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GASTROENTEROLOGY 2004;126:102–110

epatitis B Virus Maintains Its Pro-oncogenic Properties in thease of Occult HBV Infection

ERESA POLLICINO,* GIOVANNI SQUADRITO,* GIOVANNI CERENZIA,* IRENE CACCIOLA,*IUSEPPINA RAFFA,* ANTONIO CRAXI,‡ FABIO FARINATI,§ GABRIELE MISSALE,¶

NTONINA SMEDILE,� CLAUDIO TIRIBELLI,# ERICA VILLA,** and GIOVANNI RAIMONDO*Unit of Clinical and Molecular Hepatology, Department of Internal Medicine, University of Messina, Messina; ‡Department ofastroenterology, University of Palermo, Palermo; §Department of Gastroenterology, University of Padova, Padova; ¶Division of Infectiousiseases, Hospital of Parma, Parma; �Department of Gastroenterology, University of Torino, Torino; #Centro Studi Fegato, AREA Science Parknd Department BBCM, University of Trieste, Trieste; and **Department of Gastroenterology, University of Modena, Modena, Italy

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See editorial on page 347.

ackground & Aims: Occult hepatitis B virus (HBV) infec-ion is characterized by persistence of HBV DNA into theissue of hepatitis B surface antigen–negative individu-ls. The clinical relevance of this peculiar infection is stillnder debate. In particular, the impact of occult HBVnfection in cases of hepatocellular carcinoma (HCC) isncertain. We investigated the prevalence and molecu-ar status of occult HBV in patients with HCC. Methods:e tested tumor tissues from 107 patients with HCCnd the corresponding nontumor liver tissue from 72 ofhese patients for HBV DNA. We also examined liverpecimens from 192 patients with chronic hepatitis. Allases were hepatitis B surface antigen negative. Co-alently closed circular HBV genomes, HBV transcripts,nd viral integrated forms were investigated in cases ofCC found positive for occult HBV. Results: Viral DNAas detected in 68 of 107 cases of HCC (63.5%) and in3 of 192 cases of chronic hepatitis (32.8%) (P <.0001; odds ratio, 3.6; 95% confidence interval, 2.2–.9). The significant association of occult HBV with HCCas irrespective of age, sex, and contemporary hepatitisvirus infection. Both integrated viral DNA and co-

alently closed circular HBV genomes were detected inatients with occult HBV. Moreover, the presence of freeBV genomes was associated with persistence of viralranscription and replication. Conclusions: Our findingsrovide clear evidence that occult HBV is a risk factor forevelopment of HCC and show that the potential mech-nisms whereby overt HBV might induce tumor forma-ion are mostly maintained in cases of occult infection.

epatitis B virus (HBV) infection is a major healthproblem worldwide; it is one of the 10 leading

auses of mortality, with an estimated 400 million peo-le chronically infected at the turn of this millennium.1,2

BV infection may associate with a large spectrum of

linical forms, ranging from very mild and asymptomaticlinical pictures to the most severe liver diseases includ-ng fulminant hepatitis, cirrhosis, and hepatocellular car-inoma (HCC).1 In particular, chronic HBV infection isommonly considered a primary risk factor for develop-ent of HCC,3 exerting its pro-oncogenic properties

hrough both indirect and direct mechanisms.4–7 Thendirect mechanisms are related to its propensity tonduce continuous or recurrent phases of liver necroin-ammation and to promote the progression of chronicepatitis to cirrhosis, which is the step preceding theevelopment of HCC in most cases.8 Schematically, theirect carcinogenic mechanisms have been related to theapacity of HBV to integrate into the host’s genome ando produce proteins (X protein and truncated preS-Srotein) provided of potential transforming properties.9

HBV infection is usually diagnosed when the circu-ating hepatitis B surface antigen (HBsAg) is detected.owever, the availability of highly sensitive molecular

iology techniques has allowed the identification of HBVnfection in HBsAg-negative individuals with or withoutirculating antibodies to HBsAg and/or hepatitis B corentigen (anti-HBc).10–15 Much evidence suggests thathis so-called occult or cryptic HBV infection is highlyrevalent and may have a relevant clinical impact.16–18 Inarticular, we and others previously showed that occultBV infection significantly correlates with cirrhosis in

hronic hepatitis C virus (HCV) carriers.15,19–21

Since the early 1980s, a certain number of studies haveeported the detection of HBV DNA in liver tissue ofBsAg-negative patients with HCC.22,23 With the ad-

Abbreviations used in this paper: cccDNA, covalently closed circularNA; CLD, chronic liver disease; PCR, polymerase chain reaction;cDNA, relaxed circular DNA.

© 2004 by the American Gastroenterological Association0016-5085/04/$30.00

doi:10.1053/j.gastro.2003.10.048

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January 2004 OCCULT HBV AND HCC 103

ent of the most sensitive polymerase chain reactionPCR) techniques, this observation has been con-rmed24–26; however, extensive studies performed in ho-ogeneous populations and evaluating the real preva-

ence of this event are still lacking.17 Another aspect thateeds to be highlighted is the molecular status of theccult HBV in patients with HCC, which is an essentialupport for understanding the possible pathogenic mech-nisms through which the cryptic HBV might contrib-te to hepatocellular transformation.In this study, we investigated the presence and mo-

ecular features of HBV DNA in liver tissues from a largeeries of HBsAg-negative patients with HCC.

Patients and MethodsPatients

We studied liver specimens from 107 HBsAg-negativeirrhotic patients with HCC (81 men and 26 women; meange, 63.7 � 10 years) randomly collected from January 1999o December 2000 at 7 Italian liver units located in distincteographic areas of the country. Seventy-three of the 107atients were antibody to HCV (anti-HCV) positive; amonghe 34 anti-HCV–negative individuals, 5 had alcoholic liverisease and 29 had cryptogenic liver disease (Figure 1). Serumarkers of previous HBV infection were available for 82 of the

07 patients. Twenty-five of these 82 patients were anti-HBcositive and 11 of them were also antibody to HBsAg positive;he remaining 57 cases were negative for all HBV serumarkers. Tumor liver specimens from the 107 patients and the

orresponding nontumor tissue from 72 of the patients werevailable for molecular analysis. Tissue samples had been ob-ained by surgical resection or percutaneous needle liver biopsynd immediately frozen and stored at �80°C.

A total of 192 frozen liver biopsy specimens from HBsAg-egative patients (125 men and 67 women; mean age, 48.2 �

igure 1. Schematic representation of the study populations. (A)tudy group (HBsAg-negative cirrhotic patients with HCC). (B) Controlroup (HBsAg-negative patients with CLD).

1.5 years) with chronic liver disease (CLD) (143 with chronicepatitis and 43 with cirrhosis) were available for inclusion inhis study as a control group. A total of 153 of the 192 patientsad an HCV-related CLD; among the 39 anti-HCV–negativendividuals, 7 had alcohol-related liver disease and 32 hadryptogenic liver disease (Figure 1). These specimens wereollected from June 1999 to May 2000 at 3 of the previouslyentioned Italian centers (Messina, Palermo, and Turin).Serum markers of previous HBV infection were available for

19 of the 192 patients. Thirty-three of these 119 patientsere anti-HBc positive and 18 of them were also antibody toBsAg positive; the remaining 86 cases were negative for allBV serum markers. None of these patients had been treatedith antiviral or immunosuppressive drugs before undergoing

iver biopsy, and none were infected with the human immu-odeficiency virus.The study protocol was performed according to the princi-

les of the Declaration of Helsinki, and informed consent wasbtained from all patients.

HBV DNA Analyses

DNA was extracted from the frozen liver specimens ofach case by standard procedures.27 In brief, tissue specimensere homogenized in 150 mmol/L NaCl, 50 mmol/L Tris-HCl

pH 7.4), 10 mmol/L ethylenediaminetetraacetic acid, and 1%odium dodecyl sulfate and incubated overnight with protein-se K (800 �g/mL) at 37°C. After extraction with phenol/hloroform, the nucleic acids were precipitated in 2 vol ofure, cold ethanol. Nucleic acids were then resuspended andigested with pancreatic ribonuclease (100 �g/mL) followedy extraction with phenol/chloroform and reprecipitation inure cold ethanol. The DNA was resuspended in 10 mmol/Lris-HCl (pH 7.4) and 1 mmol/L ethylenediaminetetraaceticcid, and its concentration was determined by spectrophotom-try at 260 nm.

Occult HBV infection had been identified as previouslyescribed with slight modification.15 All liver DNA extractsere analyzed for the presence of HBV genomes by performingdifferent in-house single-step or nested PCR amplification

ssays to detect preS-S (S), precore-core (C), Pol, and X viralegions, respectively. We considered the cases that showedositivity in at least 2 different viral genomic regions as HBVNA positive. Appropriate negative controls were included in

ach PCR. Moreover, direct sequencing of all amplified HBVequences confirmed the specificity of the reactions. The limitf sensitivity of our single-step and nested PCR methods wasn the range of 103 and 10 genome equivalents/mL, respec-ively. The 4 sets of primers used are reported in Table 1.

Detection of HBV Covalently Closed CircularDNA

Paired tumor and nontumor liver tissues from 30ccult HBV-positive cases and tumor specimens from 12dditional positive cases were tested for the presence of HBVovalently closed circular DNA (cccDNA) by digestion of liverNA extracts with mung bean nuclease and subsequent nested

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104 POLLICINO ET AL. GASTROENTEROLOGY Vol. 126, No. 1

CR analysis.28 The primers used were selected to discriminateetween relaxed circular DNA (rcDNA) genome of HBVarticles and HBV cccDNA present in the infected cells. HBVcDNA contains 2 regions sensitive to digestion with mungean nuclease: the single-stranded area, covering about 10%–0% of the genome length, and a nick within the minus strandf the direct repeat region. When the sense primers HBV P23nd HBV P25 (Table 1), located within the single-strandedegion of HBV, are used together with the antisense primersBV P24 and HBV P26 (Table 1), amplification runs across

he minus strand nick of HBV rcDNA in both rounds ofested PCR. After digestion with mung bean nuclease of liverNA extracts used as PCR templates, positive signals are

btained only from samples containing HBV cccDNA.

Southern Blot Analysis

We analyzed both tumor and nontumor tissue from 10andomly selected occult HBV-positive cases for the presencef integrated HBV sequences. Southern blot analysis was per-ormed following standardized procedures.29 Briefly, 25 �g ofenomic DNA isolated from each liver tissue specimen wasigested with HindIII restriction enzyme, separated by elec-

able 1. Oligonucleotides Used as PCR Primers, Hybridizatio

Sense primers

Name Nucleotide sequence Positiona Na

BV 1 5�-GGTCACCATATTCTTGGGAA-3� 2815–2834 HBVBV 3b 5�-AATCCAGATTGGGACTTCAA-3� 2932–2951 HBVBV 5 5�-GCCTTAGAGTCTCCTGAGCA-3� 2021–2040 HBVBV 7b 5�-CCTCACCATACTGCACTCA-3� 2048–2066 HBVBV 9 5�-CGTCGCAGAAGATCTCAATC-3� 2416–2436 HBVBV 11b 5�-CCTTGGACTCATAAGGT-3� 2457–2473 HBVBV 13 5�-CCATACTGCGGAACTCCTAGC-3� 1266–1286 HBVBV 15b 5�-GCTAGGCTGTGCTGCCAACTG-3� 1380–1400 HBVBV P23 5�-CTGAATCCTGCGGACGACCC-3� 1441–1460 HBVBV P25b 5�-GTCTGTGCCTTCTCATCTGCC-3� 1551–1571 HBVBV 17 5�-TTGGGGTGGAGCCCTCAGGCT-3� 3037–3057 HBVBV 18b 5�-CCTGCTGGTGGCTCCAGTTCA-3� 56–76 HBVBV 5 5�-GCCTTAGAGTCTCCTGAGCA-3� 2021–2040 HBV

BV 7b 5�-CCTCACCATACTGCACTCA-3� 2048–2066 HBVBV 15 5�-GCTAGGCTGTGCTGCCAACTG-3� 1380–1400 HBV1BV 20b 5�-CGCGGGACGTCCTTTGTTTACG-3� 1408–1429 HBVCT 1 5�-GATGCATTGTTACAGGAAGT-3� 1465–1484 ACT 2BV P1 5�-CCGGAAAGCTTGGCTCTTC HBV

TTTTTCACCTCTGCCTAATCA-3� 1821–1841BV P5b 5�-CTTTTTCACCTCTGCCTAATCA-3� 1821–1841 HBVBV 17b 5�-TTGGGGTGGAGCCCTCAGGCT-3� 3037–3057 HBVEQ 1 5�-GTCTAGACTCGTGGTGGACTT-3� 246–266 SEQ

EQ 3 5�-TGTGCACTTCGCTTCACCTCT-3� 1578–1598 SEQEQ 5 5�-GTGGTATTGGGGGCCAAG-3� 748–765 HBVBV 9 5�-CGTCGCAGAAGATCTCAATC-3� 2414–243 HBVBV P23 5�-CTGAATCCTGCGGACGACCC-3� 1441–1460 HBVBV 18 5�-CCTGCTGGTGGCTCCAGTTCA-3� 56–76BV P5 5�-CTTTTTCACCTCTGCCTAATCA-3� 1821–1841 HBVBV 1 5�-GGTCACCATATTCTTGGGAA-3� 2815–2834

OTE. Heterologous nucleotides containing restriction enzyme SstI aNucleotide positions of the primers are numbered from the unique EApplied in the second round of amplification of the nested PCR.

rophoresis in a 0.8% agarose gel, and blotted onto a nylonybond N� membrane (Amersham, Buckinghamshire, En-

land). The membrane was hybridized overnight at 65°C with32P random prime-labeled (Amersham) full-length HBV

enome probe and then was washed and exposed to X-Omatlm (Kodak, Rochester, NY) at �80°C.

Isolation of Total RNA and Reverse-Transcription PCR Amplification

Good-quality material for RNA analysis was availablerom 10 pairs of tumor and nontumor tissue and from 4dditional tumor samples, all of which were positive for HBVccDNA. Total RNA was extracted from 50 mg of each liverpecimen using TRIzol reagent (Invitrogen, Paisley, Scotland)s recommended by the manufacturer. Five micrograms ofxtracted RNA was digested with ribonuclease-free deoxyri-onuclease I (Promega, Madison, WI) at 70°C for 10 minutesnd used as a template for first-strand complementary DNAynthesis by SuperScript Reverse Transcriptase (Invitrogen)nd oligo(dT) primers. The housekeeping gene �-actin wassed as an internal standard. Equilibrated amounts of comple-entary DNA were taken for HBV transcript nested PCR

bes, and Sequencing Primers

Antisense primers

Primer setdesignationNucleotide sequence Positiona

5�-AATGGCACTAGTAAACTGAG-3� 690–671 PreS-S5�-CCTTGATAGTCCAGAAGAAC-3� 459–4405�-GTCCAAGGAATACTAAC-3� 2464–2448 Precore-core5�-GAGGGAGTTCTTCTTCTAGG-3� 2385–23665�-CCTGATGTGATGTTCTCCATG-3� 174–155 Pol5�-TTGAAGTCCCAATCTGGATT-3 2951–29325�-CGTTCACGGTGGTCTCCAT-3� 1628–1608 X5�-CGTAAAGAGAGGTGCGCCCCG-3� 1540–15205�-CCCAAGGCACAGCTTGGAGG-3� 1889–1869 cccDNA5�-AGAGATGATTAGGCAGAGGTG-3� 1846–18265�-AATGGCACTAGTAAACTGAG-3� 690–672 S mRNA5�-CCTTGATAGTCCAGAAGAAC-3� 459–4405�-GTCCAAGGAATACTAAC-3� 2464–2448 Precore-core

mRNA5�-GAGGGAGTTCTTCTTCTAGG-3� 2385–23665�-GGGGAGTCCGCGTAAAGAGAGG-3� 1550–1529 X mRNA5�-GGTCGGTCGGAACGGCAGACGG-3� 1518–14975�-TCATACATCTCAAGTTGGGGG-3� 1706–1686 �-actin5�-CCGGAAAGCTTGGCTCTTC Full-length HBVAAAAAGTTGCATGGTGCTGG-3� 1796–18255�-CCTTGATAGTCCAGAAGAAC-3� 459–4405�-AAAAAGTTGCATGGTGCTGG-3� 1796–18255�-GGAGTGCGAATCCACAC-3� 2286–2270 Probes and

sequencingprimer set

5�-TTCCCAAGAATATGGTGACC-3� 2834–28155�-CGTTCACGGTGGTCTCCAT-3� 1628–16085�-CCTTGATAGTCCAGAAGAAC-3� 459–4405�-GTCCAAGGAATACTAAC-3� 2464–2448

5�-AAAAAGTTGCATGGTGCTGG-3� 1796–1825

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January 2004 OCCULT HBV AND HCC 105

mplification. HBV RNA expression was analyzed with these of 3 different sets of primers (Table 1) specific for S, C, and

transcript, respectively. In all experiments, 2 negative con-rol samples were subjected to all steps. The PCR amplificationroducts were separated by 1% agarose gel electrophoresis andisualized by ethidium bromide staining. Southern blot exper-ments were performed to show the specificity of the amplifi-ation products.

Full-Length and Subgenomic HBVAmplification

HBV DNA isolates from tissue specimens of 5 casesere full-length amplified following the method describedy Gunther et al.30 Because the first-round PCR yieldednly weak or no bands on a 1% agarose gel after electro-horesis, we performed a second-round PCR with 2 differ-nt primer pairs yielding the amplification of 2 overlappingubgenomic fragments that cover the whole length of theBV genome (Table 1).

Cloning and Sequencing of PCR Products

Cloning of PCR products was performed using theOPO TA cloning kit (Invitrogen) according to the manufac-

urer’s protocol. Nucleotide sequences of PCR products andloned DNA fragments were determined using specific prim-rs (Table 1) and the BigDye Terminator Cycle Sequencingeady Reaction Kit (Applera, Foster City, CA) according to

he manufacturer’s instructions. The sequencing products wereesolved in an automatic DNA sequencer (ABI PRISM 310enetic Analyzer; Applera).

Statistical Analysis

Data were analyzed statistically by means of �2 test,endall’s rank correlation test, and odds ratio and 95% con-dence interval test. P � 0.05 (2 tailed) was consideredignificant.

Accession Numbers

The GenBank accession numbers of HBV areY230111–AY230128, AY232836–AY232840, andY236160–AY236169.

ResultsIdentification of Occult HBV Infection inLiver Tissue

We investigated the presence of HBV DNA se-uences in liver tissue specimens from 107 HBsAg-

able 2. Prevalence of Occult HBV Infection in Patients WithHCC and in Patients With CLD

No. of cases Occult HBV positive (%)

CC 107 68a (63.5)LD 192 63a (32.8)

P � 0.0001.

egative patients with HCC and 192 HBsAg-negativeatients with CLD by nested PCR and the use of 4rimer sets, each specific for S, C, Pol, and X viralenomic regions, respectively. As we previously report-d15 and as also recommended by others,17,18 we consid-red cases in which sequences of at least 2 different HBVenomic regions were detected as occult HBV-positiveases. In this way, 68 of the 107 patients with HCC63.5%) and 63 of the 192 patients with CLD (32.8%;

� 0.0001) were positive for occult HBV (Table 2),ith a univariate odds ratio for the association betweenccult HBV and HCC of 3.6 (95% confidence interval,.2–5.9). When we divided the study populations on theasis of anti-HCV positivity, we found HBV DNA in 45f the 73 anti-HCV–positive patients with HCC61.6%) and in 56 of the 153 anti-HCV–positive pa-ients with CLD (36.6%; P � 0.001), resulting in anstimated univariate odds ratio of 2.9 (95% confidencenterval, 1.6–5.1). Among the anti-HCV–negative pa-ients (34 with HCC and 39 with CLD), we detectediral sequences in the liver of 23 patients with HCC67.6%) and 7 patients with CLD (18%; P � 0.0001)Table 3) and the univariate odds ratio was 9.6 (95%onfidence interval, 3.2–28.4). Notably, in the group ofatients with HCC, we found no correlation betweenccult HBV infection and anti-HCV positivity (P .5); in the group of patients with CLD, the presence ofntrahepatic HBV sequences was significantly associatedith HCV infection (P 0.03) (Table 3), in accordanceith previous data reported by us and others (reviewedy Torbenson and Thomas18). The prevalence of occultBV infection did not differ with sex or age either

mong patients with HCC or those with CLD. As ex-ected, the patients with HCC were older (mean age,3.7 � 10 years) than those with CLD (mean age,8.2 � 11.5 years). Consequently, to verify whether theelationship of occult HBV infection with HCC wasffected by age, we performed multivariate analysis bysing the Kendall’s rank correlation test on the total of

able 3. Prevalence of Occult HBV in Anti-HCV–Positive andAnti-HCV–Negative Patients With HCC or CLD

Anti-HCV–positivepatients (%)

Anti-HCV–negativepatients (%)

HCC(n 73)

CLD(n 153)

HCC(n 34)

CLD(n 39)

ccult HBVpositive 45 (61.6)a,b 56 (36.6)a,c 23 (67.6)b,d 7 (18)c,d

P � 0.001.P 0.5.P 0.03.P � 0.0001.

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106 POLLICINO ET AL. GASTROENTEROLOGY Vol. 126, No. 1

99 cases (107 patients with HCC and 192 patients withLD). There was a correlation between age and occultBV ( 0.2; P 0.00002), between age and HCC

0.46; P � 0.000001), and between occult HBV andCC ( 0.3; P � 0.0001). Of note, occult HBV andCC were associated regardless of age ( 0.23; P �

.000001).As previously mentioned, all patients with HCC had

irrhosis, as did 43 of the 192 individuals in the controlroup. HBV DNA was detected in 19 of these 43ubjects (44.2%). The comparison of the prevalence ofccult HBV between HCC and non-HCC cirrhotic pa-ients showed the persistence of the significant associa-ion between HBV and liver cancer (P 0.03). More-ver, the Kendall’s test showed that occult HBV isssociated with HCC independently of cirrhosis ( .18; P 0.015).Data on serum anti-HBc reactivity were available for

2 patients with HCC and 119 patients with CLD.mong patients with HCC, occult HBV was detected in1 of 25 anti-HBc–positive (84%) and 32 of 57 anti-Bc–negative cases (56.1%; P 0.01). Therefore, not

nly most of the anti-HBc–positive individuals but alsoore than half of subjects negative for all serum markers

f HBV infection carried occult HBV. The analysis ofatients with CLD showed the presence of HBV se-uences in 15 of 33 anti-HBc–positive (45.5%) and 22f 86 anti-HBc–negative patients (25.6%; P 0.04).hese results are in accordance with data that emerged

rom a recent review of the literature that, besides theignificant association between occult HBV and anti-Bc positivity, also showed that more than 20% of

atients with chronic hepatitis and negative for all HBVerum markers prove to be carriers of cryptic HBVnfection.18

Molecular Analyses of Occult HBV Infectionin Patients With HCC

Besides the tumor specimens, we were able tonalyze the corresponding nontumor liver tissue of 72 ofhe 107 patients with HCC. Fifty-two of these 72 wereositive for occult HBV. In particular, 17 cases showedositive reactivity for 3 or all 4 tested HBV genomicegions both in tumor and nontumor specimens, 8 cases

able 4. Distribution of 68 Patients With HCC Based on theand Nontumor Liver Specimens by 4 Different Prime

o. of HBV-positive regionsin tumor/nontumor liver specimens 4/4 4/3 3/4 4/2 2/4

o. of cases 4 3 2 3 3

A, not available.

nly in tumor samples, and 12 cases only in nontumorpecimens (Table 4). In addition, 16 of the 35 cases fromhich only tumor tissue was available tested positive forccult HBV, showing positivity for all 4 genomic re-ions in 7 cases, for 3 regions in 6 cases, and for 2 regionsn 3 cases (Table 4). In summary, occult HBV could beetected in most cases independently of the set of prim-rs used, suggesting the presence of viral genomes car-ying no gross rearrangements. To further confirm thepecificity of our results, we performed direct sequencingnalysis of all the PCR products. Such examination,esides the diversity of HBV DNA sequences betweenatients, showed a certain degree of sequence divergenceetween isolates from tumor and nontumor liver speci-ens of individual cases. This observation was largely

onfirmed when the entire S-gene sequences of isolatesbtained from paired tumor and nontumor tissue speci-ens of 5 randomly chosen patients were compared with

ach other, showing a nucleotide divergence rangingrom 1.5% to 3%. However, it must be noted that in noase was mutation(s) potentially responsible for the lackf serum HBsAg reactivity found. In this context, an-ther relevant finding was the presence of HBV strains ofoth genotype D and A in the tumor specimen from onef these 5 patients and only viruses of genotype A in theorresponding nontumor tissue. To study this peculiarase in depth, we performed HBV DNA full-lengthmplification, cloning, and sequencing analysis of HBVsolates from both tumor and nontumor specimens. Theequencing of 10 clones from each specimen confirmedhat only genotype A strains were present in the nontu-or tissue, whereas 6 of the clones from tumor isolatesere of genotype D and 4 of genotype A (GenBank

ccession numbers AY230111–AY230120, AY236165–Y236169, and AY232836–AY232840). Of relevance,o viral clone carried a mutation apparently able tonterfere with the HBV replication and gene expression.mplifying full-length viral genomes, we clearly showed

hat free HBV forms might infect HBsAg-negative pa-ients with HCC. Indeed, we showed that this was not anccasional event because we succeeded in amplifying thentire HBV genome in liver DNA extracts from 4 ad-itional cases randomly selected among those that were

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January 2004 OCCULT HBV AND HCC 107

ositive for all of the virus genomic regions tested byCR amplification (GenBank accession numbersY236160–AY236164).Integration of viral DNA sequences into the cellular

NA of patients with HCC with occult HBV is a knownccurrence.22,23,26,31 In this study, we could detect inte-rated HBV in 2 of 10 tumor tissues tested by Southernlot analysis (data not shown). In addition, when theost sensitive PCR amplification technique was used, we

onsidered the failure of 2 or 3 sets of primers in ampli-ying viral DNA from individual cases as indicative ofnterrupted and possibly integrated HBV genomes, asreviously suggested.32

Succeeding in the isolation of complete HBV ge-omes, we focused our attention on the molecular formsllowing occult HBV to persist as free genome withinhe liver of patients with HCC. We applied a nestedCR approach28 to discriminate between the partiallyouble-stranded HBV rcDNA present in virions that isnable to accomplish RNA transcription and the double-tranded HBV cccDNA essential for HBV expressionnd replication. By the use of this highly sensitive andpecific approach, we were able to detect HBV cccDNAn 20 of the 30 cases examined that were randomlyelected among those showing positivity for 3 or all 4BV genomic regions tested by PCR in both tumor and

ontumor tissue. In particular, HBV cccDNA could beetected both in tumor and nontumor specimens in 14ases, whereas it was shown only in tumor or nontumorissue in 4 and 2 cases, respectively. To rule out possiblertefacts, we included several controls in each experi-ent; HBV DNA–negative liver tissues were always

egative, and DNA from serum HBV particles gave noignal after digestion with mung bean nuclease. We nextought to assess the HBV transcriptional activity in

able 5. Detection of S, X, and C Transcripts by Reverse-Transcription PCR and the Use of Specific Primersin Paired Tumor and Nontumor Tissues From 10Cases Positive for HBV cccDNA

ases Tissues S RNA X RNA C RNA

1 T/NT �/� �/� �/�2 T/NT �/� �/� �/�3 T/NT �/� �/� �/�4 T/NT �/� �/� �/�5 T/NT �/� �/� �/�6 T/NT �/� �/� �/�7 T/NT �/� �/� �/�8 T/NT �/� �/� �/�9 T/NT �/� �/� �/�

10 T/NT �/� �/� �/�

OTE. Nucleotide sequences of primers used to determine the ex-ression of S, X, and C transcripts are reported in Table 1., tumor; NT, nontumor.

ccDNA-positive tumor and nontumor paired liver tis-ues by performing reverse-transcription PCR analysis.ood-quality liver RNA was available from 10 of the 14

ases showing the presence of HBV cccDNA, and HBVNA was detected in all of them. In particular, weetected transcripts corresponding to S gene in 9 tumornd 8 nontumor specimens, X transcripts were found in0 tumors and 8 nontumor specimens, and C transcriptsere found in 2 tumor and 5 nontumor specimens,

espectively (Table 5 and Figure 2). These data show thatccult HBV persisting as cccDNA maintains low levelsf transcriptional activity. Moreover, the detection of-gene transcripts corresponding to the pregenomicNA indicates ongoing viral replication.

Discussion

HCC is one of the most common cancers world-ide, and its incidence appears to be increasing in theore industrialized areas of North America, Europe, andsia.2,33 Human HCCs mostly occur in patients with

ong-lasting chronic hepatitis due to infection with HBVnd/or HCV.34–36 However, while this malignancy istill mainly related to an overt (namely, HBsAg positive)BV infection in developing countries, the increasing

ate of HCC observed in the past decade in the westernorld seems to be more frequently linked with cirrhosis.37,38 Much evidence indicates that cryptic HBV infec-

ion is a phenomenon often occurring in chronic HCVarriers, and we previously reported that about one thirdf individuals with type C CLD in our area have aasked HBV coinfection.15 In the present study, our

xamination of a large series of liver tissues from HBsAg-egative individuals with HCC showed that most ofhese patients carry occult HBV infection, with a signif-cantly higher prevalence compared with that observed inubjects with HBsAg-negative CLD; this significanceas also maintained when the comparison was performedith the subgroup of non-HCC cirrhotic patients. More-ver, the statistical analysis showed that occult HBVtrongly associated with liver cancer independently ofge, sex, and contemporary HCV infection.

Our results indicate that cryptic HBV infection is aisk factor for development of HCC. This is in agreementith data obtained in animal models showing that bothoodchucks and ground squirrels, once infected by the

orresponding hepadnaviruses (woodchuck hepatitis vi-us and ground squirrel hepatitis virus, respectively), aret high risk of developing HCC after the apparent clear-nce of the virus.39,40 The mechanisms responsible forepatocellular transformation in these rodents are essen-

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108 POLLICINO ET AL. GASTROENTEROLOGY Vol. 126, No. 1

ially related to the propensity of the viruses to act as annsertional mutagen of the host’s myc family genes.41,42

However, it was shown that besides integrating intohe host DNA, the viral genomes may also persist as freepisomal forms during occult woodchuck hepatitis virusnfection42; an interesting study on woodchucks conva-escent from acute viral hepatitis showed the lifelongersistence of small amounts of replicating virus thatnduce a mild liver necroinflammation continuing forife.43 Also, occult HBV may persist as episomal DNAfter HBsAg seroclearance,44–46 and it is able to induceild inflammation persisting for decades.47,48 Moreover,hen occult HBV infection coexists with other causes of

iver disease (HCV, alcohol abuse, iron overload, and son), it might be a cofactor in the pathogenesis of theepatic injury.17,49,50 In this context, we have to mentionhat occult HBV infection seems to favor the progressionf chronic hepatitis toward cirrhosis,15,20,21 which isnown to be the main precancerous lesion of the liver.owever, our present data indicate that occult HBV is

ssociated with HCC independently of cirrhosis. Of note,n our series of patients with HCC developed on cirrhoticivers, we found free viral genomes in a considerableumber of cases. These viruses maintain low levels ofranscription and replication, as indicated by the pres-nce of HBV cccDNA and viral messenger RNAsmRNAs) that, interestingly, were detected in both tu-or and nontumor samples. Although we cannot com-

letely rule out possible contamination between tumornd surrounding nontumor liver tissues, several consid-rations suggest that free replicative forms of HBV wereeally present in neoplastic cells: (1) all 4 HBV genomicegions were amplified in tumor extracts but not in theorresponding nontumor specimen from several cases; (2)nalogously, some cases had HBV cccDNA in tumor but

igure 2. Expression of the HBV core mRNA in liver tissues of patieontumor (NT) liver specimens of 3 individual cases (A, B, and C) wasore mRNA expression. (B) �-actin mRNA was amplified as an internA and B) M, DNA molecular-weight marker (sizes along left margin). (esults. �, positive control; �, negative control.

ot in the paired nontumor samples; (3) a discrete nu-leotide divergence was found between HBV sequencessolated from tumor and nontumor specimens of indi-idual patients; and (4) in one case, viral strains belong-ng to 2 different genotypes were detected in the tumoraliver whereas only one HBV genotype was shown in theurrounding nontumoral tissue. The sequence diver-ences observed between viral isolates from tumor andontumor tissues might reflect the heterogeneity of theBV infecting the liver in toto and tempts us to spec-

late on a possible segregation of some viral strains inransformed and clonally expanded cells. In any case,hat clearly emerged from our study is that ongoing

overt HBV replication frequently occurs in HBsAg-egative cirrhotic patients who develop HCC. The long-erm persistence of cryptic yet productive HBV infectionight exert an indirect oncogenic role by chronically

ustaining a mild necroinflammation that may contrib-te to the development of cirrhosis and liver cancer.oreover, occult HBV might play a direct oncogenic

ole through both its integration into the host genomend the maintained transcriptional activity that, al-hough at low levels, may allow the synthesis of proteinssuch as X protein) with potential pro-oncogenic prop-rties. In summary, our data show that the directnd indirect mechanisms whereby overt HBV mightnduce tumor formation are mostly maintained by occultBV.HBV has been classified as a group 1 human carcin-

gen and is considered the second most important onco-enic agent after smoking tobacco.4,51 Our study defin-tively shows that HBV also maintains its oncogenic rolen the case of occult infection. Consequently, testingBsAg-negative individuals with progressive chronic

epatitis or frank cirrhosis for HBV DNA seems to be of

ith occult HBV infection. (A) Total RNA isolated from tumor (T) andzed by reverse-transcription PCR and the use of specific primers forntrol for the integrity of the RNA and the quantity loaded per lane.uthern blot analysis was performed to confirm the specificity of the

nts wanalyal coC) So

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rimary importance for the identification of patientsith a higher risk of developing liver cancer.

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Received April 22, 2003. Accepted October 2, 2003.Address requests for reprints to: Giovanni Raimondo, M.D., Unit oflinical and Molecular Hepatology, Department of Internal Medicine,oliclinico Universitario di Messina, 98124 Messina, Italy. e-mail:aimondo@unime.it; fax: (39) 0-90-293-5162.Supported in part by a grant from the Associazione Italiana per laicerca sul Cancro.The authors thank Pierfrancesco Cacciola, M.Sc., for the statisticalnalyses as well as Prof. Cesare Campello and Dr. Marina Crovatto for

heir contribution to the collection of the samples.