 4-(2-aminoethyl)benzenesulfonamide, in the presence or absence of hexokinase II inhibitor, 3-bromopyruvate (3-BP), which has previously been demonstrated to induce HCC cell apoptosis more efficiently in hypoxic state than in normoxic state. Cell growth was assessed using the MTS assay, and apoptotic and kinase signaling were explored by immunoblot analysis. A clinico-pathological analysis in patients who had undergone surgical resection of HCC was evaluated by tissue array method and immunohistochemistry of CA-IX. Results: HCC cells expressed CA-IX, and its expression was increased in cells cultured under hypoxic condition as compared to normoxic cells. The CA-IX inhibitor alone did not induce HCC cell apoptosis, while the simultaneous treatment of this inhibitor with 3-BP enhanced 3-BP- induced apoptosis, in particular by activating caspase 9 more efficiently. This enhanced apoptosis was due to more augmented JNK activation in cells treated with both inhibitors as compared to 3-BP-treated cells. A clinico-pathological analysis revealed that tumoral CA-IX extent was positively related to Edmondson grade and its intensity was negatively correlated with recurrence free survival in HCC patients. Conclusions: This study demonstrates that the inhibition of hypoxia- inducible CA-IX enhances hexokinase II inhibitor-induced HCC cell apoptosis, and that CA-IX expression profiles may have a prognostic impli- cation in HCC patients. Thus, the blockage of this enzyme in combination with hexokinase II inhibitor may therapeutically be useful in patients with large or infiltrative hypovascular HCCs, which are aggressively growing under hypoxic environment. 446 REDUCED HEPATIC EXPRESSION OF PERFORIN AND GRANZYME B IN PATIENTS WITH HEMOPHAGOCYTIC SYNDROME M. Lemoine 1 , N. BenMena 2 , N. Ganne-Carrie 1 , N. Aras 3 , C. Laroche 4 , D. Roulot 1 , J. Stirnemann 3 , O. Fain 3 , M. Ziol 2 . 1 Service d’Hepatologie, 2 Service d’Anatomopathologie, 3 Service de Medecine interne, AP-HP, Hˆ opital Jean-Verdier, Bondy, 4 Service de Medecine interne, AP-HP, Hˆ opital Avicenne, Bondy, France E-mail: marianne.ziolbis@jvr.aphp.fr Hemophagocytic syndrome (HS) is a rare life-threatening condition char- acterized by uncontrolled macrophagic activation with hemophagocytosis. Perforin (Prf) and Granzyme B (GzmB) are cytolytic proteins stored in secretory granules of cytotoxic T lymphocytes and Natural Killer cells. Perforin mutations have been demonstrated in patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis. The pathogenesis of reactive HS and especially the involvement of cytotoxic molecules remains poorly investigated. Aims: to assess Perforin and Granzyme B expression in the liver of adult patients with reactive HS compared to control subjects with normal liver histology. Methods: From January 2000 to December 2006, adult patients with reactive HS established according to the published criteria (fever, splenomegaly, cytopenia, hypertriglyceridaemia and/or hypofibrinogene- mia, histological or cytological evidence of hemophagocytosis) who un- derwent liver biopsy at the time of diagnosis, were selected. Eleven normal liver biopsies (performed at distance from a benign nodule) were used as controls. The ratio of Perforin and Granzyme B positive lymphocytes to the number of T (CD3+) lymphocytes (Prf/CD3, GzmB/CD3) were assessed on immunostained liver biopsy sections. Results: 25 patients were selected (mean age = 52 years; M/F: 16/9). Associated diseases consisted in hematological malignancy (44%) or infection in association of immunosuppression related to HIV infection (56%). The ratio of Prf/CD3 and GzmB/CD3 were dramatically decreased compared to controls (0.14±0.17 vs 0.72±0.18, p < 0.0001 and 0.12±0.13 vs 0.41±0.32, p = 0.003 respectively). Conclusions: We provide here evidence that the ratio of Perforin and Granzyme B positive lymphocyte to the total number of T lymphocytes is profoundly reduced in the liver of patients with reactive HS, compared to control biopsies. These results suggest for the first time to our knowl- edge the involvement of Perforin/Granzyme B pathway deficiency in the pathogenesis of reactive HS. 04B. MOLECULAR AND CELLULAR BIOLOGY – B) BILIARY TRACT PATHOPHYSIOLOGY 447 SKP2 OVEREXPRESSION AND CYTOPLASMIC P27KIP1 IMMUNOREACTIVITY ARE PREDICTORS OF POOR PROGNOSIS IN PATIENTS WITH EXTRAHEPATIC BILE DUCT CANCER Y.H. Baek 1 , H.J. Kim 1 , J.H. Park 1 , D.I. Park 1 , Y.K. Cho 1 , C.I. Sohn 1 , W.K. Jeon 1 , B.I. Kim 1 , M.K. Kim 2 , D.H. Kim 2 , J.H. Sohn 2 . 1 Department of Internal Medicine, 2 Department of Pathology, Sungkyunkwan University Kangbuk Samsung Hospital, Seoul, South Korea E-mail: hongjoo3.kim@samsung.com Background and Aims: S-phase kinase associated protein 2 (SKP2) is an F-box substrate-recognition subunit of the SCF ubiquitin-protein ligase complex, which regulates progression of cell cycle by targeting regulators, such as p27kip1. Decreased levels of p27kip1 seem to be associated with high aggressiveness and poor prognosis in a variety of cancer. However, there has been no report concerning the expression status and the prognostic implication of SKP2 in extrahepatic bile duct cancer. Methods: We investigated the expression status of SKP2 and p27kip1 by immunohistochemical analyses in 50 extrahepatic bile duct cancer patients who underwent curative surgery at our institution. Nuclear staining of SKP2 and p27kip1 was scored on a semiquantitative scale by evaluating the percentage of stained nuclei within representative areas of each tumor. Expression of SKP2 was graded as 0 (<10%), 1+ (10-25%), 2+ (25-50%), and 3+ (>50% of tumor cells showing moderate to strong immunopositivity). The grading systems for the expression of nuclear and cytoplasmic p27kip1 were identical to that of nuclear SKP2.