408A A A S L D A B S T R A C T S HEPATOLOGY O c t o b e r 1995

1205 CYCLIN-DEPENDENT KINASE 4 INHIBITORS (pl61NK4A/MTS 1 and p15INK4B/MTS2) GENES STRUCTURE AND EXPRESSION IN HEPATOCELLULAR CARCINOMAS (HCC). A Delmer. C Senamand-Beaufort. V Blanquet*, P Paterlini. C Brdchot. Liver Cancer and Molecular Virology, INSERM U 370 ; *U 383, CHU NECKER, PARIS, FRANCE.

Progression through the cell cycle is driven by the cylin dependent kinases (cdk). The cdk4 inhibitors (p l6 INK4A/MTSI /CDKN2 and p l5INK4B/MTS2) genes, have appeared as putative novel tumor- suppressor genes because of the high frequency of homozygous deletion observed in numerous human cell lines. However, the results obtained from uncultured tumor samples have led to discuss the relevance of these findings. Using reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we investigated the mRNA expression of p l 6INK4A/MTS 1 in 40 samples of surgically removed HCC. p16 INK4A mRNA was expressed in all HCC samples but one, as well as in the 3 HCC cell lines we tested (HepG2, HuH7 and Hep3B). We also assessed 1?15INK4B/MTS2 mRNA expression in 29 cases. Lack o fp15 INK4~ expression was observed in the only case where p 16 INK4A was not detected. Southern blot analysis of EcoRI-digested DNA using a p16 INK4A probe was performed in 16 cases. None of them showed homozygous deletion of p16 I N K 4 A gene. Because pl.6 INK4A gene may also be inactivated through point mutations, most of them being clustered within exon 2, we performed direct sequence analysis of p16 INK4A exon 2 in 10 cases chosen at random. In 2 of these cases, we found a G to A substitution at nucleotide 436, previously reported as a polymorphism. Conclusion : Our results show that, contrarily to some other human malignancies, p16 INK4A gene alterations are very infrequent in HCC and therefore unlikely to be involved in liver oncoganesis.

1206 LIVER INFLAMMATORY ACTIVITY IN CHRONIC HEPATITIS C (CHC) IS RELATED TO HLA B8- AND DR4 ANTIGENS H DENKTAS. J GABANYI, G UHL, D VI~i-i If, R, M DO]~OEL, /VIM TONGIO Service d~Icpatogastrocntcrologic Hopital Civil 67091 STRASBOURG Ccdcx, FRANCE

Despite clear- cut evidences of immune mediation in hepatoceIlular damage of CHC, the respons~le pathways remain incompletely explained. Furthermore CHC is associated with autoantibodies, with or without consequences on hepatitis activity. In order to evaluate the possible relationships of Major Histocompatibility Complex (MHC) phenotypes with disease activity and presence of autoantibodies, HLA A, 13, and DR antigens where determined in 144 patients with a biopsy- proven, RNA + CHC, as well as histological activity (KnodeU's scoring) and presence of autoanfibodies (anti- nuclear, actin, LKM, LC, thyroid, rhumatoid factor). After multivariate analysis, the presence of HLA B8 or DR4 antigens were independently correlated with periportal and intralobular inflamm~toxy grading (i)<I).01), but net with hepatoceHular necrosis neither with fibrosis. No other correlation was found between any CMH antigens and histological activity,

] neither with the presence or absence of autoanfibodies. iT his data suggest that antigen presentation and lymphocyte- mediated liver damage are related both to DR4 and B8 anlige~ irrespectively of the presence or absence of markers of autoimmunity, and could also appear as prognostic factors in CHC.

1207 PHLEBOTOMY REDUCES TRANSAMINASE LEVELS WITHOUT INDUCING IRON DEFICIENCY IN PATIENTS WITH CHRONIC HEPATITIS C. TK Desai. Dept of Medicine, William Beaumont Hospital, Royal Oak, Michigan

When iron depletion has been used to reduce serum transaminasa levels in patients with chronic hepatitis C, the goal of phlebotomy has been to induce iron deficiency anemia with farritin values < 50 ngs/ml. There is concern regarding the effects of transient or chronic Fe deficiency. This study was undertaken to assess the effect of a conservative therapeutic phlebotomy regimen in consecutive patients with chronic hepatitis C referred to a primary gastroenterologist. Among 9 patients referred for chronic hepatitis C, 1 patient refused treatment, and 2 menstruating women were iron deficient upon presentation, 1 of whom had normal transaminases. The remaining 6 patients were treated with therapeutic phlebotomy 1 unit every 8-11 days with monitoring of serum hemoglobin to avoid a drop in serum Hb to < 11.0 gms/dl. 4 patients were former drug abusers, 2 patients had no risk factors for hepatitis C infection. All patients were positive for Hep C RNA; 2 patients had cirrhosis on biopsy, 5 pat ientshad failed prior therapy with o-interferon. Mean phlebotomy amount was 5.1 ± 1.6 units of blood (range 4-7 units of blood). Results: (mean ± SD) values given after phlebotomy of 3 units of blood and at the completion of phlebotomy. Transaminase values decreased in all patients after 3 units of phlebotomy. Age (years) 45 ± 5 Sex-1 Female/5 Male

Initial Intermediate Post Phlebotomy After 3 Units

Ferritin (ng/ml) 4 1 6 ± 4 0 7 174±181 4 2 ± 3 5 ALT(U/L) 1 7 9 ± 4 0 1 0 8 ± 2 8 9 9 ± 2 1 p<0 .05 AST (U/L) 1 2 7 ± 4 7 7 6 ± 2 0 8 3 ± 2 9 p<O.05 Hb (gms/dl) 14 .4±0 .7 13.1 ± 1 12 .6± 1 Conclusion: Phlebotomy can lower serum transaminase levels significantly without inducing frank Fe deficiency anemia. Further studies are needed tO define the extent of iron depletion that may be of maximal benefit in potentiating o-interferon therapy.

1208 ANALYSIS OF THE ANTI-HBS RESPONSE OF ELDERLY SUBJECTS TO NBV VACCINE. I Desombere. L Cobbaut. M Slaoui*. and G Leroux-Roels. Center for Evaluation and Research of Vaccines, University of Gent, Gent and *SmithKline Beecham Biologicals, Rixensart, Belgium.

Elderly subjects aged over 60 years display a poor responsd to hepatitis B vaccine. To examine whether adults, aged between 47 and 60, suffer a similar decline in immunity to HBsAg, we have analyzed retrospectively the in vivo anti-HBs response of 118 hospital employees that received the first dose of HBV vaccine between the age of 47 and 60. One month after the 3rd vaccine dose, given according to a 0, 1,2 month schedule, 14 of the 118 vaccinees did not reach an anti-HBs level of >10 U/I. This frequency of non- responsiveness is higher (p<0.05) than in a group of 100 medical students (age: 20-25) vaccinated in a similar fashion. After a 4th vaccine dose 4 elderly subjects (out of 73 tested) did not reach the 10 U/I level of enti-HBs whereas all of the students roached this troshold (p<O.05). In 17 of the 118 subjects the cellular immune response to HBsAg could be explored within 2 years following the 4th vaccine dose. Nine were non-rosponders (anti- HBs_<10 U/I after 3rd dose) and 8 were rosponders (anti-HBs>10 U/I after 3rd dose). None of these 17 subjects displayed an in vitro lymphopreliferative response to HBsAg whereas most (7•9 non-responders and 6/8 responders) reacted upon stimulation with the control antigen tetanus toxoid (TT). PBMC from these 17 vaccinees, irrespective of their responder status, were able to present HBsAg to HBsAg-specific T cell lines derived from good-rosponder veccinees. Of 8 non-responders tested 6 were HLA-DR3 + or -DR7 +. These haplotypes are known to be associated with low/non-response to HBsAg: Only 2 of the 8 responders displayed HLA- DR3 (and no DR7).

This analysis of the in vivo and in vitro anti-HBs response of healthy adults of age 47 to 60 shows that in vivo non-responsiveness after 3 and 4 vaccine doses is observed more frequently than in younger subjects. In vitro lymphoproliferation to HBsAg is completely absent in these elderly subjects, whereas anti-'l-I" responses can easily be induced. The higher incidence of HLA-DR3 and -DR7 in the non-responders suggests that hyporespon- siveness may in part be genetically determined rather than age-related.