studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1015A
1653
HBcrAg during nucleos(t)ide analog therapy are related
to intra-hepatic HBV replication and development of
hepatocellular carcinoma
Masao Honda, Takayoshi Shirasaki, Takeshi Terashima, Kazunori
Kawaguchi, Mikiko Nakamura, Naoki Oishi, Tetsuro Shimakami,
Hikari Okada, Kuniaki Arai, Taro Yamashita, Yoshio Sakai, Tatsuya
Yamashita, Eishiro Mizukoshi, Shuichi Kaneko; Gastroenterology,
Kanazawa University Graduate School of Medical Science,
Kanazawa, Japan
Background & Aim Although nucleos(t)ide analog (NA) therapy
effectively reduces hepatitis B virus (HBV)-DNA in serum and
improves liver histology in patients with chronic hepatitis B (CH-
B), it does not completely reduce the incidence of hepatocellular
carcinoma (HCC). The objective of this study is to elucidate clinical
and virological features associated with the development
of HCC during NA therapy. Material & Methods We enrolled
109 patients with CH-B who started to receive NA therapy from
2001 to 2011 at our hospital. All patients received NA therapy
for more than 2 years, and a liver function test and virological
markers before treatment and at the end of the follow-up period
were compared. HBsAg was measured quantitatively using an
Architect HBsAg-QT assay (Abbot). The amount of HBV-DNA in
serum was measured with COBAS AmpliPrep-COBAS TaqMan
HBV Test. HBcrAg was measured by CLEIA using a Lumipulse
HBcrAg assay (Fujirebio, Inc., Tokyo, Japan). HBV-DNA, HBV-
RNA and cccDNA in thirteen liver tissue samples taken before
treatment and 23 liver tissue samples taken during treatment
were analyzed. Gene expression profiling was performed by
using an Affymetrix GeneChip. Results During NA therapy for
6.5 ± 2.8 years, 36 patients (33%) developed HCC. Multivariate
cox regression analysis showed age (>56 years, HR
= 3.1) and fibrosis stage (F3–4, HR = 3.4) before treatment
and the presence of hepatitis core-related antigen (HBcrAg;
HR = 3.53) during treatment were significantly associated with
the development of HCC. There was no significant difference
in the amount of covalently closed circular DNA in HBcrAg(+)
patients (n = 16) and HBcrAg(-) patients (n = 12); however,
the amount of HBV-DNA and RNAs (pregenome, preS/S, and
HBx) were significantly higher in HBcrAg(+) patients than in
HBcrAg(-) patients, suggesting the presence of active HBV replication
in HBcrAg(+) liver. Hepatic gene expression profiling
before and during treatment (n = 13) showed a significant
improvement of inflammation signaling during treatment; however,
in HBcrAg(+) liver, viral replication-related signaling such
as pro-apoptosis and oxidative phosphorylation were up-regulated
along with cancer-related signaling (n = 11 vs. 12).
Interestingly, transcription factors that activate HBV promoter
activity, such as HNF4α and PPARα, were up-regulated in
HBcrAg(+) liver. Metformin efficiently repressed HBV-RNA and
HBcrAg levels by repressing the expression of HNF4α and
PPARα in primary human hepatocytes. Conclusions: Modulating
HBV transcription factors by metformin in combination with
NA therapy would potentiate anti-HBV activity and reduce the
incidence of HCC in HBcrAg (+) patients.
Disclosures:
Hikari Okada - Employment: Kanazawa University
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,
Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,
Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,
Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer
Japan, Eli lilly Japan
The following authors have nothing to disclose: Masao Honda, Takayoshi Shirasaki,
Takeshi Terashima, Kazunori Kawaguchi, Mikiko Nakamura, Naoki
Oishi, Tetsuro Shimakami, Kuniaki Arai, Taro Yamashita, Yoshio Sakai, Tatsuya
Yamashita, Eishiro Mizukoshi
1654
Free episomal and integrated HBV DNA In HBsAg-negative
patients with intrahepatic cholangiocarcinoma
Teresa Pollicino 1 , Cristina Musolino 2 , Gianluca Tripodi 2 , Marika
Lanza 2 , Giuseppina Raffa 2 , Carlo Saitta 2 , Salvatore Benfatto 1 ,
Concetta Beninati 1 , Giuseppe Navarra 3 , Pietro Invernizzi 4 , Domenico
Alvaro 5 , Giovanni Raimondo 2 ; 1 Pediatric, Gynecological,
Microbiological and Biomedical Sciences, University of Messina,
Messina, Italy; 2 Clinical and Experimental Medicine, University
Hospital of Messina, Messina, Italy; 3 Human Pathology, University
Hospital of Messina, Messina, Italy; 4 Medicine, Humanitas
Clinical and Research Center, Milan, Italy; 5 University of Rome
“Sapienza”, Roma, Italy
Backgroud: Intrahepatic cholangiocarcinoma (ICC) is a fatal
primary liver cancer with very poor prognosis. Genome-wide
studies have made major advances in understanding the molecular
basis of this disease, although most aspects remain unclear.
Accumulating evidence indicates that chronic HBV infection is
associated with an increased risk of ICC development and
suggests an etiological role of HBV in the development of this
tumor. Aims of the study were to investigate the prevalence of
occult HBV infection (OBI) in cases with ICC and to characterize
the molecular status of HBV in OBI-positive ICC specimens.
Methods: Frozen paired tumor and non-tumor tissue specimens
from 40 HBsAg-negative patients with ICC, who underwent
surgical resection were tested for OBI by 4 different HBV-specific
nested PCR. To reveal HBV cccDNA, DNA extracts were
digested with a plasmid-safe ATP-dependent DNase and amplified
by nested PCR with cccDNA-specific primers. Finally, for
the detection of HBV DNA integrations the Alu-PCR technique
was coupled to deep-sequence analysis. Results: HBV genomic
sequences were detected in tumor and/or non-tumor specimens
from 28 of the 40 (70%) ICC patients analysed. In particular,
20/40 (50%) tumors and 13/23 (56.5%) non-tumor tissues
were HBV DNA positive. HBV cccDNA was detected in tissue
specimens from 10/28 OBI-positive patients (36%) (both in
tumor and non-tumor specimens in 3 patients; only in tumor
tissues in 4 patients; only in non-tumor tissues in 3 patients).
HBV integrants were detected in 3 of 10 cases examined so
far, and included portions of the HBx gene sequence (including
the Basic Core Promoter/Enhancer II) in 2 cases and part of the
core gene sequence in one case. The analysis of the integration
sites revealed that the HBx sequences were located 3,374
nucleotides upstream the sequence encoding the cat eye syndrome
critical region protein 5 isoform and within the coding
sequence of the thromboxane A synthase 1, respectively, and
that the core gene sequence was located within the cystinosin
isoform 1 precursor coding sequence. Conclusion: Occult HBV
infection is highly prevalent in patients with ICC. Both free viral
genomes and integrated HBV DNA can be detected in these
cases. These results suggest an involvement of HBV in the carcinogenic
process leading to ICC development even in cases
with occult infection.
Disclosures:
Teresa Pollicino - Speaking and Teaching: Gilead, Roche, Janssen, BMS, MSD,
Bayer, Abbvie
Giovanni Raimondo - Speaking and Teaching: BMS, Gilead, Roche, Merck,
Janssen, Bayer, MSD
The following authors have nothing to disclose: Cristina Musolino, Gianluca
Tripodi, Marika Lanza, Giuseppina Raffa, Carlo Saitta, Salvatore Benfatto, Concetta
Beninati, Giuseppe Navarra, Pietro Invernizzi, Domenico Alvaro