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Hemochromatosis gene mutations, HCV infection, alcohol intake and hepatocellular carcinoma with liver cirrhosis
Hepatocellular carcinoma, liver regeneration, apoptosis I
C04/05
EXPRESSION AND FUNCTION OF COSTIMULATORY MOLECULES IN HUMAN HEPATOCELLULAR CARCINOMA R.L. Jones 1, L.S. Young2, S.G. Hubscher 3, D.H. Adams 1 1Liver Research Laboratories, University of Birmingham, UK. 2CRC Institute for Cancer Studies, University of Birmingham, UK. 3Dept. of Histopathology, University of Birmingham, UK. H C C e x p r e s s e s M H C class 1 a n d is infiltrated b y l y m p h o c y t e s (TIL), m a k i n g it a target for inmaunotherapy. T I L are partially activated m e m o r y T cells w h i c h axe functionally s u p p r e s s e d . A c t i v a t i o n v i a e o s t i m u l a t o r y m o l e c u l e s C D 2 8 a n d C D 154 m a y e n h a n c e function. Aira: To d e t e r m i n e e x p r e s s i o n o f c o s t i m u l a t o r y m o l e c u l e s o n h u m a n H C C a n d TIL. M e t h o d s : H C C sections were stained w i t h m A b to CD28, C T L A - 4 a n d C D 1 5 4 a n d l i g a n d s CDS0, C D 8 6 a n d C D 4 0 . C D 2 8 a n d C T L A - 4 o n T I L w a s investigated b y f l o w cytometry. C D 1 5 4 m R N A in T I L w a s investigated b y P C R . T I L e x p a n d e d in vitro u s i n g IL-2 a n d a n t i - C D 2 8 m A b were a n a l y s e d for proliferation a n d activation. Results: H C C cells s h o w s t r o n g e x p r e s s i o n o f C D 4 0 b u t n o C D 8 0 or C D 8 6 . T I L e x p r e s s h i g h levels o f C D 2 8 . C T L A 4 is absent. C D 1 5 4 m R N A is p r e s e n t in e x p a n d e d TIL. T I L proliferate e q u a l l y in IL-2 a n d in IL-2 +anti C D 2 8 . C D 2 8 e x p r e s s i o n is lost after 3 w e e k s in culture. C o n c l u s i o n : T h e e x p r e s s i o n o f C D 2 8 o n TIL c o n f i r m s its potential for m a n i p u l a t i o n in i m m u n o t h e r a p y in H C C . T h e lack o f C T L A - 4 e x p r e s s i o n s u g g e s t s that activation u s i n g C D 8 6 transfection will n o t result in T cell a p o p t o s i s v i a C T L A - 4 signalling. A c t i v a t e d T I L c o u l d receive additional c o s t i m u l a t o r y signals t h r o u g h CD154.
C04/06
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I
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LONG-TERM OUTCOME AFTER PERCUTANEOUS ETHANOL INJECTION FOR THE TREATMENT OF SMALL HEPATOCELLULAR CARCINOMA A. Gallego, E. Montserrat l, X. Torras, J.M. Monill 1, J. Enriouez. J. Balanz6 Gastroenterology Department, Hospital de la Sta. Creu i St. Pau, Barcelona, Spain. 1Radiology Department, Hospital de la Sta. Creu i St. Pau, Barcelona, Spain. AIM: To assess the efficacy of percutaneous ethanol injection (PEI) for the treatment of small uninodular hepatocellular carcinoma (HCC). METHODS: We analyse the outcome of 50 patients (Child: A 27 / B 18 / C 5) with uninodular HCC < 5 cm (<3 era. in 23, and 3-5 cm. in 27) treated by PEI. Diagnostic was made by ultrasonography and fine needle aspiration. All patients were treated by ultrasound-guided PEI. A second fme needle aspiration procedure was performed 1 to 2 months after treatment and, when malignant cells persisted, a second series of PEI was indicated. We considered treatment failure the persistence of malignant cells after two initial series of PEI or after one series ifa second could not be done. Hepatic ultrasound and/or hepatic helical CT were performed every 4 to 6 months of follow-up. R.ecurmnces were treated with PEI when possible. Survival and free-recurrence probabilities were calculated by Kaplan-Meier and log-rank. RESULTS: PEI achieved initial success in 39 patients (78%). Among II patients with initial treatment failure, l0 (90,9 %) had tumour of 3-5 cm and only l had turnout < 3 cm (p=0,006). The 3-5 year survival was 43% and 29 %. Patients with initial success of PEI had a 3-5 year survival of 52 % and 35 %, while those with initial treatment failure had a 24 % probability of survival at 2 years (p--0,0001). The 3-5 year survival of Child A was: 69,3 % and 39,6 % vs. 14 % and 14 % in Child B (p=0,0011). None of Child C patients reached 2 years of survival. Among patients with initial success of PEI the 3-5 year probability of free-recurrence was 31,8 % and 25,4 %. No difference was seen in the 5 year survival probability between patients with or without recurrence. CONCLUSIONS: l-Survival after PEI is marked by the degree of hepatic function (assessed by Child score), and the initial success of treatment. 2-Patiants who better benefit from PEI are Child A with a single nodule < 3 cm. 3-There is a high rate of recurrence after PEI, although this fact does not significantly worsen the 5 year survival.
HEMOCHROMATOSIS GENE MUTATIONS, HCV INFECTION, ALCOHOL INTAKE AND HEPATOCELLULAR CARCINOMA WITH LIVER CIRRHOSIS M. Abonyi, Zs.K. N~meth, T. T6th, A. Tordai l, M. Prajda 2 1st Department of Medicine, Semmelweis Medical University, Budapest. ILaboratory for Molecular Biology of National Institute of Hematology and Immunology, Budapest. 2Meditest Ltd., Budapest. In the recent years with detection o f hemochromatosis gene (HFE) provides the analysis o f the HFE mutations and its role on the development of HCC in cirrhotic patients. Patients and methods: We studied 34 patients with H C C , all patients had liver cirrhosis, 44% were heavy alcohol drinkers, 10% were HBsAg positive, 57% were anti-HCV positive, 52% were HCV-RNA PCR positive. The control groups were 49 patients with chronic viral hepatitis without HCC and 52 healthy persons. The iron status was characterized by serum iron level, by serum ferritin level, by transferrin saturation and by liver biopsy and histology. The presence of HFE mutations were determined by PCR-restriction assay. Results: Hepatic iron overload was found in 68% o f HCC patients. The C282Y mutations were 1.6% - 3.2 % and 5.3 % in healthy controls - liver cirrhosis - and HCC. The occurance o f H63D mutations were 10.5 % - 16.5% - 16.9% o f the same groups. By serum and hepatic iron grading, the frequencies of C282Y and H63D alterations were 8.2% and 25.8% in HCC with iron overload (p < 0.031) and 3.4% and 14.6% in HCC patients with normal iron in serum and liver tissue. C282Y and H63D mutations were significantly higher in cirrhotic patients (CP) with iron overload than in healthy controls (p < 0.01). We found no difference of mutations (C282Y and H63D) in patients of HCC and of liver cirrhosis with iron overload. Conclusion: iron overload could be a risk factor for HCC in cirrhotic patients, as well as HCV RNA positivity and heavy alcohol intake.
I
C04/08
]
CHOLINE IS BENEFICIAL FOR CYPROFIBRATE-INDUCED OXIDATIVE ENZYMES ACTIVATION AND LIPID PEROXIDATION R. Koci6, G. Kocic, D. Pavlovic, T. Jevtovic, D. Mikic, R. Radenkovic, D. Denovic, D. Sokolovic Clin. for Endocr. & Inst. of Biochem. Med. Fac. Nis, Yugoslavia. Ciprofibrate represents a nowel class of fibrates, now a videly used drug in the treatment of hyperlipidemic disorders. As a hepattc peroxisome proliferator it constitutes a class of chemical carcinogens~ Increased 1-120_, generation and the activation of oxidative pathways may be among the most responsible for their tumor promoting activity The aim of this study was to investigate the effect of ciprofibrate on xanthine oxidase (XO) activity as a main free radical generating pathway. Also, the activity of triptophan oxygenase (TO), susceptible enzyme to liver damage, microsome induc~ile, was measured. The activation of protein kinase C for tumor promotion is an early event of fibrate tumor promoting activity and that is why theiir effect was investigated simultaneously.. Rat hepatocytes were isolated by collagenase technique under sterile conditions, and investigated effects were studied after period of 24h of incubation on 37°C. The activity of XO increased after single ciprofibrate (50mg/ml) treatment (4.?±6.5 vs 2.76a:0.23 U 10 cells of control p<0.001). It correlates well with the i~creased lipid peroxide- MDA level (I 1.69il.69 vs 8.84±1.02 nmolMDA/10 cells of controls p<0.001), while the activity of TO was not signilicanlly changed (I ..464-0.35 vs control 156-.z0..32 Innol/ml p<0.05). At the same time the abthty ot phorbol esters (PMA 25ng/ml) to act as a tumorpromoters correlate with their ability to increase MDAlevel (9.57±0.71 p<0.05) and in smaler extent they increased XO activity (3.42±0.68) but not TO activity (1.59±0.22). Given simultaneously they did not act sinergistically neither in increasing XO (4.13±0.35) and TO activity (1.62±0.24), nor in increasing MDA level (11.82±1.94). Since recent data pointed out on limited methylation pathway during fibrate treatment, the main lbcas of this report is the protective effect of choline (0.1mM/ml) on XO activity (3.74-0.8 p<0.05). Choline appears to be especially sufficient in blocking fi'ee radicals generation, expressed as MDA level (9.61±1.53 p<0.001) and it seems that it could be effective in inducin~ TO activity (1."/2±0.34"1, while given alone choline doesnot change sigmficantly the activity of XO (3.23±0.81), TO activity (I.60±0.23), but decreased MDA level (7.6±0.68 p<0.05), compared with the controls. Obtained preliminary results are very important in the improvement of the assessment of the possible protection of human risk from peroxisome proliferators and need further experimental in vivo application.
165
C04/05
EXPRESSION AND FUNCTION OF COSTIMULATORY MOLECULES IN HUMAN HEPATOCELLULAR CARCINOMA R.L. Jones 1, L.S. Young2, S.G. Hubscher 3, D.H. Adams 1 1Liver Research Laboratories, University of Birmingham, UK. 2CRC Institute for Cancer Studies, University of Birmingham, UK. 3Dept. of Histopathology, University of Birmingham, UK. H C C e x p r e s s e s M H C class 1 a n d is infiltrated b y l y m p h o c y t e s (TIL), m a k i n g it a target for inmaunotherapy. T I L are partially activated m e m o r y T cells w h i c h axe functionally s u p p r e s s e d . A c t i v a t i o n v i a e o s t i m u l a t o r y m o l e c u l e s C D 2 8 a n d C D 154 m a y e n h a n c e function. Aira: To d e t e r m i n e e x p r e s s i o n o f c o s t i m u l a t o r y m o l e c u l e s o n h u m a n H C C a n d TIL. M e t h o d s : H C C sections were stained w i t h m A b to CD28, C T L A - 4 a n d C D 1 5 4 a n d l i g a n d s CDS0, C D 8 6 a n d C D 4 0 . C D 2 8 a n d C T L A - 4 o n T I L w a s investigated b y f l o w cytometry. C D 1 5 4 m R N A in T I L w a s investigated b y P C R . T I L e x p a n d e d in vitro u s i n g IL-2 a n d a n t i - C D 2 8 m A b were a n a l y s e d for proliferation a n d activation. Results: H C C cells s h o w s t r o n g e x p r e s s i o n o f C D 4 0 b u t n o C D 8 0 or C D 8 6 . T I L e x p r e s s h i g h levels o f C D 2 8 . C T L A 4 is absent. C D 1 5 4 m R N A is p r e s e n t in e x p a n d e d TIL. T I L proliferate e q u a l l y in IL-2 a n d in IL-2 +anti C D 2 8 . C D 2 8 e x p r e s s i o n is lost after 3 w e e k s in culture. C o n c l u s i o n : T h e e x p r e s s i o n o f C D 2 8 o n TIL c o n f i r m s its potential for m a n i p u l a t i o n in i m m u n o t h e r a p y in H C C . T h e lack o f C T L A - 4 e x p r e s s i o n s u g g e s t s that activation u s i n g C D 8 6 transfection will n o t result in T cell a p o p t o s i s v i a C T L A - 4 signalling. A c t i v a t e d T I L c o u l d receive additional c o s t i m u l a t o r y signals t h r o u g h CD154.
C04/06
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I
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LONG-TERM OUTCOME AFTER PERCUTANEOUS ETHANOL INJECTION FOR THE TREATMENT OF SMALL HEPATOCELLULAR CARCINOMA A. Gallego, E. Montserrat l, X. Torras, J.M. Monill 1, J. Enriouez. J. Balanz6 Gastroenterology Department, Hospital de la Sta. Creu i St. Pau, Barcelona, Spain. 1Radiology Department, Hospital de la Sta. Creu i St. Pau, Barcelona, Spain. AIM: To assess the efficacy of percutaneous ethanol injection (PEI) for the treatment of small uninodular hepatocellular carcinoma (HCC). METHODS: We analyse the outcome of 50 patients (Child: A 27 / B 18 / C 5) with uninodular HCC < 5 cm (<3 era. in 23, and 3-5 cm. in 27) treated by PEI. Diagnostic was made by ultrasonography and fine needle aspiration. All patients were treated by ultrasound-guided PEI. A second fme needle aspiration procedure was performed 1 to 2 months after treatment and, when malignant cells persisted, a second series of PEI was indicated. We considered treatment failure the persistence of malignant cells after two initial series of PEI or after one series ifa second could not be done. Hepatic ultrasound and/or hepatic helical CT were performed every 4 to 6 months of follow-up. R.ecurmnces were treated with PEI when possible. Survival and free-recurrence probabilities were calculated by Kaplan-Meier and log-rank. RESULTS: PEI achieved initial success in 39 patients (78%). Among II patients with initial treatment failure, l0 (90,9 %) had tumour of 3-5 cm and only l had turnout < 3 cm (p=0,006). The 3-5 year survival was 43% and 29 %. Patients with initial success of PEI had a 3-5 year survival of 52 % and 35 %, while those with initial treatment failure had a 24 % probability of survival at 2 years (p--0,0001). The 3-5 year survival of Child A was: 69,3 % and 39,6 % vs. 14 % and 14 % in Child B (p=0,0011). None of Child C patients reached 2 years of survival. Among patients with initial success of PEI the 3-5 year probability of free-recurrence was 31,8 % and 25,4 %. No difference was seen in the 5 year survival probability between patients with or without recurrence. CONCLUSIONS: l-Survival after PEI is marked by the degree of hepatic function (assessed by Child score), and the initial success of treatment. 2-Patiants who better benefit from PEI are Child A with a single nodule < 3 cm. 3-There is a high rate of recurrence after PEI, although this fact does not significantly worsen the 5 year survival.
HEMOCHROMATOSIS GENE MUTATIONS, HCV INFECTION, ALCOHOL INTAKE AND HEPATOCELLULAR CARCINOMA WITH LIVER CIRRHOSIS M. Abonyi, Zs.K. N~meth, T. T6th, A. Tordai l, M. Prajda 2 1st Department of Medicine, Semmelweis Medical University, Budapest. ILaboratory for Molecular Biology of National Institute of Hematology and Immunology, Budapest. 2Meditest Ltd., Budapest. In the recent years with detection o f hemochromatosis gene (HFE) provides the analysis o f the HFE mutations and its role on the development of HCC in cirrhotic patients. Patients and methods: We studied 34 patients with H C C , all patients had liver cirrhosis, 44% were heavy alcohol drinkers, 10% were HBsAg positive, 57% were anti-HCV positive, 52% were HCV-RNA PCR positive. The control groups were 49 patients with chronic viral hepatitis without HCC and 52 healthy persons. The iron status was characterized by serum iron level, by serum ferritin level, by transferrin saturation and by liver biopsy and histology. The presence of HFE mutations were determined by PCR-restriction assay. Results: Hepatic iron overload was found in 68% o f HCC patients. The C282Y mutations were 1.6% - 3.2 % and 5.3 % in healthy controls - liver cirrhosis - and HCC. The occurance o f H63D mutations were 10.5 % - 16.5% - 16.9% o f the same groups. By serum and hepatic iron grading, the frequencies of C282Y and H63D alterations were 8.2% and 25.8% in HCC with iron overload (p < 0.031) and 3.4% and 14.6% in HCC patients with normal iron in serum and liver tissue. C282Y and H63D mutations were significantly higher in cirrhotic patients (CP) with iron overload than in healthy controls (p < 0.01). We found no difference of mutations (C282Y and H63D) in patients of HCC and of liver cirrhosis with iron overload. Conclusion: iron overload could be a risk factor for HCC in cirrhotic patients, as well as HCV RNA positivity and heavy alcohol intake.
I
C04/08
]
CHOLINE IS BENEFICIAL FOR CYPROFIBRATE-INDUCED OXIDATIVE ENZYMES ACTIVATION AND LIPID PEROXIDATION R. Koci6, G. Kocic, D. Pavlovic, T. Jevtovic, D. Mikic, R. Radenkovic, D. Denovic, D. Sokolovic Clin. for Endocr. & Inst. of Biochem. Med. Fac. Nis, Yugoslavia. Ciprofibrate represents a nowel class of fibrates, now a videly used drug in the treatment of hyperlipidemic disorders. As a hepattc peroxisome proliferator it constitutes a class of chemical carcinogens~ Increased 1-120_, generation and the activation of oxidative pathways may be among the most responsible for their tumor promoting activity The aim of this study was to investigate the effect of ciprofibrate on xanthine oxidase (XO) activity as a main free radical generating pathway. Also, the activity of triptophan oxygenase (TO), susceptible enzyme to liver damage, microsome induc~ile, was measured. The activation of protein kinase C for tumor promotion is an early event of fibrate tumor promoting activity and that is why theiir effect was investigated simultaneously.. Rat hepatocytes were isolated by collagenase technique under sterile conditions, and investigated effects were studied after period of 24h of incubation on 37°C. The activity of XO increased after single ciprofibrate (50mg/ml) treatment (4.?±6.5 vs 2.76a:0.23 U 10 cells of control p<0.001). It correlates well with the i~creased lipid peroxide- MDA level (I 1.69il.69 vs 8.84±1.02 nmolMDA/10 cells of controls p<0.001), while the activity of TO was not signilicanlly changed (I ..464-0.35 vs control 156-.z0..32 Innol/ml p<0.05). At the same time the abthty ot phorbol esters (PMA 25ng/ml) to act as a tumorpromoters correlate with their ability to increase MDAlevel (9.57±0.71 p<0.05) and in smaler extent they increased XO activity (3.42±0.68) but not TO activity (1.59±0.22). Given simultaneously they did not act sinergistically neither in increasing XO (4.13±0.35) and TO activity (1.62±0.24), nor in increasing MDA level (11.82±1.94). Since recent data pointed out on limited methylation pathway during fibrate treatment, the main lbcas of this report is the protective effect of choline (0.1mM/ml) on XO activity (3.74-0.8 p<0.05). Choline appears to be especially sufficient in blocking fi'ee radicals generation, expressed as MDA level (9.61±1.53 p<0.001) and it seems that it could be effective in inducin~ TO activity (1."/2±0.34"1, while given alone choline doesnot change sigmficantly the activity of XO (3.23±0.81), TO activity (I.60±0.23), but decreased MDA level (7.6±0.68 p<0.05), compared with the controls. Obtained preliminary results are very important in the improvement of the assessment of the possible protection of human risk from peroxisome proliferators and need further experimental in vivo application.
165